Line Probe Assay For Detection Research Articles

Trends of drug resistance in M. tuberculosis in a reference laboratory in Central India: Forging ahead towards TB elimination

PurposeThe National Tuberculosis Elimination Programme (NTEP) of the Government of India has strived to control tuberculosis (TB) in the country through various interventions. Understanding the trends of resistance patterns may provide insights into the effectiveness of TB control activities in the country. MethodsA total of 31,144 clinical samples were received from 2013 to 2022 from presumptive drug-resistant TB patients. All the specimens were decontaminated and subjected to the line probe assay for detection of resistance to rifampicin and isoniazid. Mycobacterium tuberculosis (MTB) was detected in 28,814 samples. Autoregressive integrated moving average model (ARIMA) was fitted to assess the trend over time. ResultsA decreasing trend in multi-drug resistant TB from 19 % in 2013 to 4 % in 2022 was seen. A decreasing trend in rifampicin monoresistance from 11.2 % in 2013 to 1.5 % in 2022 was seen, though there was an increase in resistance in 2017. No significant decreasing trends were seen in the proportion of isoniazid monoresistance from 8.3 % in 2013 to 7 % in 2022. ConclusionThe findings are encouraging, and to a considerable extent, affirm that India is well on track with regard to the goal of TB elimination.

Role of line probe assay in detection of Mycobacterium tuberculosis in children with pulmonary tuberculosis

BackgroundIndia is the highest TB burden country in the world with almost 27 lakh cases reported in 2019. Pediatric tuberculosis in India accounts for almost 31% of global TB burden. Despite such high mortality and morbidity in children, diagnosis of pulmonary TB in children still remains very challenging. Material and methodsThis cross-sectional study was conducted in a tertiary care hospital in Delhi, India. Children between 1 and 12 months with clinical suspicion of pulmonary tuberculosis who had not previously taken ATT were included.Early morning gastric aspirate samples were collected after overnight fasting, on two days. Both days sputum sample were subjected to sputum smear microscopy and one of the two samples was subjected to line probe assay (LPA), cartridge based nucleic acid amplification (CBNAAT) and mycobacterium growth in tube (MGIT-960). Results84 children with pulmonary tuberculosis were enrolled. The most common presenting complaint was fever seen in 83 patients (98.8%). Only 17 (20.24%) were sputum smear positive by Ziehl- Neelsen (ZN) staining. LPA was positive in 47 (55.95%) samples and among these positive samples both INH and RIF resistance was detected in 2 (4.26%) samples. CBNAAT was positive in 53 patients (63%). Growth in liquid culture media (MGIT 960) was observed in 44 (52.38%) samples. Among 17 smear positive samples, LPA was detected in 14 (82.35%) samples and among 67 smear negative sample LPA was detected in 33 (49.25%) samples. LPA had 63.46% sensitivity, 100% specificity in detecting mycobacterium tuberculosis. DiscussionWHO's recommendation for using LPAs has been limited to culture isolates or smear-positive sputum specimens. New data has since been generated on the use of LPAs as newer versions of LPA have been developed over past few years.Previous studies conducted using LPA version 1.0 reported much lower detection rate of mycobacterium tuberculosis in smear negative specimens.With the availability of newer versions such as Hain GenoTypeMTBDRplusversion 2 and Nipro NTM + MDRTB detection kit 2, the diagnostic utility of LPA may be enhanced.

Agreement between CBNAAT, liquid culture and line probe assay for detection of Mycobacterium tuberculosis and anti-tubercular drug resistance in extrapulmonary samples

PurposeCartridge based nucleic acid amplification test (CBNAAT) has been endorsed by the WHO as the screening test for diagnosing extrapulmonary tuberculosis (EPTB). In the present study we report the agreement between CBNAAT (Xpert MTB/RIF), liquid culture (LC) and line probe assay (LPA) for diagnosis of Mycobacterium tuberculosis and detection of drug resistance among EPTB cases. MethodsThe EP samples were subjected to CBNAAT (Xpert MTB/RIF, Cepheid, USA) and wherever possible, to LC (MGIT 960, Becton Dickinson, USA) followed sequentially by first line and second line-LPA (FL-LPA, SL-LPA, Hain Lifescience, Germany) on the isolates. ResultsTotal 566/4080 (13.9%) EP samples were detected positive for M. tuberculosis on CBNAAT. Aspirates from lymph nodes were most often positive (11/30; 36.6%), followed by pus (240/873; 27.5%) and CSF samples (166/104; 15.8%). The detection of M. tuberculosis was more in adults than children except in tissue biopsy samples. Rifampicin resistance was also higher among adults except CSF in which resistance was more in children. Total 185 of 566 (32.7%) CBNAAT positive and 770 of 3510 (21.9%) CBNAAT negative samples could be cultured of which 110/185 (59.4%) and 33/770 (4.3%) respectively turned positive. FL-LPA and SL-LPA of 143 culture isolates showed that 27 isolates had drug resistance, of which 3 (2.1%) were XDR, 11 (7.7%) were Pre-XDR (FQ) and 13 (9.1%) were MDR. Of these 27 resistant isolates, 12 were negative by CBNAAT and two were mislabeled as Rifampicin sensitive or indeterminate based on the unique RpoB gene mutation patterns on LPA. The positive and negative agreements between LC and CBNAAT for detection of M. tuberculosis were 67.1% and 92.7% respectively and between LPA and CBNAAT for rifampicin resistance detection were 98.9% and 92.9% respectively. ConclusionsFor EPTB, CBNAAT should be accompanied with LC wherever possible irrespective of the CBNAAT result.

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.
 
 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).
 
 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.
 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.

Comparison of GeneXpert and line probe assay for detection of Mycobacterium tuberculosis and rifampicin-mono resistance at the National Tuberculosis Reference Laboratory, Kenya

BackgroundThe dual challenge of low diagnostic sensitivity of microscopy test and technical challenge of performing a TB culture test poses a problem for case detection and initiation of Tuberculosis (TB) second-line treatment. There is thus need for a rapid, reliable and easily accessible assay. This comparative analysis was performed to assess diagnostic performance characteristics of GeneXpert MTB/RIF and Line Probe Assay (LPA).MethodsThree hundred twenty nine sputum samples of patients across the 47 counties in Kenya suspected to have drug resistant TB were picked and subjected to GeneXpert, LPA and Culture MGIT at the National TB Reference Laboratory. Sensitivity, specificity and predictive values were then determined to assess the performance characteristics of the various assays.ResultsAgainst culture MGIT as the gold standard for TB diagnosis, GeneXpert had a sensitivity, specificity, positive predictive value, and negative predictive value of 78.5, 64.9, 59.4 and 82.2% respectively while LPA had 98.4, 66.0, 65.4 and 98.4%. For diagnosis of rifampicin mono-resistance GeneXpert had a moderate agreement (Kappa 0.59, P < 0.01) (sensitivity 62.50%, specificity 96.50%) while LPA that had almost perfect agreement (Kappa = 0.89, p < 0.01) with a (sensitivity 90.0% and specificity 99.1%).ConclusionLPA has a better performance characteristic to GeneXpert and an alternative to culture with regards to detection of RIF’s mono-resistance.

Role of line probe assay in detection of extra-pulmonary tuberculosis: Experience from a tertiary care hospital in western Maharashtra

BackgroundDiagnosis of extra pulmonary tuberculosis (EPTB) is challenging due to its atypical clinical presentation and frequently results in a delay or deprivation of treatment. Apart from rapid case detection, early determination of MDR status is imperative in such situations. The commercially available Geno Type MTBDRplus assay version 2.0 (Hain Lifescience, Nehren, Germany) detects both the presence of Mycobacterium tuberculosis (MTB) complex as well as the presence of INH and Rifampcin resistance. We aim to evaluate the role of this test in diagnosis and detection of resistance by comparing its performance against gold standard i.e. culture and against the composite reference standards (CRS) in the diagnosis of EPTB. Material and methodsThe data of 130 EPTB samples processed form January 2014 till May 2017 at Poona Hospital and Research centre were selected for the study. All the samples were processed for Ziehl–Neelsen stain, Geno Type MTBDRplus assay (LPA) and liquid automated culture (BacT/Alert) simultaneously. Geno Type MTBDRplus assay (LPA) was performed directly on the samples. The 24 samples giving positive results on LPA and grown M. tuberculosis on culture were subjected to anti mycobacterial susceptibility testing for 1st line anti-tubercular drugs by BACTEC MGIT 320 system. ResultsOut of 130 samples, 7 samples grew atypical mycobacterium and all the 7 samples turned negative on Line Probe Assay. Direct LPA on processed samples yielded 48/130 (36.9%) positivity. Geno Type MTBDRplus assay was positive for M. tuberculosis in (72.09%) 31/43 culture positive cases and (21%) 17/80 of culture negative cases. Geno Type MTBDRplus assay sensitivity and specificity results were assessed in comparison to CRS made up of culture results and clinical, radiological and histological findings. The overall sensitivity of Geno Type MTBDRplus assay was 45.19% (47/104) and specificity was 94.73 (18/19). Out of 24 samples which were compared for results between LPA and culture, Geno Type MTBDRplus assay accurately identified 3 of 3 of Rifampcin resistant strains and 20 of 21 Rifampcin sensitive strains. Geno Type MTBDRplus assay identified 4 of 4 INH resistant strains and 19 of 20 INH sensitive strains and MDR was obtained for 3 of 3 strains. ConclusionsGeno Type MTBDRplus assay can give early diagnosis and sensitivity for both INH and Rifampcin in extra pulmonary samples. More number of studies is further required to establish Geno Type MTBDRplus assay as an important tool for obtaining diagnosis and resistance to first line drugs in extra pulmonary samples.

Evaluation of Genotype MTBDRplus Line Probe Assay in Detection of Rifampicin and Isoniazid Resistance in Comparison to Solid Culture Drug Susceptibility Testing in a Tertiary Care Centre of Western Uttar Pradesh

Background: Isoniazid (INH) and rifampicin (Rif) are the key first-line antituberculosis drugs, and resistance to these drugs i.e., multi-drug-resistant tuberculosis (MDR-TB), is likely to result in treatment failure and poor clinical outcomes. India has the highest burden of TB and MDR-TB in the world, disproportionately high even for India’s population. The GenoType® MTBDRplus molecular method allows rapid detection of Rif and INH resistance. Aim: The present study was done to compare the performance of line probe assay test (GenoType® MTBDRplus) with solid culture method for an early diagnosis of MDR-TB. Methods: Totally 1503 sputum samples of MDR-TB suspects were subjected to fluorescent microscopy. Decontamination was done by N-acetyl-L-cysteine and sodium hydroxide method. Fluorescent microscopy-positive samples were subjected to GenoType® MTBDRplus (HAIN Lifescience) assay. Sixty-two random samples were compared with phenotypic drug susceptibility testing (DST) (1% proportion method) using solid culture method by Lowenstein–Jensen media. Results: The sensitivity, specificity, positive predictive value and negative predictive value for detection of resistance to Rif were 94.74%, 95.35%, 90% and 97.62% and to INH were 92.00%, 91.89%, 88.46% and 94.44%, respectively, in comparison with the phenotypic DST. Conclusion: GenoType® MTBDRplus has good sensitivity and specificity in detecting MDR-TB cases with a significantly lesser turnaround time as compared to conventional DST method and simultaneous detection of Rif and INH resistance. This technique saves several weeks of time required for culture and DST.

Line probe assay for detection of Mycobacterium tuberculosis complex: An experience from Central India.

Background & objectives:Mycobacterium tuberculosis complex may sometimes not be detected in sputum samples of suspected multidrug-resistant tuberculosis (MDR-TB) patients by line probe assay (LPA) even though they are smear positive for acid-fast bacilli (AFB). This retrospective analysis was attempted to understand and document our experience with LPA for detection of M. tuberculosis complex and diagnosis of MDR-TB under programmatic conditions.Methods:One thousand two hundred and ninety four sputum samples of MDR-TB suspects that were smear positive for AFB, and received from February to November 2013, were tested by LPA for the presence of M. tuberculosis complex and resistance to isoniazid (INH) and rifampicin as per the diagnostic mandate of an accredited reference laboratory. As per the mandate, those samples that were negative for M. tuberculosis complex were cultured, and the growth again tested by LPA. A retrospective analysis of the results was carried out.Results:M. tuberculosis complex could be detected in 1217 (94.04%) but not in 77 (5.9%) of smear-positive sputum samples. Of the 1217 positive samples, 232 (19.1%) were MDR, 130 (10.6%) were rifampicin monoresistant and 101 (8.3%) were INH monoresistant. Seven hundred and fifty four (61.9%) strains were found to be pansensitive. Overall, 5.1 per cent of the sputum samples were negative for M. tuberculosis complex by LPA and culture. In at least 10 (0.77%) sputum samples smear positive for AFB, M. tuberculosis complex could not be identified by LPA though M. tuberculosis was present, as evidenced by culture positivity.Interpretation & conclusions:LPA is a robust technique for diagnosis of drug-resistant TB that has provided the basis for rapid and effective control of drug-resistant TB in India. While the reasons for concomitantly negative LPA and culture results of smear-positive sputum samples from MDR-TB suspects may be many, the possible presence of non-tubercular mycobacteria in these samples and the likelihood of inappropriate therapy in these patients cannot be ruled out. Addition of culture to the diagnostic algorithm may enhance the diagnostic yield.

Role of line probe assay in detection of extra-pulmonary tuberculosis/multidrug-resistant tuberculosis: The experience from Kerala state, India

Background: There are limited data on use of line probe assay (LPA) for detection of TB and Rifampicin resistance among extra-pulmonary tuberculosis (EPTB) patients under specific TB control programme settings. Our aim was to compare the positivity of LPA with Lowenstein–Jensen (LJ) and Bactec MGIT and to test its utility in faster testing and in patients on treatment. Methodology: The data of all 387 EPTB samples processed in two years by the state-level accredited laboratory in 2013–2014 were selected for the study. The laboratory used standard N-acetyl-L-cysteine – sodium hydroxide processing method for all samples except for cerebrospinal fluid and urine samples before conducting Ziehl-Neelsen smear microscopy, LPA and culture on both LJ and MGIT. Those samples with a negative LPA result if culture positive was subjected to a second LPA as per laboratory protocol. Doubtful Rifampicin resistant (RR) results were cleared by phenotypic testing by MGIT. Anti-TB treatment (ATT) duration was correlated with positivity of these tests later. Results and Discussion: LPA done on 321 processed samples out of 387 EPTB samples identified eight RR cases among 136 positive results within two to four days. Performing LPA on culture positive isolates identified an additional seven RR cases, thus reducing turn-around-time by two to eight weeks for the susceptibility results. It also demonstrated far better positivity in smear negative (15.2%) and in smear positive samples (74.3%) compared to culture methods as the test demonstrated clearly, better positivity among patients on ATT for more than two months. Conclusion: LPA in association with culture is an excellent combination with very good results in rapid identification of EPTB along with detection of Rifampicin resistance. LPA gives far superior positivity compared to MGIT and solid cultures among patients under ATT.

Performance of the Genotype MTBDR Line Probe Assay for Detection of Resistance to Rifampin and Isoniazid in Strains of Mycobacterium tuberculosis with Low- and High-Level Resistance

We assessed the performance of the Genotype MTBDR line probe assay that offers the simultaneous identification of Mycobacterium tuberculosis and its resistance to rifampin (RIF) and isoniazid (INH) by detecting the most commonly found mutations in the rpoB and katG genes. One hundred thirteen M. tuberculosis isolates were tested. The nucleotide sequences of the katG and inhA genes and the mabA-inhA promoter region were also determined. The MTBDR assay detected 100% and 67% (n = 64) of the strains resistant to RIF and INH, respectively. Among the latter, 62 strains carried a Ser315Thr mutation in katG, 59 of them displaying a high level of resistance to INH. Two strains with a low level of INH resistance had a Ser315Asn mutation. No mutation was found by the MTBDR assay for 31 INH-resistant strains (33%), of which 24 showed a low level of resistance. By DNA sequencing, we found among them various mutations in the KatG protein for 7 strains, a C-->T mutation in position -15 of the mabA-inhA promoter in 17 strains, and a Ser94Ala mutation in InhA for 7 strains. In conclusion, the MTBDR assay, which fits easily in the workflow of a routine laboratory, enabled the detection of 100% of the RIF-resistant strains and 89% of the INH-resistant strains with a high level of resistance but only 17% of the strains characterized by a low level of INH resistance, indicating that the test can be used as a rapid method to detect in the same experiment the rifampin-resistant and the high-level isoniazid-resistant strains of M. tuberculosis.

Genotypic resistance to lamivudine among hepatitis B virus isolates in Mexico

Drug resistance of hepatitis B virus (HBV) is an increasing clinical problem. Resistance to lamivudine in HBV isolates in Mexico has been poorly explored. To characterize the mutation patterns associated with genotypic resistance to lamivudine and their prevalence among HBV isolates in Mexico. Thirty-nine Mexican HBV isolates were analysed by PCR and line probe assay for detection of genetic variants in the polymerase open reading frame domains B and C (INNO-LiPA HBV DR; INNOGENETICS N. V., Ghent, Belgium). This assay detects wild-type and mutations at codons 180, 204 and 207 of the HBV polymerase gene, and at codon positions 171, 172, 195, 196, 198 and 199 of the HBV surface antigen (HBsAg). HBV isolates were obtained from HBsAg-positive serum samples of 15 chronic hepatitis patients, two haemodialysis patients with chronic HBV carriage, 20 men found positive for HBsAg when seeking HIV testing and two AIDS patients with chronic HBV infection. None of the participants had received antiviral therapy. Overall, HBV wild-type was found in 37 (94.9%) out of the 39 isolates studied. Two (5.1%) out of the 39 isolates showed mixed wild-type and mutant populations. These mutations occurred in isolates from one hepatitis patient and one haemodialysis patient. The isolate from the hepatitis patient showed a double mutation at codon positions 180 (L180M) and 204 (M204V), thus a 2.6% prevalence of genotypic resistance to lamivudine was found. The isolate from the haemodialysis patient showed a single mutation at codon position 180 (L180M). The two HBV mutant isolates were further analysed for genotype and both isolates were genotype H. HBV genotypic resistance to lamivudine exists in Mexican isolates. The results highlight the importance of testing for HBV resistance before treatment and have implications for a more rational use of drugs.

Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study.

Helicobacter pylori strains from 299 patients were tested in six laboratories in different countries. Macrolide susceptibility of the strains was determined by agar dilution (17.4%) or the epsilometer test (82.6%). Mutations in the 23S ribosomal DNA (rDNA) that are associated with macrolide resistance were analyzed by PCR and reverse hybridization (PCR-line probe assay [LiPA]). This method identifies A2115G, G2141A, A2142G, A2142C, A2142T, A2143G, and A2143C mutations in the 23S rDNA. vacA s-region (s1a, s1b, s1c, and s2) and m-region (m1, m2a, and m2b) genotypes and cagA status were also determined using another PCR-LiPA system. Of the 299 strains investigated by MIC testing, 130 (43.5%) were resistant and 169 (56.5%) were susceptible to clarithromycin. Of the 130 resistant strains, 127 (97.7%) contained 23S rDNA mutations, whereas 167 (98.8%) of the 169 susceptible strains contained wild-type sequences. The predominant mutations were A2143G (45.2%) and A2142G (33.3%). Twenty-eight (19.8%) strains contained multiple 23S rDNA mutations. Only five resistant strains contained the A2142C mutation (three of these in combination with the A2142G mutation), and the A2115G, G2141A, A2142T, and A2143C mutations were not found. MICs of clarithromycin for the A2142G mutant strains were significantly higher than MICs for the A2143G strains. Although there was no significant association between 23S rDNA mutations and the vacA and cagA status, clarithromycin-susceptible strains more often contained mixed vacA genotypes, indicating the presence of multiple H. pylori strains. In conclusion, our data confirmed the very strong association between 23S rDNA mutations and macrolide resistance and showed that the PCR-LiPA permits accurate and reliable diagnosis of macrolide resistance in H. pylori.

Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1, 354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5(+)/6(+) primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.