Exposure to cetyl pyridinium chloride and loss of integrity of cell wall of mycobacteria.

Background: Cetyl pyridinium chloride (CPC) liquefied sputum was shown to reduce AFB smear positivity presumably damaging cell wall of M. tuberculosis. Settings: National Institute for Research in Tuberculosis, Chennai, (Tamil Nadu). Objective: To assess the cell wall damage of mycobacteria in CPC liquefied sputum, by Transmission Electron Microscopy (TEM) and mycobacteriophage adsorption studies. Methods: Pooled sputum sample from smear positive pulmonary TB patients was homogenized and liquefied with CPC. It was examined in TEM daily for four days, to assess cell wall damage of M. tuberculosis, and photomicrographs were taken. M. smegmatis mc2155, treated with CPC, was infected with mycobacteriophage (phAE129) to study phage adsorption on cell wall and plaque formation. CPC untreated sputum and M. smegmatis formed controls. Results: Photomicrographs showed that cell wall of M. tuberculosis was intact in controls and damaged in CPC preserved sputum for 96 hours. Plaque formation was seen and absent respectively in CPC untreated and treated M. smegmatis cells. Conclusion: Exposure to CPC damaged the cell wall of M. tuberculosis within 96 hours. Mycobacteriophage failed to form plaques after M. smegmatis mc2155 was treated with CPC implying inhibition of phage adsorption on damaged cell wall and thus providing a clue for poor staining and smear positivity in microscopy.

Research of immunogenicity of mycobacteriophage D29 toward tuberculosis therapy / 中华传染病杂志

Objective To investigate the immunogenicity of mycobacteriophage D29 (phage D 29) in guinea pig models with different delivery routes,and provide information for the application of phages in tuberculosis (TB) therapy.Methods Hartley guinea pigs were administrated with phage D29 through inhalation,intranasal drop or subcutaneous injection for 6 times within 35 days.7H9 broth aerosol inhalation and 0.85 ％ NaCl solution aerosol inhalation were set as solvent and negative controls,respectively.Anti-phage D29 neutralizing antibodies in sera collected weekly were measured by phage reduction neutralizing test (PRNT) and cytokine levels (interleukin-2,interleukin-4 and interferon-γ) were detected at day 35 by enzyme linked immunosorbent assay (ELISA).The data were analyzed by ANOVA and nonparametric test.ResultsNeutralizing antibodies were both negative in two control groups,while low-titer neutralizing antibodies (below 1 ∶ 100) appeared in inhalation and intranasal drop groups only at day 7 and day 14. Nevertheless, neutralizing antibodies were continuously detected in subcutaneous injection group,which increased rapidly and reached 1∶ 16 365.6 at day 35. After 35 days of experiments,serum concentrations of interleukin-2 (x2 =2.7605,P＞0.05),interleukin-4 (F=2.17,P＞0.05) and interferon-γ(F=0.75,P＞0.05) among three treatment groups and two control groups were all not significantly different.ConclusionsThe titer of anti-phage 29 neutralizing antibodies induced by inhalation or intranasal drop administration of phage D29 are both significantly lower than subcutaneous injection.Phage D29 administration doesn’t change the levels of cytokines,which indicates that it may not break the helper T cell (Th)1/Th2 balance.

Desenvolvimento de micobacteriófago recombinante para detecção rápida de bacilos da tuberculose / Development of recombinant micobacteriophage for rapid detection of tubercle bacillus

O diagnóstico da tuberculose por métodos tradicionais é lento e laborioso. Por outro lado, os testes moleculares são rápidos, mas com custo elevado para países em desenvolvimento. Este projeto teve o objetivo de inserir no micobacteriófago D29 o gene que codifica a proteína verde fluorescente (eGFP) e estudar o fago recombinante na detecção rápida de bacilos da tuberculose. Para tanto, foi inserido o cassete Hsp60- eGFP no genoma do fago D29 por recombinação. Micobacteriófagos recombinantes purificados foram utilizados para infectar M. smegmatis mc2 155 e M. tuberculosis H37Rv durante um período de 1- 6h nas temperaturas de 30°C, 37°C e 42°C. Bactérias fluorescentes foram observadas em um período de 2h, mas em número reduzido, indicando que o micobacteriófago lisou às células rapidamente, dificultando a expressão da eGFP e visualização em microscópio de fluorescência. A deleção do gene LysA, foi efetuada a fim de aumentar o período de latência do fago. Não foi possível a purificação de fagos recombinantes, devido à baixa quantidade de recombinantes nos halos de inibição. Será necessário a redução da atividade o gene LysA e, provavelmente, de outros genes associados a lise celular a fim de aumentar a concentração de eGFP no interior da célula.

Classical biochemical methods for Mycobacterium tuberculosis identification are lengthy and time-consuming. On the other hand, molecular assays are rapid but expensive for developing countries. This project aimed to insert into the mycobacteriophage D29, the gene coding for the green fluorescent protein (eGFP) and use the recombineered phage to detect Mycobacterium tuberculosis rapidly and less costly. For that, the Hsp-eGFP cassette was inserted into D29 genome. Recombineered mycobacteriophages was purified and used to infect M. smegmatis mc2 155 and M. tuberculosis H37Rv from 1-6 hs at 30°C, 37°C and 42°C. Observation of fluorescent bacteria was difficult and only a small number of them were seen at 2 hs of infection. This indicated that recombineered bacteriophages were lysing cells rapidly. Deletion of LysA gene, was carried out to increase the time needed for bacterial lysing. it was not possible to purify mutant mycobacteriophages due to the low concentration of recombinant phages. We conclude that might be necessary the deletion of other genes such as LysB, a gene also involved in cell lysis and reduction LysA activity to increase the concentration of eGFP inside cells.

Clinical application of first line anti-tuberculous drug susceptibility test by phage amplified biologically assay / 中华临床感染病杂志

Objective To evaluate clinical application of phage amplified biologically assay (PhaB) in susceptibility test of Mycobacterium tuberculosis (MTB) in sputum. Methods The drug susceptibility of MTB was detected by PhaB in 143 patients with sputum-positive pulmonary tuberculosis (PTB),and the chemotherapy regimens were adjusted according to the results of susceptibility test.Independent samples t-tests were used for comparison of means.Count numbers were compared with Chisquare test.If there were count number of 0,Fisher probabilities should be used.ResultsThe total positive rate of PhaB was 94.4％ (135/143) with no differences between three types of PTB (x2 =1.886,P ＞ 0.05 ).The duration of testing for PhaB group was (6.6 ± 1.8) days,while for control was (29.4 ±8.7) days (t =29.01,P ＜ 0.01 ).Compared with control group,the 2-month negative-conversion rate (63.2％ vs.35.1％,x2 =3.989,P ＜ 0.05 ) and cure rate ( 100％ vs.78.4％,P ＜ 0.05 ) of PhaB group in type Ⅱ patients were significantly higher.But there were no differences between PhaB and control groups in type Ⅰ and Ⅲ PTB patients.ConclusionThe results of PhaB drug susceptibility test can be helpful for choosing effective chemotherapy regimen for PTB patients rapidly.

Mycobacteriophages as versatile tools for genetic manipulation of mycobacteria and development of simple methods for diagnosis of mycobacterial diseases / Micobacteriófagos como herramientas versátiles para la manipulación genética y el desarrollo de métodos simples para el diagnóstico de enfermedades micobacterianas

Tuberculosis, caused by Mycobacterium tuberculosis, is responsible for over two million deaths per year worldwide. Due to its long doubling time (18 h), the microbiological detection of M. tuberculosis by conventional methods takes up to one month, unless the number of bacilli in the biological sample is high enough. Thus, drug resistance assessment requires at least one month for obtaining the primary culture and another month to determine its susceptibility to antimycobacterial drugs. Moreover, for a long time, the lack of genetic tools for mycobacteria has been a barrier for undertaking studies aimed at understanding the mechanisms of drug resistance and drug target identification, being all these topics of utmost importance considering the increase in the number of drug-resistant clones and the few therapeutic options available. Mycobacteriophages are promising as a novel source of genetic elements for mycobacteria manipulation, as well as for the development of versatile, simple, fast and cheap methods for drug resistance assessment of M. tuberculosis clinical isolates. We herein describe the background related to the use of mycobacteriophages, with emphasis placed on their utilization for drug resistance analysis in our country.

La tuberculosis, enfermedad causada por el bacilo Mycobacterium tuberculosis, es responsable de más de dos millones de muertes anuales en el mundo. Debido a su largo tiempo de duplicación (18 h), la detección bacteriológica de M. tuberculosis por métodos convencionales necesita de un mes o aun más, a menos que el número de bacilos en la muestra clínica sea suficientemente alto. Por consiguiente, se necesita un mínimo de dos meses para determinar la resistencia de este microorganismo a las drogas antituberculosas: uno para obtener el cultivo primario y otro para ensayar la sensibilidad frente a aquellas. La falta de herramientas para la manipulación genética de micobacterias ha dificultado la identificación de los blancos de acción de las drogas y el estudio de los mecanismos de resistencia a éstas, tópicos de la mayor relevancia dado el aumento mundial del número de aislamientos clínicos multirresistentes y las pocas opciones terapéuticas disponibles. Los micobacteriófagos son considerados nuevas herramientas para la manipulación de las micobacterias, así como para el desarrollo de métodos simples, rápidos y económicos para determinar la sensibilidad a drogas de los aislamientos clínicos de M. tuberculosis. En esta revisión se describen los antecedentes del uso de micobacteriófagos con énfasis en su utilización para el análisis de resistencia a drogas antituberculosas en nuestro país.

Clinical study of the application of phage amplified biologically assay in the detection of mycobacterium tuberculosis in sputum / 中国实用内科杂志

Objective To assess the application of the phage amplified biologically assay (PhaB) in the detection of the mycobacterium tuberculosis (TB) in sputum.Methods To determine the TB patients in the 53 suspected patients we detected 53 sputum samples by PhaB method,BACTEC MGIT960 rapid culture method,and conventional methods (direct auramine smear microscopy and concentrated auramine smear microscopy) individually.Results TB positive samples detected by PhaB,BACTEC MGIT960 rapid culture,direct auramine smear microscopy and concentrated auramine smear microscopy were 24,22,11 and 17 respectively.Thus the sensitivity,specificity,positive predictive value and negative predictive value were 86.4%,83.9%,79.2% and 89.7%,when the PhaB is combined with concentrated auramine smear microscopy,the sensitivity,specificity,positive predictive value and negative predictive value were increased up to 90.9%,83.9%,80.8% and 92.9%.Conclusion PhaB method is a simple and rapid method to detect the Mycobacterium Tuberculosis in sputum with a relatively higher sensitivity and specificity than the conventional methods.When combined with concentrated auramine smear microscopy,PhaB method is even more sensitive.Thus the PhaB method is accessible and applicable in the clinical use of the TB detection.

Recent advances in molecular methods for early diagnosis of tuberculosis and drug-resistant tuberculosis

Tuberculosis (TB) remains the main infectious cause of deaths in the world. Due to the slow metabolism of the causative agent, Mycobacterium tuberculosis, the isolation, identification and drug susceptibility testing requires several weeks. New techniques have improved specificity, turnaround time and cost effectiveness. Although these methods yield results within hours from sample collection, the clinical significance of each positive result requires rigorous evaluation in most cases. Herein the advantages and disadvantages of the most promising molecular techniques for detection of TB and drug resistance are discussed.

Avances recientes en métodos moleculares para el diagnóstico precoz y tuberculosis resistente al tratamiento La tuberculosis sigue siendo la principal causa de mortalidad por un agente infeccioso a escala mundial. Debido al metabolismo lento de su agente etiológico, Mycobacterium tuberculosis, el aislamiento, la identificación y las pruebas de susceptibilidad tardan varias semanas. Nuevas técnicas moleculares desarrolladas ofrecen mejorías en la especificidad, el tiempo para la obtención de resultados y su costo-efectividad. Estas pruebas producen resultados en pocas horas a partir de la toma de muestra, pero su relevancia clínica requiere aún ser evaluada rigurosamente en la mayoría de los casos. En esta revisión se discuten las ventajas y las desventajas de las pruebas moleculares más promisorias desarrolladas para el diagnóstico de la tuberculosis y la tuberculosis resistente a medicamentos.

Study on simultaneous detection of four drugs resistance anti-tuberculosis front line by phage amplified biologically assay / 中华传染病杂志

Objective Evaluating possibility of phage amplified biologically(PhaB) assay in detecting susceptibility of Mycobacterium tuberculosis(MTB) to four first-anti-tuberculosis-drugs on the same time and of 139 clinical isolates to streptomycin(S), isoniazid(H), rifampin(R) and ethambatal(E) at the same time, comparing with the results of Bactec-960 and determining the minimal inhibitory concentrations(MIC) of isolates which results were not consistent. Results Concordance rates of the susceptibility to S, H, R and E in 139 clinical isolates detected by PhaB and Bactec-960 are 97.1%, 99.3%, 95.7% and 95.0% respectively. If the results of Bactec-960 system is the golden standard, the sensitivity, specificity, positive and negative predictive value (PPV and NPV) as well as accuracy of susceptibility test to S detected by PhaB assay was 90.0%, 99.1%, 96.4%, 98.2% and 97.1% respectively, to H 97.9%, 98.9%, 97.9%, 98.9% and 95.0% , to R 86.2%, 97.3%, 89.3%, 96.4% and 95.0%, to E 81.0%, 97.5%, 85.0%, 96.6% and 95.0%. There are 19 inconsistent results of 13 isolates in comparing PhaB with Bactec-960. 18 results of 12 isolates by MIC are identical with the results of PhaB assay. 1 result of 1 isolate is identical with Bactec-960. Conclusions[KG1]The results of susceptibility to S, H, R and E detected by PhaB were highly concordance rate with the results of Bactec-960. PhaB assay can be used for rapid screening of susceptibility test for MTB.

Comparison of four kinds of method in detection isoniazid resistance in clinical isolates of mycobacterium tuberculosis / 中华传染病杂志

Objective To establish Phage Amplified Biologically Assay (PhaB) detecting isoniazid(INH) resistance rapidly and evaluate PhaB assay for drug susceptibility testing of isoniazid(INH) in clinical isolates of Mycobacterium tuberculosis(MTB). Methods Detecting the INH resistance of 167 clinical isolates of MTB by PhaB assay,comparing the results of PhaB with that of Bactec-960 system and analyzing the sensitivity, specificity and accuracy of PhaB assay. Results When the mixture of 0.2 ?g /ml INH and MTB was incubated in 37℃ for 48 h, the accurate results were obtained rapidly by calculate the reduce of plaque of PhaB assay. If the results of Bactec-960 system is the golden standard, the sensitivity, specificity, PPV, NPV and accuracy of PhaB assay was 96.4%, 96.4%, 93.1%, 98.2% and 96.4% respectively. Conclusions The PhaB assay with highly sensitivity specificity are highly consistent with Bactec-960 system. Not only it takes only three days to detect drug susceptibility of INH in clinical isolates of MTB but also it is easily to operate. We believe that this low-cost assay may be a good rapid screening of INH resistance in MTB isolates.