microRNAs in Mycobacterial Infection: Modulation of Host Immune Response and Apoptotic Pathways

Our current knowledge of mycobacterial infections in humans has progressively increased over the past few decades. The infection of Mycobacterium tuberculosis causes tuberculosis (TB) disease, which has reasoned for excessive morbidity and mortality worldwide, and has become a foremost issue of health problem globally. Mycobacterium leprae, another member of the family Mycobacteriaceae, is responsible for causing a chronic disease known as leprosy that mainly affects mucosa of the upper respiratory tract, skin, peripheral nerves, and eyes. Ample amount of existing data suggests that pathogenic mycobacteria have skilled in utilizing different mechanisms to escape or offset the host immune responses. They hijack the machinery of immune cells through the modulation of microRNAs (miRs), which regulate gene expression and immune responses of the host. Evidence shows that miRs have now gained considerable attention in the research, owing to their involvement in a broad range of inflammatory processes that are further implicated in the pathogenesis of several diseases. However, the knowledge of functions of miRs during mycobacterial infections remains limited. This review summarises recent findings of differential expression of miRs, which are used to good advantage by mycobacteria in offsetting host immune responses generated against them.

DNA sequencing by Microseq kit targeting 16S rRNA gene for species level identification of mycobacteria.

BACKGROUND & OBJECTIVES: Identification of mycobacteria to the species level is of therapeutic significance. Conventional methods are laborious and time consuming so we did 16S rRNA sequencing using a commercial MicroSeq sequencing kit, which includes DNA sequencing with software package for identification and phylogenetic analysis of clinical mycobacterial isolates. METHODS: A total of 47 mycobacteria were tested by both conventional and genotypic method using commercially available MicroSeq 500 amplification kit assay. The identification was determined by comparing the 500 bp amplified product of 16S rDNA sequence to the MicroSeq database. RESULTS: The phenotypic identification was concordant with genotypic identification in 33 (70.2%) isolates of 14 Mycobacterium tuberculosis, 11 M. fortuitum, 7 M. abscessus and 1 M. duvalii. For the discrepant isolates, identification was possible only by DNA sequencing in 14 (29.7%) isolates. The 14 discrepant isolates were 5 M. farcinogenes, 3 M. genavense, 2 M. species. nov and 1 each of M. fortuitum, M. immuogenum, M. simiae and M. wolinskyi. Of these, five were uncommon species that were difficult to identify by phenotypic method. INTERPRETATION & CONCLUSION: The MicroSeq DNA sequencing is an excellent tool for species identification of mycobacteria, which reduces the turn around time, makes repeat analysis easy as compared to phenotypic identification specially for mycobacterial isolates with ambiguous biochemical profiles.

A study of mycobacterial species causing lymphadenitis.

This prospective study evaluated the non-tuberculous mycobacteria (NTM) cases of lymphadenitis. A total of 76 isolates of mycobacteria were obtained from 200 lymph node aspirates suspected of tuberculosis, 74 of which were Mycobacterium tuberculosis, one was Mycobacterium fortuitum and one Mycobacterium kansasii. These results highlight the importance of NTM in HIV-negative patients as a case of lymphadenitis, and indicates the re-emergence of NTM as potential lymph node pathogens in this part of the country. Further studies on a larger scale are needed to delineate the association between NTM infections in HIV positive and negative subjects.

Application of PCR-based restriction fragment length polymorphism for the identification of mycobacterial isolates.

BACKGROUND AND OBJECTIVE: Conventional identification of mycobacteria is achieved by standard biochemical tests that are time consuming, laborious and is not always conclusive. This study was thus undertaken to standardize a simple, rapid and cost-effective polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) using primers coding for the 16S - 23S rRNA spacer region to identify the mycobacterial isolates to the species level. METHODS: The PCR with primers targeting the 16S-23S rRNA spacer region was standardized using the standard mycobacterial strains and applied on 51 clinical isolates. The PCR amplified products were subjected to RFLP using the restriction enzymes, Hae III, MspI and BstXI. The results obtained were compared with those of conventional biochemical tests. RESULTS: PCR was sensitive to detect 2.5 pg of H37Rv DNA (370 bp for slow grower mycobacteria) and 1.5 pg of M. fortuitum DNA (450 bp for rapid grower mycobacteria). Based on the PCRRFLP products obtained the 51 mycobacterial isolates were classified into 41 slow growers and 10 rapid growers. Among the 41 slow growers, 40 were identified as M. tuberculosis, one as M. xenopi and 10 rapid growers as M. fortuitum. INTERPRETATION AND CONCLUSION: PCR using primers targeting the 16S-23S rRNA spacer region was a reliable tool for rapid identification of mycobacterial isolates into slow and rapid growers within 4 h of isolation and further speciation by PCR-RFLP within 6-8 h.

Acid fast bacilli (AFB) in the HIV/AIDS patients hospitalized in Institute for Clinical Research in Tropical Medicine

175 specimens of expectorated sputa and cerebrospinal fluids of 106 HIV/AIDS patients hospitalized in the Institute for Clinical Research in Tropical Medicine between January 2002 and October 2003 were tested by Ziehl-Nelsen stain and cultured by MGIT media for AFB. The results showed that: 14.9% positive patients with AFB detected by microscopic stains and 49.4% positive cases with Mycobacteria detected by cultures, including 76.2% Mycobacterium tuberculosis strains and 23.8% M. non-tuberculosis strains

Cord formation and colony morphology for the presumptive identification of Mycobacterium tuberculosis complex

The identification of Mycobacterium tuberculosis complex (MT), using non-molecular methods, is time-consuming. The objective of this study was to evaluate a screening test for the presumptive identification of MT, which could potentially decrease laboratory turn-around time for reporting preliminary results. From January 1998 to December 1999, 3056 cultures were analysed at the Mycobacterial Laboratory, Instituto Adolfo Lutz, S los o Paulo, Brasil. The screening test consisted of observation of colony morphology on L que wenstein Jensen medium and evaluation of cord formation on smear microscopy from those positive cultures. After the screening test, the cultures identified as non-tuberculous mycobacteria were identified to species by conventional methods (growth on culture and biochemical tests). Those identified as MT were submitted to drug susceptibility tests. The presumptive identification of MT using the proposed screening test, when compared with conventional tests, presented 98.9, 86.9, 97.8 and 93.0 percent of sensitivity, specificity, positive and negative predictive values, respectively. The conclusion is that it is possible to make a presumptive identification of MT using visual analysis of colony morphology and cord formation on microscopy examination. This method could be used to report the presumptive identification of MT and to guide laboratory decisions regarding susceptibility and identification tests with little cost and in a very practical way.

Mycobacteria in clinical specimens of sputum smear negative adults pulmonary tuberculosis suspects in Mulago Hospital

Mycobacteria in clinical specimens of sputum smear negative adult pulmonary tuberculosis suspects in Mulago Hospital. Background: Sputum smear negative tuberculosis is a major cause of morbidity and mortality among HIV negative and HIV positive patients attending Mulago hospital. The burden of sputum smear negative tuberculosis in Uganda is not known. a few published studies have been conducted on this subject in sub-saharan Africa where the disease burden is quite high. Objectives: The aim of the study was to determine the prevalence of culture positive but sputum smear negative pulmonary TB among adult sputum smear negative pulmonary suspects admitted to Mulago Hospital and to compare the diagnostic yield of different clinical specimens. To compare the clinical and radiological features in culture positive as well as culture negative pulmonary TB suspects above. Design: A descriptive cross-sectional study was carried out on newly admitted patients aged 15-65 years inclusive; who were sputum smear negative; had abnormal X-ray and had clinical features suggestive of pulmonary TB. Consenting patients had sputum bone marrow and blood taken off for mycobacterial cultures; review of their X-rays and HIV serlogy done. Main out come measures was culture positivity on any of the specimens; X-ray disease pattern; clinical characteristics and HIV sero-status. RESULTS: Of the 50 cultures reported; 30(15) were positive for mycobacteria tuberculosis while 2 (2) had mycobcteria other than TB reported. Sputum had the highest postive culture rates with 15 of the 16 culture positive patients; positive on sputum. Of the 63 patients recruited; HIV was found in 76.2(2) had mycobcteria other than TB reported. Sputum had the highest postive culture rates with 15 of the 16 culture positive patients; positive on sputum. Of the 63 patients recruited; HIV was found in 76.2(48) and had an average WHO clinical stage 3. By clinical history and physical findings it was difficult to differentiate between culture positive and culture negative patients although culture positive patients were more likely to have a temperature 37.2o c (P_value 0.05) as oral hairy leukoplakia (P-value 0.5). On chest X-ray a trend towards more pleural effusions and adenopathy was observed in the culture positive patients. CONCLUSION: 1. Culture positive but sputum smear negative pulmonary TB is 30prevalent among newly admitted adult sputum smear negative pulmonary TB suspects in Mulago Hospital. 2. Sputum as a specimen had the highest positive culture rate and therefore if one has to culture; he/she should use the sputum in such patients. 3. it is difficult by way of clinical and radiological features to differentiate between sputum smear negative but culture negative pulmonary TB suspects although culture positive suspects are likely to have a temperature of 37.oC as well as oral hairy leukoplakia and show a trend towards more pleural effusions and hilar adenopathy