A Rapid and Accurate Detection Approach for Multidrug-Resistant Tuberculosis Based on PCR-ELISA Microplate Hybridization Assay

2020 ◽  
Vol 51 (6) ◽  
pp. 606-613
Author(s):  
Ye-Cheng Zhou ◽  
Shu-Mei He ◽  
Zi-Lu Wen ◽  
Jun-Wei Zhao ◽  
Yan-Zheng Song ◽  
...  
Keyword(s):  
Clinical Care ◽  
Pcr Amplification ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Hybridization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Mdr Tb

Abstract Rapid and accurate diagnosis of multidrug-resistant tuberculosis (MDR-TB) is important for timely and appropriate therapy. In this study, a rapid and easy-to-perform molecular test that integrated polymerase chain reaction (PCR) amplification and a specific 96-well microplate hybridization assay, called PCR-ELISA (enzyme-linked immunosorbent assay), were developed for detection of mutations in rpoB, katG, and inhA genes responsible for rifampin (RIF) and isoniazid (INH) resistance and prediction of drug susceptibility in Mycobacterium tuberculosis clinical isolates. We evaluated the utility of this method by using 32 multidrug-resistent (MDR) isolates and 22 susceptible isolates; subsequently, we compared the results with data obtained by conventional drug susceptibility testing and DNA sequencing. The sensitivity and specificity of the PCR-ELISA test were 93.7% and 100% for detecting RIF resistance, and 87.5% and 100% for detecting INH resistance, respectively. These results were comparable to those yielded by commercially available molecular tests such as the GenoType MTBDRplus assay. Based on the aforementioned results, we conclude that the PCR-ELISA microplate hybridization assay is a rapid, inexpensive, convenient, and reliable test that will be useful for rapid diagnosis of MDR-TB, for improved clinical care.

scholarly journals Assessing spatiotemporal patterns of multidrug-resistant and drug-sensitive tuberculosis in a South American setting

2010 ◽  
Vol 139 (11) ◽  
pp. 1784-1793 ◽  
Author(s):  
H. LIN ◽  
S. SHIN ◽  
J. A. BLAYA ◽  
Z. ZHANG ◽  
P. CEGIELSKI ◽  
...  
Keyword(s):  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Urban Setting ◽  
South American ◽  
Resistant Disease ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Mdr Tb ◽  
Incident Cases

SUMMARYWe examined the spatiotemporal distribution of laboratory-confirmed multidrug-resistant tuberculosis (MDR TB) cases and that of other TB cases in Lima, Peru with the aim of identifying mechanisms responsible for the rise of MDR TB in an urban setting. All incident cases of TB in two districts of Lima, Peru during 2005–2007 were included. The spatiotemporal distributions of MDR cases and other TB cases were compared with Ripley's K statistic. Of 11 711 notified cases, 1187 received drug susceptibility testing and 376 were found to be MDR. Spatial aggregation of patients with confirmed MDR disease appeared similar to that of other patients in 2005 and 2006; however, in 2007, cases with confirmed MDR disease were found to be more tightly grouped. Subgroup analysis suggests the appearance of resistance may be driven by increased transmission. Interventions should aim to reduce the infectious duration for those with drug-resistant disease and improve infection control.

scholarly journals Use of Genotype MTBDRplus Assay for Diagnosis of Multidrug-Resistant Tuberculosis in Nepal

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Elina Maharjan ◽  
Narayan Dutt Pant ◽  
Sanjeev Neupane ◽  
Jyoti Amatya ◽  
Bhawana Shrestha
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Conventional Drug ◽  
Mdr Tb ◽  
Genotype Mtbdrplus

The main aims of this study were to study the patterns of mutations in rpoB, katG, and inhA genes in Mycobacterium tuberculosis strains isolated from patients from Nepal and to evaluate the performance of genotype MTBDRplus assay, taking conventional drug susceptibility testing as gold standard for diagnosis of MDR-TB. A total of 69 Mycobacterium tuberculosis strains isolated from 73 smear positive sputum samples from patients suspected of suffering from multidrug-resistant tuberculosis were used in our study. The drug susceptibility pattern of Mycobacterium tuberculosis isolated from these sputum specimens was determined by using genotype MTBDRplus assay taking conventional drug susceptibility testing as reference. The sensitivity and specificity of the genotype MTBDRplus assay for the detection of MDR-TB were found to be 88.7% and 100%, respectively. 88.7% of the rifampicin resistant isolates had mutations in rpoB gene. Similarly, 79.7% and 9.4% of isoniazid resistant isolates had mutations in katG and inhA genes, respectively. Genotype MTBDRplus assay was found to be very rapid and highly sensitive and specific method for diagnosis of MDR-TB and will be very helpful for early diagnosis of MDR-TB in high tuberculosis burden countries.

scholarly journals Correlation of gyr Mutations with the Minimum Inhibitory Concentrations of Fluoroquinolones among Multidrug-Resistant Mycobacterium tuberculosis Isolates in Bangladesh

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1422
Author(s):  
Mohammad Khaja Mafij Uddin ◽  
Md. Fahim Ather ◽  
Rumana Nasrin ◽  
Tanjina Rahman ◽  
A. S. M. Iftekhairul Islam ◽  
...  
Keyword(s):  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Gyrb Gene ◽  
Gyra Gene ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Mdr Tb ◽  
Low Levels ◽  
High Level

Fluoroquinolone (FQ) compounds—moxifloxacin (MOX), levofloxacin (LEV), and ofloxacin (OFL)—are used to treat multidrug-resistant tuberculosis (MDR-TB) globally. In this study, we investigated the correlation of gyr mutations among Mtb isolates with the MICs of MOX, LEV, and OFL in Bangladesh. A total of 50 MDR-TB isolates with gyr mutations, detected by the GenoType MTBDRsl assay, were subjected to drug susceptibility testing to determine the MICs of the FQs. Spoligotyping was performed to correlate the genetic diversity of the gyr mutant isolates with different MIC distributions. Among the 50 isolates, 44 (88%) had mutations in the gyrA gene, one (2%) had a mutation in the gyrB gene, and five (10%) isolates had unidentified mutations. The substitutions in the gyrA region were at A90V (n = 19, 38%), D94G (n = 16, 32%), D94A (n = 4, 8%), D94N/D94Y (n = 4, 8%), and S91P (n = 1, 2%), compared to the gyrB gene at N538D (n = 1.2%). D94G mutations showed the highest MICs for MOX, LEV, and OFL, ranging between 4.0 and 8.0 μg/mL, 4.0 and 16.0 μg/mL, and 16.0 and 32.0 μg/mL, respectively; while the most common substitution of A90V showed the lowest ranges of MICs (1.0–4.0 μg/mL, 2.0–8.0 μg/mL, and 4.0–32.0 μg/mL, respectively). Spoligotyping lineages demonstrated no significant differences regarding the prevalence of different gyr mutations. In conclusion, the substitutions of codon A90V and D94G in the gyr genes were mostly responsible for the FQs’ resistance among Mtb isolates in Bangladesh. Low levels of resistance were associated with the substitutions of A90V, while the D94G substitutions were associated with a high level of resistance to all FQs.

scholarly journals Resistance to Second-Line Antituberculosis Drugs and Delay in Drug Susceptibility Testing among Multidrug-Resistant Tuberculosis Patients in Shanghai

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Yong Chen ◽  
Zhengan Yuan ◽  
Xin Shen ◽  
Jie Wu ◽  
Zheyuan Wu ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Second Line ◽  
Total Delay ◽  
Antituberculosis Drugs ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Mdr Tb

Introduction. Second-line antituberculosis drugs (SLDs) are used for treating multidrug-resistant tuberculosis (MDR-TB). Prolonged delays before confirming MDR-TB with drug susceptibility testing (DST) could result in transmission of drug-resistant strains and inappropriate use of SLDs, thereby increasing the risk of resistance to SLDs. This study investigated the diagnostic delay in DST and prevalence of baseline SLD resistance in Shanghai and described the distribution of SLD resistance with varied delays to DST.Methods. All registered patients from 2011 to 2013 in Shanghai were enrolled. Susceptibility to ofloxacin, amikacin, kanamycin, and capreomycin was tested. Total delay in DST completion was measured from the onset of symptoms to reporting DST results.Results. Resistance to SLDs was tested in 217 of the 276 MDR-TB strains, with 118 (54.4%) being resistant to at least one of the four SLDs. The median total delay in DST was 136 days. Patients with delay longer than median days were roughly twice more likely to have resistance to at least one SLD (OR 2.22, 95% CI 1.19–4.11).Conclusions. During prolonged delay in DST, primary and acquired resistance to SLDs might occur more frequently. Rapid diagnosis of MDR-TB, improved nosocomial infection controls, and regulated treatment are imperative to prevent SLD resistance.

Prevalence of Multidrug-Resistant Tuberculosis Using Phenotypic Drug Susceptibility Testing and GeneXpert MTB/RIF with Characterization of Non-tuberculous Mycobacteria Using MALDI-TOF

2021 ◽  
Vol 30 (3) ◽  
pp. 143-151
Author(s):  
Noha S. Soliman ◽  
Sahar M. Khairat ◽  
Mohamed Abdullah ◽  
Yasmin Adel El-Mahdy
Keyword(s):  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Health Concern ◽  
Maldi Tof ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Mdr Tb

Background: Multidrug-resistant tuberculosis (MDR-TB) and infections by nontuberculous mycobacteria (NTM) are diseases of major public health concern. Objective: The aim of the present work is to study the prevalence and patterns of MDRTB as well as the characterization of isolated NTM species. Methodology: All samples (1069) were subjected to smear microscopy, culture on Lowenstein-Jensen (LJ) media, and phenotypic drug susceptibility testing (DST) of MTB to isoniazid (INH), rifampin (RF), streptomycin (S), and ethambutol (E). GeneXpert was used for direct detection of MTB and RF resistance. Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) was utilized for characterizing isolated NTM species. Results: M.tuberculosis (MTB) was isolated at a rate of 95.3% (1019/1069). MDR-TB was detected at rate of 7.16% with significant patterns for INH + RF + S + E (46.5%) and INR + RF (24.6%) (P-value <0.001). RF resistance was detected at a rate of 27.2% by GeneXpert. Seven NTM species (0.6%) were isolated in culture of which M.porcinum and M.fortuitum had confident identification by MALDI-TOF (score ≥1.8). Conclusion: MDR-TB rate was found to be 7.16% with significant dominance for INH + RF + S + E and INR + RF resistance patterns, while NTM rate was 0.6%.

Evaluation of MTBDRsl for detecting resistance in Mycobacterium tuberculosis to second-line drugs

2019 ◽  
Vol 23 (12) ◽  
pp. 1257-1262 ◽  
Author(s):  
R. J. Chandak ◽  
B. Malhotra ◽  
S. Bhargava ◽  
S. K. Goel ◽  
D. Verma ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rapid Test ◽  
Drug Susceptibility ◽  
Turnaround Time ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Tb Treatment ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Mdr Tb

SETTING: Patients with presumed multidrug-resistant tuberculosis (MDR-TB) and undergoing MDR-TB treatment from Rajasthan, India.OBJECTIVE: To compare the GenoType® MTBDRsl v.1.0 (MTBDRsl) assay capacity to detect resistance to ofloxacin, amikacin, capreomycin, kanamycin and ethambutol in Mycobacterium tuberculosis with phenotypic drug susceptibility testing (DST) using MGIT™960™ in sputum samples and isolates.DESIGN: Fifty-three smear-positive sputum samples were tested directly by MTBDRsl and 205 MDR-TB isolates were processed using MTBDRsl and DST for five drugs on MGIT960. DNA sequencing was performed in isolates with discordance in the results between the two methods for the gyrA, gyrB and rrs genes.RESULT: Sensitivity and specificity of MTBDRsl was found to be respectively 93.1% and 100% for fluoroquinoline, respectively 75–78% and 100% for aminoglycosides/cyclopeptides, respectively 70% and 92% for ethambutol and respectively 92.3% and 100% for extensively drug-resistant (XDR) TB detection. On sequencing eight discordant isolates for quinolones, mutations were seen in 12.5% of the gyrB gene and among 20 discordant isolates for aminoglycosides/cyclopeptides in the rrs gene in 15% isolates. The turnaround time was 2 days for MTBDRsl vs. 10 days for MGIT960.CONCLUSIONS: MTBDRsl can be used as an initial rapid test for detecting XDR-TB, resistance to quinolones and aminogycosides/cyclopeptides in smear-positive sputum samples.

scholarly journals Risk factors associated with multidrug-resistant tuberculosis in Espírito Santo, Brazil

2017 ◽  
Vol 51 (0) ◽  
Author(s):  
Geisa Fregona ◽  
Lorrayne Belique Cosme ◽  
Cláudia Maria Marques Moreira ◽  
José Luis Bussular ◽  
Valdério do Valle Dettoni ◽  
...  
Keyword(s):  
Previous Treatment ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Espírito Santo ◽  
Factors Associated ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
History Of ◽  
Espirito Santo

ABSTRACT OBJECTIVE To analyze the prevalence and factors associated with multidrug-resistant tuberculosis in Espírito Santo, Brazil. METHODS This is a cross-sectional study of cases of tuberculosis tested for first-line drugs (isoniazid, rifampicin, pyrazinamide, ethambutol, and streptomycin) in Espírito Santo between 2002 and 2012. We have used laboratory data and registration of cases of tuberculosis – from the Sistema Nacional de Agravos de Notificação and Sistema para Tratamentos Especiais de Tuberculose. Individuals have been classified as resistant and non-resistant and compared in relation to the sociodemographic, clinical, and epidemiological variables. Some variables have been included in a logistic regression model to establish the factors associated with resistance. RESULTS In the study period, 1,669 individuals underwent anti-tuberculosis drug susceptibility testing. Of these individuals, 10.6% showed resistance to any anti-tuberculosis drug. The rate of multidrug resistance observed, that is, to rifampicin and isoniazid, has been 5%. After multiple analysis, we have identified as independent factors associated with resistant tuberculosis: history of previous treatment of tuberculosis [recurrence (OR = 7.72; 95%CI 4.24–14.05) and re-entry after abandonment (OR = 3.91; 95%CI 1.81–8.43)], smoking (OR = 3.93; 95%CI 1.98–7.79), and positive culture for Mycobacterium tuberculosis at the time of notification of the case (OR = 3.22; 95%CI 1.15–8.99). CONCLUSIONS The partnership between tuberculosis control programs and health teams working in the network of Primary Health Care needs to be strengthened. This would allow the identification and monitoring of individuals with a history of previous treatment of tuberculosis and smoking. Moreover, the expansion of the offer of the culture of tuberculosis and anti-tuberculosis drug susceptibility testing would provide greater diagnostic capacity for the resistant types in Espírito Santo.

scholarly journals Speeding up the diagnosis of multidrug-resistant tuberculosis in a high-burden region with the use of a commercial line probe assay

2019 ◽  
Vol 45 (2) ◽  
Author(s):  
Angela Pires Brandao ◽  
Juliana Maira Watanabe Pinhata ◽  
Rosangela Siqueira Oliveira ◽  
Vera Maria Neder Galesi ◽  
Helio Hehl Caiaffa-Filho ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Reference Laboratory ◽  
Line Probe Assay ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Commercial Line ◽  
Probe Assay

ABSTRACT Objective: To evaluate the rapid diagnosis of multidrug-resistant tuberculosis, by using a commercial line probe assay for rifampicin and isoniazid detection (LPA-plus), in the routine workflow of a tuberculosis reference laboratory. Methods: The LPA-plus was prospectively evaluated on 341 isolates concurrently submitted to the automated liquid drug susceptibility testing system. Results: Among 303 phenotypically valid results, none was genotypically rifampicin false-susceptible (13/13; 100% sensitivity). Two rifampicin-susceptible isolates harboured rpoB mutations (288/290; 99.3% specificity) which, however, were non-resistance-conferring mutations. LPA-plus missed three isoniazid-resistant isolates (23/26; 88.5% sensitivity) and detected all isoniazid-susceptible isolates (277/277; 100% specificity). Among the 38 (11%) invalid phenotypic results, LPA-plus identified 31 rifampicin- and isoniazid-susceptible isolates, one isoniazid-resistant and six as non-Mycobacterium tuberculosis complex. Conclusions: LPA-plus showed excellent agreement (≥91%) and accuracy (≥99%). Implementing LPA-plus in our setting can speed up the diagnosis of multidrug-resistant tuberculosis, yield a significantly higher number of valid results than phenotypic drug susceptibility testing and provide further information on the drug-resistance level.

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