Kennedy Chibesa

During Zambia’s 2023–2024 cholera outbreak, reliance on single-pathogen diagnostics risked overlooking co-circulating enteric pathogens. This study estimated the prevalence of rotavirus and described co-detected enteropathogens and rotavirus genotypes among patients admitted with suspected cholera. A sub-analysis was conducted on diarrhoeal stool specimens collected from patients who met the syndromic suspected cholera case definition. Samples were tested using the Bosphore^® Gastroenteritis Panel v2, a multiplex PCR enteric panel, to detect rotavirus and other gastrointestinal pathogens. Rotavirus-positive specimens with sufficient viral load were further genotyped by RT-PCR targeting of the VP7 and VP4 genes. Among 319 suspected cholera admissions, rotavirus was detected in 18 patients (5.6%; 95% CI 3.4–8.8%), predominantly in children aged Campylobacter. Genotyping was successful in five samples and revealed heterogenous circulating strains, including G1P[8], G2P[4], G3P[6], G12P[6], and G1P[6]. Rotavirus accounted for a modest proportion of suspected cholera admissions and was frequently detected in mixed enteric infections, underscoring the value of multi-pathogen diagnostics and continued molecular surveillance during outbreak response.

During cholera outbreaks in Zambia, diagnostic strategies that rely on single-plex or targeted assays risk overlooking concomitant infections with other clinically important enteric pathogens. We estimated the prevalence of rotavirus and described co-detected enteropathogens and rotavirus genotypes among patients admitted with clinically suspected cholera during Zambia’s 2023–2024 cholera outbreak.We conducted a sub-analysis of diarrhoeal specimens collected from patients admitted to five cholera treatment centres who met the syndromic suspected cholera case definition. Stool samples were tested using the Bosphore® Gastroenteritis Panel v2, a multiplex PCR enteric panel, to detect rotavirus and other gastrointestinal pathogens. Rotavirus-positive specimen with sufficient viral load were further characterised by RT-PCR genotyping and Sanger sequencing targeting VP7 and VP4 genes. Among 319 suspected cholera admissions, rotavirus was detected in 18 patients, yielding a prevalence of 5.6% (95% CI 3.4%, 8.8%). Rotavirus detections occurred predominantly in children aged < 5 years (87.5%) and 6-15 years (80.0%). Co-infection was common - 93.7%, (15/16) of rotavirus-positive samples showed co-infection with at least one additional enteric pathogen, primarily Campylobacter. Genotyping was successful in five samples and showed heterogenous circulating strains, including G1P[8], G2P[4], G3P[6], G12P[6], and a rare G1P[6] reassortant. During a large 2023–2024 cholera outbreak in Zambia, rotavirus accounted for a modest but clinically important fraction of the suspected cholera admissions and was typically identified within mixed enteric infections. These findings highlight the limitations of syndromic diagnosis in outbreak settings and support integrating multi-pathogen diagnostics and sustained molecular surveillance to improve case management, antimicrobial stewardship, and vaccine-era monitoring.

Introduction: Cholera remains a major public health threat in endemic settings, and oral cholera vaccine (Shanchol™) campaigns are increasingly used amid constrained global supply. However, practical decisions on revaccination require clearer, setting-specific estimates of how rapidly vaccine-induced vibriocidal antibodies peak and wane. Methods: We conducted a prospective cohort kinetics analysis in Lukanga Swamps (Central Province, Zambia), enrolling adults (18–65 years) stratified by prior Shanchol™ exposure (0, 1, or 2 previous doses). All participants received two Shanchol™ doses 14 days apart, with serum collected at baseline and days 14, 28, 60, and 90 (end of follow-up). Ogawa and Inaba vibriocidal titres were measured using a complement-based assay and analysed on the log10 scale. Serotype-specific mixed-effects models with natural cubic splines for time (knots: 14, 28, 60 days) assessed trajectories by prior-dose strata, adjusting for age, sex, and HIV status. Peak timing and post-peak half-life were derived from model-based predictions with participant-level bootstrap CIs (1000 replications). Results: The analysis included 225 participants: 68 (30.2%) with zero prior doses, 89 (39.6%) with one, and 68 (30.2%) with two; median age was 33 years (IQR 25–49), 56.4% were female, and 19.2% were HIV-positive. Modelled titres for both serotypes rose steeply after vaccination, peaking around day 36–37 across prior-dose strata. Ogawa titres reached half of peak by about day 73–78, corresponding to post-peak half-lives of 37–41 days; Inaba declined more slowly with half-lives of 42–46 days. Confidence intervals overlapped across prior-dose strata, indicating minimal differences by vaccination history. Conclusions: In this cholera-endemic adult population, Shanchol™ induced vibriocidal responses that peaked at ~5 weeks and waned over the following 5–7 weeks, with broadly similar kinetics regardless of prior vaccination and slightly slower decay for Inaba than Ogawa. These parameters can inform booster timing in hotspot settings.

Introduction. Cholera, caused by Vibrio cholerae , remains a priority public health concern, particularly in developing countries. The first cholera outbreak in Zambia was documented in the 1970s, with recurring epidemics reported since then. In 2023, a cholera outbreak affected Zambia, particularly in districts bordering Malawi, Mozambique and the Democratic Republic of Congo, with significant cases reported in these neighbouring countries. This study aims to analyse cholera cases and isolates obtained during the 2023 epidemic, focusing on geographical distribution, genetic relatedness of isolates and their antibiotic resistance profiles. Methods. Stool samples were collected from patients presenting with cholera-like symptoms across three provinces of Zambia. A total of 98 samples were cultured on thiosulphate citrate bile salts sucrose agar, resulting in 32 sequenced V. cholerae isolates. Whole-genome sequencing was performed using Oxford Nanopore Technology, and phylogenetic inference was also achieved by the analysis of SNPs. Phenotypic antimicrobial resistance testing was conducted following Clinical and Laboratory Standards Institute guidelines. The genomic data were analysed for virulence factors and antimicrobial resistance profiles. Results. Of the 98 stool samples tested, 38 confirmed cholera cases were identified. A subset of 32 confirmed V. cholerae isolates, predominantly from the Eastern Province of Zambia ( n =21), was selected for whole-genome sequencing. Genomic analysis revealed that all isolates belonged to the seventh pandemic El Tor lineage and the O1 serogroup, with two distinct clades identified corresponding to the 10th (T10) and 15th (T15) transmission events. Geographical analysis indicated a predominance of Ogawa serotypes in Eastern Province and Inaba in Northern Province. The virulence gene analysis confirmed the presence of key cholera toxin genes ( ctxA and ctxB ) and intestinal colonization factors. All isolates carried genes or mutations predicted to confer resistance to multiple antibiotics, including decreased susceptibility to ciprofloxacin, recommended for the treatment of cholera by the World Health Organization. Conclusion. The findings highlight the critical need for enhanced surveillance and targeted interventions to mitigate cholera outbreaks in Zambia. The emergence of resistant V. cholerae strains necessitates innovative strategies, including improved water sanitation, vaccination efforts and novel therapeutic approaches to combat this enduring public health threat.

Cholera remains a major public health challenge, and co-infections can complicate clinical outcomes. In a cross-sectional study, we investigated the prevalence and patterns of enteric co-infections during Zambia’s 2023–2024 cholera outbreak and evaluated their implications for disease severity. 240 suspected cholera patients were enrolled from five healthcare facilities in Lusaka. Stools were tested for 11 enteric pathogens using the Bosphore^® Gastroenteritis Panel Kit v2 on the QuantStudio 5 qPCR, with Vibrio cholerae confirmed by real-time PCR (quantitative PCR). Co-infections were highly prevalent, affecting 79.2% of participants. Campylobacter was the most frequently detected pathogen (70.0%), followed by Norovirus GI/GII (20.0%). Persons living with HIV were significantly more likely to present with co-infections than their counterparts (adjusted PR 1.27, 95% CI: 1.07–1.51; p = 0.008). Participants with confirmed V. cholerae + coinfections (N = 62) were less likely to developed moderate to severe disease compared to those with mono-infections (adjusted PR 0.59, 95% CI: 0.38–0.90; p = 0.014). These findings highlight the high prevalence and complexity of co-infections during cholera outbreaks, potentially contributing to antimicrobial resistance. They also highlight the need for targeted clinical management, particularly among persons living with HIV.

Cholera remains a major public health challenge, and co-infections can complicate out-comes and case management. In a cross-sectional study, we investigated the prevalence and patterns of enteric co-infections during Zambia’s 2023–2024 cholera outbreak and evaluated their implications for disease severity. 240 suspected cholera patients were enrolled from five healthcare facilities in Lusaka. Stools were tested for 11 enteric pathogens using the Bosphore® Gastroenteritis Panel Kit v2 on the QuantStudio 5 qPCR, with Vibrio cholerae confirmed by real-time PCR (quantitative PCR). Co-infections were highly prevalent, affecting 79.2% of participants. Campylobacter was the most frequently detected pathogen (70.0%), followed by Norovirus GI/GII (20.0%), while EIEC/Shigella, Adenovirus, VTEC, Astrovirus, Clostridioides difficile, and Salmonella occurred in < 10% of cases. Persons living with HIV were significantly more likely to present with co-infections than their counterparts (adjusted PR 1.27, 95% CI: 1.07–1.51; P = 0.008). Participants with confirmed V. cholerae + coinfections (N=62) were less likely to developed moderate to severe disease compared to those with mono-infections (adjusted PR 0.59, 95% CI: 0.38–0.90; P = 0.014). These findings highlight the high prevalence and complexity of co-infections during cholera outbreaks, potentially contributing to antimicrobial re-sistance. They also highlight the need for targeted clinical management, particularly among persons living with HIV.

Background: Enterotoxigenic Escherichia coli (ETEC) is a significant cause of diarrheal disease, particularly in low—and middle-income countries, including Zambia. ETEC pathogenesis is driven by colonisation factors (CF) and enterotoxins, and increasing antimicrobial resistance compounds the global health burden. Despite its impact, limited genomic data exists for ETEC strains in Sub-Saharan Africa. Methods: This study conducted whole-genome sequencing of 62 ETEC isolates collected from children under five years old presenting with moderate-to-severe diarrhoea in Lusaka, Zambia. Genomic DNA was extracted, sequenced using the Illumina MiSeq platform, and analysed for phylogenetic relationships, toxin profiles, CF combinations, AMR genes, and plasmid incompatibility groups. Sequences were processed using bioinformatics tools, including SPAdes for genome assembly and Abricate for virulence and AMR profiling. Results: The isolates displayed diverse phylogenetic groupings, predominantly within phylogroups A (22 isolates) and B1 (29 isolates). Forty-five serotypes and 39 sequence types were identified, with ST155, ST4, and ST847 being most prevalent. 35% of isolates lacked a known CF, but CS6-only stains were the most common (8%). The most frequent toxin profile was LTh (31%); AMR analysis revealed 350 resistance genes, with Sul2 (14%) and blaTEM-1 (10%) being predominant. ∼89% of isolates exhibited genomic multidrug resistance. Plasmid analysis identified IncFII as the most prevalent incompatibility group (19%). Conclusion: This study highlights the genomic diversity of ETEC in Zambia, revealing concerns about multidrug resistance and identifying virulence profiles. These findings reiterate the urgent need for enhanced surveillance and targeted interventions, including vaccine development, to combat ETEC-related diarrhoea effectively. Authors Summary: Diarrhoea caused by a bacteria called Escherichia coli is a significant health problem for young children in Zambia and many similar countries. Despite its profound impact, not much is known about the types of ETEC found in Zambia. In this study, we collected this type of bacteria from children under five years old who had diarrhoea and studied them in detail using advanced genetic tools. We discovered that the bacteria are very diverse, with different groups, toxin types, and ways of attaching to the host. Worryingly, most bacteria were resistant to several antibiotics, potentially making treatment challenging. This research helps us understand how ETEC spreads and causes illness in Zambia, showing the urgent need for tracking, improved treatments, and vaccines to protect children from this serious disease.

Despite the successful introduction of oral cholera vaccines, Zambia continues to experience multiple, sporadic, and protracted cholera outbreaks in various parts of the country. While vaccines have been useful in staying the cholera outbreaks, the ideal window for re-vaccinating individuals resident in cholera hotspot areas remains unclear. Using a prospective cohort study design, 225 individuals were enrolled and re-vaccinated with two doses of Shanchol™, regardless of previous vaccination, and followed-up for 90 days. Bloods were collected at baseline before re-vaccination, at day 14 prior to second dosing, and subsequently on days 28, 60, and 90. Vibriocidal assay was performed on samples collected at all five time points. Our results showed that anti-LPS and vibriocidal antibody titers increased at day 14 after re-vaccination and decreased gradually at 28, 60, and 90 days across all the groups. Seroconversion rates were generally comparable in all treatment arms. We therefore conclude that vibriocidal antibody titers generated in response to re-vaccination still wane quickly, irrespective of previous vaccination status. However, despite the observed decline, the levels of vibriocidal antibodies remained elevated over baseline values across all groups, an important aspect for Zambia where there is no empirical evidence as to the ideal time for re-vaccination.