Background: Enterotoxigenic Escherichia coli (ETEC) is one of the top aetiologic agents of diarrhea in children under the age of 5 in low-middle income countries (LMICs). The lack of point of care diagnostic tools for routine ETEC diagnosis results in limited data regarding the actual burden and epidemiology in the endemic areas. We evaluated performance of the novel Rapid LAMP based Diagnostic Test (RLDT) for detection of ETEC in stool as a point of care diagnostic assay in a resource-limited setting. Methods: We conducted a cross-sectional study of 324 randomly selected stool samples from children under 5 presenting with moderate to severe diarrhea (MSD). The samples were collected between November 2012 to September 2013 at selected health facilities in Zambia. The RLDT was evaluated by targeting three ETEC toxin genes [heat labile toxin (LT) and heat stable toxins (STh and STp)]. Quantitative PCR was used as the “gold standard” to evaluate the diagnostic sensitivity and specificity of RLDT for detection of ETEC. We additionally described the prevalence and seasonality of ETEC. Results: The study included 324 participants, 50.6% of which were female. The overall prevalence of ETEC was 19.8% by qPCR and 19.4% by RLDT. The children between 12 to 59 months had the highest prevalence of 22%. The study determined ETEC toxin distribution was LT 28/321(9%), ST 18/321(6%) and LT/ST 16/321(5%). The sensitivity and specificity of the RLDT compared to qPCR using a Ct 35 as the cut-off, were 90.7% and 97.5% for LT, 85.2% and 99.3% for STh and 100% and 99.7% for STp, respectively. Conclusion: The results of this study suggest that RLDT is sufficiently sensitive and specific and easy to implement in the endemic countries. Being rapid and simple, the RLDT also presents as an attractive tool for point-of-care testing at the health facilities and laboratories in the resource-limited settings.

Diarrhoea is a major contributor to childhood morbidity and mortality in developing countries, with diarrhoeagenic Escherichia coli being among the top aetiological agents. We sought to investigate the burden and describe the diarrhoeagenic E. coli pathotypes causing diarrhoea among children in peri-urban areas of Lusaka, Zambia. This was a facility-based surveillance study conducted over an 8-month period from 2020 to 2021. Stool samples were collected from children aged 0–3 years presenting with diarrhoea at five peri-urban health facilities in Lusaka. Stool samples were tested for diarrhoeagenic E. coli using the Novodiag bacterial GE+^® panel, a platform utilising real-time PCR and microarray technology to detect bacterial pathogens. Of the 590 samples tested, diarrhoeagenic E. coli were detected in 471 (76.1%). The top three pathogens were enteropathogenic E. coli 45.4% (n = 268), enteroaggregative E. coli 39.5% (n = 233), and enterotoxigenic E. coli 29.7% (n = 176). Our results revealed that 50.1% of the diarrhoeagenic E. coli positive samples comprised multiple pathotypes of varying virulence gene combinations. Our study demonstrates a high prevalence of diarrhoeagenic E. coli in childhood diarrhoea and the early exposure (<12 months) of children to enteric pathogens. This calls for the early implementation of preventive interventions for paediatric diarrhoea.

This study aimed to estimate the incidence and risk factors for Enterotoxigenic Escherichia coli (ETEC) diarrhea. This was a prospective cohort study of children recruited in a household census. Children were enrolled if they were 36 months or below. A total of 6828 children were followed up passively for 12 months to detect episodes of ETEC diarrhea. Diarrheal stool samples were tested for ETEC using colony polymerase chain reaction (cPCR). Among the 6828 eligible children enrolled, a total of 1110 presented with at least one episode of diarrhea. The overall incidence of ETEC diarrhea was estimated as 2.47 (95% confidence interval (CI): 2.10–2.92) episodes per 100 child years. Children who were HIV-positive (adjusted Hazard ratio (aHR) = 2.14, 95% CI: 1.14 to 3.99; p = 0.017) and those whose source of drinking water was public tap/borehole/well (aHR = 2.45, 95% CI: 1.48 to 4.06; p < 0.002) were at increased risk of ETEC diarrhea. This study found that children whose mothers have at least senior secondary school education (aHR = 0.49, 95% CI: 0.29 to 0.83; p = 0.008) were at decreased risk of ETEC diarrhea. Our study emphasizes the need for integrated public health strategies focusing on water supply improvement, healthcare for persons living with HIV, and maternal education.

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.

The occurrence of rotavirus (RV) infection among vaccinated children in high-burden settings poses a threat to further disease burden reduction. Genetically altered viruses have the potential to evade both natural infection and vaccine-induced immune responses, leading to diarrheal diseases among vaccinated children. Studies characterizing RV strains responsible for breakthrough infections in resource-limited countries where RV-associated diarrheal diseases are endemic are limited. We aimed to characterize RV strains detected in fully vaccinated children residing in Zambia using next-generation sequencing. We conducted whole genome sequencing on Illumina MiSeq. Whole genome assembly was performed using Geneious Prime 2023.1.2. A total of 76 diarrheal stool specimens were screened for RV, and 4/76 (5.2%) were RV-positive. Whole genome analysis revealed RVA/Human-wt/ZMB/CIDRZ-RV2088/2020/G1P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and RVA/Human-wt/ZMB/CIDRZ-RV2106/2020/G12P[4]-I1-R2-C2-M2-A2-N1-T2-E1-H2 strains were mono and multiple reassortant (exchanged genes in bold) respectively, whilst RVA/Human-wt/ZMB/CIDRZ-RV2150/2020/G12P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 was a typical Wa-like strain. Comparison of VP7 and VP4 antigenic epitope of breakthrough strains and Rotarix strain revealed several amino acid differences. Variations in amino acids in antigenic epitope suggested they played a role in immune evasion of neutralizing antibodies elicited by vaccination. Findings from this study have the potential to inform national RV vaccination strategies and the design of highly efficacious universal RV vaccines.

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.