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IntroductionZambia experienced a major cholera outbreak in 2017–2018, with more than 5905 cases reported countrywide, predominantly from the peri-urban slums of Lusaka city. The WHO recommends the use of oral cholera vaccines (OCVs) together with traditional control measures, including health promotion, provision of safe water and improving sanitation, in cholera endemic areas and during cholera outbreaks. In response to this outbreak, the Zambian government implemented the OVC campaign and administered the Euvichol-plus vaccine in the high-risk subdistricts of Lusaka. Although OCVs have been shown to be effective in preventing cholera infection in cholera endemic and outbreak settings, the effectiveness of the Euvichol-plus vaccine has not yet been evaluated in Zambia. This study aimed to determine the effectiveness of two doses of OCV administered during the 2017/2018 vaccination campaign.MethodsWe conducted a matched case–control study involving 79 cases and 316 controls following the mass vaccination campaign in the four subdistricts of Lusaka (Chawama, Chipata, Kanyama and Matero). Matching of controls was based on the place of residence, age and sex. Conditional logistic regression was used for analysis. Adjusted OR (AOR), 95% CI and vaccine effectiveness (1-AOR) for two doses of Euvichol-plus vaccine and any dose were estimated (p<0.05).ResultsThe AOR vaccine effectiveness for two doses of Euvichol-plus OCV was 81.0% (95% CI 66.0% to 78.0%; pConclusionThese findings show that two doses of Euvichol-plus OCV are effective in a cholera outbreak setting in Lusaka, Zambia. The findings also indicate that two doses are more effective than a single dose and thus support the use of two doses of the vaccine as part of an integrated intervention to cholera control during outbreaks.

The global malaria burden has decreased substantially, but gains have been uneven both within and between countries. In Zambia, the malaria burden remains high in northern and eastern regions of the country. To effectively reduce malaria transmission in these areas, evidence-based intervention strategies are needed. Zambia’s National Malaria Control Centre conducted targeted indoor residual spraying (IRS) in 40 high-burden districts from 2014 to 2016 using the novel organophosphate insecticide pirimiphos-methyl. The Southern and Central Africa International Centers of Excellence for Malaria Research conducted an evaluation of the impact of the IRS campaign on household vector abundance in Nchelenge District, Luapula Province. From April 2012 to July 2017, field teams conducted indoor overnight vector collections from 25 to 30 households per month using Centers for Disease Control light traps. Changes in indoor anopheline counts before versus after IRS were assessed by species using negative binomial regression models with robust standard errors, controlling for geographic and climatological covariates. Counts of Anopheles funestus declined by approximately 50% in the study area and within areas targeted for IRS, and counts of Anopheles gambiae declined by approximately 40%. Within targeted areas, An. funestus counts declined more in sprayed households than in unsprayed households; however, this relationship was not observed for An. gambiae. The moderate decrease in indoor vector abundance indicates that IRS with pirimiphos-methyl is an effective vector control measure, but a more comprehensive package of interventions is needed with sufficient coverage to effectively reduce the malaria burden in this setting.

Background While the utility of parasite genotyping for malaria elimination has been extensively documented in low to moderate transmission settings, it has been less well-characterized in holoendemic regions. High malaria burden settings have received renewed attention acknowledging their critical role in malaria elimination. Defining the role for parasite genomics in driving these high burden settings towards elimination will enhance future control programme planning. Methods Amplicon deep sequencing was used to characterize parasite population genetic diversity at polymorphic Plasmodium falciparum loci, Pfama1 and Pfcsp, at two timepoints in June–July 2016 and January–March 2017 in a high transmission region along the international border between Luapula Province, Zambia and Haut-Katanga Province, the Democratic Republic of the Congo (DRC). Results High genetic diversity was observed across both seasons and in both countries. No evidence of population structure was observed between parasite populations on either side of the border, suggesting that this region may be one contiguous transmission zone. Despite a decline in parasite prevalence at the sampling locations in Haut-Katanga Province, no genetic signatures of a population bottleneck were detected, suggesting that larger declines in transmission may be required to reduce parasite genetic diversity. Analysing rare variants may be a suitable alternative approach for detecting epidemiologically important genetic signatures in highly diverse populations; however, the challenge is distinguishing true signals from potential artifacts introduced by small sample sizes. Conclusions Continuing to explore and document the utility of various parasite genotyping approaches for understanding malaria transmission in holoendemic settings will be valuable to future control and elimination programmes, empowering evidence-based selection of tools and methods to address pertinent questions, thus enabling more efficient resource allocation.

BACKGROUND: Severe malaria resulting from Plasmodium falciparum infection is the leading parasitic cause of death in children worldwide, and severe malarial anemia (SMA) is the most common clinical presentation. The evidence in support of current blood transfusion guidelines for patients with SMA is limited. METHODS: We conducted a retrospective cohort study of 911 hospitalized children with SMA in a holoendemic region of Zambia to examine the association of whole blood transfusion with in-hospital survival. Data were analyzed in adjusted logistic regression models using multiple imputation for missing data. RESULTS: The median age of patients was 24 months (interquartile range, 16–30) and overall case fatality was 16%. Blood transfusion was associated with 35% reduced odds of death in children with SMA (odds ratio, 0.65; 95% confidence interval, .52–.81; P = .0002) corresponding to a number-needed-to-treat (NNT) of 14 patients. Children with SMA complicated by thrombocytopenia were more likely to benefit from transfusion than those without thrombocytopenia (NNT = 5). Longer storage time of whole blood was negatively associated with survival and with the posttransfusion rise in the platelet count but was not associated with the posttransfusion change in hemoglobin concentration. CONCLUSIONS: Whole blood given to pediatric patients with SMA was associated with improved survival, mainly among those with thrombocytopenia who received whole blood stored for

For a decade, the Southern and Central Africa International Center of Excellence for Malaria Research has operated with local partners across study sites in Zambia and Zimbabwe that range from hypo- to holoendemic and vary ecologically and entomologically. The burden of malaria and the impact of control measures were assessed in longitudinal cohorts, cross-sectional surveys, passive and reactive case detection, and other observational designs that incorporated multidisciplinary scientific approaches: classical epidemiology, geospatial science, serosurveillance, parasite and mosquito genetics, and vector bionomics. Findings to date have helped elaborate the patterns and possible causes of sustained low-to-moderate transmission in southern Zambia and eastern Zimbabwe and recalcitrant high transmission and fatality in northern Zambia. Cryptic and novel mosquito vectors, asymptomatic parasite reservoirs in older children, residual parasitemia and gametocytemia after treatment, indoor residual spraying timed dyssynchronously to vector abundance, and stockouts of essential malaria commodities, all in the context of intractable rural poverty, appear to explain the persistent malaria burden despite current interventions. Ongoing studies of high-resolution transmission chains, parasite population structures, long-term malaria periodicity, and molecular entomology are further helping to lay new avenues for malaria control in southern and central Africa and similar settings.

Background Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended by the World Health Organization for the prevention of malaria in pregnancy (MIP)-associated adverse outcomes in high burden areas. However, the efficacy of IPTp-SP has decreased in step with increasing parasite drug resistance. Suitable alternative strategies are needed. Methods This is a protocol for a phase IIIb open-label, two-armed randomized controlled superiority trial to assess the safety and efficacy of a hybrid approach to IPTp combining screening and treatment with dihydroartemisinin-piperaquine (DP) to the current IPTp-SP regimen at the first antenatal care clinic visit. Pregnant women without HIV infection and without signs or symptoms of malaria will be randomized to either standard IPTp-SP or hybrid IPTp-SP plus screening and treatment (IPTp-SP+). In the IPTp-SP+ arm, participants who screen positive by rapid diagnostic test for P. falciparum will be treated with DP at the first antenatal visit while those who screen negative will receive SP per current guidelines. All participants will be administered SP on days 35 and 63 and will be actively followed biweekly up to day 63 and then monthly until delivery. Infants will be followed until 1 year after delivery. The primary endpoint is incident PCR-confirmed MIP at day 42. Secondary endpoints include incident MIP at other time points, placental malaria, congenital malaria, hemoglobin trends, birth outcomes, and incidence of adverse events in infants up to the first birthday. Discussion A hybrid approach to IPTp that combines screening and treatment with an artemisinin-based combination therapy at the first visit with standard IPTp-SP is hypothesized to confer added benefit over IPTp-SP alone in a high malaria transmission area with prevalent SP resistant parasites. Trial registration Pan African Clinical Trials Registry 201905721140808. Registered retrospectively on 11 May 2019

We attempted to identify Plasmodium falciparum histidine-rich protein 2/3 (pfhrp2/3) deletions among rapid diagnostic test (RDT)-negative but PCR- or microscopy-positive P. falciparum-infected individuals in areas of low transmission (Choma District, 2009-2011) and high transmission (Nchelenge District, 2015-2017) in Zambia. Through community-based surveys, 5,167 participants were screened at 1,147 households by P. falciparum histidine-rich protein 2 (PfHRP2)-based RDTs. Slides were made and dried blood spots were obtained for molecular analysis. Of 28 samples with detectable P. falciparum DNA, none from Nchelenge District were pfhrp2/3 negative. All eight samples from Choma District had detectable pfhrp3 genes, but pfhrp2 was undetectable in three. DNA concentrations of pfhrp2-negative samples were low (< 0.001 ng/μL). These findings suggest that PfHRP2-based RDTs remain effective tools for malaria diagnosis in Nchelenge District, but further study is warranted to understand the potential for pfhrp2/3 deletions in southern Zambia where malaria transmission declined over the past decade.

Background The World Health Organization (WHO) recommendation of treating uncomplicated malaria during the second and third trimester of pregnancy with an artemisinin-based combination therapy (ACT) has already been implemented by all sub-Saharan African countries. However, there is limited knowledge on the effect of ACT on pregnancy outcomes, and on newborn and infant’s health. Methods Pregnant women with malaria in four countries (Burkina Faso, Ghana, Malawi and Zambia) were treated with either artemether–lumefantrine (AL), amodiaquine–artesunate (ASAQ), mefloquine-artesunate (MQAS), or dihydroartemisinin–piperaquine (DHA–PQ); 3127 live new-borns (822 in the AL, 775 in the ASAQ, 765 in the MQAS and 765 in the DHAPQ arms) were followed-up until their first birthday. Results Prevalence of placental malaria and low birth weight were 28.0% (738/2646) and 16.0% (480/2999), respectively, with no significant differences between treatment arms. No differences in congenital malformations (p = 0.35), perinatal mortality (p = 0.77), neonatal mortality (p = 0.21), and infant mortality (p = 0.96) were found. Conclusions Outcome of pregnancy and infant survival were similar between treatment arms indicating that any of the four artemisinin-based combinations could be safely used during the second and third trimester of pregnancy without any adverse effect on the baby. Nevertheless, smaller safety differences between artemisinin-based combinations cannot be excluded; country-wide post-marketing surveillance would be very helpful to confirm such findings. Trial registration ClinicalTrials.gov, NCT00852423, Registered on 27 February 2009, https://clinicaltrials.gov/ct2/show/NCT00852423

Background Determining gestational age in resource-poor settings is challenging because of limited availability of ultrasound technology and late first presentation to antenatal clinic. Last menstrual period (LMP), symphysio-pubis fundal height (SFH) and Ballard Score (BS) at delivery are therefore often used. We assessed the accuracy of LMP, SFH, and BS to estimate gestational age at delivery and preterm birth compared to ultrasound (US) using a large dataset derived from a randomized controlled trial in pregnant malaria patients in four African countries. Methods Mean and median gestational age for US, LMP, SFH and BS were calculated for the entire study population and stratified by country. Correlation coefficients were calculated using Pearson’s rho, and Bland Altman plots were used to calculate mean differences in findings with 95% limit of agreements. Sensitivity, specificity, positive predictive value and negative predictive value were calculated considering US as reference method to identify term and preterm babies. Results A total of 1630 women with P. falciparum infection and a gestational age > 24 weeks determined by ultrasound at enrolment were included in the analysis. The mean gestational age at delivery using US was 38.7 weeks (95%CI: 38.6–38.8), by LMP, 38.4 weeks (95%CI: 38.0–38.9), by SFH, 38.3 weeks (95%CI: 38.2–38.5), and by BS 38.0 weeks (95%CI: 37.9–38.1) (p

Since the late nineteenth century, the importance of house structure as a determinant of malaria risk has been recognized. Few studies to date have examined the association of housing and malaria in clinical populations. We conducted a cross-sectional study of febrile patients (n = 282) at two rural health clinics in a high malaria-transmission area of northern Zambia. Participants underwent testing for Plasmodium falciparum infection by PCR. Demographic and other risk factors including house structure, indoor residual spraying (IRS), bed net use, education level, and household income were collected. Data were fitted to logistic regression models for relational and mediation analyses. Residing in a house with a thatch roof was associated with higher odds of malaria than residing in a house with corrugated metal (odds ratio: 2.6; 95% CI: 1.0–6.3, P = 0.04). Lower income and educational attainment were also associated with greater odds of malaria. Living under a thatch roof accounted for 24% (95% CI: 14–82) of the effect of household income on malaria risk, and income accounted for 11% (95% CI: 8–19) of the effect of education. Neither IRS nor bed net use was associated with malaria risk despite large, local investments in these vector control interventions. The findings testify to malaria as a disease of rural poverty and contribute further evidence to the utility of housing improvements in vector control programs.

Severe malaria, caused by the protozoan Plasmodium, is the leading parasitic cause of mortality worldwide. Plasmodium falciparum, the most lethal among human malaria parasites, predominates in sub-Saharan Africa where it causes a disconcerting 1,200 child deaths per day concentrated in regions where malaria has proven recalcitrant to control measures or where control measures are tenuous or absent.1,2 In Zambia, there were an estimated 3.1 million cases of malaria (severe and uncomplicated) in 2016 among its population of 16.7 million and it was most prevalent in northern Zambia where it persists year round despite the recent scale-up of vector control efforts.2,3 Severe malaria can feature one or more of severe anemia (hemoglobin < 5 g/dL), cerebral edema, lactic acidosis, respiratory failure, or shock. In high-transmission, resource-limited settings, malaria evades easy case definition and is often clinically indistinguishable from bacterial meningitis, pneumonia, bacteremia, and other febrile diseases which can conflate or confuse diagnosis.4 Only a small proportion (∼2%) of P. falciparum infections progress to severe malaria, but in highly malarious regions where children may experience several infections a year, the absolute number of cases is large.5 Prompt initiation of treatment is vital. Rapid disease progression is fueled by an exponential expansion of parasites and an exuberant host immune response, and hospital deaths occur most often within 24 hours of admission.5,6 The preferred treatment is intravenous artesunate and supportive care as indicated (e.g., fluid resuscitation, blood transfusion, anticonvulsants, and ventilator and vasopressor support), but even with appropriate care, mortality can reach 20%.5 Delayed presentation to allopathic health providers on account of logistical constraints or health-seeking behaviors compounds the danger.7 Distinguishing factors that contribute to poor outcomes for children with malaria can inform clinical and programmatic strategies to lessen its toll. However, research is limited by the inherent challenges of conducting intrahospital epidemiologic studies in rural centers where malaria prevalence is greatest. We present results of a retrospective, time-matched, case-control study of hospitalized children with malaria who died (cases) and those who survived (controls) at a rural, district-level hospital in a high-transmission region of northern Zambia to identify and evaluate demographic and clinical factors associated with increased risk of death.

Monitoring prevalence of advanced human immunodeficiency virus (HIV) disease (i.e., CD4+ T-cell count

Background: New cases of HIV are increasing among young adults in Zambia; yet voluntary medical male circumcision (VMMC) coverage as an HIV prevention measure remains low. Despite having the highest HIV burden in the province, Ndola district had a VMMC coverage of 23% in 2015 compared to the national target of 80% among high-risk groups. Objectives: To determine predictive factors associated with circumcision status among male students in Ndola district. Methods: We conducted a cross-sectional study in May 2016 among students aged 18–35 years enrolled in five conveniently sampled colleges. We administered a structured questionnaire to assess the knowledge, attitudes and perceptions about VMMC. We used multivariable logistic regression to determine factors associated with male circumcision (MC) status. Results: Of 136 students interviewed, 63% were circumcised, and of those, 96% were medically circumcised. Half of all students were aged 21–24 years. Those who perceived the circumcision procedure to be ‘safe’ (adjusted odds ratio [aOR] = 5.13; 95% CI: 2.09–14.82), and knew that it reduced female to male HIV transmission risk (aOR = 3.65; 95% CI: 3.12–11.67), were more likely to be circumcised. The perception that MC promotes ‘promiscuous behaviour’ (aOR = 0.20; 95% CI: 0.07–0.61), and that sexual sensitivity is the ‘same’ regardless of circumcision status, were associated with not being circumcised (aOR = 0.13; 95% CI: 0.02–0.80). Conclusion: Students had adequate knowledge about the safety of medical circumcision, and the subsequent risk reduction of HIV infection. Interventions aimed at addressing negative sexual perceptions about circumcision may increase VMMC coverage among college students.

The Democratic Republic of the Congo (DRC) harbors 11% of global malaria cases, yet little is known about the spatial and genetic structure of the parasite population in that country. We sequence 2537 Plasmodium falciparum infections, including a nationally representative population sample from DRC and samples from surrounding countries, using molecular inversion probes - a high-throughput genotyping tool. We identify an east-west divide in haplotypes known to confer resistance to chloroquine and sulfadoxine-pyrimethamine. Furthermore, we identify highly related parasites over large geographic distances, indicative of gene flow and migration. Our results are consistent with a background of isolation by distance combined with the effects of selection for antimalarial drug resistance. This study provides a high-resolution view of parasite genetic structure across a large country in Africa and provides a baseline to study how implementation programs may impact parasite populations., The genome of the malaria parasite Plasmodium falciparum contains a record of past evolutionary forces. Here, using 2537 parasite sequences from the Democratic Republic of the Congo, the authors demonstrate how drug pressure and human movement have shaped the present-day parasite population.

Malaria transmission is dependent on the density and distribution of mosquito vectors, but drivers of vector abundance have not been adequately studied across a range of transmission settings. To inform intervention strategies for high-burden areas, further investigation is needed to identify predictors of vector abundance. Active household (HH) surveillance was conducted in Nchelenge district, Luapula Province, northern Zambia, a high-transmission setting with limited impact of malaria control. Between April 2012 and July 2017, mosquitoes were collected indoors during HH visits using CDC light traps. Demographic, environmental, and climatological correlates of vector abundance were identified using log-binomial regression models with robust standard errors. The primary malaria vectors in this setting were Anopheles funestus sensu stricto (s.s.) and Anopheles gambiae s.s. Anopheles funestus predominated in both seasons, with a peak in the dry season. Anopheles gambiae peaked at lower numbers in the rainy season. Environmental, climatic, and demographic factors were correlated with HH vector abundance. Higher vector counts were found in rural areas with low population density and among HHs close to roads and small streams. Vector counts were lower with increasing elevation and slope. Anopheles funestus was negatively associated with rainfall at lags of 2-6 weeks, and An. gambiae was positively associated with rainfall at lags of 3-10 weeks. Both vectors had varying relationships with temperature. These results suggest that malaria vector control in Nchelenge district should occur throughout the year, with an increased focus on dry-season transmission and rural areas.

Antibodies to Plasmodium falciparum are specific biomarkers that can be used to monitor parasite exposure over broader time frames than microscopy, rapid diagnostic tests, or molecular assays. Consequently, seroprevalence surveys can assist with monitoring the impact of malaria control interventions, particularly in the final stages of elimination, when parasite incidence is low. The protein array format to measure antibodies to diverse P. falciparum antigens requires only small sample volumes and is high throughput, permitting the monitoring of malaria transmission on large spatial and temporal scales. We expanded the use of a protein microarray to assess malaria transmission in settings beyond those with a low malaria incidence. Antibody responses in children and adults were profiled, using a P. falciparum protein microarray, through community-based surveys in three areas in Zambia and Zimbabwe at different stages of malaria control and elimination. These three epidemiological settings had distinct serological profiles reflective of their malaria transmission histories. While there was little correlation between transmission intensity and antibody signals (magnitude or breadth) in adults, there was a clear correlation in children younger than 5 years of age. Antibodies in adults appeared to be durable even in the absence of significant recent transmission, whereas antibodies in children provided a more accurate picture of recent levels of transmission intensity. Seroprevalence studies in children could provide a valuable marker of progress toward malaria elimination. IMPORTANCE As malaria approaches elimination in many areas of the world, monitoring the effect of control measures becomes more important but challenging. Low-level infections may go undetected by conventional tests that depend on parasitemia, particularly in immune individuals, who typically show no symptoms of malaria. In contrast, antibodies persist after parasitemia and may provide a more accurate picture of recent exposure. Only a few parasite antigens—mainly vaccine candidates—have been evaluated in seroepidemiological studies. We examined antibody responses to 500 different malaria proteins in blood samples collected through community-based surveillance from areas with low, medium, and high malaria transmission intensities. The breadth of the antibody responses in adults was broad in all three settings and was a poor correlate of recent exposure. In contrast, children represented a better sentinel population for monitoring recent malaria transmission. These data will help inform the use of multiplex serology for malaria surveillance.

We attempted to identify Plasmodium falciparum histidine–rich protein 2/3 (pfhrp2/3) deletions among rapid diagnostic test (RDT)–negative but PCR- or microscopy-positive P. falciparum–infected individuals in areas of low transmission (Choma District, 2009–2011) and high transmission (Nchelenge District, 2015–2017) in Zambia. Through community-based surveys, 5,167 participants were screened at 1,147 households by P. falciparum histidine-rich protein 2 (PfHRP2)-based RDTs. Slides were made and dried blood spots were obtained for molecular analysis. Of 28 samples with detectable P. falciparum DNA, none from Nchelenge District were pfhrp2/3 negative. All eight samples from Choma District had detectable pfhrp3 genes, but pfhrp2 was undetectable in three. DNA concentrations of pfhrp2-negative samples were low (< 0.001 ng/μL). These findings suggest that PfHRP2-based RDTs remain effective tools for malaria diagnosis in Nchelenge District, but further study is warranted to understand the potential for pfhrp2/3 deletions in southern Zambia where malaria transmission declined over the past decade.

A large proportion of ongoing malaria parasite transmission is attributed to low-density subclinical infections not readily detected by available rapid diagnostic tests (RDTs) or microscopy. Plasmodium falciparum gametocyte carriage is subclinical, but gametocytemic individuals comprise the parasite reservoir that leads to infection of mosquitoes and local transmission. Effective detection and quantification of these carriers can help advance malaria elimination strategies. However, no point-of-need (PON) RDTs for gametocyte detection exist, much less one that can perform noninvasive sampling of saliva outside a clinical setting. Here, we report on the discovery of 35 parasite markers from which we selected a single candidate for use in a PON RDT. We performed a cross-sectional, multi-omics study of saliva from 364 children with subclinical infection in Cameroon and Zambia and produced a prototype saliva-based PON lateral flow immunoassay test for P. falciparum gametocyte carriers. The test is capable of identifying submicroscopic carriage in both clinical and nonclinical settings and is compatible with archived saliva samples.

Additional file 1: Fig. S1. Top: Pfama1; bottom: Pfcsp. Distributions across 1000 replicates of collector’s curve analysis show the number of unique haplotypes (Y-axis) found among a randomly selected group of samples of increasing size (X-axis). Curves on the left were generated using the raw (unrarefied) dataset, while curves on the right were generated from the dataset rarefied to a depth of 200 reads per sample. Fig. S2. For each amplicon (top: Pfama1, bottom: Pfcsp), we performed 1000 re-sampling replicates of rarefaction. For each replicate we estimated MOI in each individual using the rarefied data. The distribution of MOI estimates across all rarefaction re-sampling replicates are plttoed along the Y-axis. The X-axis shows the MOI estimate from the raw data. The red dashed line is the Y = X line, or what would be expected if there was no difference between the estimate using rarefied data and the true estimate. Fig. S3. Pairwise genetic relatedness (the proportion of loci which are identical between two parasites) is plotted for all pairs of parasites from different countries or from the same country for each amplicon, Pfama1 (left) and Pfcsp (right). Comparisons between two parasites from individuals both under 5 years old are shown in pink. Comparisons between two parasites from individuals both 5 years or older are shown in blue. Comparisons between parasites from an individual 5 years or older and an individual under 5 years are shown in yellow. Fig. S4. Haplotype frequency distributions are plotted for the haplotypes present in each population (left: DRC right: Zambia; light grey bars: 2016 samples; dark grey bars: 2017 samples). Fig. S5. Left: plots the proportion of haplotypes within the four country-year “populations” considered to be rare. Here rare haplotypes are those represented at 2% or less in the “population.” Right: plots the proportion of haplotypes within the four country-year “populations” that were observed only once (singletons). Fig. S6. Dicriminatory analysis of principal components (DAPC) was performed using R package, adgenet. DAPC performs linear discriminat analysis on principal components in order to maximize separation of a priori groups. A, B: Pfama1; C, D: Pfcsp. A, C: DAPC performed using all sequences regardless of population frequency shows no linear function that can classify the principal components of the parasite seqeunces reliably by country. B, D: DAPC using only rare haplotypes (singletons) results in more refined population discrimination for Pfcsp. Fig. S7. Dicriminatory analysis of principal components (DAPC) was performed using R package, adgenet. DAPC performs linear discriminat analysis on principal components in order to maximize separation of a priori groups. A, B: Pfama1; C, D: Pfcsp. A, C: DAPC performed using all sequences regardless of population frequency shows no linear function that can classify the principal components of the parasite seqeunces reliably by country. B, D: DAPC using only rare haplotypes (2% or less frequency) results in more refined population discrimination for Pfcsp.

Standard operating procedures (PREGACT study) for obstetric ultrasound for the assessment of embryos and foetuses during pregnancy. (PDF 165 kb)

Malaria transmission in northern Zambia has increased in the past decade, despite malaria control activities. Evidence-based intervention strategies are needed to effectively reduce malaria transmission. Zambia’s National Malaria Control Centre conducted targeted indoor residual spraying (IRS) in Nchelenge District, Luapula Province, from 2014 to 2016 using the organophosphate insecticide pirimiphos-methyl. An evaluation of the IRS campaign was conducted by the Southern Africa International Centers of Excellence for Malaria Research using actively detected malaria cases in bimonthly household surveys carried out from April 2012 to July 2017. Changes in malaria parasite prevalence after IRS were assessed by season using Poisson regression models with robust standard errors, controlling for clustering of participants in households and demographic, geographical, and climatological covariates. In targeted areas, parasite prevalence declined approximately 25% during the rainy season following IRS with pirimiphos-methyl but did not decline during the dry season or in the overall study area. Within targeted areas, parasite prevalence declined in unsprayed households, suggesting both direct and indirect effects of IRS. The moderate decrease in parasite prevalence within sprayed areas indicates that IRS with pirimiphos-methyl is an effective malaria control measure, but a more comprehensive package of interventions is needed to effectively reduce the malaria burden in this setting.

The RTS,S/AS01 malaria vaccine encompasses the central repeats and C-terminal of Plasmodium falciparum circumsporozoite protein (PfCSP). Although no Phase II clinical trial studies observed evidence of strain-specific immunity, recent studies show a decrease in vaccine efficacy against non-vaccine strain parasites. In light of goals to reduce malaria morbidity, anticipating the effectiveness of RTS,S/AS01 is critical to planning widespread vaccine introduction. We deep sequenced C-terminal Pfcsp from 77 individuals living along the international border in Luapula Province, Zambia and Haut-Katanga Province, the Democratic Republic of the Congo (DRC) and compared translated amino acid haplotypes to the 3D7 vaccine strain. Only 5.2% of the 193 PfCSP sequences from the Zambia-DRC border region matched 3D7 at all 84 amino acids. To further contextualize the genetic diversity sampled in this study with global PfCSP diversity, we analyzed an additional 3,809 Pfcsp sequences from the Pf3k database and constructed a haplotype network representing 15 countries from Africa and Asia. The diversity observed in our samples was similar to the diversity observed in the global haplotype network. These observations underscore the need for additional research assessing genetic diversity in P. falciparum and the impact of PfCSP diversity on RTS,S/AS01 efficacy.

Malaria disproportionately affects young children. Clinical trials in African children showed that dihydroartemisinin–piperaquine (DP) is an effective antimalarial and has a longer posttreatment prophylactic (PTP) effect against reinfections than other artemisinin-based combination therapies, including artemether–lumefantrine (AL). Using a previously developed Markov model and individual patient data from a multicenter African drug efficacy trial, we assessed the economic value of the PTP effect of DP versus AL in pediatric malaria patients from health-care provider's perspective in low-to-moderate and moderate-to-high transmission settings under different drug co-payment scenarios. In low-to-moderate transmission settings, first-line treatment with DP was highly cost-effective with an incremental cost-effectiveness ratio of US$5 (95% confidence interval [CI] = −76 to 196) per disability-adjusted life year (DALY) averted. In moderate-to-high transmission settings, DP first-line treatment led to a mean cost saving of US$1.09 (95% CI = −0.88 to 3.85) and averted 0.05 (95% CI = −0.08 to 0.22) DALYs per child per year. Our results suggested that DP might be superior to AL for first-line treatment of uncomplicated childhood malaria across a range of transmission settings in Africa.

Objectives: To characterize antiretroviral therapy (ART) adherence across different programmes and examine the relationship between individual and programme characteristics and incomplete adherence among ART clients in sub-Saharan Africa. Design: A cross-sectional study. Methods: Systematically selected ART clients (>= 18 years; on ART >= 6 months) attending 18facilities in three countries (250 clients/facility) were interviewed. Client self-reports (3-day, 30-day, Case Index >= 48 consecutive hours of missed ART), healthcare provider estimates and the pharmacymedication possession ratio(MPR) were used to estimate ART adherence. Participants from two facilities per country underwent HIV RNA testing. Optimal adherence measures were selected on the basis of degree of association with concurrent HIV RNA dichotomized at less than or greater/equal to 1000 copies/ml. Multivariate regression analysis, adjusted for site-level clustering, assessed associations between incomplete adherence and individual and programme factors. Results: A total of 4489 participants were included, of whom 1498 underwent HIV RNA testing. Nonadherence ranged from 3.2% missing at least 48 consecutive hours to 40.1% having an MPR of less than 90%. The percentage with HIV RNA at least 1000 copies/ml ranged from 7.2 to 17.2% across study sites (mean = 9.9%). Having at least 48 consecutive hours of missed ART was the adherence measure most strongly related to virologic failure. Factors significantly related to incomplete adherence included visiting a traditional healer, screening positive for alcohol abuse, experiencing more HIV symptoms, having an ART regimen without nevirapine and greater levels of internalized stigma. Conclusion: Results support more in-depth investigations of the role of traditional healers, and the development of interventions to address alcohol abuse and internalized stigma among treatment-experienced adult ART patients. Copyright (C) 2015 Wolters Kluwer Health, Inc. All rights reserved.

Background: In Zambia, malaria is one of the leading causes of morbidity and mortality, especially among under five children and pregnant women. For the latter, the World Health Organization recommends the use of artemisinin-based combination therapy (ACT) in the second and third trimester of pregnancy. In a context of limited information on ACT, the safety and efficacy of three combinations, namely artemether-lumefantrine (AL), mefloquine-artesunate (MQAS) and dihydroartemisinin-piperaquine (DHAPQ) were assessed in pregnant women with malaria. Methods: The trial was carried out between July 2010 and August 2013 in Nchelenge district, Luapula Province, an area of high transmission, as part of a multi-centre trial. Women in the second or third trimester of pregnancy and with malaria were recruited and randomized to one of the three study arms. Women were actively followed up for 63 days, and then at delivery and 1 year post-delivery. Results: Nine hundred pregnant women were included, 300 per arm. PCR-adjusted treatment failure was 4.7% (12/258) (95% CI 2.7-8.0) for AL, 1.3% (3/235) (95% CI 0.4-3.7) for MQAS and 0.8% (2/236) (95% CI 0.2-3.0) for DHAPQ, with significant risk difference between AL and DHAPQ (p = 0.01) and between AL and MQAS (p = 0.03) treatments. Re-infections during follow up were more frequent in the AL (HR: 4.71; 95% CI 3.10-7.2; p < 0.01) and MQAS (HR: 1.59; 95% CI 1.02-2.46; p = 0.04) arms compared to the DHAPQ arm. PCR-adjusted treatment failure was significantly associated with women under 20 years [Hazard Ratio (HR) 5.35 (95% CI 1.07-26.73; p = 0.04)] and higher malaria parasite density [3.23 (95% CI 1.03-10.10; p = 0.04)], and still women under 20 years [1.78, (95% CI 1.26-2.52; p < 0.01)] had a significantly higher risk of re-infection. The three treatments were generally well tolerated. Dizziness, nausea, vomiting, headache and asthenia as adverse events (AEs) were more common in MQAS than in AL or DHAPQ (p < 0.001). Birth outcomes were not significantly different between treatment arms. Conclusion: As new infections can be prevented by a long acting partner drug to the artemisinins, DHAPQ should be preferred in places as Nchelenge district where transmission is intense while in areas of low transmission intensity AL or MQAS may be used.

ObjectivesWe assessed retention and predictors of attrition (recorded death or loss to follow-up) in antiretroviral treatment (ART) clinics in Tanzania, Uganda and Zambia. MethodsWe conducted a retrospective cohort study among adults (18years) starting ART during 2003-2010. We purposefully selected six health facilities per country and randomly selected 250 patients from each facility. Patients who visited clinics at least once during the 90days before data abstraction were defined as retained. Data on individual and programme level risk factors for attrition were obtained through chart review and clinic manager interviews. Kaplan-Meier curves for retention across sites were created. Predictors of attrition were assessed using a multivariable Cox-proportional hazards model, adjusted for site-level clustering. ResultsFrom 17 facilities, 4147 patients were included. Retention ranged from 52.0% to 96.2% at 1year to 25.8%-90.4% at 4years. Multivariable analysis of ART initiation characteristics found the following independent risk factors for attrition: younger age [adjusted hazard ratio (aHR) and 95% confidence interval (95%CI)=1.30 (1.14-1.47)], WHO stage 4 ([aHR (95% CI): 1.56 (1.29-1.88)], >10% bodyweight loss [aHR (95%CI)=1.17 (1.00-1.38)], poor functional status [ambulatory aHR (95%CI)=1.29 (1.09-1.54); bedridden aHR1.54 (1.15-2.07)], and increasing years of clinic operation prior to ART initiation in government facilities [aHR (95%CI)=1.17 (1.10-1.23)]. Patients with higher CD4 cell count were less likely to experience attrition [aHR (95%CI)=0.88 (0.78-1.00)] for every log (tenfold) increase. Sites offering community ART dispensing [aHR (95%CI)=0.55 (0.30-1.01) for women; 0.40 (0.21-0.75) for men] had significantly less attrition. ConclusionsPatient retention to an individual programme worsened over time especially among males, younger persons and those with poor clinical indicators. Community ART drug dispensing programmes could improve retention. ObjectifsNous avons evalue la retention et les predicteurs de l'attrition (deces ou pertes au suivi enregistres) dans le traitement antiretroviral (ART) dans des cliniques en Tanzanie, en Ouganda et en Zambie. MethodesNous avons mene une etude de cohorte retrospective chez les adultes (18 ans) ayant debute l'ART au cours de 2003-2010. Nous avons deliberement choisi six etablissements de sante par pays et avons choisi au hasard 250 patients dans chaque etablissement. Les patients qui ont visite les cliniques au moins une fois au cours des 90 jours avant l'extraction des donnees ont ete definis comme retenus. Les donnees sur les facteurs de risque individuels et a l'echelle du programme pour l'attrition ont ete obtenues par l'examen des dossiers et des entrevues avec les directeur des cliniques. Les courbes de Kaplan-Meier pour la retention a travers les sites ont ete creees. Les predicteurs de l'attrition ont ete evalues a l'aide d'un modele multivarie des risques proportionnels de Cox, corriges par le regroupement au niveau du site. ResultatsDe 17 etablissements, 4147 patients ont ete inclus. La retention variait de 52,0% a 96,2% a un an et de 25,8% a 90,4% a 4 ans. L'analyse multivariee des caracteristiques a l'initiation de l'ART a trouve les facteurs independants de risque pour l'attrition suivants: le plus jeune age [Hazard Ratio ajuste (aHR) et intervalle de confiance a 95% (IC 95%)=1,30 (1,14 a 1,47)], le stade 4 de l'OMS ([aHR (IC 95%): 1,56 (1,29 a 1,88)], > 10% de perte de poids corporel [aHR (IC 95%)=1,17 (1,00 a 1,38)], un mauvais etat fonctionnel [aHR ambulatoire=1,29 (1,09-1,54); aHR alite=1,54 (01,15 a 02,07)] et de plus en plus d'annees d'operations en clinique avant le debut de l'ART dans les etablissements publics [aHR(IC 95%)=1,17 (1,10 a 1,23)]. Les patients avec un taux plus eleve de cellules CD4 etaient moins susceptibles a l'attrition [aHR (IC 95%) = 0,88 (0,78 a 1,00)] pour chaque augmentation logarithmique. Les sites offrant la distribution communautaire de l'ART [aHR (IC 95%)=0,55 (0,30 a 1,01) pour les femmes; =0,40 (0,21 a 0,75) pour les hommes] avaient significativement moins d'attrition. ConclusionsLa retention des patients dans un programme individuel empirait au fil du temps surtout chez les hommes, les personnes plus jeunes et ceux avec de mauvais indicateurs cliniques. Les programmes de distribution communautaire de l'ART pourraient ameliorer la retention. ObjetivosHemos evaluado la retencion y los vaticinadores de abandono (muerte registrada o perdida durante el seguimiento) en centros de tratamiento antirretroviral (TAR) en Tanzania, Uganda y Zambia. MetodosHemos realizado un estudio retrospectivo de cohortes con adultos (18 anos) en TAR durante el 2003-2010. Seleccionamos seis centros sanitarios por pais, y de forma aleatoria a 250 pacientes de cada centro. Los pacientes que visitaron los centros al menos una vez durante los 90 dias anteriores a la fecha de obtencion de los datos se definieron como retenidos. Se obtuvieron datos sobre los factores de riesgo de abandono de los individuos y el nivel del programa mediante la revision de graficos y entrevistas con el gestor del centro. Se realizaron curvas de Kaplan-Meier para la retencion en los distintos centros. Los vaticinadores de abandono se evaluaron utilizando un modelo de riesgos proporcionales de Cox, ajustado para agrupacion a nivel de centros. ResultadosSe incluyeron 4147 pacientes de 17 centros. La retencion estaba entre 52.0%-96.2% al ano y 25.8%-90.4% a los 4 anos. En un analisis multivariante de las caracteristicas presentes en el momento de iniciar TAR, se encontraron los siguientes factores de riesgo de abandono independientes: ser mas joven [razon de riesgo ajustada (RRa) and IC 95=1.30 (1.14-1.47)], estadio 4 de la OMS ([RRa(IC 95%): 1.56 (1.29-1.88)], > 10% perdida de peso corporal [RRa (IC 95%)=1.17 (1.00-1.38)], un estatus funcional pobre [RRa ambulatorio (IC 95%)=1.29 (1.09-1.54); estar en cama RRa 1.54 (1.15-2.07) ], y un mayor numero de anos en clinicas privadas antes de comenzar TAR en un centro gubernamental [RRa (IC 95%)=1.17 (1.10-1.23)]. Los pacientes con un mayor conteo de CD4 tenian una menor probabilidad de experimentar abandono [RRa (IC 95%)=0.88 (0.78-1.00)] para un aumento logaritmico (x10). Los centros que ofrecian dispensarios comunitarios de TAR [aHR (IC95%)=0.55 (0.30-1.01) para mujeres; 0.40 (0.21-0.75) para hombres] tenian significativamente menos abandono. ConclusionesLa retencion de pacientes en un programa individual empeoro a lo largo del tiempo, especialmente entre los hombres, personas jovenes y aquellos con indicadores clinicos pobres. Los programas comunitarios de dispensacion de TAR podrian mejorar la retencion.

Background Plasmodium falciparum resistance to anti-malarial drugs remains a major obstacle to malaria control and elimination. The parasite has developed resistance to every anti-malarial drug introduced for wide-scale treatment. However, the spread of resistance may be reversible. Malawi was the first country to discontinue chloroquine use due to widespread resistance. Within a decade of the removal of drug pressure, the molecular marker of chloroquine-resistant malaria had disappeared and the drug was shown to have excellent clinical efficacy. Many countries have observed decreases in the prevalence of chloroquine resistance with the discontinuation of chloroquine use. In Zambia, chloroquine was used as first-line treatment for uncomplicated malaria until treatment failures led the Ministry of Health to replace it with artemether-lumefantrine in 2003. Specimens from a recent study were analysed to evaluate prevalence of chloroquine-resistant malaria in Nchelenge district a decade after chloroquine use was discontinued. Methods Parasite DNA was extracted from dried blood spots collected by finger-prick in pregnant women who were enrolling in a clinical trial. The specimens underwent pyrosequencing to determine the genotype of the P. falciparum chloroquine resistance transporter, the gene that is associated with CQ resistance. Results Three-hundred and two specimens were successfully analysed. No chloroquine-resistant genotypes were detected. Conclusion The study found the disappearance of chloroquine-resistant malaria after the removal of chloroquine drug pressure. Chloroquine may have a role for malaria prevention or treatment in Zambia and throughout the region in the future.

BACKGROUND Information regarding the safety and efficacy of artemisinin combination treatments for malaria in pregnant women is limited, particularly among women who live in sub-Saharan Africa. METHODS We conducted a multicenter, randomized, open-label trial of treatments for malaria in pregnant women in four African countries. A total of 3428 pregnant women in the second or third trimester who had falciparum malaria (at any parasite density and regardless of symptoms) were treated with artemether-lumefantrine, amodiaquine-artesunate, mefloquine-artesunate, or dihydroartemisinin-piperaquine. The primary end points were the polymerase-chain-reaction (PCR)-adjusted cure rates (i.e., cure of the original infection; new infections during follow-up were not considered to be treatment failures) at day 63 and safety outcomes. RESULTS The PCR-adjusted cure rates in the per-protocol analysis were 94.8% in the artemether-lumefantrine group, 98.5% in the amodiaquine-artesunate group, 99.2% in the dihydroartemisinin-piperaquine group, and 96.8% in the mefloquine-artesunate group; the PCR-adjusted cure rates in the intention-to-treat analysis were 94.2%, 96.9%, 98.0%, and 95.5%, respectively. There was no significant difference among the amodiaquine-artesunate group, dihydroartemisinin-piperaquine group, and the mefloquine-artesunate group. The cure rate in the artemether-lumefantrine group was significantly lower than that in the other three groups, although the absolute difference was within the 5-percentage-point margin for equivalence. The unadjusted cure rates, used as a measure of the post-treatment prophylactic effect, were significantly lower in the artemether-lumefantrine group (52.5%) than in groups that received amodiaquine-artesunate (82.3%), dihydroartemisinin-piperaquine (86.9%), or mefloquine-artesunate (73.8%). No significant difference in the rate of serious adverse events and in birth outcomes was found among the treatment groups. Drug-related adverse events such as asthenia, poor appetite, dizziness, nausea, and vomiting occurred significantly more frequently in the mefloquine-artesunate group (50.6%) and the amodiaquine-artesunate group (48.5%) than in the dihydroartemisinin-piperaquine group (20.6%) and the artemether-lumefantrine group (11.5%) (P

Objectives To identify the reasons patients miss taking their antiretroviral therapy (ART) and the proportion who miss their ART because of symptoms; and to explore the association between symptoms and incomplete adherence. Methods Secondary analysis of data collected during a cross-sectional study that examined ART adherence among adults from 18 purposefully selected sites in Tanzania, Uganda, and Zambia. We interviewed 250 systematically selected patients per facility (>= 18 years) on reasons for missing ART and symptoms they had experienced (using the HIV Symptom Index). We abstracted clinical data from the patients' medical, pharmacy, and laboratory records. Incomplete adherence was defined as having missed ART for at least 48 consecutive hours during the past 3 months. Results Twenty-nine percent of participants reported at least one reason for having ever missed ART (1278/4425). The most frequent reason was simply forgetting (681/1278 or 53%), followed by ART-related hunger or not having enough food (30%), and symptoms (12%). The median number of symptoms reported by participants was 4 (IQR: 2-7). Every additional symptom increased the odds of incomplete adherence by 12% (OR: 1.1, 95% CI: 1.1-1.2). Female participants and participants initiated on a regimen containing stavudine were more likely to report greater numbers of symptoms. Conclusions Symptoms were a common reason for missing ART, together with simply forgetting and food insecurity. A combination of ART regimens with fewer side effects, use of mobile phone text message reminders, and integration of food supplementation and livelihood programmes into HIV programmes, have the potential to decrease missed ART and hence to improve adherence and the outcomes of ART programmes.