Correlating clinical breakpoint concentration of moxifloxacin with gyrA mutations using the GenoType MTBDRsl assay Version 2.0.

INTRODUCTION: Widespread use of Fluoroquinolones (FQs) has led to the development of its resistance in clinical isolates of Mycobacterium tuberculosis. However, in Mycobacterium tuberculosis, phenotypic resistance to FQs has been shown to be heterogeneous, ranging from low-level resistance to high-level resistance. This stratification in resistance has important implications for the inclusion of moxifloxacin (Mfx) in the treatment regimen. The World Health Organization recommends the use of GenoType MTBDRsl assay as the initial test for detecting resistance conferring mutations (both high and low) to FQs in patients with confirmed MDR-RR TB. The present study was conducted to explore the relationship of MTBDRsl Version 2.0 detected mutations in gyrA gene and genotypic DST of Mfx at WHO defined Clinical Breakpoint (CB). MATERIALS AND METHODS: A total of 200 sputum samples from Confirmed MDR/RR TB patients were included in this study. All of these samples had mutations conferring resistance to FQ confirmed by GenoType MTBDRsl assay. These samples were further subjected to Phenotypic DST against moxifloxacin using the Bactec MGIT-960 system. RESULTS: All of the 200 representative FQ resistant isolates had mutations in gyrA gene only with no detectable mutation in gyrB gene. 109 (54.5%) of the isolates had mutations associated with high-level increase in MIC while 91 (45.5%) isolates had mutations associated with low-level increase in MIC. Phenotypic DST of these 200 isolates against Mfx at CB (1.0µg/ml) revealed that of the 109 isolates with mutations associated with high-level increase in MIC and expected to be resistant at CB, only 34 (31.2%) were resistant and the remaining 75 (68.8%) were sensitive. CONCLUSION: Moxifloxacin is an important drug in the regimen for treating Drug-resistant TB and the decision to exclude this drug from the regimen should not be taken merely on the basis of mutational patterns. It should rather be taken after considering the combined results of mutational analysis and phenotypic DST.

Effectiveness of Xpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis at various stand-alone laboratories in Delhi.

BACKGROUND: Globally, EPTB accounts for 15% of the notified incident TB cases. Laboratory confirmation of EPTB is challenging and majority of the cases remain undetected for a longer time. A major breakthrough in the diagnosis of EPTB was the introduction of nucleic acid amplification tests (NAAT). One such test-the Xpert MTB/RIF assay also known as Cartridge based nucleic acid amplification test (CBNAAT) was endorsed by the Scientific and Technical Advisory Board of the WHO for the diagnosis of Tuberculosis. The present study was conduct to evaluate the outcome of various extrapulmonary samples tested in the year 2019 at different standalone NAAT laboratories in Delhi. MATERIALS AND METHODS: A total of 20,238 samples consisting mainly of Pus (21.77%), Cerebrospinal fluid (CSF) (14.96%), Biopsies (13.87%), Pleural fluid (10.49%), Lymph node aspirations (FNAC aspirates) (6.75%), synovial fluid (0.54%) and gastric aspirates (26.4%) tested at 22 standalone NAAT laboratories were included in this study. RESULTS: Mycobacterium tuberculosis was detected in 3496 samples and resistance to rifampicin was detected in 329 of the samples. The overall yield of all the specimens combined was 17.2%. Highest yield was seen in Lymph nodes aspirates (FNAC) (36.0%), followed by pus (35.4%), tissues (15.7%), synovial fluid (13.5%), Endometrial tissues (10.7%), Pleural fluid (9.5%), Gastric aspirates (9.4%) and CSF (6.5%). The lowest yield was seen in Cavitary fluids (6.2%). CONCLUSION: The results of this study highlight the usefulness of Xpert MTB/RIF assay in the diagnosis of EPTB. In particular, this assay proved to be of great utility while testing pus samples, tissue samples and lymph node FNACs.

Laboratory diagnosis of novel corona virus (2019-nCoV)-present and the future.

BACKGROUND: In December 2019 a novel coronavirus SARS-CoV-2 emerged in the Hunan seafood market in Wuhan, China, and soon became a global health problem. Since its outbreak, SARS-CoV-2 has had a major impact on clinical diagnostic laboratories. The scientific community has quickly risen to the occasion and reports of new developments have arrived at an unprecedented scale. At present, there is a growing list of over 400 SARC-CoV-2 diagnostic tests either in development or approved for clinical use. This presentation reviews the current laboratory methods available for testing COVID- 19 in microbiology laboratories and also provides an insight into the future diagnostics approaches. METHODS: Proper respiratory specimen collected at the appropriate time and from the right anatomical site is critical in the accurate and timely diagnosis of SARSCoV2. While oropharyngeal and nasopharyngeal swabs are recommended for the detection of early infection, other lower respiratory tract specimens like the sputum and bronchoalveolar lavage are used for late detection and monitoring of patients with severe COVID-19 pneumonia. RESULTS AND CONCLUSION: Real-time RT-PCR based molecular assay remains the test of choice for the etiological diagnosis of SARS-CoV-2 while serological tests are being introduced as supplementary tools. Finally, there is an urgent need for scaling up the diagnostic capacity by the introduction of reliable and accurate point-of-care tests which will assist in effective control of this outbreak. These assays can be used in the local hospitals and clinics bearing the burden of identifying and treating patients.

Benefits and limitations of serological assays in COVID-19 infection.

Accurate and rapid diagnostic tests are critical for achieving control of coronavirus disease 2019 (covid-19), a pandemic illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnostic tests for covid-19 fall into two main categories: molecular tests that detect viral RNA, and serological tests that detect anti-SARS-CoV-2 immunoglobulins. Reverse transcriptase polymerase chain reaction (RT-PCR), a molecular test, has become the gold standard for diagnosis of covid-19; however, this test has many limitations that include potential false negative results, changes in diagnostic accuracy over the disease course, and precarious availability of test materials. Serological tests have generated substantial interest as an alternative or complement to RT-PCR and other Nucleic acid tests in the diagnosis of acute infection, as some might be cheaper and easier to implement at the point of care. A clear advantage of these tests over RT-PCR is that they can identify individuals previously infected by SARS-CoV-2, even if they never underwent testing while acutely ill. Many serological tests for covid-19 have become available in a short period, including some marketed for use as rapid, point-of-care tests. The pace of development has, however, exceeded that of rigorous evaluation, and important uncertainty about test accuracy remains.

Advances in the diagnosis of tuberculosis- Journey from smear microscopy to whole genome sequencing.

The laboratory plays an important role in diagnosing tuberculosis (TB) and the identification and drug sensitivity testing (DST) of Mycobacterium tuberculosis. With a timely diagnosis and treatment with appropriate anti-TB drugs, most people who develop TB can be cured and onward transmission of infection curtailed. For a long time, laboratories used only microscopy and conventional culture-based diagnosis, however these procedures are slow and may require 3-4 weeks to yield results. Given the increasing rate of drug resistance, it has been necessary to look for new and rapid diagnostic methods. Various molecular based diagnostic technologies became available in the beginning of early 90s, providing rapid detection, identification and DST of M. tuberculosis. Molecular technologies offer the greatest potential for laboratories because they have the highest sensitivity and specificity. The present article will review some of the new methodology that has been introduced in the clinical laboratory.

Enteric opportunistic parasites among HIV infected individuals: associated risk factors and immune status.

Data on various etiologic agents causing diarrhea in human immunodeficiency virus type-1 (HIV-1) infected individuals are sparse in Delhi, India. The present study was undertaken to identify various causative agents, the role of associated risk factors and immune status. A case-control study was conducted among 75 HIV-1 infected individuals, 50 with and 25 without diarrheal infection. Fecal samples were screened for coccidian parasites, enteric protozoa, and helminthes by using various staining techniques. The CD4+ T-lymphocyte count was estimated. Enteric parasites were identified among 62.7% individuals, of which Cryptosporidium emerged as the single largest pathogen predominant among 33% of the individuals (P < 0.025). Other parasites diagnosed that were significantly associated with diarrhea were Giardia lamblia (13.3%), microsporidia (6.7%), and Isospora belli (2.7%). Chronic infected diarrheal cases were found to have polyparasitic infections. The mean CD4+ cell count was found to be lower among the diarrheal cases when compared with the non-diarrheal cases (mean, 141 cells/mm(3) versus 390 cells/mm(3)). Similarly, among diarrheal individuals, the chronic diarrheal cases had a comparatively lower CD4+ cell count than the acute cases (mean, 123 cells/mm(3) versus 265 cells/mm(3)). Risk factors found significant during multivariate analysis were: residence in a slum, exposure to pets and animals, use of public toilets, and practice of unsafe homosexual activity. Enteric coccidian parasites were identified as significant agents associated with diarrhea, especially among those with improper hygiene, multiple infections and a lower CD4+ cell count. Thus, this study emphasizes the need for routine screening of enteric parasites as well as education about practicing personal hygiene and taking timely and appropriate prophylactic measures.