National genomic profiling of Plasmodium falciparum antimalarial resistance in Zambian children participating in the 2018 Malaria Indicator Survey.

The emergence of antimalarial drug resistance is a major threat to malaria control and elimination. Using whole genome sequencing of 282 P. falciparum samples collected during the 2018 Zambia National Malaria Indicator Survey, we determined the prevalence and spatial distribution of known and candidate antimalarial drug resistance mutations. High levels of genotypic resistance were found across Zambia to pyrimethamine, with over 94% (n=266) of samples having the Pfdhfr triple mutant (N51 I , C59 R , and S108 N ), and sulfadoxine, with over 84% (n=238) having the Pfdhps double mutant (A437 G and K540 E ). In northern Zambia, 5.3% (n=15) of samples also harbored the Pfdhps A581 G mutation. Although 29 mutations were identified in Pfkelch13 , these mutations were present at low frequency (<2.5%), and only three were WHO-validated artemisinin partial resistance mutations: P441 L (n=1, 0.35%), V568 M (n=2, 0.7%) and R622 T (n=1, 0.35%). Notably, 91 (32%) of samples carried the E431 K mutation in the Pfatpase6 gene, which is associated with artemisinin resistance. No specimens carried any known mutations associated with chloroquine resistance in the Pfcrt gene (codons 72-76). P. falciparum strains circulating in Zambia were highly resistant to sulfadoxine and pyrimethamine but remained susceptible to chloroquine and artemisinin. Despite this encouraging finding, early genetic signs of developing artemisinin resistance highlight the urgent need for continued vigilance and expanded routine genomic surveillance to monitor these changes.

Entomological effects of attractive targeted sugar bait station deployment in Western Zambia: vector surveillance findings from a two-arm cluster randomized phase III trial.

BACKGROUND: Attractive targeted sugar bait (ATSB) stations are a novel tool with potential to complement current approaches to malaria vector control. To assess the public health value of ATSB station deployment in areas of high coverage with standard vector control, a two-arm cluster-randomized controlled trial (cRCT) of Sarabi ATSB® stations (Westham Ltd., Hod-Hasharon, Israel) was conducted in Western Province, Zambia, a high-burden location were Anopheles funestus is the dominant vector. The trial included 70 clusters and was designed to measure the effect of ATSBs on case incidence and infection prevalence over two 7-month deployments. Reported here are results of the vector surveillance component of the study, conducted in a subset of 20 clusters and designed to provide entomological context to guide overall interpretation of trial findings. METHODS: Each month, 200 paired indoor-outdoor human landing catch (HLC) and 200 paired light trap (LT) collections were conducted to monitor An. funestus parity, abundance, biting rates, sporozoite prevalence, and entomological inoculation rates (EIR). RESULTS: During the study 20,337 female An. funestus were collected, 11,229 from control and 9,108 from intervention clusters. A subset of 3,131 HLC specimens were assessed for parity: The mean non-parous proportion was 23.0% (95% CI 18.2-28.7%, total n = 1477) in the control and 21.2% (95% CI 18.8-23.9%, total n = 1654) in the intervention arm, an OR = 1.05 (95% CI 0.82-1.34; p = 0.688). A non-significant reduction in LT abundance (RR = 0.65 [95% CI 0.30-1.40, p = 0.267]) was associated with ATSB deployment. HLC rates were highly variable, but model results indicate a similar non-significant trend with a RR = 0.68 (95%CI 0.22-2.00; p = 0.479). There were no effects on sporozoite prevalence or EIR. CONCLUSIONS: Anopheles funestus parity did not differ across study arms, but ATSB deployment was associated with a non-significant 35% reduction in vector LT density, results that are consistent with the epidemiological impact reported elsewhere. Additional research is needed to better understand how to maximize the potential impact of ATSB approaches in Zambia and other contexts. TRIAL REGISTRATION NUMBER: This trial was registered with Clinicaltrials.gov (NCT04800055, 16 March 2021).

Diversity and selection analyses identify transmission-blocking antigens as the optimal vaccine candidates in Plasmodium falciparum.

BACKGROUND: A highly effective vaccine for malaria remains an elusive target, at least in part due to the under-appreciated natural parasite variation. This study aimed to investigate genetic and structural variation, and immune selection of leading malaria vaccine candidates across the Plasmodium falciparum's life cycle. METHODS: We analysed 325 P. falciparum whole genome sequences from Zambia, in addition to 791 genomes from five other African countries available in the MalariaGEN Pf3k Database. Ten vaccine antigens spanning three life-history stages were examined for genetic and structural variations, using population genetics measures, haplotype network analysis, and 3D structure selection analysis. FINDINGS: Among the ten antigens analysed, only three in the transmission-blocking vaccine category display P. falciparum 3D7 as the dominant haplotype. The antigens AMA1, CSP, MSP119 and CelTOS, are much more diverse than the other antigens, and their epitope regions are under moderate to strong balancing selection. In contrast, Rh5, a blood stage antigen, displays low diversity yet slightly stronger immune selection in the merozoite-blocking epitope region. Except for CelTOS, the transmission-blocking antigens Pfs25, Pfs48/45, Pfs230, Pfs47, and Pfs28 exhibit minimal diversity and no immune selection in epitopes that induce strain-transcending antibodies, suggesting potential effectiveness of 3D7-based vaccines in blocking transmission. INTERPRETATION: These findings offer valuable insights into the selection of optimal vaccine candidates against P. falciparum. Based on our results, we recommend prioritising conserved merozoite antigens and transmission-blocking antigens. Combining these antigens in multi-stage approaches may be particularly promising for malaria vaccine development initiatives. FUNDING: Purdue Department of Biological Sciences; Puskas Memorial Fellowship; National Institute of Allergy and Infectious Diseases (U19AI089680).

Detection of Human Adenovirus and Rotavirus in Wastewater in Lusaka, Zambia: Demonstrating the Utility of Environmental Surveillance for the Community.

Enteric infections due to viral pathogens are a major public health concern. Detecting the risk areas requires a strong surveillance system for pathogenic viruses in sources such as wastewater. Towards building an environmental surveillance system in Zambia, we aimed to identify group A rotavirus (RVA) and human adenovirus (HAdV) in wastewater. Convenient sampling was conducted at four study sites every Tuesday for five consecutive weeks. The research team focused on three different methods of viral concentration to determine the suitability in terms of cost and applicability for a regular surveillance system: the bag-mediated filtration system (BMFS), polyethylene glycol-based (PEG) precipitation, and skimmed milk (SM) flocculation. We screened 20 wastewater samples for HAdV and RVA using quantitative polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR). Of the 20 samples tested using qPCR, 18/20 (90%) tested positive for HAdV and 14/20 (70%) tested positive for RVA. For the genetic sequencing, qPCR positives were subjected to cPCR, of which 12 positives were successfully amplified. The human adenovirus was identified with a nucleotide identity range of 98.48% to 99.53% compared with the reference genome from GenBank. The BMFS and SM flocculation were the most consistent viral concentration methods for HAdV and RVA, respectively. A statistical analysis of the positives showed that viral positivity differed by site (p < 0.001). SM and PEG may be the most appropriate options in resource-limited settings such as Zambia due to the lower costs associated with these concentration methods. The demonstration of HAdV and RVA detection in wastewater suggests the presence of the pathogens in the communities under study and the need to establish a routine wastewater surveillance system for the identification of pathogens.

Diversity and selection analyses identify transmission-blocking antigens as the optimal vaccine candidates in Plasmodium falciparum.

Background: A highly effective vaccine for malaria remains an elusive target, at least in part due to the under-appreciated natural parasite variation. This study aimed to investigate genetic and structural variation, and immune selection of leading malaria vaccine candidates across the Plasmodium falciparum's life cycle. Methods: We analyzed 325 P. falciparum whole genome sequences from Zambia, in addition to 791 genomes from five other African countries available in the MalariaGEN Pf3k Rdatabase. Ten vaccine antigens spanning three life-history stages were examined for genetic and structural variations, using population genetics measures, haplotype network analysis, and 3D structure selection analysis. Findings: Among the ten antigens analyzed, only three in the transmission-blocking vaccine category display P. falciparum 3D7 as the dominant haplotype. The antigens AMA1, CSP, MSP119 and CelTOS, are much more diverse than the other antigens, and their epitope regions are under moderate to strong balancing selection. In contrast, Rh5, a blood stage antigen, displays low diversity yet slightly stronger immune selection in the merozoite-blocking epitope region. Except for CelTOS, the transmission-blocking antigens Pfs25, Pfs48/45, Pfs230, Pfs47, and Pfs28 exhibit minimal diversity and no immune selection in epitopes that induce strain-transcending antibodies, suggesting potential effectiveness of 3D7-based vaccines in blocking transmission. Interpretations: These findings offer valuable insights into the selection of optimal vaccine candidates against P. falciparum. Based on our results, we recommend prioritizing conserved merozoite antigens and transmission-blocking antigens. Combining these antigens in multi-stage approaches may be particularly promising for malaria vaccine development initiatives. Funding: Purdue Department of Biological Sciences; Puskas Memorial Fellowship; National Institute of Allergy and Infectious Diseases (U19AI089680).

Genomics reveals heterogeneous Plasmodium falciparum transmission and selection signals in Zambia.

BACKGROUND: Genomic surveillance is crucial for monitoring malaria transmission and understanding parasite adaptation to interventions. Zambia lacks prior nationwide efforts in malaria genomic surveillance among African countries. METHODS: We conducted genomic surveillance of Plasmodium falciparum parasites from the 2018 Malaria Indicator Survey in Zambia, a nationally representative household survey of children under five years of age. We whole-genome sequenced and analyzed 241 P. falciparum genomes from regions with varying levels of malaria transmission across Zambia and estimated genetic metrics that are informative about transmission intensity, genetic relatedness between parasites, and selection. RESULTS: We provide genomic evidence of widespread within-host polygenomic infections, regardless of epidemiological characteristics, underscoring the extensive and ongoing endemic malaria transmission in Zambia. Our analysis reveals country-level clustering of parasites from Zambia and neighboring regions, with distinct separation in West Africa. Within Zambia, identity by descent (IBD) relatedness analysis uncovers local spatial clustering and rare cases of long-distance sharing of closely related parasite pairs. Genomic regions with large shared IBD segments and strong positive selection signatures implicate genes involved in sulfadoxine-pyrimethamine and artemisinin combination therapies drug resistance, but no signature related to chloroquine resistance. Furthermore, differences in selection signatures, including drug resistance loci, are observed between eastern and western Zambian parasite populations, suggesting variable transmission intensity and ongoing drug pressure. CONCLUSIONS: Our findings enhance our understanding of nationwide P. falciparum transmission in Zambia, establishing a baseline for analyzing parasite genetic metrics as they vary over time and space. These insights highlight the urgency of strengthening malaria control programs and surveillance of antimalarial drug resistance.

Malaria is caused by a parasite that is spread to humans via mosquito bites. It is a leading cause of death in children under five years old in sub-Saharan Africa. Analysis of the malaria parasite's complete set of DNA (its genome) can help us to understand transmission of the disease and how this changes in response to different strategies to control the disease. We analyzed the genomes of malaria parasites from children across Zambia. Our study revealed that 77% of children harbored multiple parasite strains, which suggests that local transmission (transmission between people within the same local area) is high. Genetic evidence for long-distance transmission was rarer. Furthermore, our findings suggest parasites are evolving in response to antimalarial drugs. Our study enhances our understanding of malaria dynamics in Zambia and may help to inform strategies for improved surveillance and control.

Genomics reveals heterogeneous Plasmodium falciparum transmission and population differentiation in Zambia and bordering countries.

Genomic surveillance plays a critical role in monitoring malaria transmission and understanding how the parasite adapts in response to interventions. We conducted genomic surveillance of malaria by sequencing 241 Plasmodium falciparum genomes from regions with varying levels of malaria transmission across Zambia. We found genomic evidence of high levels of within-host polygenomic infections, regardless of epidemiological characteristics, underscoring the extensive and ongoing endemic malaria transmission in the country. We identified country-level clustering of parasites from Zambia and neighboring countries, and distinct clustering of parasites from West Africa. Within Zambia, our identity by descent (IBD) relatedness analysis uncovered spatial clustering of closely related parasite pairs at the local level and rare cases of long-distance sharing. Genomic regions with large shared IBD segments and strong positive selection signatures identified genes involved in sulfadoxine-pyrimethamine and artemisinin combination therapies drug resistance, but no signature related to chloroquine resistance. Together, our findings enhance our understanding of P. falciparum transmission nationwide in Zambia and highlight the urgency of strengthening malaria control programs and surveillance of antimalarial drug resistance.

Flexible and cost-effective genomic surveillance of P. falciparum malaria with targeted nanopore sequencing.

Genomic surveillance of Plasmodium falciparum malaria can provide policy-relevant information about antimalarial drug resistance, diagnostic test failure, and the evolution of vaccine targets. Yet the large and low complexity genome of P. falciparum complicates the development of genomic methods, while resource constraints in malaria endemic regions can limit their deployment. Here, we demonstrate an approach for targeted nanopore sequencing of P. falciparum from dried blood spots (DBS) that enables cost-effective genomic surveillance of malaria in low-resource settings. We release software that facilitates flexible design of amplicon sequencing panels and use this software to design two target panels for P. falciparum. The panels generate 3-4 kbp reads for eight and sixteen targets respectively, covering key drug-resistance associated genes, diagnostic test antigens, polymorphic markers and the vaccine target csp. We validate our approach on mock and field samples, demonstrating robust sequencing coverage, accurate variant calls within coding sequences, the ability to explore P. falciparum within-sample diversity and to detect deletions underlying rapid diagnostic test failure.

Reactive focal drug administration associated with decreased malaria transmission in an elimination setting: Serological evidence from the cluster-randomized CoRE study.

Efforts to eliminate malaria transmission need evidence-based strategies. However, accurately assessing end-game malaria elimination strategies is challenging due to the low level of transmission and the rarity of infections. We hypothesised that presumptively treating individuals during reactive case detection (RCD) would reduce transmission and that serology would more sensitively detect this change over standard approaches. We conducted a cluster randomised control trial (NCT02654912) of presumptive reactive focal drug administration (RFDA-intervention) compared to the standard of care, reactive focal test and treat (RFTAT-control) in Southern Province, Zambia-an area of low seasonal transmission (overall incidence of ~3 per 1,000). We measured routine malaria incidence from health facilities as well as PCR parasite prevalence / antimalarial seroprevalence in an endline cross-sectional population survey. No significant difference was identified from routine incidence data and endline prevalence by polymerase chain reaction (PCR) had insufficient numbers of malaria infections (i.e., 16 infections among 6,276 children) to assess the intervention. Comparing long-term serological markers, we found a 19% (95% CI = 4-32%) reduction in seropositivity for the RFDA intervention using a difference in differences approach incorporating serological positivity and age. We also found a 37% (95% CI = 2-59%) reduction in seropositivity to short-term serological markers in a post-only comparison. These serological analyses provide compelling evidence that RFDA both has an impact on malaria transmission and is an appropriate end-game malaria elimination strategy. Furthermore, serology provides a more sensitive approach to measure changes in transmission that other approaches miss, particularly in very low transmission settings. Trial Registration: Registered at www.clinicaltrials.gov (NCT02654912, 13/1/2016).

What is the prevalence of COVID-19 detection by PCR among deceased individuals in Lusaka, Zambia? A postmortem surveillance study.

OBJECTIVES: To determine the prevalence of COVID-19 postmortem setting in Lusaka, Zambia. DESIGN: A systematic, postmortem prevalence study. SETTING: A busy, inner-city morgue in Lusaka. PARTICIPANTS: We sampled a random subset of all decedents who transited the University Teaching Hospital morgue. We sampled the posterior nasopharynx of decedents using quantitative PCR. Prevalence was weighted to account for age-specific enrolment strategies. INTERVENTIONS: Not applicable-this was an observational study. PRIMARY OUTCOMES: Prevalence of COVID-19 detections by PCR. Results were stratified by setting (facility vs community deaths), age, demographics and geography and time. SECONDARY OUTCOMES: Shifts in viral variants; causal inferences based on cycle threshold values and other features; antemortem testing rates. RESULTS: From 1118 decedents enrolled between January and June 2021, COVID-19 was detected among 32.0% (358/1116). Roughly four COVID-19+ community deaths occurred for every facility death. Antemortem testing occurred for 52.6% (302/574) of facility deaths but only 1.8% (10/544) of community deaths and overall, only ~10% of COVID-19+ deaths were identified in life. During peak transmission periods, COVID-19 was detected in ~90% of all deaths. We observed three waves of transmission that peaked in July 2020, January 2021 and ~June 2021: the AE.1 lineage and the Beta and Delta variants, respectively. PCR signals were strongest among those whose deaths were deemed 'probably due to COVID-19', and weakest among children, with an age-dependent increase in PCR signal intensity. CONCLUSIONS: COVID-19 was common among deceased individuals in Lusaka. Antemortem testing was rarely done, and almost never for community deaths. Suspicion that COVID-19 was the cause of deaths was highest for those with a respiratory syndrome and lowest for individuals <19 years.

Performance evaluation of RDT, light microscopy, and PET-PCR for detecting Plasmodium falciparum malaria infections in the 2018 Zambia National Malaria Indicator Survey.

BACKGROUND: Zambia continues to advance on the path to elimination with significant reductions in malaria morbidity and mortality. Crucial components that have contributed to progress thus far and are necessary for achieving the national malaria elimination goals include properly identifying and treating all malaria cases through accurate diagnosis. This study sought to compare and assess the diagnostic performance of Rapid Diagnostic Tests (RDT) and Light Microscopy (LM) with photo-induced electron transfer polymerase chain reaction (PET-PCR) as the gold standard using 2018 Malaria Indicator Survey (MIS) data across Zambia to better understand diagnostic accuracy metrics and how these vary across a transmission gradient. METHODS: Cross-sectional samples collected in a nationally representative survey from 7 provinces in Zambia were tested for the presence of malaria parasites by light microscopy (LM), rapid diagnostic test (RDT) and the gold standard PET-PCR. Diagnostic performance was assessed including sensitivity, specificity, negative- and positive-predictive values across a wide malaria transmission spectrum. Diagnostic accuracy metrics were measured, and statistically significant differences were calculated between test methods for different outcome variables. RESULTS: From the individuals included in the MIS, the overall prevalence of Plasmodium falciparum malaria was 32.9% by RDT, 19.4% by LM, and 23.2% by PET-PCR. Herein, RDT and LM diagnostic performance was compared against gold standard PET-PCR with LM displaying a higher diagnostic accuracy than RDTs (91.3% vs. 84.6% respectively) across the transmission spectrum in Zambia. However, the performance of both diagnostics was significantly reduced in low parasitaemia samples. Consistent with previous studies, RDT diagnostic accuracy was predominantly affected by a high rate of false positives. CONCLUSIONS: RDTs and LM both perform well across a range of transmission intensities within their respective target applications, i.e., in the community, for the former, where ease of use and speed of result is critical, and at the health facility, for the latter, where accuracy is prioritized. However, the performance of both diagnostic methods is adversely affected by low parasitaemia infections. As Zambia moves towards elimination more sensitive tools may be required to identify the last cases.

Detection of B.1.351 SARS-CoV-2 Variant Strain - Zambia, December 2020.

The first laboratory-confirmed cases of coronavirus disease 2019 (COVID-19), the illness caused by SARS-CoV-2, in Zambia were detected in March 2020 (1). Beginning in July, the number of confirmed cases began to increase rapidly, first peaking during July-August, and then declining in September and October (Figure). After 3 months of relatively low case counts, COVID-19 cases began rapidly rising throughout the country in mid-December. On December 18, 2020, South Africa published the genome of a SARS-CoV-2 variant strain with several mutations that affect the spike protein (2). The variant included a mutation (N501Y) associated with increased transmissibility.†,§ SARS-CoV-2 lineages with this mutation have rapidly expanded geographically.¶,** The variant strain (PANGO [Phylogenetic Assignment of Named Global Outbreak] lineage B.1.351††) was first detected in the Eastern Cape Province of South Africa from specimens collected in early August, spread within South Africa, and appears to have displaced the majority of other SARS-CoV-2 lineages circulating in that country (2). As of January 10, 2021, eight countries had reported cases with the B.1.351 variant. In Zambia, the average number of daily confirmed COVID-19 cases increased 16-fold, from 44 cases during December 1-10 to 700 during January 1-10, after detection of the B.1.351 variant in specimens collected during December 16-23. Zambia is a southern African country that shares substantial commerce and tourism linkages with South Africa, which might have contributed to the transmission of the B.1.351 variant between the two countries.

Data on selected antimalarial drug resistance markers in Zambia.

This article describes data on selected resistance markers for antimalarial drugs used in Zambia. Antimalarial drug resistance has hindered the progress in the control and elimination of malaria. Blood samples were collected during a cross-sectional household survey, conducted during the peak malaria transmission, April to May of 2017. Dried blood spots were collected during the survey and transported to a laboratory for analysis. The analysed included polymerase chain reaction (PCR) followed by high resolution melt (HRM) for mutations associated with Sulfadoxine-pyrimethamine resistance in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps) genes. Mutations associated with artemether-lumefantrine resistance in falciparum multi-drug resistance gene 1 (Pfmdr1) were also assessed using PCR and HRM analysis, whereas the P. falciparum Kelch 13 (PfK13) gene was assessed using nested PCR followed by amplicon sequencing.

Surveillance of molecular markers for antimalarial resistance in Zambia: Polymorphism of Pfkelch 13, Pfmdr1 and Pfdhfr/Pfdhps genes.

Antimalarial resistance is an inevitable feature of control efforts and a key threat to achieving malaria elimination. Plasmodium falciparum, the deadliest of several species causing human malaria, has developed resistance to essentially all antimalarials. This study sought to investigate the prevalence of molecular markers associated with resistance to sulfadoxine-pyrimethamine (SP) and artemether-lumefantrine (AL) in Southern and Western provinces in Zambia. SP is used primarily for intermittent preventive treatment during pregnancy, while AL is the first-line antimalarial for uncomplicated malaria in Zambia. Blood samples were collected from household members of all ages in a cross-sectional survey conducted during peak malaria transmission, April to May of 2017, and amplified by polymerase chain reaction (PCR). Amplicons were then analysed by high-resolution melt following PCR to identify mutations associated with SP resistance in the P. falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps) genes and lumefantrine resistance in the P. falciparum multi-drug resistance 1 (Pfmdr1) gene. Finally, artemether resistance was assessed in the P. falciparum Kelch 13 (PfK13) gene using nested PCR followed by amplicon sequencing. The results showed a high frequency of genotypic-resistant Pfdhps A437G (93.2%) and Pfdhfr C59R (86.7%), N51I (80.9%), and S108N (80.8%) of which a high proportion (82.4%) were quadruple mutants (Pfdhfr N51I, C59R, S108N +Pfdhps A437G). Pfmrd1 N86Y, Y186F, and D1246Y - NFD mutant haplotypes were observed in 41.9% of isolates. The high prevalence of quadruple dhps/dhfr mutants indicates strong antifolate drug pressure from SP or other drugs (e.g., co-trimoxazole). Three samples contained PfK13 mutations, two synonymous (T478 and V666) and one non-synonymous (A578S), none of which have been associated with delayed clearance. This suggests that artemisinin remains efficacious in Zambia, however, the moderately high prevalence of approximately 40% Pfmdr1 NFD mutations calls for close monitoring of AL.

Pyrethroid and Carbamate Resistance in Anopheles funestus Giles along Lake Kariba in Southern Zambia.

Whereas data on insecticide resistance and its underlying mechanisms exist for parts of Zambia, data remain limited in the southern part of the country. This study investigated the status of insecticide resistance, metabolic mechanisms, and parasite infection in Anopheles funestus along Lake Kariba in southern Zambia. Indoor-resting mosquitoes were collected from 20 randomly selected houses within clusters where a mass drug administration trial was conducted and raised to F1 progeny. Non-blood-fed 2- to 5-day-old female An. funestus were exposed to WHO insecticide-impregnated papers with 0.05% deltamethrin, 0.1% bendiocarb, 0.25% pirimiphos-methyl, or 4% dichloro-diphenyl-trichloroethane (DDT). In separate assays, An. funestus were pre-exposed to piperonyl butoxide (PBO) to determine the presence of monooxygenases. Wild-caught An. funestus that had laid eggs for susceptibility assays were screened for circumsporozoite protein of Plasmodium falciparum by ELISA, and sibling species were identified by polymerase chain reaction. Anopheles funestus showed resistance to deltamethrin and bendiocarb but remained susceptible to pirimiphos-methyl and DDT. The pre-exposure of An. funestus to PBO restored full susceptibility to deltamethrin but not to bendiocarb. The overall sporozoite infection rate in An. funestus populations was 5.8%. Detection of pyrethroid and carbamate resistance in An. funestus calls for increased insecticide resistance monitoring to guide planning and selection of effective insecticide resistance management strategies. To prevent the development of resistance and reduce the underlying vectorial capacity of mosquitoes in areas targeted for malaria elimination, an effective integrated vector management strategy is needed.

A Longitudinal Cohort to Monitor Malaria Infection Incidence during Mass Drug Administration in Southern Province, Zambia.

Rigorous evidence of effectiveness is needed to determine where and when to apply mass drug administration (MDA) or focal MDA (fMDA) as part of a malaria elimination strategy. The Zambia National Malaria Elimination Centre recently completed a community-randomized controlled trial in Southern Province to evaluate MDA and fMDA for transmission reduction. To assess the role of MDA and fMDA on infection incidence, we enrolled a longitudinal cohort for an 18-month period of data collection including monthly malaria parasite infection detection based on polymerase chain reaction and compared time to first infection and cumulative infection incidence outcomes across study arms using Cox proportional hazards and negative binomial models. A total of 2,026 individuals from 733 households were enrolled and completed sufficient follow-up for inclusion in analysis. Infection incidence declined dramatically across all study arms during the period of study, and MDA was associated with reduced risk of first infection (hazards ratio: 0.36; 95% CI: 0.16-0.80) and cumulative infection incidence during the first rainy season (first 5 months of follow-up) (incidence rate ratio: 0.34; 95% CI: 0.12-0.95). No significant effect was found for fMDA or for either arm over the full study period. Polymerase chain reaction infection status at baseline was strongly associated with follow-up infection. The short-term effects of MDA suggest it may be an impactful accelerator of transmission reduction in areas with high coverage of case management and vector control and should be considered as part of a malaria elimination strategy.

Prevalence of Plasmodium falciparum and Non-falciparum Infections by Photo-Induced Electron Transfer-PCR in a Longitudinal Cohort of Individuals Enrolled in a Mass Drug Administration Trial in Southern Province, Zambia.

Malaria burden in Zambia has significantly declined over the last decade because of improved coverage of several key malaria interventions (e.g., vector control, case management, bed net distributions, and enhanced surveillance/responses). Campaign-based mass drug administration (MDA) and focal MDA (fMDA) were assessed in a trial in Southern Province, Zambia, to identify its utility in elimination efforts. As part of the study, a longitudinal cohort was visited and tested (by PCR targeting the 18s rRNA and a Plasmodium falciparum-specific rapid diagnostic test [RDT] from SD Bioline) every month for the trial duration (18 months). Overall, there was high concordance (> 97%) between the PCR and RDT results, using the PCR as the gold standard. The RDTs had high specificity and negative predictive values (98.5% and 98.6%, respectively) but low sensitivity (53.0%) and a low positive predictive value (53.8%). There was evidence for persistent antigenemia affecting the low specificity of the RDT, while false-negative RDTs were associated with a lower parasite density than true positive RDTs. Plasmodium falciparum was the dominant species identified, with 98.3% of all positive samples containing P. falciparum. Of these, 97.5% were mono-infections and 0.8% coinfections with one other species. Plasmodium malariae was found in 1.4% of all positive samples (50% mono-infections and 50% coinfections with P. falciparum), whereas Plasmodium ovale was found in 1.1% of all positive samples (90% mono-infections and 10% coinfections with P. falciparum). Although MDA/fMDA appeared to reduce P. malariae prevalence, P. ovale prevalence appeared unchanged.

Evidence for Reduced Malaria Parasite Population after Application of Population-Level Antimalarial Drug Strategies in Southern Province, Zambia.

A mass drug administration trial was carried out in Southern Province, Zambia, between 2014 and 2016, in conjunction with a standard of care package that included improved surveillance, increased access to malaria case management, and sustained high levels of vector control coverage. This was preceded by mass test and treatment in the same area from 2011 to 2013. Concordant decreases in malaria prevalence in Southern Province and deaths attributed to malaria in Zambia over this time suggest that these strategies successfully reduced the malaria burden. Genetic epidemiological studies were used to assess the consequences of these interventions on parasite population structure. Analysis of parasite material derived from 1,620 rapid diagnostic test (RDT)-positive individuals obtained from studies to evaluate trial outcomes revealed a reduction in the average complexity of infection and consequential increase in the proportion of infections that harbored a single parasite genome (monogenomic infections). Highly related parasites, consistent with inbreeding, were detected after interventions were deployed. Geographical analysis indicated that the highly related infections were both clustered focally and dispersed across the study area. These findings provide genetic evidence for a reduced parasite population, with indications of inbreeding following the application of comprehensive interventions, including drug-based campaigns, that reduced the malaria burden in Southern Province. Genetic data additionally revealed the relationship between individual infections in the context of these population-level patterns, which has the potential to provide useful data for stratification and targeting of interventions to reduce the malaria burden.

The use of spatial and genetic tools to assess Plasmodium falciparum transmission in Lusaka, Zambia between 2011 and 2015.

BACKGROUND: Zambia has set itself the ambitious target of eliminating malaria by 2021. To continue tracking transmission to zero, new interventions, tools and approaches are required. METHODS: Urban reactive case detection (RCD) was performed in Lusaka city from 2011 to 2015 to better understand the location and drivers of malaria transmission. Briefly, index cases were followed to their home and all consenting individuals living in the index house and nine proximal houses were tested with a malaria rapid diagnostic test and treated if positive. A brief survey was performed and for certain responses, a dried blood spot sample collected for genetic analysis. Aggregate health facility data, individual RCD response data and genetic results were analysed spatially and against environmental correlates. RESULTS: Total number of malaria cases remained relatively constant, while the average age of incident cases and the proportion of incident cases reporting recent travel both increased. The estimated R0 in Lusaka was < 1 throughout the study period. RCD responses performed within 250 m of uninhabited/vacant land were associated with a higher probability of identifying additional infections. CONCLUSIONS: Evidence suggests that the majority of malaria infections are imported from outside Lusaka. However there remains some level of local transmission occurring on the periphery of urban settlements, namely in the wet season. Unfortunately, due to the higher-than-expected complexity of infections and the small number of samples tested, genetic analysis was unable to identify any meaningful trends in the data.

En-route to the 'elimination' of genotypic chloroquine resistance in Western and Southern Zambia, 14 years after chloroquine withdrawal.

BACKGROUND: Anti-malarial resistance is, and continues to be a significant challenge in the fight against malaria and a threat to achieving malaria elimination. In Zambia, chloroquine (CQ), a safe, affordable and well-tolerated drug, was removed from use in 2003 due to high levels of resistance evidenced with treatment failure. This study sought to investigate the prevalence of chloroquine resistance markers in Southern and Western Provinces of Zambia 14 years after the withdrawal of CQ. METHODS: Data from a cross-sectional, all-age household survey, conducted during the peak malaria transmission season (April-May 2017) was analysed. During the all-age survey, socio-demographic information and coverage of malaria interventions were collected. Consenting individuals were tested for malaria with a rapid diagnostic test and a spot of blood collected on filter paper to create a dried blood spot (DBS). Photo-induced electronic transfer-polymerase chain reaction (PET-PCR) was used to analyse the DBS for the presence of all four malaria species. Plasmodium falciparum positive samples were analysed by high resolution melt (HRM) PCR to detect the presence of genotypic markers of drug resistance in the P. falciparum chloroquine resistance transporter (Pfcrt) and P. falciparum multi-drug resistance (Pfmdr) genes. RESULTS: A total of 181 P. falciparum positive samples were examined for pfcrt K76T and MDR N86. Of the 181 samples 155 successfully amplified for Pfcrt and 145 for Pfmdr N86. The overall prevalence of CQ drug-resistant parasites was 1.9% (3/155), with no significant difference between the two provinces. No N86Y/F mutations in the Pfmdr gene were observed in any of the sample. CONCLUSION: This study reveals the return of CQ sensitive parasites in Southern and Western Provinces of Zambia 14 years after its withdrawal. Surveillance of molecular resistant markers for anti-malarials should be included in the Malaria Elimination Programme so that resistance is monitored country wide.