Utility Of Bacteriophage D29 In Diagnosis And Treatment Of Tuberculosis

ObjectiveThe best way of tuberculosis control and prevention need rapid diagnosis and appropriate treatment based on the results of drug susceptibility testing, which can lead to few opportunities for dissemination. The phage amplified biologically (PhaB) assay was developed, using phage D29, to detect viable M. tuberculosis, and screen for resistance to rifampin and isoniazid among clinical isolates of M. tuberculosis. The PhaB assay has been compared with conventional proportion method and rapid molecular techology for the detection of rifampin, isoniazid resistance in M.tuberculosis. We evaluated the clinical use of the PhaB assay in China. There is still urgent requirement for new anti-tuberculosis drug, because drug-resistant tuberculosis remains a serious public health problem. Phage D29 is a lytic phage that infects and destroys both fast and slow-growing mycobacterial species, and likely to therapeutic efficacy. We explore the effect of phage D29 to M.tuberculosis in vivo and in vitro, and evaluate the possibility of it as anti-tuberculosis agent.Methods(1) We study the biological character of phage D29, including size, configuration, the replication cycle, preservation condition and others. The PhaB assay was compared with standard culture technique for detection of M. tuberculosis in simulated clinical samples.(2) 52 isolates of M. tuberculosis were identified the resistance to rifampin and isoniazid by PhaB assay and proportion method, and 26 isolates by DNA sequencing technology. We evaluate the PhaB assay in sensitivity, specificity and expense.(3) Macrophages, which had been infected mycobacteria, were randomly divided into control (i.e. saline) group, high-dose phage treated group, moderate-dose phage treated group and low-dose phage treated group. After treated with phage D29 for 24 or 48 hours, the number of intracellular viable bacteria was determined on agar or egg culture, and the intracellular changes were observed by electron microscopy.(4) For each infection experiment, guinea pigs separately were infected with sensitive M. tuberculosis or resistant M. tuberculosis. The animal model of tuberculosis were randomly divided into 3 or 4 groups: control (i.e. non-therapy) group, rifampin treated group, phage treated group, and (or) multi-drug treated group. Experiments were done in which either rifampin 25 mg per kilogram of weight through gastroesophagus, or phage 108 PFU through nasal, or four drugs (including rifampin, ethambutol, pyrazinamide and levofloxacin) through gastroesophagus by the interval of 3 times per 1 week. All guinea pigs were weighted weekly, and 3 to 11 pigs per group were sacrificed at week 4 and 7 post therapy, including pathological degree, lung or liver or spleen weight index, and quantitative cultures to assess the growth of M.tuberculosis in lung or spleen. In addition, the lungs, livers, and spleens from one guinea pig were used to assess the histopathologic changes induced by different therapy method at each sacrifice time. Moreover, blood samples from different therapy duration were collected to test the titer of serum phage-neutralizing antibody, and the concentration of IL-2, IL-4, IFN-gamma.Results(1) Mycobacteriophage D29 is a lytic phage, which could infect and destroy M. smegmatis and M. tuberculosis. The diameter of virus granule is about 75 to 150 nm. In M.smegmatis, the replication cycle is completed within 90 minutes to 180 minutes, whereas lysis takes approximately more than 3 hours to 6 hours in M.tuberculosis.(2) The phage preserved at 4℃with liquid formation was stable, and the declined titer was not more than 101 . when the preserver was phage's host bacteria and stored at 4℃, it had the same stable with dehydration form in 28 weeks.(3) The results of all bacteria lived in the respiratory tract with PhaB assay were negative. Compared with the results of L-J culture method, the sensitivity, specificity and accordance of the PhaB assay in rapid detection of M.tuberculosis respectively were 86.2%, 66.7% and 84.4%. When the sample included more than 100 viable M. tuberculosis, the PhaB assay could detect them and appear positive result in 3 days.(4) The result of L-J proportion method was seen as a standard for evaluation of PhaB assay in testing drug susceptibility of 52 M. tuberculosis isolates. When used to test the susceptibility to rifampin, the sensitivity, specificity and accordance of PhaB assay respectively were 94.1%, 94.4%, and 94.2%. When used to test the susceptibility to isoniazide, the sensitivity, specificity and accordance of PhaB assay respectively were 92.1%, 78.6%, and 88.5%. DNA sequencing technology was used to detect the resistance to rifampin or isoniazide, the sensitivity, specificity and accordance were respectively 82.4%, 100%, 88.5%, and 85.7%, 100%, 88.5%.(5) After macrophages were treated with high or moderate-dose phage D29 for 24 hours, the number of intracellular viable M. smegmatis is significantly less than control group(p<0.05). That of low-dose phage group was not significant compared to control group (p>0.05). Through electron microscopy, we searched that intracellular M.smegmatis was infected with phages and filial phages within intracellular bacteria appeared in 24 hours later.(6) Compared with control group, the number of decreased intracellular viable M.tuberculosis of high or moderate-dose phage treated group in 24 or 48 hours was significant(p<0.05), and the low dose group was not significant(p>0.05). Moreover, the number of decreased intracellular viable mycobacteria is related with time dependence and phage titer dependence.(7) When guinea pigs with tuberculosis had been treated with rifampin or phage D29 for 4 weeks, the pathological degree were significantly less than control group (p<0.05), and the quantitative cultures of M.tuberculosis in lung from phage treated group was significantly less than control group (p<0.05). However, the lung or liver or spleen weight index between various groups were not significant (p>0.05). There are mainly fibrosis in lung of phage treated guinea pigs and little necrosis, but most hyperplasia and Langhans cells in lung and caseous necrosis in spleen were seen in guinea pigs of control group in histopathologic examination.(8) The cytokine IL-2 secretion capability of phage treated group, which was infected resistant M. tuberculosis for 4 weeks, was statistically significant difference compared to control group(p<0.05). Other cytokine (IL-4, IFN-gamma) was no statistically significant difference.(9) The phage-neutralizing antibody in serum appeared after guinea pigs had been treated with phage D29 for 18 days, and it had titer–time dependence.(10) The guinea pigs'weights of rifampin treated group and multi-drugs treated group were significantly less than that of control or phage treated group (p<0.05) during treatment for 4 weeks. 2 pigs from multi-drugs treated group, 1 pig from rifampin treated group died during therapy. Histopathological examinations indicated that choletelin appeared in liver of guinea pigs from rifampin or multi-drug treated group. However, none of pig from control or phage group died unpredictly, which indicated phage therapy had no adverse effect.Conclusions(1) The method and process of phage amplification were easy. PhaB assay could detect viable M.tuberculosis in 3 days, and there is a feasibility of clinical utility for rapid diagnosis of tuberculosis.(2) PhaB assay is a simple and convenient, rapid, sensitive method and has high accordance with proportion method. The result of drug susceptibility with PhaB assay could obtain in 48 to 72 hours. It has lower-cost than molecular technology and has a good perspective in Chinese clinical laboratory.(3) Infected-mycobacteria macrophages could phagocyte phage D29, and phage D29 was effective in lysis intracellular mycobacteria without any deliver system in vitro. It might be a novel and hopeful strategy to combat mycobacteria infection diseases.(4) Phage D29 resulted in a significant reduction in organ pathological degree and quantitative cultures of mycobacteria, so the therapeutic efficacy of it approached to even than that of rifampin. None of adverse effect appeared in the bodies of guinea pigs treated with phage. The results reinforced the view that phage D29 has potential in the management of mycobacteria infection.(5) The phage-neutralizing antibodies appeared during therapy, which could hamper the effectiveness of subsequent treatments by the same phage. However, it could activate cell immunity which is important in host's mechanisms of defense against TB.(6) In conclusion, the results of this study reinforce the utility of PhaB assay in diagnosis of TB and drug susceptibility testing, which is helpful to the establishment of chemotherapy project and few opportunities for dissemination of resistant TB. Moreover, phage D29 has potential in becoming a novel and hopeful strategy to combat mycobacteria infection diseases. But additional problems, such as delivery system, may restrict the use of phage in the treatment of TB.