Understanding the composition, host feeding patterns, and infection status of all potential vectors is essential to understand changing disease transmission dynamics and inform future vector control strategies. This study assessed anopheline species composition, abundance, distribution, and host feeding preferences in Southern Province, Zambia to identify potential contributors to residual malaria transmission beyond primary vectors. Adult mosquitoes were collected along a 100 km transect between the districts of Pemba, Monze and Gwembe in Southern Province, Zambia, an area of low malaria transmission. Mosquitoes were sampled from 12 randomly selected households at 5 km intervals along the transect. Centers for Disease Control and Prevention light traps were used to collect adult mosquitoes indoors and outdoors. Mosquitoes were identified using morphological keys and molecular approaches. A multiplexed PCR assay was utilized to identify mammalian hosts and Plasmodium falciparum (Pf) sporozoite infection was determined using an enzyme linked immunosorbent assay. A total of 5697 female anopheline mosquitoes were collected from both indoor and outdoor traps, revealing a diverse range of species. Nine species of Anopheles mosquitoes were identified, dominated by Anopheles rufipes (n = 1527, 26.8

Background:Combination antiretroviral therapy (cART) reduces morbidity among children living with HIV (CHIV) but does not restore measles immunity. Many CHIV now initiate cART before measles vaccination but the impact on measles-specific immunity is not understood. This study compared measles antibodies between CHIV initiating cART before and after 9 months of age in Zambia. Methods:This retrospective study was nested within an open cohort study of CHIV and community-based malaria studies in Macha, Zambia conducted from 2007-2020. Samples were tested for measles IgG antibodies from CHIV defined by age at cART initiation (aged 0-8, 9-23, and 24-59 months) and age-matched community-based controls. A cross-sectional analysis compared antibody concentrations between CHIV groups after 6-12 months of cART and aged >12 months and controls. A longitudinal analysis evaluated antibody trajectories from 9 months of age through 48 months of cART and during the 2010-11 measles outbreak. Results:In the cross-sectional analysis, 31.9% of 301 CHIV were seropositive after 6-12 of cART, with no significant difference by group (0-8: 30.8%; 9-23: 37.0%; 24-59 months: 26.6%). Within each CHIV group, the proportion seropositive was significantly lower than age-matched controls. Among 242 CHIV included in the longitudinal analysis, 46-56% had an antibody response, with an estimated time to seroreversion of 1.8-3.2 years. Among CHIV followed in 2010-11, 50% seroconverted or boosted; the estimated time to seroreversion was 2.1 years. Conclusions:Low levels of measles antibodies were found among CHIV, even when cART was started before 9 months of age, supporting a recommendation that CHIV, including infants, would benefit from revaccination.

The African kinship system facilitates the movement of children and adults between households to mitigate hardship, yet few studies have documented membership changes in rural African households over time and addressed the effects of these household dynamics on the developmental outcomes of children. We used a household inventory instrument to document the number of changes in household membership experienced by 93 households in rural Zambia between two timepoints (∼2 years apart). We then used multilevel models to evaluate the effects of migration and household change on the academic learning of the children affected by HIV residing in these households, positing migration between households as a proxy for household connectivity and a potential protective factor for children. Our inventoried households experienced on average 4.12 changes in membership over the measured period. About half of these changes were due to children under 10 moving between relatives' households. No effects of these migration-related variables were found on children's academic progress at the family level. Household connectivity, as operationalized by the movement of individuals between households, may be typical to these children's home life and were not shown to be detrimental to their academic learning.

Post-campaign coverage surveys estimate the proportion of previously unvaccinated children vaccinated during supplementary immunization activities (SIA) but cannot address whether susceptible children seronegative to measles and rubella viruses were vaccinated during the SIA. We conducted a cross-sectional serosurvey nested within the November 2020 measles-rubella SIA in Zambia, systematically sampling children aged 9 months to 5 years at 30 SIA sites in Choma and Ndola Districts. A questionnaire administered to caregivers collected the child's demographic information and vaccination history. Dried blood spot specimens were collected from child participants and for Immunoglobulin-G antibodies to measles and rubella viruses by enzyme immunoassay. We used the expected vaccination probability by age derived from Demographic and Health Surveys to quantify the value of the immunization campaign, estimating the proportion of children who would not have otherwise received routine MR doses. Among children enrolled with MR vaccination data (N = 2,364), 2,214 (94%) reported at least one routine MR dose before the SIA. We estimate 5.0% [95%CI, 4.2-5.9%] (n = 118/2364) of children would not have otherwise received routine MR dose 1 without the SIA and 23.4% [95%CI, 21.7-25.1] (n = 553/2364) would not have otherwise received routine MR dose 2. Thus, 1 in 3.5 doses were given to an MR un- or under-vaccinated child who may not have received that dose in the absence of an SIA. Eighty-six percent and 90% of children were measles and rubella seropositive before vaccination during the SIA. Thirty-six percent of children with no prior routine MR dose were measles seropositive, while nine percent of children with two prior routine MR doses were measles seronegative. Although children vaccinated during this SIA were highly likely to have previously received routine vaccinations, the SIA reached a considerable number of susceptible children. Monitoring SIA effectiveness and efficiency through standardized metrics and formats is critical for impactful vaccine delivery strategies.

The emergence of antimalarial drug resistance is an impediment to malaria control and elimination in Africa. Analysis of temporal trends in molecular markers of resistance is critical to inform policy makers and guide malaria treatment guidelines. In a low and seasonal transmission region of southern Zambia, we successfully genotyped 85.5% (389/455) of Plasmodium falciparum samples collected between 2013 and 2018 from 8 spatially clustered health centres using molecular inversion probes (MIPs) targeting key drug resistance genes. Aside from one sample from 2016 carrying K13 622I, no other World Health Organization-validated or candidate artemisinin partial resistance (ART-R) mutations were observed. However, in the more recent years (2016-2017) five novel K13-propeller-domain mutations, C532S, A578S, Q613E, D680N and G718S were identified at low prevalence. Moreover, 13% (CI, 9.6-17.2) of isolates had the AP2MU 160N mutation, which has been associated with delayed clearance following artemisinin combination therapy in Africa. This mutation increased in prevalence between 2015 and 2018 and bears a genomic signature of selection. During this time period, there was an increase in the MDR1 NFD haplotype that is associated with reduced susceptibility to lumefantrine. Sulfadoxine-pyrimethamine polymorphisms were near fixation. While validated ART-R mutations are rare, a mutation associated with slow parasite clearance in Africa appears to be under selection in southern Zambia.

Risk perception, its relationship to preventive health behaviors and other factors (e.g., direct experience of a disease) are important for determining effective targets for disease prevention. Whether these relationships are the same for various diseases has not been well-researched. Drawing on a holistic model of risk perception, this study compares levels of perceived risk for COVID-19 and HIV in a rural Zambian community, examines hypothesized correlates of perceived risk, and evaluates whether higher perceived risk and/or its correlates are associated with practicing preventive behaviors for each disease. The sample included 118 adults participating in a larger study of families affected by HIV. Via surveys, information about risk perception, preventive behaviors, knowledge about, trust in information sources, direct experience and hearing about each disease, and prosociality, were collected. For each disease, perceived risk was not related to its preventive behaviors. Levels of perceived risk for COVID-19 and HIV differed significantly, as did their correlates. Having trusted sources of information about HIV was related to higher perceived risk of HIV. Direct experience of COVID-19 was related to higher perceived risk of COVID-19, but only at a level of interest. Although practicing preventive behaviors for each condition was related to higher levels of knowledge about each disease at a level of interest, willingness to engage in behavior beneficial to others (prosociality) was significantly related only to COVID-19 preventive behaviors. Different diseases provoke different levels of perceived risk and engagement with preventive behaviors and may be correlated with distinct factors. These differences may be due to history of experience with a disease, as well as cultural factors.

Nested serosurveys within routine service delivery platforms such as planned supplemental immunization activities (SIAs) provide an opportunity to collect information that can be used to answer valuable questions on the effectiveness and efficiency of the delivery model to inform future activities. However, integrating research data collection in SIAs is rarely done due to concerns it will negatively impact the program. We conducted a serosurvey nested within the November 2020 measles-rubella SIA integrated with the Child Health Week activities in Zambia to evaluate this approach. In-depth interviews with the study teams and vaccination campaign staff at the vaccination sites were conducted. Recorded interviews were transcribed, transcripts were coded and then grouped into themes based on a process evaluation framework. A multi-methods analytical approach was used to assess the feasibility and acceptability of collecting dried blood spots from children during the SIA. This included a quantitative assessment of participant enrollment. The serosurvey successfully enrolled 90% of children from Child Health Week due to close coordination and teamwork between the vaccination teams and serosurvey team, in addition to substantial social mobilization efforts. Continually adjusting the sampling interval that was used to select eligible children allowed us to enroll throughout the SIA and capture a representative sample of children in attendance although it was challenging for the staff involved. As vaccination programs aim to tailor their approaches to reach the hardest-to-reach children, embedding research questions in SIAs will allow evaluation of the successes and challenges and compare alternative approaches. Lessons learned from this experience collecting data during an SIA can be applicable to future research activities embedded in SIAs or other delivery platforms.

The emergence of antimalarial drug resistance is an impediment to malaria control and elimination in Africa. Analysis of temporal trends in molecular markers of resistance is critical to inform policy makers and guide malaria treatment guidelines. In a low and seasonal transmission region of southern Zambia, we successfully genotyped 85.5% (389/455) of Plasmodium falciparum samples collected between 2013-2018 from 8 spatially clustered health centres using molecular inversion probes (MIPs) targeting key drug resistance genes. Aside from one sample carrying K13 R622I, none of the isolates carried other World Health Organization-validated or candidate artemisinin partial resistance (ART-R) mutations in K13. However, 13% (CI, 9.6-17.2) of isolates had the AP2MU S160N mutation, which has been associated with delayed clearance following artemisinin combination therapy in Africa. This mutation increased in prevalence between 2015-2018 and bears a genomic signature of selection. During this time period, there was an increase in the MDR1 NFD haplotype that is associated with reduced susceptibility to lumefantrine. Sulfadoxine-pyrimethamine polymorphisms were near fixation. While validated ART-R mutations are rare, a mutation associated with slow parasite clearance in Africa appears to be under selection in southern Zambia.

IntroductionMalaria surveillance in Africa is conducted largely through health facility-based health management information systems (HMIS) which provide aggregated data to malaria control programs. Supplementation of HMIS surveillance with other routinely collected hospital data can provide vital statistics on malaria control in regions of high burden.MethodsTo assess the utility of supplementing HMIS data, we implemented a pilot program of enhanced malaria surveillance in a district hospital in northern Zambia over a five-year period. Data were tabulated from existing nursing records, central pharmacy inventories, laboratory logbooks, and ward registers and cross-referenced with routinely collected HMIS data.ResultsThe additional data collections captured excess malaria deaths resulting from pharmacy and blood bank stockouts (10.3 excess deaths/year) and revealed small but significant changes over time in the age distribution of patients that likely reflect underlying shifts in the local epidemiology due to malaria control programming or other factors (median age from 1.9 to 2.4 months old, P=0.001).DiscussionReadily available data can supplement existing HMIS surveillance in high malaria burden areas to provide actionable information about the local epidemiology and impacts of control efforts. Excess malaria deaths due to health systems factors can be feasibly captured and tracked and fed back to national malaria control programs and the World Health Organization to present a fuller picture of malaria burden.

ABSTRACT Understanding temporal and spatial dynamics of ongoing malaria transmission will be critical to inform effective interventions and elimination strategies in low transmission regions approaching elimination. Parasite genomics are being used as a tool to monitor epidemiologic trends, including assessing residual transmission across seasons or importation of malaria into these regions. Southern Province, Zambia is a low-transmission setting with seasonal malaria. We genotyped 441 Plasmodium falciparum samples using molecular inversion probes at 1,832 positions across the genome, using dried blood spots collected from 2012-2018 from 8 health centers in the catchment area of Macha Hospital in Choma District. We show that highly related parasites persist across multiple seasons, suggesting that the persistence of malaria is at least in part fueled by parasites “seeding” across the dry season. In addition, we identify clusters of clonal parasites that are dissimilar to the general population, suggesting that introduction of parasites from elsewhere may contribute to the continued malaria burden. We identified signals of population size fluctuation over the course of individual transmission seasons, suggesting a ramp-up of malaria transmission from a season’s beginning. Despite the small spatial scale of the study (2,000 sq km), we identified an inverse relationship between genetic relatedness of parasite pairs and distance between health centers, as well as increased relatedness between specific health centers. These results, leveraging both genomic and epidemiological data, provide a comprehensive picture of fluctuations in parasite populations in this pre-elimination setting of southern Zambia.

The Southern Province of Zambia has been afflicted with malaria for many years, but in recent times, with the success of the national malaria control programme, malaria transmission has declined significantly and is now at low ebb. This success has been attributed to the effective use of indoor residual spraying, proper use of insecticide-treated mosquito nets provided to the people, as well as the use of artemisinin combination therapies in all rural health centres (RHCs) to treat clinical malaria cases at health facilities as well as in the communities by trained community health workers (CHWs). Despite this success and in trying to understand the epidemiology and control of malaria in rural areas of sub-Saharan Africa, it has been clear that local elimination is most unlikely due many challenges such as lack of incentives for CHWs who are the first link of the community to the health care system, inability of the officially approved rapid diagnostic tests to detect low level parasitemia in asymptomatic individuals, and anti-malaria stock-outs in RHCs, especially during the rainy season when most areas are unreachable due to impassable roads. This paper discusses the role of RHCs in the detection and management of malaria outbreaks in low transmission settings and the challenges of achieving elimination in Choma District, Southern Province, Zambia. These challenges could adversely impact malaria elimination if they remain unanswered.

BACKGROUND:Remarkable progress has been made in expanding access to services addressing the pediatric HIV epidemic, including programs to prevent mother-to-child transmission, early diagnosis and treatment for children living with HIV. Few long-term data are available from rural sub-Saharan Africa to assess implementation and impact of national guidelines. METHODS:Results from 3 cross-sectional studies and 1 cohort study conducted at Macha Hospital in Southern Province, Zambia from 2007 to 2019 were summarized. For infant diagnosis, maternal antiretroviral treatment, infant test results and turnaround times for results were evaluated by year. For pediatric HIV care, the number and age of children initiating care and treatment, and treatment outcomes within 12 months were evaluated by year. RESULTS:Receipt of maternal combination antiretroviral treatment increased from 51.6% in 2010-2012 to 93.4% in 2019, and the proportion of infants testing positive decreased from 12.4% to 4.0%. Turnaround times for results returning to clinic varied but were shorter when labs consistently used a text messaging system. The proportion of mothers receiving results was higher when a text message intervention was piloted. The number of children living with HIV enrolled into care and the proportion initiating treatment with severe immunosuppression and dying within 12 months decreased over time. CONCLUSIONS:These studies demonstrate the long-term beneficial impact of implementing a strong HIV prevention and treatment program. While expansion and decentralization brought challenges, the program succeeded in decreasing the rate of mother-to-child transmission and ensuring that children living with HIV benefit from access to life-saving treatment.

ABSTRACT. Obtaining accurate malaria surveillance data is challenging in low-transmission settings because large sample sizes are required to estimate incidence and prevalence precisely. Serology is an additional tool to document progress toward malaria elimination. An enzyme immunoassay to Plasmodium falciparum lysate was used to estimate age-specific seroprevalence among residents of southern Zambia, where malaria transmission has declined to pre-elimination levels during the past two decades. Plasma was eluted from 3,362 dried blood spots collected during five cross-sectional surveys conducted between 2009 and 2012, and again in 2018. Annual seroconversion rates (SCRs), an estimate of the force of infection, were calculated using a reversible catalytic model. The SCR decreased by two thirds from a level of approximately 0.15/year in 2009 and 2010 to approximately 0.05/year in 2011 and 2012, and then decreased 5-fold to 0.01/year by 2018, demonstrating the utility of serology in documenting progress toward elimination.

Background Factors associated with whether individuals choose to participate in serosurveys are not well understood. Understanding perceptions from multiple perspectives, including the perspectives of both data collectors and participants, through a holistic model such as the socio-ecological model contextualizes individual, interpersonal, and structural level influences on survey research participation. We used a multiple methods approach to characterize reasons for serosurvey participation in communities in Southern Province, Zambia where a serosurvey was conducted in 2016. Methods The first phase conducted focus group discussions and in-depth interviews with 24 data collectors who participated in a measles-rubella serosurvey in 2016. The second phase surveyed 34 caregivers at health facilities to identify barriers and facilitators to serosurvey participation. Emergent themes were then classified into a socio-ecological model using individual, interpersonal, and structural level constructs. Results Common themes emerged from data collectors as well as caregivers surveyed. At the individual level, providing incentives was a facilitator, and some religious beliefs were described as a barrier to serosurvey participation. At the interpersonal level, family dynamics and community peer influences could help or hinder serosurvey participation. Community health workers were consistently named as facilitators of participation. At the structural level, concerns about specimen collection, who was selected for serosurveys, and not receiving test results arose as potential barriers. The most frequently reported facilitator was provision of information about the purpose of the serosurvey (85% of respondents). The most frequently reported barrier was lack of clarity regarding use of their blood specimen (53% of respondents). For specimen collection type, caregivers consistently preferred finger prick blood collection over both venous blood draw and oral swabs. Conclusion Serosurvey participation was deemed acceptable to most study participants. The socio-ecological model revealed barriers and facilitators for participation to guide strategies to improve participation which can be applied to ongoing serosurveys for SARS-CoV-2. Serosurveys should continue to develop engagement plans to provide information about blood collection ahead of the serosurvey and communicate the objectives of such studies through trusted sources such as community health workers and traditional leaders.

The African malaria mosquito Anopheles gambiae exhibits a strong innate drive to seek out humans in its sen-sory environment, classically entering homes to land on human skin in the hours flanking midnight. To gain insight into the role that olfactory cues emanating from the human body play in generating this epidemiolog-ically important behavior, we developed a large-scale multi-choice preference assay in Zambia with infrared motion vision under semi-field conditions. We determined that An. gambiae prefers to land on arrayed visual targets warmed to human skin temperature during the nighttime when they are baited with carbon dioxide (CO2) emissions reflective of a large human over background air, body odor from one human over CO2, and the scent of one sleeping human over another. Applying integrative whole body volatilomics to multiple humans tested simultaneously in competition in a six-choice assay, we reveal high attractiveness is associ-ated with whole body odor profiles from humans with increased relative abundances of the volatile carboxylic acids butyric acid, isobutryic acid, and isovaleric acid, and the skin microbe-generated methyl ketone ace-toin. Conversely, those least preferred had whole body odor that was depleted of carboxylic acids among other compounds and enriched with the monoterpenoid eucalyptol. Across expansive spatial scales, heated targets without CO2 or whole body odor were minimally or not attractive at all to An. gambiae. These results indicate that human scent acts critically to guide thermotaxis and host selection by this prolific malaria vector as it navigates towards humans, yielding intrinsic heterogeneity in human biting risk.

Studies focusing on children affected by HIV have shown that they have generally lower academic performance, however, few studies separate children who are HIV exposed and infected (CHEI) and those who are HIV exposed but uninfected (CHEU). Importantly, in rural sub-Saharan Africa, the majority of studies on CHEI and CHEU examine academic performance indirectly based on cognitive test scores. Therefore, studies assessing the effects of HIV on academic achievement directly for CHEI and CHEU are needed. This article evaluates the effects of HIV-infection on cognitive and academic performance by comparing CHEI (n = 82) and CHEU (n = 1045) aged 7-17 years old using cross-sectional data from an ongoing longitudinal study in a rural area of Zambia. Youth completed cognitive and academic assessments; their height and weight were assessed to generate Body Mass Index (BMI). Caregiver questionnaires provided information on youths' years in school and household socio-economic status (SES). Results indicated that while HIV infection status did explain some of the variance in performance between CHEI and CHEU, age, BMI, years of schooling and SES accounted for additional variance. The effect of years of schooling on both cognitive and academic performance demonstrated that CHEI's performance may be greatly improved by consistent school enrollment.

Abstract: This study aimed to investigate the psychometric properties of the Pediatric Symptom Checklist-17 (PSC-17) in a sample of children orphaned or made vulnerable (OVC) by HIV in Zambia. Caregivers of 1,076 OVC (55.1% boys; Mage = 12.91 years) completed the PSC-17. Competing models, including confirmatory factor analysis (CFA), hierarchical CFA, bifactor CFA, exploratory structural equation modeling (ESEM), and bifactor ESEM, were tested to evaluate the optimal factor structure of the PSC-17. Results showed that the bifactor ESEM provided the best approximation of the PSC-17 data with a well-defined general psychosocial problems factor explaining 72% of the reliable variance in the total score and an internalizing factor containing 63% of the reliable variance unique from the general factor. The observed overall psychosocial problems score was associated with lower academic achievement and working memory (with small effect sizes), supporting the discriminant validity of score interpretation. Results of multiple indicators multiple causes (MIMIC) analyses revealed that all items functioned equivalently across child gender and age.

AbstractVariability in the chemical composition of human scent has the potential to modulate mosquito attraction to certain humans. We have engineered a large-scale, semi-field system in Zambia for quantifying mosquito olfactory preferences towards whole body odor sourced from different humans under naturalistic conditions. In a flight cage arena with infrared tracking, we document that the African malaria mosquitoAnopheles gambiaehierarchically prefers to land on heated targets mimicking human skin temperature when they are baited with carbon dioxide (CO2) over background air, human body odor over CO2, and the scent of one individual over another. In a six-choice assay configuration, we further identify humans at both ends of the attractiveness spectrum whose scent is differentially attractive toAn. gambiaerelative to other individuals. We demonstrate integrative use of this multi-choice olfactory assay with whole body volatilomics, establishing a powerful method for discovery of human odorants modulating heterogeneity in biting risk at enhanced throughput.

Background Although vector-borne zoonotic diseases are a major public health threat globally, they are usually neglected, especially among resource-constrained countries, including those in sub-Saharan Africa. This scoping review examined the current knowledge and identified research gaps of vector-borne zoonotic pathogens in Zambia. Methods and findings Major scientific databases (Web of Science, PubMed, Scopus, Google Scholar, CABI, Scientific Information Database (SID)) were searched for articles describing vector-borne (mosquitoes, ticks, fleas and tsetse flies) zoonotic pathogens in Zambia. Several mosquito-borne arboviruses have been reported including Yellow fever, Ntaya, Mayaro, Dengue, Zika, West Nile, Chikungunya, Sindbis, and Rift Valley fever viruses. Flea-borne zoonotic pathogens reported include Yersinia pestis and Rickettsia felis . Trypanosoma sp. was the only tsetse fly-borne pathogen identified. Further, tick-borne zoonotic pathogens reported included Crimean-Congo Haemorrhagic fever virus, Rickettsia sp., Anaplasma sp., Ehrlichia sp., Borrelia sp., and Coxiella burnetii . Conclusions This study revealed the presence of many vector-borne zoonotic pathogens circulating in vectors and animals in Zambia. Though reports of human clinical cases were limited, several serological studies provided considerable evidence of zoonotic transmission of vector-borne pathogens in humans. However, the disease burden in humans attributable to vector-borne zoonotic infections could not be ascertained from the available reports and this precludes the formulation of national policies that could help in the control and mitigation of the impact of these diseases in Zambia. Therefore, there is an urgent need to scale-up “One Health” research in emerging and re-emerging infectious diseases to enable the country to prepare for future epidemics, including pandemics.

The International Centers of Excellence for Malaria Research (ICEMR) were established by the National Institute of Allergy and Infectious Diseases more than a decade ago to provide multidisciplinary research support to malaria control programs worldwide, operating in endemic areas and contributing technology, expertise, and ultimately policy guidance for malaria control and elimination. The Southern and Central Africa ICEMR has conducted research across three main sites in Zambia and Zimbabwe that differ in ecology, entomology, transmission intensity, and control strategies. Scientific findings led to new policies and action by the national malaria control programs and their partners in the selection of methods, materials, timing, and locations of case management and vector control. Malaria risk maps and predictive models of case detection furnished by the ICEMR informed malaria elimination programming in southern Zam-bia, and time series analyses of entomological and parasitological data motivated several major changes to indoor resid-ual spray campaigns in northern Zambia. Along the Zimbabwe-Mozambique border, temporal and geospatial data are currently informing investigations into a recent resurgence of malaria. Other ICEMR findings pertaining to parasite and mosquito genetics, human behavior, and clinical epidemiology have similarly yielded immediate and long-term policy implications at each of the sites, often with generalizable conclusions. The ICEMR programs thereby provide rigorous scientific investigations and analyses to national control and elimination programs, without which the impediments to malaria control and their potential solutions would remain understudied.