HIV drug resistance (HIVDR) testing was included in the 2017 South African national HIV household survey. We describe the prevalence of HIVDR by drug class, age, sex and antiretroviral drugs (ARV) status.Dried blood were spots tested for HIV, with Viral load (VL), exposure to ARVs and HIVDR testing among those HIV positive. HIVDR testing was conducted on samples with VL ≥1000 copies/ml using Next Generation Sequencing. Weighted percentages of HIVDR are reported.697/1,105 (63%) of HIV positive samples were sequenced. HIVDR was detected in samples from 200 respondents  22.8–32.6)). Among these 130 (18.9% (95% CI 14.8–23.8)), had resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) only, 63 (7.8% (95% CI 5.6–10.9)) resistance to NNRTIs and nucleoside reverse transcriptase inhibitors, and 3 (0.5% (95% CI 0.1–2.1)) resistance to protease inhibitors. Sixty-five (55.7% (95% CI 42.6–67.9) of ARV-positive samples had HIVDR compared to 112 (22.8% (95% CI 17.7–28.7)), in ARV-negative samples. HIVDR was found in 75.6% (95% CI 59.2–87.3), n = 27, samples from respondents who reported ARV use but tested ARV-negative, and in 15.3% (95% CI 6.3–32.8), n = 7, respondents who reported no ARV use and tested ARV-negative. There were no significant age and sex differences in HIVDR.27% of virally unsuppressed respondents had HIVDR, increasing to 75% among those who had discontinued ARV. Our findings support strengthening first-line ARV regimens by including drugs with a higher resistance barrier and treatment adherence strategies, and close monitoring of HIVDR. Access to antiretroviral therapy (ART) has expanded globally in the last decade, reaching 25.4 million individuals in 2017 , which aUniversal test and treat and reduced mortality in PLHIV  have meaData indicates increasing levels of HIVDR across the world. A systematic review of 26 studies in low- and middle-income countries (LMIC) conducted between 2014 and 2017, found that 9.7% of adults on ART had HIVDR –11. A prThis study presents the profile of HIVDR among PLHIV enrolled and tested in a nationally representative HIV household survey conducted in South Africa in 2017 [The national HIV household survey was a national cross-sectional survey that included people of all ages in all provinces of South Africa, in 2017. The survey included people living in hostels while those living in educational institutions, old-age homes, hospitals, and uniformed-service barracks were excluded. All members of the selected households were invited to participate. The design was similar to that implemented in four previous HIV surveys conducted in 2002, 2005, 2008, and 2012 –17, as wSurvey interviews and collection of blood samples were undertaken in the household after written or verbal consent . Effort was made to ensure confidentiality of the interview process, data transmission and processing. The questionnaires included a household questionnaire for household information and age targeted questionnaires, one for parents/guardians of children aged 0 to 11 years, one for adolescents aged 12 to 14 years, and one for those aged 15 years and older. The questionnaires included a question about daily use of ARVs.Dried blood spot (DBS) samples were collected by finger prick (or heel prick in infants) and were tested for HIV antibodies using an algorithm with two different enzyme immunoassays (EIAs) and a nucleic acid amplification test (NAAT) for validating all positives. The HIV status of children under two years old was confirmed with the NAAT. Testing for HIV antibodies was conducted at Global Clinical and Viral laboratories in Durban South Africa, with quality assurance testing conducted at the South African Medical Council. HIV-positive (HIV+ve) specimens were further tested for: i) antiretroviral drugs (ARVs), ii) HIV VL, iii) Limiting Antigen (LAg) Avidity EIA  as part of a multisassay incidence estimation algorithm that took into account ARV treatment and HIV VL , in sampTesting for ARVs was by High Performance Liquid Chromatography coupled with Tandem Mass Spectrometry , and incVL testing used the Abbott platform , and was undertaken at the National Institute for Communicable Diseases (NICD) in Johannesburg South Africa. Samples with VL ≥ 1000 copies/ml were classified as virally unsuppressed and were submitted for HIVDR testing.HIVDR testing was undertaken at the NICD and was conducted by Next Generation Sequencing (NGS). DBS were excised, immersed in 2 ml of NucliSENS lysis buffer  and lysed on a roller mixer at room temperature. Total nucleic acid was extracted using the NucliSENS EasyMAG® automated system according to the manufacturer’s instructions. Amplification of a 1,084 base pair polymerase chain reaction (PCR) fragment consisting of codons 1–99 of protease and codons 1–250 of reverse transcriptase was performed as previously described , with thData from samples tested for HIVDR are presented. Survey weights were applied to generate nationally representative estimates, and the analyses took into account the multi-level stratified design of the survey. The final individual weights were benchmarked against the 2017 mid-year population estimates by age, race, age, and province [The survey was approved by the Human Sciences Research Council (HSRC) Research Ethics Committee (REC: 4/18/11/15). The study was also reviewed in accordance with the Centers for Disease Control and Prevention (CDC) human research protection procedures and was determined to be research. CDC investigators did not interact with human subjects or have access to identifiable data or specimens for research purposes (CGH 2016-143a).Samples from 23,836 participants were tested for HIV and among these 2,994 (median age 36 years (interquartile range (IQR) 26–48) tested HIV+ve translating to an estimated HIV prevalence of 14.0% (95% CI 13.1–15.0) [Among the HIV+ve samples with VL results , 1,105 (37.4%) were virally unsuppressed and among these, 697 (63.1%) were successfully genotyped see , median Resistance was identified in 200/697 samples, giving a prevalence of 27.4% (95% CI 22.8––32.6). Median VL in these samples was 18,913 . The most frequent mutation by drug class was resistance to NNRTIs only, found in 18.9% (95% CI 14.8–23.8), n = 130 of samples . ResistaOverall, resistance was detected in 29.4% of samples from males and in 25.8% of samples from females, p = 0.473 . There wResistance was detected in 29.5% of samples from respondents in urban areas, and in 23.1%, p = 0.077 of samples from rural areas, Of the 200 samples with HIVDR, 177 had laboratory results for exposure to ARVs, with (65) 29.4% ARV+ve. HIVDR was found in 55.7% of the ARV+ve samples compared to 22.8% in ARV-ve samples p <0.001 . When stNinety-four samples tested for HIVDR were from respondents who reported daily ARV use at the time of the survey . Those who reported taking ARVs, but tested ARV-ve were classified as ART defaulters, . HIVDR was detected in 75.9% of these samples, ; 56.4% oThirty-three samples tested for HIVDR were from respondents who reported they were not taking ARVs and tested ARV-ve in the laboratory . HIVDR was detected in 15.3% (95% CI 6.3–32.8) of these samples and all had NNRTI-only resistance. The proportion of HIVDR in this group was significantly lower than in respondents who were taking ARVs, 15.3% vs 55.7% p = 0.004.HIVDR was detected in 7of 32 samples (21.8% unweighted percentage) recently infected with HIV, and with genotype data available. All seven samples had resistance to NNRTIs  and wereWith the largest public sector ART programme in the world, monitoring of population level HIVDR is critical in South Africa since HIVDR levels can impact achievement of the UNAIDS 90–90–90 targets . The HIVHIVDR levels were high. Resistance was detected in more than a quarter 27.4%) of samples from virally unsuppressed respondents. Nearly a fifth of the respondents had resistance to NNRTIs. This is consistent with the lower genetic barrier of NNRTIs to resistance . The hig% of sampWe found low levels of resistance to second-line therapy which includes PIs, and this could indicate appropriate limited prescription of second-line therapy, since PI mutations are much less common than NNRTI and NRTI mutations because PIs have a much higher genetic barrier to resistance . HoweverWe found NNRTI-monoresistance in samples from 18.9% respondents, and this was the most frequent resistance type as expected in settings where NNRTIs are a key part of first-line treatment regimens. NNRTI mono-resistance also occurred in 15.3% of samples from respondents who tested ARV–ve and reported not taking ARVs. These findings further indicate the low resistance barrier of efavirenz, the most widely used NNRTI in South Africa. Mbuagbaw et al  showed tHIVDR were most prevalent in samples from respondents with prior exposure to ART (those who reported taking ARVs but tested ARV-ve in the laboratory and were classified as ART defaulters):– 75.9% had DRMs, 56.4% of them with NNRTI mono-resistance. This estimate is much higher than previously reported in South Africa, where HIVDR was found in 37.5% of samples of patients with prior exposure to ART who were enrolled as part of sentinel surveillance in public clinics in three provinces . This diOur findings of high HIVDR levels among people with prior ART exposure compared to those who reported not taking ARVs and tested ARV-ve are consistent with previous reports that PDR is more than two-fold higher among people starting first-line ART with prior ARV drug exposure, compared to ARV drug-naive individuals , 33. TheOur estimated HIVDR of 55.7% among virally unsuppressed people on ART is lower than reported from facility-based studies in South Africa. Hunt et al, 2017  found DRAlthough the numbers are small our finding of 7 samples with DRMs out of 32 samples from respondents recently infected with HIV (21.8%) could suggest higher levels of transmitted HIVDR than found in studies in KZN –40. It iRates of successful PCR amplification from dried blood spots were lower than expected. The WHO recommends that DBS specimens with a viral load of >1,000copies/ml be used for HIVDR testing, but acknowledges poor amplification rates at lower VL levels. As we tested all specimens with VL>1,000 copies/ml, this could have contributed to lower overall PCR non-amplification rates . In addiThis is the first analysis of HIVDR in a nationally-representative sample of PLHIV in South Africa, and therefore includes PLHIV who are not in care, and those accessing care from the private sector who would be excluded from HIVDR surveillance that utilizes data from public health facilities as recommended by WHO . HoweverAnalysis of a nationally representative sample of PLHIV in South Africa, found high levels of resistance to first-line ART regimens among virally unsuppressed PLHIV. HIVDR among people who have defaulted from ART are particularly high. Our findings support strengthening first-line ARV regimens by including drugs with a higher resistance barrier, and treatment adherence strategies, and close monitoring of HIVDR.


Plasmodium serology data are not available for Suriname and even extremely scarce within the region, therefore malaria serology testing was introduced, country customized cut-off values were determined and a study was performed to explore the antibody status for Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae.Suriname has accomplished a steep decline in malaria burden, even reaching elimination levels. Plasmodium antigens was detected using a multiplex bead-based, IgG antibody assay. Demographic information was gathered through a questionnaire. Country customized cut-off values were generated from a Surinamese malaria-naïve reference population (n = 50).A cross-sectional survey was conducted between July 2017 and March 2018 in two areas of the interior with different malaria settings: Stoelmanseiland, representing Maroon villages and Benzdorp, a gold mining area, with mostly Brazilian miners. Dried blood spots (DBS) were collected (n = 197) and antibody presence against seven 19. Seroprevalence against any of the three MSP-119 antibodies was similar in both regions and surpassed 75%. Single seropositivity against PfMSP-119 antibodies was higher in Stoelmanseiland (27.0%) than Benzdorp (9.3%), in line with the historical malaria burden of Stoelmanseiland, while the reverse was observed for PvMSP-119 antibodies. Despite sporadic reports of P. malariae infections, PmMSP-119 antibody presence was 39.6%. A more detailed examination of P. falciparum serology data displayed a higher seroprevalence in villagers (90.7%) than in Brazilians (64.6%) and a highly diverse antigenic response with 22 distinct antibody combinations.Serological analysis for the reference population revealed cut-off values ranging from 14 MFI for LSA-1 to 177 MFI for PmMSP-1Plasmodium seroprevalence, especially for P. falciparum in villagers, still reflecting the historical malaria burden. The seroprevalence data for both regions and the observed combinations of P. falciparum antibodies provided a valuable dataset from a historically important region to the international malaria serology knowledge. First insight in malaria serology data for Suriname indicated that the use of other target groups and assessment of age-dependent seroprevalence are required to successfully use malaria serology as tool in the national elimination strategy.The results on the malaria antibody signature of Maroon villagers and Brazilian miners living in Suriname displayed a high  Plasmodium falciparum infections, with a drop from more than 10,000 cases in 2001, to fewer than 800 cases since 2010. In April 2016, Suriname was identified by WHO  as a country with the potential to reach elimination of local malaria transmission by 2020. However, the road towards malaria elimination is paved with challenges. Firstly, the looming threat of emerging artemisinin resistance. The Guiana Shield, comprised of the interior of Guyana, Suriname, French Guiana, and bordering areas of Brazil, Colombia and Venezuela, has been recognized by the PAHO  [Malaria incidence and mortality have declined significantly in Suriname since 2006, due to stringent measures from the National Malaria Programme, such as the use of artemisinin combination therapy, widespread distribution of free long-lasting insecticide-impregnated mosquito nets, awareness campaigns and improved case management , special programme for risk populations including tailored activities for miners scattered in the interior). A marked decrease was noted, particularly for P. falciparum-infected patients, treated with Coartem® were still positive on day 3 [pfATP6 [pfmdr1 [K13 genes [pfmdr1 copy numbers in P. falciparum isolates [Indeed, several warning signals for emerging resistance were reported in studies conducted in Suriname. An in vivo efficacy study conducted in 2011 demonstrated that 31% of  [pfATP6 , pfmdr1  [pfmdr1  and K13 isolates  have beeThe second challenge in the process of malaria elimination is the country’s geographical location within the Guiana Shield, situated between two different, unfavourable epidemiological settings. In the east, French Guiana with a malaria prevalence of 22.3% in gold miners , and in C580Y mutation in the K13 gene [The majority of the cases of malaria, diagnosed and treated in Suriname in 2018, originated across borders, predominantly from French Guiana . Spill-oK13 gene . SurinamThirdly, malaria transmission occurs almost exclusively in hard-to-reach, mobile, migrant, artisanal gold mining populations in the remote areas of the interior and the illegal nature of the mining activities, on top of poor compliance with national treatment policies, further hampers the already logistically difficult and expensive operational processes.Lastly, the lack of suitable monitoring procedures for malaria elimination, as most of the current field activities evolved from historical malaria control programmes, which are not yet optimized for low malaria numbers and a zero tolerance strategy.Plasmodium parasite especially in hypo-endemic malaria settings [These challenges underscore the need to implement innovative elimination strategies and appropriate tools and procedures for vigilant monitoring of every case in order to reach malaria elimination in the country. The common malaria detection tools, such as microscopy, rapid diagnostic tests and PCR detection, do not suffice for monitoring of elimination, especially within the mobile migrant mining population in the interior. Malaria serology alone or in conjunction with other malariometric indicators has proved to be a useful tool for malaria control and especially for malaria elimination programmes. It has been demonstrated that serological data can aid countries striving for malaria elimination through a better understanding of the transmission dynamics of the settings –14, wherPlasmodium serology data are not available for Suriname and are extremely scarce within the region.As malaria infection imposes a lasting antibody ‘footprint’, malaria serology data reflects cumulative malaria exposure and can serve various purposes. Serology data can be utilized to evaluate changes in exposure, to monitor long-term trends, besides geographic mapping and assessments of potential effects of control interventions. International studies in various geographical settings have been conducted to determine seroconversion rates (antibody acquisition rates of a population) and seroreversion rates (loss of antibody) from age-dependent seroprevalence data , 13. HowPlasmodium species  in Suriname, with the introduction of a serological bead-based malaria multiplex assay, offering the possibility to simultaneously detect IgG antibodies against different Plasmodium species.This study aimed to gain first insight into the antibody status for the three circulating Plasmodium IgG distributions were analysed for persons from two distinctly different malaria environments: Stoelmanseiland, representing Maroon villages in Suriname, and Benzdorp with a mostly migrant gold mining population. Dried blood spots (DBS) and demographic information were collected from persons from both sites and serological assessment was performed using seven Plasmodium antibodies.Country customized cut-off values were generated from a Surinamese malaria-naïve reference population and P. falciparum for the Guiana Shield prompted a more detailed investigation for the presence of P. falciparum antibodies in two sub-populations of interest: the Surinamese participants from Stoelmanseiland (n = 86) and the Brazilians from Benzdorp (n = 65).In order to achieve a representative cut-off value for Suriname, a malaria-naïve reference population was generated, consisting of 50 adults living in Suriname  and cut-off values were calculated. Secondly, a cross-sectional investigation was conducted in two areas in the interior of Suriname with a different malaria environment, Stoelmanseiland and Benzdorp and their surroundings, to gain first insight in malaria seroprevalence in different regions in Suriname. Participant data and samples for serological testing were collected between July 2017 and March 2018. Thirdly, the historical significance of P. vivax.Suriname is part of the Guiana Shield and is located along the northeastern coast of South America bordering French Guiana to the east, Guyana to the west and Brazil to the south. Suriname is the smallest sovereign country in South America with just over 575,000 inhabitants, most of whom live in the coastal area in and around the capital, Paramaribo . MalariaAnopheles darlingi is the principal vector for malaria transmission. Historically, P. falciparum had been the predominant malaria species in Suriname until 2006, after which P. vivax became the main species. Plasmodium malariae has not been detected in Suriname since 2013.The scarcity of serology data in the region necessitated the compilation of initial serology data, starting with the determination of country customized cut-off values and a subsequent investigation of variations in antibody status in persons from two regions with a different malaria environment.P. falciparum and P. vivax), including import cases was registered annually, by the National Malaria Programme.Stoelmanseiland is a village located at the junction of Tapanahony River and Lawa River at the border of Suriname with French Guiana. Selection of this area was based on its historical malaria incidence, the highest in the period between 1999 and 2003, representing more than 10% of the overall national malaria burden. The inhabitants, mainly Maroons, live in small villages and camps along the river. Healthcare in this region is provided free for all by the Medical Mission and in 2016, the number of Stoelmanseiland inhabitants registered for healthcare was 1010. In the past 5 years however, an average of three cases  are still recorded for this area, while diagnosis of indigenous malaria cases has been sporadic in the last 5years.The Benzdorp region is a mining area in the interior with a population of approximately 2500–3000 persons in several settlements, some directly along Lawa River bordering French Guiana. This region was selected for the composition of the population, consisting of service providers and mobile gold miners, mainly Brazil nationals, entering the country primarily through French Guiana. The Malaria Programme of the Ministry of Health performs surveillance and provides all malaria services free of charge to these high-risk populations, supported by the Global Fund. Positive import cases  [Without existing data on antibody prevalence of the three  (48.8%)  and Pará (48.8%) . The reqFor both regions, all consenting adults (≥ 18 years), male or female, living or working in the respective areas were eligible for participation in the period between July 2017 and March 2018. Fever or microscopically confirmed malaria was applied as exclusion criteria. For the Benzdorp region, participants were recruited during ACD conducted by the National Malaria Programme. For Stoelmanseiland and surroundings, all villagers who could be reached within a 4-day enrolment period were requested to participate. In order to achieve a representative sample of actual villagers, Brazilians either entering via Brazil or French Guiana were excluded for this site.® 903 protein saver cards from all participants. Samples were individually packed in plastic ziplock bags containing desiccant and were transported from the interior at room temperature and subsequently stored at −20 °C until processing.Sampling and data collection were performed by trained field workers who visited each area alongside personnel from the National Malaria Programme or staff from the Medical Mission. Information about the study was provided in the native language of the participant through local translators. Participant information was gathered using an interviewer-administered, pre-tested questionnaire comprised of questions regarding basic demographic information and questions concerning history of malaria, mining and travel. Finger-prick dried blood spots (DBS) were collected onto WhatmanPlasmodium detection with photo-induced electron transfer (PET) primers [For the sub-set of participants from Benzdorp (n = 108), recruited during ACD, additional testing common during ACD, such as malaria rapid diagnostic tests (RDTs), thick and thin smears for malaria microscopy as well as real-time PCR for  primers  was perf® bead-based multiplex immunoassay. IgG responses were measured for the merozoite surface protein-119 antigens of P. falciparum (PfMSP-119), P. vivax (PvMSP-119) and P. malariae (PmMSP-119). In addition, the responses for four other P. falciparum antigens: apical membrane antigen‐1 (AMA‐1), glutamate-rich protein (GLURP), circumsporozoite protein (CSP), and liver stage antigen-1 (LSA-1) were studied, next to tetanus-toxoid. The 19 kD fragment of the MSP-119 was fused to glutathione S-transferase (GST), cloned and coupled to the beads by CDC, Atlanta . For AMA-1, produced at Walter Reed Army Institute of Research, 20 µg/ml beads; GLURP, CDC, 30 µg/ml beads; CSP, CDC, 30 µg/ml beads; LSA-1, CDC, 60 µg/ml beads and tetanus-toxoid, Massachusetts Biologic Laboratories, 12.5 µg/ml beads were used. The coupled beads were provided by V. Udhayakumar from CDC, Atlanta.Blood spot elution and immunoassays were performed according to protocols described elsewhere  with theP. falciparum antigens are all vaccine candidates and included two pre-erythrocytic and three blood-stage antigens. AMA‐1 and MSP-119 are blood-stage antigens and both are considered to induce long-lived humoral immune responses after malaria infection. AMA-1 is highly immunogenic and has been suggested to be involved in red blood cell invasion by the merozoite, while MSP-119 is regarded as moderately immunogenic. GLURP is a key merozoite surface antigen, which is expressed at each stage of parasite life in the human host. The pre-erythrocytic CSP and LSA-1 are considered to induce short-living antibodies.The selected ® COOH beads  per analyte. Secondary antibodies were tagged with biotin ) and incubated for 90 min at room temperature on a shaker. Subsequently, 50 μl of 1:200 dilution streptavidin, R–phycoerythrin conjugate  was added and incubated for 30 min at room temperature on a shaker. After various washing steps, beads were analysed on a MAGPIX® system  using the xPONENT 4.2 software for assay design and data acquisition. The reader was set to read a minimum of 50 beads/analyte of unique fluorescent signature per region.A final dilution (1:100), from whole blood eluted for each sample from DBS, was incubated in flat-bottom plates with antigen-coupled BioPlexIn each experiment, positive and negative controls and all samples were run in duplicate to monitor intra-experimental variation. Pooled malaria-positive sera, obtained from V. Udhayakumar from CDC Atlanta, served as positive control to assess day-to-day variability, while the negative control consisted of an eluate from blank filter paper. Blanks (PBS) were also included to determine the background signal within each run. The median fluorescence intensity (MFI) as output for each sample was calculated as the mean value of the duplicate wells after subtraction of MFI values originating from the blank background beads (MFI-bg). The net MFI was calculated by subtracting the respective cut-off value from the MFI-bg. Retesting was performed for samples with poor elution, or for replicates with a CV higher than 20% for their MFI-bg values.The majority of malaria serology studies utilize cut-off values either obtained from ELISA or earlier reported values for multiplex assays, derived from non-immune reference populations, consisting of US citizens with no travel history to malaria-endemic countries . More rePlasmodium species.However, conditions in Suriname are quite different, with a highly multi-ethnic population, distinctive population genetics, heterogeneous malaria history, varying environments  and a dissimilar distribution of Therefore, a malaria-naïve reference population was generated, consisting of 50 adults living in the coastal area of Suriname for at least 10 years, with no fever and no reported history of malaria infection .Plasmodium antigens. Determination of the ‘true negative’ serodistribution for Suriname was calculated for each antigen as the mean signal from the non-immune reference population for the respective antigen without outliers + 3SD, according to calculations described elsewhere [The background signal intensity for the DBS from the non-immune reference cohort, eluted and processed within the study setting was utilized to determine the specific background signal  for each of the seven lsewhere .Plasmodium antibodies, tetanus-toxoid was included to serve as possible internal positive control for the non-immune population, as substantial responses to this antigen were expected, based on the high national immunization coverage.The acquired cut-off values were applied for the corresponding study results, thus generating the true seropositivity value for each experiment. In addition to the seven 19 and PmMSP-119 antibodies with age. The data were analysed using Statistical Packages for Social Sciences (SPSS version 22.0). Statistical significance was set at p < 0.05.The Chi square test was used to study associations between malaria antibodies and variables, such as history of malaria and travel, and for seroprevalence comparison between groups. Logistic regression was used to correlate seropositivity against PfMSP-1Serological analysis for the non-immune cohort (n = 50) revealed no anti-malarial antibodies as expected, and the generated values validated the selection criteria used for the non-immune Surinamese cohort. Calculation of the true negative serodistribution for each malaria antigen resulted in the cut-off values depicted in Fig. Plasmodium antibody cut-off values ranged from 14 for LSA-1 to 177 for PmMSP-119. The cut-off value determined for P. falciparum MSP-119 antibodies was lower than those for P. malariae and P. vivax. The presence of tetanus-toxoid antibodies in the malaria non-immune participants from Suriname was 90.0%, coinciding with the WHO estimated immunization coverage for Suriname in 2018 for DPT1/DPT3 (95%) [The Plasmodium antigens  was successfully conducted for all collected blood samples (n = 197). The detected antibody intensities were highly variable for the seven malaria analytes and positive MFI values even exceeded 30,000, both for PfMSP-119 and AMA-1. The presence of Plasmodium antibodies was positively linked to the documented number of infections, in line with the common belief that cumulative exposure will elicit a long-lived response. Data on malaria history revealed that the majority of participants (75.6%) reported one or more past malaria episodes, of which only 21.5% could recollect the responsible Plasmodium species. Furthermore, 70.5% of the participants with a malaria history reported multiple infections, some surpassing more than 20 infections during their lifetime. The results revealed a positive association for all seven Plasmodium antigens between self-reported malaria history and actual presence of Plasmodium antibodies (p < 0.05). However, the timeline of previous malaria episodes of the participants and the correlation with the current presence of antibodies could not be examined, since information was gathered on the number of past episodes, but details were only collected for the most recent infection, anticipating recall bias.Serological analysis for the seven utilized 19 antibodies. The low presence of P. vivax antibodies in the mainly Maroon population in Stoelmanseiland prohibited this analysis for P. vivax antibodies. Age was positively associated with seropositivity against PfMSP-119 antibodies for every 10-year increase, in age increments between 20 and 69 years. The odds ratio per 10-year increase was 1.7 . The lack of all age groups within this study population prohibited the determination of seroconversion rates.The correlation of the seropositivity with age was studied only in the Surinamese participants from Stoelmanseiland for PfMSP-1Plasmodium vivax parasites seem to require the Duffy Antigen Receptor for Chemokines on the erythrocytes for their attachment and P. vivax infection should therefore be uncommon in persons with the Duffy-negative (FyFy) blood group, which is widespread in populations with African ancestors. To investigate this concept, the total population with known ancestry was grouped as participants from African descent (n = 124) and participants without African ancestry (n = 63). Indeed, general occurrence of P. vivax antibodies was significantly lower in persons from African descent (p < 0.001).Plasmodium detection revealed no Plasmodium presence in any of these participants. The mean age of the study population was similar for both areas  as could be anticipated with mostly male participants in gold mining activities. On the other hand, the gender distribution for the Stoelmanseiland area (M/F: 0.21) was skewed towards women, which could be for a number of reasons including differences in earlier documented health-seeking behaviour, the perceived reluctance of local men to participate in investigations, especially when blood collection is required and the sampling during day time when the majority of men were engaged in activities  outside the village.The ratio of males to females (M/F) for the Benzdorp population was higher than the general Surinamese population  were evaluated, to prevent skewing towards P. falciparum. The distribution of MFI values for each of the antibodies used in this comparative survey is shown in the Additional file 19 antibodies was similar in both regions and surpassed 75%. The prevalence of antibodies against more than one Plasmodium species was higher than single-species antibody prevalence in both regions. The Plasmodium MSP-119 antibody prevalence in participants from Stoelmanseiland and Benzdorp is illustrated in Fig. In the comparison of seroprevalence between Stoelmanseiland and Benzdorp, with their diverging malaria environment, only the three MSP-119 antibodies displayed a significantly higher occurrence in Stoelmanseiland (27.0%) versus Benzdorp region (9.3%) (p < 0.05), while the reverse was observed for PvMSP-119 antibodies (p < 0.001) . The single-species P. malariae seroprevalence did not exhibit a significant difference between Stoelmanseiland and Benzorp participants (p = 0.182). The overall multiple-species antibody presence did not exhibit a significant variation within the two regions (p = 0.374) , while the P. falciparum/P. vivax combination was more common in Benzdorp (40.4%).In Stoelmanseiland, MSP-1P. vivax antibodies in combination with other malaria species antibodies, even though carriage of single species antibodies to P. vivax was only observed in one participant from Stoelmanseiland  was observed in conjunction with antibodies against other malaria species  were more often detected in Benzdorp (22.2%) than in Stoelmanseiland (14.6%), although not reaching significance (p = 0.292).Antibodies against all three 19 antibodies revealed that travel was not associated with the presence of PvMSP-119, PfMSP-119 and PmMSP-119 antibodies in Stoelmanseiland. In Benzdorp, travel was associated only with the presence of PfMSP-119 antibodies and more specifically for the sub-group (n = 33) of mobile Surinamese miners in this region (p < 0.05).Travel to a neighbouring country within the past 5 years was reported by 55.8% of all participants. Analysis of the seropositivity against MSP-1P. falciparum within Suriname and the Guiana Shield, alongside the expectation that elimination of P. falciparum will precede elimination of the relapse-prone P. vivax species, prompted a more detailed examination of P. falciparum serology data with five antigens for two sub-populations of interest, namely Surinamese villagers from Stoelmanseiland and Brazilian miners from the Benzdorp region  was statistically higher for the Surinamese participants compared to the Brazilians, as was the presence of the frequently investigated PfMSP-119 and AMA-1, either alone or in combination . The long-lasting antibodies were leading the overall prevalence in both sub-groups, while the presence of LSA-1 and CSP antibodies was significantly lower (p < 0.001). To gain some insight into the combined occurrence of specific long- and short-lasting P. falciparum antibodies in Suriname, all possible combinations of antibodies in the Surinamese participants from Stoelmanseiland were analysed. Twenty-two distinctive patterns were detected and the various combinations with their frequency of occurrence are depicted in Fig. The In accordance with the high overall seroprevalence of the long-lasting antibodies, the two leading combinations consisted of the three long-lasting antibodies with and without LSA-1. As expected, the presence of long-lasting antibodies was detected in 86.0% of the participants, but despite a high prevalence for all three long-lasting antibodies, only 54.1, 25.7 and 20.3% of these positive cases carried all three, two or just one of these predominant antibodies, respectively. Furthermore, no correlation could be established between the two short-lasting antibodies CSP and LSA-1.Plasmodium species  in Suriname, a country in the Guiana Shield with low malaria transmission in the last decade. Country customized cut-off values were determined for antibodies against seven common Plasmodium antigens, applicable for the utilized multiplex assay and to counteract population-specific differences in immune responses. The use of a reference population of Surinamese with no malaria history, instead of the general reference population consisting of US citizens with no travel history to malaria-endemic countries, enabled the generation of a true representation of the actual ‘negative’ serodistribution in this setting. The generated cut-off values in this study were lower than cut-off values for the commonly used reference population consisting of US citizens [19 for P. falciparum compared to MSP-119 for the other two Plasmodium species was in line with recent results [The current study provided the first malaria seroprevalence data for the three circulating citizens , probablcitizens . The obs results .P. falciparum seroprevalence in villagers from Stoelmanseiland compared to the mobile migrants from the Benzdorp region. The expectations that both malaria history and higher age of an individual would be associated with presence of antibodies could be confirmed. The observed positive association between age and seropositivity against PfMSP-119 antibodies for every 10-year increase in the Surinamese participants substantiated the receding local malaria transmission trend in Stoelmanseiland. Nevertheless, P. falciparum IgG positivity was still very high in Stoelmanseiland, despite scarce reports during the last decade. This outcome pointed to a slow decay of these antibodies and could be used to extrapolate the longevity of the P. falciparum antibody responses in this population in the absence of re-infection. However, it should be noted that antibody half-lives increase with age and that age is related to the presence of long-lived IgG antibody secreting cells that can maintain a sustained P. falciparum antibody response in the absence of re-infection [Prior to 2006, Suriname had a very high malaria burden and was even considered as the country in the Americas with the highest concentration of falciparum malaria , which wnfection .P. falciparum only in mobile Surinamese miners from Benzdorp, in concert with the increasing trend of imported malaria cases in Suriname, highlighted the importance of keeping detailed records of patient travel history.The finding that a travel history to a malaria-endemic country was a risk-factor for Plasmodium species are further discussed in more detail per species. Comparative serological analysis is quite complex and malaria serology prevalence is influenced by a variety of factors, including the dynamic mechanism of antibody formation and decay for each antibody type, the target population, the geographic location, and the historical malaria burden of the area. Furthermore, available data of possible serological biomarkers of malaria transmission intensity have to be retrieved from studies conducted in quite different endemic settings, each employing only a few of the assortment of Plasmodium antibodies. Caution is therefore warranted for associations and comparisons, as confounding conclusions can be reached, if only a single mechanism aspect is considered.The results on seroprevalence of the three circulating 19 in Brazilian miners in the Benzdorp region (64.6%) was in line with reports from the Brazilian Amazon, where nearly 70% of the studied population carried PvMSP-119 antibodies [19 antibodies, relatively uniform seropositivity against PvMSP-119 has been reported in the Brazilian Amazon, with or without travel between areas with markedly different malaria transmission levels [The overall seroprevalence of the highly immunogenic PvMSP-1tibodies . In accon levels .P. vivax antibodies detected in persons from African descent provided support for the general belief that P. vivax parasites use the Duffy Antigen Receptor for Chemokines on the erythrocytes for their attachment. The results that P. vivax antibodies were still detected in more than 20% of the Maroon population from Suriname seem to concur with the observation from the African West Coast that the Duffy-negative antigen no longer seems to be an insurmountable barrier for P. vivax merozoites [P. vivax antibodies in conjunction with other Plasmodium antibodies with just one exception. This finding could also suggest that Maroons living in co-endemic regions may have an increased risk of a P. vivax malaria infection, following another malaria episode, which augmented an earlier hypothesis that P. falciparum infections are a risk factor for P. vivax relapses in co-endemic regions [The significantly lower occurrence of rozoites . On the  regions .P. malariae (39.6%) was in contrast with the sporadic P. malariae infections registered in South America. In the last decade, P. malariae infections rarely surpassed 1–2% in South America [P. malariae infections of 43.8 [P. malariae infections may have long been under-detected in Suriname, due to difficult microscopic differentiation, although it should be noted that P. malariae infections can be long-lasting, even up to 40 years without re-infection [19 provided additional support for a reflection of archaic history. The observation that the majority of PmMSP-119 antibodies (94.9%) appeared in conjunction with other malaria antibodies, corroborated results from Zimbabwe, where the frequency of IgG responses to both PmMSP-119 and PfMSP-119 ranged between 78 and 87% [Plasmodium antibodies was regarded unlikely as it has been demonstrated that IgG antibody responses to the utilized malaria MSP-119 antigens appear to be species-specific, even in high transmission settings with multiple circulating species [P. malariae antibodies.The remarkably high presence of antibodies against  America . Interna of 43.8  and 11.9 of 43.8 . The stunfection . The lac and 87% . Possibl species , besidesP. falciparum malaria serology prevalence have been reported, but originating from different endemic settings and achieved with varying types and numbers of antibodies tested, evidenced by the heterogeneity of published data. In this first malaria serology study in Suriname, a highly diverse antigenic response against P. falciparum was revealed, with an assortment of distinct combinations of long-lasting and short-lasting antibodies. The observed overall P. falciparum antibodies prevalence  was significantly higher in the Stoelmanseiland area (90.7%) than in the Brazilian participants  and also substantially higher than the 24.7% from the neighbouring state of Pará in northern Brazil, although their IgG positivity results were obtained by testing only PfMSP-119 and AMA-1 [P. falciparum seropositivity in the population from Stoelmanseiland. The assessment of five different P. falciparum antibodies allowed for a more scrutinized examination of the combined occurrence of the long- and short-lasting antibodies in the Surinamese participants from Stoelmanseiland. The observed predominance of AMA-1, PfMSP-119 and GLURP in the patterns reflected the high P. falciparum burden of the Surinamese population and the ability of the corresponding antigens to elicit long-lived humoral immune responses after malaria infection.Internationally, several findings on nd AMA-1 . The dis19 and AMA-1 antigens was shown, with just one of these two long-lasting antibodies present in 22.1% of the cases. Other studies also observed a skewed antibodies distribution [19, the use of multiplex serology allowed simultaneous examination of GLURP with the two other long-lasting antibodies. The seroprevalence of GLURP in the Surinamese sub-group (62.8%) was in line with a Brazilian study on GLURP antibody response revealing a high prevalence (79.0%) [An uneven distribution of antibodies against the PfMSP-1ribution , 32. In  (79.0%) , and did (79.0%) .P. falciparum negative. Nonetheless, others also report the presence of CSP antibodies in persons long after malaria infection and even postulated that CSP antibodies, despite their short-lived nature, do not necessarily correlate with recent exposure [The mere presence in the study population of antibodies against the non-erythrocytic short-lived CSP with an estimated half-life ranging from 3 months  to aboutexposure .Despite the insight gained in the various combinations of the long-, and short-lasting antibodies exhibited in this study group, the intricate aspects regarding lasting presence of specific antibodies challenged straightforward conclusions.Plasmodium antibodies and their combinations and provided first insight into the antibody status of two different populations in Suriname (Maroon villagers and Brazilian miners). The high antibody response for the three long-lasting P. falciparum antibodies indicated an earlier antibody saturation, thus suggesting a limited value for these antibodies to detect significant recent transmission shifts in similar study settings with a heterogeneous historic malaria burden. However, the newly introduced serological assay in the country paved the way for future studies, possibly even using MSP-119, but with different target groups, as it has been demonstrated that utilization of MSP-119 in age-stratified surveys in northeastern Tanzania allowed differentiation of hyper- to hypo-endemic villages [P. vivax and P. falciparum [The malaria seroprevalence data in this study added to the international knowledge of different villages . Moreovelciparum .Plasmodium-specific antibodies from persons living in the interior of Suriname. Moreover, serological data are reported for persons from two countries, Surinamese (mainly Maroon villagers) and Brazilian miners. Other strengths of the study are the determination of representative cut-off values for Plasmodium antibodies for Suriname and providing some insight in profile patterns of P. falciparum antibodies.The major strength of this cross-sectional study is the presentation of first data on both short- and long-lasting Plasmodium species were obtained from self-reports of participants, probably resulting in recall bias. However, there are no indications that any recall bias would be different amongst participants from both areas. Second, the link between the burden of consecutive infections and malaria antibodies could not be addressed properly, since details of each previous malaria infection were unavailable. Third, the enrolment of only adults limited the extent of the gathered information and did not enable estimation of seroconversion rates. Lastly, this study was performed in just two study areas in the interior of Suriname. Hence, care should be taken to draw conclusions about malaria serology regarding children or persons residing in other geographic areas with historically different temporal malaria transmission. Follow-up studies in other areas with varying malaria histories and the inclusion of children are required to estimate seroconversion rates, and studies among other ethnic groups, such as the Amerindians, will provide further insight into the complex malaria serology in Suriname. Nevertheless, the current study results contributed to the international serology knowledge on malaria antibodies and introduced preliminary requirements for possible future use of this tool in Suriname for stratification of absence of transmission per region, which is particularly important for other countries also striving for malaria elimination.The study has limitations. First, data on malaria history, number of infections and P. falciparum MSP-119 seropositivity was recorded in the Stoelmanseiland area, in line with the historical malaria burden of this region, while the reverse was demonstrated for P. vivax antibodies, presumably because of the high proportion of Maroons in Stoelmanseiland. The finding that the Maroons from Stoelmanseiland harboured P. vivax antibodies predominantly in conjunction with other Plasmodium antibodies suggested that Maroons living in co-endemic regions may have an increased risk of a vivax malaria infection, following another malaria episode.Prevalence of indigenous malaria has declined dramatically over the past years in Suriname, transforming the country from the area with the highest concentration of falciparum malaria in the Americas, to a country aiming for elimination. A multiplex bead-based assay was introduced and country customized cut-off values were determined, enabling future studies to utilize this panel of samples from the country specific malaria-naïve reference population. A cross-sectional seroprevalence study was conducted in two regions with a differing malaria environment. Higher P. malariae added to the data of the region that P. malariae infections may have long been under-detected in South America.The high presence of antibodies against P. falciparum antibodies with a multitude of combinations of long-lasting and short-lasting antibodies provided a valuable dataset from a historically important region to the international P. falciparum malaria serology knowledge. The high P. falciparum antibody response measured in the low transmission setting implied that a serological approach with any of the five studied antibodies may not detect significant recent deviations from the historic pattern of transmission in similar study settings. Additional studies with other target groups and assessment of age-dependent seroprevalence in other geographical areas of the country are required, prior to introduction of malaria serology as integral part of a multi-pronged elimination strategy for Suriname.This study not only presented the first results on malaria and tetanus-toxoid antibody presence in Suriname, but the highly diverse profile patterns of Additional file 1: Fig S1. Distribution of MFI values for MSP-119 antibodies in Benzdorp and Stoelmanseiland.


Plasmodium falciparum and P. vivax, parasitology and entomology have been combined to survey malaria transmission in two nearby villages.Malaria is still a heavy public health concern in Madagascar. Few studies combining parasitology and entomology have been conducted despite the need for accurate information to design effective vector control measures. In a Malagasy region of moderate to intense transmission of both Plasmodium sporozoites was assessed by TaqMan assay.Community-based surveys were conducted in the villages of Ambohitromby and Miarinarivo at three time points  during a single malaria transmission season. Human malaria prevalence was determined by rapid diagnostic tests (RDTs), microscopy and real-time PCR. Mosquitoes were collected by human landing catches and pyrethrum spray catches and the presence of P. falciparum and to a lesser extent to P. vivax. However, there was a significantly higher prevalence rate as determined by PCR at T2 (P = 0.025). Likewise, mosquitoes were significantly more abundant at T2 (P < 0.001), especially in Ambohitromby. At T1 and T3 mosquito abundance was higher in Miarinarivo than in Ambohitromby (P < 0.001). Of 1550 Anopheles mosquitoes tested, 28 (1.8%) were found carrying Plasmodium sporozoites. The entomological inoculation rate revealed that Anopheles coustani played a major contribution in malaria transmission in Miarinarivo, being responsible of 61.2 infective bites per human (ib/h) during the whole six months of the survey, whereas, it was An. arabiensis, with 36 ib/h, that played that role in Ambohitromby.Malaria prevalence was not significantly different between villages, with an average of 8.0% by RDT, 4.8% by microscopy and 11.9% by PCR. This was mainly due to An. coustani and An. arabiensis to malaria transmission in each village. Importantly, the suspected secondary malaria vector An. coustani, was found playing the major role in malaria transmission in one village. This highlights the importance of combining parasitology and entomology surveys for better targeting local malaria vectors. Such study should contribute to the malaria pre-elimination goal established under the 2018–2022 National Malaria Strategic Plan. Despite a similar malaria prevalence in two nearby villages, the entomological survey showed a different contribution of  Plasmodium falciparum being the most prevalent. Across the ecozones, the average prevalence of P. falciparum varies from 2 to 12% [Anopheles species described in the country, six have been reported as malaria vectors with different role according to geography and behaviour [Anopheles gambiae complex: Anopheles gambiae (sensu stricto), An. arabiensis and An. merus, the latter having a minor role in malaria transmission and being restricted to the most southern region of Madagascar [An. gambiae complex, An. arabiensis is prevalent throughout Madagascar and plays a major role in malaria transmission along with An. funestus [Anopheles mascarensis, endemic to Madagascar, and An. coustani, act as local or minor vectors [Malaria remains a major public health concern in Madagascar, with an increase in the number of cases and deaths in years 2017 and 2018, compared to the year 2016 . All fou2 to 12%  but can 2 to 12% . Among tehaviour . Three s vectors .P. falciparum and P. vivax circulate. In this area, malaria prevalence and Anopheles species distribution have been described in detail [P. falciparum and P. vivax. The study was conducted in two neighbouring villages in Andriba, a rural commune located at the transition between the western fringe of the Central Highlands  and the northwestern ecozone  according to Howes et al. [Anopheles species contribute to the local malaria transmission with the aim for providing targeted vector control recommendation to the local authorities. To our knowledge, no such combined parasitological and entomological survey has ever been performed in that region.Recent surveys of malaria incidence and prevalence between 2010–2016 confirm the heavy malaria burden for the population living in the western part of Madagascar , 4, 11. n detail , 12, 13.The study was conducted in two villages of the rural commune of Andriba  which is located in the tropical northwest region of Madagascar. Andriba is characterised by a dry season that generally lasts from April to October and a rainy season from November to April; the average annual temperature is 24 °C and the average annual rainfall is 1828 mm . The twoStudy participants included village residents among whom volunteers were involved in human landing catches (HLCs). The week before each survey, a community sensitisation was organized with the help of the staff of the Andriba health centre and the presence of the head of the village explaining the benefit of malaria parasite detection. On the day of the survey, people gathered at a central point proposed by the head of the village, usually the local school. Each participant was provided a short questionnaire to collect name, age, sex, and any health conditions that might exclude them from the survey . Blood samples were collected from both children and adults without clinical signs of malaria at the time of the survey . According to the study protocol, any individual who would have come with malaria symptoms would have been referred to the Andriba health centre. We did not encounter such a situation. Temperature and weight where recorded for each participant showing a positive rapid diagnostic test (RDT) for malaria. Participant’s names were used for longitudinal follow-up of their participation at the next surveys, but names were encoded for data analysis.P. falciparum, and any of P. vivax, P. ovale and P. malariae, as all four species are present in Madagascar. Plasmodium species, parasite stages and parasite density were further determined by microscopic observation of the blood smears stained with 10% Giemsa, using a light microscope (100×). A thin blood smear slide was declared malaria-negative when Plasmodium parasites were not detected after examination of 100 high power microscopic fields. Slides were read for asexual parasites and gametocytes, enumerated against 500 leucocytes and expressed as density/μl assuming an average leucocyte count of 8,000/μl of blood (data not shown). Individuals with positive RDT were treated with artemisinin-based combination therapy (ACT), according to national guidelines.Blood samples were obtained by finger prick to perform RDTs , thick and thin blood smears, and blood spots on filter paper (standard Whatman 3MM filter paper). The Bioline RDT enabled specific detection of Plasmodium parasites were performed in two steps as previously described by Canier et al. [Plasmodium spp. were first detected by a real-time PCR using genus-specific primers targeting the Plasmodium cytochrome b gene. Then, Plasmodium species identification was performed on DNA samples identified as positive for Plasmodium using a nested real-time PCR assay [Dried blood spots (DBS) were lysed overnight at 4 °C in 150 μl per microtube of 1× HBS buffer  supplemented with 0.5% saponin, final concentration. Samples were then washed twice with 1× PBS and DNA extracted with Instagene® Matrix resin  according to manufacturer’s instructions. Molecular detection and species identification of Mosquitoes were collected by HLCs and indoor pyrethrum spray catches (PSCs), following WHO protocols . For botPSCs were conducted in 5 houses/day/village, choosing houses that were not used for HLCs and in which no insecticide or repellent had been used during the previous week. Some of the houses were using insecticide-treated bed nets. The GPS  coordinates of each house was recorded as well as the date at which the PSC was performed. PSCs were done on 3 consecutive days from 06:00 h to 08:00 h each morning following the HLCs. No insecticide residual spraying had occurred in the villages from 2016 till 2018. PSCs were performed using a pyrethroid mixture of prallethrin, tetramethrin, and deltamethrin. Spraying was performed from outside of the houses, into openings, holes in walls and eaves, then in the rooms, following WHO procedures. Knockdown mosquitoes were then collected by hand picking.An. gambiae from An. arabiensis, a TaqMan assay was used targeting the intergenic spacer region of rDNA as described by Walker et al. [All mosquitoes were identified morphologically using the determination keys of Grjebine  and De Mr et al. , followir et al. . PrimersAnopheles head-thorax was extracted using the DNAzol® Reagent . Briefly, the head-thorax from each mosquito was put individually in a tube; care was taken to rinse the dissecting equipment in 70% ethanol between each mosquito. A volume of 150 µl of DNAzol was added and mosquito tissues were crushed using an individual conical plastic pestle. DNA was then extracted following the manufacturer’s protocol. After precipitation, the DNA pellet was suspended in a final volume of 50 µl of nuclease-free water. DNA quality was controlled using a SYBR Green real-time PCR assay targeting the ribosomal S7 protein encoding gene. This gene is highly conserved among species belonging to the same genus. Primers previously designed against the An. gambiae S7 gene [Anopheles S7 sequences to ensure that those primers will efficiently amplify the S7 gene fragment from any Anopheles captured in the field. Amplification conditions were validated on a subset of laboratory and field-collected mosquito samples including species of the subgenera Anopheles, Cellia and Nyssorhynchus (not shown). Amplification using the PowerSYBER® Green Master mix (Applied Biosystems) was performed as follows: an initial step at 95 °C for 15 min; followed by 40 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 30 s and elongation at 60 °C for 45 s. Specificity of the amplification was assessed by viewing the melting curves.Genomic DNA from  S7 gene  were aliPlasmodium gDNA in mosquitoes was performed in 2 steps. The first step used a TaqMan PCR assay targeting a region of the 18S rRNA gene conserved among the human infecting Plasmodium species. For this assay, primers and probe previously described [Plasmodium detection in mosquitoes [Plasmodium TaqMan probe was labelled with 5’ NED. PCR reactions (20 μl) contained 5 μl of mosquito genomic DNA, 4 μl of 5× HOT FIREPol® Probe qPCR Mix Plus/no ROX (Solis Biodyne), 300 nM of each primer and 200 nM of probe. Reactions were run on a StepOnePlus (Applied Biosystems) using the following conditions: an initial step at 95 °C for 10 min; followed by 50 cycles of denaturation at 95 °C for 20 s and annealing/elongation at 60 °C for 1 min. Plasmodium falciparum genomic DNA extracted from NF54 parasite cultures was used as a positive control. Any sample with amplification signal before the 38th cycle was considered positive. For high throughput screening, a pool strategy was used [Plasmodium TaqMan assay was run using 5 µl of each DNA pool in triplicate. Mosquitoes from positive pools were then analysed individually using the same protocol as for pools. All samples positive in the TaqMan assay were then analysed for the identification of P. falciparum and P. vivax species. For each positive sample, 2 distinct real-time SYBR Green PCR assays were done using species-specific primers targeting the cytochrome b gene. Purified gDNA from P. falciparum and P. vivax were used as positive controls. Each reaction was run in triplicate. The real-time PCR conditions were as previously described [Plasmodium detection in Anopheles mosquitoes are listed in Additional file The detection of human escribed  have beeescribed ; this cosquitoes . The Plawas used ; equal vescribed : an initPlasmodium spp. prevalence rates, as determined by microscopy, RDT and PCR were analysed by fitting a generalized linear mixed model (GLMM) with binomial error structure (i.e. a logistic regression) with individual as the random factor and village, time point (and their interaction), age and sex as explanatory variables. Total mosquito numbers from HLC were analysed by fitting a GLMM with Poisson error structure (i.e. a loglinear regression) with house identification number as the random factor and village, time point (and their interaction) as explanatory variables. For endophagy, mosquito species-specific analyses were similarly performed individually for all mosquito species for which more than a grand total of 50 individual mosquitoes were collected. In addition to village and time point (and their interaction), place of capture (indoors vs outdoors) was also included in the model. All GLMM analyses were performed in GenStat version 15 . To account for any underdispersion or overdispersion in the data, a dispersion heterogeneity was used in the analyses.Plasmodium spp. prevalence rates when determined by microscopy or RDT. However, there was a significantly higher prevalence rate as determined by PCR at T2 (P = 0.025). There were no differences between villages, nor by age or sex. Except for T3 in Miarinarivo, the PCR technique, as expected, was able to detect a greater number of parasite carriers over RDT and microscopy, revealing a substantial proportion of sub-microscopic parasite carriers. The lower proportion of PCR-positive samples at T3 in Miarinarivo, compared to T1 and T2, might result from inadequate conservation of the blood spots (12 of them) before PCR processing.The parasitological survey involved 380 individuals  followed by Culex , Mansonia , Aedes  and Coquillettidia . Among Anopheles, An. coustani was by far the most abundant representing 52.99% (751/1417) of the known potential malaria vectors in Madagascar, followed by An. arabiensis , An. funestus  and An. mascarensis ; An. gambiae was barely represented . Detailed data covering all collected species are presented in Additional file In total, 2407 mosquitoes were collected during 72 HNs in Ambohitromby and Miarinarivo. As presented in Table Anopheles caught by HLC revealed significant associations with time point and an interaction with village. The number of Anopheles was higher at T2 (P < 0.001), especially in Ambohitromby, whereas at T1 and T3, numbers were higher in Miarinarivo (interaction term P < 0.001). The human-biting rate (HBR), corresponding to the number of collected mosquitoes per number of human-nights, was determined for the known potential malaria vectors, but An. gambiae due to the low number of captured mosquitoes (n = 8). Results over time and for each village are depicted in Fig. Anopheles HBR varies in each village over the time course of the survey, from 8 bites per human and per night (b/h/n) to 34.3 b/h/n. However, the bite frequency was higher at T2 in Ambohitromby, while being the highest at T3 in Miarinarivo. Strikingly, An. arabiensis was the malaria vector species with the highest HBR in Ambohitromby, with 17.3 b/h/n at T2, while in Miarinarivo, it was An. coustani with 25.9 b/h/n at T3. Nevertheless, An. coustani exhibited the highest biting frequency in both villages at T3, which corresponds to the time where the rice reached full maturity providing high shade on the padding fields suitable for An. coustani larval development.Analysis of total An. coustani and An. arabiensis. Considering An. coustani, the HBR in the two villages was higher at T3 compared to T1 and T2. The highest HBR peak at T1 was between 22:00–23:00 h in Ambohitromby and between18:00–20:00 h in Miarinarivo. The HBR peak at T2 was higher in Miarinarivo compared to Ambohitromby and occurred between 03:00–04:00 h. At T3, the biting pattern over the night was overall similar between the two villages. The An. arabiensis HBR in the two villages was higher at T2 compared to T1 and T3. The highest HBR peak occurred between 19:00–20:00 h in Ambohitromby and between 21:00–22:00 h in Miarinarivo. An. funestus and An. mascarensis showed the lowest HBR peak in the two villages with the same biting pattern along the night.Looking at the hourly biting rate of the four potential malaria vectors across time-points in the two villages, the four mosquito species mostly bit all night long Fig. . There wAnopheles funestus exhibited the highest endophagy rate in both villages (58.00 ± 4.94% in Ambohitromby and 53.66 ± 5.51% in Miarinarivo). The lowest endophagy rate in Ambohitromby was exhibited by An. coustani (5.73 ± 1.39%), while it was An. mascarensis that exhibited the lowest endophagy rate in Miarinarivo (11.76 ± 7.81%). Anopheles arabiensis and An. coustani, known as zoo-anthropophilic species, exhibited higher endophagy rates in Miarinarivo  compared to Ambohitromby . Comparing the endophagy rate by mosquito species, village and time point revealed a number of associations. All Anopheles species, except for An. funestus which did not differ significantly between indoors and outdoors, were collected in higher numbers outdoors, while Anopheles arabiensis and An. coustani were collected in higher numbers at T2 and T3, respectively. From 17 PSCs, a total of 70 mosquitoes were collected resting indoors  and An. mascarensis (3.84%) were collected resting indoors in Miarinarivo.Endophagy rates, representing the proportion of mosquitoes collected indoors over the total number of mosquitoes collected indoors and outdoors by HLCs, are summarized in Table n = 1650) and PSCs (n = 65), 1550 were tested for the presence of Plasmodium sporozoites by TaqMan and SYBR Green assays. As described in the methods section, DNA was first extracted from the head-thorax of individual mosquitoes and its quality assessed by amplification of the S7 gene. Using an equal volume of gDNA from at most 6 mosquitoes of the same species, 261 pools were assembled and tested for the presence of Plasmodium DNA, using the Plasmodium TaqMan assay. A total of 23 pools were found positive for Plasmodium DNA. Deconvolution of each positive pool to individual mosquito revealed that 28 mosquitoes carried Plasmodium DNA in their head-thorax, all of which had been captured by HLCs only. However, the SYBR Green assay for P. falciparum/P. vivax species detection was conclusive only for 13 (out 28) mosquitoes carrying either P. falciparum or P. vivax parasite  . As there are increased reports on its role in malaria transmission in other countries [Plasmodium sporozoite carriage in future surveillance programs.Among 1715 anopheline mosquitoes captured by HLCs  and the mid-term (T2) of transmission season, with EIRs of 0.27 and 0.31 ib/h/n respectively, followed by An. coustani at T2 (0.28 ib/h/n). By contrast, in Miarinarivo, An. coustani was the main vector at T2 and T3 (EIRs of 0.43 and 0.61 ib/h/n respectively), followed by An. arabiensis that played a vector role at T2 only (EIR of 0.25 ib/h/n). Plotting the number of An. arabiensis and An. coustani, and their respective EIR, show that the EIRs are not proportional to the number of captured mosquitoes and that this is evidenced in the two villages over the transmission season ; while their density at T2 and T3 was greatly different, but the EIR was similar for both species. In Miarinarivo, despite similar density of An. arabiensis and An. coustani at T2, An. coustani contributed most to malaria transmission and maintained this role at T3 with increased EIR possibly associated to its higher density.When looking at the entomological inoculation rate (EIR) as a proxy for malaria transmission, it appears that me Table . Indeed,The objective of this study, conducted in two neighbouring villages in a region of Madagascar where malaria is still a high public health problem, was to estimate the level of malaria transmission and to identify the mosquito vector species involved. Indeed, in that region, no such study had ever been conducted despite the high number of patients diagnosed with malaria at the local health centres. To our knowledge, the only study carried out at the same site, just provided entomological data and goes back to 1992 .P. falciparum, in addition to low levels of P. vivax and P. malariae. The overall malaria prevalence throughout the study period was 11.9% as determined by PCR, and 4.8% by microscopy. These values are similar to the ones reported in the Tsiroanomandidy study performed in March 2014 [Plasmodium carriage which represents 50–75% of the investigated cases .Parasitological data in the asymptomatic villagers revealed malaria infection cases mainly due to rch 2014 . Like thP = 0.019, Chi-square test) in malaria RDT prevalence was observed between Ambohitromby  and Miarinarivo , but not in 2017 (this study) nor 2018 . Indeed, in 2016 a significant difference  in Ambohitromby, while An. coustani was the one with the highest HBR at the onset (December) and at the late-term (April) in both villages. This change in HBR over the malaria transmission season could be explained by changes in the ecological environment, precisely the rice fields which constitute the main Anopheles larval habitats in the two villages. Indeed, the growing phases of the rice determine important changes in the characteristics of the Anopheles breeding sites [An. gambiae (s.l.) larvae. As the rice plants grow, they shade the water of the rice fields which become less favourable for An. gambiae (s.l.) larvae, giving way to An. coustani larvae that prefer shaded breeding sites. This transition from An. gambiae (s.l.) to An. coustani in breeding sites by increasing vegetation cover, was also demonstrated for borrow pits in Ethiopia [Entomological data from HLCs and PSCs, showed that d Tables , 4. Howeng sites . Fields Ethiopia .Anopheles vector feeding behaviour, only An. funestus showed a strong tendency for both biting and resting indoor, not departing from its known behaviour in Andriba [An. arabiensis and An. coustani exhibited a significant outdoor biting preference in each village towards increased endophagy in Miarinarivo . All mosquitoes positive for Plasmodium spp were collected by HLCs, with 20/28 collected outdoors. Among the 28 Plasmodium-positive mosquitoes, 14 were An. coustani, 9 An. arabiensis, 3 An. funestus and 2 An. rufipes. Whereas An. funestus and An. arabiensis are well known malaria vectors in Madagascar, the contribution of An. coustani to malaria transmission has been suspected on several occasions due to its high density and propensity to anthropophily [An. coustani samples were detected CSP-positive by ELISA [An. coustani in malaria transmission in Andriba. Anopheles coustani is also known to be a malaria vector in continental Africa: in Cameroon [An. rufipes analysed by the TaqMan assay were found possibly carrying Plasmodium sporozoites. To date, An. rufipes has never been reported naturally infected with Plasmodium in Madagascar. In continental Africa, it was found naturally infected with P. falciparum in Burkina Faso [An. mascarensis samples (n = 59) were found positive for Plasmodium although it is known as a malaria vector in other Malagasy areas [Among 1550 mosquitoes tested, 28 were found positive for bearing opophily . It was Cameroon ; Zambia Cameroon , 41; andCameroon . This woina Faso , 43 and sy areas , 44, 45.An. coustani, was mainly responsible for malaria transmission in Miarinarivo, despite the presence of An. funestus and An. arabiensis. In that village, people were exposed to 61.2 ib/h infected bites per human (ib/h) from An. coustani during the study period (6 months), compared to only 5.4 ib/h in Ambohitromby, despite its relatively high abundance in this latter village. Plasmodium infected An. coustani (n = 12) were captured equally outdoor and indoor in Miarinarivo. By contrast, An. arabiensis was mainly responsible for malaria transmission in Ambohitromby, with 36 ib/h during the same study period, while it was responsible for 14.4 ib/h in Miarinarivo. The majority of infected An. arabiensis (9/10) were found outdoors. Anopheles funestus contributed to a minor extent to malaria transmission in both villages, being responsible for 5.4 and 7.2 ib/h during the whole survey in Ambohitromby and Miarinarivo respectively. Infected An. funestus (n = 3) were captured either indoors or outdoors. Overall, these results are similar to those observed in a study conducted in the Taveta district in Kenya, where malaria transmission, due to An. coustani, An. arabiensis and An. funestus occurred both indoors and outdoors [An. coustani recorded in Miarinarivo.Surprisingly, this study revealed that outdoors . The difoutdoors . Indeed,An. coustani as the major vector in Miarinarivo. Detailed analysis of the EIR over time  compared to 84.6 ib/h in Miarinarivo.In summary, the results of this study show that in neighbouring villages with a similar malaria prevalence in the human population, malaria transmission was driven by two different mosquito species and notably involved me Table  shows thAn. coustani has been clearly demonstrated as playing a major contribution in malaria transmission in an area of Madagascar, despite the presence of An. arabiensis and An. funestus known as major malaria vectors in the country. This finding was quite surprising as An. coustani is being known as a zoophilic and exophilic species in most areas of Madagascar where it was found. The results of this study can be used to better describe the epidemiology and transmission of malaria in Madagascar and to provide relevant information as guidance for adapted malaria vector control strategies. In an epidemiological context such as Madagascar, marked by the presence of both P. falciparum and P. vivax in combination with presence of several vector species, understanding the vector-specific contributions to the transmission of these two main Plasmodium species constitutes a challenge for malaria elimination.Overall, this study demonstrates the variability of vector biology dynamics between two neighbouring villages with similar ecological settings. This is the first time that Additional file 1: Table S1. Sequences of the primers and TaqMan probes used for the morphological identification of An. gambiae/An. arabiensis and for Plasmodium detection in Anopheles mosquitoes. Table S2. Human population that participated in the study categorised by age group and sex. Table S3. Mosquitoes collected by HLCs inside and outside houses, in Ambohitromby and Miarinarivo at the three time points. Table S4. Number of mosquitoes collected resting indoor by PSC.