Line Probe Assay Research Articles

Effectiveness of GenoType MTBDRsl in excluding TB drug resistance in a clinical trial.

OBJECTIVES: To assess the performance of the GenoType MTBDRsl v1, a line-probe assay (LPA), to exclude baseline resistance to fluoroquinolones (FQs) and second-line injectables (SLIs) in the Standard Treatment Regimen of Anti-tuberculosis Drugs for Patients With MDR-TB 1 (STREAM 1) trial.METHODS: Direct sputum MTBDRsl results in the site laboratories were compared to indirect phenotypic drug susceptibility testing (pDST) results in the central laboratory, with DNA sequencing as a reference standard.RESULTS: Of 413 multidrug-resistant TB (MDR-TB) patients tested using MTBDRsl and pDST, 389 (94.2%) were FQ-susceptible and 7 (1.7%) FQ-resistant, while 17 (4.1%) had an inconclusive MTBDRsl result. For SLI, 372 (90.1%) were susceptible, 5 (1.2%) resistant and 36 (8.7%) inconclusive. There were 9 (2.3%) FQ discordant pDST/MTBDRsl results, of which 3 revealed a mutation and 5 (1.3%) SLI discordant pDST/MTBDRsl results, none of which were mutants on sequencing. Among the 17 FQ- and SLI MTBDRsl-inconclusive samples, sequencing showed 1 FQ- and zero SLI-resistant results, similar to frequencies among the conclusive MTBDRsl. The majority of inconclusive MTBDRsl results were associated with low bacillary load samples (acid-fast bacilli smear-negative or scantily positive) compared to conclusive results (P < 0.001).CONCLUSION: MTBDRsl can facilitate the rapid exclusion of FQ and SLI resistances for enrolment in clinical trials.

Molecular Methods and Culture in Diagnosis of Tuberculous Meningitis in Children

Objectives: This study aimed to detect the incidence of tuberculous meningitis (TM) in pediatric patients using GenoType MTBDRplus, GenoType MTBDRsl, and Mycobacteria Growth Indicator Tube (MGIT). Additionally, it aimed to evaluate the utility of GeneXpert MTB/RIF assay in the detection of Mycobacterium tuberculosis in comparison to MGIT culture. Methods: Children under or equal to 15 years of age who were clinically suspected of tuberculous meningitis (TBM) were included in the study. Cerebrospinal Fluid (CSF) samples were collected and tested for GeneXpert MTB/RIF assay and MGIT culture. Culture-positive samples were further analyzed using Line Probe Assay (LPA) to detect drug-resistant mycobacteria. Results: Out of 272 CSF samples, 28 (10.3%) samples were positive for MTB, and 5 (17.8%) were MDR-TB. GeneXpert MTB/RIF assay detected 23 (8.4%) MTB cases of which 3 (13%) were rifampicin-resistant. MGIT culture detected the presence of MTB organism in 19 (6.9%) cases, of which 6 (31.6%) were isoniazid-resistant and 2 (10.5%) were rifampicin-resistant by first line LPA. None of the samples had Extensively Drug-Resistant TB (XDR-TB). GeneXpert MTB/RIF assay had 73.6% sensitivity and 96.4% specificity taking MGIT as a gold standard. Conclusion: Total positivity for MTB was seen in 10.3% cases, among which 17.8% were MDR-TB; no XDR-TB was detected in pediatric patients. GeneXpert MTB/RIF assay is a rapid and reliable method for diagnosis of tuberculous meningitis but may miss some cases, so samples should also be cultured in MGIT to enhance yield and for extended sensitivity panel. J Microbiol Infect Dis 2021; 11(3):140-146.

Genotyping of human papillomavirus DNA in Wielkopolska region.

Human papillomavirus infection is one of the most common sexually transmitted diseases. Long-term exposure to the HPV leads to development of high-grade squamous intraepithelial lesions that can eventually transform into cervical cancer. The aim of the study was to assess the HPV genotype distribution in patients with abnormal pap smear and provide prospective study. We obtained material from 674 women who registered to Specialist Medical Practice in the years 2008-2020. The sample for the molecular test was collected using combi brush and forwarded to the independent, standardized laboratory. HPV detection was done using PCR followed by DNA enzyme immunoassay and reverse hybridization line probe assay for virus genotyping. Sequence analysis was performed to characterize virus genotypes in HPV - positive samples. We found that 53% of patients tested positive for HPV. The percentage decreased with age. The following HPV types were the most common: HPV - 16 (24.5%), HPV - 53 (13.1%), HPV - 31 (10.3%), HPV - 51 (9.7%), HPV - 56 (9.5%). To our knowledge, this study is the largest assessment of HPV genotypes in Poland. Our results suggest that type-specific, high-risk HPV DNA - based screening should focus on HPV types 16, 31, 51, 56.

Prevalence of Human Papillomavirus (HPV) Genotypes in Cervicovaginal Secretions of Human Immunodeficiency Virus (HIV) Positive Indian Women and Correlation With Clinico-Virological Parameters.

Introduction and Background: Both human papillomavirus (HPV) and the human immunodeficiency virus (HIV) are sexually transmitted. High-risk (HR) HPV types are a causal factor in cervical cancer. Persistent HPV infection in this subset of immunocompromised women results in faster disease progression. The study determined the prevalence of HPV genotypes in cervicovaginal secretions of HIV seropositive women and the correlation with CD4 counts and cytology.Method: One hundred, non-pregnant, HIV-positive women of 18 years of age and above were enrolled in this cross-sectional study following approval by the institutional ethical committee. A written consent, questionnaire, followed by sample collection including a Papanicolaou (Pap) smear for cytology was undertaken. Cervicovaginal secretion samples were collected in the Digene® specimen transport medium (STM) (Qiagen Gaithersburg Inc., MD, USA). HPV genotyping was carried out with PCR amplification of a 65-base pair (bp) fragment in the L1 region of the HPV genome using the short PCR fragment (SPF10) primers followed by reverse hybridization by line probe assay (LPA) using the INNOLiPA HPV Genotyping Extra kit (Fujirebio, Belgium). Quantitation of HPV-16 and−18 viral loads (VLs) was done by real-time PCR. Results of Pap smear cytology were correlated with CD4 counts and HPV-16 and−18 VLs.Results: Mean age of the subjects was 34.9 years ± 7.2 years (median 33.0 years, range 24–60 years). HPV was detected in 62 of 93 (66.6%) samples. Twenty (32.25%) of these 62 samples harbored a single HPV genotype. Multiple genotypes (more than two) were detected in 38 (61.3%) samples. HPV-16 was the commonest genotype detected in 26 (27.9%) of all samples and 41.9% of HPV positive samples. Pap smear cytology was reported for 93 women included in the study. Women who had normal cytology were reported as negative for intraepithelial malignancy or lesion (NILM; n = 62; 71.36%), two women had a high-grade squamous intraepithelial lesion (HSIL), low-grade squamous intraepithelial lesion (LSIL; n = 11), atypical squamous cells of undetermined significance (ASCUS; n = 12). Those smears with inadequate material were reported as scant (n = 6). The median CD4 count was 363/cu.mm (range 39–787) in HPV-positive women compared to 423/cu.mm (range 141–996) in those HPV-negative women. Quantitation of HPV-16 and−18 VL was done in duplicate for samples positive by PCR reverse hybridization (INNOLiPA). Of these 20 samples (65%), 12 samples were positive by real-time PCR. The normalized HPV-16 VL ranged between 18 and 240,000 copies/cell. The normalized HPV-18 VL in cervical samples ranged between ~24 and 60,000 copies/cell.Conclusion: HIV-positive women may be infected with multiple genotypes other than HPV-16 and−18. This may have implications on the vaccines available currently which target few specific genotypes only. Studies are required to determine the predictive role of HR HPV genotypes, in significant copy numbers especially in HIV seropositive women. It would be clinically relevant if the HPV VLs, cervical cytology, and CD4 counts are considered into cervical cancer screening programs for triage and follow-up of these women.

The Use of Molecular Genetic Diagnostic Tests to Improve MDR TB Treatment Outcomes in Arkhangelsk Region

The use of molecular genetic tests as a part of tuberculosis patients examination made it possible to reduce the time for TB diagnosis and determination of drug resistance (DR) of M. tuberculosis (MTB) in Arkhangelsk Region. Early detection of multiple drug resistant tuberculosis (MDR TB) made it possible to prescribe the adequate chemotherapy regimen promptly and thus to improve treatment outcomes.The objective of the study: to evaluate the results of treatment of MDR TB patients in whom MDR TB was diagnosed by molecular genetic tests. It was assumed that the introduction of molecular genetic tests would result in improved treatment outcomes in MDR TB patients [(the research project of the International Union Against Tuberculosis and Lung Diseases and Tuberculosis Control Program of Arkhangelsk Region of the PROVE-IT LPA (Policy Relevant Outcomes from Validating Evidence on Impact of Line Probe Assays)].Subjects: 295 MDR TB patients detected in Arkhangelsk Region were enrolled in the study. MDR TB was detected by molecular genetic tests in the main group (132 patients) and by culture in the control group (163 patients). Patients from both groups received the standard chemotherapy regimen. Chemotherapy outcomes were compared in both groups.Results. Treatment outcomes were better in the group (MGT group) where molecular genetic tests were used for drug susceptibility testing (p = 0.003) versus the comparison group where the culture was used. Effective treatment was documented more frequently (65.2%) in the MGT group versus the comparison group (44.8%). All-cause mortality was lower in the MGT group (7.6%) than in the comparison group (15.9%). There were no statistically significant differences between the groups in the time when sputum conversion (by smear and culture) was achieved.

Comparison of Line Probe Assay (LPA) and Mycobacterium Growth Indicator Tubes (MGIT) Assay for Second-line TB Drug Susceptibility Testing

BACKGROUND: Tuberculosis (TB) infection is one of the most prominent health issues in the world, including in Indonesia. TB is evolving into multidrug-resistant tuberculosis (MDR-TB) and requiring second-line TB drugs. Mycobacterium growth indicator tube (MGIT) is the gold standard for susceptibility testing of second-line TB drugs. Alternatively, line probe assay (LPA), which detects genes resistant to second-line TB drugs, takes a shorter time to run. This study aims to compare MGIT and LPA's ability to detect TB resistance to second-line TB drugs and observe mutation patterns of genes encoding second-line TB drugs.METHODS: This was an observational analytic study, using cross-sectional method. The data were acquired from the MDR-TB clinic’s medical records at the Dr. Hasan Sadikin Hospital from September to December 2019. LPA and MGIT test were conducted at the Health Laboratory Hall of West Java Province, then tested using Kolmogorov-Smirnov and chi-square statistic.RESULTS: From 121 subjects, 113 people were not resistant to the second-line TB drugs, which was examined using both LPA and MGIT (93.4%), p=0.991. Mutations were found in gyrA and rrs gene. There was no significant difference between the proportion of subjects resistant to the second-line of TB drugs tested using LPA and MGIT.CONCLUSION: LPA is an alternative method to MGIT because it requires a shorter time and reduces the risk of exposure that will improve MDR-TB patients management.KEYWORDS: line probe assay (LPA), multidrug-resistant TB, mycobacterium growth indicator tube (MGIT), second-line TB drugs

Estimation of genotype MTBDRPLUS line probe assay in detection of rifampicin and isoniazid resistance in comparison to liquid culture (BACTEC-960) drug susceptibility testing in a tertiary care hospital from Eastern India

Introduction and Aim: India has the uppermost trouble of Multidrug resistant tuberculosis (MDR-TB) is a major challenge controlling resistance, reducing transmission and improving handling outcomes in MDR-TB patients is dependent on susceptibility testing. Isoniazid (INH) and rifampicin (Rif) are the key first-line antituberculosis drugs, and resistance to these drugs i.e., MDR-TB, is likely to result in treatment failure and poor clinical outcomes. The present study was done to compare the performance of line probe assay test (GenoType® MTBDRplus) with liquid culture (MGIT 960) system for the detection of resistance to first-line drugs.&#x0D; &#x0D; Materials and Methods: We estimate the performance of LPAs to BACTEC MGIT 960 system for susceptibility testing of bacterial resistance to first-line drugs: rifampicin (RIF), isoniazid (INH).&#x0D; &#x0D; Results: We performing Drug susceptibility testing (DST), 219/258 MTB cultures were viable after subculture the results of DST using the MGIT 960 system were compared to those obtained by line probe assay. LPA detected a total 46/258 (17.81%) samples as drug resistant, of which 35/258 (13.70%) were resistant to both rifampicin and isoniazid (MDR), 6/258 (2.28%) were rifampicin mono?resistant samples and 11/258 (4.11%) were isoniazid mono?resistant. Out of the culture?positive samples (219), LPA detected 39/219 (17.83%) as drug?resistant, of which 31/219 (14.2%) were resistant to both rifampicin and isoniazid, 5/193 (2.08%) were rifampicin mono?resistant and 8/219 (3.7%) were isoniazid mono?resistant.&#x0D; Conclusion: Drug resistant TB poses an enormous threat to TB control programs worldwide. Effective treatment of MDR-TB is very expensive, particularly in middle income countries such as India.

Impact of Lockdown on Tuberculosis Management during SARS-Cov-2 Pandemic: Urban &amp; Rural Scenario of Surat

Introduction: India has highest burden of tuberculosis cases both drug sensitive as well as drug resistance in the world. Covid 19 pandemic had affected infectious diseases like TB, HIV, Malaria in developing countries like India. Here, we aimed to study the impact of lockdown on diagnosis and management of tuberculosis in Surat, Gujarat, India due to SARS Cov 2. Method: Cross sectional survey of Tuberculosis patients regarding problems faced during lockdown involving all 18 Tb unitof SMC was done, 15 patients were randomly selected by simple random sampling of line list of patients of each unitand secondary data analysis, data was collected from District Tuberculosis centre for rural part and SMC for urban part. Results: In this study we compared data 5 months before and after lockdown i.e. November 2019 to March 2020 and April 2020 to August 2020 , comparison of diagnostic tests like sputum examination by staining, CBNAAT, and first line Line Probe Assay (LPA) which shows significant reduction in number of test. Also the number of cases of pulmonary as well as drug sensitive, drug resistance and no of referral cases too were significantly decreased during and after lockdown. However, treatment success rate and case fatality rate remain the same as five months before and after the lockdown. Conclusion: Comparison of diagnostic test of TB showed significant reduction in number of tests done during and after lockdown. It was also observed that no major issues were faced by patients from health care provider side of National Tuberculosis Elimination Programme.

Early detection of Pre-XDR TB with line probe assay in a high TB burden country.

Worldwide, tuberculosis (TB) is one of the top 10 causes of death. Drug resistant tuberculosis has lately become a major public health problem that threatens progress made in Tuberculosis (TB) care and control worldwide. The aim of this study was to determine the prevalence of Pre-extensive drug resistant TB among MDR TB in North Central of Nigeria. This study was conducted from October, 2018 to August, 2019 with 150 samples. In Nigeria, guidelines for DR-TB as recommended by WHO is followed. All the samples from the patients who gave their consent were transported to a zonal reference TB laboratory (ZRL). Mean age was 38.6 ± 13.4 years with peak age at 35-44. Out of these 103 samples processed with LPA, 101(98%) were rifampicin resistant and 2 were rifampicin sensitive, 99(96%) were INH resistant and 4 (4%) were INH sensitive, 5(5%) were fluoroquinolone resistant, 98(95%) were fluoroquinolone sensitive, 12 (12%) were Aminoglycoside + Capreomycin resistant, 91(83%) were Aminoglycoside + Capreomycin sensitive. Multidrug resistant TB and its severe forms (Pre-extensive & extensively drug resistant TB) can be detected early with rapid tool- Line Probe Assay rapid and prevented timely by early initiation on treatment.

Misidentification of Mycobacterium smegmatis as Mycobacterium fortuitum by DNA line probe assay

Optimal management of infection with mycobacterial species requires accurate identification down to complex/species level due to variations in outcomes. Over the last few decades, there have been significant advances in laboratory diagnostics with development of newer and rapid molecular methods. Here we describe a case of Mycobacterium smegmatis that was misidentified as Mycobacterium fortuitum by DNA line probe assay.

Unlocking the health system barriers to maximise the uptake and utilisation of molecular diagnostics in low-income and middle-income country setting

BackgroundEarly access to diagnosis is crucial for effective management of any disease including tuberculosis (TB). We investigated the barriers and opportunities to maximise uptake and utilisation of molecular diagnostics in...

English

The control of tuberculosis (TB) has become a global health challenge due to the emergence of multidrug-resistant tuberculosis (MDR-TB) in Mycobacterium tuberculosis (MTB). This highlights the need for faster and more accurate detection of tuberculosis cases. The study aims to detect MDR-TB strains of pulmonary tuberculosis using resistance ratio method and to compare the diagnostic value of drug susceptibility testing (DST) with line probe assay (LPA) using Genotype MTBDRplus. All the sputum samples were tested for Acid Fast Bacilli (AFB) by Ziehl-Neelsen&rsquo;s staining method and were cultured on Lowenstein-Jensen (L-J) media. The identification and confirmation of M. tuberculosis were done using various biochemical tests. DST was carried against the first line of anti-TB drugs. MTB positive samples were subjected to LPA. A total of 57 samples were subjected to DST and LPA for the detection of drug resistance of MTB to RIF and INH after conventional detection methods were applied to all the samples. Among these 57 MTB samples, 11 (19.29%) were resistant to INH, 4 (7.14%) were resistant to RIF; eleven (19.29%) isolates were identified as MDR-TB. LPA revealed 54 MTB positive among 57 MTB culture-positive samples and 3 showed invalid results. In LPA, MDR-TB was found in10 samples (17.54%) in which one was RIF-resistant. The study concludes risk factors that resulted in the development of TB are biomedical, socio-cultural, and behavioral interactions. LPA can be used as a rapid diagnostic technique for the detection of MDR-TB. Key words: Tuberculosis (TB), multidrug resistance (MDR-TB), drug susceptibility testing (DST), line probe assay (LPA). &nbsp

A Systematic Review on the Diagnostic Accuracy of Line Probe Assay in the Rapid Diagnosis of Drug Resistant Tuberculosis in Indian Scenario

Owing to the drastic increase in the number of patients with drug resistant TB around the world, it is important to increase the testing for it. Line probe assay (LPA) is the rapid diagnostic tool to detect drug resistant TB and it was endorsed by WHO for testing first line drugs such as Isoniazid (INH) and Rifampicin (RIF). This systematic review evaluated the accuracy of this LPA by analysing its sensitivity and specificity against the phenotypic drug susceptibility testing (DST) methods like LJ and liquid culture DST. A total of 4774 samples were included in this review from 19 articles. The average sensitivity and specificity for the detection of RIF resistance from 17 articles was 95.79% and 96.71% and for INH resistance it was 89.85% and 97.33% respectively when compared to phenotypic DST. Out of 19 articles included, 2 articles have mentioned the sensitivity and specificity for multi-drug resistant TB (MDR TB) and the average was 98.50 % and 97 % respectively. The accuracy for RIF resistance detection through first line LPA was good and the sensitivity detection for INH was less across the studies. This could be improved further in future generation assays. Our finding supports the use of LPA especially on smear positive specimens but use on smear negative specimens still be considered as studies have shown some interpretable results.

Performance of QuantaMatrix Microfluidic Agarose Channel system integrated with mycobacteria growth indicator tube liquid culture.

The QuantaMatrix Microfluidic Agarose Channel (QMAC) system was used for rapid drug susceptibility testing (DST). Here, we performed DST using QMAC integrated with the mycobacteria growth indicator tube (MGIT) liquid culture employing a specially designed cross agarose channel for the tuberculosis chip. MGIT-, QMAC-, and Löwenstein-Jensen (LJ)-DSTs were performed using 13 drugs. The protocol for QMAC-DST was optimized using the inoculum obtained after the disaggregation of Mycobacterium tuberculosis clumps in MGIT culture. The completion times of QMAC-DST and MGIT-DST were analyzed, and the results of all three DSTs were compared. Discrepant results were analyzed using line probe assays and DNA sequencing. Nontuberculous mycobacteria were distinguished using the ρ-nitrobenzoic acid inhibition test. The overall agreement rate of QMAT-DST and LJ-DST was 97.0% and that of QMAT-DST and MGIT-DST was 86.3%. An average turnaround time for DST was 5.4 days, which was considerably less than the time required for MGIT-DST. The overall time required to obtain DST results using QMAC-DST integrated with MGIT culture was an average of 18.6 days: 13.2 days for culture and identification and 5.4 days for DST. Hence, QMAC-DST integrated with liquid culture can be used to perform DSTs with short turnaround times and effective detection. KEY POINTS: • QMAC system can simultaneously perform phenotypic DST with 13 anti-TB drugs and PNB. • An optimized DST protocol led to a marked decrease in clumping in MGIT culture. • QMAC system integrated with MGIT liquid culture system reduced the turnaround time.

Whole-Genome Sequencing Analysis to Identify Infection with Multiple Species of Nontuberculous Mycobacteria.

Mixed infection with multiple species of nontuberculous mycobacteria (NTM) is difficult to identify and to treat. Current conventional molecular-based methods for identifying mixed infections are limited due to low specificity. Here, we evaluated the utility of whole-genome sequencing (WGS) analysis to detect and identify mixed NTM infections. Analytical tools used included PubMLST, MetaPhlAn3, Kraken2, Mykrobe-Predictor and analysis of heterozygous SNP frequencies. The ability of each to identify mixed infections of NTM species was compared. Sensitivity was tested using 101 samples (sequence sets) including 100 in-silico simulated mixed samples with various proportions of known NTM species and one sample of known mixed NTM species from a public database. Single-species NTM control samples (155 WGS samples from public databases and 15 samples from simulated reads) were tested for specificity. Kraken2 exhibited 100% sensitivity and 98.23% specificity for detection and identification of mixed NTM species with accurate estimation of relative abundance of each species in the mixture. PubMLST (99% and 96.47%) and MetaPhlAn3 (95.04% and 83.52%) had slightly lower sensitivity and specificity. Mykrobe-Predictor had the lowest sensitivity (57.42%). Analysis of read frequencies supporting single nucleotide polymorphisms (SNPs) could not detect mixed NTM samples. Clinical NTM samples (n = 16), suspected on the basis of a 16S–23S rRNA gene sequence-based line-probe assay (LPA) to contain more than one NTM species, were investigated using WGS-analysis tools. This identified only a small proportion (37.5%, 6/16 samples) of the samples as mixed infections and exhibited only partial agreement with LPA results. LPAs seem to be inadequate for detecting mixed NTM species infection. This study demonstrated that WGS-analysis tools can be used for diagnosis of mixed infections with different species of NTM.

Nontuberculous mycobacterium: An emerging pathogen: Indian perspective.

Nontuberculous mycobacteria (NTM), considered as mere contaminants, are off late, being reported as potential pathogens through various studies. The infections due to NTM range from pulmonary to extra pulmonary including skin and soft-tissue infections, traumatic and surgical wound infections, and catheter and implant-associated infections. Although extrapulmonary infections are extensively explored, pulmonary infections are scarcely reported due to their misdiagnosis as tuberculosis caused by M. tuberculosis (MTB). Appropriate detection methods are essential in order to facilitate the differential diagnosis of NTM from MTB infections. We aimed to collate the data available on NTM diagnosis and its epidemiology in India in this review. While diagnosis of MTB itself is more challenging in India, for appropriate treatment of NTM, special training and attention is needed for differential diagnosis of the former from latter. Currently, in India, in addition to the available techniques for identification of NTM, line probe assay (Hains life sciences) is proving to be a promising tool for the detection of NTM (common mycobacteria/additional species kit) and their antimicrobial resistance (Genotype NTM-DR VER 1.0). In future, with the price of sequencing steadily coming down, with proper monitoring, whole-genome sequencing could be the test of choice to predict the species, drug resistance, outbreaks in hospitals, and transmission dynamics.

Diagnostic Performance of Line Probe Assay for the Diagnosis of Rifampicin and Isoniazid ‎Resistant Tuberculosis in a Resource-Poor Country ‎

Background and study aim: The use of LPA is still new in Nigeria and only available in TB reference laboratories. In this ‎study, the performance of LPA version 2.0 was evaluated for the detection of resistant to first-line ‎anti-TB drugs‎‎‎. Patients and Methods: ‎We evaluated the performance of LPA version 2.0 for the detection of rifampicin (RIF) and ‎isoniazid (INH) resistance. Sputum samples from 223 participants were subjected to phenotypic ‎drug susceptibility testing (PDST) and LPA. Statistical analyses included calculation of sensitivity, ‎specificity, positive and negative predictive values. Cross tabulation was done along the kappa test ‎to measure the degree of agreement between PDST and LPA. P-Value > 0.05 was considered ‎significant‎.‎ Results: The overall sensitivity and specificity of 89.6% (95% C.I 82.5-94.5%) and 65.4% (95% C.I 44.3-‎‎82.7%) for detection of RIF resistance; for INH they were 76.6 (95% C.I 67.5-84.5%) and 76.7% ‎‎(95% C.I 49.5-82.6%); and for MDR-TB, they were 67.0% (95% C.I 56.4-76.5%) and 72.0% ‎‎(95% C.I 57.6-83.7%). The kappa values were 0.53 (0.001), 0.38 (p = 0.000) and 0.36 (p = 0.000) ‎for the detection of RIF, INH and MDR-TB. There was moderate agreement between PDST and ‎LPA for detection of RIF (κ = 0.57; P = 0.0001), INH (κ = 0.44; P = 0.0001), MDR-TB (κ = 0.43; ‎P = 0.001)‎‎. Conclusion: The Line probe assay has good sensitivity and specificity for detecting rifampicin and isoniazid. ‎However, the overall performance is moderate; this should be considered when interpreting the ‎assay’s results‎‎‎‎.

Import and transmission of Mycobacterium orygis and Mycobacterium africanum, Norway

BackgroundThe aim of the current study was to improve our understanding of the origins and transmission of Mycobacterium africanum (MAF) in Norway.MethodsWhole-genome sequences (WGS) were generated for all (n = 29) available clinical isolates received at the Norwegian National Reference Laboratory for Mycobacteria (NRL) and identified as MAF in Norway, in the period 2010–2020. Phylogenetic analyses were performed.ResultsThe analyses indicated several imports of MAF lineage 6 from both East and West African countries, whereas MAF lineage 5 was restricted to patients with West African connections. We also find evidence for transmission of MAF in Norway. Finally, our analyses revealed that a group of isolates from patients originating in South Asia, identified as MAF by means of a commercial line-probe assay, in fact belonged to Mycobacterium orygis.ConclusionsMost MAF cases in Norway are the result of import, but transmission is occurring within Norway.

Compatibility of a novel filter paper-based bio-safe sputum transport kit with line probe assay for diagnosing drug-resistant tuberculosis: a single-site evaluation study.

BackgroundNear-patient access to appropriate tests is a major obstacle for the efficient diagnosis of tuberculosis (TB) and associated drug resistance.MethodsWe recently developed the “TB Concentration & Transport” kit for bio-safe, ambient-temperature transportation of dried sputum on Trans-Filter, and the “TB DNA Extraction” kit for DNA extraction from Trans-Filter for determining drug resistance by DNA sequencing. In the present study, we evaluated the compatibility of Kit-extracted DNA with Hain's line probe assays (LPAs), which are endorsed by National TB programmes for the detection of drug resistance in sputum collected from presumptive multidrug-resistant TB patients (n=207).ResultsTrans-Filter-extracted DNA was seamlessly integrated with the LPA protocol (Kit-LPA). The sensitivity of Kit-LPA for determining drug resistance was 83.3% for rifampicin (95% CI 52–98%), 77.7% for isoniazid (95% CI 52–94%), 85.7% for fluoroquinolones (95% CI 42–100%) and 66.6% for aminoglycosides (95% CI 9–99%), with a specificity range of 93.7% (95% CI 87–97) to 99.1% (95% CI 95–100) using phenotypic drug susceptibility testing (DST) as a reference standard. A high degree of concordance was noted between results obtained from Kit-LPA and LPA (99% to 100% (κ value: 0.83–1.0)).ConclusionsThis study demonstrates successful integration of our developed kits with LPA. The adoption of these kits across Designated Microscopy Centres in India can potentially overcome the existing challenge of transporting infectious sputum at controlled temperature to centralised testing laboratories and can provide rapid near-patient cost-effective “Universal DST” services to TB subjects residing in remote areas.

Molecular Characterization of Drug-Resistant Mycobacterium tuberculosis Isolated from HIV-Positive Patients in Rivers State, Nigeria

Aim: Drug resistant tuberculosis is a major challenge in the global bid to control the disease burden and improve treatment outcomes of DRTB infected individuals.&#x0D; Methods: The Line Probe Assay (LPA) was used to assess the drug resistance pattern and gene mutations in 260 sputum specimens collected consecutively from 260 adult, HIV sero-positive subjects presenting with symptoms suggestive of TB in clinical settings across Rivers State, Nigeria.&#x0D; Results: The results showed a 61.2% (n = 159) prevalence of TB among all study subjects. LPA analysis showed that 16 (10.1%) were multidrug resistant strains, 17 (10.7%) Rifampicin (RIF) monoresistant strains, 24 (15.1%) Isonaizid (INH) monoresistant strains and 102 (64.2%) drug susceptible strains. Among all 32 RIF-resistant strains, 24 (75%) had a mutation in rpoB S531L at the MUT3 band. Another mutation was observed at rpoB H526D (1/17) in RIF-monoresistant strains but not in MDR-TB strains. In INH resistant strains, mutations were observed at the katG gene [32/39 (82.1%)] which constituted 13/15 (86.7%) in MDR-TB strains and 19/24 (79.2%) in INH-monoresistant strains (p =0.002). Overall frequency of inhA mutation was 6/39 (15.4%); MDR-TB strains [2/15 (13.3%)] and INH-monoresistant strains [4/24 (16.7%), p = 0.2]. Combined KatG and inhA mutation was found in 1/39 (2.6%) of INH-monoresistant strains and in none of MDR-TB strains. Of the single inhA mutation bands, only 1/39 (4.2%) MUT 3B (T8A mutation) was observed in INH-monoresistant strains.&#x0D; Conclusion: The results showed that drug-resistant TB is prevalent in Rivers State with various gene mutations observed in the different TB strains isolated, making LPA analysis a necessity in the bid to improving prevention and control efforts in the region.