Insight into the genetic diversity of Mycobacterium bovis isolated from cattle in Malawi.

We describe the genetic diversity and phylogenetic relationships of Mycobacterium bovis, isolated from cattle in Malawi. Deletion analysis, spoligotyping, and MIRU-VNTR typing were used to genotype the isolates. Combined with a larger dataset from neighboring countries, the overall M. bovis diversity in Southern Africa was contextualized. From the southern and northern regions of Malawi, 24 isolates were confirmed as M. bovis. We pooled data for the central region (60 isolates) from our recent publication to conceptualize the genetic and phylogenetic relationships of M. bovis in Malawi. European 1 was the dominant M. bovis clonal complex, with 10 unique spoligotype patterns, and SB0131 was ubiquitous. High genetic diversity, a low clustering rate, and many singletons, coupled with a low mutation transmission index, infer a low level of recent transmission, and suggest an endemic status of bovine tuberculosis (bTB) in Malawi. M. bovis isolates from Zambia, Mozambique, and South Africa were genetically related to Malawian isolates, whereas Tanzanian isolates were distantly related. The diversity and phylogenetic analysis suggest earlier introductions and maintenance of M. bovis by constant reinfection from reservoir animals. These findings are fundamental to understanding the source and route of infection in order to establish alternative management strategies for bTB.

Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia.

The emergence of pre-extensively drug-resistant tuberculosis (pre-XDR-TB) is a threat to TB control programs in developing countries such as Zambia. Studies in Zambia have applied molecular techniques to understand drug-resistance-associated mutations, circulating lineages and transmission patterns of multi-drug-resistant (MDR) Mycobacterium tuberculosis. However, none has reported genotypes and mutations associated with pre-XDR TB. This study characterized 63 drug-resistant M. tuberculosis strains from the University Teaching Hospital between 2018 and 2019 using targeted gene sequencing and conveniently selected 50 strains for whole genome sequencing. Sixty strains had resistance mutations associated to MDR, one polyresistant, and two rifampicin resistant. Among MDR strains, seven percent (4/60) had mutations associated with pre-XDR-TB. While four, one and nine strains had mutations associated with ethionamide, para-amino-salicylic acid and streptomycin resistances, respectively. All 50 strains belonged to lineage 4 with the predominant sub-lineage 4.3.4.2.1 (38%). Three of four pre-XDR strains belonged to sub-lineage 4.3.4.2.1. Sub-lineage 4.3.4.2.1 strains were less clustered when compared to sub-lineages L4.9.1 and L4.3.4.1 based on single nucleotide polymorphism differences. The finding that resistances to second-line drugs have emerged among MDR-TB is a threat to TB control. Hence, the study recommends a strengthened routine drug susceptibility testing for second-line TB drugs to stop the progression of pre-XDR to XDR-TB and improve patient treatment outcomes.

Detection of Mutations in pncA in Mycobacterium tuberculosis Clinical Isolates from Nepal in Association with Pyrazinamide Resistance.

Without the proper information on pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis (MTB), PZA is inappropriately recommended for the treatment of both susceptible and multidrug-resistant tuberculosis (MDR-TB) in Nepal. This study aimed to collect information regarding PZA susceptibility in MTB isolates from Nepal by analyzing pncA and its upstream regulatory region (URR). A total of 211 MTB isolates were included in this study. Sequence analysis of pncA and its URR was performed to assess PZA resistance. First-line drug susceptibility testing, spoligotyping, and sequence analysis of rpoB, katG, the inhA regulatory region, gyrA, gyrB, and rrs were performed to assess their association with pncA mutation. Sequencing results reveal that 125 (59.2%) isolates harbored alterations in pncA and its URR. A total of 57 different mutation types (46 reported and 11 novel) were scattered throughout the whole length of the pncA gene. Eighty-seven isolates (41.2%) harbored mutations in pncA, causing PZA resistance in MTB. There was a more significant association of pncA alterations in MDR/pre-extensively drug-resistant (Pre-XDR) TB than in mono-resistant/pan-susceptible TB (p < 0.005). This first report on the increasing level of PZA resistance in DR-TB in Nepal highlights the importance of PZA susceptibility testing before DR-TB treatment.

Characterization of embB mutations involved in ethambutol resistance in multi-drug resistant Mycobacterium tuberculosis isolates in Zambia.

BACKGROUND: Ethambutol (EMB) is an important anti-tuberculosis drug used in the management of multi-drug resistant tuberculosis (MDR-TB). Mutations in embB are the major mechanism of resistance. This study investigated embB mutations among MDR-TB isolates and analyzed their correlations with phenotypic drug susceptibility testing (DST) in Zambia. METHOD: A total of 132 MDR-TB isolates were collected from January 2014 to April 2017 and characterized using MGIT 960 systems, embB sequencing, and spoligotyping. RESULTS: Out of 61 phenotypically EMB resistant isolates, 53 had mutations in embB. Among the 71 EMB susceptible isolates, 47 had embB mutations. Sensitivity of embB mutations was 86.9% while specificity was 33.8%. CAS1_Kili (SIT21) had high odds of having embB mutations, particularly, G918A (Met306eIl) (Odds ratio 16.7, p < 0.0001). CONCLUSION: Molecular EMB resistance testing by DNA sequencing can improve detection of EMB resistance among MDR-TB patients in Zambia. Additionally, CAS1_Kili was associated with embB amino acid substitution Met306Ile suggesting transmission. A detailed investigation to track and determine transmission hotspot area for MDR-TB could help optimize control strategies.

Genetic Diversity and Transmission of Multidrug-Resistant Mycobacterium tuberculosis strains in Lusaka, Zambia.

OBJECTIVE: Zambia is among the 30 high tuberculosis burden countries in the world. Despite increasing reports of multidrug-resistant tuberculosis (MDR-TB) in routine surveillance, information on the transmission of MDR Mycobacterium tuberculosis strains is largely unknown. This study elucidated the genetic diversity and transmission of MDR M. tuberculosis strains in Lusaka, Zambia. METHODS: Eighty-five MDR M. tuberculosis samples collected from 2013 to 2017 at the University Teaching Hospital were used. Drug-resistance associated gene sequencing, spoligotyping, 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), and multiplex PCR for RD-Rio sub-lineage identification were applied. RESULTS: The identified clades were LAM (48%), CAS (29%), T (14%), X (6%) and Harlem (2%). Strains belonging to SITs 21/CAS1-Kili and 20/LAM1 formed the largest clonal complexes. Combined spoligotyping and 24 loci-MIRU-VNTR revealed 47 genotypic patterns with a clustering rate of 63%. Ninety-five percent of LAM strains belonged to the RD-Rio sub-lineage. CONCLUSION: The high clustering rate suggested that a large proportion of MDR-TB was due to recent transmission rather than the independent acquisition of MDR. This spread was attributed to clonal expansion of SIT21/CAS1-Kili and SIT20/LAM1 strains. Therefore, TB control programs recommending genotyping coupled with conventional epidemiological methods can guide measures for stopping the spread of MDR-TB.

Characterization of Mutations Associated with Streptomycin Resistance in Multidrug-Resistant Mycobacterium tuberculosis in Zambia.

Streptomycin (STR) is recommended for the management of multidrug-resistant tuberculosis (MDR-TB). Streptomycin resistance-conferring mutation types and frequency are shown to be influenced by genotypes of circulating strains in a population. This study aimed to characterize the mutations in MDR-TB isolates and examine their relationship with the genotypes in Zambia. A total of 138 MDR-TB isolates stored at the University Teaching Hospital Tuberculosis Reference Laboratory in Zambia were analyzed using spoligotyping and sequencing of STR resistance-associated genes. Streptomycin resistance was observed in 65.9% (91/138) of MDR-TB isolates. Mutations in rpsL, rrs, and gidB accounted for 33%, 12.1%, and 49.5%, respectively. Amino acid substitution K43R in rpsL was strongly associated with the CAS1_Kili genotype (p < 0.0001). The combination of three genes could predict 91.2% of STR resistance. Clustering of isolates based on resistance-conferring mutations and spoligotyping was observed. The clustering of isolates suggests that the increase in STR-resistant MDR-TB in Zambia is largely due to the spread of resistant strains from inadequate treatment. Therefore, rapid detection of STR resistance genetically is recommended before its use in MDR-TB treatment in Zambia.

Prevalence and Characterization of Quinolone-Resistance Determinants in Escherichia coli Isolated from Food-Producing Animals and Animal-Derived Food in the Philippines.

Antimicrobial resistance to quinolones, which constitutes a threat to public health, has been increasing worldwide. In this study, we investigated the prevalence of quinolone-resistant determinants in Escherichia coli not susceptible to quinolones and isolated from food-producing animals and food derived from them, in the Philippines. A total of 791 E. coli strains were isolated in 56.4% of 601 beef, chicken, pork, egg, and milk samples, as well as environmental, cloacal, and rectal swab-collected samples from supermarkets, open markets, abattoirs, and poultry, swine, and buffalo farms. Using the disc diffusion method, it was determined that 78.6% and 55.4% of the isolates were resistant to at least one antimicrobial and multiple drugs, respectively. In 141 isolates not susceptible to quinolones, 115 (81.6%) harbored quinolone-resistant determinants and had mutations predominantly in the quinolone-resistance determining regions (QRDRs) of gyrA and parC. Plasmid-mediated, quinolone resistance (PMQR) and Qnr family (qnrA1, qnrB4, and qnrS1) genes were detected in all isolates. Forty-eight sequence types were identified in isolates harboring mutations in QRDR and/or PMQR genes by multilocus sequence typing analysis. Moreover, 26 isolates harboring mutations in QRDR and/or PMQR genes belonged mostly to phylogroup B1 and Enteroaggregative E. coli. In conclusion, a high prevalence of E. coli was found in food-producing animals and products derived from them, which could potentially spread high-risk clones harboring quinolone-resistance determinants.

Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis.

Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.

Whole-Genome Sequencing Reveals Recent Transmission of Multidrug-Resistant Mycobacterium tuberculosis CAS1-Kili Strains in Lusaka, Zambia.

Globally, tuberculosis (TB) is a major cause of death due to antimicrobial resistance. Mycobacterium tuberculosis CAS1-Kili strains that belong to lineage 3 (Central Asian Strain, CAS) were previously implicated in the spread of multidrug-resistant (MDR)-TB in Lusaka, Zambia. Thus, we investigated recent transmission of those strains by whole-genome sequencing (WGS) with Illumina MiSeq platform. Twelve MDR CAS1-Kili isolates clustered by traditional methods (MIRU-VNTR and spoligotyping) were used. A total of 92% (11/12) of isolates belonged to a cluster (≤12 SNPs) while 50% (6/12) were involved in recent transmission events, as they differed by ≤5 SNPs. All the isolates had KatG Ser315Thr (isoniazid resistance), EmbB Met306 substitutions (ethambutol resistance) and several kinds of rpoB mutations (rifampicin resistance). WGS also revealed compensatory mutations including a novel deletion in embA regulatory region (-35A > del). Several strains shared the same combinations of drug-resistance-associated mutations indicating transmission of MDR strains. Zambian strains belonged to the same clade as Tanzanian, Malawian and European strains, although most of those were pan-drug-susceptible. Hence, complimentary use of WGS to traditional epidemiological methods provides an in-depth insight on transmission and drug resistance patterns which can guide targeted control measures to stop the spread of MDR-TB.

Characterization of Mycobacterium tuberculosis genotypes and their correlation to multidrug resistance in Lusaka, Zambia.

OBJECTIVES: The burden of multidrug-resistant tuberculosis (MDR-TB) has been reported to be increasing in Zambia. The reasons for the increase are still unclear. This study determined the diversity of Mycobacterium tuberculosis genotypes among isolates in Lusaka, the capital city, and investigated their association with MDR-TB. METHODS: Spoligotyping, large sequence polymorphism (LSP) analysis, and sequencing of MDR associated genes were performed on a total of 274 M. tuberculosis clinical isolates stored at the University Teaching Hospital from 2013 to 2017. Of these, 134 were MDR-TB while 126 were pan-susceptible. RESULTS: Spoligotyping showed the LAM family as the most predominant genotype (149/274, 54.4%) followed by the CAS family (44/274, 16.1%), T family (39/274, 14.2%), and minor proportions of X, S, Harleem, EAI and Beijing spoligofamilies were identified. Three M. bovis isolates were also observed. Among those, CAS1-Kili (SIT 21) and LAM1 (SIT 20) subfamilies showed a propensity for MDR-TB with p = 0.0001 and p = 0.001, respectively. CONCLUSIONS: This phenomenon might explain the future increase in the MDR-TB burden caused by specific lineages in Zambia. Therefore, it is recommended that the National TB control program in the country complements conventional control strategies with molecular analysis for monitoring and surveillance of MDR-TB epidemiology.

Mutations in rpoB and katG genes and the inhA operon in multidrug-resistant Mycobacterium tuberculosis isolates from Zambia.

OBJECTIVES: It is established that resistance to rifampicin (RIF) in 90% of RIF-resistant Mycobacterium tuberculosis isolates is attributable to point mutations in the rpoB gene, whilst 50-95% of M. tuberculosis resistance to isoniazid (INH) is caused by mutations in the katG gene. However, the patterns and frequencies of mutations vary by geographical region. In Zambia, the genetic mechanisms of resistance of M. tuberculosis to RIF and INH were unreported before this study. METHODS: Using gene sequencing, the rpoB, katG and inhA genes of 99 multidrug-resistant M. tuberculosis (MDR-TB) and 49 pan-susceptible M. tuberculosis isolates stored at a tuberculosis reference laboratory from 2013 to 2016 were analysed and were compared with published profiles from other African countries. RESULTS: Of the 99 MDR-TB isolates, 95 (96.0%) carried mutations in both rpoB and katG. No mutations were detected among the pan-susceptible isolates. The most common mutations among RIF- and INH-resistant isolates were in codon 531 of the rpoB gene (55.6%; 55/99) and codon 315 of the katG gene (94.9%; 94/99), respectively. Distinctly, katG mutations were predominantly high among Zambian isolates (96.0%) compared with other countries in the region. CONCLUSION: Resistance-associated mutations to RIF and INH circulating in Zambia are similar to those reported globally, therefore these data validate the applicability of molecular diagnostic tools in Zambia. However, katG mutations were predominantly high among M. tuberculosis isolates in this study compared with other regional countries and might distinguish cross-boundary transmission of MDR-TB from other African nations.

Genotypic characterization of pyrazinamide resistance in Mycobacterium tuberculosis isolated from Lusaka, Zambia.

Pyrazinamide forms a core part of treatment for all types of tuberculosis (TB) in Zambia. Due to challenges associated with pyrazinamide testing, little information is available to indicate the frequency of resistance to this drug in Zambia. To determine the frequency of pyrazinamide (PZA) resistance and its correlation with mutation in pncA in Mycobacterium tuberculosis isolated from patients in Lusaka, Zambia, BACTEC MGIT M960 was used for phenotypic PZA susceptibility testing while sequencing was used to determine resistance-conferring mutations in the pncA. Of the 131 isolates analyzed, 32 were phenotypically resistant to PZA. Among multidrug-resistant (MDR) M. tuberculosis isolates, the frequency of PZA resistance was 21 of 35 (58.3%). And 27 of 32 PZA resistant isolates had mutations in the pncA that seem to confer resistance. With BACTEC MGIT 960 as the reference standard, gene sequencing showed 84.4% sensitivity and 100% specificity. Nine new mutations were identified and the single nucleotide substitution T104G and C195T were the most frequent mutations. However, they were observed in both susceptible and resistant strains and indicating that they are non-resistance conferring mutations. This study has demonstrated that PZA susceptibility testing is necessary especially in patients suffering from MDR-TB as approximately half of the patients have PZA resistant TB. Similar studies will have to be carried out in other provinces to get an accurate estimate of PZA resistance in Zambia. Mutations in pncA were the major mechanism of PZA resistance with no involvement of rpsA and panD genes. However, the presence of mutations among phenotypically PZA susceptible M. tuberculosis isolates makes it challenging to independently use genotyping method for the determination of PZA resistance.

Evaluation of in-house loop-mediated isothermal amplification for tuberculosis diagnosis compared with Xpert MTB/RIF.

INTRODUCTION: To evaluate the diagnostic performances of an in-house loop-mediated isothermal amplification (LAMP) kit and the Xpert MTB/RIF test for the diagnosis of pulmonary tuberculosis in a resource-limited setting, this study was performed at the University Teaching Hospital, Ministry of Health, the Republic of Zambia. METHODOLOGY: Two hundred sputum specimens obtained from new tuberculosis (TB) suspects were used for the evaluation of the diagnostic performance of an in-house LAMP kit in comparison with the Xpert MTB/RIF kit. RESULTS: The sensitivity of in-house LAMP and Xpert MTB/RIF was 96.9% and 95.4% in smear-positive samples, 96.8% and 100% in smear-positive/culture-positive samples, and 39.1% and 73.9% in smear-negative/culture-positive samples, respectively. The specificity of in-house LAMP and MTB/RIF kits with culture was 96.5% and 94.5%, respectively. This indicated the superiority of the Xpert MTB/RIF kit; however, mechanical errors during sample processing and the insufficient quantity of samples by Xpert MTB/RIF kit occurred at 2.0% and 19.7%, respectively, comparing to the 100% accessibility of in-house LAMP. CONCLUSIONS: Considering the results obtained in this study together with the easy setup with much simpler equipment, such as an aluminum heat block or water bath, in in-house LAMP compared with real-time polymerase chain reaction equipment in Xpert MTB/RIF kit, the applicability of in-house LAMP for the screening of tuberculosis directly from sputum in resource-limited setting seemed to be high.