Knowledge, awareness and practices of healthcare workers regarding antimicrobial use, resistance and stewardship in Zambia: a multi-facility cross-sectional study.

Background: Antimicrobial resistance (AMR) poses a threat to public health globally. Despite its consequences, there is little information about the knowledge, awareness, and practices towards AMR among healthcare workers (HCWs). Therefore, this study assessed the knowledge, awareness and practices regarding antimicrobial use (AMU), AMR and antimicrobial stewardship (AMS) among HCWs who are involved in the implementation of AMS activities across eight hospitals in Zambia. Methods: A cross-sectional study was conducted among 64 HCWs from October to December 2023 using a semi-structured questionnaire. Data were analysed using IBM SPSS version 25.0. Results: Of the 64 HCWs, 59.4% were females, 60.9% were aged between 25 and 34 years, 37.5% were nurses, 18.7% were pharmacists, 17.2% were medical doctors and only one was a microbiologist. Overall, 75% of the HCWs had good knowledge, 84% were highly aware and 84% had good practices regarding AMU, AMR and AMS. Most of the HCWs (90.6%) responded that they had a multidisciplinary AMS team at their hospitals and were implementing the use of the WHO AWaRe classification of antibiotics. Conclusion: This study found good knowledge levels, high awareness and good practices regarding AMU, AMR and AMS among HCWs who were involved in the implementation of AMS activities in hospitals in Zambia. Additionally, most hospitals have been conducting AMS training and implementing the use of the WHO AWaRe classification of antibiotics. However, there is still a need to address some identified gaps in AMU and AMR through the strengthening of AMS activities in hospitals.

Trends, patterns and relationship of antimicrobial use and resistance in bacterial isolates tested between 2015-2020 in a national referral hospital of Zambia.

Increased antimicrobial resistance (AMR) among bacteria underscores the need to strengthen AMR surveillance and promote data-based prescribing. To evaluate trends and associations between antimicrobial usage (AMU) and AMR, we explored a dataset of 34,672 bacterial isolates collected between 2015 and 2020 from clinical samples at the University Teaching Hospital (UTH) in Lusaka, Zambia. The most frequently isolated species were Escherichia coli (4,986/34,672; 14.4%), Staphylococcus aureus (3,941/34,672; 11.4%), and Klebsiella pneumoniae (3,796/34,672; 10.9%). Of the 16 drugs (eight classes) tested, only amikacin and imipenem showed good (> 50%) antimicrobial activity against both E. coli and K. pneumoniae, while nitrofurantoin was effective only in E. coli. Furthermore, 38.8% (1,934/4,980) of E. coli and 52.4% (2,079/3,791) of K. pneumoniae isolates displayed multidrug resistance (MDR) patterns on antimicrobial susceptibility tests. Among S. aureus isolates, 44.6% (973/2,181) were classified as methicillin-resistant (MRSA). Notably, all the MRSA exhibited MDR patterns. The annual hospital AMR rates varied over time, while there was a weak positive relationship (r = 0.38, 95% CI = 0.11-0.60) between the monthly use of third-generation cephalosporins (3GCs) and 3GC resistance among Enterobacterales. Overall, the results revealed high AMR rates that fluctuated over time, with a weak positive relationship between 3GC use and resistance. To our knowledge, this is the first report to evaluate the association between AMU and AMR in Zambia. Our results highlight the need to strengthen antimicrobial stewardship programs and optimize AMU in hospital settings.

Antibiotic prescribing patterns and carriage of antibiotic-resistant Escherichia coli and Enterococcus species in healthy individuals from selected communities in Lusaka and Ndola districts, Zambia.

Objectives: This study assessed antibiotic prescribing patterns in primary healthcare facilities and antimicrobial resistance (AMR) profiles of commensal Escherichia coli and enterococci isolated from pregnant women and children under 5 years of age. Materials and methods: This cross-sectional study was conducted in Lusaka and Ndola districts of Zambia. Prescription pattern data were obtained from hospital pharmacies. Identification and antimicrobial susceptibility profiles of E. coli and enterococci were determined by conventional methods, while confirmation of both pathogens and AMR genes were determined by PCR. Data were analysed using WHONET and SPSS version 25.0. Results: Most prescribed antibiotics at the primary healthcare facilities belonged to the Access group of the WHO Access, Watch and Reserve (AWaRe) classification. All the primary healthcare facilities adhered to the AWaRe framework of ≥60% prescribed antibiotics belonging to the Access group. However, resistance was highest in the Access group of antibiotics. E. coli resistance to ampicillin ranged from 71% to 77% and to co-trimoxazole from 74% to 80%, while enterococcal resistance to tetracycline was 59%-64%. MDR was highest in E. coli (75%) isolates, while XDR was highest in enterococcal isolates (97%). The identified AMR genes in E. coli included blaCTX-M, sul2 and qnrA, while those of enterococci included erm(B), erm(C) and erm(A). Conclusions: Resistance was highest in the prescribed WHO Access group of antibiotics. These findings highlight the need to use local susceptibility data to formulate country-specific treatment guidelines in line with WHO AWaRe classification and enforce regulations that prohibit easy access to antibiotics.

Antifungal resistance and stewardship: a knowledge, attitudes and practices survey among pharmacy students at the University of Zambia; findings and implications.

Introduction: Antifungal resistance (AFR) is a growing global public health concern. Little is currently known about knowledge, attitudes and practices regarding AFR and antifungal stewardship (AFS) in Zambia, and across the globe. To address this evidence gap, we conducted a study through a questionnaire design starting with pharmacy students as they include the next generation of healthcare professionals. Methods: A cross-sectional study among 412 pharmacy students from June 2023 to July 2023 using a structured questionnaire. Multivariable analysis was used to determine key factors of influence. Results: Of the 412 participants, 55.8% were female, with 81.6% aged between 18 and 25 years. Most students had good knowledge (85.9%) and positive attitudes (86.7%) but sub-optimal practices (65.8%) towards AFR and AFS. Overall, 30.2% of students accessed antifungals without a prescription. Male students were less likely to report a good knowledge of AFR (adjusted OR, AOR = 0.55, 95% CI: 0.31-0.98). Similarly, students residing in urban areas were less likely to report a positive attitude (AOR = 0.35, 95% CI: 0.13-0.91). Fourth-year students were also less likely to report good practices compared with second-year students (AOR = 0.48, 95% CI: 0.27-0.85). Conclusions: Good knowledge and positive attitudes must translate into good practices toward AFR and AFS going forward. Consequently, there is a need to provide educational interventions where students have low scores regarding AFR and AFS. In addition, there is a need to implement strategies to reduce inappropriate dispensing of antifungals, especially without a prescription, to reduce AFR in Zambia.

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.

Knowledge, attitude, and practices toward infection prevention and control among undergraduate pharmacy students in Zambia: findings and implications.

Background: Infection prevention and control (IPC) measures are critical in preventing the risk of acquiring and transmitting nosocomial infections. In Zambia, there is little information concerning IPC practices among pharmacy students who are exposed to potentially infectious areas both in public and private healthcare settings. Therefore, this study assessed the knowledge, attitude, and practices of undergraduate pharmacy students toward infection prevention and control at the University of Zambia. Materials and methods: This cross-sectional study was conducted among 290 undergraduate pharmacy students at the University of Zambia using a structured questionnaire from August 2022 to October 2022. Data analysis was performed using SPSS version 25.0, and statistical significance was set at a 95% confidence level. Results: Of the 290 participants, 166(57.2%) were female and the majority were aged between 18 and 23 years. Overall, 252(86.9%) had good knowledge, 167(57.6%) had positive attitudes, and 248(85.5%) had good practices toward IPC measures. These results indicate lower attitude scores compared to knowledge and practices. Having good knowledge of IPC was associated with being a Christian by religion compared to other religions (OR = 5.314, 95% CI: 1.141-24.745). There was no association between sociodemographics and attitude and practice concerning IPC. Conclusion: This study found that pharmacy students had good knowledge, positive attitudes, and good practices toward IPC measures. Consequently, more emphasis is needed to improve the student's knowledge, attitudes, and practices toward IPC, especially in areas where gaps were identified. Additionally, there is a need to improve curricula on IPC measures in the training of pharmacy programs.

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.

Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.

Direct detection of Mycobacterium bovis by a dry loop-mediated isothermal amplification assay in cattle samples collected during routine abattoir examination in Malawi.

The lack of quick, accurate, and low-cost detection methods has hindered the active control strategies for bovine tuberculosis (bTB) in resource-limited countries with a high burden of disease. We developed a dry loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Mycobacterium bovis, the principal causative agent of bTB, and evaluated the efficacy of the assay using suspected bTB samples collected during routine meat inspection at major regional abattoirs in Malawi. Template genomic DNA was extracted directly from the granulomatous bTB-like lesion (crude extracted DNA), as well as growth from the incubated mycobacterial growth indicator tubes (MGIT). Field results were visualized by the naked eye within 40 min following a color change of the amplified products. The sensitivity and specificity of the dry LAMP assay while using 152 DNA samples extracted from MGIT with confirmed M. bovis results were 98% and 88%, respectively. When 43 randomly selected crude DNA samples from lesions were used, the sensitivity and specificity of the dry LAMP assay were 100% and 75%, respectively. Our LAMP assay offers the potential to meet the demands for a low-cost and rapid field detection tool for bTB in resource-limited countries in which bTB is endemic.

Prevalence and risk factors of bovine tuberculosis in slaughtered cattle, Malawi.

Bovine tuberculosis (bTB) is an infectious disease with significant socioeconomic, animal, and public health impacts. However, the prevalence of bTB remains largely unclear in Malawi due to a paucity of information. Additionally, the existence of multiple risk factors is postulated to enhance bTB transmission in animals. A cross-sectional survey to estimate the prevalence of bTB, animal characteristics and identify associated risk factors was conducted from slaughtered cattle at three major regional abattoirs (southern, central and northern regions) in Malawi. Out of a total of 1547 cattle examined, 154 (9.95%) had bTB-like lesions in various visceral organs and lymph nodes; one sample per animal was collected, processed, and cultured in the in the BACTEC Mycobacterial growth indicator tube (MGIT) 960 system. From the 154 cattle that showed tuberculous like lesions, only 112 were positive on MGIT and 87 were confirmed to have M. bovis based on multiplex PCR. Cattle from the southern region (odds ratio (OR) = 1.96, 95% CI: 1.03-3.85) and central region (OR = 2.00, 95% CI: 1.16-3.56) were more likely presented with bTB-like lesions at slaughter than from the northern region. The risk of having bTB-like lesions was higher in females (OR = 1.51, CI: 1.00-2.29), older cattle (OR = 2.17, CI: 1.34-3.37), and crossbreeds (OR = 1.67, 95% CI: 1.12-2.47) than in males, younger animals, and Malawi Zebu breed, respectively. The high prevalence of bTB is of critical concern and necessitates active surveillance and strengthening of the current control strategies under a One Health (OH) approach at the animal-human interface.

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.

An unusually long Rift valley fever inter-epizootic period in Zambia: Evidence for enzootic virus circulation and risk for disease outbreak.

Rift valley fever (RVF) is a mosquito-borne disease of animals and humans. Although RVF outbreaks are usually reported at 5-15-year intervals in sub-Saharan Africa, Zambia has experienced an unusually long inter-epizootic/-epidemic period of more than three decades. However, serological evidence of RVF virus (RVFV) infection in domestic ruminants during this period underscores the need for comprehensive investigation of the mechanisms of virus perpetuation and disease emergence. Mosquitoes (n = 16,778) captured from eight of the ten provinces of Zambia between April 2014 and May 2019 were pooled (n = 961) and screened for RVFV genome by a pan-phlebo RT-PCR assay. Aedes mosquito pools (n = 85) were further screened by nested RT-PCR assay. Sera from sheep (n = 13), goats (n = 259) and wild ungulates (n = 285) were screened for RVFV antibodies by ELISA while genome detection in pooled sera (n = 276) from domestic (n = 248) and wild ungulates (n = 37) was performed by real-time RT-PCR assay. To examine the association between the long inter-epizootic period and climatic variables, we examined El Niño-Southern Oscillation indices, precipitation anomalies, and normalized difference vegetation index. We then derived RVF risk maps by exploring climatic variables that would favor emergence of primary RVFV vectors. While no RVFV genome could be detected in pooled mosquito and serum samples, seroprevalence was significantly high (OR = 8.13, 95% CI [4.63-14.25]) in wild ungulates (33.7%; 96/285) compared to domestic ruminants (5.6%; 16/272). Retrospective analysis of RVF epizootics in Zambia showed a positive correlation between anomalous precipitation (La Niña) and disease emergence. On risk mapping, whilst northern and eastern parts of the country were at high risk, domestic ruminant population density was low (< 21 animals/km2) in these areas compared to low risk areas (>21 animals/km2). Besides evidence of silent circulation of RVFV and the risk of disease emergence in some areas, wildlife may play a role in the maintenance of RVFV in Zambia.

Autochthonous Leishmania infantum in Dogs, Zambia, 2021.

Leishmaniases are neglected tropical diseases of humans and animals. We detected Leishmania infantum in 3 mixed-breed dogs in Zambia that had no travel history outside the country. Our findings suggest presence of and probable emergence of leishmaniasis in Zambia, indicating the need for physicians and veterinarians to consider the disease during diagnosis.

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.

First report of Mycobacterium bovis in wild chacma baboons (Papio ursinus) at the human-wildlife interface area in Zambia.

Mycobacterium bovis (M. bovis) causes tuberculosis in mammals and is a major public health threat worldwide. While M. bovis has been reported in humans, domestic and wild ruminants at the human-wildlife-livestock interface area in Zambia, there is paucity of information on the role of primates as reservoir hosts. We screened seven wild chacma baboons (Papio ursinus) for tuberculosis at the human-wildlife interface area in Lochinvar National Park in the Kafue Flats, Zambia. Following necropsy, lung tissue and associated lymph nodes with tuberculous-like lesions collected from four adult male baboons were prepared for Mycobacterium culture. The isolates were initially typed using the Mycobacterium tuberculosis complex-discrimination multiplex PCR assay and further characterized by spoligotyping and 26-loci MIRU-VNTR. Mycobacteria were isolated from all four animals and identified as M. bovis by PCR. On Spoligotyping, all isolates belonged to SB 0120 spoligotype, which is similar to what was previously reported in humans, cattle and Kafue lechwe antelopes in Kafue Flats ecosystem. Furthermore, on MIRU-VNTR typing, the baboon isolates clustered with cattle and Kafue lechwe isolates from the same catchment area. This finding intimates probable cross-species transmission of M. bovis in the Kafue Flats ecosystem. Due to the close interaction of baboons and humans at interface areas in Zambia, our results have potential implications for public health. Equally, this finding raises concerns for conservation.

Molecular epidemiology of Mycobacterium bovis in central parts of Malawi.

Bovine tuberculosis (bTB) is a neglected disease that affects cattle and humans. The burden of bTB is higher in developing countries as compared to industrialized countries. The reasons behind this discrepancy include the fact that bTB control measures, such as testing and slaughter of infected cattle and pasteurization of milk, are not usually practised in developing countries largely because of their high cost. To improve our understanding of bTB in developing countries, molecular typing studies are essential, in particular in terms of transmission dynamics, infection sources and knowledge of circulating strains of the principal causative agent, Mycobacterium bovis. In this study, we applied a suite of molecular typing techniques encompassing deletion analysis, spoligotyping and MIRU-VNTR to isolates recovered from samples collected during the routine post-mortem of cattle at the cold storage abattoir in Lilongwe, Malawi. Out of 63 isolates, 51 (81%) belonged to the European 1. M. bovis clonal complex. Spoligotyping identified 8 profiles, with SB0131 being the predominant type (56% of isolates). Spoligotypes SB0273 and SB0425 were identified in 14% and 13%, respectively, of the isolates. MIRU-VNTR showed a high discriminatory power of 0.959 and differentiated the 8 spoligotypes to 31 genotypes. The high diversity of M. bovis within the study area suggests the infection has been circulating in the area for a considerable period of time, likely facilitated by the lack of effective control measures. We also observed genetic similarities between isolates from Malawi (this study) to isolates described in previous studies in Zambia and Mozambique, suggesting transmission links in this region. The information provided by this study provides much needed evidence for the formulation of improved bTB control strategies.

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.

ERRATUM: Rapid and Simple Detection of Isoniazid-Resistant Mycobacterium tuberculosis Utilizing a DNA Chromatography-Based Technique.

Volume 74, no.3, p.214-219, 2021. Page 214, affiliation "1TBA Co., LTD, Sendai; 2Hokkaido University Research Center for Zoonosis Control, Sapporo; 3Hokkaido University, GI-CoRE Global Station for Zoonosis Control, Sapporo; 4Zambia National Public Health Institute, Ministry of Health, Lusaka, Zambia; 5Department of Pathology and Microbiology, University Teaching Hospital Ministry of Health, Lusaka, Zambia; and 6Ministry of Health, Ndeke House, Lusaka, Zambia." should read "1TBA Co., LTD, Sendai, Japan; 2Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan; 3Hokkaido University, GI-CoRE Global Station for Zoonosis Control, Sapporo, Japan; 4Zambia National Public Health Institute, Ministry of Health, Lusaka, Zambia; 5Department of Pathology and Microbiology, University Teaching Hospital Ministry of Health, Lusaka, Zambia; and 6Ministry of Health, Ndeke House, Lusaka, Zambia".

Interaction of Quinolones Carrying New R1 Group with Mycobacterium leprae DNA Gyrase.

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and the treatment of choice is ofloxacin (OFX). Specific amino acid substitutions in DNA gyrase of M. leprae have been reported leading to resistance against the drug. In our previous study, WQ-3810, a fluoroquinolone with a new R1 group (6-amino-3,5-difluoropyridin-2-yl) was shown to have a strong inhibitory activity on OFX-resistant DNA gyrases of M. leprae, and the structural characteristics of its R1 group was predicted to enhance the inhibitory activity. Methodology/Principal Finding: To further understand the contribution of the R1 group, WQ-3334 with the same R1 group as WQ-3810, WQ-4064, and WQ-4065, but with slightly modified R1 group, were assessed on their activities against recombinant DNA gyrase of M. leprae. An in silico study was conducted to understand the molecular interactions between DNA gyrase and WQ compounds. WQ-3334 and WQ-3810 were shown to have greater inhibitory activity against M. leprae DNA gyrase than others. Furthermore, analysis using quinolone-resistant M. leprae DNA gyrases showed that WQ-3334 had greater inhibitory activity than WQ-3810. The R8 group was shown to be a factor for the linkage of the R1 groups with GyrB by an in silico study. Conclusions/Significance: The inhibitory effect of WQ compounds that have a new R1 group against M. leprae DNA gyrase can be enhanced by improving the binding affinity with different R8 group molecules. The information obtained by this work could be applied to design new fluoroquinolones effective for quinolone-resistant M. leprae and other bacterial pathogens.