Molecular Characterization of Drug-Resistant Mycobacterium Tuberculosis and Evaluation of the Performance of Genexpert Mtb/Rif® Assay Using Urine Specimen to Diagnose Pulmonary Tuberculosis in Sputum-Scarce Patients in Addis Ababa, Ethiopia

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Addis Ababa University


Multidrug drug-resistant tuberculosis (MDR-TB) is a major health problem and seriously threatens TB control globally. Ethiopia is among the 30th highest TB burden countries. MDR-TB is a result of unsuccessful TB control programs and associated with mutations in drug target genes. Another challenge of TB is that about one-third of TB/HIV co-infected patients fail to produce a sputum specimen for diagnosis. Understanding of the pattern of resistance and genotyping of strains, and identify alternative specimen for diagnosis are important for TB/MDR-TB control. Thus, the aim of this study was to determine the pattern of mutations associated with resistance, genotype, and performance of GeneXpert MTB/RIF using urine specimens. A cross-sectional study was conducted in Addis Ababa from June 2015 to December 2016. Sputum specimens and socio-demographic data were collected from 358 MDR-TB suspected cases, and 150 urine specimens from sputum scarce cases and cases capable of giving sputum suspected of pulmonary TB. Specimens were analyzed using Ziehl-Neelsen, GeneXpert MTB/RIF assay, and cultures. All culture-positive specimens were analyzed for drug susceptibility tests. Sequence analysis for rpoB, katG, fabG1-inhA, embB, pncA, rrs and rpsL genes, and genotyping was performed using 24 MIRU-VNTR and spoligotype. Data were analyzed using SPSS version 23. Of 358 MDR-TB suspected cases, 226 were culture positive for Mycobacterium tuberculosis, of these, 162 (71.7%) had been previously treated for tuberculosis, while 128 (56.6%) were TB/HIV co-infected. An analysis of drug resistance indicated that 110 (48.7%) strains were resistant to isoniazid, 94 (41.6%) to streptomycin, 89 (39.4%) to rifampicin, 72 (31.9%) to ethambutol, and 70 (30.9%) to pyrazinamide. Among the 226 study participants, 89 (39.4%) were determined to be MDR-TB with TB/HIV co-infection, cigarette smoking, alcohol drinking, and admissions and/or visits to hospitals significantly (p<0.044) associated with the observed MDR-TB prevalence. Among the 209 isolates submitted for drug target gene sequencing, 101 (48.3%) isolates had the mutations in a katG gene, of these, 98 (98.0%) mutations were found at codon S315T, and other XIII mutations were found 90 (43.1%) isolates in rpoB gene. Out of these, 61 (67.8%) and 15 (16.7%) mutations occurred at codons S531L/W and H526Y/S/D/L respectively. Moreover, 73 (34.9%) isolates had a mutation in embB gene, and majority 48 (65.8%) of the mutations were found at codon M306I/V/L. In another finding, 68 (30.1%) isolates had a mutation in pncA gene with high mutation at codon 65 in 19 (27.9%) isolates. Likewise, mutations in rrs and rpsl genes were found in 56 (26.8%) isolates and the highest 50 (89.3%) proportion of mutation was found in rpsL gene at codon K88R/T and K43R. Furthermore, the least frequency of mutation was observed in fabG1-inhA promoter region in 7 (3.3%) isolates. A total of 167 isolates had a valid genotyping results, of thse 157 (94.0%) were classified as known lineages; Dehli/CAS 50 (29.9%), TUR 44 (26.3%), H37Rv like 25 (15.0%), TUR_Ethiopia_3 17 (10.2%), Haarlem 14 (8.4%), Ural 4 (2.4%), LAM 1 (0.6%), X-type 1 (0.6%), and EIA 1 (0.6%). The remaining 10 (6.0%) isolates were undefined. Among MDR-TB, the highest predominant genotype was TUR with 35 (46.1%). Cluster analysis showed that a total of 103 (61.7%) strains shared a genotyping pattern, and a recent transmission index was 50.3%. In another finding, of the 150 urine specimens tested, 5 (3.3%) were positive by GeneXpert MTB/RIF and culture. The sensitivity and specificity of GeneXpert MTB/RIF were 100% and 99.3% (95%CI: 97.4-100%) respectively, and overall performance agreement was 99.3% (95% CI: 97.4-100%). In conclusion, a highly diverse M. tuberculosis population structure was found, with a predominance of the Dehli/CAS genotype. The prevalence of MDR-TB in the study population was significantly high and the predominant lineage among MDR-TB strains was TUR, and there was a high rate of recent transmission among MDR-TB strains. A substantial number of mutations were observed at codons 531, 315, 306, 65, and 88 in rpoB, katG, embB, pncA, and rpsl genes respectively. TB/HIV co-infection, smoking of a cigarette, alcohol drinking, admission and visit of health facility were identified as risk factors for developing MDR-TB. A good sensitivity and specificity of GeneXpert MTB/RIF were observed using urine specimen, thus, urine can be used XIV as an alternative specimen for diagnosis of PTB from sputum scare patients. In general, our findings support us to conclude that there is weak health system for prevention and control of MDR-TB/TB in Ethiopia. Therefore, effective strategies should be designed considering the identified risk factors for control of MDR-TB.



Mycobacterium Tuberculosis, Mdr-Tb, Risk Factors Rpob, Katg, Embb, Pnca, Rpsl, Mutation, Isoniazid, Rifampicin, Ethambutol, Pyrazinamide, Streptomycin, Genexpert Mtb/Rif, Urine, Sensitivity, Specificity, Genotyping