Phage lysin to control the overgrowth of normal flora in processed sputum samples for the rapid and sensitive detection of Mycobacterium tuberculosis by luciferase reporter phage assay

BACKGROUND: Phage lysin, extracted from three bacteriophages was used in place of antibiotics to control the overgrowth of normal flora in processed sputum samples leading to the sensitive detection of Mycobacterium tuberculosis using diagnostic luciferase reporter phage assay (DLRPA). METHODS: A total of 129 sputum samples were processed by modified Petroff’s method. Two Lowenstein Jensen slopes were inoculated from the processed sputum deposit thus obtained. The remaining deposits were transferred to 7 ml of Middlebrook 7H9 complete medium supplemented with phage lysin and incubated at 37°C. DLRPA was done using phAE129 at days 7, 9, 14 and 21. At the end of day 21, the samples were centrifuged and the pellets were inoculated on to 2 more LJ slopes to validate DLRPA results. RESULTS: The sensitivity and specificity of DLRPA in detecting M. tuberculosis from sputum specimens was 90% and 81% respectively compared to conventional LJ culture. The agreement between the methods was 87%. The rate of contamination for DLRPA using phage lysin was 9.3%. CONCLUSION: Phage lysin can be used to decontaminate sputum samples for the detection of M. tuberculosis by DLRPA directly from processed sputum specimens

Drug resistance among extrapulmonary TB patients: Six years experience from a supranational reference laboratory.

BACKGROUND & OBJECTIVES There is limited information available about the drug resistance patterns in extrapulmonary tuberculosis (EPTB), especially from high burden countries. This may be due to difficulty in obtaining extrapulmonary specimens and limited facilities for drug susceptibility testing. This study was undertaken to review and report the first and second-line anti-TB drug susceptibility patterns in extrapulmonary specimens received at the National Institute for Research in Tuberculosis (NIRT), Chennai, India, between 2005 and 2012. METHODS Extrapulmonary specimens received from referring hospitals were decontaminated and cultured using standard procedures. Drug susceptibility testing (DST) for Mycobacterium tuberculosis was done by absolute concentration or resistance ratio methods for the first and the second line anti-TB drugs. RESULTS Between 2005 and 2012, of the 1295 extrapulmonary specimens, 189 grew M. tuberculosis, 37 (19%) cases were multidrug resistant (MDR) while one was extensively drug resistant (XDR). Specimen-wise MDR prevalence was found to be: CSF-10 per cent, urine-6 per cent, fluids and aspirates-27 per cent, pus-23 per cent, lymph nodes-19 per cent. Resistance to isoniazid and ethionamide was found to be high (31 and 38%, respectively). INTERPRETATION & CONCLUSIONS Drug resistance including MDR-TB was observed in a significant proportion of extrapulmonary specimens referred for DST. Access to culture and DST for extrapulmonary specimens should be expanded. Guidelines for MDR-TB management should have explicit sections on extra-pulmonary tuberculosis and training on laboratory techniques is urgently required

Ofloxacin resistance in Mycobacterium tuberculosis: An increasing concern

Multidrug resistance tuberculosis (MDR-TB) associated with the development of resistance to fluoroquinolones (FQs) especially ofloxacin is a matter of concern, as they had been earlier recommended drugs for usage in the MDR-TB treatment regimens, and moxifloxacin and other quinolones are still on the list. Mycobacterium tuberculosis acquires resistance to FQs mainly through mutations in the quinolone resistance determining regions (QRDRs) of the gyrA gene and less frequently in the gyrB gene. A literature search on the geographical distribution of ofloxacin resistance in TB shows that there is a mild surge in reporting of the resistance to ofloxacin in tuberculosis patients. Molecular tests demonstrating mutations in gyrA and gyrB genes is widely used to detect ofloxacin resistance and the broadly available commercial assay for the rapid detection of second-line-drug resistance, including FQ resistance, the GenoType MTBDRsl assay (Hain Life science, Nehren, Germany), detects the most common mutations found in the QRDR of gyrA while its new version 2.0 detects mutations in the gyrB as well. It has been shown that on reviewing the frequency and geographic distribution of gyrA and gyr B mutations associated with FQ resistance, there do exist geographic differences in the frequencies within and across countries. Cross-resistance to FQs is an area of concern, although some studies show that concordance in resistance among the FQ agents, lower level of cross-resistance has also been reported. The presence of ofloxacin resistance is an alarm signal while Moxifloxacin and other FQs are still the recommended drugs for the resistant TB cases. The WHO recommendation that ofloxacin be phased out from MDR-TB regimens is well justified. It is important that rationale usage of ofloxacin is needed for preventing ofloxacin resistance, to aid in the management of tuberculosis

Extrapulmonary Tuberculosis—An Update on the Diagnosis, Treatment and Drug Resistance

Pathogenic Mycobacterium tuberculosis complex organisms (MTBC) primarily cause pulmonary tuberculosis (PTB); however, MTBC are also capable of causing disease in extrapulmonary (EP) organs, which pose a significant threat to human health worldwide. Extrapulmonary tuberculosis (EPTB) accounts for about 20–30% of all active TB cases and affects mainly children and adults with compromised immune systems. EPTB can occur through hematogenous, lymphatic, or localized bacillary dissemination from a primary source, such as PTB, and affects the brain, eye, mouth, tongue, lymph nodes of neck, spine, bones, muscles, skin, pleura, pericardium, gastrointestinal, peritoneum, and the genitourinary system as primary and/or disseminated disease. EPTB diagnosis involves clinical, radiological, microbiological, histopathological, biochemical/immunological, and molecular methods. However, only culture and molecular techniques are considered confirmatory to differentiate MTBC from any non-tuberculous mycobacteria (NTM) species. While EPTB due to MTBC responds to first-line anti-TB drugs (ATD), drug susceptibility profiling is an essential criterion for addressing drug-resistant EPTB cases (DR-EPTB). Besides antibiotics, adjuvant therapy with corticosteroids has also been used to treat specific EPTB cases. Occasionally, surgical intervention is recommended, mainly when organ damage is debilitating to the patient. Recent epidemiological studies show a striking increase in DR-EPTB cases ranging from 10–15% across various reports. As a neglected disease, significant developments in rapid and accurate diagnosis and better therapeutic interventions are urgently needed to control the emerging EPTB situation globally. In this review, we discuss the recent advances in the clinical diagnosis, treatment, and drug resistance of EPTB

Retrieval of Mycobacterium tuberculosis cultures suspended in phosphate buffered saline

AbstractOne hundred and twenty-seven of 130 isolates of Mycobacterium tuberculosis, suspended in phosphate buffered saline (PBS) and stored at ambient conditions in the laboratory for 14days, and another 55 of 60 cultures, suspended as above, transported from reference laboratories within 7days, were successfully retrieved on LJ medium. Considering the maximum retrieval of M. tuberculosis, use of PBS can be explored further for transportation of M. tuberculosis cultures across laboratories

Lytic Efficiency of Mycobacteriophages

Abstract: Mycobacteriophages D29 and TM4 are the two virulent phages widely used for the study of mycobacterial genetics. Both the phages are capable of killing Mycobacterium tuberculosis but the efficiency of these phages in killing has not been evaluated and compared. There are reports based on codon usage analysis where TM4 is predicted to be a better killing phage over D29 which corroborated with the whole genome in silico analysis. In addition a kill assay using 5 wild type virulent mycobacteriophages viz. D29, TM4, I3, Che7 and Che11 was performed to study the killing efficiency of these phages using LRP assay. Based on the results, D29 was found to infect all the 10 clinical strains of M. tuberculosis and significantly reduced RLU at 3 hours and this effect continued up to 24 hours. Thus, D29 is found to have better killing efficiency than TM4 contradicting the earlier predictions. In silico analysis of holin and lysin genes of TM4 and D29 substantiated our findings

An alternative sputum processing method using chitin for the isolation of Mycobacterium tuberculosis

An alternative bio-friendly sputum processing method is the need of the hour to augment the rate of detection of TB cases and to improve the sensitivity of rapid growth based diagnostic methods. Chitin, mucolytic in nature and present ubiquitously in animal kingdom, was found to have decontaminating activity when used for processing sputum specimens. The aim of the present study is to develop an alternative bio friendly sputum processing method using chitin. Smear microscopy was done on direct sputum samples and on the deposits obtained after processing with modified Petroff’s method as well as Chitin method. Two direct smears were made from each of the sputum samples and stained by Ziehl Neelsen and Auramine phenol (AP) method. The samples were divided in to two aliquots and processed by chitin and modified Petroff’s method. Smears were made from each of the deposits and stained by both methods. The deposits were inoculated on to two Lowenstein Jensen slopes. AP method showed a sensitivity of 95% in direct smear. Samples processed by chitin and the deposit smears stained by AP method showed a sensitivity of 80% and a specificity of 89% compared to that of modified Petroff’s method. The sensitivity of chitin culture is 87% and the specificity is 85%. Chitin–H2So4 solution took less time compared to 4% NaOH to homogenize the mucopurulent sputum specimens. Chitin–H2So4 can be used as an alternative method of sputum processing for the detection of M. tuberculosis