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Клінічні випробування препаратів "Екомеду" при лікуванні туберкульозу (оригінал на англ.мові)

TB basic science / Clinical trials and TB treatment

P14, 91121 ADJUNCT IMMUNOTHERAPY OF XDR-TB, GA Kutsyna,1 N Nathalia Prihoda,2 O Olga Arjanova,2 V Volodymyr Pylypchuk3 'Lubansk State Medical University, Luhansk, Ukraine; 2Lisichansk TB Dispensary, Lisichansk, Ukraine; 3EkomedLLC, Kiev, Ukraine. Fax: (380) 1234S678 e-mail: kutsyna@list ru

Keywords: immunotherapy; drug-resistant TB; XDR-TB According to the latest study patients with extensively drug-resistant tuberculosis (XDR-TB) had higher mortality rate than patients with MDR-TB [1], Only 29,3% of those with XDR-TB were cured compared to 46 2% of patients with MDR-TB [2] TB therapy success rate in Russian patients with XDR-TB as reported by Keshavjee et al., was 48.3% [3]. Earlier reported cure rates in Europe, USA, Peru and Korea were between 37.5%-67% indicating that XDR-TB poses serious clinical challenge [4­7], We have shown that immune-modulating interventions can significantly improve TB treatment outcomes [8]. We treated twelve XDR-TB individuals, seven of which in addition to standard chemotherapy received Dzherelo (Immunoxel), Svitanok and Lisorm - over-the-counter herbal immunomodulators available in Ukraine (Table 1) All these seven patients improved clinically and radiologically and were discharged after 3.7±0.8 months, with average/median time to mycobacterial clearance 28/25 days. None of five patients on TB drugs alone improved and one had died. These results reveal statistically different treatment outcomes due to immune intervention (Mantel Haenszel odds ratio= 11; P= 0.0009 at 95% CI). Adjunct herbal immunotherapy is safe, shortens treatment duration, and can overcome drug resistance even in patients with extremely poor prognosis. Further studies are needed to confirm our findings.

Treatment of cavitary and infiltrating pulmonary tuberculosis with and without the immunomodulator Dzherelo

S. I. Zaitzeva1, S. L. Matveeva1, T. G. Gerasimova1, Y. N. Pashkov1, D. A. Butov1, V. S. Pylypchuk2 and G. A. Kutsyna3
1) Department of Phtysiatry and Pulmonology, Kharkov National Medical University, Kharkov, 2) Ekomed LLC, Kiev and 3) Department of Xxx, Luhansk 7 State Medical University, Luhansk, Ukraine
Abstract
An open-label, 60-day trial was conducted in 75 newly diagnosed tuberculosis (TB) patients to assess the adjunctive effect of the oral immunomodulator Dzherelo with standard anti-TB chemotherapy (ATT) consisting of izoniazid, rifampicin, pyrazinamide and streptomycin (HRZS) administered as directly observed therapy. Group 1 (n = 28) with cavitary TB and group 2 (n = 17) with infiltrating pulmonary TB received 50 drops of Dzherelo twice daily in addition to HRZS. Group 3 (n = 30), which served as a control, received ATT Oonly. Liver damage indicators, bilirubin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) decreased to normal levels in groups 1 and 2, but increased significantly in group 3. Kidney failure markers, urea and creatinine, normalized in Dzherelo recipients, but were unchanged or worsened in the ATT-only group. The changes in serum lactate dehydrogenase, catalase, malondialdehyde and diene conjugates suggested that Dzherelo downregulates TB-associated inflammation. The anti-inflammatory property of Dzherelo was further supported by a favourable haematology profile, reduced erythrocyte sedimentation rate and faster defervescence. Radiological recovery was significant in both Dzherelo groups, but not in the control group (p = 0.0085, p = 0.025 and p = 0.23, respectively). 6|These findings correlated positively with sputum smear conversion and clinical findings (r = 0.94; p < 0.05). Mycobacterial clearance at day 30 was observed in 77%, 72% and 40% of patients in groups 1, 2 and 3, respectively. After 2 months sputum conversion rates in groups 1, 2 and 3 were 93%, 89% and 70%, respectively. Sixty-day treatment outcomes in groups 1, 2 and 3 as assessed by improvement in clinical features and respiratory function attained respective p-values of 0.008, 0.25 and 0.72, and 0.013, 0.48 and 0.0015. Dzherelo is thus useful as an immunotherapeutic adjunct in the management of TB.
Keywords: Botanical, cavitary, DOT, hepatotoxicity, herbal, immunotherapy, Mycobacterium, phytoconcentrate, phytomedicine, phytotherapy, X-ray
Original Submission: 11 June 2008; Revised Submission: 30 September 2008; Accepted: 23 October 2008 Edited by M. Drancourt Clin Microbiol Infect
Introduction
Tuberculosis (TB) is a re-emerging global public health problem, the incidence of which has increased in the Ukraine, as it has in other countries [1]. The incidence of TB in the Odessa region was 178 cases per 100 000 individuals in 1962. It gradually declined to 73.0, 42.0 and 41.6 cases per 100 000 in 1972, 1982 and 1992, respectively. This trend, however, then reversed, and by 2002 the incidence and prevalence of TB had risen to 80.4 and 330 cases per 100 000, respectively. Mortality caused by TB more than doubled from 10.2 per 100 000 to 21.6 per 100 000 between 1990 and 2001 [2]. The success rates of therapy in Eastern Europe, including the Ukraine, as in Africa, are substantially below those in other regions of the world [1]. In addition, the Ukraine has worsening epidemics of drug-resistant TB, which is increasingly converging with HIV. Despite the availability of anti-TB drugs the situation is far from ideal and better therapeutic interventions are clearly needed to reverse the current trend.
The oral immunomodulator
Dzherelo, which contains extracts of many plants (see below), is used in the Ukraine for the management of both TB and HIV infections, including simultaneous infections by both. Dzherelo was approved in 1997 by the Ukrainian Ministry of Health as an immunomod-ulating supplement and has been used extensively for various indications, including chronic bacterial and viral infections, autoimmune diseases and malignancy. In 1999 Dzherelo was recommended by the Ukrainian health authorities as an immune adjunct in the treatment of tuberculosis [3]. Previous clinical studies have indicated that Dzherelo can increase CD4 T-lymphocytes and decrease viral load, and that it improves the clinical response when combined with standard anti-retroviral (ART) or anti-tuberculosis therapy (ATT).
Dzherelo has been found to reduce the incidence of opportunistic infections and reverse TB-associated wasting. Dzherelo has also been found to alleviate the hepatotoxicity associated with ATT, as evidenced by improvement in liver function tests [4-10]. However, these studies have not dealt with the effect of Dzherelo on other clinical parameters associated with TB. Our study was thus aimed at defining  the adjunct effect of Dzherelo on clinical and radiological symptoms as well as on selected biochemical and blood parameters among patients with cavitary and infiltrating pulmonary TB. The addition of Dzherelo to standard ATT was compared with a treatment regimen consisting of ATT alone.
Materials and Methods 
Patients and ethical approval
The study involved 75 patients with newly diagnosed pulmonary TB. All patients were males aged between 19 and 68 years. All patients who presented with newly diagnosed pulmonary TB were enrolled in this study. No restrictive exclusion criteria for study enrolment were set up except that we disallowed patients who tolerated chemotherapy poorly or had resistant strains of Mycobacterium tuberculosis.
Patients were divided into three groups. Group 1 contained 28 individuals with cavitary or destructive forms of pulmonary TB; their mean ± standard deviation (SD) age was 46.5 ± 11.9 years (median 48 years). Group 2 contained 17 TB patients with the disseminated form of pulmonary TB but without cavitary lesions; their mean age was 46.4 ± 13.7 years (median 47 years). The third group, which received standard TB therapy without Dzherelo and served as a comparison population, contained 30 patients with a mean age of 42.7 ± 11.3 years (median 44 years) and included patients with the cavitary and the infiltrating forms of pulmonary TB. Patients were allocated to the Dzherelo and ATT-only groups without formal randomization. All study patients presented with the active form of pulmonary TB. The most common symptoms were prolonged heavy cough, pain in the chest, high fever, profuse night sweats, fatigue, dyspnoea, haemoptysis, and loss of weight and appetite. Active pulmonary tuberculosis was defined by a medical history and clinical findings compatible with tuberculosis, a chest X-ray showing lung involvement, and a positive sputum smear for acid-fast bacilli (AFB) and positive culture for M. tuberculosis. Participation in this study was voluntary and conduct of the trial was approved by the internal review board of the TB dispensary.
Treatment regimen
All patients received standard ATT, consisting of orally administered izoniazid (H; 300 mg), rifampicin (R; 600 mg) and pyrazinamide (Z; 2000 mg), and intramuscular injection of streptomycin (S; 1000 mg). The anti-TB drugs were procured through the Ukraine’s centralized national supply system. Patients in groups 1 and 2 also received 50 drops of Dzherelo, given in a glass of water twice daily, usually 2 h after breakfast and 30 min before supper. The treatment was administered for 60 days as directly observed therapy (DOT) to patients hospitalized in our department at Kharkov National Medical University, between March and May 2006.
The over-the-counter phytoconcentrate Dzherelo (Immunoxel) was generously supplied by Ekomed LLC (Kiev, Ukraine). It contains a concentrated aqueous alcohol extract from medicinal plants, including: aloe (Aloe arborescens); common knotgrass (Polygonum aviculare); yarrow (Achillea millefolium); centaury (Centaurium erythraea); snowball tree berries (Viburnum opulus); nettle (Urtica dioica); dandelion (Taraxacum officinale); sweet-sedge (Acorus calamus); oregano (Origanum majorana); marigold (Calendula officinalis); sea buckthorn berries (Hippophae rhamnoides); elecampane (Inula helenium); tormentil (Potentilla erecta); greater plantain (Plantago major); wormwood (Artemisia sp.); Siberian golden root (Rhodiola rosea); cudweed (Gnaphalium uliginosum); licorice (Glycyrrhiza glabra); fennel (Foeniculum vulgare); chaga (Inonotus obliquus); thyme (Thymus vulgaris); three-lobe beggarticks (Bidens tripartita); sage (Salvia officinalis); dog rose (Rosa canina), and juniper berries (Juniperus communis). Dzherelo was approved in 1997 by the Ukrainian Ministry of Health as a dietary supplement. In 2006 it received the status of a ‘functional food’, placing it in a superior category of herbal supplements which can carry medical claims substantiated by clinical evidence.
Clinical endpoints
Primary endpoints of interest in this study were the effect of prescribed therapy on clinical features and radiological and microbiological findings. The clinical manifestations of TB that were considered comprised a complex of so-called ‘intoxication’ symptoms and signs, including fever, night sweats, loss of appetite, weight loss, weakness, depression, cough, and lung crepitations, etc. [11,12]. The second aspect of clinical evaluation concerned respiratory function and covered symptoms such as dyspnoea and lung function tests, including total lung capacity (TLC), forced vital capacity (FVC), forced expiratory volume (FEV), residual volume (RV), carbon monoxide transfer factor, O2UQ and CaO2 according to the Dembo and Liberman classification [11,12]. In both cases, the features or tests were given a score ranging from 0 (severely affected) to 3 (absent or normal) and the mean of these scores was used to define the overall result as indicative of no, mild or severe abnormality. In addition, information on the potential adverse effects of Dzherelo administered in combination with chemotherapy was sought via questionnaires completed by the patients.
Laboratory evaluation
A standard microbiological examination of a sputum smear stained by the Ziehl–Neelsen method was conducted prior to study entry and at days 30 and 60 from the start of treatment. Isolates of M. tuberculosis were tested for sensitivity to first- and second-line anti-TB drugs with a commercially available kit (Tulip Diagnostics Pvt Ltd, Goa, India) [7]. These tests were supplemented by regular examination of haematological and biochemical parameters. Plasma levels of catalase, malondialdehyde (MDA), lactate dehydrogenase (LDH) and diene conjugates (DC) were measured as markers of oxidative stress, as described earlier [7].
Statistical analysis
The results were analysed with the statistical software GraphPad (GraphPad Software, Inc., La Jolla, CA, USA). Baseline quantitative values were compared with end-of-study values by paired or unpaired Student’s t-test. Non-parametric or categorical values of treatment outcomes were compared by McNemar or chisquare contingency tables. All statistical analyses were performed on an intent-to-treat basis, involving the total number of patients without subgrouping them into responders and non-responders. The resulting probability values were considered significant at p < 0.05.
Results
Lack of adverse reactions
During the entire follow-up no adverse reactions attributable to Dzherelo were identified. In particular, no dyspepsia, malaise, intolerance or allergic reactions were evident at any time (data not shown).
Effect on body temperature
The dynamics of body temperature among treated patients are shown in Fig. 1. All three groups showed a decline in mean body temperature during the investigation. Group 1, however, showed a clear tendency towards more rapid normalization: the mean axillary temperature on day 30 reached a ‘normal’ level, at 36.65 ± 1.24 C, compared with 37.17 ± 2.02 C in group 3 (p < 0.005). Group 2 had a similar rate of decline to group 1, whereas group 3 showed a slower rate of decrease.
Effect on haematological parameters
The effects of ATT and Dzherelo on blood cell counts, erythrocyte sedimentation rate (ESR) and haemoglobin are shown in Table 1. Patients in groups 1 and 2 reacted in a similar manner and displayed changes which appeared to be specific to Dzherelo intervention as opposed to the effect of ATT alone in group 3 patients. In particular, the relative content of lymphocytes almost tripled among Dzherelo-treated patients, reaching normal levels, whereas patients in the control group failed to attain normal values. Elevated counts of cells, such as neutrophils, eosinophils and total leukocytes, commonly associated with inflammation, were reduced to a greater extent in Dzherelo-treated patients, although the decrease was also statistically significant in group 3 patients. By contrast, monocyte levels were almost unchanged among patients who received Dzherelo, whereas patients in the comparison group had a greater reduction in these cells. Other cells such as erythrocytes behaved in a similar manner in all three groups. Erythrocyte sedimentation rate declined faster in the Dzherelo groups. Haemoglobin concentrations decreased at the same rate in all patients. In general, Dzherelo appeared to have greater effect in normalizing the haematology profile than ATT treatment alone.
Serum biochemistry markers of liver and kidney function improved markedly among Dzherelo recipients, whereas patients in group 3 showed a decline in liver function (Table 2). All three evaluated hepatic markers, bilirubin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), decreased in groups 1 and 2 but increased in group 3 in a statistically significant manner. Levels of urea and creatinine normalized in Dzherelo recipients but were unchanged or worsened in ATT-alone patients.
 
Effect on oxidative stress markers
The effect of therapy on plasma levels of catalase, MDA, LDH and DC as markers of oxidative stress and lipid peroxidation are presented in Table 3. There is a clear impression that Dzherelo helps to slow down the pathological processes associated with TB and its treatment. For example, concentrations of LDH, a marker indicative of cell injury and inflammation, in patients receiving Dzherelo remained within the normal range (0.8–2.5 IU), but almost tripled in patients on ATT alone. Increases in levels of DC and MDA were lower in Dzherelo recipients than in ATT-alone recipients. The decrease in antioxidant catalase levels was smaller in the Dzherelo groups, whereas, among those on ATT alone, these were more than halved.
Effect on mycobacterial clearance
Bacterial clearance, evaluated by repeated sputum Ziehl–Neelsen staining at monthly intervals, is shown in Fig. 2. At day 30, 77% and 72% of patients in groups 1 and 2, respectively, had negative findings, whereas only 40% of patients in group 3 showed sputum clearance of bacteria. After 60 days the corresponding figures were 93%, 89% and 70%, respectively. The difference between the two groups receiving Dzherelo was not significant at either test-point (p = 0.42, p = 0.32, respectively, as tested by non-parametric chisquare analysis). When groups 1 and 2 were compared with group 3, the statistical difference was highly significant at 30 days (p < 0.0000001, p < 0.000005, respectively) and 60 days (p = 0.000028, p = 0.00088, respectively).
Effect on clinical and respiratory symptoms
The response rates of patients receiving chemotherapy with or without immunotherapy are shown in Figs 3 and 4.
Changes in two categories of clinical manifestations were evaluated: the first was a complex of so-called ‘intoxication’
symptoms and the second represented ‘respiratory function’. At study entry the degree of severity of both categories of clinical manifestations was similar in all three groups. Contingency table analysis comparing baseline results between groups 1 vs. 2, 1 vs. 3, and 2 vs. 3 reveals p-values of 0.40, 0.28 and 0.002, respectively, for the intoxication and 0.22, 0.21, and 0.0008, respectively, for the respiratory function categories of clinical features. The inter-group differences between Dzherelo recipients and ATT-alone recipients became greater by the end of the first month. After 60 days of treatment, the p-values for comparisons between groups 1 vs. 2, 1 vs. 3 and 2 vs. 3 reached 0.13, 0.004 and 0.22, and 0.13, 0.00002 and 0.13, respectively, for the intoxication and respiratory function categories of clinical features, respectively. The intra-group differences between baseline and 30 days were not significant by McNemar test. The p-values for groups 1, 2 and 3 were 0.07, 0.61 and 0.7, respectively, for intoxication, and 0.11, 1.0 and 0.5, respectively, for respiratory function categories. However, intra-group changes between baseline status and 60-day treatment outcomes for groups 1, 2 and 3 for intoxication and respiratory function attained p-values of 0.008, 0.25 and 0.72, respectively, and 0.013, 0.48 and 0.0015, respectively. These findings indicate that the addition of Dzherelo to ATT enhances the efficacy of ATT and achieves statistically significant favourable clinical responses at 2 months after treatment initiation.
Effect on radiological manifestations
Results of chest X-ray evaluation of lung segments affected by cavitary and non-cavitary infiltrating TB are presented in Fig. 5. Chi-square contingency table analysis comparing baseline differences between groups 1 vs. 2, 1 vs. 3 and 2 vs. 3 showed that p-values were 0.39, 0.82 and 0.18, respectively, indicating that at study entry patients in different treatment groups had similar levels of pulmonary invasion.
After 60 days the differences in response to treatment became highly significant for Dzherelo recipients, but not for ATT-treated patients. Observed radiological improvement as determined by the McNemar categorical test for groups 1, 2 and 3 attained p-values of 0.0085, 0.025 and 0.23, respectively. Thus, the healing rate was
greater among patients on Dzherelo than among those who received ATT only. These observations correlated positively with microscopy and clinical findings (r = 0.94; p < 0.05).

FIG. 5. Radiological findings prior to and after 60 days of therapy, giving the number of pulmonary segments involved. Numbers over columns represent numbers of patients.
Discussion
This open-label, comparative study in a group of newly diagnosed TB patients at our dispensary reveals that when standard, first-line anti-TB drugs are combined with Dzherelo, significant clinical and radiological improvements and clearance of M. tuberculosis take place at a higher rate than in patients on ATT alone. Biochemical and haematological analyses of blood samples supported this favourable adjunctive effect of Dzherelo, which did not show any adverse effects throughout the investigation. Our findings support earlier clinical studies of Dzherelo demonstrating similar results [3–10]. The proportion of TB patients cured according to sputum culture and radiology was two- to four-fold higher among Dzherelo recipients. Furthermore, the combination therapy gave results after a much shorter period.
Earlier indications showed Dzherelo to exhibit anti-inflammatory properties [5–7]. This notion is reinforced by the findings of this study. The decline in elevated body temperature to < 36.6 C occurred in the Dzherelo-treated groups within 30 days, as opposed to the control group, where this happened only after 60 days. As fever is an independent indicator associated with an aggressive form of TB, the outcome of treatment is likely to be influenced positively by Dzherelo [13]. Similarly, the indices of inflammation such as leukocytosis, neutrophilia and eosinophilia tended to return to normal at an earlier date and more strikingly in Dzherelo recipients than in patients on ATT alone. Elevated numbers of these cells are known to be associated with active pulmonary TB and their normalization is considered to indicate a favourable effect on the course of disease [14,15]. The observed changes in the blood profile are paralleled by findings from previous studies; in particular, lymphocyte counts almost tripled among Dzherelo recipients, whereas in control patients they failed to reach normal levels. Restoration of lymphocyte counts is considered to have a favourable prognostic significance [15]. Although Dzherelo did not affect erythrocyte counts, it did reduce the ESR (considered to be a marker of TB-associated inflammation) much more efficiently than ATT alone [16].
Lactate dehydrogenase, another indicator of inflammation, had a three-fold slower rate of accumulation in the Dzherelo group than in the ATT-alone group, suggesting a
favourable anti-inflammatory property of Dzherelo [17]. During pulmonary inflammation in patients with active TB increased amounts of reactive oxygen species are produced as a consequence of the phagocyte respiratory burst. Among the manifestations of these free radical-mediated processes are higher concentrations of DC and MDA [18,19]. Increases in DC and MDA were lower in Dzherelo recipients than in ATT-alone patients, suggesting that herbal components in this preparation can downregulate the inflammatory process. Observed changes in antioxidant catalase levels in TB patients are in line with these findings as the rate of decline of this enzyme was twice as slow in the Dzherelo groups as in the ATT-alone group. The oxidative injury is an essential component of inflammatory damage and it is clear that the reversal of excessive peroxidation favourably influences the outcome of the disease by lessening the impact on pulmonary tissue damage and respiratory distress [20].
The hepatotoxicity induced by anti-TB drugs has serious adverse consequences for treated patients and imposes limitations on treatment options [21]. The addition of Dzherelo reversed ATT-associated liver damage, as evidenced by the two- to three-fold reduction of baseline bilirubin and AST and ALT levels in the Dzherelo groups compared with the ATT-alone group, in which all three liver function parameters continued to rise. These observations confirm previous clinical studies in which Dzherelo has been shown to counter the hepatotoxicity of anti-TB as well as anti-HIV drugs [5–7].
Renal failure characteristics as assessed by urea and creatinine output seemed to improve upon intake of Dzherelo, whereas in group 3 patients there was practically no improvement.
The conversion of sputum smear from positive to negative is considered a critical indicator of the efficacy of anti-TB intervention [22]. We observed that Dzherelo accelerated and significantly enhanced bacillary clearance (Fig. 2). A large, prospective Canadian study evaluating 428 TB cases showed that 48% of patients had converted after 4 weeks [22], a reduction which seems to be in line with our 40% conversion rate in the ATT-alone group, but is smaller than the 77% and 72% clearance rates in groups 1 and 2, respectively. In a recently reported study from India, the 2-month sputum conversion rate in newly diagnosed TB was 58%, which is also lower than our Dzherelo-facilitated conversion rates at 60 days (i.e. 93% and 89%, respectively) [23]. The difference between ATT-alone and ATT-plus-Dzherelo recipients was highly significant at both time-points, indicating that administration of this botanical immunomodulator produces better results than standard treatment. A longer follow-up study is needed to assess the bacteriological relapse rate in our patients.
The mycobacterial clearance results agree with our clinical findings. Based on two categories of clinical evaluation, features of intoxication and respiratory function, it became apparent that the combination of Dzherelo with ATT produces a faster and more profound beneficial effect than ATT alone. As Figs 3 and 4 show, only 50.0% and 33.3%, respectively, of patients on ATT alone became free of indicators after 60 days of treatment. By contrast, in groups 1 and 2, respectively, 85.7% and 94.1% were alleviated of intoxication features, and 85.7% and 76.5% became free of respiratory function problems. The proportion of patients with mild problems also fell significantly in Dzherelo-treated patients. Although the classification of clinical features into the two categories used in this study is not employed in countries outside the former Soviet Union, these categories continue to be commonly used in the Ukraine and have been proven reliable by several generations of Ukrainian tuberculosis physicians since the 1950s [11,12,24]. These categories, especially when used in combination with bacteriological and chest X-ray evaluations, appear to facilitate the dependable assessment of the efficacy of anti-TB intervention.
Indeed, radiographic examination of lung segments affected by cavitary and non-cavitary TB lesions unequivocally supported the benefits of adding Dzherelo to the standard TB treatment regimen. As Fig. 5 shows, there was clear improvement in groups 1 and 2 after 60 days compared with group 3, in which 30% of patients still had three or more affected pulmonary segments. Our findings indicate that, although radiographic involvement in one or two segments persisted in all treated patients at 60 days, the length of follow-up was perhaps too short to see complete pulmonary recovery. For this reason a longer study is required.
Throughout the study we observed statistically significant discrepancies between groups 1 and 2 in terms of response at certain therapy endpoints, for which we do not have valid explanations. It is likely that these differences are attributable to differences in pulmonary lesions in these groups. It has been reported that a predominant Th1 immune response is observed in non-cavitary patients, whereas cavitary-involved segments exhibit the presence of Th2 lymphocyte subsets [25]. This needs to be explored further in a larger population of patients.
Several immunomodulators have been tested as adjuvants for TB therapy. These include likopid, interferon-c (IFN-c) and environmental Mycobacterium vaccae, all of which have shown promising results in preliminary trials. Likopid is a synthetic lipid analogue of bacterial cell wall which has been developed in Russia [26]. The combination of likopid with ATT in patients with pulmonary TB resulted in positive clinical effects, including bacterial culture conversion in 80% of cases, a lower rate of positive sputum staining, lack of intoxication, and accelerated resolution of pulmonary infiltrations. Such effects were not observed in control patients who received conventional ATT. Interferon-c has been used as an adjunct immunotherapy for TB since 1997 [27]. Several clinical trials of inhaled or injected IFN-c have shown positive clinical outcomes, although to less extent than in likopid-treated cases [27–29]. Environmental M. vaccae has shown encouraging results in some trials, but
was ineffective in others [30–32]. For example, treatment of drug-susceptible pulmonary TB with M. vaccae did not improve radiological responses or resolve cavitary disease [32].
In conclusion, the phytopreparation Dzherelo, which has been used as an immunomodulating adjuvant to standard TB therapy, has been shown to be safe and capable of reversing ATT-associated hepatotoxicity. In addition, Dzherelo seems to reduce inflammation, as evidenced by several haematological and biochemical markers, and significantly accelerated and enhanced the sputum smear conversion rate. Clinical and radiological improvements resulting from the combination of Dzherelo and ATT occurred earlier and at higher rates than those from ATT alone. It is hoped that when herbal medicines such as Dzherelo are validated through rigorous scientific and clinical research, their integration into modern medical practice will be more readily accepted and treatment options for TB will be expanded as a result.
Acknowledgements
We thank all volunteers who participated in this study, the clinicians, nurses and laboratory personnel, whose wholehearted support made the study possible, and our colleagues, who shared their experience with botanicals and the tuberculosis drugs used in the study. The final stage of this study was supported by compassionate financial support graciously provided by the MAPI Research Trust, Lyon, France, a non-profit organization that advances the art and use of scientific methods to patient-reported outcome measures. This work was presented in part at the Keystone Symposia on HIV Pathogenesis and HIV Vaccines, 27 March to 1 April 2008, Banff, AB, Canada, through a grant from the Bill and Melinda Gates Foundation’s Global Health Travel Award.
Transparency Declaration
All authors, except VSP, declare no conflicts of interest. VSP developed Dzherelo and is director of Ekomed LLC.
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Clinical validation of sublingual formulations of Immunoxel (Dzherelo) as an adjuvant immunotherapy in treatment of TB patients

Yuri V Efremenko1, Olga V Arjanova1, Natalia D Prihoda1, Larisa V Yurchenko1, Nina I Sokolenko1, Igor V Mospan2, Volodymyr S Pylypchuk2, John Rowe3, ai Jirathitikal4, Aldar ourinbaiar5 & Galyna A Kutsyna*
1Lisichansk Tuberculosis Dispensary, Lisichansk, Ukraine 2Ekomed LLC, Kiev, Ukraine 3Island Abbey Foods Ltd, ottetown, Prince Edward Island, da 4Immunitor Thailand Co., LLC, Bangpakong Industrial Park, hoengsao, Thailand unitor USA Inc., College Park, MD, USA
*Author for correspondence: Luhansk State Medical University, k, Ukraine kutsynagalyna@yahoo.com

Immunoxel (Dzherelo) is a water-alcohol extract of medicinal plants used in Ukraine as an adjunct immunotherapy to TB and HIV therapy. Four types of solid sublingual formulations of Immunoxel were made: sugar dragées, sugar-coated pills, gelatin pastilles and dried-honey lozenges. They were administered once-daily along with TB drugs. After 1 month, 84.1 % of TB patients became sputum-negative with rates in individual groups of 89.5, 70, 76.9 and 100%, respectively. The conversion rate was independent of bodyweight, age, gender, differences in chemotherapy regimens or whether subjects had newly diagnosed TB, re-treated TB, multidrug-resistant TB or TB with HIV coinfection. Patients experienced earlier clinical improvement, faster defervescence, weight gain, a higher hemoglobin content and reduced inflammation as evidenced by lower leukocyte counts and erythrocyte sedimentation rate. By contrast, in the placebo group, only 19% of patients had converted. These findings imply that mucosal delivery of solid Immunoxel is equivalent to the original liquid formula given per os twice-daily for 2-4 months.

KEYWORDS: biomarker ■ immunomodulator ■ immunotherapy ■ inflammation ■ mycobacterium ■ phytomedicine

TB is a global killer. Approximately 2 billion people, or a third of the global population, are latently infected with Mycobacterium tuberculosis. Annually, nearly 9 million of these people develop active TB and 2 million die. Current tuberculosis drugs have existed for more than 40 years and must be taken for 6—9 months for drug-sensitive disease and for up to 24 months for drug-resistant strains such as multidrug-resistant (MDR)-TB and extremely drug-resistant TB [1]. As new TB drugs currently under development will be not available in the near future, new approaches are urgently needed [2]. One of these approaches is immunotherapy, which is believed to enhance the efficacy of chemotherapy and can potentially shorten treatment duration [3-5].
The multiherbal immunomodulator Immunoxel (Dzherelo), manufactured by Kiev-based botanical company Ekomed, has been sold in Ukraine for the last 15 years. More than a dozen clinical trials have been published over the past 10 years showing its safety and efficacy in approximately 1200 TB and HIV patients [6]. This extensive experience can be summarized as follows: Dzherelo produced higher levels of IL-2, whereas in the control group given TB drugs alone the levels declined; the production of IL-6 increased in the control group but declined in the immune intervention group; levels ofTNF-a were suppressed in the immunotherapy group, but had risen in control patients; the pattern of production of IFN-g was the opposite to that of TNF-a; moderately decreased levels of IFN-a were observed in both treatment arms, but differences were not significant. These changes in cytokine profile were accompanied by the following clinical changes: the typical sputum conversion rate among MDR-TB, extremely drug-resistant TB and TB with HIV coinfection (TB/HIV) patients after 2—4 months of herbal immunotherapy ranged from 85 to 100%, but in chemotherapy controls it took 6—24 months to reach 48—85%. While these results are clearly in favor of Immunoxel, they are not ideal since the duration of adjunct treatment is still not optimal and faster-acting regimens would be more advantageous. In order to overcome this drawback, the authors have made four types of solid, sublingual formulations of Immunoxel and tested them clinically to see if they performed better than original water—alcohol extract of herbs.

Materials & methods

■ Patients
The experimental group involved 69 hospitalized patients with TB (76.8%) and TB/HIV (23.2%). They were randomly assigned to four different sublingual formulations of Immunoxel given once per day (Tables 1 & 2).

The significant proportion of patients presented with newly diagnosed TB (37.7%); remaining patients had re-treated TB (RTB), treatment-failed TB, MDR-TB and extra-pulmonary TB at a 31:6:4:2 ratio. Most of the patients were treated with a standard isoniazid, rifampicin, pyrazinamide, ethambu-tol and streptomycin combination (84.9%) or were on individualized chemotherapy regimens (15.1%). The duration of chemotherapy prior to immunotherapy initiation ranged between 1 and 11 months with a mean ± standard deviation (SD) of 2.6 ± 2.4 (median: 2.0) months for the group, comprising 42% of patients who were treated for 1 month or less, and the rest were treated for a mean ± SD of 3.8 ± 2.7 (median: 2.5) months. The study group had 15 females and 54 males between the ages of 20 and 61 years with a mean ± SD age of 38.7 ± 10.5 (median: 36.0) years. The baseline bodyweight and BMI ranged between 40 and 98 kg and 15.2 and 36.0 kg/m2, respectively, with a mean ± SD of 56.9 ± 9.2 (median: 56.0) and 19.1 ± 3 (median: 18.5), respectively. At study initiation, only six out of 69 (8.7%) patients had normal 36.8°C axillary body temperature, with an overall mean ± SD of 38.0 ± 0.7 (median: 38.0)°C.
Twenty one patients in the control chemotherapy group received placebo sugar pills (Tables3 &4). The group had afemale:male ratio of 2:19 with an age range between 21 and 58 years and a mean ± SD of 36.6 ± 9.8 (median: 35.0) years. The baseline bodyweight and BMI ranged between 47 and 72 kg and 16.3 and 22.5 kg/m2 with a mean ± SD of 60.9 ± 6.7 (median: 61.0) and 19.9 ±1.6 (20.5), respectively. The baseline distribution of TB categories was according to the ratio 5:11:5:6 for newly diagnosed TB (first diagnosis), RTB, MDR-TB andTB/HIV, respectively. Most patients (57.1%) received standard first-line TB chemotherapy while the remaining patients were on an individualized TB drug regimen. At the beginning of the study, four out of 21 patients (19%) had normal body temperature with an intragroup mean ± SD of 37.7 ± 0.5 (median: 38.0)°C. Participation in this study was voluntary and patients were eligible to enroll only after signing the written consent. The conduct of the trial was approved by the internal review board of the Lisichansk TB Dispensary [101].

■ Solid formulations of Immunoxel
Four different solid formulations were designed for sublingual absorption:
■ Sugar-based dragees
■ Sugar-coated pills
■ Gelatin pastilles
■ Dried-honey lozenges


Sugar-based dragées were made by Ekomed. Sugar-coated pills were made in collaboration with Immunitor at their manufacturing plant in Chachoengsao, Thailand. The gelatin-based formulation was made by a local confectionery factory specializing in the production of gummy bear-like candies. The dried-honey lozenges were made in collaboration with Island Abbey Foods Ltd (Charlottetown, Prince Edward Island, Canada). This company has a unique proprietary technology for making dried-honey lozenges.
Each sublingual preparation contained either five or ten drops (125 or 250 pl) of Immunoxel, which was administered once per day for a month. Immunoxel, an improved and simplified version of the herbal immunomodulator Dzherelo, contains an aqueous—alcohol cold extract from medicinal plants including aloe (Aloe arborescens), licorice (Glycyrrhiza glabra), dog rose fruit (Rosa canina), oregano (Oreganum majorana), sage (Salvia officinalis), thyme (Thymus vulgaris), fennel (Foeniculum vulgare), purple coneflower (Echinacea purpurea), dandelion (Taraxacum officinale), nettle (Urtica dioica), marigold (Calendula officinalis), greater plantain (Plantago major), wormwood (Artemisia sp.), common knotgrass (Polygonum aviculare), yarrow (Achillea millefolium), cen-taury (Centaurium erythraea), elecampane (Inula helenium), tormentil (Potentilla erecta), cudweed (Gnaphalium uliginosum), three-lobe beggarticks (Bidens tripartita), Siberian golden root (Rhodiola rosea), chaga (Inonotus obliquus), snowball tree berries (Viburnum opulus), seabuckthorn berries (Hippophae rhamnoides) and juniper berries ( Juniperus communis). This multiherbal extract is manufactured by Ekomed according to international Hazard Analysis Critical Control Point (HACCP) standards and has been approved by the Ministry of Health of Ukraine as an immunomodulating herbal supplement belonging to a functional food category.

■ Laboratory evaluation
The sputum microscopy on acid-fast bacilli smears was conducted in a blinded fashion at baseline and 1 month later. TB drug resistance was determined by a commercially available kit (Tulip Diagnostics, Goa, India) in approximately a third of the patients. The failure to test every patient for drug resistance was due to a temporary lack of funds for laboratory services. MDR-TB status was assigned when resistance to both isoniazid and rifampicin, with or without resistance to other drugs, was present.
The hematology parameters such as hemoglobin content, leukocyte counts and erythrocyte sedimentation rate (ESR) were evaluated by standard routine techniques at baseline and repeated 1 month later.

■ Statistical analysis
The obtained results were analyzed with commercially available statistical software (GraphPad Software Inc., La Jolla, CA, USA). The paired Student's t-test was used to compare before and after mean values by assuming that the distribution of the differences was in accord with the Gaussian distribution. Side-by-side comparison between two independent populations was performed by an unpaired t-test or by a Mann-Whitney U test. The Wilcoxon ranking test was used to compare paired before and after nonparametric values such as conversion rates arranged in a 'yes' or 'no' binary manner. Fisher's exact two-tailed test was employed for analysis of data arranged in a contingency table. Comparison across multiple groups was tested either by analysis of variance or a categorical Kruskal-Wallis test. All statistical analyses were performed on an intent-to-treat basis, involving all initially enrolled patients including fatalities. The resulting probability values were considered significant at p < 0.05.

Results
■ Sugar dragées
The first trial of a solid form of Immunoxel involved small-size (~4 mm) sugar dragées to which gradually increasing doses of Immunoxel were added, so that ten of them contained a total of ten drops of Immunoxel. The test involved 21 patients who were instructed to place dragées under the tongue until fully absorbed. Half of the patients received five drops and the other half a dose of ten drops. After 1 month, every measured end point had improved significantly (p < 0.0001). These and other results described below are summarized in Tables 1 & 2 and Figure 1. The percentage of patients with negative sputum conversion was 81% — results from which the authors did not exclude the three patients who died. In two of these patients post-treatment sputum samples were not analyzed and were assumed to remain positive at the time of death. One, a female patient, was in a very unfavorable condition and died a few days after checking into the hospital. Another female patient was obese (BMI = 36 kg/m2) and had a fatal stroke before her sputum could be analyzed. The third patient, whose sputum had converted to negative, unfortunately died from an alcohol overdose after the 1-month study was concluded. If the two female patients without post-treatment sputum results are excluded, then the conversion rate in this group stands at 89.5%. No difference was seen in sputum outcome between five- and ten-drop doses.
■ Sugar-coated pills
Following the initial trial with sugar dragées, which were found to be inconvenient owing to the high moisture absorption property and cumbersome dosing procedure, conventional sugar-coated, oval-shaped pills were made, each containing the same ten-drop dose of Immunoxel and standard excipients such as lactose, talc, magnesium stéarate and menthol flavoring. Ten patients were enrolled to evaluate the effect. Of these ten patients, seven became negative after 1 month (p < 0.0001). Bodyweight and temperature, as well as BMI, demonstrated statistically significant improvements. Other end points, such as hemoglobin content, ESR and leukocyte counts, had improved as well, but statistical significance was not reached owing to sample size (Tables 1 &2 & Figure 1). This formulation had no additional active ingredients that could have biased the outcome and was essentially the same as the sugar dragées described above. If sugar-coated pill results are combined with the outcome from sugar dragées, then the overall conversion rate for sugar-based formulations is 79.8%.


■ Gelatin pastilles
The batch of Immunoxel formulated into so-called 'gummy candies' contained a ten-drop dose per pastille, also containing in addition sugar, glucose syrup, starch, food coloring, citric acid and porcine gelatin, according to their standard procedure. Out of 20 patients, 13 were smear-positive at baseline, of whom ten (76.9%) became negative after 1 month (p < 0.0001). All other end points except leukocyte counts had improved in a statistically significant manner (Tables 1 & 2 & Figure 1).

■ Dried-honey lozenges
This preparation was made by Island Abbey Foods Ltd. This company has a unique proprietary technology for slowly drying liquid honey into solid dried-honey lozenges. Two batches were manufactured, one with four drops and another with ten drops of Immunoxel per lozenge, which were then given to two equal groups of patients (n = 18). After 1 month, every smear-positive patient in both groups had converted. All other measured end points were also improved in a statistically significant manner (Tables 1 & 2 & Figure 1).


■ Comparison of outcomes from different Immunoxel formulations
The baseline characteristics of four groups of patients were tested by the analysis of variance or Kruskal—Wallis test, as appropriate, and were found to be matched by age (p = 0.25), BMI (p = 0.83), and hematological parameters such as hemoglobin (p = 0.36) and ESR (p = 0.12), but not by gender distribution (p = 0.03) or leukocyte counts (p = 0.03). The primary end point of the study is sputum conversion (Figures 1 & 2). When results from the four formulations were compared by the nonparametric Kruskal—Wallis test, the outcomes were not different from each other (p = 0.15), even though, at baseline, there were differences between individual groups (p = 0.02). This indicates that solid formulations have an equivalent biological activity (Tables 1 & 2 & Figure 1). The effective dose was equivalent to ten drops of liquid Immunoxel. However, it is possible that smaller doses could be equally effective, since sugar-based dragées and dried-honey lozenges tested at five- and four-drop doses produced the same favorable outcome. The stratified analysis of data comparing outcomes between newly diagnosed TB (n = 26), RTB (n = 31), TB/HIV (n = 16) and MDR-TB (n = 4) also failed to reveal any difference (p = 0.17). Similar results were obtained when outcomes of patients on standard isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin chemotherapy (n = 57) were compared with the conversion of patients receiving individualized drug regimens (n = 12). The Mann-Whitney U test shows that the two treatment regimes produced identical favorable outcomes (p =0.83). The duration of chemotherapy prior to immunotherapy also had no impact on outcome. Those who were treated for 1 month or less were converting as readily as those who were treated for longer; for example, 1 versus 3.8 months (p = 0.67). The same Mann-Whitney U test failed to reveal gender- or age-dependent differences. Males and females, as well as younger or older patients (the age threshold was equal to highest mean age + highest SD, i.e., > 48 years), converted at the same rate without any gender or age preference (p = 0.5 and p = 0.29, respectively). A baseline BMI higher or lower than the normal 18.5 kg/ m2 value had no effect on conversion rate either (p = 0.6). However, when these data were compared with outcomes from control patients, there was an obvious and highly significant difference (p < 0.0001) irrespective of formulation type, discrepancies in baseline diagnosis, gender, age, BMI or treatment regimen.

■ Treatment outcome in the placebo group
As a control, the authors used sugar-coated pills without Immunoxel, which were administered to 21 randomly chosen patients who were concurrently receiving TB drugs. Despite a sufficient sample size that was equivalent to or larger than any of the Immunoxel-treated populations, the only statistically significant results were obtained with ESR and sputum smear conversion (Tables 3 & 4). As can be seen from Figure 1, the proportion of patients responding to chemotherapy was adequate; however, the magnitude of response was smaller. For example, control group patients gained less weight than those who received various Immunoxel formulations; the mean weight and BMI accruals were 0.5 kg and 0.2 kg/m2 versus 1.9 kg and 0.6 kg/m2 in the immune intervention arm. Similar trends were observed with other end points indicating that, even though chemotherapy alone has been beneficial to TB patients, its potency was lower than that of chemotherapy with adjunct immunotherapy.

■ Discussion
This study reveals that all tested solid formulations of Immunoxel produced similar favorable outcomes. The conversion rates in patients were identical regardless of their baseline diagnosis, such as first diagnosis, RTB, MDR-TB or TB/HIV. The success of therapy was not affected by concomitant chemotherapy regimens, and both standard and individualized treatments had comparable favorable outcomes. Similarly, the duration of chemotherapy prior to immunotherapy had no impact either. Those who were treated for 1 month or less converted at the same rate as those who were receiving TB drugs for 4 months on average. Neither gender nor age was a significant factor influencing sputum conversion. Underweight patients converted at the same rate as those in the normal weight range. In addition to bacterial clearance, Immunoxel has shown clear improvement in clinical manifestations, weight gain, defervescence, reduction of inflammation such as leukocytosis and ESR, and increase in hemoglobin content. All these benefits were described in prior studies of liquid Immunoxel, except that solid formulations acted faster and much smaller and less frequent doses were required.
A bewildering range of botanical supplements, especially in traditional Chinese and Ayurvedic medicine, is used today with the intention of boosting immunity and curing various diseases including tuberculosis [7]. However, the clinical or experimental evidence is lacking in most cases [8]. To the best of the authors' knowledge, clinical results from only three plant-derived preparations with anti-TB properties have been reported in peer-reviewed literature [9-11]. The oldest known is umck-aloabo - a traditional South African medicine from the roots of Pelargonium sidoides — used to treat TB and respiratory tract infections since the late 19th century [9]. Another report concerns inhaled tea tree oil from Australian Melaleuca alternifolia, which has produced remarkable recovery in a few cases of advanced TB [10]. Finally, a multiherbal water-infusion cocktail based on Russian folk medicine has been reported to reduce the duration of TB treatment to 6.4 months, instead of 8.6 months in controls [11]. For other phytomedicines, there is only anecdotal evidence without any systematic clinical studies [12,13]. Immunoxel stands out in this crowd since more than a dozen clinical trials involving a total of 1200 patients have been published by the authors and their colleagues [14-26]. During the past couple of years the authors have attended several conferences on TB topics and Immunoxel has become quite familiar in professional circles. While the TB research community outside of Ukraine has been slow to embrace this approach, Immunoxel clinical data have attracted genuine interest and calls have been made at various venues to test it independently [3,4].


In the course of the present study several advantages of solid Immunoxel were uncovered. First, sublingual formulations appeared to act very quickly, producing, within 1 month, the conversion rate that usually occurs after 2—4 months of dosing with liquid formulas. Second, the effective dose was equivalent to ten drops and possibly even less, given once-daily as opposed to 30—60 drops of liquid immunomodulator given twice-daily, showing at least six- to 12-fold enhancement in activity. Third, sublingual administration as opposed to liquid formulation was simple and convenient; there was no need to count drops and mix them in drinking water, which can be an issue in countries without ready access to clean water. Fourth, the elimination of an alcohol component from the preparation has improved patient compliance. Many of the authors' patients have a tendency to abuse alcohol and were often inclined to overdose with a herbal tincture containing 50% ethanol. As a result, the monthly supply of Immunoxel was consumed at once causing treatment default. Finally, another advantage of solid formulations relates to shipping cost and logistics, especially for export purposes. These issues are now defunct owing to the elimination of bulky glass bottles and potentially flammable liquid, which complicated clearing procedures with cargo airlines and customs. In summary, the solid form of Immunoxel has major advantages over the original formulas and is more appealing as the next generation of botanical immunomodulator from Ukraine.

The evaluation of clinical results in the context of commercial feasibility of product manufacture leads us to two choices; one is sugar-based tablets and the other dried-honey lozenges. Even though Immunoxel pastilles performed no worse than other formulations they have limited appeal owing to the presence of gelatin from animal sources. Sugar-based tablets are the cheapest and simplest delivery vehicle, and in two separate tests produced reliable conversion results. However, this formulation needs to be improved, which will be achieved by adding appropriate excipients such as those found in slow-release tablets. On the other hand, dried-honey lozenges, which produced 100% conversion, may be more attractive than sugar-coated pills. We do not know whether this outcome was random or if the higher efficacy was due to synergy with honey. The antimycobacterial activity of honey has been known since ancient times and it is used as a folk medicine even today. Several in vitro studies have demonstrated the direct inhibition of tubercle bacilli growth with honey [27-30]. Although inhibitory doses of honey were rather high, there are clinical studies showing its benefit in healing tuberculous lesions and counteracting the toxicity of TB drugs [31,32]. The only drawback in this approach is the price of honey. If Immunoxel lozenges can be made at a low cost, this, perhaps, will be an ideal formulation, especially considering the beneficial antimycobacterial and hepatoprotective properties of honey [27-32]. Both formulations will be pursued in future clinical studies expanding on these preliminary findings.

Executive summary
Four different versions of solid Immunoxel were tested: sugar dragées, sugar-coated pills, gelatin pastilles and dried-honey lozenges, which produced negative sputum conversion equal to 89.5% (n = 21), 70% (n = 10), 76.9% (n = 20) and 100% (n = 18) of patients, respectively. The differences in outcome were not statistically significant.
Concurrent administration of various solid forms of Immunoxel with first- or second-line TB drugs resulted in the clearance of Mycobacterium tuberculosis in sputum smears in a mean 84.1% of patients versus 19% among placebo recipients. Sputum conversion occurred very fast - only 1 month of treatment was needed.
No difference was seen when first-diagnosed TB was compared with re-treated TB, multidrug-resistant TB or TB with HIV coinfection; the proportion of converted patients and time to conversion were the same. Differences in gender, age and bodyweight had no influence on conversion rates.
A 1-month pretreatment time with chemotherapy had the same favorable outcome as a 3.8-month pretreatment time.
One sublingual dose of Immunoxel given once-daily produced the same effect as six- to 12-times higher doses of liquid formula
administered for 2-4 months.
Immunoxel reversed TB-associated wasting; the average weight gain was 1.9 kg in 89.8% of Immunoxel-treated patients versus 0.5 kg in 61.9% of placebo recipients.
Immunoxel eliminated TB-associated fever in 97.5% of patients versus 42.9% of placebo recipients.
Immunoxel demonstrated marked anti-inflammatory effects. Erythrocyte sedimentation rate and leukocyte counts reverted back to normal over 1 month. However, patients on TB drugs alone also experienced reduced inflammation. Immunoxel is affordable, easy to administer and made from renewable sources. Immunoxel is safe; it has not produced any adverse effects or caused reactivation of TB.

Conclusion
Many medicinal plants have been identified as having antimycobacterial activity, but they have to go through lengthy development processes before, and if, they are made available to patients [33]. The integration of herbal medicines into modern medical practice has to be validated through rigorous scientific and clinical research in order to be accepted by the medical establishment. Ekomed started developing herbal immune modulators in the late 1980s and today both Dzherelo and Immunoxel are commonly found in Ukrainian pharmacies. Present findings further support the value of Immunoxel and may allow us to persuade public opinion and health authorities that new sublingual formulations are worth being considered as an integral part of TB management strategy. As a result, lives of many thousands of individuals may be saved in Ukraine and elsewhere. Our dream of contributing to the reduction of the global TB burden may then materialize.

Future perspective
Despite increasing attention, the future for TB immunotherapy is uncertain. We cannot predict whether, in the next 5 years, solid forms of Immunoxel will be used widely as an adjunct to immune therapy for TB or will remain outside of mainstream TB management and control strategies, without adequate funding or interest. However, the emergence of drug-resistant TB and TB/HIV has become a great concern to TB caregivers and policymakers. If this trend continues we may see changes that will be constructive and may result in adoption of immune interventions on a larger scale than exists today.

Acknowledgements
We thank all patients who participated in this study. The assistance of Ekomed in generously providingfree supplies of Immunoxel is very much appreciated.

Financial & competing interests disclosure
This work was supported in part by the STEP Business Partnership Grant UKB1-9017-LK-09 awarded by the US Civilian Research and Development Foundation (CRDF), a nonprofit organization authorized by the US Congress and established in 1995 by the National Science Foundation. This study was conducted under the auspices of the regional health authorities of Ukraine as a part of the routine clinical care at the TB Dispensary (Lisichansk, Ukraine). IV Mospan, VS Pylypchuk, J Rowe, VJirathitikal and AS Bourinbaiar are employees of their respective companies. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research
The authors have obtained the Institutional Review Board approval of Lisichansk TB Dispensary and have followed the principles of the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved. Participation in this trial was voluntary and participants were free to withdraw from this study at any time. This study is registered with ClinicalTrials.gov, identifier NCT01061593.

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16 Nikolaeva LG, Pylypchuk VS, Volyanskii YL, Masyuk LA, Maystat TV, Kutsyna GA. Effect of immunomodulator Dzherelo on CD4+ T-lymphocyte counts and viral load in HIV infected patients receiving anti-retroviral therapy. Res. J. Pharmacol.
2, 8—12 (2008).
17 Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YL, Masyuk LA, Kutsyna GA. Effect of oral immunomodulator Dzherelo (Immunoxel) in TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOTS. Int. Immunopharmacol. 8, 845—851 (2008).
18 Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YL, Masyuk LA, Kutsyna GA. Changes in CD4 + T-cells and HIV RNA resulting from combination of anti-TB therapy with Dzherelo in TB/HIV dually infected patients. Drug Des. Dev. Ther. 2, 87-93 (2008).
19 Prihoda ND, Arjanova OV, Yurchenko LV et al. Adjuvant immunotherapy of tuberculosis in drug-resistant TB and TB/HIV co-infected patients. Int. J. Biomed. Pharm. Sei. 2, 59-64 (2008).
20 Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YuL, Frolov VM, Kutsyna GA. Cytokine profiles of patients with pulmonary tuberculosis resulting from adjunct immunotherapy with herbal phytoconcentrates Dzherelo and Anemin. Cytokine 44, 392-396 (2008).
21 Zaitzeva SI, Matveeva SL, Gerasimova TG et al. Efficacy and safety of phytoconcentrate Dzherelo (Immunoxel) in treatment of patients with multi-drug resistant TB (MDR-TB) in comparison to standard chemotherapy. Res. J. Med. Sei. 3, 36—41 (2009).
22 Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YL, Frolov VM, Kutsyna GA. Effect of immunomodulating adjuvant Dzherelo (Immunoxel) in HIV infected patients receiving standard antiretroviral therapy. Open J. Virol. 3, 31-36 (2009).
23 Zaitzeva SI, Matveeva SL, Gerasimova TG et al. Treatment of cavitary and infiltrating pulmonary TB with or without immunomodulator Dzherelo. Clin. Microbiol. Infect. 15, 1154-1162 (2009).
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26 Arjanova OV, Prihoda ND, Sokolenko NI et al. Impact of adjunct immunotherapy with multi-herbal supplement Dzherelo (Immunoxel) on treatment outcomes in end-stage TB/HIV patients. J. Antivir. Antiretrovir. 1, 86-88 (2009).
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32 Sharma M, Khayyam KU, Kumar V, Imam F, Pillai KK, Behera D. Influence of honey on adverse reactions due to anti-tuberculosis drugs in pulmonary tuberculosis patients. Cont. J. Pharm. Tox. Res. 2, 6-11 (2008).
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■ Website
101 Adjunct Immunotherapy With Immunoxel in
Patients With TB and TB/HIV.
http://clinicaltrials.gov/ct2/show/
NCT01061593

Immune approaches in tuberculosis therapy: a brief overview

Expert Rev. Anti Infect. Ther. 10(3), 1–xxx  (2012)Aldar S Bourinbaiar*1, Marina V Mezentseva 2, Dmitry A Butov 3, Peter S Nyasulu 4, Yuri V Efremenko 5, Vichai Jirathitikal 1, Vladimir V Mishchenko6 and Galyna A Kutsyna 71Immunitor Inc., Vancouver, BC, Canada, 2 Gamaleya Institute for Epidemiology & Microbiology, Moscow, Russia, 3 Department of Phtysiatry and Pulmonology, Kharkov National Medical University, Kharkov, Ukraine, 4 Faculty of Health Sciences, Witwatersrand University, Johannesburg, South Africa,  5 Lisichansk Regional Tuberculosis Dispensary, Lisichansk, Ukraine,  6 Central Institute for Tuberculosis, Moscow, Russia, 7 Luhansk State Medical University, Luhansk, Ukraine.*Author for correspondence: aldar@immunitor.com
TB is typically caused by Mycobacterium tuberculosis, a symbiotic bacterium present in one-third of the world’s population. There any many factors triggering overt clinical disease in a small proportion of humans. In our view the major role in the process is played by the host’s immune response, especially self-directed, destructive inflammation. Conventional chemotherapy produces bactericidal or bacteriostatic effects, but immunopathological changes can only be corrected by immunotherapy. Various attempts have been made to identify the optimal immune intervention. Some have shown promising effects, but many have failed. It is commonly believed that the field started in 1890: the year Robert Koch announced his tuberculin therapy. In the Pên Ts’ao Kang Mu, classical Chinese materia medica, published during Ming dynasty, Li Shi Chen (1518–1593) recommended, as a remedy for hemoptysis, to collect from the sputum “… blood lumps, roast them till they are black, and take then them as a powder”. In retrospect, this is perhaps the earliest recorded reference relating to immunotherapy of TB with heat-killed mycobacteria. Modern science is obviously geared toward more palatable approach, but without hindsight from often disdained empirical evidence no progress can be made. The clinical experience from various trial and error processes is briefly discussed in this review.

Keywords: AIDS • antibody • autoimmune • cytokine • HIV • immunomodulator • inflammation • lung •multidrug-resistant TB • mycobacterial • pulmonary • therapeutic vaccine • tubercle bacillus • tuberculin 

Abundant literature exists concerning immune factors contributing to the development of TB. As Mycobacterium tuberculosis is an intracellular parasite the role of humoral, that is antibody- mediated immunity, is far less important than that of cell-mediated immunity. The survey of published studies concerning immunol- ogy of TB reveals a great deal of confusion. Contradicting opinions coexist even today and this perhaps is one of the main reasons why we still have no unanimous consensus with regard to the immunotherapy of TB. What is  worse is that prevailing opinion is bent towards the incorrect concept, which postulate that TB arises from an insufficient immune response. This concept lacks logic; two main diagnos- tic tools for identifying active TB, tuberculin test and IFN-g release assay, are based on the contrary principle. Indeed, individuals with TB do mount a vigorous immune response against tubercle bacilli [1]. Progressive pulmonary TB is caused by a continuous host response to myco- bacterial products and is not due to increasing numbers of viable bacilli in a host [2]. The goal for immunotherapy is to prevent a self-damaging  inflammatory response, instead of instigating more of the same. This goal is closely related to figuring out the immune response that correlates with protective immunity. Over the past 90 years, this gap in our understanding of TB immunology has been responsible for the failure of developing a safe and effective vaccine, since introduction of BCG in   1921. Recently, the research efforts in TB have intensified. However, it is difficult to appreciate the difference or distinguish immunopathologies that are associated with, and those that are causing TB. Without this there is an impression that we are ‘flying without a compass’ a term that Gene Shearer used in his comment with regard to the effort of developing AIDS vaccines. Conventional chemotherapy approaches are straightforward with clear goals of suppressing mycobacterial replication. Bacteriostatic or bactericidal agents seldom restore the immune status. This task cannot be achieved without proper immune intervention. The emergence of multidrug-resistant TB and TB–HIV coinfection, together with advances in immunology, has led to renewed interest of exploiting the immune response against M. tuberculosis. This review is intended to provide a short excurse into the history of TB immunotherapy and analysis of the current state-of-the-art. We hope that understanding the past and present experiences will provide valuable insight and may result in a better grasp of the problem. As Kaufmann quoted in one of his articles on immunol- ogy of TB “those who don’t remember the past are condemned to repeat it” [3].

Early days of immunotherapy Robert Koch, discoverer of M. tuberculosis, can be considered as the first modern TB immunotherapist when in 1890 he announced his tuberculin therapy. Koch’s original preparation of tuberculin was a crude extract from heat-killed cultures of M. tuberculosis. Koch’s idea was based on the assumption that the reaction of the organism to tuberculin would cause the elimination of bacilli-infected tissue. It must be said that 300 years earlier, renowned Chinese scholar, Li Shi Chen (Figure 1) in his encyclopedical treatise Pên Ts’ao Kang Mu (1595) described methods of treating TB, which were not too far from Koch’s principle. Koch’s initial results of treating skin and lymphatic forms of TB were encouraging. Unfortunately, the massive trial that followed shortly after his announcement had produced disastrous results and the hopes he raised were not fulfilled.

Figure 1. The portrait rendition of Li Shi Chen (1518-1593). Li Shi Chen is the author of most comprehensive Chinese pharmacopoeia Pên Ts’ao Kang Mu, describing the earliest recorded TB immunotherapy based on ingestion of heat- inactivated tubercle bacilli derived from sputum. Taken from [101].

The medical opinion settled firmly against immunotherapy and against the founder of modern microbiology. The implications stemming from this trial, which was clearly indicative of exaggerated and deleterious immune reaction, known today as Koch reaction, have been ignored. It is clear that Koch knew of this and he was calling for caution, pointing out that tuberculin should not be used in patients with virulent or acute forms of TB and he argued against high starting doses (Figure 2). Despite negative attitudes prevailing in the medical establishment, Koch’s approach has not been totally ignored. The late 19th and early 20th centuries were replete with success and failure. Several dozen tuberculin-based approaches were promoted by their proponents [4]. A wide range of ingenious modifications of tubercle bacillus were made including passage through refractory animals, attenuation and killing by various methods. Furthermore, inoculation of nonpathogenic or atypical mycobacteria came into wide use. Names of many physicians in Europe and the USA, practicing various tuberculin- derived therapies, are commonly found in the medical literature and lay media of those days. Among them were Lichtheim (1891), Hunter (1892), Klebs (1892), Trudeau (1893), Viquerat (1894), Maragliano (1895), Paquin (1895), Fisch (1897), De Scweinitz and Dorset (1897), Smith and Baldwin (1898), Murphy (1898), Cantacuzino (1901), Goetsch (1901), Armand-Delille  (1902), Marmorek (1903), Wright (1903), Behring (1905),   Calmette (1906), Matthew (1908), Hunter (1908), von Ruck (1908), Leber and Steinharter (1908), Vallée (1909), Webb (1911), Wolff-Eisner (1912), Sahli (1912), Friedmann (1914), Wyeth (1914),   Dreyer (1919), Spahlinger (1922), Josset (1924), Dryer (1924), Reenstierna (1934) and many others. This highly active period in the history of immunotherapy had dwindled down by 1940s when first, streptomycin, and then para-aminosalicylic acid and isoniazid, were introduced as TB drugs and chemotherapy research became the next trend.

Nevertheless, Koch’s idea has greatly influenced the field. The tuberculin skin test (TST) became a critically important diagnos- tic tool in the management of TB. Von Pirquet and Schick (1907) developed a method of cutaneous scratch, Moro and Doganoff (1907) made percutaneous patch, and Calmette and Wolff-Eisner (1907) a conjunctival probe. Intracutaneous injection of tubercu- lin was refined by Mendel and Mantoux (1908) and this method became widespread because of the reproducibility of the results. In the 1930s Florence Seibert prepared purified protein derivative from ‘old tuberculin’, which now serves as a standard reference material. Jules Freund’s emulsified suspension of killed M. tuberculosis cells in mineral oil is nothing more than a super-tuberculin and most potent experimental vaccine adjuvant known, which is not used in humans owing to an extreme reactogenicity. Another outcome that remains as a tribute to Koch’s greatness is BCG vaccine, the only prophylactic TB vaccine available today. At the International Congress on TB, held in Washington, USA, in 1908, Albert Calmette stated that, “he had prepared for thera-peutic use [sic] a particularly active and relatively pure tuberculin called Tuberculin CL, which could be introduced intravenously into the body of a healthy animal…which evidently delayed   the  progress of the disease, and endowed the organism with resistance to the infection.” The fact that Calmette was inspired by Koch and started initially developing BCG as a therapy, which ended up as a preventive vaccine for cattle and was then applied in people is seldom known or is ignored by most TB  vaccinologists. 

It should be remembered that even though BCG is used widely with a relatively good safety record, it does not provoke the immune reaction that one would expect from a classical vaccine. As TB is a disease of mucosal origin, the route of administration of BCG makes a critical difference [5]. The massive transition from Calmette’s oral version to parenteral BCG may have been respon- sible for what is now blamed as a loss of vaccine’s effectiveness. It is also usually ignored that BCG is seldom administered alone as most children receive a vaccine after having been tested with the TST, which in vaccine terminology equals to priming with tuberculin. Thus, in this setting BCG is used as a booster vaccine. In neonatal direct BCG vaccination the efficacy of a vaccine is tested by TST – a situation where the prime–boost sequence is reversed. One needs to closely evaluate failed and successful BCG trials to determine whether the outcomes were influenced by these ignored but important factors. Aronson’s study in children in Indian reservations, which had shown one of the best known success rate for BCG vaccination, offers clues to that. Furthermore, depending on particular species of environmental mycobacteria, the BCG vaccination can be more efficacious or not effective at all in certain endemic areas [6,7]. The combination of BCG with tuberculin has been used with success in the treatment of TB [8]. The combination of BCG priming with heat inactivated M. vaccae therapeutic vaccine was reported to prevent TB incidence [9]. These examples imply that vaccines for therapeutic and prophylactic purposes have a common mechanism. Successes with both types of vaccines were usually associated with the lack of or lower delayed-type hypersensitivity, that is, tuberculin reactogenicity, either pre- or post-vaccination.

Tuberculin continued It is commonly held that the reputation of tuberculin therapy has been deeply affected by a large TB trial held at the Charité hospital in Berlin. Robert Koch’s tuberculin trial, which included 1010 patients with pulmonary TB, resulted in 1.3% cured, 36.1% improved, 4.6% dead, and the rest without apparent clinical benefit. Among 707 patients with extrapulmonary TB, including skin, bone and lymphatic forms, the outcomes were 2.1, 54.5, 1.3 and 42.1% for cured, improved, dead and without clinical benefit patients, respectively [10]. The way these finding are usually interpreted is that therapeutic vaccination against TB is ineffective and even harmful [11]. Considering that the mortality among untreated TB cases in 19th century Berlin was approximately 25%, this is clearly not an example of a failed treatment, but rather a case of misin- terpreted results, which was orchestrated by Koch’s rival, Rudolf Virchow. Today, in the 21st century, the global TB death rate has not greatly improved, even though chemotherapy is widely avail- able. Thus, even by today’s standards, if one looks at mortality as an unequivocal clinical end point, since cure and improvement end points were subject to arbitrary bias, then Koch’s trial had an outstanding outcome.

Figure 2. Robert Koch (1843–1910), discoverer of Mycobacterium  tuberculosis  and tuberculin immunotherapy. A portrait by unknown artist (most likely German portraitist Gustav Graef – mentor of Hedwig Freiberg – Koch’s second wife) made from the original photograph taken in 1890 [102] – the year he announced tuberculin remedy. The hastened clinical trial at Charité and misplaced interpretation of the results had tarnished his reputation. His later method of making pure tuberculin, as opposed to the original crude reactogenic tuberculin, was based on washing out the  lipid fraction with ethanol and salt-precipitating proteinaceous  content, which resulted in apparently safe and effective preparation. Normal healthy volunteers, including his students  von Behring and Kitasato, without signs of TB, had only transient mild increase in temperature and malaise. However, one volunteer with suspected TB, had more pronounced adverse reaction and Koch had overcome this by reducing further the starting dose of tuberculin. Unfortunately, his ingenious foresight of decreasing undesirable reactogenicity by removing lipids and using small initial doses, which were gradually increased, was somehow forgotten and lost to the TB vaccinologists of today. Image courtesy of Vera Seehausen of the Institute of History of the Medicine, Centre for Human and Health Sciences, at Charité University of Medicine, Berlin, Germany. Taken from [102].

Note that Calmette’s preventive BCG trial in children resulted in an almost identical mortality rate and yet his results were hailed as a pioneering breakthrough. However, owing to Virchow’s domineering influence on medical establishment in Germany at the time, his negative opinion perpetuated and carried over to modern day. Judging from various contemporary  sources, Virchow did not seem to be overly preoccupied with praising clini- cal benefits of tuberculin, in fact he did not discuss it at all – he was primarily concerned with fatal ‘injection pneumonias’ – found in approximately half of the deceased patients and attributed to Koch reaction.

Nevertheless, the renewed interest in this approach became apparent by the 1950s, perhaps due to the realization that chemo- therapy was not a panacea and doctors started seeing drug-resist- ant cases. By then most of the work had been carried out in the Soviet Union. The important clues to successfully managing tuberculin therapy outcome were revealed by several groups of Soviet phthysiatists who started using immune intervention in combination with chemotherapy. Most prominent among them was Edvard Mirzoian whose deductive work in the 1960s has laid basis to the currently used tuberculin treatment scheme common in Russia [12]. The main message resulting from their extensive work spanning over decades was simple: dilute! This concept was not entirely new, heated debates were raging in the early part of 20th century as to which dose was most optimal. Koch himself noted that smaller doses were producing tolerizing effects that could eventually heal guinea pigs and high doses were caus- ing massive, occasionally lethal, immune reactions, which have become known as the ‘Koch phenomenon’.

The Soviet school has adopted an approach consisting of a series of increasing doses of subcutaneous or intradermal tuberculin starting with initial dilutions as low as 1:10,000,000. The initial dose was one that produced minimal induration and this was incrementally scaled-up by injecting a tenfold higher dose twice every week until it reached a 1:10 dilution after a 2–3-month treatment course. The typical results were quite convincing. In a large-scale trial involving 1380 patients, sputum clearance at the end of 3 months stood at 82 versus 61.9% of those who received chemotherapy alone (Table 1). Clinical symptoms, healing of cavities were clearly better in the tuberculin group. The healing of cavities was observed in 73.7% (n = 752) as opposed to 49% (n = 422) patients in the control group. This and other trials spanning over the five decades (beginning in 1950) have been, unfortunately, totally ignored in the west.

It must be said that the use of gradually increasing doses has been known since Koch times. His contemporaries such as Ziegler, Pottenger, Petruschky, Römer, Pearson, Gilliland, Jurgens and Neumann were promoting this approach, but to no avail. It is perhaps worthwhile to reassess the value of ‘similia similibus curantur’, the term used in homeopathic practice that relies on tiny doses of antigen. It is well known that immune tolerance can be produced by two means; either by injecting very small doses or by oral administration of antigen. Interestingly, both of these approaches had, historically, higher success rates in TB immunotherapy than other modalities. Indeed, Koch’s tenth experiment, described in his seminal work Die Ätiologie der Tuberkulose (1882), refers to oral feeding of tubercular meat to rats, a proce- dure that completely protected them from subsequent challenge without provoking reactivity, but when feeding was stopped for several weeks animals became susceptible to repeated challenge. The famous tenth experiment was certainly a most curious one, as it almost defied Koch’s own Postulates, but it might have inspired another Nobelist, Charles Richet, of promoting zomotherapy and René Dubos’ eloquent example of lower TB incidence among meat-eating Masai than in vegetarian tribe Akikuyu.

Cytokines, Th1/Th2 & rational empiricism

With rapid advances in immunology resulting in the discovery of various humoral factors secreted by immune cells and domination of Th1/Th2 concept in cellular immunity, attempts have been made to apply this new knowledge to immunotherapeutic interventions. Without going into details of several clinical trials, employing the extensive range of cytokines and anticytokines, as reviewed elsewhere it can be said that the value of these immuno- modulatory molecules remained unclear and their clinical utility has been dismal [13]. Immune intervention such as inhaled IFN-g, corticosteroids, Type 1 interferons, IL-2 or anti-TNF regimens have all been quite disappointing and were largely responsible for the cautious attitude toward immunotherapy that prevails today in the field. In fact calls were made to abandon immunotherapy altogether. Thus, there is a significant gap between a priori (rational reasoning) and a posteriori (empirical evidence), a problem that has been plaguing not only the TB community but the philosophy in general, long before Immanuel Kant’s reconciliation attempt in his Critique of Pure Reason (1781). Despite the progress the correlates of protective immunity are still unknown and without it the rational science has to rely on evidence from empirical ‘poking’. Without mutual feedback and cooperation between bench versus bedside camps there will be no true progress.

Other mycobacterial vaccines

In addition to tuberculin, there are several preparations of mycobacterial origin, which have been or are still being used as adjunct therapy for TB. Among these are Mycobacterium chelonae or ‘turtle vaccine’ developed by Friedrich Friedmann, sold until recently under brand name Anningzochin (Laves-Arzeimittel Gmbh, Ronnenberg, Germany) [4]; Mycobacterium microti, which has been tested in UK and used widely in The Czech Republic and Slovakia, but mostly as a prophylactic vaccine [14]; Mycobacterium phlei from Sanum-Kehlbeck in Germany, also manufactured in China (Utilin S, Chengdu Jin Xing Sanum-Kehlbeck Medicine Co., Ltd, China) [15]; Mycobacterium w or Mycobacterium indicus originally developed as a leprosy vaccine (Immuvac, Cadila Pharmaceuticals, India) [16]; Mycobacterium bovis (standard BCG vaccine), which is also used as an adjunct immunotherapy [8,17]; and Mycobacterium vaccae, a therapeutic vaccine developed by Stanford et al., (Immodulon Therapeutics, London, UK) [18], approved in China under the Vaccae® brand name for adjuvant therapy of TB (Anhui Longcom, China) [19]. The oral tableted form of heat-killed of M. vaccae (V7) is now being tested in a Phase II trial by Immunitor Inc., (Canada), this trial is aimed to confirm beneficial clinical findings reported by Dlugovitzky et al., of their own oral formulation [20]. Preliminary results from the V7 trial are shown in Table 1. A therapeutic vaccine, RUTI®, containing delipidated fragments of M. tuberculosis, as a modern day Dreyer’s ‘defatted’ or diaplyte tuberculin [21] and (Figure 3),was developed by Archivel Farma (Spain), but no data regarding its efficacy in humans are yet available [22].

Figure 3. Box containing ampoules with the ‘defatted’ tuberculin vaccine of Georges Dreyer (1924).

Of special interest is the extremely small quantity of    tubercular  antigen – only 10 ng per dose. Georges Dreyer, born in Shanghai from Danish parents, was, between 1907 and 1934, the first full Professor of Pathology at Oxford University, UK. Of note is that after Dreyer’s death his place was taken by Howard Florey – who with Chain and Heatley succeeded in producing clinical quantities of penicillin from the sample of the original mold strain, which was actually given to Dreyer by none other than Alexander Fleming himself. The principle of action of Dreyer’s vaccine is similar to ‘delipidated’ or detoxified RUTI vaccine, currently being developed by Archivel Farma, Spain. This principle follows the proposal made by Auclair (1900) and Sternberg (1902) that adverse effects of tuberculin were due to the presence of the lipid fraction of the bacterial cell wall, which supports Koch’s description of the same phenomenon described in Über neue Tuberkulinpräparate  (1896). 

Image courtesy of Lorenzo Iozzi (History and Technology  Department, Museum Victoria, Victoria, Australia).

Last year, Immunitor made a similar vaccine, V5; containing circulating M. tuberculosis antigens derived from heat and chemically inactivated blood of donors, which has already been through several Phase II trials [23–26]. Finally, the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) reported therapeutic vaccine based on acellular Mycobacterium smegmatis that appeared to be safe in patients with TB  [27]. The efficacy of the above vaccines is variable, ranging from unknown to excellent, but what is important is that all of them reported to be safe. Furthermore, these vaccines appeared to be equally effective against drug-resistant TB and TB with HIV, the major challenge for current chemotherapeutic interventions. Curiously, most of these vaccines, some of which have been in commercial use for decades, are known to only small number of TB researchers. Nevertheless, they exist and resources need to be devoted to investigate them further, since ignoring or dismissing them a priori will be a disservice to patients, especially to those who have no treatment options left.

Other immunotherapeutics  This section summarizes TB trials in which various immuno- modulators from Russia and other former Soviet republics were used as an adjunct therapy to conventional chemotherapy. Russia has an exceptionally diverse repertoire of immunomodulating agents, with over 130 unique chemical entities currently sold for a wide range of clinical indications. We have previously published a review on many of these agents and refer to it for the detailed description of composition and putative mechanisms of action [28]. Most studies relating to their use against TB are either published in the Russian language or exist as a technical information sheet or treatment recommendations from the manufacturers [29–32]. A review article that summarizes TB trials with select immuno modulators was recently published by Mezentseva et al. [33]. See also Table 1 for the outcome of some of these immune interventions. It can be seen that in all cases the immunomodulators enhance the efficacy of TB drugs and in less time than the mandatory 6 months required for chemotherapy. Admittedly, many of them are inconvenient in terms of administration, or have potential side effects, but one thing is clear – there is plenty of choice. This is in stark contrast to the situation in western countries, there are no immunomodulators to choose from, since none exist.

We and our clinical collaborators in the Ukraine have worked on the immunotherapy of TB over the past 10 years resulting in nearly 20 published clinical trials involving close to 1500 indi- viduals. Initially, we have used locally-produced, herbal immu- nomodulator Dzherelo or Immunoxel [34–36]. Sputum conversion among multidrug-resistant-TB, extremely drug-resistant-TB and TB–HIV patients after 2–4 months of treatment were approximately 85–100%, while in controls on TB drugs alone it took 6–24 months to reach the 48–85% range. However, what was of interest to us is that inflammation and other TB symptoms were improved by Dzherelo in a manner that was strikingly similar to V5 [23–26]. Similar anti-inflammatory properties were reported by our colleagues in Russia in investigations of their own immuno modulators [29–33]. This suggests that an effective immunotherapy ought to have a beneficial effect on inflammation, which can be, for example, measured by two unsophisticated tests, that is, eryth- rocyte sedimentation rate and leukocyte counts. Measuring fever reduction or bodyweight gain are even more simple tests. These simple biomarkers combined with immunological tests evaluating cytokine profile and Th1/Th2 balance can provide reliable correlates of immune protection [37]. The effective immune intervention must possess similar if not identical properties in terms of anti-inflam matory effects and other clinical improvements we have observed in our trials.

Conclusion  The emergence of TB, especially drug-resistant TB and TB–HIV is of great concern to global public health. As new prophylactic vaccines, currently under investigation, are not expected before 2020, the elimination of TB must rely on existing immune inter- ventions to be used together with currently available TB drugs. In recent times, the immunotherapy of TB has been mostly a disappointing experience with high rates of failure, and it is not surprising that many physicians are skeptical about this concept. We have been working with various immune therapies during the past 10 years and can attest that this approach merits more atten- tion than it has previously received. However, caution is required so that protective and not harmful traits of immunity are induced [11,38]. TB is a classic example of chronic infectious disease char acterized by persistent inflammation with autoimmune features. This simple concept is still struggling to gain wider acceptance [1–2,38–40]. Conventional chemotherapy cannot correct protective immunity. Without adjunct immunotherapy one cannot speed up bacterial clearance and the healing process. Without immuno therapy one cannot overcome drug resistance and HIV coinfec tion. Clinical experience with various immunomodulators has not always been encouraging, but those that have shown clinical benefit must be urgently adopted and more resources must be allocated to understand their mechanism and introduce existing immunomodulators into wider clinical use.

Expert commentary  Medical practitioners have a set of 40-year-old antibiotics which they use according to outdated regimens and they seldom venture outside indoctrinated confines. With expansion of drug resistant strains and HIV the therapeutic choices are becoming even more limited. It is thus imperative that basic science, especially immuno logy, is brought back to iatrochemist masses. Phtysiatrists must understand that their enemy is not tubercle bacilli only, but misdirected immune response. They often hear and read that a third of the population carries M. tuberculosis, but they seldom question why 95% of them never get sick. They know well that every 25 seconds a person dies from TB, but they never question what the real cause of death was. Everyone has been taught in their medical school to equate mycobacterium with death. In our opinion this is wrong. The death results from self-destructive inflammatory responses triggered by mycobacterial infection. The division of TB bacilli into virulent and avirulent types is based on in vivo assays, which is clearly dependent from the host immune response, since in vitro assays based on measurement of cell death in infected macrophages or dendritic cells, the virulent TB strains are usually less cytotoxic than avirulent ones [41,42]. To the best of our knowledge there is only one reference in the literature, which implies M. tuberculosis being directly cytopathic to human epithelial cells and even then this might have been an artifact due to HIV coinfection [43]. Furthermore, the symbiotic relationship between mycobacteria and host is not necessarily harmful, one can find in the literature examples of beneficial coexistence [44]. In order to increase our ability to control infectious diseases and prevent epidemics, it is vital that doctors are properly educated. It is important to allocate more resources toward existing immune interventions so that clinical outcomes are improved. The current gaps in our knowledge must be identified and explored according to their relevance not paradigmatic convenience.Five-year view  Basic TB research has intensified in the last decade. The TB pandemic that started in early 1990s has shown that even the most developed countries are vulnerable. TB will continue to be a threat to the global community as it has been for mil- lennia. Every country should take measures by implementing a national preparedness and response plan, by empowering the local community, by training the medical personnel and out- reach health staff in the identification and management of TB, and by creating new tools for diagnosis and treatment. The establishment of truly novel protocols for management of TB needs to be implemented. If the scarcely populated field of immune interventions is invigorated with renewed enthusiasm then in the next 5 years we may witness the renaissance of the immune therapy that was dominating TB research prior to introduction of the chemotherapy.

Acknowledgements  We would like to thank many experts who have contributed their comments and critiques. In particular, we are indebtful to John Stanford, Graham Rook, John Grange, Ali Zumla, Gene Shearer, Feng-li Tao, Shilong Yang, Jiang Pu, Chuanyou Li, Wing-wai Yew, CC Leung, Ying Zhang, Satoshi Makino, Ray Spier, Nataliya Kozhan, Vasilyi Petrenko, Elena Rekalova, Irina Il’ inskaya, Petr Rytik, Felix Ershov, Volodymyr Pylypchuk, Pere-Joan Cardona, Jim Johnston, Carl Feng, Christoph Lange, Bob Wallis, Keertan Dheda, Gavin Churchyard, Tony Hawkridge, Tom Evans, Robert Loddenkemper, Stefan Kaufmann and Mel Spigelman for sharing with us their views on TB issues. The authors are also grateful to patients who participated in trials of immunotherapeutics discussed in this review.  

Key issues 

•   The immune approach in TB therapy has been promoted since 1890, starting with Robert Koch himself, but there are even earlier empirical precedents such as those found in Li Shi Chen’s Pên Ts’ao Kang Mu

•   Immunotherapy is still skeptically viewed due to the failure of various approaches, especially involving cytokines. 

•   Failures are linked to the belief that TB arises as a result of a ‘weakened’ immune response. 

•   We believe that TB, as a disease, occurs when the immune tolerance to mycobacterium is replaced by an exaggerated immune response, characterized by inflammatory reaction against own tissues harboring the tubercle bacilli. 

•   By consequence, TB is an autoimmune disease triggered by mycobacterial infection. 

•   In order to produce a favorable clinical outcome one needs to manage immunity with the aim of restoring immune tolerance. 

•   Immune tolerance does not mean immune suppression or anergy, it’s an active process, which is as potent as classical immune activation. 

•   If this concept is true, then immune correlates of protective immunity have to be searched for in places that were overlooked by current dogmas. 

•   There are many immunotherapeutics that have been successful and they need to be investigated so that clues emerge, which could help not only the therapy but also prophylaxis of TB.

Financial & competing interests disclosure 

AS Bourinbaiar and V Jirathitikal are officers of the Immunitor company, involved in developing TB immunotherapies. Some of the trials of immuno- therapeutics described in this review were supported by Business Partnership Grant UKB1-9017-LK-09 awarded by the US Civilian Research & Development Foundation – a nonprofit organization authorized by the US Congress and established in 1995 by the National Science Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.  No writing assistance was utilized in the production of this manuscript.

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The use of immunomodulator likopid in the combined treatment of pulmonary tuberculosis. Probl. Tuberk. 3, 21–25 (2002).  31 Borzenko AS, Antonov YuV, Popkova NL. Use of polyoksidonium in adjunct therapy of patients with pulmonary tuberculosis. Immunologia 2, 41–43 (1999).  32  Knoring BE, El’kin AV, Smirnov MN, Sakharova IYa, Basek TS, Zhuravleva VYu. Immunocorrection of pulmonary tuberculosis with Ronkoleukin. Probl. Tuberk. 5, 26–29 (1999).  33 Mezentseva MV, Stakhanov VA, Zakharova MV et al. Prospects for immunotherapy in adjunct treatment of infiltrating pulmonary tuberculosis. Biopreparaty 2, 20–25 (2011).  · Describes some of the Russian immunomodulators used as adjunct therapy for TB. 34 Zaitzeva SI, Matveeva SL, Gerasimova TG et al. Treatment of cavitary and infiltrating pulmonary tuberculosis with and without the immunomodulator Dzherelo. Clin. Microbiol. Infect. 15, 1154–1162 (2009). 35  Arjanova OV, Prihoda ND, Yurchenko LV et al. Enhancement of efficacy of tuberculosis drugs with Immunoxel (Dzherelo) in HIV-infected patients with active pulmonary tuberculosis. Immunotherapy 1, 549–556 (2009).  36 Prihoda ND, Arjanova OV, Yurchenko LV et al. Adjuvant immunotherapy of extensively drug-resistant tuberculosis (XDR-TB) in Ukraine. Curr. Res. TB 1, 1–6 (2009).  37  Mezentseva  MV,  Stakhanov VA, Zakharova MV, Zotova IF, Tregubova MI, Shapoval IM. Cytokines as the markers of the infiltrative pulmonary tuberculosis progress. Infect. Immun. 1, 367–372 (2011).  38 Grange JM, Brunet LR, Rieder HL. Immune protection against tuberculosis – when is immunotherapy preferable to vaccination? Tuberculosis 91, 179–185  (2011). 39 Ellner JJ. Immunoregulation in TB; observations and implications. Clin. Transl. Sci. 3, 23–28 (2010).  •    Links TB with inflammatory reaction.  40 Kivity S, Agmon-Levin N, Blank M, Shoenfeld Y. Infections and autoimmunity  friends or foes? Trends Immunol. 30, 409–411 (2009).41 Danelishvili L, McGarvey J, Li YJ, Bermudez LE. Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells. Cell Microbiol. 5, 649–660 (2003). 42 Ryan RC, O’Sullivan MP, Keane  J. Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells. BMC Microbiol. 11, 237 (2011). 43 Gadea I, Zapardiel J, Ruiz P,    Gegúndez MI, Esteban J, Soriano F. Cytopathic effect mimicking virus culture due to Mycobacterium tuberculosis. J. Clin. Microbiol. 31, 2517–2518 (1993). 44 Lehmann D, Ben-Nun A. Bacterial agents protect against autoimmune disease. I. Mice pre-exposed to Bordetella pertussis or Mycobacterium tuberculosis are highly refractory to induction of experimental autoimmune encephalomyelitis.  J. Autoimmun. 5, 675–690 (1992).• Demonstrates that tubercular infection can protect against autoimmunity.  Websites 101Classics of traditional Chinese medicine. www.nlm.nih.gov/exhibition/ chinesemedicine/emperors.html  • Refers to Ch’en Hsueh-lou, Chung-kuo li tai ming i t’u chua: Biographies and Portraits of Chinese Famous Doctors in Past Dynasties, Nan-ching, 1987. 102 The natural history of pulmonary tuberculosis, facilitator guide. www.emro.who.int/STB/media/pdf/ HARRIS+coverpage.pdf  103 Cycloferon (Polysan) St-Peterburg, Russia. www.polysan-ru.com  104 Amiksin (Pharmstandart) Tomsk, Russia. http://pharmstd.ru  105 Galavit (Medicor) Moscow, Russia. www.medicor.ru  106 Polyoxidonium (Petrovax) Moscow, Russia. www.petrovax.ru  107 Likopid (Peptec) Moscow, Russia. www.licopid.ru  108 Glutoxim (Farma VAM) St-Peterburg,  Russia www.glutoxim.ru109 Neovir (Pharmsynthez) St-Peterburg, Russia.  www.pharmsynthez.com110 Bestim (Verta) St-Peterburg, Russia. www:hpb-spb.com  111 Ronkoleukin (Biotech) St-Peterburg, Russia.  www.biotech.spb.ru112 Dzherelo Immunoxel (Ekomed) Kiev, Ukraine.  www.ekomed.com.ua113 Tuberculin (SPbNIIVS - Institute of Vaccines and Sera) St-Peterburg, Russia. www.spbniivs.ru 114 V7 (Immunitor) Vancouver, Canada. www.immunitor.com 

Adjuvant Immunotherapy of Extensively Drug-Resistant Tuberculosis (XDR-TB) in Ukraine

'NafhaliaD. Prihoda, 'Olga V. Arjanova, 'Larisa V. Yurchenko, 'Nina I. Sokolenko, 2Lyudmila A. Vihrova, 3Volodymyr S. Pylypchuk, 4Valery M. Frolov and4GalynaA. Kutsyna 'Lisichansk Regional Tuberculosis Dispensary, Lisichansk, Ukraine 2Lisichansk Regional Hospital, Lisichansk, Ukraine 3Ekomed LLC, Prospect Pravdy 80-A, Kiev, Ukraine 4Luhansk State Medical University, Luhansk, Ukraine

Abstract: Conventional TB chemotherapy success rates are very low in patients with Extensively Drug-Resistant Tuberculosis (XDR-TB). We treated twelve XDR-TB individuals, seven of which in addition to standard Anti-TB Therapy (ATT) received Immunoxel (Dzherelo), Svitanok and Lisorm - ove - the-counter herbal immunomodulators. All seven patients, who received adjunct immunotherapy improved clinically and radiologically and were discharged after 3.7 ± 0.8 months, with average/median time to mycobacterial clearance 28/25 days. None of five patients on TB drugs alone improved and one had died. Patients on immune intervention gained 9.6 kg (p = 0.0001) while those on ATT lost 1.4 kg. The levels of total bilirubin had decreased from 15.6 to 10.7 uM L-1, similarly the values of alanine transaminase (ALT) have declined from abnormally high 42.6 IU L"1 to normal levels 22. IU L"1 (p = 0.23). Patients on ATT had unchanged levels of bilirubin, but their ALT declined from 29.6 to 12 IU L"1 (p = 0.02). The levels of hemoglobin had risen from 104.1 to 118 g L-1 (p = 0.07), whereas, leukocyte counts descended to normal levels from 8.9 to 7.3x10s cells L-1 (p = 0.003). Conversely, inpatients on ATT leukocyte counts had risen from 8.7 to 13.8x10s cells L-1 (p = 0.21), whereas, hemoglobin declined to below normal levels from 116.4 to 96.6 g L-1 (p = 0.18). These results show that immune-modulating interventions can favorably influence the effect of TB drugs. The difference between two treatment outcomes was highly significant (Mantel Haenszel odds ratio = 11; p = 0.0009 at 95% CI). Thus, adjunct herbal immunotherapy is safe, shortens treatment duration and can overcome drug resistance even in patients with XDR-TB.

Key words: Immunomodulator, MDR-TB, XDR-TB, phytoconcentrates, Mycobacterium

INTRODUCTION
The extensively resistant TB (XDR-TB) is diagnosed whenM tuberculosis bacilli in addition to lack of sensitivity to isoniazid (H) and rifampicin (R), two most commonly-used, first-line TB drugs, are also resistant to any one of fluoroquinolones and at least one of second-line injectable drugs, e.g., capreomycin, kanamycin and amikacin (Migliori et a!., 2008). This emerging form of TB caused worldwide concern after outbreak in Kwazulu Natal province of South Africa where 52 of 53 patients with XDR tuberculosis and HIV co-infection had died within 2 weeks from the time of diagnosis (Gandhi et a!., 2006). Success rates in treating XDR-TB are significantly lower than among drugsensitive cases ranging between 29 and 67%. In additionit takes much longer (18-24 months) to achieve a cure and concerns over adverse effects of drugs became more prominent since second-line drugs are more toxic. The cost is another factor as the deployment of second-line drugs increases treatment cost by about hundred-fold. Clearly there is an urgent need to find additional therapeutic interventions that could overcome these problems.
 

Immunomodulators Immunoxel (Dzherelo), Svitanok and Lisorm are made from a proprietary combination of medicinal plants and are commonly used in Ukraine for the management of TB and HIV infections, including patients with dual infection (Arjanova et a!., 2009; Chkhetiany et a!., 2007; Melnik et al., 1999; Nikolaeva et al., 2008; Prihoda et al., 2007; Zaitzeva et al., 2008). They have been approved in 1997 by the Ministry of Health of Ukraine as functional supplements with therapeutic indications. Dzherelo and Svitanok were specifically recommended as immune adjuvants to the therapy of pulmonary tuberculosis (Melnik et al., 1999). So far, the phytoconcentrates we have decided to use in this study have been taken safely by several hundred thousand individuals for various indications including chronic bacterial and viral infections such as TB and HIV, autoimmune diseases and malignancy (Chkhetiany et al., 2007). In this retrospective study, conducted at Lisichansk TB Dispensary, we have compared the adjunct effect of herbal immunomodulators to outcome of treatment with conventional TB therapy.

MATERIALS AND METHODS

Patients
Lisichansk TB Dispensary is within Luhansk administrative region of the Eastern Ukraine with total population 2.5 million. Approximate population of registered TB patients in this region is 2 thousand. Lisichansk TB dispensary has turnover of about 600-800 patients per year. The dispensary has six medical doctors and approximately 15 medical nurses and lab technicians who care for hospitalized patients and perform the lab work. Twelve patients with pulmonary XDR-TB were identified retrospectively, five of which received individualized TB drugs regimen and seven who received in addition to ATT an immunomodulating phytopreparations Dzherelo, Svitanok and Lisorm. All patients were males with age range between 25 and 67 years. Five presented with first-diagnosed TB and the rest were previously treated, relapsed cases of TB. All study patients presented with acute symptoms of pulmonary TB that required hospitalization. Most common symptoms were prolonged heavy cough, pain in the chest, high fever, profuse night sweats, fatigue and loss of weight and appetite. Active pulmonary tuberculosis was certified by a medical history and clinical findings compatible with tuberculosis, a chest X-ray showing lung involvement and positive sputum smear for Acid-Fast Bacilli (AFB) and the culture of M. tuberculosis. The conduct of the study was approved by the Internal Review Board (IRB) of Lisichansk TB dispensary in accordance with the Helsinki Declaration.
Treatment Regimen
All anti-TB drugs were procured free-of-charge through centralized national supply system of Ukraine. The over-the-counter phytoconcentrates, Dzherelo, Lisorm and Svitanok were generously supplied by Ekomed LLC. Individualized, first- and second-line anti-TB drugs were administered to all hospitalized patients based on physician's decision prior to or after results of drug susceptibility tests. In the immunotherapy group, in addition to ATT, patients received a daily dose of Dzherelo which was given as 30 drops diluted in a half-glass of water 30 min before breakfast. Some patients received Immunoxel-slightly modified form of Dzherelo. The same dose, 30 drops, of Lisorm and Svitanok were given before lunch and supper, respectively. The exact formula of phytoconcentrates has been described by Prihoda et al. (2008). Sputum smear and culture examinations for AFB were performed at monthly intervals. The decision to discharge was based on at least twice-repeated negative culture outcome and satisfactory clinical and radiological findings.
TB Drug Resistance Testing
The drug resistance to first- and second-line TB drugs was tested with commercially supplied kit (Tulip Diagnostics, Goa, India). The cultures ofM tuberculosis derived from sputum of each patient were inoculated into ready-to-use tubes containing TB drugs incorporated at manufacturer-predetermined concentrations into standard Lowenstein-Jensen agar slants. The cultures were incubated at 37°C and checked periodically until appearance of colonies in control tubes without drugs.
The calculation of the proportion of resistant bacilli was done by comparing counts on drug free and drug-containing Lowenstein-Jensen medium.
Statistical Analysis
The obtained results were analyzed with available online statistical software (GraphPad Software, Inc., La Jolla, CA). All statistical analysis were done on intent-to-treat basis, involving the total number of patients without subgrouping them into responders and non-responders. The stratification analysis of patients was conducted to reveal the difference between distinct treatment categories. Parametric baseline values relative to the end of study values were evaluated by paired or unpaired Student t-test. The categorical test was done by Mantel Haenszel's odds ratio calculation. The probability values were considered as significant at p< 0.05 cut-off value.
RESULTS AND DISCUSSION
None of five patients on conventional TB drugs regimen had positive outcome after 9 months of treatment and one patient died after 9.5 months. The duration of treatment in immunotherapy group ranged between 10.6-20.4 weeks with average/median 15.7/16.7 weeks (Table 1). The treatment lasted until patients were discharged from the dispensary upon twice-repeated negative culture findings and clinical and radiological improvements. The time to negative culture conversion ranged between 20-37 days with mean/median 28/25 days. Mycobacterial clearance was confirmed by repeated cultures at monthly intervals.
There appears to be no difference between first-diagnosed TB cases versus chronic, previously treated TB in terms of median days to discharge, i.e., 117 vs. 105.6 or days to mycobacterial clearance, 23 vs. 30. However, sample size was too small to reveal statistically significant difference.
At the end of study every patient had gained substantial lean body mass, ranging between 6 and 13 kg. The average accrual in lean body mass was 9.6 kg, which was statistically highly significant as evidenced by a paired Student's t-test (p = 0.0001) -an effect that was evident early as one month from initiation of the therapy. In contrast, patients on ATT had lost on average 1.4 kg (p = 0.4).
The potential hepatotoxicity of ATT combination with herbal preparations was monitored by quantitative liver function tests. Surprisingly, despite intensive chemotherapy patients have shown signs of better liver function. The level of total bilirubin had decreased from mean 15.6 to 10.7 uM L-1 -a favorable change that was not statistically significant (p= 0.16). Similarly, the values of alanine transaminase (ALT), another marker of liver damage, have declined from abnormally high (42.6 IU L-1) to normal levels (22. IU L-1) -a change that was not statistically significant (p = 0.23). Patients on ATT had same levels of bilirubin but their ALT declined from 29.6 to 12 IU L"1 (p = 0.02).Another phenomenon observed during therapy is a reversal of baseline anemic state and proinflammatory condition-symptoms very common in TB. Most patients at study entry displayed signs of anemia and had abnormally elevated leukocyte counts. At the end of treatment these parameters were improved in a statistically significant manner. The levels of hemoglobin had risen from 104.1 to 118 g L-1 (p = 0.07), whereas leukocyte counts descended to quasi-normal levels from 8.9 to 7.3x10s cells L 1 (p = 0.003). Inpatients on ATT the reverse trend was observed. Leukocyte counts had risen from 8.7 to 13.8x10s cells L-1 (p =0.21) whereas hemoglobin declined to below normal levels from 116.4 to 96.6 g L"1 (p = 0.18).  
Table1: Baseline and outcome characteristics of XDR-TB patients treated with TB drugs without or in combination with Dzherelo (Immunoxel), Svitanok and Lisorm   
   These results show that immune-modulating interventions can favorably influence the efficacy of TB drugs (Arjanova et al., 2009; Chkhetiany et al., 2007; Nikolaeva et ah, 2008; Prihoda et ah, 2007; Zaitzeva, 2008). All seven patients who received ATT and immunotherapy improved clinically and radiologically and were discharged after 3.7±0.8 months, with average/median time to mycobacterial clearance 28/25 days. None of five patients on TB drugs alone improved and one had died. The difference between two treatment outcomes was statistically significant (Mantel Haenszel odds ratio = 11; p = 0.0009 at 95% CI).
Present results compare favorably to XDR-TB chemotherapy outcomes reported in several recent papers. According to study by Kim et al. (2008) only 29.3% of those with XDR-TB were cured. TB therapy success rate in Russian patients with XDR-TB as reported by Keshavjee et al. (2008) was 48.3%. Earlier reported cure rates in Europe, USA, Peru and Korea were between 37.5-67% indicating that XDR-TB poses serious clinical challenge (Edward et al., 2008; Kwon et al., 2008; Migliori et al., 2008; Mitnicke/o/., 2008). In conclusion, adjunct herbal immunotherapy is safe, enhances significantly treatment outcome and can overcome drug resistance even in patients with extremely poor prognosis. Further studies are needed to confirm present findings.
ACKNOWLEDGMENTS     
We thank all participants who volunteered in this study. The support of clinical staff and technicians who contributed to this study has been of tremendous help to us. We are grateful to other colleagues who shared their insight and provided helpful suggestions based on their own experience with phytoconcentrates used in present study.
Kwon, Y.S., Y.H. Kim, G.Y. Suh, M.P. Chung and H. Kim et a!., 2008. Treatment outcomes for HlV-uninfected patients with multidrug-resistant and extensively drug-resistant tuberculosis. Clin. Infect. Dis.,47: 496-502.
Melnik, V.P., O.V. Panasyuk, V.S. Pylypchuk, O.P. Moshich, N.M. Procenko and O.M. Leonenko,1999. Deployment of herbal preparations Dzherelo and Svitanok for combination therapy of pulmonary tuberculosis. Medical Institute of Ukrainian Association of People's Medicine. Information Bulletin of the Ministry of Health of Autonomous Republic of Crimea. UDK:616.24-002.5-085-038:615.017. 1999. Kiev, Ukraine.
Migliori, G.B., C. Lange, R. Centis, G. Sotgiu and R. Mutterlein et al., 2008. TBNET Study Group. Resistance to second-line injectables and treatment outcomes in multidrug-resistant and extensively drug-resistant tuberculosis cases. Eur. Respir. J., 31: 1155-1159.
Mitnick, CD., S.S. Shin, K.J. Seung, M.L. Rich and S.S. Atwood et al., 2008. Comprehensive treatment of extensively drug-resistant tuberculosis. New Engl. J. Med., 563: 563-574.
Nikolaeva, L.G., T.V. Maystat, V.S. Pylypchuk, Y.L. Volyanskii, L.A. Masyuk and G.A. Kutsyna, 2008. Effect of oral immunomodulator Dzherelo (Immunoxel) in TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOTS. Intl. Immunopharmacol., 8: 845-851.
Prihoda, N.D., O.V. Arjanova, L.V. Yurchenko, N.I. Sokolenko, L.A. Vihrova, V.S. Pylypchuk and G.A. Kutsyna, 2007. Open label trial of adjuvant immunotherapy with Dzherelo, Svitanok and Lisorm, in MDR-TB, XDR-TB and TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOT. J. Med. Plant Res., 1: 117-122. Prihoda, N.D., O.V. Arjanova, L.V. Yurchenko, N.I. Sokolenko, L.A. Vihrova, V.S. Pylypchuk and GA. Kutsyna, 2008. Adjuvant immunotherapy of tuberculosis in drug-resistant TB and TB/HIV co-infected patients. Int. J. Biomed. Pharm. Sci., 2: 59-64.
Zaitzeva, S.I., S.L. Matveeva, T.G. Gerasimova, Y.N. Pashkov, D.A. Butov, V.S. Pylypchuk V.M. Frolov and G.A. Kutsyna, 2008. Efficacy and safety of phytoconcentrate Dzherelo (Immunoxel) in treatment of patients with multi-drug resistant TB (MDR-TB) in comparison to standard chemotherapy. Res. J. Med. Sci.
 

Efficacy and Safety of Phytoconcentrate Dzherelo (Immunoxel) in Treatment of Patients with Multi-Drug Resistant TB (MDR-TB) in Comparison to Standard Chemotherapy

MEDWELL PUBLISHING Research Journal of Medical Sciences 3 (2): 36-41, 2009 ISSN: 1815-9346 © Medwell Journals, 2009
'S.I. Zaitzeva, 'S.L. Matveeva, 'T.G. Gerasimova, 'Y.N. Pashkov, 'D.A. Butov, 2V.S. Pylypchuk, 3V.M. Frolov and 3G.A. Kutsyna 'Department of Phtysiatry and Pulmonology, Kharkov National Medical University, Kharkov, Ukraine 2Ekomed LLC, Kiev Ukraine 3Luhansk AIDS Center and Luhansk State Medical University, Luhansk, Ukraine
Abstract: Safety and efficacy of phytoconcentrate Dzherelo (Immunoxel) as anadjunct immunotherapy for treatment of TB was compared to a standard anti-mycobacterial chemotherapy by enrolling 66 patients with active pulmonary tuberculosis. Among them 48 individuals presented with Multi-Drug Resistant (MDR) form of TB who were divided into 2 matching groups to receive either individualized Anti-TB Therapy (ATT) or ATT with 50 drops of Dzherelo twice-per-day. Patients were followed for 3 months and then assessed for sputum smear clearance rate, radiological findings and liver damage markers such as bilirubin, ALT, cholinesterase, gamma-glutamyl transpeptidase and thymol turbidity test. At the end of the 3rd month of follow-up 81.8% (27/33) of patients on Dzherelo had negative sputum smear, whereas, only 7 out 33 patients (21.2%) on ATT had converted (p = 0.0002). Administration of Dzherelo resulted in complete healing of pulmonary infiltrations and cavities in 12 (36.4%) patients in Dzherelo group, while only one patient (3%) treated with TB drugs had improved (p = 0.0008). Our results indicate that when  Dzherelo is added to ATT it can produce significant clinical, microbiological and radiological improvements. Based on liver function tests  Dzherelo was found to be safe and improved or reversed liver damage caused by ATT. Our findings support clinical studies by other investigators showing that immune intervention with Dzherelo accelerates and enhances the outcome of tuberculosis therapy. 
Key words: MDR-TB, Mycobacterium tuberculosis, immunotherapy, immuno-modulator, herbal, phytomedicine, medicinal plants.
INTRODUCTION
Nearly 9 million people develop Tuberculosis (TB) each year and 2 million die from the disease. Tuberculosis has become an emerging global public health priority. The incidence of TB continues to increase in Ukraine as well (Dye et al, 2005). In 1962, the incidence of TB in Ukraine was 178 cases/100,000 individuals, which then gradually declined to 73, 42 and 41.6 cases/100,000 in 1972, 1982 and 1992, respectively. However this trend reversed and by 2002, TB incidence and prevalence became 80.4 and 330 per 100,000, respectively. TB mortality almost doubled from 10.2/100,000-21.6/100,000 between 1990 and 2001 (Drobniewski et al., 2005).
Similarly to situation in Africa the success rates of therapy in Eastern Europe, including Ukraine, are substantially   below   average   when   compared with other regions of the world (Atun and Olynik, 2008). In addition the Ukraine has worsening epidemics of drug resistant TB that is increasingly converging with HIV (Dubrovina et al., 2008). Despite availability of TB drugs the situation is far from ideal and better therapeutic interventions are clearly needed to reverse the current trend.
Oral immunomodulator Dzherelo (Immunoxel) is used in Ukraine for the management of both TB and HIV infections, including patients co-infected with TB and HIV. Dzherelo was approved in 1997 by the Ministry of Health of Ukraine as an immunomodulating supplement, which so far has been used by hundreds of thousands individuals for various indications including chronic bacterial and viral infections, autoimmune diseases and malignancy. In 1999  Dzherelo was recommended by the health authorities of Ukraine as an immune adjunct for
treatment of tuberculosis (Melnik et al., '999). Clinical studies have indicated that Dzherelo helps to achieve better clinical response when combined with standard Anti-Retroviral (ART) or Anti-Tuberculosis Therapy (ATT) (Chechitiany et al., 2007; Prihoda et al, 2007, 2008; Nikolaeva et al, 2008a, b; Zaitzeva et al., 2008).  Dzherelo has been found to reduce the incidence of opportunistic infections and reverse TB-associated wasting.  Dzherelo has also been found to alleviate the hepatotoxicity associated with ATT as evidenced by improvement of liver function tests. However, these studies have not dealt with the effect of Dzherelo on other clinical parameters associated with TB. Our study was, thus, aimed at defining the adjunct effect of  Dzherelo on sputum conversion and radiological symptoms as well as select biochemical and liver function among patients with active pulmonary TB. The advantage of adjunct Dzherelo immunotherapy was compared to a treatment regimen consisting of ATT alone.
MATERIALS AND METHODS
Patients: The study involved 66 patients most of whom had with first-diagnosed pulmonary TB. Only 9% of patients had been treated previously with anti-TB drugs. The ratio of males/females was 54/'2. All study patients presented with active form of pulmonary TB. Most common symptoms were prolonged heavy cough, pain in the chest, high fever, profuse night sweats, fatigue, dyspnea, hemoptysis and loss of weight and appetite. Active pulmonary tuberculosis was certified by a medical history and clinical findings compatible with tuberculosis, a chest X-ray showing lung involvement and positive sputum smear for Acid-Fast Bacilli (AFB) and the culture of M. tuberculosis. The prevailing majority of these patients 48/66 (72.2%) had Multi-Drug Resistant TB (MDR-TB). The categories of observed pulmonary involvement on chest X-ray were infiltrating (72%), milliary ('8%) and cavitary ('0%) forms of TB. Every patient was positive for Mycobacterium tuberculosis by sputum smear and culture. Patients' age ranged between '9-60 years. Patients were divided into 2 groups matched by gender, age and disease severity. The conduct of the trial was approved by the internal review board of the University. The participation in this study was voluntary and patients were eligible to enroll only after signing the written consent.
Treatment regimen: All patients received standard individualized TB therapy. The anti-TB drugs were procured through the centralized national supply system of Ukraine. Patients in adjunct immunotherapy group in addition to the same regimen of TB drugs received 50 drops of Dzherelo, given in a glass of water twice daily; usually 2 h after breakfast and 30 min before supper. The treatment was administered for 3 months as Directly Observed Therapy (DOT) to patients hospitalized in our TB dispensary. The over-the-counter phytoconcentrate, Dzherelo (Immunoxel) was generously supplied by Ekomed company. It contains concentrated aqueous-alcohol extract from medicinal plants such as aloe (Aloe arborescens), common knotgrass (Polygonum aviculare), yarrow (Achillea millefolium), centaury (Centaurium erythraea), snowball tree berries (Viburnum opulus), nettle (Urtica dioica), dandelion (Taraxacum officinale), sweet-sedge (Acorus calamus), oregano (Oreganum majorana), marigold (Calendula officinalis), seabuckthorn berries (Hippophae rhamnoides), elecampane (Inula helenium), tormentil (Potentilla erecta), greater plantain (Plantago major), wormwood (Artemisia sp.), siberian golden root (Rhodiola rosea), cudweed (Gnaphalium uliginosum), licorice (Glycyrrhiza glabra), fennel (Foeniculum vulgare), chaga (Inonotus obliquus), thyme (Thymus vulgaris), 3-lobe beggarticks (Bidens tripartite), sage (Salvia officinalis), dog rose (Rosa canina) and juniper berries (Juniperus communis). Dzherelo approved in 1997 by the Ministry of Health of Ukraine as dietary supplements. In 2006, it received status of a functional food-a superior category of herbal supplements which can carry medical claims as substantiated by clinical evidence.
Clinical endpoints and exclusion/inclusion criteria: Every patient who presented with active pulmonary TB was eligible for this study. No restrictive exclusion criteria for study enrollment were set up except disallowing patients who tolerated poorly chemotherapy. Primary endpoints of interest in this study were the effect of prescribed therapy on microbiology and radiological findings. Secondary endpoints were changes in liver biochemistry.
Laboratory evaluation: In addition to clinical and radiological evaluation a standard microbiology examination of sputum smear staining by Ziehl Neelsen method was conducted prior to study entry and at 2nd and 3rd months post-treatment. Liver function was assessed by measuring ALT transaminase, total bilirubin, cholinesterase, Gamma-Glutamyl Transpeptidase (GGT) and thymol turbidity test.
Statistical analysis: The obtained results were analyzed with statistical software GraphPad (GraphPad Software, Inc., La Jolla, CA 92037). The baseline quantitative values relative to the end of study values were evaluated by paired or unpaired Student t-test.  Other statistical calculations such as determination of standard deviation, mean and median, were performed with the same software. The non-parametric or categorical values of treatment outcomes were compared by Fisher's exact 2x2 test. All statistical analyses were done on intent-to-treat basis, involving the total number of patients without subgrouping them into responders and non-responders. The resulting probability values were considered as significant at p« 0.05.
RESULTS AND DISCUSSION
Clinical improvements: The improvement of baseline clinical symptoms such as night sweats, dyspnea, nausea, fatigue and general malaise was observed within 2-3 weeks. By 8-10 weeks increase in body weight, disappearance of fever and weakness were clearly established. The improvement in quality of life signs among patients on ATT was less common and usually correlated with other endpoints. Due to subjective nature of clinical symptoms and difficulty of measuring them in an objective manner these effects are not presented in this study.
Sputum smear conversion: At the end of 2nd month of follow-up 21 out of 33 patients (63.6%) on Dzherelo had negative sputum smear conversion, whereas only 6 patients (18.2%) on ATT had converted (Fig. 1). The difference by Fisher's exact two-way test was highly significant (p = 0.0002). Further discrepancy was observed at the end of 3rd month. Negative smear test has been found in additional 6 patients on Dzherelo whereas only one individual converted in ATT arm, bringing total to 8'.8 and 2'.2% (p = 0.00000').
Radiological findings: Administration of Dzherelo was characterized by remarkable clinical response as judged by treating physicians. In ATT group these improvements were seldom observed. Chest X-ray analysis confirmed these subjective impressions (Fig. '). The healing of pulmonary lesions and cavities was seen in '2 (36.4%) patients in Dzherelo group while only one patient (3%) treated with TB drugs had shown resolution by third month (p = 0.0008).
Liver function test: Since TB chemotherapy causes hepatotoxicity we compared liver function markers in 2 groups of patients. The results are presented in Table 1. The levels of ALT appeared slightly to increase above normal in Dzherelo group (p = 0.09) while they had almost doubled in ATT group (p<0.0001). The levels of total bilirubin descended to normal in Dzherelo recipients but increased in patients treated with chemotherapy alone. The activity of cholinesterase deteriorated in ATT group but improved in immune intervention group. Levels of gamma-glutamyl transpeptidase were not much affected by either of treatment modalities. Thymol turbidity test appeared to show twice-higher inflammation process in ATT but no differences were seen in Dzherelo recipients.
Our results indicate that when Dzherelo is combined with ATT significant clinical, microbiological and radiological improvements are produced. Dzherelo was found to be safe and has improved or even reversed liver damage produced by ATT. These findings support earlier clinical investigations of Dzherelo by other investigators (Melnik et al'., 1999; Chechitiany et all., 2007; Prihoda et al, 2007, 2008; Nikolaeva et al, 2008a, b; Zaitzeva et al., 2008).
MDR-TB is diagnosed when Mycobacterium tuberculosis are resistant to at least izoniazid (H) and Rifampicin (R)-two most common first-line drugs. Nikolayevskyy et al. (2007) indicated that in the southern Ukraine the Multi-Drug Resistant form of TB (MDR-TB) was found in 27.3% of TB patients and was twice higher among formerly incarcerated individuals. According to Dubrovina et al. (2008) MDR-TB rates among civilian population in Donetsk region of the eastern Ukraine were 15.5 and 41.5% in newly diagnosed and previously treated TB patients.


Among prisoners, these figures were 2'.8% for new cases and 52.8% in previously treated TB cases. Treating drug-resistant TB is more difficult than drug-sensitive TB (Dye et al., 2005). Due to high failure and relapse rate of TB therapy in such patients the immune intervention with Dzherelo has been evaluated. In the prior studies Dzherelo has been shown to enhance the success rate of ATT and shorten significantly the duration of treatment even among those who had MDR or XDR forms of TB. However, as the number of patients with drug-resistant TB in the prior study was small, we evaluated Dzherelo in a larger population of patients. We had 48 patients with multi-drug resistant TB, which were equally divided into two treatment regimens along with 12 drug susceptible individuals.
Our results indicate that 81.8% of patients on Dzherelo became negative by sputum smear as opposed to 21.2% in ATT arm as early as three months after initiation of treatment. The difference in outcome was highly significant (p = 0.00000'). The proportion of sputum smear conversion appears to be not inferior of rates reported in the literature. The average successful treatment outcome of MDR-TB in Dominican Republic, Hong Kong, Italy, Russian Federation, Korea and Peru was 57% (Espinal et al., 2000). The best reported 85% success rate was reported by Chan et al. (2004) who
studied 205 MDR-TB patients in USA. However, such
results were reached after prolonged therapy which lasted on average '8 months or more. Recent study by Chinese colleagues, who treated MDR-TB for 3 months, revealed 50.8% sputum conversion rate (Fu et al., 2008). These findings are more relevant to our situation since we had identical treatment duration.
Sputum smear conversion is regarded as a reliable indicator of the efficacy of interventional therapy. However, this endpoint alone is not sufficient to reveal the extent of positive clinical outcome. To TB physicians the chest X ray remains the most dependable tool for gauging the outcome of treatment. The complete resolution of pulmonary infiltrations and cavities was seen in '2 (36.4%) patients in Dzherelo group, while only one patient (3%) treated with ATT had shown resolution by the 3rd month (p = 0.0008). The Chinese study has shown cavity closure and marked foci absorption in 2'.3 and 50.8% of 6' patients after 3 months (Fu et al, 2008). The rate of healing of local lesions was somewhat better than ours, but we had accounted only those who had shown the complete resolution. Most patients in Dzherelo had markedly improved radiological features but we did not include them due to subjective bias in evaluation of such assessments.
Adverse effects caused by TB drugs are common and can occur in up to 75% of patients (Greinert et al., 2007; Shin et al., 2007). We have measured the levels of liver damage markers such as total bilirubin, ALT and AST, gamma-glutamyl transpeptidase, cholinesterase and thymol turbidity. The results of liver function tests indicate that patients who received adjunct immunotherapy were better-off than those on ATT alone. The hepatoprotective effect of Dzherelo has been previously demonstrated in patients who were receiving anti-TB as well as anti-HIV therapy (Chechitiany et al., 2007; Prihoda et al., 2007, 2008). However, we do not know the exact mechanism of action and which specific components in the herbal preparation are responsible for this effect. Considering that on average it takes 18-24 months to treat MDR-TB the addition of Dzherelo can favorably enhance the quality of life and drug adherence of patients. The neutralization of hepatotoxic effect of TB drugs by Dzherelo represents a significant bonus in long-term management of MDR-TB patients.
The immunotherapy needs to be the indispensable part of interventional therapies against tuberculosis (Kaufmann, 2006). Several potentially effective immunomodulators were tested as adjuvant TB therapy (Wallis, 2005). These include interferon-gamma and environmental Mycobacterium vaccae, which have shown promising results in preliminary MDR-TB trials (Condos et al., 1997; Grahmann and Braun, 2008; Stanford et al, 2001; Zheng et al, 2004). While often effective their mechanism is not well understood (Wallis, 2005). This drawback should be balanced against clinically confirmed benefits.
Very few medicinal plants, such as Pelargonium sidoides, were shown to be clinically effective against TB (Kolodziej, 2008). The prevailing majority of other herbs exerted direct or indirect antimycobacterial activity only in in vitro studies (Tomioka, 2004; Newton et al., 2000). Unfortunately, we do not know which active ingredients in our multi-herbal preparation are responsible for the observed clinical effect. It is unlikely that Dzhereloacts as a tuberculostatic agent since in vitro growth of M. tuberculosis reference strains H32 and H37Rv was not affected directly and diverse diseases etiologically unrelated to TB were responsive to this preparation (Pylypchuk, 2003).
CONCLUSION
Our study provides additional evidence of safety and efficacy of Dzherelo (Immunoxel), which is recommended in Ukraine as an immune adjunct to TB therapy (Melnik et al., 1999). Depending on specific clinical endpoint the addition of Dzherelo enhanced the therapeutic efficacy of ATT by about 3-10 fold (Fig. 1).
Further studies are needed to develop better understanding of this unique herbal preparation and to increase treatment options for TB patients, especially with drug-resistant forms of tuberculosis, such as MDR-TB and XDR-TB (Prihoda et al., 2008).
ACKNOWLEDGEMENT
We thank all participants who volunteered in this study. The enthusiastic support of clinical staff and technicians made this study possible. We are grateful to other colleagues who shared their insight and provided helpful suggestions based on their own experience with Dzherelo.
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Adjuvant immunotherapy of extensively drug-resistant tuberculosis (XDR-TB) in Ukraine

Nathalia D. Prihoda1, Olga V. Arjanova1, Larisa V. Yurchenko1, Nina I. Sokolenko1, Lyudmila A. Vihrova2, Volodymyr S. Pylypchuk3, Valéry M. Frolov4, Galyna A. Kutsyna4 1Lisichansk Regional Tuberculosis Dispensary, Lisichansk, Ukraine, 2Lisichansk Regional Hospital, Lisichansk, Ukraine; 3Ekomed LLC, Prospect Pravdy 80-A, Kiev, Ukraine; 4Luhansk State Medical University, Luhansk, Ukraine

Abstract

Conventional TB chemotherapy success rates are low in patients with extensively drug- resistant tuberculosis (XDR-TB). We treated twelve XDR-TB individuals, seven of which in addition to standard anti-TB therapy (ATT) received Immunoxel (Dzherelo), Svitanok and Lisorm - over-the-counter herbal immunomodulators. All seven patients who received adjunct immunotherapy improved clinically and radiologically and were discharged after 3.7±0.8 months, with average/median time to mycobacterial clearance 28/25 days. None of five patients on TB drugs alone improved after 9 months and one had died. Patients on immune intervention gained 9.6 kg (P=0.0001) while those on ATT lost 1.4 kg. The levels of total bilirubin had decreased from 15.6 to 10.7 umol/L. Similarly, the values of alanine transaminase (ALT) have declined from abnormally high 42.6 IU/L to normal levels 22. IU/L (P=0.23). Patients on ATT had unchanged levels of bilirubin, but their ALT declined from 29.6 to 12 IU/L (P=0.02). The levels of hemoglobin had risen from 104.1 to 118 g/L (P=0.07), whereas leukocyte counts descended to normal levels from 8.9 to 7.3 * 10 cells/L (P=0.18). Conversely, in patients on ATT leukocyte counts had risen from 8.7 to 13.8 * 109 cells/L (P=0.21), whereas hemoglobin declined to below normal levels from 116.4 to 96.6 g/L (P=0.18). These results show that immune-modulating interventions can favorably influence the effect of TB drugs. The difference between two treatment outcomes was highly significant (Mantel Haenszel odds ratio=11; P=0.0009 at 95% CI). Thus, adjunct herbal immunotherapy is safe, shortens dramatically treatment duration, and can overcome drug resistance even in patients with XDR-TB.

Keywords: Immunomodulator; MDR-TB; XDR-TB; phytoconcentrates; Mycobacterium
Running title: Immunotherapy of XDR-TB

"Corresponding author:
Ms. Galyna Kutsyna
Tel: +380508093822
Fax: +3806454347106
E-mail: kutsyna@list.ru

Introduction

The extensively resistant TB (XDR-TB) is diagnosed when M. tuberculosis bacilli in addition to lack of sensitivity to isoniazid (H) and rifampicin (R), two most commonly-used, first-line TB drugs, are also resistant to any one of fluoroquinolones, and at least one of second-line injectable drugs, e.g., capreomycin, kanamycin, and amikacin (Migliori et al., 2008). This emerging form of TB caused worldwide concern after outbreak in Kwazulu Natal province of South Africa where 52 of 53 patients with XDR tuberculosis and HIV co-infection had died within 2 weeks from the time of diagnosis (Gandhi et al., 2006). Success rates in treating XDR-TB are significantly lower than among drug-sensitive cases ranging between 29% and 67%. In addition it takes much longer (18-24 months) to achieve a cure and concerns over adverse effects of drugs became more prominent since second-line drugs are more toxic. The cost is another factor as the deployment of second-line drugs increases treatment cost by about hundred-fold. Clearly there is an urgent need to find additional therapeutic interventions that could overcome these problems. Lisichansk TB Dispensary is within Luhansk administrative region of the eastern. Ukraine with total population 2.5 million. Approximate population of registered TB patients in this region is 2 thousand. Lisichansk TB dispensary has turnover of about 600-800 patients per year. The dispensary has six medical doctors and approximately 15 medical nurses and lab technicians who care for hospitalized patients and perform the lab work. Immunomodulators Immunoxel (Dzherelo), Svitanok and Lizorm are made from a proprietary combination of medicinal plants and are commonly used in Ukraine for the management of TB and HIV infections, including patients with dual infection (Arjanova et al., 2009; Chkhetiany et al., 2007; Melnik et al., 1999; Nikolaeva et al., 2008; Prihoda et al., 2007; Zaitzeva 2008). They have been approved in 1997 by the Ministry of Health of Ukraine as functional supplements with therapeutic indications. In 1999 Dzherelo and Svitanok were specifically recommended as immune adjuvants to the therapy of pulmonary tuberculosis (Melnik et al., 1999). So far, the phytoconcentrates we have decided to use in this study have been taken safely by several hundred thousand individuals for various indications including chronic bacterial and viral infections such as TB and HIV, autoimmune diseases, and malignancy (Chkhetiany et al., 2007). In this retrospective study, conducted at Lisichansk TB Dispensary, we have compared the adjunct effect of herbal immunomodulators to outcome of treatment with conventional TB therapy.

Materials and Methods
Patients

Twelve patients with pulmonary XDR-TB were identified retrospectively, five of which received individualized TB drugs regimen and seven who received in addition to ATT an immunomodulating phytopreparations Dzherelo, Svitanok and Lizorm. All patients were males with age range between 25 and 67 years. Five presented with first-diagnosed TB and the rest were previously treated, relapsed cases of TB. All study patients presented with acute symptoms of pulmonary TB that required hospitalization. Most common symptoms were prolonged heavy cough, pain in the chest, high fever, profuse night sweats, fatigue, and loss of weight and appetite. Active pulmonary tuberculosis was certified by a medical history and clinical findings compatible with tuberculosis, a chest X-ray showing lung involvement, and positive sputum smear for acid-fast bacilli (AFB) and the culture of M. tuberculosis. The conduct of the study was approved by the internal review board (IRB) of Lisichansk TB dispensary in accordance with the Helsinki Declaration.

Treatment regimen All anti-TB drugs were procured free-of-charge through centralized national supply system of Ukraine. The over-the-counter phytoconcentrates, Dzherelo, Lizorm and Svitanok were generously supplied by Ekomed LLC. Individualized, first- and second-line anti-TB drugs were administered to all hospitalized patients based on physician's decision prior to or after results of drug susceptibility tests. In the immunotherapy group, in addition to ATT, patients
received a daily dose of Dzherelo which was given as 30 drops diluted in a half-glass of water 30 minutes before breakfast. Some patients received Immunoxel - slightly modified form of Dzherelo. The same dose, 30 drops, of Lizorm and Svitanok were given before lunch and supper respectively. The exact formula of phytoconcentrates has been described by us earlier (Prihoda et al., 2008). Sputum smear and culture examinations for AFB were performed at monthly intervals. The decision to discharge was based on at least twice- repeated negative culture outcome and satisfactory clinical and radiological findings.

TB drug resistance testing

The drug resistance to first- and second-line TB drugs was tested with commercially supplied kit (Tulip Diagnostics, Goa, India). The cultures of M. tuberculosis derived from sputum of each patient were inoculated into ready-to-use tubes containing TB drugs incorporated at manufacturer-predetermined concentrations into standard Lowenstein-Jensen agar slants. The cultures were incubated at 37° C and checked periodically until appearance of colonies in control tubes without drugs. The calculation of the proportion of resistant bacilli was done by comparing counts on drug free and drug-containing Lowenstein-Jensen medium.

Statistical analysis

The obtained results were analyzed with available online statistical software (GraphPad Software, Inc., La Jolla, CA). All statistical analyses were done on intent-to-treat basis, involving the total number of patients without subgrouping them into responders and non-responders. The stratification analysis of patients was conducted to reveal the difference between distinct treatment categories. Parametric baseline values relative to the end of study values were evaluated by paired or unpaired Student t-test. The categorical test was done by Mantel Haenszel's odds ratio calculation. The probability values were considered as significant at P<0.05 cut-off value.

Results and Discussion

None of five patients on conventional TB drugs regimen had positive outcome after 9 months of treatment and one patient died after 9.5 months. The duration of treatment in immunotherapy group ranged between 10.6-20.4 weeks with average/median 15.7/16.7 weeks (Table 1). The treatment lasted until patients were discharged from the dispensary upon twice-repeated negative culture findings and clinical and radiological improvements. The time to negative culture conversion ranged between 20-37 days with mean/median 28/25 days. Mycobacterial clearance was confirmed by repeated cultures at monthly intervals. There appears to be no difference between first-diagnosed TB cases versus chronic, previously treated TB in terms of median days to discharge, i.e., 117 vs. 105.6 or days to mycobacterial clearance, 23 vs. 30. However sample size was too small to reveal statistically significant difference.

At the end of study every patient had gained substantial lean body mass, ranging between 6 and 13 kg. The average accrual in lean body mass was 9.6 kg, which was
statistically highly significant as evidenced by a paired Student's t-test (P=0.0001) - an effect that was evident early as one month from initiation of the therapy. In contrast, patients on ATT had lost on average 1.4 kg (P=0.4).

The potential hepatotoxicity of ATT combination with herbal preparations was monitored by quantitative liver function tests. Surprisingly, despite intensive chemotherapy patients have shown signs of better liver function. The level of total bilirubin had decreased from mean 15.6 to 10.7 umol/L - a favorable change that was not statistically significant (P=0.16). Similarly, the values of alanine transaminase (ALT), another marker of liver damage, have declined from abnormally high (42.6 IU/L) to normal levels (22. IU/L) - a change that was not statistically significant (P=0.23). Patients on ATT had same levels of bilirubin but their ALT declined from 29.6 to 12 IU/L (P=0.02).

Another phenomenon observed during therapy is a reversal of baseline anemic state and pro-inflammatory condition - symptoms very common in TB. Most patients at study entry displayed signs of anemia and had abnormally elevated leukocyte counts. At the end of treatment these parameters were improved in a statistically significant manner. The levels of hemoglobin had risen from 104.1 to 118 g/L (P=0.07), whereas leukocyte counts descended to quasi-normal levels from 8.9 to 7.3 * 109 cells/L (P=0.003). In patients on ATT the reverse trend was observed. Leukocyte counts had risen from 8.7 to 13.8 * 109 cells/L (P=0.21). Whereas hemoglobin declined to below normal levels from 116.4 to 96.6 g/L (P=0.18). These results show that immune-modulating interventions can favorably influence the efficacy of TB drugs (Arjanova et al., 2009; Chkhetiany et al., 2007; Nikolaeva et al., 2008; Prihoda et al., 2007; Zaitzeva 2008). All seven patients who received ATT and immunotherapy improved clinically and radiologically and were discharged after 3.7±0.8 months, with average/median time to mycobacterial clearance 28/25 days. None of five patients on TB drugs alone improved and one had died. The difference between two treatment outcomes was statistically significant (Mantel Haenszel odds ratio=11; P=0.0009 at 95% CI).

Our results compare favorably to XDR-TB chemotherapy outcomes reported in several
recent papers. According to study by Kim et al., (2008) only 29.3% of those with XDR-TB were cured. TB therapy success rate in Russian patients with XDR-TB as reported by Keshavjee et al., (2008) was 48.3%. Earlier reported cure rates in Europe, USA, Peru and Korea were between 37.5%-67% indicating that XDR-TB poses serious clinical challenge (Edward et al., 2008; Kwon et al., 2008; Migliori et al., 2008; Mitnick et al., 2008). In conclusion, adjunct herbal immunotherapy is safe, enhances significantly treatment outcome, and can overcome drug resistance even in patients with extremely poor prognosis. Further studies are needed to confirm our findings.

Acknowledgements

We thank all participants who volunteered in this study. The support of clinical staff and technicians who contributed to this study has been of tremendous help to us. We are grateful to other colleagues who shared their insight and provided helpful suggestions based on their own experience with phytoconcentrates used in present study.

References

Arjanova OV, Prihoda ND, Sokolenko Nl, Yurchenko LV, Vihrova LA, Pylypchuk VS, Frolov VM, Kutsyna GA. Impact of adjunct immunotherapy with multi-herbal supplement Dzherelo (Immunoxel) on treatment outcomes in end-stage TB/HIV patients. 2009 (in press J Appl Res Clin Exp Ther. Chechitiany R, Pylypchuk V, Argzanova O, Prihoda N, Vichrova L, Zagaydanova E, Kutsyna G. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naive HIV infected individuals. Intl J Biotechnol 2007;9:267-276. Edward D. Chan ED, Strand MJ, Iseman MD. Treatment outcomes in extensively resistant tuberculosis. New Engl J Med 2008;359:657-9. Keshavjee S, Gelmanova IY, Farmer PE, Mishustin SP, Strelis AK, Andreev YG, Pasechnikov AD, Atwood S, Mukherjee JS, Rich ML, Furin JJ, Nardell EA, Kim JY, Shin SS. Treatment of extensively drug-resistant tuberculosis in Tomsk, Russia: a retrospective cohort study. Lancet 2008;372:1403-9. Kim DH, Kim HJ, Park SK, Kong SJ, Kim YS, Kim TH, Kim EK, Lee KM, Lee SS, Park JS, Koh WJ, Lee CH, Kim JY, Shim TS. Treatment outcomes and long-term survival in patients with extensively drug-resistant tuberculosis. Am J Respir Crit Care Med 2008;178:1075-1082. Kwon YS, Kim YH, Suh GY, Chung MP, Kim H, Kwon OJ, Choi YS, Kim K, Kim J, Shim YM, Koh WJ. Treatment outcomes for HIV-uninfected patients with multidrug-resistant and extensively drug-resistant tuberculosis. Clin Infect Dis 2008;47:496-502. Melnik VP, Panasyuk OV, Pylypchuk VS, Moshich OP, Procenko NM, Leonenko OM. Deployment of herbal preparations Dzherelo and Svitanok for combination therapy of pulmonary tuberculosis. Medical Institute of Ukrainian Association of People's Medicine. Information Bulletin of the Ministry of Health of Autonomous Republic of Crimea. UDK:616.24-002.5-085-038:615.017. 1999. Kiev, Ukraine. Migliori GB, Lange C, Centis R, Sotgiu G, Mütterlein R, Hoffmann H, Kliiman K, De laco G, Lauria FN, Richardson MD, Spanevello A, Cirillo DM, TBNET Study Group. Resistance to second-line injectables and treatment outcomes in multidrug-resistant and extensively drug-resistant tuberculosis cases. Eur Respir J 2008;31:1155-9. Mitnick CD, Shin SS, Seung KJ, Rich ML, Atwood SS, Furin JJ, Fitzmaurice GM, Alcantara Viru FA, Appleton SC, Bayona JN, Bonilla CA, Chalco K, Choi S, Franke MF, Fräser HS, Guerra D, Hurtado RM, Jazayeri D, Joseph K, Llaro K, Mestanza L, Mukherjee JS, Muñoz M, Palacios E, Sanchez E, Sloutsky A, Becerra MC. Comprehensive treatment of extensively drug-resistant tuberculosis. New Engl J Med 2008;359:563-74. Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YuL, Masyuk LA, Kutsyna GA. Effect of oral immunomodulator Dzherelo (Immunoxel) in TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOTS. Intl Immunopharmacol 2008;8:845-851. Prihoda ND, Arjanova OV, Yurchenko LV, Sokolenko Nl, Vihrova LA, Pylypchuk VS, Kutsyna GA. Open label trial of adjuvant immunotherapy with Dzherelo, Svitanok and Lizorm, in MDR-TB, XDR-TB and TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOT. J Med Plant Res 2007;1:117-122. Prihoda ND, Arjanova OV, Yurchenko LV, Sokolenko Nl, Vihrova LA, Pylypchuk VS, Kutsyna GA. Adjuvant immunotherapy of tuberculosis in drug-resistant TB and TB/HIV co-infected patients. Intl J Biomed Pharm Sei 2008;2:59-64. Shah NS, Pratt R, Armstrong L, Robison V, Castro KG, Cegielski JP. Extensively drug- resistant tuberculosis in the United States, 1993-2007. JAMA 2008;300:2153-60. Zaitzeva SI, Matveeva SL, Gerasimova TG, Pashkov YuN, Butov DA, Pylypchuk VS, Frolov VM, Kutsyna GA. Efficacy and safety of phytoconcentrate Dzherelo (Immunoxel) in treatment of patients with multi-drug resistant TB (MDR-TB) in comparison to standard chemotherapy. In press Res J Med Sei. 2008.
Conclusions
  • Immunoxel is safe and reverses hepatotoxicity of TB drugs
  • Favorably affects anemia: increases hemoglobin levels
  • Has clear anti-inflammatory property: reduces high leukocyte counts
  • Helps to gain weight: mean gain 9.6 kg
  • Reduces time to negative culture conversion: median 25 days
  • Time to complete cure: 4 months
  • Success rate in our study is 100%
  • Standard TB therapy is not effective against XDR-TB: no result after 9 months. Further studies are required to confirm our findings

Baseline and outcome characteristics of XDR-TB patients treated with TB drugs without or in combination with Dzherelo, Lizorm and Svitanok

No.

Sex

Age

Type of
TB
infection
at
baseline
Resis
tance
to
TB
drugs3

Prescribed
TB drugs
regimen

Days
until
dischar
ge
Days
to
negative
culture
Weight
change
kg
Leukocyte
x 109L
Hb
g/L
Total
bilirubin
umol/L
ALT
IU/L

before

after

before

after

before

after

before

after

before

after

1

M

47

Relapse

H/R/S/K/
L

H/R/Z/S/E
Proth

Died
after
9.5
Months

No
conversion

67

55

8.9

4.0

115

90

10

12

37

12

2

M

52

Relapse

H/R/Z/S/
K/O

H/R/Z/S/E
PAS/L/A/
Cs
Still
treated
12 Mo

No
conversion

66

68

10.9

21

100

101

10

11

12

12

3

M

32

Relapse

H/R/Z/S/
K/L

H/R/Z/S/E
PAS/A

Still
treated
10 Mo

No
conversion

70

68

8,5

13.4

162

102

13

14

37

12

4

M

46

Relapse

H/R/Z/E/
S/K/L
H/R/Z/S/E/
PAS/Cs/
RFB
Still
treated
9 Mo
No
conversion

65

63

10.5

11.4

119

95

14

14

37

12

5

M

67

Relapse

H/R/E/
K/L
H/R/Z/S/E
Proth/PAS/
RFB
Still
treated
9 Mo
No
conversion

73

75

4.8

19.4

86

95

18

14

25

12



48.8
±12.
6






68.2
±3.3

66.8
±7.4

8.7
±2.4

13.8
±6.8

116.4
±28.6

96.6
±4.9

13.0
±3.3

13.0
±1.4

29.6
±11.1

12.0
±0









Mean loss
=1.4 kg
P=0.40
Mean gain
=5.1 x 109L
P=0.21
Mean loss
=19.8 g/L
P=0.18
Mean loss
=0 umol/L
P=1.0
Mean loss
=17.6 IU/L
P=0.02

6

M

42

First Rx

H/R/E/
K/O/PAS
H/R/Z/S/E/
Eth/RFB
+Dzh/Sv/Li

74

23

59

68

11.6

8.1

122

114

10.5

11.7

25

50

7

M

44

First Rx

H/R/K/L/
Eth/PAS
H/R/Z/S/E/
Eth
+Dzh/Sv/Li

143

34

63

69

4.5

6.8

120

118

18.6

10.5

12

50

8

M

35

First Rx

H/R/K/
A/C/P
H/R/Z/S/E/
Proth
+Dzh/Sv/Li

93

20

50

63

9

10

88

118

32.4

10.5

25

12

9

M

47

First Rx

H/R/S/
K/L/P
H/R/Z/S/E/
Proth/PAS
+Dzh/Sv/Li

133

22

52

62

9.1

9.1

108

116

11.7

10.7

62

12

10

M

25

Relapse

H/R/Z/O/
K/A/PAS
H/R/Z/S/E/
Proth/RFB
+Dzh/Sv/Li

89

25

65

78

8.2

6

109

120

10.5

10.5

62

12

11

M

52

First Rx

H/R/A/
P/PAS
H/R/Z/S/E/
Proth
+Dzh/Sv/Li

117

37

64

74

11

6

100

118

11.7

10.4

75

12

12

M

48

Relapse

H/R/K/O
A/PAS
H/R/Z/S/E/
Proth/RFB
+Dzh/Sv/Li

122

35

72

78

8.8

5.3

82

122

14

10.5

37

12



41.9
±9.2




110.1
±25.3
28
±7.1
60.7
±7.7
70.3
±6.6
8.9
±2.3
7.3
±1.8
104.1
±15.1

118.0
±2.6

15.6
±7.9

10.7
±0.5

42.6
±23.8

22.9
±18.5








Mean gain
=9.6 kg
P=0.0001
Mean loss
=1.6 x 109L
P=0.18
Mean gain
=13.9 g/L
P=0.07
Mean loss
=4.9 umol/L
P=0.16
Mean loss
=19.7 IU/L
P=0.23
aCriteria for definition of XDR are as per the WHO recommendations. TB drugs are
abbreviated as follows: Isoniazid (H), Rifampicin (R), Pyrazinamide (Z), Ethambutol (E), Streptomycin (S), Levofloxacin (L), Ofloxacin (O), Ciprofloxacin (C), Pefloxacin (P), Kanamycin (K), Amikacin (A), Cs (Cycloserine), Para-aminosalicylic acid (PAS), Ethionamide (Eth), Prothionamide (Proth), Rifabutin (RFB), Dzh (Dzherelo), Sv (Svitanok), Li (Lizorm).
 

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