Ram Awadh Singh Kushwaha

Role of genotype® mycobacterium common mycobacteria/additional species assay for rapid differentiation between Mycobacterium tuberculosis complex and different species of non-tuberculous mycobacteria

Background: Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) may or may not have same clinical presentations, but the treatment regimens are always different. Laboratory differentiation between MTBC and NTM by routine methods are time consuming and cumbersome to perform. We have evaluated the role of GenoType® Mycobacterium common mycobacteria/additional species (CM/AS) assay for differentiation between MTBC and different species of NTM in clinical isolates from tuberculosis (TB) cases. Materials and Methods: A total of 1080 clinical specimens were collected from January 2010 to June 2012. Diagnosis was performed by Ziehl-Neelsen staining followed by culture in BacT/ALERT 3D system (bioMerieux, France). A total of 219 culture positive clinical isolates (BacT/ALERT® MP cultures) were selected for differentiation by p-nitrobenzoic acid (PNB) sensitivity test as and BIO-LINE SD Ag MPT64 TB test considering as the gold standard test. Final identification and differentiation between MTBC and different species of NTM were further confirmed by GenoType® Mycobacterium CM/AS assay (Hain Lifescience, Nehren, Germany). Results: Out of 219 BacT/ALERT® MP culture positive isolates tested by PNB as 153 MTBC (69.9%) and by GenoType® Mycobacterium CM/AS assay as 159 (72.6%) MTBC and remaining 60 (27.4%) were considered as NTM species. The GenoType® Mycobacterium CM/AS assay was proved 99.3% sensitive and 98.3% specific for rapid differentiation of MTBC and NTM. The most common NTM species were; Mycobacterium fortuitum 20 (33.3%) among rapid growing mycobacteria and Mycobacterium intracellulare 11 (18.3%) among slow growing mycobacteria. Conclusion: The GenoType® Mycobacterium assay makes rapid and accurate identification of NTM species as compared with different phenotypic and molecular diagnostic tool and helps in management of infections caused by different mycobacteria.

The advantage of using IS6110-PCR vs. BACTEC culture for rapid detection of Mycobacterium tuberculosis from pleural fluid in northern India.

Pleural tuberculosis is an extra-pulmonary disease which poses a diagnostic dilemma. The detection of mycobacterial DNA by IS6110 polymerase chain reaction (PCR) in clinical samples is a promising approach for the rapid diagnosis of pleural tuberculosis infections. The aim of the present study is to evaluate the advantage of using IS6110 PCR for rapid detection of Mycobacterium tuberculosis (M. tuberculosis) from pleural fluid. 102 clinically suspected cases of pleural tuberculosis cases were enrolled from inwards and outwards of the Department of Pulmonary Medicine at Chattrapati Shahuji Maharaj Medical University, Lucknow from April 2007 to April 2010. The pleural fluids were processed at the Mycobacteriology Laboratory of Department of Microbiology at Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Pleural fluid samples were processed and examined by Ziehl Neelsen (ZN) staining for acid fast bacilli and detection of M. tuberculosis by BACTEC culture. We applied IS6110 PCR to detect specific M. tuberculosis complex in pleural fluid samples. We found a significant difference in sensitivity of different tests, acid fast bacilli were detected in 17 (16.6%) samples by ZN Staining , 47 (46.1%) by BACTEC culture and using IS6110 PCR, 62 (60.7%) were positive for IS6110 PCR for M. tuberculosis. We found IS6110 PCR was much more sensitive than ZN staining and BACTEC culture. IS6110 PCR detection of M. tuberculosis may be very useful in cases that are highly suspect as pleural tuberculosis and those that are negative for AFB and culture. IS6110 PCR may gain an immense prospective to better clinicians ability to improve diagnosis of pleural tuberculosis.

Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India.

BACKGROUND The problem of multi-drug resistance tuberculosis (MDR-TB) is growing in several hotspots throughout the world. Rapid and accurate diagnosis of MDR-TB is crucial to facilitate early treatment and to reduce its spread in the community. The aim of the present study was to evaluate the new, novel GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing (DST) of MDR-TB cases in Northern India. MATERIALS AND METHODS A total of 550 specimens were collected from highly suspected drug resistant from pulmonary and extra-pulmonary TB cases. All the specimens were processed by Ziehl- Neelsen staining, culture, differentiation by the GenoType® CM assay, first line DST using BacT/ALERT 3D system and GenoType® MTBDRplus assay. The concordance of the GenoType® MTBDRplus assay was calculated in comparison with conventional DST results. RESULTS Overall the sensitivity for detection of rifampicin, isoniazid and MDR-TB resistance by GenoType® MTBDRplus assay was 98.0%, 98.4% and 98.2% respectively. Out of 55 MDR-TB strains, 45 (81.8%), 52 (94.5%) and 17 (30.9%) strains showed mutation in rpoB, katG and inhA genes respectively (P < 0.05). The most prominent mutations in rpoB, katG and inhA genes were; 37 (67.3%) in S531L, 52 (94.5%) in S315T1 and 11 (20%) in C15T regions respectively (P < 0.05). CONCLUSIONS Our study demonstrated a high concordance between the GenoType® MTBDRplus assay resistance patterns and those were observed by conventional DST with good sensitivity, specificity with short turnaround times and to control new cases of MDR-TB in countries with a high prevalence of MDR-TB.

Expression of Toll-like Receptor 2 and 4 in Peripheral Blood Neutrophil Cells from Patients with Chronic Obstructive Pulmonary Disease

Objectives Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. Preliminary studies have evaluated the association between innate immunity including Toll-like receptors (TLRs) and airway samples of patients with COPD. The role of TLRs in peripheral blood neutrophils is poorly understood. Hence, this study aimed to investigate the role of TLR2 and TLR4 in peripheral blood neutrophils of COPD patients. Methods A total of 101 COPD cases and an equal number of healthy controls participated in this case-control study. Peripheral blood neutrophils were isolated from all participants and cultured for 24 hours through lipopolysaccharide (LPS) stimulation. The gene expressions of TLR2 and TLR4 were assessed by real-time polymerase chain reaction. The protein levels of interleukin (IL)-8 and matrix metalloproteinase (MMP)-9 were measured in neutrophils cell culture supernatants using enzyme-linked immunosorbent assay (ELISA). Results The levels of IL-8 and MMP-9 were significantly higher in patients with COPD compared to healthy controls. Similarly, the gene expression of TLR2 and TLR4 were increased in LPS stimulated peripheral blood neutrophils of patients with COPD. Smoke pack years was positively correlated with IL-8 levels and negatively correlated with forced expiratory volume in the first second % (r = -0.33; p = 0.023) and FEV1/forced vital capacity (FVC) (r = -0.27; p = 0.011). Conclusions The increased expression of TLR2 and TLR4 suggests its role in disease pathogenesis of COPD. Smoke pack years was negatively associated with spirometric parameters in COPD patients. This may help to predict the smokers without COPD who risk developing the condition in the future.

A study to compare the diagnostic efficacy of closed pleural biopsy with that of the thoracoscopic guided pleural biopsy in patients of pleural effusion.

Background: The diagnostic approach to exudative pleural effusion remains an underappreciated aspect of modern thoracic medicine. 15-20% of the pleural effusions remain undiagnosed. The most efficient approach to pleural exudates remains uncertain and controversial particularly if acquisition of pleural tissue is required. The clinician needs to consider various factors when confronted with the choice between closed pleural biopsy (CPB) and thoracoscopy. Hence this study was planned to compare the diagnostic efficacy of CPB and Thoracoscopic pleural biopsy (TPB). Materials and Methods: This was a prospective interventional study in patients of exudative pleural effusion. CPB was performed by Cope′s biopsy needle. Then inspection of the pleural cavity was performed by single port rigid thoracoscope (KARL, STORZ TELECAM DX II 20 2330 20) with viewing angle of zero (0) degrees and biopsy taken from the diseased or unhealthy parietal pleura. Accordingly we compared the results of CPB and TPB. Results: 46 Patients underwent this study. In all 46 patients both CPB and TPB were performed.TPB was diagnostic in 36 cases (78.2%) while CPB was diagnostic only in 10 cases i.e. 21.7%. 10 (21,7%) cases remained undiagnosed. On thoracoscopic examination 30 patients were having nodularity, 25 (54.3%) were having adhesions and 20 (43.5%) were having hyperemia. 79.3% of the patients with nodularity turned out to be malignant and 71.4% of patients with adhesions and hyperemia tubercular. Conclusions: TPB has much greater diagnostic efficacy than CPB.

Detection of anaplastic lymphoma kinase gene re-arrangement in non-small cell lung carcinoma in the indian population: comparison of techniques and immunohistochemistry clones

OBJECTIVE Predictive and prognostic markers have revolutionized personalized therapy in non-small cell lung carcinoma (NSCLC). Crizotinib is now approved for locally advanced or metastatic NSCLC that is anaplastic lymphoma kinase (ALK) positive by either Fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC). The current study aimed to detect the incidence of ALK gene re-arrangement in the Indian population, to compare the various IHC antibodies with FISH as a gold standard, and to analyze the morphology of cases with ALK phenotype. MATERIAL AND METHOD A case series of 614 cases of NSCLC were included. IHC for detection of ALK phenotype was compared with FISH using 5A4 clone (Labvision, USA), ALK-1(Dako, Denmark) and D5F3 clone (Ventana, USA). RESULTS ALK gene rearrangement was evident in 4.07% of the cases. Cases with ALK phenotype had unique histomorphology with presence of mucin or signet ring cells in association with necrosis, high tumour grade and poor differentiation. Comparison of various antibody clones used in IHC revealed that the sensitivity and specificity using the D5F3 clone (100%, 100%) and 5A4 clone (87.5%, 100%) were similar while the ALK-1 clone had the lowest sensitivity and specificity (50%, 95.5%). CONCLUSION The incidence of ALK gene rearrangement in NSCLC in the current Indian study is within the worldwide reported range of 3-5%. This is the first study from the Indian subcontinent to compare various IHC antibodies used for detection of ALK phenotype. IHC using D5F3 clone and 5A4 clone may be considered as a rapid reliable and inexpensive method for detection of ALK gene rearrangement.

Multiple abdominal abscesses – A not so common presentation of NTM

Non-tuberculous Mycobacteria/Mycobacterium other than tuberculosis (MOTT) are ubiquitous organisms. They are acid fast bacilli often giving trouble to the physician to distinguish it from Mycobacterium tuberculosis. These organisms are a menace for the treating physician as when to treat and when not to treat. They are often difficult to diagnose and may present in a variety of forms with propensity to cause number of infections of different body parts and organs. They are more common in immunocompromised individuals e.g. HIV infection. Here we are reporting a not so common manifestation of NTM which presented as multiple abdominal abscesses in a middle aged female probably secondary to surgical site infection, however she responded dramatically to the designed treatment.

Two cases of eyelid tuberculosis – An uncommon presentation of ocular tuberculosis

Mycobacterium tuberculosis apart from being the causative agent of pulmonary tuberculosis is also notorious to cause tuberculosis at various sites in the human body and ocular tuberculosis is one of the extra pulmonary manifestations of this organism. The most common presentation of ocular tuberculosis is anterior uveitis or choroiditis caused by hematogenous infection or hypersensitivity after another organ infection. Eyelid involvement by tuberculosis is most of the times secondary to orbital involvement and often seen in the form of drainage sinus. Isolated eyelid tuberculosis is however uncommon. Here we report two such cases of eyelid tuberculosis in different age groups; first case in a young female and second case of an old aged female with different presentation. Fortunately both of them responded well to the antitubercular treatment.

Recent methods for diagnosis of nontuberculous mycobacteria infections: Relevance in clinical practice

Nontuberculous mycobacteria (NTM) infections are ever more important in recent years for leading causes of morbidity and mortality worldwide. Clinical appearance of Mycobacterium tuberculosis (TB) complex and NTM is same, but the treatment regimen is always different. NTM is challenging for both diagnostic and therapeutic with reason that it mimic pathological, microbiological, immunological, and radiological findings of TB. Newer molecular diagnostic methods allow for a better identification of NTM infections in patients not responding to antitubercular treatment and falsely categorized as drug-resistant TB. This article will explore the recent methods for the diagnosis and identification of NTM infections in clinical practice. In the future, the molecular-based diagnosis will significantly reduce the turnaround time of the diagnosis and thereby improving patient outcome.

Frequency of adverse events observed with second-line drugs among patients treated for multidrug-resistant tuberculosis.

BACKGROUND Multidrug-resistant tuberculosis (MDR-TB) is considered to be a worldwide problem with notoriously difficult and challenging treatment. Adverse events associated with second-line drugs (SLDs) can have severe impact on efficient management. OBJECTIVE To know the frequency of adverse events due to SLDs in patients of MDR-TB. DESIGN A prospective cohort analysis of 98 MDR-TB patients enrolled between June 2009 to February 2010 was conducted in Department of Pulmonary Medicine, King George Medical University, Lucknow, India. All the patients were provided standardized regimen. Adverse events associated with treatment were recognized primarily by clinical evidence and/or laboratory investigations that were advised at baseline and whenever clinically indicated during course of treatment. Adverse events were considered major if required permanent discontinuation or substitution of drugs. RESULTS 119 adverse events were reported in 46 (46.9%) patients. The grouped adverse events were most commonly gastrointestinal that was observed with a frequency of 48 (40.3%) followed by ototoxicity in 28 (23.6%), and neurological in 21 (17.6%). 17 (17.4%) patients had major adverse events requiring permanent discontinuation or substitution of drugs that included deafness and tinnitus in 5 (5.1%) followed by psychosis in 4 (4.1%). None of the patients stopped complete regimen due to adverse events. The treatment success rate was observed to be 71 (72.4%). CONCLUSIONS MDR-TB can be cured successfully with appropriate combination of drugs if adverse events associated with them can be managed aggressively and timely. Newer and less toxic drugs are urgently needed to treat MDR-TB patients.