Single-test syphilis serology: A case of not seeing the forest for the trees.

INTRODUCTION: There have been few empirical studies for diagnostic test accuracy of syphilis using a sequence of rapid tests in populations with low prevalence of syphilis such as pregnant women. This analysis describes syphilis test positivity frequency among pregnant women at an antenatal clinic in Zambia using a reverse-sequence testing algorithm for antenatal syphilis screening. METHODS: Between August 2019 and May 2023, we recruited 1510 pregnant women from a peri-urban hospital in Lusaka, Zambia. HIV positive and HIV negative women were enrolled in a 1:1 ratio. Blood collected at recruitment from the pregnant mothers was tested on-site for syphilis using a rapid treponemal test. Samples that tested positive were further tested at a different laboratory, with rapid plasma reagin using archived plasma. RESULTS: Of the total 1,421 sera samples which were screened with a rapid treponemal test, 127 (8.9%) were positive and 1,294 (91.1%) were negative. Sufficient additional samples were available to perform RPR testing on 114 of the 127 (89.8%) RDT positive specimens. Thirty-one (27.2%) of these 114 were reactive by RPR and 83 (72.8%) were negative, resulting in a syphilis overtreatment rate of 3 fold (i.e, 84/114). Insufficient sample or test kit availability prevented any testing for the remaining 89 (5.9%) participants. CONCLUSION: Use of only treponemal tests in low prevalence populations, like pregnant women, subjects individuals with non-active syphilis to the costs and possible risks of overtreatment. The use of the dual treponemal and non-treponemal tests would minimize this risk at some additional cost.

New Vibrio cholerae sequences from Eastern and Southern Africa alter our understanding of regional cholera transmission.

Despite ongoing containment and vaccination efforts, cholera remains prevalent in many countries in sub-Saharan Africa. Part of the difficulty in containing cholera comes from our lack of understanding of how it circulates throughout the region. To better characterize regional transmission, we generated and analyzed 118 Vibrio cholerae genomes collected between 2007-2019 from five different countries in Southern and Eastern Africa. We showed that V. cholerae sequencing can be successful from a variety of sample types and filled in spatial and temporal gaps in our understanding of circulating lineages, including providing some of the first sequences from the 2018-2019 outbreaks in Uganda, Kenya, Tanzania, Zambia, and Malawi. Our results present a complex picture of cholera transmission in the region, with multiple lineages found to be co-circulating within several countries. We also find evidence that previously identified sporadic cases may be from larger, undersampled outbreaks, highlighting the need for careful examination of sampling biases and underscoring the need for continued and expanded cholera surveillance across the African continent.

The Incidence and Risk Factors for Enterotoxigenic E. coli Diarrheal Disease in Children under Three Years Old in Lusaka, Zambia.

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.

COVID-19 mortality sentinel surveillance at a tertiary referral hospital in Lusaka, Zambia, 2020-2021.

Deaths from COVID-19 likely exceeded official statistics in Zambia because of limited testing and incomplete death registration. We describe a sentinel COVID-19 mortality surveillance system in Lusaka, Zambia. We analyzed surveillance data on deceased persons of all ages undergoing verbal autopsy (VA) and COVID-19 testing at the University Teaching Hospital (UTH) mortuary in Lusaka, Zambia, from April 2020 through August 2021. VA was done by surveillance officers for community deaths and in-patient deaths that occurred <48 hours after admission. A standardized questionnaire about the circumstances proximal to death was used, with a probable cause of death assigned by a validated computer algorithm. Nasopharyngeal specimens from deceased persons were tested for COVID-19 using polymerase chain reaction and rapid diagnostic tests. We analyzed the cause of death by COVID-19 test results. Of 12,919 deceased persons at UTH mortuary during the study period, 5,555 (43.0%) had a VA and COVID-19 test postmortem, of which 79.7% were community deaths. Overall, 278 (5.0%) deceased persons tested COVID-19 positive; 7.1% during waves versus 1.4% during nonwave periods. Most (72.3%) deceased persons testing COVID-19 positive reportedly had fever, cough, and/or dyspnea and most (73.5%) reportedly had an antemortem COVID-19 test. Common causes of death for those testing COVID-19 positive included acute cardiac disease (18.3%), respiratory tract infections (16.5%), other types of cardiac diseases (12.9%), and stroke (7.2%). A notable portion of deceased persons at a sentinel site in Lusaka tested COVID-19 positive during waves, supporting the notion that deaths from COVID-19 might have been undercounted in Zambia. Many had displayed classic COVID-19 symptoms and been tested before death yet nevertheless died in the community, potentially indicating strained medical services during waves. The high proportion of cardiovascular diseases deaths might reflect the hypercoagulable state during severe COVID-19. Early supportive treatment and availability of antivirals might lessen future mortality.

Human Cytomegalovirus Seropositivity and Its Influence on Oral Rotavirus Vaccine Immunogenicity: A Specific Concern for HIV-Exposed-Uninfected Infants.

Oral rotavirus vaccines demonstrate diminished immunogenicity in low-income settings where human cytomegalovirus infection is aquired 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 titres 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 titres at 12 months (geometric mean ratio 1.01, 95%CI: 0.70,1.45; p=0.976) or fold-increase in RV-IgA titre 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 titres 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.

Application of a Novel Proteomic Microarray Reveals High Exposure to Diarrhoeagenic Escherichia coli among Children in Zambia Participating in a Phase I Clinical Trial.

Diarrhoeagenic E. coli (DEC) significantly contributes to the burden of diarrhoea among children. Currently, there is no approved vaccine against DEC, but several vaccines against the enterotoxigenic E. coli (ETEC) pathotype are in advanced clinical trial stages, including the ETVAX® vaccine, undergoing evaluation in Zambia. This study reports on the reactivity of antibodies from ETVAX® vaccine and placebo recipients in a phase I clinical trial to proteins derived from (DEC) other than ETEC. Plasma samples collected at two time points (prior to any vaccination and post-third dose vaccination) from 16 vaccinated and 4 placebo participants in a phase 1 clinical trial examining the safety, tolerability, and immunogenicity of ETVAX® with dmLT adjuvant were evaluated for IgG response to E. coli antigens other than ETEC using the Pan-DEC protein microarray. This was the first field application of the novel pan-DEC array as a new tool in assessing the antigenic breadth of antibody responses induced by the ETVAX vaccine, as well as to assess early life exposure to DEC pathotypes and other bacterial enteric pathogens. We observed that plasma obtained from ETVAX® and placebo recipients had high antibody reactivity to Ipa, SseC and EspB proteins. These findings suggest that there is high exposure early in life to DEC pathogens, like EPEC, EHEC, EAEC and EIEC in addition to ETEC, in the Zambian population. These immunological observations are consistent with the results of recent epidemiological studies assessing the etiology of diarrheal disease among infants and young children in Zambia.

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

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

Test negative case-control study of COVID-19 vaccine effectiveness for symptomatic SARS-CoV-2 infection among healthcare workers: Zambia, 2021-2022.

OBJECTIVES: The study aim was to evaluate vaccine effectiveness (VE) of COVID-19 vaccines in preventing symptomatic COVID-19 among healthcare workers (HCWs) in Zambia. We sought to answer the question, 'What is the vaccine effectiveness of a complete schedule of the SARS-CoV-2 vaccine in preventing symptomatic COVID-19 among HCWs in Zambia?' DESIGN/SETTING: We conducted a test-negative case-control study among HCWs across different levels of health facilities in Zambia offering point of care testing for COVID-19 from May 2021 to March 2022. PARTICIPANTS: 1767 participants entered the study and completed it. Cases were HCWs with laboratory-confirmed SARS-CoV-2 and controls were HCWs who tested SARS-CoV-2 negative. Consented HCWs with documented history of vaccination for COVID-19 (vaccinated HCWs only) were included in the study. HCWs with unknown test results and unknown vaccination status, were excluded. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was VE among symptomatic HCWs. Secondary outcomes were VE by: SARS-CoV-2 variant strains based on the predominant variant circulating in Zambia (Delta during May 2021 to November 2021 and Omicron during December 2021 to March 2022), duration since vaccination and vaccine product. RESULTS: We recruited 1145 symptomatic HCWs. The median age was 30 years (IQR: 26-38) and 789 (68.9%) were women. Two hundred and eighty-two (24.6%) were fully vaccinated. The median time to full vaccination was 102 days (IQR: 56-144). VE against symptomatic SARS-CoV-2 infection was 72.7% (95% CI: 61.9% to 80.7%) for fully vaccinated participants. VE was 79.4% (95% CI: 58.2% to 90.7%) during the Delta period and 37.5% (95% CI: -7.0% to 63.3%) during the Omicron period. CONCLUSIONS: COVID-19 vaccines were effective in reducing symptomatic SARS-CoV-2 among Zambian HCWs when the Delta variant was circulating but not when Omicron was circulating. This could be related to immune evasive characteristics and/or waning immunity. These findings support accelerating COVID-19 booster dosing with bivalent vaccines as part of the vaccination programme to reduce COVID-19 in Zambia.

Characterization of Rotavirus Strains Responsible for Breakthrough Diarrheal Diseases among Zambian Children Using Whole Genome Sequencing.

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.

Association of biomarkers of enteric dysfunction, systemic inflammation, and growth hormone resistance with seroconversion to oral rotavirus vaccine: A lasso for inference approach.

BACKGROUND: Rotavirus gastroenteritis remains a leading cause of morbidity and mortality despite the introduction of vaccines. Research shows there are several factors contributing to the reduced efficacy of rotavirus vaccines in low- and middle-income settings. Proposed factors include environmental enteric dysfunction (EED), malnutrition, and immune dysfunction. This study aimed to assess the effect of these factors on vaccine responses using a machine learning lasso approach. METHODS: Serum samples from two rotavirus clinical trials (CVIA 066 n = 99 and CVIA 061 n = 124) were assessed for 11 analytes using the novel Micronutrient and EED Assessment Tool (MEEDAT) multiplex ELISA. Immune responses to oral rotavirus vaccines (Rotarix, Rotavac, and Rotavac 5D) as well as a parenteral rotavirus vaccine (trivalent P2-VP8) were also measured and machine learning using the lasso approach was then applied to investigate any associations between immune responses and environmental enteric dysfunction, systemic inflammation, and growth hormone resistance biomarkers. RESULTS: Both oral and parenteral rotavirus vaccine responses were negatively associated with retinol binding protein 4 (RBP4), albeit only weakly for oral vaccines. The parenteral vaccine responses were positively associated with thyroglobulin (Tg) and histidine-rich protein 2 (HRP2) for all three serotypes (P8, P6 and P4), whilst intestinal fatty acid binding protein (I-FABP) was negatively associated with P6 and P4, but not P8, and soluble transferrin receptor (sTfR) was positively associated with P6 only. CONCLUSION: MEEDAT successfully measured biomarkers of growth, systemic inflammation, and EED in infants undergoing vaccination, with RBP4 being the only analyte associated with both oral and parenteral rotavirus vaccine responses. Tg and HRP2 were associated with responses to all three serotypes in the parenteral vaccine, while I-FABP and sTfR results indicated possible strain specific immune responses to parenteral immunization.

Prevalence of Diarrhoeagenic Escherichia coli among Children Aged between 0-36 Months in Peri-Urban Areas of Lusaka.

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.

Safety, tolerability, and immunogenicity of an oral inactivated ETEC vaccine (ETVAX®) with dmLT adjuvant in healthy adults and children in Zambia: An age descending randomised, placebo-controlled trial.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is an important cause of moderate to severe diarrhoea in children for which there is no licensed vaccine. We evaluated ETVAX®, an oral, inactivated ETEC vaccine containing four E. coli strains over-expressing the major colonization factors CFA/I, CS3, CS5, and CS6, a toxoid (LCTBA) and double mutant heat-labile enterotoxin (dmLT) adjuvant for safety, tolerability, and immunogenicity. METHODS: A double-blind, placebo-controlled, age-descending, dose-finding trial was undertaken in 40 adults, 60 children aged 10-23 months, and 146 aged 6-9 months. Adults received one full dose of ETVAX® and children received 3 doses of either 1/4 or 1/8 dose. Safety was evaluated as solicited and unsolicited events for 7 days following vaccination. Immunogenicity was assessed by evaluation of plasma IgA antibody responses to CFA/I, CS3, CS5, CS6, and LTB, and IgG responses to LTB. RESULTS: Solicited adverse events were mostly mild or moderate with only 2 severe fever reports which were unrelated to the vaccine. The most common events were abdominal pain in adults (26.7 % in vaccinees vs 20 % in placebos), and fever in children aged 6-9 months (44 % vs 54  %). Dosage, number of vaccinations and decreasing age had no influence on severity or frequency of adverse events. The vaccine induced plasma IgA and IgG responses against LTB in 100 % of the adults and 80-90 % of the children. In the 6-23 months cohort, IgA responses to more than 3 vaccine antigens after 3 doses determined as ≥2-fold rise was significantly higher for 1/4 dose compared to placebo (56.7 % vs 27.2 %, p = 0.01). In the 6-9 months cohort, responses to the 1/4 dose were significantly higher than 1/8 dose after 3 rather than 2 doses. CONCLUSION: ETVAX® was safe, tolerable, and immunogenic in Zambian adults and children. The 1/4 dose induced significantly stronger IgA responses and is recommended for evaluation of protection in children. CLINICAL TRIALS REGISTRATION: The trial is registered with the Pan African Clinical Trials Registry (PACTR Ref. 201905764389804) and a description of this clinical trial is available on: https://pactr.samrc.ac.za/Trial Design.

Characterizing Epstein-Barr virus infection of the central nervous system in Zambian adults living with HIV.

The significance of Epstein-Barr virus (EBV) detection in the cerebrospinal spinal fluid (CSF) in people living with HIV (PLWH) is not entirely understood. The detection of EBV DNA may represent active central nervous system (CNS) infection, reactivation in the setting of another CNS pathogen or due to impaired immunity, or detection of quiescent virus. We screened 470 adult PLWH in Zambia with neurological symptoms for the presence of EBV DNA in the CSF. We performed quantitative EBV PCR on the CSF and blood. We then performed quantitative EBV DNA PCR on the blood of controls with documented HIV viral suppression without CNS symptoms. The prevalence of EBV DNA in the CSF of patients with CNS symptoms was 28.9% (136/470). EBV DNA positivity was associated with younger age, shorter duration of HIV diagnosis, lower CSF glucose levels, higher CSF protein and white blood cell levels, and a positive CSF Mycobacterium tuberculosis result. The median EBV DNA load was 8000 cps/mL in both the CSF and blood with a range of 2000-2,753,000 cps/mL in the CSF and 1000 to 1,871,000 cps/mL in the blood. Molecular screening of CSF for other possible causes of infection identified Mycobacterium tuberculosis in 30.1% and cytomegalovirus (CMV) in 10.5% of samples. EBV DNA load in the blood and CSF was not associated with mortality. Our results suggest that even though EBV DNA was commonly detected in the CSF of our population, it appears to have limited clinical significance regardless of EBV DNA load.

Ecological Niche Modeling of Aedes and Culex Mosquitoes: A Risk Map for Chikungunya and West Nile Viruses in Zambia.

The circulation of both West Nile Virus (WNV) and Chikungunya Virus (CHIKV) in humans and animals, coupled with a favorable tropical climate for mosquito proliferation in Zambia, call for the need for a better understanding of the ecological and epidemiological factors that govern their transmission dynamics in this region. This study aimed to examine the contribution of climatic variables to the distribution of Culex and Aedes mosquito species, which are potential vectors of CHIKV, WNV, and other arboviruses of public-health concern. Mosquitoes collected from Lusaka as well as from the Central and Southern provinces of Zambia were sorted by species within the Culex and Aedes genera, both of which have the potential to transmit viruses. The MaxEnt software was utilized to predict areas at risk of WNV and CHIKV based on the occurrence data on mosquitoes and environmental covariates. The model predictions show three distinct spatial hotspots, ranging from the high-probability regions to the medium- and low-probability regions. Regions along Lake Kariba, the Kafue River, and the Luangwa Rivers, as well as along the Mumbwa, Chibombo, Kapiri Mposhi, and Mpika districts were predicted to be suitable habitats for both species. The rainfall and temperature extremes were the most contributing variables in the predictive models.

Protocol-driven primary care and community linkage to reduce all-cause mortality in rural Zambia: a stepped-wedge cluster randomized trial.

Introduction: While tremendous progress has been made in recent years to improve the health of people living in low- and middle-income countries (LMIC), significant challenges remain. Chief among these are poor health systems, which are often ill-equipped to respond to current challenges. It remains unclear whether intensive intervention at the health system level will result in improved outcomes, as there have been few rigorously designed comparative studies. We present results of a complex health system intervention that was implemented in Zambia using a cluster randomized design. Methods: BHOMA was a complex health system intervention comprising intensive clinical training and quality improvement measures, support for commodities procurement, improved community outreach, and district level management support. The intervention was introduced as a stepped wedge cluster-randomized trial in 42 predominately rural health centers and their surrounding communities in Lusaka Province, Zambia. Baseline survey was conducted between January-May 2011, mid-line survey was conducted February-November, 2013 and Endline survey, February-November 2015.The primary outcome was all-cause mortality among those between 28 days and 60 years of age and assessed through community-based mortality surveys. Secondary outcomes included post-neonatal under-five mortality and service coverage scores. Service coverage scores were calculated across five domains (child preventative services; child treatment services; family planning; maternal health services, and adult health services). We fit Cox proportional hazards model with shared frailty at the cluster level for the primary analysis. Mortality rates were age-standardized using the WHO World Standard Population. Results: Mortality declined substantially from 3.9 per 1,000 person-years in the pre-intervention period, to 1.5 per 1,000 person-years in the post intervention period. When we compared intervention and control periods, there were 174 deaths in 49,230 person years (age-standardized rate = 4.4 per 1,000 person-years) in the control phase and 277 deaths in 74,519 person years (age-standardized rate = 4.6 per 1,000 person-years) in the intervention phase. Overall, there was no evidence for an effect of the intervention in minimally-adjusted [hazard ratio (HR) = 1.18; 95% confidence interval (CI): 0.88, 1.56; value of p = 0.265], or adjusted (HR = 1.12; 95% CI: 0.84, 1.49; value of p = 0.443) analyses.Coverage scores that showed some evidence of changing with time since the cluster joined the intervention were: an increasing proportion of children sleeping under insecticide treated bed-net (value of p < 0.001); an increasing proportion of febrile children who received appropriate anti-malarial drugs (value of p = 0.039); and an increasing proportion of ever hypertensive adults with currently controlled hypertension (value of p = 0.047). No adjustments were made for multiple-testing and the overall coverage score showed no statistical evidence for a change over time (value of p = 0.308). Conclusion: We noted an overall reduction in post-neonatal under 60 mortality in the study communities during the period of our study, but this could not be attributed to the BHOMA intervention. Some improvements in service coverage scores were observed. Clinical Trial Registration: clinicaltrials.gov, Identifier NCT01942278.

Maternal and Infant Histo-Blood Group Antigen (HBGA) Profiles and Their Influence on Oral Rotavirus Vaccine (RotarixTM) Immunogenicity among Infants in Zambia.

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.

Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia.

The emergence of pre-extensively drug-resistant tuberculosis (pre-XDR-TB) is a threat to TB control programs in developing countries such as Zambia. Studies in Zambia have applied molecular techniques to understand drug-resistance-associated mutations, circulating lineages and transmission patterns of multi-drug-resistant (MDR) Mycobacterium tuberculosis. However, none has reported genotypes and mutations associated with pre-XDR TB. This study characterized 63 drug-resistant M. tuberculosis strains from the University Teaching Hospital between 2018 and 2019 using targeted gene sequencing and conveniently selected 50 strains for whole genome sequencing. Sixty strains had resistance mutations associated to MDR, one polyresistant, and two rifampicin resistant. Among MDR strains, seven percent (4/60) had mutations associated with pre-XDR-TB. While four, one and nine strains had mutations associated with ethionamide, para-amino-salicylic acid and streptomycin resistances, respectively. All 50 strains belonged to lineage 4 with the predominant sub-lineage 4.3.4.2.1 (38%). Three of four pre-XDR strains belonged to sub-lineage 4.3.4.2.1. Sub-lineage 4.3.4.2.1 strains were less clustered when compared to sub-lineages L4.9.1 and L4.3.4.1 based on single nucleotide polymorphism differences. The finding that resistances to second-line drugs have emerged among MDR-TB is a threat to TB control. Hence, the study recommends a strengthened routine drug susceptibility testing for second-line TB drugs to stop the progression of pre-XDR to XDR-TB and improve patient treatment outcomes.

Impact of antibiotics on gut microbiome composition and resistome in the first years of life in low- to middle-income countries: A systematic review.

BACKGROUND: Inappropriate antimicrobial usage is a key driver of antimicrobial resistance (AMR). Low- and middle-income countries (LMICs) are disproportionately burdened by AMR and young children are especially vulnerable to infections with AMR-bearing pathogens. The impact of antibiotics on the microbiome, selection, persistence, and horizontal spread of AMR genes is insufficiently characterized and understood in children in LMICs. This systematic review aims to collate and evaluate the available literature describing the impact of antibiotics on the infant gut microbiome and resistome in LMICs. METHODS AND FINDINGS: In this systematic review, we searched the online databases MEDLINE (1946 to 28 January 2023), EMBASE (1947 to 28 January 2023), SCOPUS (1945 to 29 January 2023), WHO Global Index Medicus (searched up to 29 January 2023), and SciELO (searched up to 29 January 2023). A total of 4,369 articles were retrieved across the databases. Duplicates were removed resulting in 2,748 unique articles. Screening by title and abstract excluded 2,666 articles, 92 articles were assessed based on the full text, and 10 studies met the eligibility criteria that included human studies conducted in LMICs among children below the age of 2 that reported gut microbiome composition and/or resistome composition (AMR genes) following antibiotic usage. The included studies were all randomized control trials (RCTs) and were assessed for risk of bias using the Cochrane risk-of-bias for randomized studies tool. Overall, antibiotics reduced gut microbiome diversity and increased antibiotic-specific resistance gene abundance in antibiotic treatment groups as compared to the placebo. The most widely tested antibiotic was azithromycin that decreased the diversity of the gut microbiome and significantly increased macrolide resistance as early as 5 days posttreatment. A major limitation of this study was paucity of available studies that cover this subject area. Specifically, the range of antibiotics assessed did not include the most commonly used antibiotics in LMIC populations. CONCLUSION: In this study, we observed that antibiotics significantly reduce the diversity and alter the composition of the infant gut microbiome in LMICs, while concomitantly selecting for resistance genes whose persistence can last for months following treatment. Considerable heterogeneity in study methodology, timing and duration of sampling, and sequencing methodology in currently available research limit insights into antibiotic impacts on the microbiome and resistome in children in LMICs. More research is urgently needed to fill this gap in order to better understand whether antibiotic-driven reductions in microbiome diversity and selection of AMR genes place LMIC children at risk for adverse health outcomes, including infections with AMR-bearing pathogens.

Use of an ETEC Proteome Microarray to Evaluate Cross-Reactivity of ETVAX® Vaccine-Induced IgG Antibodies in Zambian Children.

Developing a broadly protective vaccine covering most ETEC variants has been elusive. The most clinically advanced candidate yet is an oral inactivated ETEC vaccine (ETVAX®). We report on the use of a proteome microarray for the assessment of cross-reactivity of anti-ETVAX® IgG antibodies against over 4000 ETEC antigens and proteins. We evaluated 40 (pre-and post-vaccination) plasma samples from 20 Zambian children aged 10-23 months that participated in a phase 1 trial investigating the safety, tolerability, and immunogenicity of ETVAX® adjuvanted with dmLT. Pre-vaccination samples revealed high IgG responses to a variety of ETEC proteins including classical ETEC antigens (CFs and LT) and non-classical antigens. Post-vaccination reactivity to CFA/I, CS3, CS6, and LTB was stronger than baseline among the vaccinated compared to the placebo group. Interestingly, we noted significantly high post-vaccination responses to three non-vaccine ETEC proteins: CS4, CS14, and PCF071 (p = 0.043, p = 0.028, and p = 0.00039, respectively), suggestive of cross-reactive responses to CFA/I. However, similar responses were observed in the placebo group, indicating the need for larger studies. We conclude that the ETEC microarray is a useful tool for investigating antibody responses to numerous antigens, especially because it may not be practicable to include all antigens in a single vaccine.

Superimposed Pulmonary Tuberculosis (PTB) in a 26-Year-Old Female with No Underlying Co-Morbidities Recovering from COVID-19-Case Report.

Tuberculosis before the COVID-19 pandemic is said to have killed more people globally than any other communicable disease and is ranked the 13th cause of death, according to the WHO. Tuberculosis also still remains highly endemic, especially in LIMCs with a high burden of people living with HIV/AIDS, in which it is the leading cause of mortality. Given the risk factors associated with COVID-19, the cross similarities between tuberculosis and COVID-19 symptoms, and the paucity of data on how both diseases impact each other, there is a need to generate more information on COVID-19-TB co-infection. In this case report, we present a young female patient of reproductive age with no underlying comorbidities recovering from COVID-19, who later presented with pulmonary tuberculosis. It describes the series of investigations performed and treatments given during the follow-up. There is a need for more surveillance for possible COVID-19-TB co-infection cases and further research to understand the impact of COVID-19 on tuberculosis and vice versa, especially in LMICs.