Genetic Diversity and Drug Resistance Pattern of Mycobacterium Species Among Smear Negative Pulmonary Tuberculosis Patients in Addis Ababa, Ethiopia
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Date
2025-01
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Addis Ababa University
Abstract
Tuberculosis (TB) is being treated empirically in about 38% of patients globally due to difficulty in detecting the Mycobacterium tuberculosis complex (MTBC) bacilli with the routine laboratory diagnostic tests, including smear microscopy, GeneXpert and culture. As a result, the situation with smear negative pulmonary tuberculosis (SNPTB) has remained challenging, leading to diagnostic delays and making monitoring treatment outcomes difficult. However, there is scarce information on the actual burden, drug resistance pattern and the strains of MTBC involved in SNPTB. Therefore, this study aimed to evaluate the genetic diversity and drug resistance pattern of MTBC isolates recovered SNPTB patients. A health facility-based case control study was conducted in Addis Ababa, Ethiopia, involving 313 pulmonary TB patients (173 SNPTB and 140 Smear positive PTB (SPPTB)). Laboratory data was generated by collecting sputum specimens from consenting study participant and acid fast staining, GeneXpert and culture were done at Armauer Hansen Research Institute (AHRI) laboratory. Molecular analysis, spoligotyping and whole genome sequencing (WGS) as well as phenotypic drug susceptibility test were done for culture grown MTBC isolates. The revised online MTBC molecular marker database SITVIT2 and appropriate bioinformatics tools were applied to assign lineages and sublineages of MTBC strains. Structured questionnaires were used to assess TB related factors, clinical and imaging findings associated among SNPTB and SPPTB patients. SPSS version 25 and Stata version 17 software were used for data analysis and a P-value < 0.05 was considered statistically significant. Of the 173 SNPTB patients 75 were culture positive. The spoligotyping and WGS analysis of MTBC isolates identified
four major MTBC lineages including lineage 4 (Euro-American), lineage 3 (East-African-Asian), lineage 1 (Indo-Oceanic) and lineage7 (Ethiopian) which had 80.2%, 16.7%, 2.4% and 0.8% proportion, respectively. Lineage 3 MTBC strains were higher among SNPTB patients compared to SPPTB patients while the proportion of lineage 4 MTBC strain was higher among SPPTB than SNPTB (P-value <0.05). A higher proportion of MTBC isolates from SNPTB 16.7% were resistant to one or more first-line anti-TB drugs than SPPTB 12.9% (P-value >0.05). MDR-TB was detected from single SNPTB patients 5.6% (1/18) by both phenotypic and molecular methods whereas none in SPPTB patients (0/33) (P-value >0.05). Although, overall clinical presentations of SNPTB patients resemble those seen in SPPTB patients, prior history of TB was strongly associated with SNPTB (P-value <0.05). There was a significant difference in MTBC strain distribution among SNPTB and among SPPTB patients, which may be responsible for difficulty in MTBC laboratory detection that may need further advanced analysis of sequenced isolates. There was also primary drug resistant TB among culture positive SNPTB patients, which would be otherwise missed by current national protocols. An additional study with larger sample size and more sensitive TB detection approach is recommended for better understanding of SNPTB.
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Keywords
GeneXpert, MTBC, NTM, SNPTB, Spoligotyping, WGS