Line Probe Assay Research Articles

Clinical presentations of nontuberculous mycobacteria as suspected and drug-resistant tuberculosis: Experience from a tertiary care center in Eastern India.

Nontuberculous mycobacteria (NTM) are on the rise worldwide. The diagnosis and treatment of NTM disease create a dilemma for physicians as their clinical features often overlap with that of tuberculosis (TB). The present study aims to report a series of NTM infections presenting as suspected TB. It was a prospective observational study starting from December 2018 to January 2022. A total of 1850 suspected TB patients (pulmonary = 522 and extrapulmonary = 1328) were included in this study. Clinical features, radiological findings, microbiological diagnosis, treatment, and outcome were recorded. Clinical specimens were processed for Ziehl-Neelsen staining, GeneXpert MTB/Rif assay by cartridge-based nucleic acid amplification test, and culture. The culture-positive isolates were categorized as Mycobacterium tuberculosis complex or NTM depending on the detection of MPT64 antigen by immunochromatographic test. The NTM isolates were speciated by line probe assay using GenoType® Mycobacterium common mycobacteria kit. The criteria of the American Thoracic Society/Infectious Diseases Society of America were applied to confirm NTM disease. Of 1850 suspected TB patients, NTM disease was diagnosed in 20 patients (pulmonary = 9, nonpulmonary = 11). Eight NTM cases presented as suspected drug-resistant-TB with a history of antitubercular therapy. Among pulmonary NTM cases, Mycobacterium scrofulaceum (n = 7) was the most common species followed by Mycobacterium kansasii (n = 1) and Mycobacterium intracellulare (n = 1). In nonpulmonary cases, Mycobacterium abscessus (n = 8) was involved in majority of cases followed by Mycobacterium fortuitum (n = 3). Cavitary lung disease and laparoscopic port site infections were most frequent pulmonary and non-pulmonary manifestations respectively. Hence, there is an urgent need for better diagnostic and drug susceptibility testing facility along with standardized treatment protocol for NTM disease.

Isoniazid and Rifampicin Tuberculosis Drug Resistance in HIV Endemic Region of Western Kenya

Background: Tuberculosis drug resistance is often associated with inadequate anti-tuberculosis treatment regimen resulting to mutations that confers resistance to anti-tuberculosis agents. This is aggravated by synergetic relationship between Tuberculosis and HIV (Human Immunodeficiency Virus). Over 25% of Global Tuberculosis deaths occur in Africa and Kenya is one of the 30 high burden countries that together account for more than 80% of the world’s TB cases. According to World Health Organization, in 2018, Multi drug resistant Tuberculosis prevalence in Kenya was 1.3% in new cases and 4.4% in retreatment cases. Kisumu County recorded the second highest HIV prevalence at 18.6% against the national prevalence of 4.5% in 2020. The extent of regional burden of DR-TB and HIV co-infection has not been exactly well-defined in Western Kenya. Methods: This was a prospective cross sectional study that aimed to explore the association between Tuberculosis drug resistance and HIV status among new and previously treated pulmonary tuberculosis cases in Kisumu County, Kenya. Tuberculosis clinical suspects were recruited into the study and classified as HIV positive or negative based on their clinical data. Sputum samples from tuberculosis clinical suspects were subjected to fluorescent microscopy, phenotypic culture and line probe assay. Results: Out of a sample of 256, response rate was 216 of which HIV positive cases were 119(55.1%) and negative were 97 (44.9%). The study found that out of 11 that were phenotypic Isoniazid resistance 8(6.7%) were from HIV positive cases while 3 (3.2%) were from HIV negative cases. Phenotypic rifampicin resistance among the HIV positive were 8 (6.7%) while HIV negative were 2 (2.1%). All the 2(1.7%) MDR cases were from HIV positive participants. The study found out that HIV status and Tuberculosis cases were significantly associated at p<.05. HIV positive cases were more likely associated with retreatment cases (OR=1.4,95CI:1.00-1.90) compared to new cases. The study found out that the common mutant probe among the HIV positive was katG MUT1 4(2.6%), while mutant probes among the HIV negative were in hA MUT1 1(0.7%), katG MUT1 1(0.7%) and roB MUT2A 1(0.7%). Wild type gene deletion among the HIV positive cases were observed in probes katG WT 3(2.1%), roB WT7, katG WT 1(0.7%) while wild type gene deletion among the HIV negative cases were inhA WT1 1(0.7%), in hA WT1/inhAWT2 1(0.7%), katG WT 1(0.7%). Conclusion: Interventions specific to HIV-endemic areas are urgently needed to block tuberculosis drug resistance transmission. Development and improvement of the efficacy of interventions will require a greater understanding of the transmission of multidrug-resistant tuberculosis in HIV-endemic settings like Kisumu County, Western Kenya.

The clinician, the lab and the patient: Understanding lab diagnostics to eradicate tuberculosis

BackgroundDiagnostic modalities for diagnosing Tuberculosis caused by Mycobacterium group of organisms include mainly AFB smear by Ziehl Neelsen carbol fuchsin smear microscopy, GeneXpert (CB NAAT) molecular method, Line probe assay (Molecular method) and AFB culture (Liquid automated systems and solid media) methods. MethodsThis study was initiated to understand and prioritize TB lab diagnosis, with reference to selection of lab diagnostic tests and its order of preference for MTC and NTM/MOTT closely associating it with the TB irradication program initiated by the Government of India. Result and conclusionThe results and discussion bring to light the importance of each test and the purpose of their requisition. When diagnosis is handled half heartedly eradication of TB becomes a challenge. All the efforts including planning, resources in the form of manpower, infrastructure, finances, education, time etc., would be hampered. This challenge is not only for India but the globe. For countries harboring TB, Correct diagnostic request and timely diagnosis and treatment is the key to eradication of tuberculosis.

Trends in cocirculation of oncogenic HPV genotypes in single and multiple infections among theunvaccinated community.

Cocirculation of multiple human papillomavirus (HPV) infections with low, probably high, and high‐risk genotypes are to be associated with various grades of infections and cancer progression. The oncogenic high‐risk HPVs are distributed and cocirculated throughout the world. This study was investigated to identify HPV genotypes related to genital disorders in unvaccinated women. The subjects were referred from clinics to a molecular lab for HPV testing in Iran as a low‐coverage vaccinated country. HPVs DNAs of cervical scrapping and genital tissue specimens of 1,133 un‐vaccinated women were genotyped using an in vitro diagnostic line probe (reverse hybridization) assay. In addition, phylogenetic trees were constructed on 100 MY09/MY11 polymerase chain reaction (PCR) amplicons of common genotypes of HPV L1 gene by Sanger sequencing. The mean age of the population study was 32.7 ± 8.0 and the mean age of HPV‐positive cases was 31.6 ± 7.8. HPV DNA was detected in 57.8% (655/1133) of women subjects and 42.2% (478/1133) of cases were undetected. Among 655 HPV‐positive cases, 639 subjects (56.4%) were related to defined genotypes and 16 subjects (1.4%) were untypeable. The highest prevalence rate of HPV genotypes was identified in the 25–34 years. The top 6 dominant HPVs in single and multiple genotypes were HPV6 (284/655 [43.4%]), HPV16 (111/655 [16.9%]), HPV31 (72/655 [11%]), HPV53 (67/655 [10.2%]), HPV11 (62/655 [9.5%]), and HPV52 (62/655 [9.5%]). Moreover, single, multiple and untypeable HPV genotypes were diagnosed as follows: 1 type (318/655 [48.5%]), 2 types (162/655 [24.8%]), 3 types (83/655 [12.7%]), 4 types (42/655 [6.5%]), more than 5 types (34/655 [5.3%]), and 1.4% un‐typeable subjects. The sequenced partial L1 gene of HPV genotypes (GenBank databases under the accession numbers: MH253467‐MH253566) confirmed and determined the cocirculated HPV genotypes' origins and addressed helpful insights into the future viral epidemiology investigations. Multiple HPV infections and cocirculation of various oncogenic HPV genotypes among the normal population (women and men) with asymptomatic forms are still challenging in unvaccinated communities. The preventive and organized surveillance programs for HPV screening are needed to be considered and compiled by health policy makers of low or unvaccinated countries.

Accuracy of Xpert MTB/RIF assay in diagnosis of tuberculosis a comparison to culture

Culture method is the most sensitive and specific method for the detection of Mycobacterium tuberculosis. Despite this, cultures are highly prone to contamination and the process can still take several days and does require expensive equipment, strict biosafety practices and well trained technical staff .Among the currently available Nucleic Acid Amplification Tests (NAAT), the Xpert MTB/RIF Assay (CB-NAAT), the LINE Probe Assay (LPA) and the Loop-Mediated Isothermal Amplification (LAMP) are endorsed by WHO for in vitro diagnosis of TB. The present study was carried out to assess the performance of CB-NAAT (Cepheid GeneXpert®) system for the diagnosis of MTB in both pulmonary and extra pulmonary specimens, within the demographic area of south Kashmir, India. Materials and methods: In this study Pulmonary & extra pulmonary specimens received at the Microbiology laboratory, from the new clinically suspected tuberculosis patients were used. The samples received at the laboratory were divided into three portions, one part each was used for AFB direct smear preparation, CB-NAAT and culture, respectively. A smear was reported positive if either the direct or the concentrated smear showed the presence of AFB. The concentrates were cultured on LJ media and incubated for up to 8 weeks.

Kinetics of Circulating Human Papillomavirus (cHPV) DNA in Plasma and Oral Gargles From Patients with HPV-Positive Oropharyngeal Cancer (OPC) Treated with Definitive Radiation Therapy

<h3>Purpose/Objective(s)</h3> Changes in cHPV DNA copy numbers and absence of detectable oral HPV DNA during and after treatment may be biomarkers of treatment response and recurrence. The goal of this study was to assess the kinetics of oral and plasma HPV DNA among HPV positive OPC patients pre, during and post radiation treatment. <h3>Materials/Methods</h3> Patients with T0-2 N0-1 p16 positive OPC were enrolled to a personalized radiation treatment trial where a total dose of 60-70 Gy based on size of largest lesion was delivered. Plasma and oral gargle was collected prior to treatment, during week 4 of treatment, at end of treatment (EOT), and 2-3 months post-treatment. Tumor-tissue-modified human papillomavirus (TTMV) DNA copy numbers were quantified in plasma (Naveris, Natick, MA). HPV genotyping of oral cell DNA was performed utilizing the HPV SPF₁₀ PCR-DEIA-LiPA₂₅ line probe assay system. Statistics were performed via Fisher's exact test. <h3>Results</h3> 33 patients with circulating TTMV and oral HPV genotyping were included. Patients with tonsil primaries were more likely to have detectable oral HPV (9/13) compared with BOT (6/20) (p=0.038). Of the 30 patients that were plasma TTMV positive, 15 were oral gargle positive. Of the 30 pretreatment TTMV positive patients, 11 of 30 (36.7%) had undetectable TTMV levels at week 4 of radiation therapy, 18 of 29 patients (62.1%) were TTMV negative by EOT, and 26 of 27 (96.3%) patients were negative at 3 months post-treatment. The one patient who remained TTMV positive at 3 months had a bone metastasis. Retrospective review of pretreatment imaging revealed that this was an undiagnosed metastasis in the bone at the time of trial enrollment. Oral HPV DNA was cleared in 12 of the 15 initially positive patients (80%) at 4 weeks, 14 of 15 patients (93.3%) at EOT, and 15 of 15 patient (100%) at 3 months. All patients had no evidence of disease clinically and radiologically at their 3 month post-treatment follow up visit, except for the one patient who was metastatic at presentation. TTMV clearance at 4 weeks was associated with a >30% volumetric imaging response at 4 weeks with 13 of 14 patients who had undetectable TTMV levels at 4 weeks also having with a >30% volumetric response as compared to 5 of 13 patients who did not have TTMV HPV clearance at 4 weeks but having a >30% volumetric imaging response (p=0.003). Clearance of oral HPV DNA was not associated with a partial response at 4 weeks. <h3>Conclusion</h3> Circulating TTMV copy numbers are detected in the majority of p16 positive OPC at diagnosis. TTMV clearance at 4 weeks is associated with a robust radiological response, suggesting radiosensitivity. These patients may represent a subset that would be candidates for de-escalation strategies.

The utility of repeat Xpert MTB/RIF testing to diagnose tuberculosis in HIV-positive adults with initial negative result

Background: Amongst HIV-positive adults in South Africa with initial negative Xpert results, we compared the yield from repeating Xpert MTB/RIF (“Xpert”) on sputum to guideline-recommended investigation for tuberculosis (TB). Methods: A systematic sample of adults attending for HIV care were enrolled in a cohort exploring TB investigation pathways. This substudy was restricted to those at highest risk of TB (CD4&lt;200 cells/mm3 or unknown) who had a negative initial Xpert result. At attendance for the Xpert result, a repeat sputum sample was stored, and further investigations facilitated per national guidelines. Participants were reviewed monthly, with reinvestigation if indicated, for at least three months, when sputum and blood were cultured for mycobacteria, and the stored sputum tested using Xpert. We defined TB as “confirmed” if Xpert, line probe assay or Mycobacterium tuberculosis culture within six months of enrolment were positive, and “clinical” if TB treatment was started without microbiological confirmation. Results: Amongst 227 participants with an initial negative Xpert result (63% female, median age 37 years, median CD4 count 100 cells/mm3), 28 (12%) participants had TB diagnosed during study follow-up (16 confirmed, 12 clinical); stored sputum tested positive on Xpert in 5/227 (2%). Amongst 27 participants who started TB treatment, the basis was bacteriological confirmation 11/27 (41%); compatible imaging 11/27 (41%); compatible symptoms 2/27 (7%); and unknown 3/27 (11%). Conclusions: Amongst HIV-positive individuals at high risk of active TB with a negative Xpert result, further investigation using appropriate diagnostic modalities is more likely to lead to TB treatment than immediately repeating sputum for Xpert. TB diagnostic tests with improved sensitivity are needed.

Isoniazid resistance in patients with Extrapulmonary Tuberculosis using line probe assay in a private multispecialty hospital in Bangalore, India

We aimed to study the rate of isoniazid (INH) resistance in Extrapulmonary Tuberculosis samples from a private care setting.A Line probe assay was performed on 74 culture isolates of Mycobacterium tuberculosis or directly on extrapulmonary samples received in our laboratory from 2018 to 2021.The INH mono-resistance among these extrapulmonary samples was 6.7%. (5 among 74) (95% CI: 1.04%–12.48%) Resistance to rifampicin was not detected.Increasing the availability and leveraging public private partnerships in hospitals for universal testing for INH resistance may increase detection of INH monoresistance in EP-TB and improve the strategy for TB elimination.

Diagnostic Modalities of Tuberculosis- Then and Now

Tuberculosis is one of the most ancient diseases known to affect humans and according to WHO remains major cause of death after HIV/AIDS. It remains a global public health problem due to increasing number of undiagnosed and drug resistant cases. Early diagnosis and immediate initiation of treatment is crucial to prevent the extensive spread of this deadly disease. Nowadays advancements in molecular based test imparted better and rapid diagnosis of TB. Several commonly used methods to screen and diagnose TB are clinical, immunological, microscopy, radiography, and bacterial culture. Molecular diagnostic methods including loop-mediated isothermal amplification (LAMP), line probe assays (LPA), GeneXpert, whole genome sequencing (WGS) etc have been employed to diagnose and characterize TB. Here, we reviewed use of these currently available and other future promising diagnostic methods along with their sensitivity, specificity, advantage and disadvantage of each method. Although new diagnostic methods have been developed, they require extensive amount of studies to validate their confirmatory role in the diagnosis of TB. There is increased requirement for biomarker discovery, validation and translation into clinical tools. High-burden countries will need to improve their efficiency of health care delivery and ensure better uptake of new technologies.

Non-tuberculous mycobacteria, not Mycobacterium bovis, are a significant cause of TB-like lesions observed in slaughtered cattle in Ghana

ObjectivesThe aim was to isolate and identify the species of mycobacteria causing tuberculous-like (TB-like) lesions in cattle in Ghana. MethodsBetween 2019 and 2020, 68 bovine tissue samples with TB-like lesions, identified during post slaughter examination, were obtained from four major abattoirs close to border towns in Ghana. The samples were cultured on Lowenstein–Jensen medium. Isolated bacteria were characterized by Ziehl–Neelsen staining and observation for acid-fast bacilli (AFB) under a microscope. DNA was extracted from AFB-positive isolates, and mycobacterial speciation was performed by line probe assay using GenoType Mycobacterium CM and also with mycobacterial 16S rRNA gene amplification and sequencing. ResultsNo Mycobacterium bovis was identified; however 53 bacterial isolates were obtained, of which 41 were non-tuberculous mycobacteria (NTM) strains and 12 were gram-positive bacteria. The predominant NTM species was M. fortuitum (43.9%, 18/41), with the rest being M. novocastrense, M. terrae, M. flavescens, M. holsaticum, M. cosmeticum, M. virginiense, M. intracellulare, M. mageritense, M. minnesotensis, M. duvalii, M. lehmannii, and M. koreense. ConclusionsIn cattle, NTM contribute significantly to lesions observed during slaughter examination and may be an important cause of zoonotic tuberculosis. A One Health surveillance of NTM in Ghana would provide insights into their clinical significance.

Human papillomavirus genotyping in high-grade squamous intraepithelial lesion.

Human papillomavirus infection is one of the most common sexually transmitted infections. Long-term exposure to the HPV leads to high-grade squamous intraepithelial lesions affecting cervical cancer. Knowledge about the distribution of HPV genotypes is crucial to guide the introduction of prophylactic vaccines. We aimed the genotype distribution in patients reporting due to abnormal Pap - smear tests. We provide a prospective observational cohort study. We obtained material from 428 women registered to Provincial Hospital in Poznan and Specialist Medical Practice in 2018-2020. In the current study, we analyze results from the first 110 inclusions with the diagnosis of HSIL from a cervical biopsy. The probe for the molecular test was collected with a combi brush and passed to an independent, standardized laboratory. HPV detection was done using PCR followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay. Sequence analysis was performed to characterize HPV-positive samples with unknown HPV genotypes. The molecular test detected DNA of 41 HPV genotypes. We performed statistical analyzes using the STATISTICA package 13.3. We found that 98.2% of patients received HPV-positive test results. The most frequent HPV genotype was 16, which assumed for 54.1%. In patients negative for HPV 16, the percentage decreased with increasing age. We detected that the following HPV types are next most common: HPV 31 (16.2 %), HPV 52 (11.7%), HPV 51 (9.9%), HPV 18 (9.0%), HPV 33 (9%). Moreover, thyroid diseases were the most common comorbidities and occurred in 15 patients CONCLUSIONS: To our knowledge, this study is the most extensive assessment of HPV genotypes in HSIL diagnoses in Poland.

Human papillomavirus genotyping in low-grade squamous intraepithelial lesions.

Human papillomavirus infection is one of the most common sexually transmitted infections. Histological LSIL in 70-80% of cases will regress spontaneously, while a subset is associated with residual risk for a future precancerous lesion. This study evaluates the performance of HPV genotypes for LSIL preceded by normal or mildly abnormal Pap smear. We provide a prospective observational cohort study. We obtained material from 428 women registered to Specialist Medical Practice and Provincial Hospital in Poznań in 2018-2021. In the current study, we analyze results from the first 112 inclusions with the diagnosis of LSIL from a cervical biopsy. The probe for the molecular test was collected with a combi brush and passed to the independent, standardized laboratory. HPV detection was done using PCR followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay. Sequence analysis was performed to characterize HPV - positive samples with unknown HPV genotypes. The molecular test detected DNA of 42 HPV genotypes. We performed statistical analyzes using the STATISTICA package 13.3. We found that 77.7% of patients received HPV-positive test results. The most frequent HPV genotype was 16, which was assumed for 22.3%. We detected that following HPV types are next most common: HPV 56 (11.6%), HPV 52 (8.9%), HPV 31 (8.0%) and HPV 51 (8.0%). Among HPV 16-negative women, the vast majority are those living in the town (p = 0.048). Moreover, thyroid diseases were the most common comorbidities. To our knowledge, this study is the most extensive assessment of HPV genotypes in LSIL diagnoses in Poland.

Diagnostic and public health investigation of Mycobacterium tuberculosis infection in a dog in Ontario, Canada

A 4-y-old, female mixed-breed dog was presented to the Ontario Veterinary College for further evaluation of multiple pulmonary and hepatic masses, intrathoracic lymphadenitis, and recent development of a pyogranulomatous pleural effusion. Along with other comprehensive tests, a thoracic lymph node biopsy was performed, and Mycobacterium tuberculosis complex infection was confirmed by real-time PCR. The dog’s condition declined post-operatively, and euthanasia was elected. Postmortem examination confirmed severe granulomatous pneumonia, hepatitis, intrathoracic and intraabdominal lymphadenitis, omentitis, and nephritis. Line-probe assays performed on samples collected postmortem confirmed the species as M. tuberculosis. 24-loci MIRU-VNTR genotyping, spoligotyping, and whole-genome sequencing revealed relations to known human isolates, but no epidemiologic link to these cases was investigated. Given the concern for potential human exposure during this animal’s disease course, a public health investigation was initiated; 45 individuals were tested for M. tuberculosis exposure, and no subsequent human infections related to this animal were identified. Our case highlights the need for more readily available, minimally invasive testing for the diagnosis of canine mycobacteriosis, and highlights the ability of canid species to act as potential contributors to the epidemiology of M. tuberculosis infections.

Investigation of Hepatitis C Virus (HCV) Genotypes by "Reverse Hybridisation Strip Assay" and DNA Sequence Analysis Methods

Hepatitis C Virus (HCV) is a virus that is estimated to infect approximately 185 million people worldwide and causes 350 000 deaths each year. The target in HCV treatment is the sustained virological response (SVR), and the most important virological factors determining SVR are genotype and baseline HCV-RNA levels. In this study, it was aimed to compare the HCV genotypes and subtypes detected by the "line probe assay (LIPA)" method with sequence analysis. HCV genotypes and subtypes were investigated by line probe assay (LIPA) (NLM analytica, Italy) and sequence analysis methods in 212 patients with chronic HCV diagnosis and with HCV RNA viral load of 10⁴ IU/ml and above. 5'UTR and core regions were studied in LIPA method and NS5B gene region was studied in the sequencing method. After amplifying 340 bp region in the NS5B gene region with the hemi-nested PCR method, sequence analysis (ABI 3500 Genetic Analyzer [Applied Biosystems, USA]) was performed. Statistical analysis of the study was determined by using TURCOSA Analytics program. In the study, HCV genotypes and subtypes that were determined in 212 patients by LIPA method were; 40 (18.8%) genotype 1a, 97 (45.75%) genotype 1b, 7 (3.3%) genotype 2, 12 (5.6%) genotype 2a/c, 2 (0.94%) genotype 2b, 19 (8.96%) genotype 3, 16 (7.55%) genotype 4, 1 (0.47%) genotype 4a, 15 (7.5%) genotype 4c/d, 1 (0.47%) genotype 4h and 2 (0.94%) genotype 5. The HCV genotypes and subtypes determined in 212 patients by sequence analysis were; 20 (9.43%) genotype 1a, 118 (55.6%) genotype 1b, 16 (7.55%) genotype 2a, 4 (1.89%) genotype 2b, 1 (0.47%) genotype 2k, 21 (9.91%) genotype 3a, 2 (0.94%) genotype 4a, 28 (13.21%) genotype 4d and 2 (0.94%) genotype 5a. The difference in results between the two methods was found to be statistically significant (p< 0.001). According to the viral load-genotype relationship, the highest viral load was detected in genotype 1a patients (p< 0.001). In conclusion, in order to determine HCV genotypes and subtypes in our study, LIPA and sequence analysis methods were compared and the genotype compatibility between the two methods were determined as 97.5% on the basis of genotype and 19% on the basis of subtype. Since the direct-acting antiviral (DEA) treatment protocol in chronic HCV patients is planned according to the genotype/subtype determination, the inconsistency of the results obtained in the LIPA method, which is routinely widely used, with the sequence analysis method is remarkable, and it was concluded that these results should be supported by studies containing more samples.

Drug susceptibility testing of Mycobacterium tuberculosis using next generation sequencing and Mykrobe software

Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis. Drug susceptibility testing is performed by phenotypic and molecular tests. Commonly used for phenotypic drug susceptibility testing is the automated BACTEC system in a liquid culture medium. Drug susceptibility by line probe molecular tests was introduced almost 15 years ago. Recently whole genome sequencing (WGS) analysis of M. tuberculosis strains demonstrated that genotyping of drug-resistance could be accurately performed. Several software tools were developed.Our study aimed to perform whole-genome sequencing on phenotypically confirmed multi-drug resistant (MDR) M. tuberculosis strains, to identify drug-resistant mutations and to compare whole-genome sequencing profiles with line probe assay and phenotypic results.Materials and methods. We performed analysis on 34 MDR M. tuberculosis Bulgarian strains. Phenotypic drug susceptibility testing was performed on the BACTEC system. For molecular testing of drug susceptibility to first- and second-line tuberculostatics, we applied line probe assay Geno Type MTBDR plus v.1.0 и Geno Type MTBDR sl v.1.0. Sequencing was performed on MiSeq. Generated FASTQ files were analyzed for known drugresistant mutations with the software platform Mykrobe v.0.8.1.Results. All three methods — phenotypic analysis using the BACTEC system, genetic analysis of strains applying the Geno Type test and Mykrobe software gave comparable sensitivity/resistance results for the studied strains. All phenotypically proven rifampicin and isoniazid-resistant strains were 100% confirmed using Mykrobe software. The C-15T mutation is a marker for isoniazid resistance in strains of the SIT41 spoligotype. We observed a 75% (21/28) agreement between BACTEC and Mykrobe for ethambutol resistance. Phenotypically, 87% (n = 27) of the strains are resistant to streptomycin, but only 59% (n = 19) are proven by Mykrobe software. Comparing phenotypic and genotypic resistance to ofloxacin, amikacin and kanamycin, we observed 100% coincidence of results.Conclusions. Whole-genome sequencing approach is relatively expensive and laborious but useful for detailed analysis such as epidemiological genotyping and molecular drug susceptibility testing.

Clinical Impact of the Line Probe Assay and Xpert® MTB/RIF Assay in the Presumptive Diagnosis of Drug-Resistant Tuberculosis in Brazil: A Pragmatic Clinical Trial.

ABSTRACTBackground: Rapid molecular methods such as the line probe assay (LPA) and Xpert® MTB/RIF assay (Xpert) have been recommended by the World Health Organization for drug-resistant tuberculosis (DR-TB) diagnosis. We conducted an interventional trial in DR-TB reference centers in Brazil to evaluate the impact of the use of LPA and Xpert. Methods: Patients with DR-TB were eligible if their drug susceptibility testing results were available to the treating physician at the time of consultation. The standard reference MGITTM 960 was compared with Xpert (arm 1) and LPA (arm 2). Effectiveness was considered as the start of the appropriate TB regimen that matched drug susceptibility testing (DST) and the proportions of culture conversion and favorable treatment outcomes after 6 months. Results: A higher rate of empirical treatment was observed with MGIT alone than with the Xpert assay (97.0% vs. 45.0%) and LPA (98.2% vs. 67.5%). Patients started appropriate TB treatment more quickly than those in the MGIT group (median 15.0 vs. 40.5 days; p<0.01) in arm 1. Compared to the MGIT group, culture conversion after 6 months was higher for Xpert in arm 1 (90.9% vs. 79.3%, p=0.39) and LPA in arm 2 (80.0% vs. 83.0%, p=0.81). Conclusions: In the Xpert arm, there was a significant reduction in days to the start of appropriate anti-TB treatment and a trend towards greater culture conversion in the sixth month.

Assessment of resistance to second line anti-tubercular drugs by line probe assay at a tertiary care hospital

Context: Emergence and spread of extensively drug resistance (XDR) tuberculosis (TB) is a challenge in resource-limited settings. Although India accounts for one-fourth burden of multi-drug resistant cases of TB, the prevalence of XDR TB varies widely. Timely detection of resistance to anti-tubercular drugs is important for optimal case management. Aims: Characterization of resistance mutations to second-line anti-tubercular therapy (ATT) using line probe assay (LPA). Materials and Methods: In this cross-sectional, conducted at a Tertiary Care Teaching Hospital in Western Maharashtra, we assessed resistance to second-line antitubercular drugs in 100 consecutive sputum specimens from patients failing first-line ATT as per national guidelines by LPA. Results: Of the 100 specimens studied, a total of 94 assay results were valid. Of these, no resistance was seen in 52 specimens. Resistance to fluoroquinolones (FLQ) was detected in 30% of specimen with a predominance of gyrA gene mutations. A total of 12% isolates were resistant to aminoglycosides with mutations observed in rrs gene. Conclusions: The study demonstrates the utility of LPA as a tool to support the diagnosis of drug-resistant TB in clinical setting.

Rifampicin Resistance by Xpert MTB/RIF Assay in Pulmonary Tuberculosis- Is there a Need for Confirmation by Retesting?

Introduction: Xpert Mycobacterium tuberculosis/ resistance to Rifampicin (MTB/RIF) assay detects MTB complex (MTBC) and rifampicin resistance simultaneously. In high prevalence countries like India, detection of rifampicin resistance in sputum specimen of a newly diagnosed case of pulmonary TB with a low pretest probability needs to be confirmed by retesting. Aim: To evaluate the results of retesting of rifampicin resistant specimens in newly diagnosed pulmonary TB cases. Materials and Methods: A retrospective analysis of the data of Xpert assay was performed on specimens received in Department of Microbiology, Seth G.S. Medical College and KEM Hospital, Mumbai, Maharashtra, India. If rifampicin resistance was detected in a newly diagnosed case of Tuberculosis (TB), a second specimen was retested by Xpert assay for confirmation. Concordance of retesting was seen with results of Line Probe Assay (LPA). Results: Total 27,429 specimens were processed by Xpert assay of which 803 specimens showed rifampicin resistance, 157 sputum specimens fulfilling criteria of Programmatic Management of Drug resistant Tuberculosis (PMDT) guidelines were retested. High, medium, low and very low bacterial load was observed in 30, 51, 34 and 42 specimens’ respectively. All specimens having high or medium bacillary load showed rifampicin resistant result on retesting. On retesting 34 sputum specimens with low bacterial load, rifampicin resistance was confirmed in 30 specimens. LPA done after growing them by liquid culture confirmed rifampicin resistance in remaining four specimens. Conclusion: Xpert assay is recommended when the bacterial load identified by Xpert assay is very low and when there is discordance between Xpert results of rifampicin resistance and the reflex LPA testing.

Utility of line probe assay in detecting drug resistance and the associated mutations in patients with extrapulmonary tuberculosis in Addis Ababa, Ethiopia.

Introduction:Molecular tests allow rapid detection of Mycobacterium tuberculosis and drug resistance in a few days. Identifying the mutations in genes associated with drug resistance may contribute to the development of appropriate interventions to improve tuberculosis control. So far, there is little information in Ethiopia about the diagnostic performance of line probe assay (LPA) and the M. tuberculosis common gene mutations associated with drug resistance in extrapulmonary tuberculosis. Thus, this study aimed to assess the frequency of drug resistance-associated mutations in patients with extrapulmonary tuberculosis (EPTB) and to compare the agreement and determine the utility of the genotypic in the detection of drug resistance in Addis Ababa, Ethiopia.Methods:A cross-sectional study was conducted on stored M. tuberculosis isolates. The genotypic and phenotypic drug susceptibility tests were performed using LPA and BACTEC-MGIT-960, respectively. The common mutations were noted, and the agreement and the utility of the LPA were determined using the BACTEC-MGIT-960 as a gold standard.Results:Of the 151 isolates, the sensitivity and specificity of MTBDRplus in detecting isoniazid resistance were 90.9% and 100%, respectively. While for rifampicin, it was 100% and 99.3% for sensitivity and specificity, respectively. The katG S315Tl was the most common mutation observed in 85.7% of the isoniazid-resistant isolates. In the case of rifampicin, the most common mutation (61.9%) was observed at position rpoB S531L. Mutations in the gyrA promoter region were strongly associated with Levofloxacin and Moxifloxacin resistance.Conclusion:Line probe assay has high test performance in detecting resistance to anti-TB drugs in EPTB isolates. The MTBDRplus test was slightly less sensitive for the detection of isoniazid resistance as compared to the detection of rifampicin. The most prevalent mutations associated with isoniazid and rifampicin resistance were observed at katG S315Tl and rpoB S531L respectively. Besides, all the fluoroquinolone-resistant cases were associated with gyrA gene. Finally, a validation study with DNA sequencing is recommended.

Role of Mycobacterial Culture and Drug Sensitivity Testing Laboratory under National Tuberculosis Elimination Program for the Abolition of Tuberculosis in India by 2025

India has made a bold promise to eradicate Tuberculosis (TB) by 2025 five years ahead of the global target. Although, one-fourth of the global burden with highest new cases of TB is shown by the country. So yet, no comprehensive analysis has been published on India’s National Tuberculosis Elimination Program (NTEP) (2017-2025). The current review details the advanced diagnostic methods like Fluorescence Microscopy (FM), culture, nucleic acid amplification test (Cartridge Based Nucleic Acid Amplification Test (CBNAAT) and True Nucleic Acid amplification Test (TrueNAT)) and line probe assay as well as the role and network of Mycobacterial culture and Drug Sensitivity Testing (C and DST) laboratories in national scaling-up of evidence-based policies and facilities, which is a critical component in India's fight against TB. The material of this study was mostly obtained from policy and program making documents of World Health Organisation (WHO) and annual TB reports of India. India’s TB annual report 2021 says that only half of the patients were successfully treated in the period of conventional longer care regimens. The interventions to achieve the factors related patient’s care have been implemented through universal drug sensitivity testing through C and DST laboratories, which has driven therapy with a shorter regimen, newer medications, and social protection. In one hand, the comprehensive monitoring scheme through C and DST laboratories for TB including all possible drug-resistance cases and other hand, patient’s systemic treatment through shorter, more reliable, and safer first- or second-line drug regimens are all necessary milestones to achieve the goal of our government for abolition of TB in India by 2025.