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.

Rapid detection of extensively drug-resistant (XDR-TB) strains from multidrug-resistant tuberculosis (MDR-TB) cases isolated from smear-negative pulmonary samples in an Intermediate Reference Laboratory in India.

BACKGROUND: Direct sputum smear microscopy is commonly used for diagnosing tuberculosis (TB). The objectives of the study were first, to determine the recovery of Mycobacterium tuberculosis in smear-negative sputum samples through liquid culture (using MGIT 960) and solid culture (using LJ slant) and second, to screen multidrug-resistant isolates through line probe assay and further third, to identify XDR isolates through MGIT second-line DST from these positive MDR cultures in Delhi region. METHODS: In this study, the sample size was 717 (sputum smear AFB negative and culture positive for M. tuberculosis complex by both solid and liquid culture methods) MDRTB suspects who were enrolled from January 2014 to December 2014 at the Intermediate Reference Laboratory in New Delhi Tuberculosis Centre, New Delhi. Rapid line probe assay was performed on all culture-positive samples, which were direct smear-negative specimens, and LPA-confirmed MDR samples were tested on MGIT 960 second-line DST for identification of XDR strains. RESULTS: An overall increase in the culture positivity (9.4%) among these smear-negative cases shows a good sign of recovery from M. tuberculosis infection in these samples. 717 (9.4%) positive cultures (MGIT+LJ) were subjected to line probe assay. Out of these 717 cultures, 9 (1.2%) were confirmed as NTM, 50 (7%) were MDR, 4 (0.6%) were mono-rifampicin resistant and 654 (91.2%) cultures were sensitive to both drugs Rif and Inh, respectively. Out of these 54 (50 MDR +4 mono-RIF resistant) cultures as screened by LPA, 1 (1.8%) was XDR, 10 (18.6%) were mono-ofloxacin resistant and 1 (1.8%) was mono-Kanamycin resistant. Sensitivity to both drugs KAN and OFX was seen in 42 (77.8%) cultures. CONCLUSIONS: Since the bacterial load in direct smear-negative suspected MDR samples is less, it is important to recover mycobacteria by rapid liquid culture method in such samples. Initial screening for MDRTB is to be done in such cases by performing rapid molecular genotypic drug susceptibility test such as LPA. Baseline second-line DST is also done to rule out the XDR cases among them for rapid and better management of XDRTB patients.

Multi-drug resistant tuberculosis among category I treatment failures--a retrospective study.

BACKGROUND: Tuberculosis (TB) remains a major global health problem and ranks as the second leading cause of death worldwide. An important cause of TB epidemic is the emergence of multi drug resistant (MDR) strains of Mycobacterium tuberculosis. Despite the availability of treatment that is expected to cure most cases of TB, levels of MDR-TB remain worryingly high in India. OBJECTIVE: This study was carried out to ascertain the prevalence of MDR-TB among category I pulmonary TB treatment failure patients. METHODS: This was a retrospective study involving 750 pulmonary tuberculosis patients enrolled at six district centres of Delhi State under RNTCP who failed to respond to CAT I treatment and whose sputum samples were submitted for culture and drug sensitivity testing (DST) over a period of three years (2009-2012). MDR-TB was defined as TB caused by bacilli showing resistance to at least isoniazid and rifampicin. RESULTS: Out of the total 750 patients included in the study, 470 (62.6 %) were culture positive. Of these, 377 (80.2%) were subjected to DST and rest 93 (19.7%) were excluded. Ultimately, DST result was available for 353 (93.6 %) cases. 239 (68%) cases were detected as multi drug resistant TB. CONCLUSION: High proportion of MDR-TB (68%) among culture positive CAT I treatment failure cases highlights the need for rapid diagnostic tests which will enable the detection of MDR-TB at an early stage and will thus minimize the risk of transmission as well as the possible errors associated with the treatment.

Predominance of Beijing genotype in extensively drug resistant Mycobacterium tuberculosis isolates from a tertiary care hospital in New Delhi, India.

Out of a total of 311 Mycobacterium tuberculosis isolates from sputum specimens subjected to first- and second-line drug-susceptibility testing (DST) at a hospital serving as a referral center for chronic tuberculosis (TB) cases in New Delhi, 232/311 (74.6%) isolates were found to be resistant to isoniazid and rifampicin. Among multidrug-resistant (MDR) isolates, 119/232 (51.3%) were resistant to four first-line drugs (streptomycin, isoniazid, rifampicin and ethambutol). Mono-resistance to isoniazid was observed in 18 (5.7%) isolates, while none of the isolates tested showed mono-resistance to rifampicin. 50/232 (21.5%) isolates met the definition of extensively drug resistant (XDR) TB, i.e., additional resistance to a fluoroquinolone and at least one of the three injectable second-line drugs: kanamycin, capreomycin, or amikacin. Spoligotyping of the XDR-TB isolates revealed 14 patterns; 39/50 (78%) isolates being grouped in three clusters vs. 11/50 (22%) isolates being unique. SIT1/Beijing represented the largest cluster (n=21, 42%), followed by SIT26/CAS1-Delhi (n=10, 20%) and SIT 53/T1 (n=8 isolates; 16%). This study corroborates recent observations from North India suggesting that both Beijing and CAS1-Delhi lineages constitute the bulk of XDR-TB isolates that are disseminating rapidly across a large geographical region in and around the capital city of India.

Performance of light-emitting diode fluorescence microscope for diagnosis of tuberculosis.

BACKGROUND: Fluorescence microscopy (FM) over the years has shown the potential for increasing the performance of microscopy. The present study was aimed to access the performance of the LED microscope for the detection of acid fast bacilli in a tuberculosis (TB) endemic country. METHODS: The study was conducted at a National Reference Laboratory (NRL) in New Delhi, India. Sputum samples were collected from suspected TB patients. Each sample was processed with Auramine O and ZN methods. Auramine O stained smears were evaluated using two different excitatory light sources (MVP and LED); and ZN stained smears were examined under light microscope. The mean time required to read the smears with different modalities was recorded. Bacterial cultures provided the reference standard. RESULTS: A total of 200 patients were included in this study. Sensitivity and specificity for the LED assessment, MVP assessment and light microscopy were 83.1% and 82.4%, 78.5% and 87.5% and 81.6% and 83.5%, respectively. Mean reading time was approximately three times faster than ZN microscopy. The mean time to read a negative smear was 2min with fluorescence microscopy and 5min with light microscopy with time savings of 60%. CONCLUSION: Although the use of LED-FM only marginally increased sensitivity, the considerable time saving ability combined with very good acceptance and ease of use makes it a reliable alternative to other conventional methods available.