Cost analysis of GenoType® MTBDRplus and GenoType® MTBDRsl at the State Laboratory of São Paulo, Brazil

ABSTRACT Background: We aimed to evaluate the costs of GenoType® MTBDRplus and MTBDRsl incurred during the diagnosis of first- and second-line drug-resistant tuberculosis (TB) in São Paulo, Brazil. Methods: Mean and activity-based costs of GenoType® were calculated in a referral laboratory for TB in Brazil. Results: The mean cost value and activity-based cost of GenoType® MTBDRplus were USD 19.78 and USD 35.80 and those of MTBDRsl were USD 54.25 and USD 41.85, respectively. Conclusions: The cost of GenoType® MTBDRplus was reduced owing to the high number of examinations performed and work optimization.

Clinical outcomes and molecular characterization of drug-resistant tuberculosis in pre- and extensively drug-resistant disease based on line probe assays

ABSTRACT Multidrug-resistant tuberculosis (MDR-TB) represents a significant impact in transmission, outcome, and health costs. The World Health Organization recommends implementation of rapid diagnostic methods for multidrug-resistance detection. This study was performed to evaluate the frequency of pre- and extensively drug resistant tuberculosis (pre-XDR-TB and XDR-TB) among MDR-TB patients, the pattern of resistance mutations for fluoroquinolones and the clinical outcome. Adult patients followed at a Brazilian regional reference center for TB, from January 2013 to June 2019 were included. Stored Mycobacterium tuberculosis (Mtb) cultures were recovered, the DNA was extracted, and the susceptibility test was performed using the line probe assay for second line antimycobacterial drugs, Genotype MTBDRsl version 2.0 (Hain Lifescience, CmbH, Germany). Among 33 MDR-TB included patients, we diagnosed XDR-TB or pre-XDR in five (15%) cases. Of these, mutations related to fluoroquinolones resistance were observed in four Mtb isolates, including one who had no phenotypic resistance profile. In two other patients with phenotypic resistance to ofloxacin, genotypic resistance was not found. Case fatality rate was 60% in pre/XDR-TB group, compared to 3.6% in the remaining of patients. This study observed few cases of pre-XDR and XDR-TB among a MDR-TB cohort. Phenotypic and genotypic assays presented good agreement. Clinical outcome was more favorable for patients with susceptibility to fluoroquinolones and injectable drugs.

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

Background - 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. Methods - 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). Results - 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. Conclusion - 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.

Evaluation of GenoType® MTBDRplus VER2.0 kit for detecting mycobacterium tuberculosis complex in sputum / 公共卫生与预防医学

Objective To evaluate the performance of GenoType®MTBDRplus VER2.0 kit for detecting Mycobacterium tuberculosis complex (MTBC) in sputum. Methods Sputum samples from 177 patients with suspected tuberculosis were collected, and tested by smear, MGIT liquid culture and GenoType® MTBDRplus VER2.0. When the liquid culture was positive, identification of strains was carried out. The results were statistically analyzed using SPSS 22.0, and the consistency of the count data was compared using the Kappa test. Good consistency was defined as K≥0.75. The sensitivity and specificity were applied to evaluate the performance of GenoType® MTBDRplus VER2.0 kit for detecting MTBC in sputum. Results Based on the MGIT liquid culture results,，the sensitivity, specificity, positive predictive value, negative predictive value and total coincidence rate of the GenoType® MTBDRplus VER2.0 kit was 91.67%, 95.58%, 91.67%, 95.58% and 94.22%，respectively．The Kappa test was performed on both methods, K=0.872 (P® MTBDRplus VER2.0 kit had high sensitivity and specificity for detecting MTBC in sputum, and it has good application value for early diagnosis of tuberculosis.

Role of line probe assay in diagnosis and detection of drug resistance in Mycobacterium tuberculosis

Tuberculosis caused by Mycobacterium tuberculosis has remained a major global health problem worldwide. TB requires prolonged period of time for isolation by conventional culture methods. The emergence and spread of multi drug resistant (MDR-TB) poses great threats and challenges in controlling the infection. MDR-TB is resistant to both first line drugs rifampicin and isoniazid. PCR tests are based on targeting the mutation in rpoB, katG and inhA genes which can detect resistance to these drugs. To compare microscopy, conventional culture and Line probe assay for the detection of M. tuberculosis & detect rifampicin and isoniazid resistance using Lineprobe assay in various clinical samples. A total of 347 suspected patients of tuberculosis were included in the study. Demographic details & clinical presentation was noted. Various samples were received & processed for ZN staining, culture on LJ media and Line probe assay. Out of 347 cases, majority of cases were in the age group of 51-60 years (18.4%). Majority of the population was males (65.1%). Among suspected tuberculosis patients, cough with expectoration (55.9%) was the commonest complaint. Microscopy was positive in 17.3%, conventional culture was positive in 16.1% and line probe assay was positive in 26.2%. Out of 347, 91 were diagnosed with MTB, out of which 85.7% were sensitive to both rifampicin and isoniazid whereas 14.3% showed resistance to either rifampicin / isoniazid or both. LPA & direct microscopy are a good screening method for early diagnosis and detection of drug resistance but are not a complete replacement of conventional culture which is still a gold standard.

Isolation, Identification and Drug Resistance Testing of Mycobacterium tuberculosis by Recent Diagnostic Modalities at Teaching Hospital in Moradabad (UP).

Objective: Tuberculosis is the second leading cause of death in developing countries among all infectious diseases. Globally, drug resistance strain of Mycobacterium tuberculosis is a public threat. Due to diagnostic delay, inadequate infection control and poor drug supply there is a emergence of MDR- TB and XDR- TB. Our aim was to isolate and identify the drug resistant strain of M. tuberculosis by using newer diagnostic modalities. Methods: Sputum sample and BAL fluid from 70 suspected cases were collected and analysed for Mycobacterium by Ziehl – Neelsen staining and liquid culture with molecular detection of drug resistant strain of M. tuberculosis. Result: In our study, among the 70 patients 27 (38.5%) were positive for AFB by microscopy. On testing for Mycobacterium by BacT/Alert 3D system, 54 were found to be positive. On performing further identification and susceptibility of 54 isolates towards rifampicin and isoniazid by molecular method, 5 isolates (9.25%) were resistant to both rifampicin and isoniazid confirming as multidrug resistant. 5 isolates (9.25%) were sensitive to rifampicin and resistant to isoniazid and 2 isolates (3.70%) were resistant to rifampicin and sensitive to isoniazid. Whereas 5 isolates (9.25%) found to be negative for M. tuberculosis. Conclusion: Our investigation highlights the importance of newer diagnostic modalities for isolation and identification of drug resistant strain of M. tuberculosis. Which ensure early and accurate diagnosis of patients with prevention of further transmission of disease.

Detection of rifampicin resistance in tuberculosis by molecular methods: A report from Eastern Uttar Pradesh, India.

Diagnosis of drug resistance tuberculosis (TB) by the gold standard method is labour intensive and time consuming. Hence, there is an urgent need for introduction of rapid diagnostic techniques. Line probe assay (LPA) and cartridge‑based nucleic acid amplification test (CBNAAT) have been introduced in India under Revised National Tuberculosis Control Program. Spot and morning sputum samples of previously treated patients by anti‑TB drugs were subjected to LPA or CBNAAT. Total 682/1253 (54.4%) were diagnosed as rifampicin‑resistant. The patients could be diagnosed early by molecular methods and put on second line treatment.

Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay.

Purpose: There is scarcity of prevalence data of multi-drug–resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in fi ve districts of New Delhi were referred to the laboratory from 1st October 2011 to 31st December 2012 as per criterion defi ned by Programmatic Management of Drug Resistant Tuberculosis (PMDT). GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF) and katG and inhA genes for isoniazid (INH) were analyzed. Results: A total of 366 (16.8%) MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0%) and for INH was S315T1 (88.3%). Mutations S531L and S315T1 occurred signifi cantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT) for dispatch of result to districts for test conducted on samples was 4.4 days. Conclusion: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed signifi cantly for both RIF and INH.

Detection of multi-drug resistance & characterization of mutations in Mycobacterium tuberculosis isolates from North- Eastern States of India using GenoType MTBDRplus assay.

Background & objectives: information on drug resistance tuberculosis is sparse from North-East (N-E) States of Iindia. We undertook this study to detect multi-drug resistant tuberculosis (MDR-TB) among MDR-TB suspects, and common mutations among MDR-TB cases using GenoType MTBDRplus. Methods: All MDR suspect patients deposited sputum samples to peripheral designated microscopy centres (DMC) in North-East States. The district TB officers (DTOs) facilitated the transport of samples collected during January 2012 to August 2012 to our laboratory. The line probe assay to detect common mutations in the rpoB gene for rifampicin (RIiF) and katG and inhA genes for isoniazid (IiNH), respectively was performed on 339 samples or cultures. Results: A total of 553 sputum samples from MDR suspects were received of which, 181 (32.7%) isolates were found to be multi-drug resistant. Missing WT8 along with mutation in codon S531L was commonest pattern for rifampicin resistant isolates (65.1%) and missing WT along with mutations in codon S315T1 of katG gene was commonest pattern for isoniazid resistant isolates (86.2%). Average turn-around time for dispatch of LPA result to these sStates from cultures and samples was 23.4 and 5.2 days, respectively. Interpretations & conclusions: The MDR-TB among MDR-TB suspects in North-Eastern States of Iindia was found to be 32.7 per cent. The common mutations obtained for RIiF and IiNH in the region were mostly similar to those reported earlier.

Comparison between the conventional method & molecular line probe assay for identification & drug sensitivity of mycobacteria tuberculosis from clinical specimens.

Rapid susceptibility testing of Mycobacterium tuberculosis strains is imperative for therapy selection but traditional drug susceptibility tests take weeks or are expensive. Classical drug susceptibility (DST) may take up to 2 to 4 months. The line probe assay is a commercially available line-probe assay that rapidly detects Mycobacterium tuberculosis (MTB) complex, as well as the most common mutations associated with rifampicin and isoniazid. In this study we assessed the sensitivity and specificity of the rapid molecular method in comparison with the conventional method.

Detection of drug resistant Mycobacterium tuberculosis among patients with and without HIV infection in a rural setting / Detección de Mycobacterium tubersulosis resistente a los antibióticos entre pacientes con y sin infección por VIH en un área rural

OBJECTIVE: To analyse the sensitivity of Mycobacterium tuberculosis by nitrate reductase assay (NRA) and the Hain molecular line probe assay (LPA) in sputa of tuberculosis (TB)/HIV co-infected patients in Guyana. DESIGN: Sputum samples were collected from known TB patients at Georgetown Chest Clinic and were analysed at the Reference Laboratory, Guyana, over the period April 2010 to April 2011. RESULTS: Both methods recorded greater sensitivity for rifampin (RIF) than of isoniazid (INH). Both methods detected four RIF resistant, two INH resistant and two multi-drug resistant (MDR) strains and they had greater negative agreement indices than positive agreement indices. CONCLUSION: It was established that the sensitivity of Mycobacterium tuberculosis by the NRA and Hain LPA in TB/HIV co-infected patients has acceptable correlation and that HIV infection does not affect drug susceptibility testing.

OBJETIVO: Analizar la sensibilidad de Mycobacterium tuberculosis por medio del ensayo de nitrato reductasa (NRA) y el ensayo de sonda lineal (LPA) molecular de Hain en esputos de pacientes co-infectados TB/VIH en Guyana. DISEÑO: Muestras de esputo de pacientes de la Clínica del Tórax en Georgetown diagnosticados con tuberculosis, fueron analizadas en el Laboratorio de Referencias, en Guyana, en el período de abril de 2010 a abril de 2011. RESULTADOS: Ambos métodos registraron una mayor sensibilidad a la rifampicina (RIF) que a la isoniacida (INH). Ambos métodos detectaron cuatro cepas resistentes a RIF, dos resistentes a INH, y dos resistentes a mútiples medicamentos (RMM). Asimismo, presentaban mayores índices de concordancia negativa que de concordancia positiva. CONCLUSIÓN: Se estableció que la sensibilidad de Mycobacterium tuberculosis por medio del ensayo de NRA y el LPA de Hain en pacientes co-infectados TB/VIH, guarda una correlación aceptable, y que la infección por VIH no afecta la prueba de susceptibilidad a los medicamentos.

Clinical Usefulness of the Line Probe Assay for Rapid Detection of Rifampicin-resistant Tuberculosis / 결핵

BACKGROUND: RpoB gene mutations have been found in about 96-98% of rifampicin (RMP)-resistant Mycobacterium tuberculosis. Recent reports confirm that the in laboratory settings a rpoB gene mutation can be used as a surrogate marker for multi-drug resistant tuberculosis. However, its usefulness in clinical applications has not been evaluated. This study was performed to confirm whether mutation analysis of the rpoB gene of M.tuberculosis is useful in clinical settings. METHODS: The medical records of 33 patients in whom rpoB gene analysis was conducted using an INNOLiPA Rif. TB assay (LiPA) from June, 1998, to July, 2000, at the Asan Medical Center were retrospectively reviewed in 33 patients. the clinical characteristics in addition to the drug susceptibility and LiPA results were analyzed. The drug susceptibility test was considered as a gold standard method for M/ tuberculosis susceptibility and these results were compared with those of the rpoB gene study and sequencing analysis. sequencing analysis of the rpoB gene was done in cases where there was a discrepancy between the results of the drug susceptibility and rpoB gene study. RESULTS: The mean age and sex ratio was 42±18, and 24:9(M:F), respectively. there were 19 RMP susceptible (58%) and 14 RNP-resistant cases (42%) according to the rpoB gene study. The mean time from the request to reporting the results of the rpoB gene study was 5.2±2.6 days. The mean gap from reporting the rpoB gene study to reporting the susceptibility was 56±35 days. Twenty-eight cases (85%) showed identical results compared with the drug susceptibility results, wheres five cases (15%) showed contradictory results. When compared with the sequencing analysis, of the five cases that showed contradictory results, two had LiPA analysis errors and the remaining three were identical to the sequencing results. The rpoB gene study was of assistance in choosing the appropriate drugs in 28 cases (85%). CONCLUSIONS: An rpoB gene study using an LiPA assay was useful in rapidly diagnosing RMP-resistant tuberculosis, which enabled a proper choice of the appropriate drugs in clinical practices. However, an LiPA assay always should be performed in conjunction with microscopy, culture, and susceptibility tests.

Rapid detection of rpoB mutation in rifampin-resistant mycobacterium tuberculosis strains with Line Probe Assay / 中华传染病杂志

Objective To detect rpoB mutations in rifampin resistant mycobacterium tuberculosis strains isolated from Shanghai, and to evaluate the implication of applicating line probe assay (LiPA). Methods A fragment (213bp) of rpoB gene of 58 Mycobacterium tuberculosis isolates was amplified and sequenced, 18 rifampin resistant strains and 10 susceptible strains were selected to detect mutation by LiPA. Results Mutations of rpoB gene in 17 strains of the 18 rifampin resistant isolates were found by LiPA, and there were no mutations in any of the 10 susceptible strains. The sensitivity of LiPA was 94.4% and the concordance with drug susceptibility of Mycobacterium tuberculosis was 96.4%. Conclusions LiPA is a useful method for the rapid detection of mutations of rpoB gene in rifampin resistant Mycobacterium tuberculosis with high sensitivity.

Early Diagnosis of Rifampin-Resistant Mycobacterium tuberculosis by Gene Analysis of RNA Polymerase B Subunit

PURPOSE: The control of tuberculosis is seriously threatened worldwide by the recently emerging multidrug-resistant Mycobacterium tuberculosis. As a result, early detection of drug resistant M.tuberculosis strain has become very important but conventional laboratory methods are time consuming and delayed results often affect patients adversely in controlling tuberculosis. The authors studied the usefulness of the line probe assay to determine the mutaion in rpoB gene of rifampin resistant M.tuberculosis and to find out if this method can substitute conventional methods in the detection of resistant strain. METHODS: This study employed 40 clinical samples of M.tuberculosis which had been determined by culture and drug sensitivity test. After amplification of rpoB-the gene for the B subunit of the RNA polymerase-by PCR, the amplified products were hybridized with specific oligonucleotide probes immobilized on nitrocellulose strip and direct DNA sequencing was also performed. The results were compared with those of the classical susceptibility test. RESULTS: Among the 40 samples, 10 were identified as drug resistant strain by classical drug susceptibility test. Three of the ten resistant samples were rifampin resistant strains, which were identified by either method. All mutations were clustered within the region of 69bp of rpoB and all were single nucleotide mutations. Two isolates had a TCG->TTG(serine->leucine) mutation in codon 522. One isolate had a CAC->CTC(histidine->leucine) mutation in codon 526. CONCLUSION: In contrast to culture and sensitivity tests, line probe assay is an easy and speedy method for detecting rifampin resistant M.tuberculosis in clinical samples as well as a helpful tool for choosing antituberculosis drug in children.

Comparison of PCR-Line Probe and PCR-SSCP Methods for the Detection of Rifampicin Resistant Mycobacterium Tuberculosis / 결핵

BACKGROUND: Rifampicin (RFP) is a key component of the antituberculous short-course chemotherapy and the RFP resistance is a marker of multi-drug resistant (MDR) tuberculosis. RpoB gene encodes the beta-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. And the mutations of rpoB gene have been found in about 96% of rifampicin resistant clinical isolates of M. tuberculosis. So in order to find a rapid and clinically useful diagnostic method in identifying the REP resistance, we compared the PCR-line probe method with PCR-SSCP for the detection of the rpoB gene mutation in cultured M. tuberculosis. METHODS: 45 clinical isoLates were collected from patients who visited Sung Kyun Kwan University Hospital. The REP susceptibility test was referred to the referral laboratory of the Korean Tuberculosis Institute. 33 were rifampicin resistant and 12 were rifampicin susceptible. The susceptibility results were compared with the results of the PCR-SSCF and PCR-line probe method. RESULTS: We could find rpoB mutations in 27/33(81.8%) RFP-resistant strains by PCR-line probe method, and in 23/33(69.7%) by PCR-SSCP and there was no significant difference between two methods. There was no mutation in rifampicinn susceptible strains by both methods.: Comparison of PCR-line probe and PCR-SSCP methods for detection rifampicin resistance CONCLUSION: PCR-Iine probe method would be a rapid, sensitive and specific method for the detection of rifampicin resistant Mycobacterium tuberculosis.

Direct Detection of Rifampin-Resistant Mycobacterium Tuberculosis by Polymerase Chain Reaction and Line Probe Assay in Sputums / 대한임상병리학회지

BACKGROUND: Recently, the control of tuberculosis may be threatened by widespread emergence of multidrug-resistant tuberculosis. Rifampin resistance is considered as a useful marker of multidrug-resistant tuberculosis and is developed by missense mutations in region of the rpoB gene encoding the beta-subunit of RNA polymerase. This study aimed to evaluate the line probe assay for direct detection of rifampin-resistant Mycobacterium tuberculosis in clinical samples. METHODS: Twenty-seven smear-positive and 13 smear-negative clinical samples, 13 M. tuberculosis strains and 2 nontuberculous mycobacteria strains were analyzed with line probe assay (Innogenetics, Belgium) and results were compared with conventional susceptibility test. I also reviewed conventional antimicrobial susceptibility testing patterns of 128 M. tuberculosis isolates from January 1996 to March 1997. RESULTS: All (100%) of total 30 patients who had rifampin-resistant strains revealed 3 or more drug resistances, but 5 (5%) out of 88 patients who had rifampin-sensitive strains revealed 2 or more drug resistances. By the line probe assay, 3 out of 13 smear-negative samples, 6 out of 27 smear-positive samples and 6 out of 13 M. tuberculosis strains were rifampin-resistant. Overall concordance of the line probe assay with conventional susceptibility test was 100%. Most common mutation site of rpoB gene was codon 531 (53.3%) followed by codon 526 (40%) and codon 516 (6.7%). CONCLUSIONS: These suggest that rifampin resistance will be a useful marker of multidrug- resistant tuberculosis and line probe assay will be a rapid and direct diagnostic tool for the detection of rifampin-resistant M. tuberculosis in clinical samples, especially AFB smear-negative samples.

Rapid Detection of Rifampin Resistant Mycobacterium tuberculosis Using the Line Probe Assay / 대한임상병리학회지

BACKGROUND: Tuberculosis continues to be a major threat to health throughout the world, with an estimated 8 to 10 million new cases and 3 million deaths annually. And control of the disease is further threatened by the emergence of drug resistance. Recent major advances have been made in unravelling the molecular basis of M. tuberculosis resistance to isoniazid, streptomycin, quinolones and rifampin. And rifampin resistance is the useful indicator for the occurance of the multi-drug resistance. Hence, the rapid detection of rifampin resistant strain of M. tuberculosis is the key to have successful anti-tuberculosis therapy. Here we present our experience using PCR and line probe assay (INNO-LiPA) for easy and rapid detection of rifampin resistance of M. tuberculosis. METHODS: Thirty rifampin resistant and twenty susceptible strains of M. tuberculosis were collected from the routine culture and analyzed with INNO-LiPA. And results were compared with conventional antibiotic susceptibility testing. After amplification of the region of the RNA polymerase(rpoB), the amplified product is hybridized with a set of 10 oligonucleotides immobilized onto a membrane strip. From the pattern obtained the presence or not of rifampin resistance M. tuberculosis can be assessed. RESULTS: Ninety three percent of patients who had rifampin resistant strain revealed the multidrug resistance while only two showed resistance to rifampin only. The INNO-LiPA test results were generally agreeable with that of the conventional susceptibility testing(90%). The mutations in codon 531 (absence of 55 probe) were most commonly observed. In 55.2% of the 31 rifampin resistance M. tuberculosis confirmed on mutation by R-probes on the INNO-LiPA strips. CONCLUSIONS: The line probe assay after polymerase chain reaction is a fast and convenient method to detect both presence of M. tuberculosis complex strains and its resistance to rifampin in clinical specimens. We have suggested that detection of rifampin resistance may play a key role in monitoring multi-drug resistance. Consequently, the INNO-LiPA test may constitute an important tool for the control of tuberculosis.