Research activity information

• Detection of carbapenem resistance among third-generation cephalosporin-resistant Enterobacterales from small-scale poultry farms in peri-urban Lusaka, Zambia
  Situmbeko J. Nasilele; Misheck Shawa; Harvey K. Kamboyi; Bruno S. J. Phiri; Tapiwa Lundu; Mike Nundwe; Angela Lungu; Ladslav Moonga; Shohei Ogata; Masahiro Kajihara; Hirofumi Sawa; Yasuhiko Suzuki; Hideaki Higashi; Ntombi B. Mudenda; Mudenda B. Hang’ombe; Kaampwe Muzandu
  Access Microbiology, 8, 1, Microbiology Society, 01 Jan. 2026
  Scientific journal, Background. Carbapenem and third-generation cephalosporin (3GC) resistance among Gram-negative bacteria poses a serious threat to human and animal health. This study aimed to identify and characterize carbapenem- and 3GC-resistant Enterobacterales isolated from poultry in Lusaka Province, Zambia.
  
  Methods. Ninety pooled cloacal samples were collected from market-ready broiler chickens in the Chongwe and Chilanga districts of Lusaka Province. The isolates were screened for 3GC and carbapenem resistance using the disc diffusion and broth microdilution methods. PCR and Sanger sequencing were performed for species identification and detection of β-lactamase-encoding ( bla ) genes, including bla _CTX-M , bla _TEM , bla _OXA-1 and bla _SHV . Hierarchical clustering was used to assess phenotypic and genotypic relationships.
  
  Results. A total of 83 3GC-resistant Gram-negative isolates were recovered, of which 12% were also carbapenem resistant. Escherichia coli was the most prevalent species, followed by Klebsiella pneumoniae and Enterobacter spp., then Pseudomonas aeruginosa, other Pseudomonas spp., Acinetobacter baumannii , Citrobacter freundii and Aeromonas caviae . Multidrug resistance occurred in 84.3% of the isolates, with the highest resistance to ampicillin, tetracycline and co-trimoxazole. Overall, 80.7% of the isolates harboured at least one of the four tested bla genes, with bla _CTX-M and bla _TEM being the most common. Hierarchical clustering revealed that isolates from both districts shared similar phenotypic and genotypic resistance patterns.
  
  Conclusions. The presence of multidrug- and carbapenem-resistant Enterobacterales from poultry highlights the emergence of carbapenem resistance in Zambia’s food production sector. The detection of imipenem-resistant isolates indicates the potential for transmission of resistance genes between animals and humans. These findings underscore the need for prudent antimicrobial use, strengthened stewardship and a One Health surveillance approach to contain the spread of carbapenem resistance genes.
• Diagnostic Stewardship Trends and Antimicrobial Resistance Profiles of Bacteria Isolated in Zambia: A Five-Year Retrospective Study (2020-2024).
  Joseph Yamweka Chizimu; Aubrey Chichonyi Kalungia; Steward Mudenda; Zoran Muhimba; Chileshe Lukwesa; Webrod Mufwambi; Amon Siame; Kelvin Mwangilwa; Priscilla Gardner; Jimmy Hangoma; Victor Daka; Chikwanda Chileshe; Ntombi B Mudenda; Maisa Kasanga; Misheck Shawa; Herman Chambaro; Duncan Chanda; Taona Sinyawa; Bertha Chibwe; Kaunda Kaunda; Kaunda Yamba; O-Tipo Shikanga; Freddie Masaninga; Mpela Chibi; Sandra Diana Mwadetsa; Doreen Mainza Shempela; Mwendalubi Albert Hadunka; Samson Mukale; Andrew Bambala; Malambo Mutila; Loveness Sakalimbwe; Fred Mulako Simwinji; Geoffrey Mainda; Chie Nakajima; Ruth Nakazwe; Fusya Goma; Nyambe Sinyange; Yasuhiko Suzuki; John Bwalya Muma; Roma Chilengi
  Antibiotics (Basel, Switzerland), 14, 11, 10 Nov. 2025, [International Magazine]
  English, Scientific journal, BACKGROUND/OBJECTIVES: The right diagnostic tests, for the right patient, at the right time, are key to optimising antimicrobial use (AMU) and preventing antimicrobial resistance (AMR). This study evaluated diagnostic stewardship trends and AMR patterns in Zambian surveillance sentinel sites from 2020 to 2024. METHODS: This descriptive, retrospective study analysed routine laboratory data from January 2020 to December 2024 at seven designated AMR surveillance sentinel sites across Zambia. Data on clinical specimens submitted for antimicrobial susceptibility testing were extracted from WHONET and analysed by year, specimen type, and antimicrobial susceptibility profile. RESULTS: A total of 184,788 bacteriology specimens were processed over five years. Urine was the most frequently collected specimen, peaking above 20,000 in 2024. Escherichia coli was the most commonly isolated organism among 15 priority pathogens, comprising 25.9% of the 30,013 isolates. Though a statistically significant increasing trend in total organism isolations was observed annually, only Shigella sp. demonstrated a substantial increase in non-susceptibility to azithromycin (p = 0.027). High resistance was observed with doxycycline, azithromycin, clindamycin, trimethoprim/sulfamethoxazole, ciprofloxacin, and rifampicin, exhibiting resistance ranging from 50% to 80%. Critical AMR alerts included 65% for Vancomycin-Resistant Enterococcus (VRE), 72% for linezolid, and 44% for carbapenems, and possible ESBL-producing Enterobacterales showing the highest overall resistance at 35%, across sentinel sites. CONCLUSIONS: Zambia faces a concerning and significant increase in AMR, with high resistance prevalence across commonly used antibiotics. Critical resistance alerts for VRE, linezolid, carbapenems, and possible ESBL-producing Enterobacterales underscore an urgent need for robust antimicrobial stewardship and continuous diagnostic surveillance.
• Impact of Multidisciplinary-Led Implementation of Antimicrobial Stewardship Programs in Zambia: Findings and Implications.
  Joseph Yamweka Chizimu; Steward Mudenda; Victor Daka; Webrod Mufwambi; Zoran Muhimba; Kaunda Yamba; Misheck Shawa; Kelvin Mwangilwa; Jimmy Hangoma; Sombo Fwoloshi; Amon Siame; Kaunda Kaunda; Andrew Bambala; Kenneth Kapolowe; Priscilla Nkonde Gardner; Duncan Chanda; Shempela Doreen; Charles Chileshe; Paul Simujayang Ombe; Ntombi B Mudenda; Loveness Sakalimbwe; Aubrey C Kalungia; Chikwanda Chileshe; Taona Sinyawa; Maisa Kasanga; Raphael Chanda; Samson Mukale; Shikanga O-Tipo; Evelyn Wesangula; Cephas Sialubanje; Adrian Muwonge; Fred Simwinji; Chie Nakajima; Freddie Masaninga; Fusya Goma; Nyambe Sinyange; Yasuhiko Suzuki; John Bwalya Muma; Roma Chilengi
  Antibiotics (Basel, Switzerland), 14, 11, 07 Nov. 2025, [International Magazine]
  English, Scientific journal, Background/Objectives: Antimicrobial resistance (AMR) is a critical global health threat, with sub-Saharan Africa disproportionately affected. Antimicrobial stewardship (AMS) programs are essential in combating AMR; however, data on their implementation in resource-limited settings like Zambia remain scarce. This study assessed the post-implementation status of World Health Organization (WHO) AMS core elements in selected public hospitals in Zambia. Methods: A cross-sectional pre- and post-intervention survey was conducted in 11 public hospitals across Zambia's 10 provinces. Baseline (pre-implementation) and 12-month follow-up (post-implementation) assessments were carried out using the WHO-adapted Periodic National and Healthcare Facility Assessment Tool. The six AMS core elements evaluated included leadership, accountability, AMS actions, education, monitoring, and feedback. Results: The average AMS program score increased from 59% at pre-implementation to 81% at post-implementation. Significant improvements were observed in education and training (+36%) and accountability (+31%). While leadership and monitoring also showed positive trends, gaps persisted in AMS actions (63%) and feedback/reporting mechanisms (68%). Drug and Therapeutics Committee (DTC) functionality improved by 23%, with 90% of facilities now holding regular DTC meetings. Implementation of AMS actions, such as ward rounds, rose from 0% to 73%. Challenges remained in clinical audit feedback, resource mobilization, and prescribing optimization. Variability across facilities highlighted differences in leadership, resources, and technical capacity. Conclusions: AMS implementation in Zambia improved substantially across key domains. However, sustained leadership, adequate financing, and continuous capacity-building are needed to address persistent gaps and ensure long-term success in mitigating AMR.
• A high prevalence of dogs seropositive to Leishmania in Zambia.
  Herman M Chambaro; Kyoko Hayashida; Lavel C Moonga; Misheck Shawa; Walter Muleya; Joseph Chizimu; David Squarre; Tatsuki Sugi; Junya Yamagishi; Shohei Ogata; Masahiro Kajihara; Hirofumi Sawa; Chizu Sanjoba; Enala T Mwase; Roma Chilengi; Gilbert H Munsaka; Kelvin L Sarenje; Namwiinga R Mulunda; Mable M Mutengo; Boniface Namangala; Yasuyuki Goto
  Parasitology international, 108, 103081, 103081, Oct. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis in humans. Although the disease is widespread in the world, the burden of visceral or any other disease form of leishmaniasis is poorly documented in Zambia, largely due to lack of surveillance. Recently, three cases of autochthonous canine leishmaniasis (CanL) were reported in Zambia following decades of presumed disease absence. This finding intimated probable disease emergence, raising the need for the identification of infection foci. Thus, in this study, we conducted the first mass serological survey for Leishmania infections in domestic dogs from two densely populated urban areas of Zambia in July 2022. In some of the study sites, seropositivity was up to ∼17 %, suggesting probable presence of Leishmania transmission hot spots. Moreover, on follow-up surveys of seropositive dogs, presence of antileishmanial antibodies was a risk factor for dog survival (relative risk = 7.9; odds ratio = 42.5). Our findings implies that Leishmania infection can be a health issue in domestic dogs in Zambia. Considering the risk of zoonotic transmission, the need for improved disease diagnosis and surveillance in both humans, dogs and sand fly vectors is highlighted in Zambia.
• In silico characterization of chromosomally integrated blaCTX-M genes among clinical Enterobacteriaceae in Africa: insights from whole-genome analysis
  Misheck Shawa; Herman Chambaro; Harvey K. Kamboyi; Clement Sulwe; Joseph Y. Chizimu; Situmbeko J. Nasilele; Shohei Ogata; Mulemba Samutela; Tuvshinzaya Zorigt; Steward Mudenda; Manyando Simbotwe; Mwamba Nsofwa; Jedidiah Chanda; Freeman Chabala; Mike Nundwe; Joseph Ndebe; Msangwa Sinjani; Kyoko Hayashida; Naganori Nao; Roma Chilengi; Hirofumi Sawa; Yasuhiko Suzuki; Bernard Hang'ombe; Masahiro Kajihara; Hideaki Higashi
  Frontiers in Microbiology, 16, Frontiers Media SA, 12 Sep. 2025, [Peer-reviewed], [Lead author, Corresponding author], ［Internationally co-authored］, [International Magazine]
  Scientific journal, Antimicrobial resistance (AMR) mediated by extended-spectrum β-lactamases (ESBLs) is a growing global concern, particularly among Enterobacteriaceae. The CTX-M-type ESBLs, encoded by the bla_CTX-M gene, are of significant public health importance due to their high prevalence and broad geographic distribution. Typically located on plasmids and often co-occurring with other AMR genes, bla_CTX-M contributes to multidrug resistance (MDR). However, increasing evidence suggests secondary chromosomal integration of bla_CTX-M, sometimes alongside other resistance determinants. The extent and implications of this mechanism remain poorly characterized, especially in Africa, where genomic surveillance is limited. In this study, we retrieved 295 chromosomal sequences of Enterobacteriaceae of African origin from the GenBank and performed in silico predictions of bla_CTX-M and other AMR genes. bla_CTX-M-carrying sequences were further characterized by in silico multilocus sequence typing and genome annotation. Chromosomal insertions were identified through alignment with reference genomes. Overall, 47 of 295 sequences (15.9%) harbored the bla_CTX-M gene, with the highest prevalence in Klebsiella pneumoniae (29/157, 18.5%), followed by Escherichia coli (13/72, 18.1%), Enterobacter spp. (4/38, 10.5%), and Shigella spp. (1/12, 8.3%). The most common allele was bla_CTX-M-15 (31/47, 66.0%), followed by bla_CTX-M-14 (12/47, 25.5%), bla_CTX-M-55 (3/47, 6.4%), and bla_CTX-M-27 (1/27, 3.7%). Co-occurrence of bla_CTX-M with additional AMR genes was frequently observed, with integration events often associated with mobile genetic elements such as ISEcp1 and IS26. Notably, strains from the same hospital setting were phylogenetically related and shared sequence types and AMR gene profiles, suggesting local clonal dissemination. These findings reveal a notable presence of chromosomally integrated bla_CTX-M among African Enterobacteriaceae, frequently in association with other resistance genes, thereby facilitating stable MDR propagation independent of plasmid maintenance. This evolutionary adaptation may have significant implications for the persistence and spread of MDR in clinical settings.
• Bacteriological quality of fresh and processed black soldier fly Hermetia illucens larvae reared on chicken manure in Kitwe, Zambia.
  Prudence Mapiki; Ester Laiser; Joyce Mufungwe; Misheck Shawa; Mazuba Siamujompa; Todd Johnson; Ngawo Namukonde; Phenny Mwaanga; Bernard Mudenda Hang'ombe
  Microbiology spectrum, 13, 8, e0057024, 05 Aug. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, UNLABELLED: Black soldier fly larvae (BSFL) have high nutrient content and are rapidly becoming an alternative protein source for animal feed. However, microbial contamination is a potential risk due to the environment in which they are reared. This study assessed the bacteriological quality of fresh and processed BSFL by comparing the processed BSFL using two traditional methods (oven-dried and sun-dried), on their effectiveness at reducing the bacterial load and further elucidated bacterial composition. PCR was used to identify extended-spectrum β-lactamase (ESBL) and mecA genes in Escherichia coli and Staphylococcus spp., respectively. A total of 51 fresh BSFL samples were collected from a commercial poultry farm in Kitwe, Zambia. The results showed various bacterial genera, with a higher diversity among gram-positive isolates. The comparison of the effectiveness of two traditional processing methods for BSFL, sun-drying and oven-drying, showed that both methods significantly reduced the bacterial load, with oven-drying causing a larger reduction. While various genera were identified, we focused on E. coli and Staphylococcus spp. This is because some E. coli harbor ESBLs that hydrolyze β-lactam antibiotics like cephalosporins and penicillin, leading to resistance. Similarly, the genus Staphylococcus was selected since some strains are potentially pathogenic and contain the mecA gene that encodes resistance to β-lactam antibiotics. Molecular characterization of the isolated strains revealed blaCTX-M and blaTEM genes among E. coli, but the mecA gene was not detected among Staphylococcus. This study revealed that BSFL harbor bacteria of zoonotic significance, emphasizing the need for good processing methods to eliminate potential risks. IMPORTANCE: Isolation and identification of Escherichia coli and Staphylococcus spp. in processed black soldier fly larvae (BSFL) samples meant for animal feed indicate insufficient processing methods and pose a public health risk. For instance, some E. coli harbor extended-spectrum β-lactamases (ESBLs) that hydrolyze β-lactam antibiotics like cephalosporins and penicillin, leading to resistance. In addition, some E. coli commensals can transfer antimicrobial resistance genes to pathogenic bacteria through horizontal gene transfer using various mobile genetic elements, leading to resistance. Similarly, for Staphylococcus spp., some strains of the genus Staphylococcus are potentially pathogenic and contain the mecA gene that encodes resistance to β-lactam antibiotics. In this study, we used PCR to screen E. coli isolates for the two commonly reported ESBL genes in Zambia, blaCTX-M and blaTEM, and Sanger sequencing was used to reveal blaCTX-M gene alleles. Our results highlight the importance of using adequate processing methods for BSFL to eliminate potential health risks to animal feed.
• Prevalence and antibiotic resistance profiles of ESKAPE pathogens in the neonatal intensive care unit of the women and newborn hospital in Lusaka, Zambia.
  Sharon Namukonda; Misheck Shawa; Amon Siame; James Mwansa; Gina Mulundu
  Antimicrobial resistance and infection control, 14, 1, 96, 96, 05 Aug. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, BACKGROUND: Bacterial contamination of the Neonatal Intensive Care Unit (NICU) poses a significant risk for cross-transmission, potentially leading to infections in vulnerable neonates. Key pathogens involved in NICU-acquired infections such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. are collectively known as ESKAPE pathogens. They are known for their antibiotic resistance, posing challenges for treatment. This study aimed to investigate the prevalence and antibiotic resistance profiles of ESKAPE pathogens in the NICU at the Women and Newborn Hospital (WNH). METHODS: A total of 344 Samples were collected from different medical equipment, inanimate, animate surfaces and indoor air using standard microbiological methods. Antimicrobial susceptibility testing was then performed using the Kirby-Bauer method. RESULTS: Bacterial contamination rate was 323/344 (93.9%), with 83/323 (25.7%) of samples containing ESKAPE pathogens. Antimicrobial susceptibility varied among ESKAPE pathogens with a total of 75/83 (90%) of the ESKAPE isolates being multi-drug resistant (MDR). Gram-negative isolates exhibited high resistance to β-lactams, carbapenems, and fluoroquinolones, with susceptibility to aminoglycosides, while Gram-positive isolates showed resistance to β-lactams and macrolides but remained largely susceptible to linezolid, clindamycin, and vancomycin. CONCLUSION: There was a high level of contamination with MDR ESKAPE pathogens in the NICU. This highlights the need for improved infection prevention and control measures as well as antimicrobial stewardship to prevent further resistance.
• Occurrence of cefotaxime-resistant and beta-lactamase-producing Enterobacterales in poultry from small-scale farms in the Copperbelt province of Zambia.
  Situmbeko J Nasilele; Misheck Shawa; Harvey K Kamboyi; Bruno S J Phiri; Herman Chambaro; Tapiwa Lundu; Mike Nundwe; Angela Lungu; Ladslav Moonga; Masahiro Kajihara; Hirofumi Sawa; Yasuhiko Suzuki; Hideaki Higashi; Amon Siame; Ntombi B Mudenda; Mudenda B Hang'ombe; Kaampwe Muzandu
  JAC-antimicrobial resistance, 7, 4, dlaf125, Aug. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, BACKGROUND: The inappropriate use of antimicrobials in poultry farming is associated with the emergence of antimicrobial-resistant Enterobacterales. This cross-sectional study aimed to identify various cefotaxime-resistant and β-lactamase-producing Enterobacterales and characterize their antimicrobial resistance profiles. METHODS: Pooled cloacal and meat samples collected from market-ready broiler chickens in Kitwe and Ndola districts of Zambia were screened for cefotaxime-resistant Enterobacterales. The samples were inoculated on MacConkey agar supplemented with 1 mg/L cefotaxime. The cefotaxime-resistant isolates were further subjected to antimicrobial susceptibility tests. Further, the isolated cefotaxime-resistant Enterobacterales were analysed for bla CTX-M, bla TEM, bla OXA-1-like and bla SHV genes using PCR and Sanger sequencing. RESULTS: From a total of 114 pooled samples, 81 (71.1%) cefotaxime-resistant Gram-negative strains were isolated. These were dominated by Escherichia coli (77.8%) followed by Klebsiella pneumoniae (6.2%), Pseudomonas spp. (6.2%), Acinetobacter baumannii (4.9%), Pseudomonas aeruginosa (2.5%), Enterobacter spp. (1.2%) and Comamonas aquatica (1.2%). Furthermore, 64.2% of the 81 isolates exhibited multidrug resistance with high resistance (>64%) to ampicillin, co-trimoxazole and tetracycline. The results also showed that 66.7% of the isolates harboured at least one of the four tested bla genes (bla CTX-M, bla TEM, bla OXA-1-like and bla SHV), with the commonest being bla CTX-M (58%) and bla TEM (45.7%). CONCLUSIONS: The study revealed a high prevalence of cefotaxime-resistant Enterobacterales and multidrug resistance involving medically important antibiotics. Four bla genes (bla CTX-M, bla TEM, bla OXA-1-like and bla SHV) were identified. Our results highlight the need to strengthen antimicrobial stewardship programmes and optimize antimicrobial use in poultry farming.
• Development of a PCR-dipstick DNA chromatography-based tool for the detection of CTX-M- and TEM-producing Escherichia coli and Klebsiella pneumoniae isolated from patients in Kafue and Katete districts of Zambia.
  Misheck Shawa; Kyoko Hayashida; Naganori Nao; Atmika Paudel; Harvey Kamboyi; Herman Chambaro; Malala Mulavu; Cynthia Sipho Khumalo; Mike Nundwe; Tuvshinzaya Zorigt; Chinatsu Nakamura; Yongjin Qiu; Naoko Kawai; Maisa Kasanga; Joseph Chizimu; Lavel Moonga; Joseph Ndebe; Manyando Simbotwe; Shohei Ogata; Mulemba Samutela; Chie Nakajima; Roma Chilengi; Mable Mutengo; Masahiro Kajihara; Hirofumi Sawa; Bernard Hang'ombe; Yasuhiko Suzuki; Hideaki Higashi
  BMC infectious diseases, 25, 1, 541, 541, 16 Apr. 2025, [Peer-reviewed], [Lead author, Corresponding author], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, In Zambia, 40% of clinical Gram-negative bacteria are either Escherichia coli or Klebsiella pneumoniae, with a high third-generation cephalosporin (3GC) resistance prevalence. Therefore, 3GC resistance surveillance is a crucial indicator for guiding focused intervention policies. However, the lack of genotypic diagnostic tools limits the ability to elucidate trends, especially in peri-urban and rural areas of developing countries. This study aimed to develop a rapid, cost-effective tool for the genotypic surveillance of 3GC resistance. Here, 900 stool samples collected from patients in Kafue (peri-urban, n = 400) and Katete (rural, n = 500) districts of Zambia were used for bacterial isolation on MacConkey agar supplemented with 1 μg/ml cefotaxime. Isolated 3GC-resistant strains were characterized by sequencing the 16S rRNA gene and screening for blaCTX-M and blaTEM genes using single polymerase chain reaction (PCR). Furthermore, selected 3GC-resistant strains were subjected to whole-genome sequencing (WGS) using MiSeq/HiSeq (n = 34) and MinION (n = 1). Using the data from this and other previous studies, we developed a rapid PCR-dipstick DNA chromatography-based tool for detecting blaCTX-M, blaTEM, E. coli-specific yaiO, and K. pneumoniae-specific khe genes. The prevalence of isolated 3GC resistant strains was 15.4% (139/900), dominated by E. coli (102/139, 73.4%). On PCR, the blaCTX-M gene was detected in 72.7% (101/139) of the isolates, while blaTEM was found in 46.8% (65/139) of the strains. The developed tool displayed a high level of agreement with WGS and single PCR/Sanger sequencing, with sensitivity and specificity ≥ 95% and Kappa ≥ 0.95 for each of the four target genes. We envisage that the simplicity and adaptability of this tool will be a significant advantage for the surveillance of 3GC resistance in Zambia and elsewhere.
• Antimicrobial Resistance Patterns and Serological Diversity of Shigella Species from Patient Isolates at University Teaching Hospital in Lusaka, Zambia
  Mike Nundwe; Joseph Yamweka Chizimu; John Mwaba; Misheck Shawa; Rodrick S. Katete; Mable Mwale Mutengo; Ruth Nakazwe; Namwiinga R. Mulunda; Cephas Sialubanje; Mox Malama Kalumbi; Yamba Kaunda; Rapheal Chanda; Herman Chambaro; Harvey K. Kamboyi; Thoko Kapalamula; Steward Mudenda; Freeman W. Chabala; Bernard M. Hang’ombe; Roma Chilengi; Chie Nakajima; Yasuhiko Suzuki
  Bacteria, 4, 2, 18, 18, MDPI AG, 02 Apr. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  Scientific journal, Background: Shigella species are the leading cause of human shigellosis. In Zambia, more than 30% of children experiencing diarrhea are infected with Shigella species. The increasing resistance of Shigella species to the recommended therapy is of great concern. Therefore, this study investigated the antibiotic resistance profiles and phenotypic and genotypic characteristics of Shigella isolates at the largest referral hospital in Zambia. Methodology: Of the forty-eight archived presumptive Shigella isolates, thirty-two were serologically confirmed and subjected to antimicrobial susceptibility testing using the Kirby Bauer disk diffusion method. Thereafter, polymerase chain reaction was performed to detect the bla genes. Results: Most isolates were Shigella flexneri (16/32, 50%) and Shigella sonnei (14/32, 44%), while Shigella boydii and Shigella dysenteriae were rare. High resistance rates were noted for sulfamethoxazole/trimethoprim (78%) and tetracycline (75%), while 15.6% of the isolates showed resistance to ciprofloxacin and/or azithromycin. The blaTEM gene encoding beta-lactamase was detected in 7/32 (22%) of isolates. Conclusions: In this study, a significant number of multidrug-resistant isolates were identified. Additionally, Shigella species resistant to the World Health Organization-recommended drugs call for strengthened laboratory diagnosis and close monitoring of these pathogens to guide the clinical management of shigellosis.
• Evaluating infection prevention and control programs in Zambian hospitals using the WHO infection prevention and control assessment framework tool.
  Steward Mudenda; Joseph Yamweka Chizimu; Linus Ndegwa; Maisa Kasanga; Ilunga Mutwale; Aubrey Chichonyi Kalungia; Evelyn Wesangula; Adriano Focus Lubanga; James C L Mwansa; Martha Mwaba; Amos Yared Massele; Nyambe Sinyange; Tapfumanei Mashe; Malambo Mutila; Paul Simujayang'ombe; David Lowrance; Kaunda Yamba; Misheck Shawa; Chie Nakajima; Yasuhiko Suzuki; John Bwalya Muma; Massimo Sartelli; Brian Godman; Roma Chilengi
  Frontiers in public health, 13, 1642119, 1642119, 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, BACKGROUND: Infection Prevention and Control (IPC) is key to preventing healthcare-associated infections (HAIs) and the spread of antimicrobial resistance (AMR). This study evaluated the implementation of IPC in Zambian hospitals. MATERIALS AND METHODS: We conducted a multicentric cross-sectional study in nine hospitals across Zambia using the WHO IPCAF tool. Data were collected from September 1 to 30, 2024 and analyzed using the self-scoring Excel and IBM SPSS version 25.0. RESULTS: Out of the nine hospitals assessed, four were tertiary-level hospitals while the rest were secondary-level hospitals. Overall, the implementation of IPC core components was intermediate (IPCAF Score of 594 out of 800). Four hospitals had IPCAF scores between 401 and 600, indicating an intermediate level of IPC implementation. Five hospitals scored between 601 and 800, indicating an advanced implementation of IPC in these hospitals. Three tertiary hospitals scored between 601 and 800, demonstrating their advanced implementation of IPC core components. CONCLUSION: This study found that the overall implementation of IPC in the surveyed hospitals was intermediate, indicating that further improvements were needed. There is a need to provide peer-learning support and strengthen IPC implementation to respond to new or re-emerging infections and AMR in the country and beyond.
• Antibiotic use and adherence to the WHO AWaRe guidelines across 16 hospitals in Zambia: a point prevalence survey.
  Joseph Yamweka Chizimu; Steward Mudenda; Kaunda Yamba; Chileshe Lukwesa; Raphael Chanda; Ruth Nakazwe; Misheck Shawa; Herman Chambaro; Harvey K Kamboyi; Aubrey Chichonyi Kalungia; Duncan Chanda; Sombo Fwoloshi; Elimas Jere; Tiza Mufune; Derick Munkombwe; Peter Lisulo; Tebuho Mateele; Jeewan Thapa; Kenneth Kapolowe; Nyambe Sinyange; Cephas Sialubanje; Nathan Kapata; Mirfin Mpundu; Freddie Masaninga; Khalid Azam; Chie Nakajima; Makomani Siyanga; Nathan Nsubuga Bakyaita; Evelyn Wesangula; Martin Matu; Yasuhiko Suzuki; Roma Chilengi
  JAC-antimicrobial resistance, 6, 5, dlae170, Oct. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, BACKGROUND: The inappropriate use of antibiotics in hospitals contributes to the development and spread of antimicrobial resistance (AMR). This study evaluated the prevalence of antibiotic use and adherence to the World Health Organization (WHO) Access, Watch and Reserve (AWaRe) classification of antibiotics across 16 hospitals in Zambia. METHODS: A descriptive, cross-sectional study employing the WHO Point Prevalence Survey (PPS) methodology and WHO AWaRe classification of antibiotics was conducted among inpatients across 16 hospitals in December 2023, Zambia. Data analysis was performed using STATA version 17.0. RESULTS: Of the 1296 inpatients surveyed in the 16 hospitals, 56% were female, and 54% were aged between 16 and 50 years. The overall prevalence of antibiotic use was 70%. Additionally, 52% of the inpatients received Watch group antibiotics, with ceftriaxone being the most prescribed antibiotic. Slightly below half (48%) of the inpatients received Access group antibiotics. Compliance with the local treatment guidelines was 53%. CONCLUSIONS: This study found a high prevalence of prescribing and use of antibiotics in hospitalized patients across the surveyed hospitals in Zambia. The high use of Watch group antibiotics was above the recommended threshold indicating non-adherence to the WHO AWaRe guidelines for antibiotic use. Hence, there is a need to establish and strengthen antimicrobial stewardship programmes that promote the rational use of antibiotics in hospitals in Zambia.
• EsxA, a type VII secretion system-dependent effector, reveals a novel function in the sporulation of Bacillus cereus ATCC14579.
  Harvey K Kamboyi; Atmika Paudel; Misheck Shawa; Misa Sugawara; Tuvshinzaya Zorigt; Joseph Y Chizimu; Tomoe Kitao; Yoshikazu Furuta; Bernard M Hang'ombe; Musso Munyeme; Hideaki Higashi
  BMC microbiology, 24, 1, 351, 351, 17 Sep. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, BACKGROUND: Bacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA's roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished. METHODS: We used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar. RESULTS: Through LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3' end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567. CONCLUSION: B. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA.
• Pan-genome analysis reveals novel chromosomal markers for multiplex PCR-based specific detection of Bacillus anthracis.
  Tuvshinzaya Zorigt; Yoshikazu Furuta; Atmika Paudel; Harvey Kakoma Kamboyi; Misheck Shawa; Mungunsar Chuluun; Misa Sugawara; Nyamdorj Enkhtsetseg; Jargalsaikhan Enkhtuya; Badgar Battsetseg; Musso Munyeme; Bernard M Hang'ombe; Hideaki Higashi
  BMC infectious diseases, 24, 1, 942, 942, 09 Sep. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, BACKGROUND: Bacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis. METHODS: The complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes. RESULT: The distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361. CONCLUSION: The study identified thirty chromosome-encoded genes specific to B. anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis.
• Detection of Human Adenovirus and Rotavirus in Wastewater in Lusaka, Zambia: Demonstrating the Utility of Environmental Surveillance for the Community.
  Ngonda Saasa; Ethel M'kandawire; Joseph Ndebe; Mulenga Mwenda; Fred Chimpukutu; Andrew Nalishuwa Mukubesa; Fred Njobvu; Doreen Mainza Shempela; Jay Sikalima; Carol Chiyesu; Bruce Muvwanga; Sarah M Nampokolwe; Clement Sulwe; Thokozile Khondiwa; Todd Jennings; Ameck Kamanga; Edgar Simulundu; Conceptor Mulube; Wizaso Mwasinga; Jalaimo Mumeka; John Simwanza; Patrick Sakubita; Otridah Kapona; Chilufya Susan Aneta Mulenga; Musole Chipoya; Kunda Musonda; Nathan Kapata; Nyambe Sinyange; Muzala Kapina; Joyce Siwila; Misheck Shawa; Masahiro Kajihara; Ayato Takada; Hirofumi Sawa; Simulyamana A Choonga; Roma Chilengi; Earnest Muyunda; King S Nalubamba; Bernard M Hang'ombe
  Pathogens (Basel, Switzerland), 13, 6, 07 Jun. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, Enteric infections due to viral pathogens are a major public health concern. Detecting the risk areas requires a strong surveillance system for pathogenic viruses in sources such as wastewater. Towards building an environmental surveillance system in Zambia, we aimed to identify group A rotavirus (RVA) and human adenovirus (HAdV) in wastewater. Convenient sampling was conducted at four study sites every Tuesday for five consecutive weeks. The research team focused on three different methods of viral concentration to determine the suitability in terms of cost and applicability for a regular surveillance system: the bag-mediated filtration system (BMFS), polyethylene glycol-based (PEG) precipitation, and skimmed milk (SM) flocculation. We screened 20 wastewater samples for HAdV and RVA using quantitative polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR). Of the 20 samples tested using qPCR, 18/20 (90%) tested positive for HAdV and 14/20 (70%) tested positive for RVA. For the genetic sequencing, qPCR positives were subjected to cPCR, of which 12 positives were successfully amplified. The human adenovirus was identified with a nucleotide identity range of 98.48% to 99.53% compared with the reference genome from GenBank. The BMFS and SM flocculation were the most consistent viral concentration methods for HAdV and RVA, respectively. A statistical analysis of the positives showed that viral positivity differed by site (p < 0.001). SM and PEG may be the most appropriate options in resource-limited settings such as Zambia due to the lower costs associated with these concentration methods. The demonstration of HAdV and RVA detection in wastewater suggests the presence of the pathogens in the communities under study and the need to establish a routine wastewater surveillance system for the identification of pathogens.
• Antimicrobial Use Survey and Detection of ESBL-Escherichia coli in Commercial and Medium-/Small-Scale Poultry Farms in Selected Districts of Zambia.
  Taona Sinyawa; Misheck Shawa; Geoffrey M Muuka; Fusya Goma; Paul Fandamu; Joseph Yamweka Chizimu; Cynthia Sipho Khumalo; Malala Mulavu; Masuzyo Ngoma; Herman Moses Chambaro; Harvey Kakoma Kamboyi; Masahiro Kajihara; Hirofumi Sawa; Yasuhiko Suzuki; Hideaki Higashi; Geoffrey Mainda; Musso Munyeme; John Bwalya Muma; Christian Owusu Nyantakyi; Beverly Egyir; Bernard Mudenda Hang'ombe
  Antibiotics (Basel, Switzerland), 13, 5, 20 May 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, Antimicrobial resistance (AMR) among Escherichia coli from food animals is a rising problem, and heavy antimicrobial use in poultry is a contributing factor. In Zambia, studies linking poultry-associated AMR and antibiotic use (AMU) are rare. This study aimed to investigate commercial and medium-/small-scale poultry farmers' usage of antimicrobials based on a questionnaire survey in ten districts of Zambia. In addition, the study characterized extended-spectrum β-lactamase (ESBL)-producing E. coli isolates obtained from poultry in the same districts. Data regarding knowledge and usage of antimicrobials were collected from commercial and medium-/small-scale poultry farmers using a pre-tested structured questionnaire. At the same time, cloacal samples were collected and analyzed. One hundred and fifty E. coli isolates were tested for antimicrobial susceptibility using eight antibiotic classes. The isolates were further screened for ESBL production by streaking them on cefotaxime (CTX)-supplemented MacConkey agar, then subjecting them to sequencing on a NextSeq. The questionnaire survey showed that more medium-/small-scale than commercial poultry farmers used antimicrobials (OR = 7.70, 95% CI = 2.88-20.61) but less prescriptions (OR = 0.02, 95% CI = 0.00-0.08). Susceptibility testing revealed that resistance was highest to ampicillin (128/148, 86.5%) and tetracycline (101/136, 74.3%) and that the prevalence of multidrug resistance (MDR) (28/30, 93.3%) was high. Whole-genome sequencing (WGS) of eight (8/30, 26.7%) isolates with CTX Minimum Inhibitory Concentration (MIC) ≥ 4 µg/mL revealed the presence of ESBL-encoding genes blaCTX-M-14, blaCTX-M-55, and blaTEM. WGS also detected other AMR genes for quinolones, aminoglycosides, phenicols, tetracycline, macrolides, and folate-pathway antagonists. Altogether, the questionnaire survey results showed a higher proportion of AMU and lower prescription usage among medium-/small-scale farmers. In addition, our results emphasize the circulation of ESBL-producing E. coli strains with associated MDR. It is critical to educate farmers about AMR risks and to encourage responsible usage of antimicrobials. Furthermore, there is a need to strengthen regulations limiting access to antimicrobials. Finally, there is a need to establish a one health system to guide public health response.
• Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacteriaceae from Diseased Broiler Chickens in Lusaka District, Zambia.
  Chikwanda Chileshe; Misheck Shawa; Nelson Phiri; Joseph Ndebe; Cynthia Sipho Khumalo; Chie Nakajima; Masahiro Kajihara; Hideaki Higashi; Hirofumi Sawa; Yasuhiko Suzuki; Walter Muleya; Bernard Mudenda Hang'ombe
  Antibiotics (Basel, Switzerland), 13, 3, 15 Mar. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, Poultry products in Zambia form an integral part of the human diet in many households, as they are cheap and easy to produce. The burden of poultry diseases has, however, remained a major challenge. Growing consumer demand for poultry products in Zambia has resulted in non-prudent antimicrobial use on farms, intending to prevent and treat poultry diseases for growth optimisation and maximising profits. This cross-sectional study aimed to identify the different types of bacteria causing diseases in chickens in Lusaka and to detect the extended-spectrum lactamase (ESBL)-encoding genes. We collected 215 samples from 91 diseased chickens at three post-mortem facilities and screened them for Gram-negative bacteria. Of these samples, 103 tested positive for various clinically relevant Enterobacteriaceae, including Enterobacter (43/103, 41.7%), Escherichia coli (20/103, 19.4%), Salmonella (10/103, 9.7%), and Shigella (8/103, 7.8%). Other isolated bacteria included Yersinia, Morganella, Proteus, and Klebsiella, which accounted for 21.4%. E. coli, Enterobacter, Salmonella, and Shigella were subjected to antimicrobial susceptibility testing. The results revealed that E. coli, Enterobacter, and Shigella were highly resistant to tetracycline, ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole, while Salmonella showed complete susceptibility to all tested antibiotics. The observed resistance patterns correlated with antimicrobial usage estimated from sales data from a large-scale wholesale and retail company. Six (6/14, 42.9%) E. coli isolates tested positive for blaCTX-M, whilst eight (8/14, 57.1%) Enterobacter samples tested positive for blaTEM. Interestingly, four (4/6, 66.7%) of the E. coli isolates carrying blaCTX-M-positive strains were also positive for blaTEM. Sanger sequencing of the PCR products revealed that five (5/6, 83.3%) of the abovementioned isolates possessed the blaCTX-M-15 allele. The results suggest the presence of potentially pathogenic ESBL-producing Enterobacteriaceae in poultry, threatening public health.
• Verbal Autopsy and Outbreak Investigation of Anthrax in Livestock and Wildlife at the Virunga National Park Interface Area, Democratic Republic of the Congo
  Selemani M. Désire; Lombe P. Boniface; Syaluha K. Eddy; Kamboyi K. Harvey; Bushu M. Ézéchiel; Banze M. Tatiana; Malengela Léon; Ngumuna Florent; Abdel-Amir D. Halawi; Misheck Shawa; Masumu Justin
  Fortune Journal of Health Sciences, 07, 02, Fortune Journals, 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  Scientific journal
• Trends, patterns and relationship of antimicrobial use and resistance in bacterial isolates tested between 2015-2020 in a national referral hospital of Zambia.
  Misheck Shawa; Atmika Paudel; Herman Chambaro; Harvey Kamboyi; Ruth Nakazwe; Luke Alutuli; Tuvshinzaya Zorigt; Taona Sinyawa; Mulemba Samutela; Joseph Chizimu; Manyando Simbotwe; Kyoko Hayashida; Naganori Nao; Masahiro Kajihara; Yoshikazu Furuta; Yasuhiko Suzuki; Hirofumi Sawa; Bernard Hang'ombe; Hideaki Higashi
  PloS one, 19, 4, e0302053, 2024, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, 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.
• Antimicrobial stewardship situation analysis in selected hospitals in Zambia: findings and implications from a national survey.
  Joseph Yamweka Chizimu; Steward Mudenda; Kaunda Yamba; Chileshe Lukwesa; Raphael Chanda; Ruth Nakazwe; Bwalya Simunyola; Misheck Shawa; Aubrey Chichonyi Kalungia; Duncan Chanda; Uchizi Chola; Tebuho Mateele; Jeewan Thapa; Kenneth Kapolowe; Mazyanga Lucy Mazaba; Mirfin Mpundu; Freddie Masaninga; Khalid Azam; Chie Nakajima; Yasuhiko Suzuki; Nathan Nsubuga Bakyaita; Evelyn Wesangula; Martin Matu; Roma Chilengi
  Frontiers in public health, 12, 1367703, 1367703, 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, BACKGROUND: Antimicrobial stewardship (AMS) programs are critical in combating antimicrobial resistance (AMR). In Zambia, there is little information regarding the capacity of hospitals to establish and implement AMS programs. The objective of this study was to conduct a baseline assessment of WHO core elements for an AMS program implementation in eight hospitals in Zambia. MATERIALS AND METHODS: We conducted an exploratory cross-sectional study from September 2023 to December 2023 using a self-scoring Periodic National and Healthcare Facility Assessment Tool from the World Health Organization (WHO) policy guidance on integrated AMS activities in human health. Eight public hospitals were surveyed across the five provinces of Zambia. Data was analyzed using the WHO self-scoring tool and thematic analysis. RESULTS: Overall, 62.5% (6/8) of the facilities scored low (below 60%) in implementing AMS programs. Most facilities had challenges with reporting AMS feedback within the hospital (average score = 46%), Drugs and Therapeutics Committee (DTC) functionality (average score = 49%), AMS actions (average score = 50%), education and training (average score = 54%), and leadership commitment to AMS activities (average score = 56%). The overall score for all AMS core elements was average (56%). All the hospitals (100%) did not have an allocated budget for AMS programs. Finally, there were neither antibiograms to guide antimicrobial utilization nor AMS-trained staff in more than 50% of the hospitals surveyed. CONCLUSION: This study found low AMS implementation in these public hospitals, especially where DTCs were non-functional. The identified challenges and gaps require urgent attention for sustainable multidisciplinary AMS programs.
• Prevalence and Genomic Characterization of Rotavirus A from Domestic Pigs in Zambia: Evidence for Possible Porcine-Human Interspecies Transmission.
  Joseph Ndebe; Hayato Harima; Herman Moses Chambaro; Michihito Sasaki; Junya Yamagishi; Annie Kalonda; Misheck Shawa; Yongjin Qiu; Masahiro Kajihara; Ayato Takada; Hirofumi Sawa; Ngonda Saasa; Edgar Simulundu
  Pathogens (Basel, Switzerland), 12, 10, 27 Sep. 2023, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, Rotavirus is a major cause of diarrhea globally in animals and young children under 5 years old. Here, molecular detection and genetic characterization of porcine rotavirus in smallholder and commercial pig farms in the Lusaka Province of Zambia were conducted. Screening of 148 stool samples by RT-PCR targeting the VP6 gene revealed a prevalence of 22.9% (34/148). Further testing of VP6-positive samples with VP7-specific primers produced 12 positives, which were then Sanger-sequenced. BLASTn of the VP7 positives showed sequence similarity to porcine and human rotavirus strains with identities ranging from 87.5% to 97.1%. By next-generation sequencing, the full-length genetic constellation of the representative strains RVA/pig-wt/ZMB/LSK0137 and RVA/pig-wt/ZMB/LSK0147 were determined. Genotyping of these strains revealed a known Wa-like genetic backbone, and their genetic constellations were G4-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1 and G9-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1, respectively. Phylogenetic analysis revealed that these two viruses might have their ancestral origin from pigs, though some of their gene segments were related to human strains. The study shows evidence of reassortment and possible interspecies transmission between pigs and humans in Zambia. Therefore, the "One Health" surveillance approach for rotavirus A in animals and humans is recommended to inform the design of effective control measures.
• Multidrug-Resistant Escherichia coli from Raw Cow Milk in Namwala District, Zambia: Public Health Implications.
  Wizaso Mwasinga; Misheck Shawa; Patrick Katemangwe; Herman Chambaro; Prudence Mpundu; Ethel M'kandawire; Chisoni Mumba; Musso Munyeme
  Antibiotics (Basel, Switzerland), 12, 9, 08 Sep. 2023, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, Escherichia coli (E. coli), a major foodborne disease-causing pathogen found in raw cow milk, has even far more reaching public health ramifications as it encodes for antimicrobial resistance (AMR). This study aimed to identify multidrug-resistant (MDR) E. coli from raw cow's milk and evaluate their antimicrobial-resistant profiles. In total, 418 pooled raw cow milk samples were collected from milk collection centers and analysed using standard culture methods to isolate E. coli. Antimicrobial Susceptibility Testing (AST) was conducted using the Kirby Bauer disk diffusion method and PCR was used to identify cefotaxime (CTX) resistant genes. Overall isolation of E. coli was 51.2% (214/418) with MDR observed in 21% (45/214) of isolates across different antibiotic combinations. Resistance was observed towards ampicillin (107/214, 50%), tetracycline (86/214, 40.1%), trimethoprim/sulfamethoxazole (61/214, 28.5%), and amoxicillin/clavulanic acid (CTX) (50/214, 23.4%). Notably, 15% (32/214) resistance to CTX was observed, while 12.6% (27/214) exhibited resistance to imipenem. The blaCTX-M and blaTEM genes were detected in CTX-resistant isolates. The findings of MDR E. coli that harbour blaCTX-M and blaTEM genes in raw cow's milk indicate serious public health risks for consumers.
• Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia.
  Joseph Yamweka Chizimu; Eddie Samuneti Solo; Precious Bwalya; Thoko Flav Kapalamula; Kaemba Kunkuta Mwale; David Squarre; Misheck Shawa; Patrick Lungu; David Atomanyi Barnes; Kaunda Yamba; Tiza Mufune; Herman Chambaro; Harvey Kamboyi; Musso Munyeme; Bernard Mudenda Hang'ombe; Nathan Kapata; Victor Mukonka; Roma Chilengi; Jeewan Thapa; Chie Nakajima; Yasuhiko Suzuki
  Antibiotics (Basel, Switzerland), 12, 7, 29 Jun. 2023, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, 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.
• Prevalence and risk factors of bovine tuberculosis in slaughtered cattle, Malawi.
  Thoko Flav Kapalamula; Francis Kawonga; Misheck Shawa; Joseph Chizimu; Jeewan Thapa; Mirriam Ethel Nyenje; Rajhab Sawasawa Mkakosya; Kyoko Hayashida; Stephen Gordon; Chie Nakajima; Musso Munyeme; Bernard M Hang'ombe; Yasuhiko Suzuki
  Heliyon, 9, 2, e13647, Feb. 2023, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, 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.
• Seroprevalence and Associated Risk Factors of Rift Valley Fever in Livestock from Three Ecological Zones of Malawi.
  Henson Kainga; Marvin Collen Phonera; Elisha Chatanga; Simegnew Adugna Kallu; Prudence Mpundu; Mulemba Samutela; Herman Moses Chambaro; Masahiro Kajihara; Doreen Mainza Shempela; Jay Sikalima; Walter Muleya; Misheck Shawa; Julius Chulu; Gilson Njunga; Martin Simuunza; Ayato Takada; Hirofumi Sawa; Edgar Simulundu; Ngonda Saasa
  Pathogens (Basel, Switzerland), 11, 11, 14 Nov. 2022, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, The epidemiology of Rift Valley fever (RVF) is poorly understood in Malawi. Here, a cross-sectional study was conducted (March-June 2020) to investigate the seroprevalence and potential risk factors of RVF virus (RVFV) in cattle, goats, and sheep in three ecological zones of Malawi. A total of 1523 serum samples were tested for anti-RVFV IgG and IgM antibodies by ELISA. Additionally, a questionnaire survey was used to assess potential RVF risk factors. The overall seroprevalence was 17.14% (261/1523; 95% CI = 15.33-19.11) for individual livestock and 33.24% (120/361; 95% CI = 28.18-38.11) for the livestock herd. Seroprevalence was significantly high in sheep (25.68%, 95% CI = 19.31-33.26) compared with cattle (21.35%, 95% CI = 18.74-24.22) and goats (7.72%, 95% CI = 5.72-10.34), (p = 0.047). At the individual livestock level, the risk was elevated in female livestock (OR: 1.74, 95% CI = 1.08-12.82) (p = 0.016), while at the herd level, areas receiving approximately 1001-1500 mm of rainfall (OR: 2.47, 95% CI = 1.14-5.37) (p = 0.022), areas of rainfall amount greater than approximately 1600 mm (OR: 2.239, 95% CI = 1.07-8.82) (p = 0.023), and mixed species herds (OR: 10.410, 95% CI = 3.04-35.59) (p = 0.001), were significant risk factors. The detection of IgM antibodies confirmed active circulation of RVFV in Malawi. Therefore, monitoring of RVF in animals, humans, and vectors using a "One Health" approach, along with community sensitization among the high-risk populations, could help mitigate the threat posed by this zoonotic disease in Malawi.
• An unusually long Rift valley fever inter-epizootic period in Zambia: Evidence for enzootic virus circulation and risk for disease outbreak.
  Herman M Chambaro; Kazuyo Hirose; Michihito Sasaki; Brigadier Libanda; Yona Sinkala; Paul Fandamu; Walter Muleya; Fredrick Banda; Joseph Chizimu; David Squarre; Misheck Shawa; Yongjin Qiu; Hayato Harima; Yuki Eshita; Edgar Simulundu; Hirofumi Sawa; Yasuko Orba
  PLoS neglected tropical diseases, 16, 6, e0010420, Jun. 2022, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, 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.
• Complete Genome Sequences of 24 Strains of Bacillus cereus Isolated from Nosocomial Infection and Bacteremia Cases in Japan.
  Yoshikazu Furuta; Mai Tsujinouchi; Misheck Shawa; Tuvshinzaya Zorigt; Yuya Miyajima; Atmika Paudel; Satowa Suzuki; Hideaki Higashi
  Microbiology resource announcements, 11, 4, e0120321, 21 Apr. 2022, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, Bacillus cereus is mainly associated with foodborne illness but sometimes causes nosocomial infections. We previously reported that B. cereus strains of a specific sequence type, ST1420, were associated with nosocomial infection. Here, we determined the complete genome sequences of B. cereus strains isolated from nosocomial infection cases in Japanese hospitals.
• Clonal relationship between multidrug-resistant Escherichia coli ST69 from poultry and humans in Lusaka, Zambia.
  Misheck Shawa; Yoshikazu Furuta; Atmika Paudel; O'Brian Kabunda; Evans Mulenga; Maron Mubanga; Harvey Kamboyi; Tuvshinzaya Zorigt; Herman Chambaro; Manyando Simbotwe; Bernard Hang'ombe; Hideaki Higashi
  FEMS microbiology letters, 368, 21-24, 12 Feb. 2022, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, Multidrug-resistant (MDR) Escherichia coli in food animals such as chickens is an emerging public health concern in Zambia. Additionally, the country's high demand for poultry products necessitates further investigation into the link between poultry and human MDR E. coli. Twenty cefotaxime-resistant E. coli isolates collected from poultry in Lusaka, Zambia, were screened for multidrug resistance and sequenced on MiSeq and MinION platforms. Genomes were assembled de novo and compared with 36 previously reported cefotaxime-resistant E. coli isolates from inpatients at the University Teaching Hospital, Lusaka. All (20/20, 100%) poultry isolates exhibited resistance to ampicillin, chloramphenicol and doxycycline. Phylogenetic analysis and hierarchical clustering showed a high degree of genetic relatedness between E. coli O17:H18-ST69 from poultry and humans. The E. coli O17:H18-ST69 clone accounted for 4/20 (20%) poultry- and 9/36 (25%) human-associated isolates that shared two plasmids harboring 14 antimicrobial resistance (AMR) genes. However, comparison analysis showed that the isolates also had other AMR plasmids distinct for each niche. Our results suggested clonal transmission of MDR E. coli between poultry and humans, with the potential acquisition of niche-specific AMR plasmids. Thus, the control of MDR E. coli requires a One Health approach involving both human and animal health sectors.
• Whole-Genome Sequencing Reveals Recent Transmission of Multidrug-Resistant Mycobacterium tuberculosis CAS1-Kili Strains in Lusaka, Zambia.
  Joseph Yamweka Chizimu; Eddie Samuneti Solo; Precious Bwalya; Wimonrat Tanomsridachchai; Herman Chambaro; Misheck Shawa; Thoko Flav Kapalamula; Patrick Lungu; Yukari Fukushima; Victor Mukonka; Jeewan Thapa; Chie Nakajima; Yasuhiko Suzuki
  Antibiotics (Basel, Switzerland), 11, 1, 28 Dec. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, 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.
• Hepatitis E virus infection in pigs: a first report from Zambia.
  Herman M Chambaro; Michihito Sasaki; Walter Muleya; Masahiro Kajihara; Misheck Shawa; Kabemba E Mwape; Hayato Harima; Yongjin Qiu; William W Hall; Paul Fandamu; David Squarre; Edgar Simulundu; Hirofumi Sawa; Yasuko Orba
  Emerging microbes & infections, 10, 1, 2169, 2172, Dec. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, While evidence suggests presence of HEV infection in humans in Zambia, currently, there is no information on its occurrence in domestic pigs. Here, we investigated the presence of HEV antibodies and genome in domestic pigs in Zambia. Sera (n = 484) from domestic pigs were screened for antibodies against HEV by ELISA while genome detection in fecal (n = 25) and liver (n = 100) samples from slaughter pigs was conducted using nested RT-PCR assay. Overall, seroprevalence was 47.7% (231/484) while zoonotic genotype 3 HEV RNA was detected in 16.0% (20/125) of slaughtered pigs. This is the first report to highlight occurrence of HEV infection in domestic pigs in Zambia. This finding suggests possible contamination of the pork supply chain. Moreover, there is a potential risk of zoonotic transmission of HEV to abattoir workers, pig farmers and handlers.
• Novel chromosomal insertions of ISEcp1-blaCTX-M-15 and diverse antimicrobial resistance genes in Zambian clinical isolates of Enterobacter cloacae and Escherichia coli.
  Misheck Shawa; Yoshikazu Furuta; Gillan Mulenga; Maron Mubanga; Evans Mulenga; Tuvshinzaya Zorigt; Christone Kaile; Manyando Simbotwe; Atmika Paudel; Bernard Hang'ombe; Hideaki Higashi
  Antimicrobial resistance and infection control, 10, 1, 79, 79, 10 May 2021, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, BACKGROUND: The epidemiology of extended-spectrum β-lactamases (ESBLs) has undergone dramatic changes, with CTX-M-type enzymes prevailing over other types. blaCTX-M genes, encoding CTX-M-type ESBLs, are usually found on plasmids, but chromosomal location is becoming common. Given that blaCTX-M-harboring strains often exhibit multidrug resistance (MDR), it is important to investigate the association between chromosomally integrated blaCTX-M and the presence of additional antimicrobial resistance (AMR) genes, and to identify other relevant genetic elements. METHODS: A total of 46 clinical isolates of cefotaxime-resistant Enterobacteriaceae (1 Enterobacter cloacae, 9 Klebsiella pneumoniae, and 36 Escherichia coli) from Zambia were subjected to whole-genome sequencing (WGS) using MiSeq and MinION. By reconstructing nearly complete genomes, blaCTX-M genes were categorized as either chromosomal or plasmid-borne. RESULTS: WGS-based genotyping identified 58 AMR genes, including four blaCTX-M alleles (i.e., blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, and blaCTX-M-55). Hierarchical clustering using selected phenotypic and genotypic characteristics suggested clonal dissemination of blaCTX-M genes. Out of 45 blaCTX-M gene-carrying strains, 7 harbored the gene in their chromosome. In one E. cloacae and three E. coli strains, chromosomal blaCTX-M-15 was located on insertions longer than 10 kb. These insertions were bounded by ISEcp1 at one end, exhibited a high degree of nucleotide sequence homology with previously reported plasmids, and carried multiple AMR genes that corresponded with phenotypic AMR profiles. CONCLUSION: Our study revealed the co-occurrence of ISEcp1-blaCTX-M-15 and multiple AMR genes on chromosomal insertions in E. cloacae and E. coli, suggesting that ISEcp1 may be responsible for the transposition of diverse AMR genes from plasmids to chromosomes. Stable retention of such insertions in chromosomes may facilitate the successful propagation of MDR clones among these Enterobacteriaceae species.
• Development of ELISA based on Bacillus anthracis capsule biosynthesis protein CapA for naturally acquired antibodies against anthrax.
  Tuvshinzaya Zorigt; Yoshikazu Furuta; Manyando Simbotwe; Akihiro Ochi; Mai Tsujinouchi; Misheck Shawa; Tomoko Shimizu; Norikazu Isoda; Jargalsaikhan Enkhtuya; Hideaki Higashi
  PloS one, 16, 10, e0258317, 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) of B. anthracis are mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen of B. anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Using in silico analyses, we screened coding sequences encoded on pXO2 plasmid, which is absent in the veterinary vaccine strain Sterne 34F2 but present in virulent strains of B. anthracis. Among the 8 selected antigen candidates, capsule biosynthesis protein CapA (GBAA_RS28240) and peptide ABC transporter substrate-binding protein (GBAA_RS28340) were detected by antibodies in infected horse sera. Of these, CapA has not yet been identified as immunoreactive in other studies to the best of our knowledge. Considering the protein solubility and specificity of B. anthracis, we prepared the C-terminus region of CapA, named CapA322, and developed CapA322-ELISA based on a horse model. Comparative analysis of the CapA322-ELISA and PAD1-ELISA (ELISA uses domain one of the PA) showed that CapA322-ELISA could detect anti-CapA antibodies in sera from infected horses but was non-reactive to sera from vaccinated horses. The CapA322-ELISA could contribute to the anthrax surveillance in endemic areas, and two immunoreactive proteins identified in this study could be additives to the improvement of current or future vaccine development.
• Risk factors and spatio-temporal patterns of livestock anthrax in Khuvsgul Province, Mongolia.
  Tuvshinzaya Zorigt; Satoshi Ito; Norikazu Isoda; Yoshikazu Furuta; Misheck Shawa; Natsagdorj Norov; Baasansuren Lkham; Jargalsaikhan Enkhtuya; Hideaki Higashi
  PloS one, 16, 11, e0260299, 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, Anthrax is a worldwide zoonotic disease. Anthrax has long been a public health and socio-economic issue in Mongolia. Presently, there is no spatial information on carcass burial sites as a potential hazard of future anthrax outbreaks and possible risk factors associated with anthrax occurrences in Mongolia. Here, we analyze retrospective data (1986-2015) on the disposal sites of livestock carcasses to describe historical spatio-temporal patterns of livestock anthrax in Khuvsgul Province, which showed the highest anthrax incidence rate in Mongolia. From the results of spatial mean and standard deviational ellipse analyses, we found that the anthrax spatial distribution in livestock did not change over the study period, indicating a localized source of exposure. The multi-distance spatial cluster analysis showed that carcass sites distributed in the study area are clustered. Using kernel density estimation analysis on carcass sites, we identified two anthrax hotspots in low-lying areas around the south and north regions. Notably, this study disclosed a new hotspot in the northern part that emerged in the last decade of the 30-year study period. The highest proportion of cases was recorded in cattle, whose prevalence per area was highest in six districts (i.e., Murun, Chandmani-Undur, Khatgal, Ikh-Uul, Tosontsengel, and Tsagaan-Uul), suggesting that vaccination should prioritize cattle in these districts. Furthermore, size of outbreaks was influenced by the annual summer mean air temperature of Khuvsgul Province, probably by affecting the permafrost freeze-thawing activity.
• Evidence for exposure of asymptomatic domestic pigs to African swine fever virus during an inter-epidemic period in Zambia.
  Herman M Chambaro; Michihito Sasaki; Yona Sinkala; Gabriel Gonzalez; David Squarre; Paul Fandamu; Caesar Lubaba; Liywalii Mataa; Misheck Shawa; Kabemba E Mwape; Sarah Gabriël; Mwelwa Chembensofu; Michael J Carr; William W Hall; Yongjin Qiu; Masahiro Kajihara; Ayato Takada; Yasuko Orba; Edgar Simulundu; Hirofumi Sawa
  Transboundary and emerging diseases, 67, 6, 2741, 2752, Nov. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
  English, Scientific journal, African swine fever (ASF) causes persistent outbreaks in endemic and non-endemic regions in Zambia. However, the epidemiology of the disease is poorly understood, particularly during the inter-epidemic periods. We conducted surveillance for ASF in asymptomatic domestic pigs and soft ticks in selected Zambian provinces. While serum samples (n = 1,134) were collected from crossbred pigs from all study sites between 2014 and 2017, whole blood (n = 300) was collected from both crossbred and indigenous pigs in Eastern Province (EP) in 2017. Soft ticks were collected from Mosi-oa-Tunya National Park in Southern Province (SP) in 2019. Sera were screened for antibodies against ASF by ELISA while genome detection in whole blood and soft ticks was conducted by PCR. Ticks were identified morphologically and by phylogenetic analysis of the 16S rRNA gene. Seroprevalence was highest in EP (50.9%, 95% CI [47.0-54.9]) compared to significantly lower rates in SP (2.9%, 95% CI [1.6-5.1]). No antibodies to ASFV were detected in Lusaka Province. In EP, the prevalence of ASFV genome was 11.7% (35/300), significantly higher (OR = 6.2, 95% CI [2.4-16.6]) in indigenous pigs compared to crossbred pigs. The pooled prevalence of ASFV genome in ticks was 11.0%, 95% CI [8.5-13.9]. Free-range husbandry system was the only factor that was significantly associated with seropositive (p < .0001, OR = 39.3) and PCR-positive results (p < .001, OR = 5.7). Phylogenetically, based on the p72 gene, ASFV from Ornithodoros moubata ticks detected in this study belonged to genotype I, but they separated into two distinct clusters. Besides confirming ASF endemicity in EP and the presence of ASFV-infected ticks in SP, these results provide evidence for exposure of domestic pigs to ASFV in non-endemic regions during the inter-epidemic period.