Whole genome sequencing of Mycobacterium tuberculosis isolates and clinical outcomes of patients treated for multidrug-resistant tuberculosis in Tanzania

- Whole genome sequencing of.
- Whole genome sequencing (WGS) is a current alternative to the WHO-approved probe-based methods for TB diagnosis and detection of drug resistance, genetic diversity and transmission dynamics of Mycobacterium tuberculosis complex (MTBC).
- Drug resistance was determined by Xpert® MTB/RIF assay and phenotypic culture-based drug-susceptibility-testing (DST).
- MDR-TB participants had MTBC with multiple mutant genes, compared to those with mono or polyresistant TB, and the majority belonged to lineage 3 Central Asian Strain (CAS).
- Also, MDR- TB was associated with delayed culture-conversion (median: IQR vs.
- WGS had high concordance with both culture-based DST and Xpert® MTB/RIF assay in detecting drug resistance (kappa = 1.00)..
- 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- Full list of author information is available at the end of the article.
- Sub-Saharan Africa carries a disproportionate burden of HIV, accounting for more than 70% of the global burden of infection [4].
- The recent introduction of the Xpert®MTB/Rif assay (Cepheid, USA) has shown an increase in detection of drug resistance TB patients and the detection of multidrug-resistant TB (MDRTB) has increased three- to eight-fold compared to conventional testing [6].
- Despite the gaps in documentation of MDR- TB cases in several Sub-Saharan African countries (SSA), pooled analysis that involved several studies re- ported a prevalence of 2.1% of MDR-TB in new cases, signifying a low prevalence of MDR-TB cases in SSA [7]..
- The National anti- tuberculosis drug resistance survey conducted in 2010 in Tanzania found the resistance to any of the four first- line drugs in new patients to be 8.3%, while the preva- lence of MDR-TB was 1.1%.
- However, the crude preva- lence for any resistance and for MDR-TB in retreated cases increased to 20.6 and 3.9% respectively [10]..
- Globally, MDR-TB claims over 580,000people ’ s lives annually [12].
- About 10% of MDR- TB isolates exhibit resistance to fluoroquinolones (FQs) and second-line-injectable drugs (SLIDs) (aminoglycosides/.
- Clinical man- agement of both MDR-TB and XDR-TB is very complex and there are different treatment regimens [15].
- About 50 and 70% of patients treated for MDR-TB and XDR-TB have unfavourable treatment outcomes, respectively..
- Although XDR-TB is rare in Tanzania, cohort review re- ports of patients treated for MDR-TB show that treatment success is only 75%.
- early, rapid and accurate diagnostic methods are required to detect and decipher susceptibility profile of the MTBC to anti-TB drugs in TB endemic settings such as Tanzania..
- These assays have po- tential to guide implementation of the new WHO shorter regimen for treating MDR-TB [15, 19].
- WGS can identify genotypes predictive of drug-resistance phenotype within the en- tire region of microbial genome and has potential to determine genetic relatedness and identify transmis- sion dynamics necessary in guiding clinical decisions..
- Based on this, we performed WGS for the first time on isolates sourced from patients treated for MDR-TB at Kibong ’ oto Infectious Diseases Hospital (KIDH) in Tanzania.
- These data will provide a baseline set of the types and variations in TB in Tanzania.
- Association of clinical information with genetic drug resistance.
- We found association between previous history of TB treatment and phenotypic drug resistance (OR, 0.01 95% CI p = <.
- Analysis of mutations associated with phenotypic drug resistance to anti-TB drugs.
- Of the 87 MTBC isolates sequenced had at least one mutation in a gene that was predictive for 7.
- The common mutation associated with phenotypic resistant to RIF was S450L (substitution of serine to leucine) accounted for of the detected RIF resistance.
- Of the 57 isolates, 29 (33%) had mutations in the embB gene and Q497R was the common mutation (8/29.
- In addition, our analysis revealed pncA deletion involving Rv2044c in two drug resistance isolates (MDRTB &.
- Patients with MDR-TB had highly diverse mutations as compared to monoresistance (Table 3).
- The drug resistance mutations involving either rrs or gyrB genes against AMK and FQs respectively were detected in low proportions among DR resistance isolates (Table 3).
- Treatment outcome and culture conversion rates among the drug resistance TB isolates.
- Of the 57 study participants with mutation (s) to at least one of the anti-TB drugs had successful treatment, 3 (5.3%) died, and 7 (12.3%) defaulted (Table 4).
- Phylogenetic analysis of the drug resistance TB isolates Phylogenetic analysis revealed that the DR-TB strains were heterogeneously distributed in lineages 1 to 7 (Fig.
- The Central Asian Strains (CAS) (lineage 3) pre- dominated in the MTBC strains and accounted for of the isolates, followed by lineage 4 (32/.
- Of the 24 MDR isolates Table 1 Demographic and clinical information of study.
- In this study, we found high concordance between WGS and conventional culture-based DST in predicting phenotypic drug resistance to anti-TB drugs, ranging from 81% for INH to 97% for RIF, and 95% concordance with Xpert® MTB/RIF (Cepheid, USA) for RIF.
- The ability of Xpert® MTB/RIF and phenotypic detection of TB drug resistance cannot be underscored as it can pave a way to complementary and confirmatory WGS in early detection of resistance.
- Although the facilities are not yet available the approach can be advocated in future clinical settings to enable reduction in unnecessary laboratory diagnostic time delays be- tween identification of patients suspected of MDR-TB and initiation of treatment [29].
- The potential for WGS to provide a rapid and comprehensive view of the geno- type and reliable prediction of the drug susceptibility phenotype has been reported elsewhere .
- In our study, the WGS uncovered 2 pre patient har- bouring XDR-TB isolates, which were also detected by phenotypic DST at our hospital and later cured after MDR-TB treatment.
- RIF (Cepheid, USA) MDR-TB results for informed Table 2 Demographic and clinical characteristics of drug resistance TB patients.
- We also found that the majority of the MTBC isolates from DR-TB participants had mutations in genes predict- ive of phenotypic resistance to all 7 anti-TB drugs tested including INH (katG), RIF (rpoB), EMB (embB, embC- embA), FQs (gyrB) and second line injectable drugs (rrs) (Table 3).
- As expected, patients with MDR-TB had multiple mutations in these genes, confirming the previous concept that drug resistance in mycobacteria spp., and other bac- teria evolves as they accumulate mutations either de novo or after multiple and longer exposure to antibiotics [37, 38].
- This high diversity of mutations among DR-TB pa- tients suggests on-going transmission of MDR-TB strains rather than acquisition through random mutation and se- lection of drug resistance strains [37, 39].
- In addition, our analysis revealed a large deletion that involved Rv2044c in Table 3 Frequency and distribution of mutations associated with drug resistance M.
- in MDR-TB strains n.
- Such isolates also possessed mutations either in drug resistance genes for RIF or INH..
- INH, FQS and SLIDs are used for shorter MDR-TB treat- ment regimes.
- INH and EMB have been in use for several decades either as part of the standard treatment regimen for drug suscep- tible TB and INH monotherapy as infection preventive therapy for HIV infected individuals [40].
- tuberculosis drug resistance strains, and pattern of drug resistance.
- Table 5 Description of lineages, spoligotype, cluster of drug resistance M.
- 7 lineage3 CAS MDR S450L S315T.
- LAM MDR S450L S315T.
- CAS MDR S450L S315T.
- CAS MDR S450L S315T V128G.
- CAS MDR S450L S315T V128G Q497R.
- In this study, participants with MDR-TB were cured using the.
- Time to culture conversion was Table 5 Description of lineages, spoligotype, cluster of drug resistance M.
- CAS MDR S450L S315T V128G Q497R,.
- This success rate is 30% higher than the global MDR-TB treatment success rate [15], positioning the WGS method along with culture based DST as a useful testing algorithm for rigor- ous microbiological monitoring that will accelerate the 2035 strategic END-TB vision to create a world popula- tion free of TB [45].
- In this study, 29.9% of the study participants had low BMI and 23% were infected with HIV but had no effects on MDR-TB treatment outcomes..
- In addition to that, the WGS enables earlier use of the most appropriate drug regimen, thus improving patient outcomes and reducing overall healthcare costs [33]..
- Phenotypic culture-based DST for PZA, and cycloserine, which were part of the standardized treatment regimen for all partic- ipants was missing.
- In addition, concordance between line probe assays (LPA) genotype MDBTDRplus and MTBDRsl and WGS could not be done, which would have given a full comparison of the robustness of the WGS since the former had not been in use at the time of the study.
- The drug resistance was either mono/.
- polyresistance-resistant or MDR-TB.
- MDR-TB was treated for at least 20 months and monitored monthly with culture and smear microscopy for AFB while DS TB was monitored with smear microscopy only to deter- mine microbiological treatment response and program- matic outcomes.
- Thereafter, the volume of the mixture was adjusted to 50 mLs in a falcon tube and concentrated by centrifugation at 3000 g for 15 min.
- Phenotypic drug susceptibility testing of the MTBC iso- lates was performed using a standard proportion method on LJ media [53, 54]..
- The concentration and quality of the DNA were measured using Qubit™ 4 Fluorometer (Invi- trogen.
- The QIAseq FX DNA Library Kit covers DNA fragmentation for 15 min, adapter ligation and a final purification of the samples using AMPure XP beads (Beckman-Coulter).
- The median depth and the percentage of the reference covered by a minimum of 10 reads were calculated using SAMtools depth [56] and custom scripts.
- Variant calling and in-silico resistance and lineage typing Variants in drug resistance gene candidates were called using LoFreq version v2.1.2 [57].
- tbdr.lshtm.ac.uk/) as it does not perform its own variant calling but annotates existing calls as drug resistance mutations using a database of mutations.
- maximum likelihood phylogeny was constructed using ExaML version v3.0.21, a state of the art tool for phylo- genomic analyses [60].
- This was visualized with drug resistance phenotypes and lineages using iTOL [62].
- Descriptive statistics was performed for demographic characteristics, frequency and distribution of mutation patterns in MDR-TB and other resistance patterns in MTBC.
- Genotype drug resistance was defined as any mutations that are known to confer decreased anti tuberculosis drug susceptibility..
- Kappa statistics (K) were used to determine agreement between drug susceptibility testing (DST) and genetic drug resistance using four levels of agreement for kappa: <.
- MDR-TB: Multidrug resistant Tuberculosis;.
- We acknowledge laboratory staff of the Central Referral Tuberculosis Laboratory, Biochemistry Department at Muhimbili University of Health and Allied Science and the Director of Kibong ’ oto hospital for their logistic support during the execution of this study..
- The Senate Research and Publications Committee of the Muhimbili University of Health and Allied Sciences (MUHAS) in Tanzania approved the study (Ref.
- All study participants received MDR-TB treatment according to existing National and global MDR-TB treatment guidelines in Tanzania [23, 27]..
- National anti- tuberculosis drug resistance study in Tanzania.
- Comparison of TaqMan® Array card and MYCOTBTM with conventional phenotypic susceptibility testing in MDR-TB.
- National anti-tuberculosis drug resistance study in Tanzania.
- Applicability of the shorter &#x2018;Bangladesh regimen&#x2019.
- Whole genome sequencing of clinical strains of mycobacterium tuberculosis from Mumbai, India: A potential tool for determining drug-resistance and strain lineage.
- sequencing and reporting of drug resistance profiles on clinical cases of mycobacterium tuberculosis in New York state.
- A standardised method for interpreting the association between mutations and phenotypic drug resistance in mycobacterium tuberculosis.
- Whole genome sequencing of Mycobacterium tuberculosis for detection of drug resistance: a systematic review.
- Removing the bottleneck in whole genome sequencing of mycobacterium tuberculosis for rapid drug resistance analysis: a call to action.
- Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti- tuberculosis drug resistance.
- Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda.
- Evolution of drug resistance in mycobacterium tuberculosis: a review on the molecular determinants of resistance and implications for personalized care.
- Achieving high treatment success for multidrug-resistant TB in Africa: initiation and scale-up of MDR TB care in Ethiopiaâ € an observational cohort study.
- Genotypic drug resistance using whole-genome sequencing of mycobacterium tuberculosis clinical isolates from North-Western Tanzania.
- Rapid determination of anti-tuberculosis drug resistance from whole-genome sequences

Xem thử không khả dụng, vui lòng xem tại trang nguồn
hoặc xem Tóm tắt