Genetic markers, genotyping methods & next generation sequencing in mycobacterium tuberculosis.

Molecular epidemiology (ME) is one of the main areas in tuberculosis research which is widely used to study the transmission epidemics and outbreaks of tubercle bacilli. It exploits the presence of various polymorphisms in the genome of the bacteria that can be widely used as genetic markers. Many DNA typing methods apply these genetic markers to differentiate various strains and to study the evolutionary relationships between them. The three widely used genotyping tools to differentiate Mycobacterium tuberculosis strains are IS6110 restriction fragment length polymorphism (RFLP), spacer oligotyping (Spoligotyping), and mycobacterial interspersed repeat units - variable number of tandem repeats (MIRU-VNTR). A new prospect towards ME was introduced with the development of whole genome sequencing (WGS) and the next generation sequencing (NGS) methods, where the entire genome is sequenced that not only helps in pointing out minute differences between the various sequences but also saves time and the cost. NGS is also found to be useful in identifying single nucleotide polymorphisms (SNPs), comparative genomics and also various aspects about transmission dynamics. These techniques enable the identification of mycobacterial strains and also facilitate the study of their phylogenetic and evolutionary traits.

Polymerase chain reaction targeting insertion sequence for the diagnosis of extrapulmonary tuberculosis.

Background & objectives: Diagnosis of extrapulmonary tuberculosis (EPTB) is difficult using conventional diagnostic methods. This study was conducted to evaluate the use of polymerase chain reaction (PCR) in diagnosis of definitive and probable extrapulmonary tuberculosis patients, and to assess the performance of insertion sequence (IS) 6110 based PCR assay as compared to conventional culture by Lowenstein-Jensen (LJ) method for the diagnosis of EPTB. Methods: A total of 178 non repeated clinical specimens were collected from clinically suspected extrapulmonary tuberculosis patients. The specimens included 59 ascitic fluid, 54 pleural fluid, 25 cerebrospinal fluid (CSF), 12 fine needle aspiration (FNA), 8 urine, 7 pus, 6 synovial fluid, 2 skin tissue, one pericardial fluid, one liver abscess, one pancreatic cyst fluid, one omental biopsy and one semen sample. All these clinical samples were subjected to Ziehl-Neelsen staining (ZN) for acid fast bacilli (AFB) and culture on LJ medium. PCR was performed by targeting 123bp fragment of insertion sequence IS6110 of Mycobacterium tuberculosis (MTB). Results: Of the 178 specimens, 10 (5.61%) were ZN smear positive for AFB, six (3.37%) were L-J culture positive from 10 AFB smear positive cases and 48 (26.96%) were PCR IS 6110 positive for M. tuberculosis. Interpretation & conclusions: PCR using IS6110 primer was able to pick up more EPTB patients compared to conventional L-J culture method for detection of M. tuberculosis. False positive PCR IS6110 in three CSF samples may be due to latent TB infection which was limitation in this study.

Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes.

CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

Evaluating PCR, culture & histopathology in the diagnosis of female genital tuberculosis.

Background & objectives: Genital tuberculosis (GTB) is one of the major causes for severe tubal disease leading to infertility. Unlike pulmonary tuberculosis, the clinical diagnosis of GTB is difficult because in majority of cases the disease is either asymptomatic or has varied clinical presentation. Routine laboratory values are of little value in the diagnosis. An absolute diagnosis cannot be made from characteristic features in hysterosalpingogram (HSG) or laparoscopy. Due to the paucibacillary nature of GTB, diagnosis by mycobacterial culture and histopathological examination (HPE) have limitations and low detection rate. The objective of this study was to evaluate the efficacy of PCR technique, culture and histopathological examination in the diagnosis of GTB in female infertility. Methods: This study included 72 infertile women who met the inclusion and exclusion criteria. After a detailed history and clinical examination all patients were subjected to investigations including pelvic sonogram, HSG and laparoscopy. Endometrial samples from were allocated for AFB smear, culture and HPE examination. Only 49 samples were available for PCR using IS 6110 and TRC4 primers. In seven patients peritoneal fluid was also taken for culture and PCR. Based on the clinical profile and laparoscopic findings, a diagnostic criteria was derived to suspect GTB. Specific diagnostic tests were evaluated against this diagnostic criterion. Results: Laparoscopy was suggestive of tuberculosis in 59.7 per cent of cases, AFB smear was positive in 8.3 per cent, culture was positive in 5.6 per cent, HPE positive in 6.9 per cent and PCR was positive in 36.7 per cent of cases. Based on the diagnostic criteria, GTB was suspected in 28 of the 49 cases. On evaluating against the diagnostic criteria, the sensitivity of PCR, HPE and culture were 57.1, 10.7, 7.14 per cent respectively. The concordance of results between the clinical criteria and specific diagnostic tests were analysed by Kappa measure of agreement. The culture and HPE showed mild agreement with the clinical criteria, whereas PCR showed a moderate agreement. PCR was positive in Two of the 21 cases in whom GTB was not suspected. False positive PCR in these two cases were ruled out by multiple areas of sampling and re-sampling in one case. The PCR results were negative in 12 of the 28 cases. PCR using TRC4 primers had a higher sensitivity (46.4%) than IS 6110 primers (25%) in detecting clinically suspected GTB. Interpretation & conclusions: Our results showed that conventional methods of diagnosis namely, HPE, AFB smear and culture have low sensitivity. PCR was found to be useful in diagnosing early disease as well as confirming diagnosis in clinically suspected cases. False negative PCR was an important limitation in this study.

Bilateral variability of the quadriceps angle [Q angle] in an adult Indian population

The objective of this study was to document and explain bilateral differences in the Q angle. Two hundred limbs of healthy adult Indian volunteers were studied. The Q angle was measured using a goniometric method with the subjects supine, quadriceps relaxed and lower limbs in neutral rotation. The relative lateral placement of the tibial tuberosity with respect to the centre of the patella was measured. Appropriate statistical tests were used to determine the bilateral variability in the Q angle and the lateral placement of the tibial tuberosity. Inter-observer variation of the above mentioned parameters were studied in twenty limbs. The average Q angle value of all the 200 limbs was 12.73 °C; the mean value on the right was 12.86 °C and 12.60 °C on the left. When the Q angle and the lateral placement of the tibial tuberosity were considered in pairs a significant difference was noted in males. The Q angle value on the right side was more often greater than the left. The relative lateral placement of the tibial tuberosity showed a significant positive correlation with the Q angle. The intra-class correlation coefficient was 0.66 for the Q angle and 0.8 for the lateral placement of the tibial tuberosity. The present study shows that bilateral variability in the Q angle could be attributed to an alteration of the relative placement of the tibial tuberosity with respect to the centre of the patella

Identification & differentiation of Mycobacterium avium & M. intracellulare by PCR- RFLP assay using the groES gene.

BACKGROUND & OBJECTIVE: We report a new polymerase chain reaction (PCR) - restriction fragment length polymorphism (RFLP) assay using mycobacterial groES as a target to identify Mycobacterium avium and M. intracellulare in clinical samples. METHODS: The assay was standardized using M. avium and M. intracellulare standard strains obtained from ATCC and was tested with 45 M. avium-M. intracellulare complex (MAC) clinical isolates (Of which 31 were from HIV(+) individuals). The standard and clinical strains were typed with HPLC based mycolic acid fingerprinting. RESULTS: Three polymorphisms (BamHI, BstNI and HgaI) were identified for inter-species differentiation among standard strains; of which, only HgaI was found to be useful in clinical isolates. Of the 45 isolates, 25 were M. avium and 20 were M. intracelluare. MAC isolates, which could not be differentiated by HPLC analysis, were also typed by this method. INTERPRETATION & CONCLUSION: The use of mycobacterial groES as a PCR-RFLP target for M. avium and M. intracellulare is a simple and rapid method that can complement HPLC in their differentiation.

Molecular epidemiology of tuberculosis.

Molecular epidemiology (ME), a blend of molecular biology and epidemiology, is very useful to study the spread of tubercle bacilli in mini epidemics, outbreaks, to analyse the transmission dynamics of tuberculosis (TB) and to determine the risk factors for TB transmission in a community. ME has a great role in distinguishing between exogenous reinfection and endogenous reactivation. In the laboratory, molecular epidemiology can be used to identify cross contamination. Many new DNA typing methods have been introduced after the initial introduction of restriction fragment length polymorphism (RFLP) in 1993. An internationally accepted, standardized protocol for RFLP typing of the Mycobacterium tuberculosis complex using IS6110 was published in 1993 and is still used today. Most of the newer DNA typing methods are PCR based and microarray based methods are also available. This will enable individual strains of M. tuberculosis or clonal groups to be identified by specific phenotypic traits. ME will continue to be a useful tool in future to measure the impact of any public health intervention strategy for control of tuberculosis in the community.

Effect of oral exposure of Mycobacterium avium intracellular on the protective immunity induced by BCG.

The relative protective efficacy of oral administration of mycobacteria as compared to the conventional intradermal route of vaccination has been assessed in guinea pigs. Skin test reactivity to partially purified protein derivative and protective immunity to challenge with virulent Mycobacterium tuberculosis were used as parameters of protective immunity. Oral immunisation of guinea pigs either with BCG or with Mycobacterium avium intracellulare induces skin test reactivity and protective immunity comparable to that induced by intradermal route of vaccination. Oral exposure of Mycobacterium avium intracellulare prior to oral or intradermal dose of BCG did not interfere with the protective immunity induced by BCG in guinea pigs challenged with Mycobacterium tuberculosis H37Rv.