Natasha Makabilo Laban

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.

Background: Seroprevalence estimates of endemic human coronaviruses (HCoV) and novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited in Zambia. Information on development of acquired immunity to endemic HCoV in early life is also scarce and the potential cross-protective effect of HCoV immunity on SARS-CoV-2 remains debatable. We investigated seroprevalence of endemic HCoV in mother-child dyads and SARS-CoV-2 in children and explored the association between HCoV and SARS-CoV-2 antibodies in children to elucidate coronavirus seroepidemiology in Zambia. Methods: We measured endemic HCoV NL63, 229E, OC43 and HKU1 and SARS-CoV-2 Spike protein subunit 1 (S1) specific immunoglobulin G (IgG) using an immunoassay. We tested plasma samples collected from Zambian mother-child dyads before the coronavirus disease 2019 pandemic in 2018 and 2019, and during the pandemic in 2020 and 2021. We determined HCoV S1 IgG seropositivity in mothers and children and SARS-CoV-2 S1 IgG seropositivity in children. We correlated HCoV antibodies in the mother-child pairs and longitudinally profiled HCoV antibodies in children to investigate development of HCoV immunity and contribution of maternal immunity. We compared child HCoV S1 IgG and SARS-CoV-2 S1 IgG antibodies before and during the pandemic to explore cross-reactivity. Results: HCoV and SARS-CoV-2 S1 IgG antibodies were detected among mothers and children. Child HCoV seroconversion occurred following waning of maternal immunity but there was no significant correlation between HCoV and SARS-CoV-2 S1 IgG before and during the pandemic. A rise in SARS-CoV-2 S1 IgG seroprevalence among children was observed following the second and third epidemic waves of the COVID-19 pandemic in Zambia. Conclusions: Endemic HCoV NL63, 229E, OC43 and HKU1 widely circulated and are acquired early in Zambia. SARS-CoV-2 seroprevalence in children speaks to the susceptibility of this population to infection that necessitates their inclusion in control measures.

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.

Oral rotavirus vaccines demonstrate diminished immunogenicity in low-income settings where human cytomegalovirus infection is acquired early in childhood and modulates immunity. We hypothesized that human cytomegalovirus infection around the time of vaccination may influence immunogenicity. We measured plasma human cytomegalovirus-specific immunoglobulin M antibodies in rotavirus vaccinated infants from 6 weeks to 12 months old and compared rotavirus immunoglobulin A antibody titers between human cytomegalovirus seropositive and seronegative infants. There was no evidence of an association between human cytomegalovirus serostatus at 9 months and rotavirus-specific antibody titers at 12 months (geometric mean ratio 1.01, 95% CI: 0.70, 1.45; P = 0.976) or fold-increase in RV-IgA titer between 9 and 12 months (risk ratio 0.999, 95%CI: 0.66, 1.52; P = 0.995) overall. However, HIV-exposed-uninfected infants who were seropositive for human cytomegalovirus at 9 months old had a 63% reduction in rotavirus antibody geometric mean titers at 12 months compared to HIV-exposed-uninfected infants who were seronegative for human cytomegalovirus (geometric mean ratio 0.37, 95% CI: 0.17, 0.77; P = 0.008). While the broader implications of human cytomegalovirus infections on oral rotavirus vaccine response might be limited in the general infant population, the potential impact in the HIV-exposed-uninfected infants cannot be overlooked. This study highlights the complexity of immunological responses and the need for targeted interventions to ensure oral rotavirus vaccine efficacy, especially in vulnerable subpopulations.

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 individual’s resident in cholera hotspot areas remains unclear. Using a prospective cohort study design, 225 individuals were enrolled, re-vaccinated with two doses of Shanchol™ regardless of previous vaccination and followed-up for 90 days. Bloods 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 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.

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.

Live-attenuated, oral rotavirus vaccines have significantly reduced rotavirus-associated diarrhoea morbidity and infant mortality. However, vaccine immunogenicity is diminished in low-income countries. We investigated whether maternal and infant intrinsic susceptibility to rotavirus infection via histo-blood group antigen (HBGA) profiles influenced rotavirus (ROTARIX^®) vaccine-induced responses in Zambia. We studied 135 mother–infant pairs under a rotavirus vaccine clinical trial, with infants aged 6 to 12 weeks at pre-vaccination up to 12 months old. We determined maternal and infant ABO/H, Lewis, and secretor HBGA phenotypes, and infant FUT2 HBGA genotypes. Vaccine immunogenicity was measured as anti-rotavirus IgA antibody titres. Overall, 34 (31.3%) children were seroconverted at 14 weeks, and no statistically significant difference in seroconversion was observed across the various HBGA profiles in early infant life. We also observed a statistically significant difference in rotavirus-IgA titres across infant HBGA profiles at 12 months, though no statistically significant difference was observed between the study arms. There was no association between maternal HBGA profiles and infant vaccine immunogenicity. Overall, infant HBGAs were associated with RV vaccine immunogenicity at 12 months as opposed to in early infant life. Further investigation into the low efficacy of ROTARIX^® and appropriate intervention is key to unlocking the full vaccine benefits for U5 children.

Live-attenuated, oral rotavirus vaccines have significantly reduced rotavirus-associated diarrhoea morbidity and infant mortality. However, vaccine immunogenicity is diminished in low-income countries. We investigated whether maternal and infant intrinsic susceptibility to rotavirus infection via histo-blood group antigen (HBGA) profiles influenced rotavirus (ROTARIX®) vaccine-induced responses in Zambia. We studied 135 mother-infant pairs under a rotavirus vaccine clinical trial aged 6 to 12 weeks at pre-vaccination up to 12 months old. We determined maternal and infant ABO/H, Lewis, and secretor HBGA phenotypes, and infant FUT2 HBGA genotypes. Vaccine immunogenicity was measured as anti-rotavirus IgA antibody titres. Overall, 34 (31.3%) of children were seroconverted at 14 weeks, and no statistically significant difference in seroconversion was observed across the various HBGA profiles in early infant life. We also observed a statistically significant difference in rotavirus-IgA titres across infant HBGA profiles at 12 months though no statistically significant difference was observed between study arms. There was no association between maternal HBGA profiles and infant vaccine immunogenicity. Overall, infant HBGA was associated with RV-Vaccine immunogenicity at 12 months as opposed to early infant life. Further investigation into the low efficacy of ROTARIX® and appropriate intervention is key to unlocking full vaccine benefits for U5 children.

Oral rotavirus vaccines show diminished immunogenicity in low-resource settings where rotavirus burden is highest. This study assessed the safety and immune boosting effect of a third dose of oral ROTARIX^® (GlaxoSmithKline) vaccine administered at 9 months of age. A total of 214 infants aged 6 to 12 weeks were randomised to receive two doses of ROTARIX^® as per standard schedule with other routine vaccinations or an additional third dose of ROTARIX^® administered at 9 months old concomitantly with measles/rubella vaccination. Plasma collected pre-vaccination, 1 month after first- and second-dose vaccination, at 9 months old before receipt of third ROTARIX^® dose and/or measles/rubella vaccination, and at 12 months old were assayed for rotavirus-specific IgA (RV-IgA). Geometric mean RV-IgA at 12 months of age and the incidence of clinical adverse events 1 month following administration of the third dose of ROTARIX^® among infants in the intervention arm were compared between infants in the two arms. We found no significant difference in RV-IgA titres at 12 months between the two arms. Our findings showed that rotavirus vaccines are immunogenic in Zambian infants but with modest vaccine seroconversion rates in low-income settings. Importantly, however, a third dose of oral ROTARIX^® vaccine was shown to be safe when administered concomitantly with measles/rubella vaccine at 9 months of age in Zambia. This speaks to opportunities for enhancing rotavirus vaccine immunity within feasible schedules in the national immunization program.