Plasmodium vivax parasitaemia in communities of the Peruvian Amazon. Four cross-sectional active case detection surveys were conducted between May and July 2015 in four riverine communities in Mazan district. Analysis of 2785 samples of 820 individuals nested within 154 households for Plasmodium parasitaemia was carried out using light microscopy and qPCR. The spatio-temporal distribution of Plasmodium parasitaemia, dominated by P. vivax, was shown to cluster at both household and community levels. Of enrolled individuals, 47% had at least one P. vivax parasitaemia and 10% P. falciparum, by qPCR, both of which were predominantly sub-microscopic and asymptomatic. Spatial analysis detected significant clustering in three communities. Our findings showed that communities at small-to-moderate spatial scales differed in P. vivax parasite prevalence, and multilevel Poisson regression models showed that such differences were influenced by factors such as age, education, and location of households within high-risk clusters, as well as factors linked to a local micro-geographic context, such as travel and occupation. Complex transmission patterns were found to be related to human mobility among communities in the same micro-basin.Malaria has steadily increased in the Peruvian Amazon over the last five years. This study aimed to determine the parasite prevalence and micro-geographical heterogeneity of  According to data after World War II, malaria transmission in Peru has been dominated by Plasmodium vivax. Since the 1990s P. falciparum, in addition to P. vivax, emerged as a major epidemic, with continued outbreaks and rising incidence continuing to the present6. In the last decades, five years (2005–2010) of funding from the Global Fund for AIDS, TB and Malaria  was used to scale-up comprehensive control strategies, which included improving early diagnosis and treatment of malaria cases in endemic areas, strengthening of health services systems, training health promoters and microscopists, distributing insecticide-treated bed nets and environmental management through community participation8. As a result, the number of passively reported cases of malaria in 2010 was the lowest in the previous two decades .Progress in reducing the malaria burden in the last decade has been substantial worldwide, yet key challenges remain, particularly with a global emphasis on malaria eliminationP. vivax relapse, change in Anopheles darlingi populations and behaviours 12, sub-microscopic infection reservoirs14, general weather-related factors (such as flooding in Loreto in recent years) and reduced access of rural dwellers to health services15.Since then, Malaria control activities in the Iquitos region fall within the purview of the Regional Health Directorate of Loreto (RHDL), under the auspices of the national Ministry of Health (MOH). As a matter of operational policy, passive and reactive case detection is based on light microscopy which then determines treatment of confirmed infections. The increased numbers of malaria cases reported since 2011 in Loreto department are due to a variety of factors: expansion of residual foci of transmission with new infections; reintroductions from 2) of total Peruvian territory, encompassing 3.3%  of the total population of Peru16, and 95% of the countrywide malaria burden. Despite the fact that malaria transmission is perennial in the Peruvian Amazon basin, with a high transmission season between November and June5, RHDL data suggest that Loreto has a heterogeneous spatio-temporal malaria distribution3.Located in the Amazon Region, Loreto department covers 28.7%  that would not otherwise come for treatment due to lack of symptoms. In this scenario, it would be expected that different spatial aggregation patterns would be identified for malaria disease  and malaria parasitaemia (with or without symptoms)19.In this context, it is sometimes possible to distinguish groups with greater malaria riskP. vivax includes a dormant liver stage (i.e. hypnozoites) that remains undetectable until unknown mechanisms35 reactivate the parasite cycle and cause relapses from the original infection36. Importantly, the treatment of P. vivax in Peru was based on a regimen of 7 days of primaquine (PQ) at 0.5 mg/kg/day. However this regimen is not typically supervised and an adherence rate of only 62% has previously been reported37. Tools to distinguish between new infections and hypnozoite relapses are still in development41; therefore, here the term parasitaemia refers to indicate the presence of Plasmodium parasites in the blood, as suggested by the World Health Organization42, regardless of its origin from new infection or hypnozoite relapse43.The particular biology of 45. Nonetheless, the complex dynamics of malaria transmission in riverine communities – the typical malaria context in Amazonia – require accurate identification of these high-risk clusters of P. vivax parasitaemia and the associated factors at the person, household and community levels, to improve the implementation of intervention strategies in these foci of infections47. This study aimed to determine the parasite prevalence and the heterogeneity of risk factors for P. vivax parasitaemia between communities, taking into account the clustering of parasitaemia within households and within communities. Thus, a spatial and multilevel analysis was carried out using spatial information in four riverine communities of the Peruvian Amazon Region.Previous studies in communities near Iquitos  show spatial and micro-epidemiological heterogeneity of malaria transmission both in terms of incidence of disease and parasite prevalenceOf the 935 censused inhabitants in the four communities, a total of 820 individuals (Ind) from 154 households (HHs) were included in the analysis  and Urco Miraño ; and two smaller communities, Gamitanacocha  and Primero de Enero . Of all communities, 60 households (39.5%) had an open structure , with higher proportions in Gamitanacocha and Primero de Enero, than in Libertad and Urco Miraño. The predominant materials in walls were cement or wood (78.6% of HHs) and in roofs were palm, leaf or straw (77.6% of HHs). Approximately one quarter of the households in each community had an electricity supply, except Urco Miraño where three-fourths (76.3%) of HHs used solar panels or diesel-powered generators. Inhabitants of most households (90.7%) reported that the household received indoor residual spraying (IRS) by the RHDL in the last five years, and one-third (34.2%) of households had livestock inside the dwelling.Plasmodium spp. parasitaemia by qPCR and 2,768 slides (99.4% of samples collected) were examined by light microscopy. The cumulative number of individuals with P. vivax parasitaemia over the study period and the P. vivax parasite prevalence detected by light microscopy and qPCR in each survey round are presented in Fig. Throughout the four ACD surveys, 515 inhabitants had samples drawn at four visits, 172 at three visits, 76 at two visits and 57 at one visit. In total, 2,785 blood samples were examined for Plasmodium spp.; 589 P. vivax and 122 P. falciparum. In contrast, 95 (3.4%) of the available slides were positive for Plasmodium spp.; 89 P. vivax and 6 P. falciparum  were light microscopy negative, i.e. sub-microscopic parasitaemia (there were slight variations between surveys)  than in P. vivax . The sensitivity and specificity of light microscopy using qPCR as the reference method was 12.9%  and 99.8% , respectively. However, qPCR did not detect 4 Plasmodium spp. parasitaemia  that were identified by light microscopy.Of the total of blood spots tested, 711 25.5%) were positive for 5.5% wereys) Fig. . The proP. vivax and 85 (10.4%) P. falciparum parasitaemia during the study period. In the surveys, qPCR melting analysis did not identify mixed species parasitaemia, probably because the subjects in the active surveillance screens were predominantly asymptomatic  had at least one % had at Plasmodium spp. parasitaemia by qPCR across location and time (survey rounds), was 55% (451/820). By light microscopy, only 86 (10.5%) participants had P. vivax and 6 (0.7%) had P. falciparum parasitaemia (Table P. vivax parasitaemia detected by qPCR (44.6–49.5%) and light microscopy (1–16.5%) decreases in older people. By contrast, the proportion of P. falciparum parasitaemia by qPCR (9.2–14.1%) and light microscopy (0.7–1.2%) increases across age.The overall cumulative parasite prevalence, meaning the proportion of individuals with at least one 55% 451/80. By ligPlasmodium spp. parasitaemia during the study, and 85.7% and 39.0% had ≥1 participants with at least one P. vivax or P. falciparum parasitaemia in the follow-up, respectively. Given the small number of P. falciparum qPCR-positive parasitaemia, spatial and risk factors analyses were carried out only for P. vivax parasitaemia and P. falciparum parasitaemia were considered as negative for P. vivax. The household-level spatial distribution of individuals with at least one P. vivax parasitaemia during the study period is presented in Fig. Nearly all households 90.3%) had at least one participant with a % had at I = 0.26; p-value = 0.04) while in other communities, global Moran’s I values were statistically not significant. FDR-corrected LISA analysis detected statistically significant clusters in three of the four communities  and Urco Miraño , than in Gamitanacocha. Other epidemiological variables possibly associated  with higher P. vivax parasite prevalence were observed with the community-specific approach: education level in Gamitanacocha (primary school) and Primero de Enero (secondary and higher); presence of fever at time of visit and livestock in dwelling in Urco Miraño, a travel record, occupation and location in a high-risk cluster in Primero de Enero, and wall material and age between 15 and 39 in Gamitanacocha  remained associated with a lower P. vivax parasite prevalence, even after adjusting for community. Furthermore, significant interaction terms demonstrated that the effects of travel, and occupation on P. vivax parasite prevalence varied by community. The P. vivax parasite prevalence was higher in loggers, fishermen and farmers from Urco Miraño , and Gamitanacocha  than those from Libertad. Whereas travel in a period of a month was associated with higher P. vivax parasite prevalence only in Primero de Enero   living in HHs with walls made of palm, leaf, straw or corrugated plastic , which had not been treated with indoor residual spraying  after adjusting for whether HH is located in a high-risk cluster. In Libertad, no significant variables were retained in the multivariate model, showing a predominant effect of age over 40 years  related with lower P. vivax parasite prevalence after adjusting for household structure.The multivariate community-specific models Table  show remP. vivax parasite prevalence after adjusting for occupation and gender. Finally, in Urco Miraño, the presence of fever at time of visit was the most important variable  associated with a higher P. vivax parasite prevalence after adjusting for occupation , and whether HH is located in a high-risk cluster .In Primero de Enero inhabitants that reported travel in the last month , and living in a high-risk cluster  were associated with higher P. vivax parasitaemia was highly heterogeneous both within and among riverine Amazonian communities where a large proportion of inhabitants carried sub-microscopic P. vivax and P. falciparum parasitaemia51. This study further found that communities at small-to-moderate spatial scales (1 to 15 km) differed in P. vivax parasite prevalence, and that such differences were influenced by factors such as age, educational attainment, and location of households within high-risk clusters, as well as factors that were tied to a local micro-geographic context, such as travel and occupation. We observed that spatial clustering patterns of P. vivax parasitaemia differed in communities with different occupation-related activity profiles. The complex transmission patterns observed in this area seem to be determined by human movement among communities in the same micro-basin (i.e. tributary streams network that flows into the Amazon River). This concept is key yet neglected and must be understood to be a critical intervention point for malaria control in the Peruvian Amazon, a concept generalizable in similar contexts elsewhere.In the Peruvian Amazon, as in other malaria endemic regions, micro-geographical variation in malaria parasitaemia risk factors are consistently under-appreciated by public health policy makers as important for malaria control strategies. Using molecular tools and active case detection, this study demonstrated that proportion of P. vivax or P. falciparum in Amazonia—can lead to clinical immunity in this region that is manifested by asymptomatic parasitaemia53. Our data suggest that the relatively high prevalence of malaria parasitaemia in the study populations leads to the maintenance of transmission on a micro-geographic scale. The differences between the age-stratified proportions of P. vivax and P. falciparum parasitaemias detected by qPCR and light microscopy seems to be the result of a combination of exposure and immunity as observed elsewhere55. One unavoidable issue with the present analysis is that we cannot differentiate the contribution of acute, new infections from hypnozoite relapses from a previous mosquito bite to transmission quantitatively. However, previous studies indicate that in the Peruvian Amazon, most parasitaemias originated from relapses are asymptomatic and hence capable of transmission to mosquitoes56. Furthermore, in tropical regions such as the Peruvian Amazon, relapses tend to occur rapidly—at a frequency of three to ten weeks60. In any event, our data are consistent with the notion that asymptomatic P. vivax parasitaemia —whether from new or distant infection— leads to maintenance of malaria transmission on a scale larger than previously known33. One limitation of this analysis is the actual demonstration and quantification of the transmissibility of asymptomatic, low-level parasitaemia to mosquitoes; such work is in progress in our study sites/laboratory in Loreto.The magnitude of asymptomatic parasitaemia in this region challenges the traditional definition of hypoendemicity with regard to transmission from humans to mosquitoes. Previous studies indicate that only a few antecedent infections—whether with P. vivax parasitaemia in these communities, which enabled us to properly understand the transmission potential of patent/sub-patent, and symptomatic/asymptomatic malaria parasitaemia, regardless if it was a new acute infection or a relapse from hypnozoites. If exposure and infection are indeed highest over small geographical areas, most inhabitants will harbor low parasitaemia —likely due to host acquired immunity— with important implications for malaria control strategies62, especially if inhabitants frequently come into contact with the high transmission environments as observed in other studies33. Our within-community spatial clustering analysis shows that communities such as Gamitanacocha and Urco Miraño, which were found to have larger clusters of P. vivax parasitaemia, were identified as closed and isolated communities. Indeed, the data showed that nearly all households had at least one asymptomatically infected individual; suggesting that at larger scale (i.e. micro-basin), these communities are foci of parasites. Nonetheless, community level spatial clustering may be a consequence of the spatial distribution of dwellings and mosquito breeding sites. As previously demonstrated, An. darlingi has adapted to a diverse habitats64, with focal activity related to high density nearby breeding sites66. In tropical riverine communities, such favourable conditions are shaped by seasonal flooding and intermittent heavy rains68, allowing multiple breeding sites mainly in the large number of slow-flow streams proximate to households71. In contrast, communities with higher occupational-related population movement, like Libertad and Primero de Enero, had smaller or non-clustering of parasitaemia.The comprehensive spatio-temporal follow-up data allowed for a detailed assessment of An. darlingi in rural Amazon74. Urco Miraño is a geographically isolated community on the Napo River, and is the settlement of the Yagua ethnic group75. Traditional lifestyle and customs may play a role in community interactions; thus, population movement was found to be less common than communities in the Mazan River micro-basin. The population movement in this community, mainly towards Mazan community and the city of Iquitos, seems to be shaped by work activities related to visitor’s ecological tourism activities as in other communities of the Napo River micro-basin. A similar clustering pattern was observed in one of the most distant communities in the Mazan River micro-basin, Gamitanacocha. In this community the high-risk clusters were located near the ‘cocha‘ (lagoon), where the stagnant water ecosystem likely contributes to suitable An. darlingi breeding sites. Consistently, in Gamitanacocha, a high population movement was observed towards Maucallacta, a closer community with low risk of malaria during the study period according to RHDL records.In Urco Miraño, the high-risk clusters were observed in a highly deforested area with substantial surrounding vertical vegetation. Studies have shown that such deforested areas are preferred breeding sites for et al.44 as hyperendemic malaria transmission areas, were the most important destinations of occupational-related travels by inhabitants of Libertad and Primero de Enero. This liability to the frequent influx of infected individuals suggests a high vulnerability  in these communities77, as in other Amazonian Regions78.The remaining communities in the Mazan River micro-basin showed a different spatial clustering of malaria parasitaemia and population movement patterns. Libertad and Primero de Enero are contiguous communities with the highest malaria transmission in the Mazan River micro-basin; nevertheless, both are the biggest and most developed communities of this micro-basin. These communities are mainly devoted to extractive activities, and thus, the occupational-related mobility was higher than in Gamitanacocha or Urco Miraño. Interestingly, the occupational-related localities reported by Parker P. vivax predominated over P. falciparum, consistent with RHDL routine surveillance data. In addition to communities in different micro-basins showing a high heterogeneity of P. vivax parasite prevalence, significant differences were observed between the neighbouring communities of Libertad and Primero de Enero. Our findings in riverine communities from the Amazon region are consistent with recent studies that showed a high heterogeneity between villages at a micro-geographical scale along the China-Myanmar border, where P. vivax predominated80; in western Kenya, where most P. falciparum infections were sub-microscopic and asymptomatic81; and in peri-urban communities in the Loreto Region of Peru14. In addition to human population movement, vector populations and behaivours might be other contributing factors to the observed micro-geographical heterogeneities in malaria tranmission. Moreno et al.10 reported a high spatio-temporal heterogeneity in the entomological inoculation rate (EIR) among riverine and semi-urban communities in the Amazon Region; and Lainhart et al.9 reported two major An. darlingi subpopulations among peri-Iquitos communities, including Mazan samples collected in Libertad. Those genetically differentiated populations might be associated with different An. darlingi biting behaviors82.Our findings show that P. vivax parasite prevalence. This age-dependent parasitaemia effect is consistent in general with studies carried out in Kenya83, Papua New Guinea54, Botswana84, Ethiopia86, and Brazil48, where parasite prevalence and density decreased with age. Consistently, the presence of fever at time of visit remained a specific symptom for P. vivax parasite prevalence particularly in young children, indicating a positive relationship between age and acquired immunity. In the Peruvian Amazon region, individuals’ control of parasite density may be fostered by the high clonality of parasite populations observed at a local level in the Peruvian Amazon Region87, and the high EIR, as described in similar communities upriver in the Mazan River micro-basin44.The multilevel analysis provided important insights about malaria transmission potential in these micro-basins. Across all communities (i.e. multi-community model), our findings showed that young inhabitants had higher P. vivax parasite prevalence in Libertad was higher than in other communities from the same micro-basin or the Napo River micro-basin, taking into account the within-community heterogeneity, thus confirming the high heterogeneity between communities. In addition, heterogeneity in risk factors for P. vivax parasite prevalence was observed between communities. The significance of the interaction terms of community versus travel and occupation indicates that the communities did not behave as a single population. Instead, these results suggest that these risk factors seems to be tied to a local micro-geographic context. These risk factors for malaria parasitaemia were previously reported in the Amazon Region88. However, this study demonstrates a community-specific effect of these variables. This spatial non-stationarity association between P. vivax parasitaemia and risk factors was observed in several scenarios where a global risk factor estimate can become inappropriate since they averaged relationships that are not transferable to all local contexts92.Importantly, the P. vivax parasite prevalence only in Urco Miraño. This clinical manifestation was typically observed in susceptible populations not previously exposed to current parasite populations94, suggesting a high receptivity  in this community. A further factor associated with P. vivax parasite prevalence in Gamitanacocha using this approach was that inhabitants who reported that their households had not received IRS by the MOH had higher P. vivax parasite prevalence. In 2014, RHDL started an IRS campaign in several communities along the Mazan and Napo Rivers, including all communities in our study area. This protective effect of IRS has also been reported in previous studies95 and is recommended as an important component of control and elimination programs97. Nevertheless, this effect was not significant in other communities, supporting the hypothesis that mobile individuals acquire infection outside their residence communities.Using the community-specific approach, we observed particular risk factors for each community, such as occupation in Gamitanacocha and Urco Miraño. Interestingly, the presence of fever at time of visit was associated with higher 99, that over space and time would be associated with increased heterogeneity of transmission101, facilitating parasite population admixture32. Delgado-Ratto et al.34 showed the importance of human population movement on the geographic distribution of parasites and consequently the development of sub-structured parasite populations observed in peri-Iquitos communities. The genotyping analysis of isolates from the Mazan community reported by Van den Eede et al.32 showed high heterogeneity and strong differentiation from other peri-Iquitos isolates, suggesting multiple geographic sources of parasite sub-populations. Activation of heterologous hypnozoites acquired over the lifetime of the adult population102 could not be excluded as an explanation for the sub-structured parasite population observed in this communities. However, relapses in these areas presumably arose from infections in the near past (3–10 weeks)60, suggesting that the origin of the parasite population admixture is mainly the human population movement between communities with clustered transmission. As this riverine context is common in Amazonia, it is likely that the malaria rebound observed since 2011 was accelerated based on the high vulnerability and receptivity of riverine communities104. Thus, further work is needed to determine ‘source’ and ‘sink’ areas for malaria parasites in the Amazonian riverine network107 and to develop a comprehensive framework of human population movement in Amazonian communities109 to prevent malaria importation during transit (from hypo- to hyper- endemic areas) or upon return100.The intense flow of infected and non-infected people between communities of these micro-basins, plus introductions due to population movement to location outside the study area, has likely established a tangled yet open transmission network, generating complex community transmission patterns111, and believe this assumption to be appropriate since all factors in our dataset associated with missingness were included in the final models, and did not show a relevant effect (>10% change) on the risk factor estimates. We thus opted for the most parsimonious models, as presented in Tables 61, infected individuals can harbor very low parasitaemia, and at those parasite densities the probability of detecting a malaria parasite DNA molecule is random. That being said, PCR based on filter papers only underestimates the potential effect of asymptomatic parasitaemia on parasite mobility and transmission, and, as discussed above, the transmission potential of extremely low parasitaemia remains to be determined.This study has some limitations. Despite efforts in each survey to include all community members, we achieved a rate of at least 80% participation at each survey point. When we were able to contact a person, we did not observe any refusal to participate. Instead, the most important reason for incomplete or missing data was the fact that individuals were away from the area for different reasons, such as work, national army recruitment, and festivities. We used generalized linear mixed models which are valid under the assumption of missingness at random (MAR)P. vivax, and spatial clustering of transmission, strongly suggests that public health authorities should reformulate malaria control strategies based on sensitive parasite detection and active surveillance. Larger, comprehensive assessments are needed to advance the understanding of the impact of human mobility on malaria transmission in riverine communities before decisions of mass drug administration on a community or population level can be made, for example issues of community acceptability and which drugs to use and their safety.In conclusion, this study highlights that to tailor strategies for effective malaria control and future elimination in riverine communities of the Amazon Region, it will be essential to take local geography and human socio-demographics into account to estimate dynamics of ongoing malaria transmission. Evidence from this and other studies regarding the large reservoir of sub-microscopic infections, high heterogeneity in risk factors for The study was approved by the Ethics Review Board of the Universidad Peruana Cayetano Heredia in Lima and the Regional Health Direction of Loreto. Prior to study enrollment, every adult participant signed an informed consent and every participant under 18 years old provided informed assent with parental or guardian written approval. All the methods were carried out in accordance with the approved guidelines.67. Mazan district is comprised of several small communities, and the capital and largest community is the community of Mazan , located near the confluence of the Mazan and Napo Rivers, about 55–60 km from the city of Iquitos (1 hour by boat).The present study was conducted in the district of Mazan (North of Iquitos), province of Maynas in Loreto department. The population is mainly mestizo (i.e. mixed-race identity), very poor, and lives in semi-open wooden houses without screens. The local economy depends on agriculture, lumber, and fish extraction. The weather is tropical, with minimum temperatures of 17 °C to 20 °C in the months from December to March, and maximum temperatures up to 36 °C in June and July. The average humidity is 84% with heavy rains from November to MayAn. darlingi, highly anthropophilic10 and about 85% of malaria cases reported by the RHDL are caused by P. vivax  distributed across all ages. Mazan is considered a very high-risk district for malaria transmission; 1,954 cases were reported in 2014, indicating an annual parasite index (API) of 141.1 cases per 1,000 inhabitants.Malaria transmission in Mazan district has an unstable and seasonal epidemiological pattern, with a peak between May and September. The primary vector is In March 2015, a comprehensive census and household geo-referencing was conducted in ten high-risk malaria communities  in Mazan district. From March to April 2015, these communities were under weekly surveillance to detect communities where any unusual increase of cases might occur. Surveillance was based on monitoring the slide positivity rate (SPR) from passive case detection (PCD) of the RHDL. Study communities were selected based on two criteria: a) the proportion of the population screened for malaria (by thick or thin blood films) was larger than 20% in the previous 8 weeks, and b) the SPR of the previous 8 weeks remained above 5% for two consecutive weeks. These criteria resulted in the selection of four communities  surveys rounds were conducted in each of the four communities, one ACD survey every 10 days. To look for risk factors and heterogeneities in risk factors for In the census, each household in the communities was encoded (6-digit numeric code) and geo-referenced using a Global Positioning System (GPS) hand device . All members of each household were invited to participate, and, upon consent, a unique 8-digit numeric code was assigned to each participant. A structured questionnaire designed and previously pilot-tested was used to collect individual and household data .In each ACD survey, data regarding socio-demographic characteristics and clinical manifestations at time of visit were collected. Each participant was clinically examined for fever or any other malaria symptom. Blood samples were collected on every household member (regardless of symptoms) by finger-prick for microscopic examination (thick and thin blood films), and for PCR testing . Blood samples were labeled during fieldwork with pre-printed labels with 9-digit numerical codes. This code was used in the fieldwork and at the Institute of Tropical Medicine “Alexander von Humboldt”, (ITM-AvH) in Lima, where samples were processed. New participants were enrolled during the ACD surveys. Participants with malaria diagnosis by microscopy at ACD surveys were treated following national guidelines.113 on mobile devices without network connection. Data were synchronized with the project´s server in Lima, and linked to laboratory results using the 9-digit unique numerical codes.During the census and the ACD surveys, houses were visited up to 3 times in a period of 3 days to maximize subject participation. Data were collected using Open Data Kit (ODK)114. Quality control was done blindly on all positive slides and 10% of randomly chosen negative slides by a senior technician at ITM-AvH.Microscopy examination was performed the same day of sample collection in each ACD survey. Thick and thin blood smears were stained with 10% Giemsa solution for 10 minutes and were examined to determine the parasite density by counting the number of parasites on asexual stage and gametocytes by 200 leucocytes (L) and assuming a concentration 8,000 L/μl of blood. A sample was considered negative if no malaria parasite was detected after the examination of 100 fields of microscopy2 sections for the parasite genomic DNA extraction using E.Z.N.A.® Blood DNA Kit , following manufacturer instructions with slight modifications – addition of TEN  buffer, supplemented with SDS 10% w/v – and stored at 4 °C for immediate use and at −20 °C for further analysis.Filter paper areas containing blood were cut into ~6 mmet al.115 using PerfeCta SYBR Green Fast Mix . Briefly, this method amplifies the 18SSU rRNA gene sequence of the Plasmodium species-specific region. After the amplification, analysis of the differences in melting curves provided an accurate differentiation between Plasmodium species.Quantitative real-time polymerase chain reaction (qPCR) testing was done following a modified protocol from Mangold P. vivax parasitaemia occurred in the study area, a global Moran’s I statistics116 was computed to describe the overall spatial dependence of P. vivax parasitaemia in each community and Anselin’s Local Moran’s I  was performed with a correction for multiple and dependent tests using the false discovery rate (FDR)118 to identify local patterns and high-risk areas. These analyses were performed with the proportion of individuals with P. vivax parasitaemia at household level as outcome. The time-series data for each participant were collapsed to a single register defined as at least one P. vivax parasitaemia (positive result by qPCR) of the 4 ACD surveys. For the spatial data processing and visualization, QGIS 2.16  was used.To assess whether spatial autocorrelation of 119. Two factors guided the criterion for choosing distance threshold/band. The first is the flight range of mosquitoes in the Amazon region. The longest reported flight range of An. darlingi in the Brazilian Amazon is 7.2 km120, but most mosquitoes fly within 400–500 m66. Second, in these communities, buffers larger than 2,000 m cover the entire community. A non-parametric spatial autocorrelation analysis was performed in GeoDa to evaluate neighborhood distance bands from 100 m to 2,000 m. As result, a band of 550 m displays the maximum level of spatial autocorrelation and ensure that each household has a neighbor. With this distance band the global Moran’s I was used to test the null hypothesis that measured values at one household location are independent of values at other households locations, and LISA analyses identify significant relations between the household values and the neighborhood-lagged values in each community as: high-risk clusters (high-high) where both proportion in household and neighborhood were high, low-risk clusters (low-low) where both proportion in household and neighborhood were low, and outliers (high-low or low-high) where the proportion in household and neighborhood were different.Distance weighted neighborhoods were defined in GeoDa v1.8 P. vivax parasitaemia as the dependent variable in the models, and the subsequent observations were excluded from the analysis. We modeled the presence of P. vivax parasitaemia  regardless of symptoms and/or previous treatment. All P. falciparum parasitaemia were excluded from the analysis. Poisson regression models were used to estimate the prevalence ratio (PR) of the outcome, assuming a binomial distribution for the error term, and a log link function123.The multilevel design of the study consisted of repeated observations of individuals (up to 4 ACD surveys), nested within households, nested in the four selected communities  or its 550m-neighborhood buffer, household structure (houses with at least one closed room or not), walls material, roof material, whether household received indoor residual spraying (IRS) by the RHDL, had electricity supply, and had livestock inside dwelling; 4 at the individual-level: subjects’ gender, age , education level , and outdoor occupation , and 2 time-dependent variables: travel history in the last month, and fever symptom at each survey round , two approaches were used: The first consisted in the construction of a single model including all communities and exploring interaction terms between each factor and community (from here on termed “multi-community” model), and in the second approach, separate regression models were built for each community (from here on termed “community-specific” models). Therefore, three levels  were included in the equation for our multilevel model of the dependent variable P. vivax parasitaemia (yijk):ijk subscript refers to the ith observation of the jth individual from the kth household; δ000 represents the fixed effect of the intercept and (Vk00 + Ujk0) represents the intercept random effects of households and individuals within households, respectively; βjk1 represents the fixed effects of the slope for the covariates terms, and εijk refers to the random error term.To evaluate if factors associated with 126, the bivariate and multivariate analyses were performed using the same multilevel structure. In the bivariate multilevel analysis, the relationship between each factor in all levels  and time-dependent P. vivax parasitaemia was analyzed for the multi-community model, and the community-specific models.All data analyses were done in STATA 14 . Statistical significance was defined at p-value < 0.05 and 95% confidence intervals (CI) were estimated as appropriate. As in other epidemiological studies that applied multilevel models127, using the ‘MuToss’ package128 in R software v3.2 .Construction of the multivariate multilevel models was performed using a forward stepwise process, and a likelihood ratio test (LRT) to compare nested models. Multi-community model was constructed adding factors ordered according to the log likelihood in the bivariate model and retained if they had a p-value < 0.2 in the adjusted model. Systematically, interaction terms between each factor in the final model and community were evaluated, and significant  interactions were retained. Community-specific model construction consisted of a stand-alone process for each community. Factors with a p-value < 0.2 in the bivariate model for the corresponding community were introduced and retained if they had a p-value < 0.2 in the adjusted model. A correction for multiple and dependent tests was done with the adaptive false discovery rate (AFDR)Supplementary information


Blood film microscopy is the gold standard approach for malaria diagnosis, and preferred method for routine patient diagnosis in health facilities. However, the inability of laboratory professionals to correctly detect and identify malaria parasites microscopically leads to an inappropriate administration of anti-malarial drugs to the patients and incorrect findings in research areas. This study was carried out to evaluate the performance of laboratory professionals in malaria diagnosis in health facilities under the Defense Health Main Department in Addis Ababa and its surroundings, Ethiopia.A cross sectional study was conducted from June to July 2015. Totally, 60 laboratory professionals out of the selected 16 health facilities were included in the study. Data were collected by distributing standardized pre-validated malaria slide-panels and self-administered questionnaires among professionals, onsite in each study facility. Sensitivity, specificity, and strength of agreement (with kappa score) in performance among the study participants against WHO-certified expert malaria microscopists were calculated.Of the 60 study participants, 8.3% (5/60) correctly read all the distributed slides in terms of parasite detection, species identification and parasite counting; whereas, each of the remaining 55(91.7%) interpreted at least two slides incorrectly. The overall sensitivity and specificity of participants’ performance in detection of malaria parasites were 65.7% and 100%, respectively. Overall, fair agreement  in detection of malaria parasite was observed between the study subjects and expert readers. The overall sensitivity and specificity of participants in species identification of malaria parasites were respectively 41.3% and 100%. Overall, slight agreement  in identification of malaria species was observed. Generally, agreement was lower in parasite detection and species identification at low parasite density and mixed infection cases.The general agreement between the study participants and expert microscopists in malaria parasite detection and species identification was very low, particularly in the cases of low-parasite density and mixed infections. Therefore, regular external quality assessments and further refreshment trainings are crucial to enhance the skill of professionals in malaria microscopy; particularly for those in non-malarious areas where exposure to malaria diagnosis is low. Plasmodium parasites by light microscopy is yet the primary method of malaria diagnosis in most health care facilities throughout the world  gives a single number to indicate screening performance. This may be the weighted average of the sensitivity and specificity or the weighted average of the positive predictive value and negative predictive value ,13. FishThe study was ethically reviewed and approved by the ethical clearance committee of Addis Ababa University. Informed consent forms were signed by the participating health professionals and blood donors. To ensure confidentiality, the participants’ data was linked to a code number only.Total of 60 laboratory professionals, who were available in the facility during data collection, participated in the study. The mean age of participants was 32 ± 5.43 years and 48(80%) of participants were males. Regarding educational status, 32(53.3%) were bachelor degree and 28(46.7%) were diploma holders. Majority of the study participants that is 38(63.3%) had an experience on malaria microscopy for more than two years and 22(36.7%) had less than two years. Of the total participants, 20(33.3%) took refreshment training on malaria microscopy at least once. Those individuals who participated in an external quality assurance (EQA) program accounted for 36(60%) .Of the 60 study participants, only 5(8.3%) interpreted all the distributed slides correctly in terms of parasite detection, species identification and parasite counting; whereas, 55(91.7%), each of which, interpreted at least two slides incorrectly. Based on WHO grading system, 40(66.7%) of participants were at “In-training”, five (8.3%) at “Advanced”, 10 (16.7%) at “Reference”, and five (8.3%) at an “Expert level” in malaria parasite detection . From thP.falciparum, 240 P.vivax, 120 mixed Pf + PV, and 120 negative controls were used for evaluation. From the total 720 slides, 514 (71.3%) were correctly reported for the detection of malaria parasites. Of the 600 positive slides (60 participants x 10 slides), 248 (41.3%) on species identification and only 45 slides (7.5%) on quantification were reported correctly. In the case of negative slides, all of the 120 (60 x 2) slides were reported acceptable  which is 'fair agreement' . The oveP. falciparum, 45% of slides with P.vivax, and 16.6% of slides with mixed infections were identified correctly. A total of 100 (83.3%) of slides with mixed infection were identified wrongly as P. falciparum, 97 (40.4%) of slides with P. falciparum were identified wrongly as negative and 109 (45.4%) of slides with P. vivax were identified wrongly as negative , which is almost ‘slight agreement’  interpreted all the distributed slides correctly in terms of parasite detection & species identification; and the rest 55 (91.6%), each of which, interpreted at least two slides incorrectly. This is in accordance to study finding from Hawassa, Ethiopia .P. falciparum slides, and 109(45.4%) slides with P. vivax were identified wrongly as negative. The number of participants who failed to correctly report P. falciparum in the current finding was higher than the 27% failure rate in identification of P. falciparum in Canada [The sensitivity and specificity of the professionals in detecting malaria parasites were 65.7% and 100%, respectively. The current study showed lower performance in parasite detection when compared to a respective sensitivity and specificity reports of 82% and 96% from Hawassa, Ethiopia , 88% andn Canada  and in cn Canada .P. facliparum and P. vivax infections, 100 (83.3%) slides were identified wrongly as P. falciparium mono-infections. This finding is similar to low performance in Hawassa Ethiopia [The general performance of participants in species identification is lower than in parasite detection in the current study. This matches the findings from other studies ,18,19. FEthiopia , Hong koEthiopia . The posEthiopia . SecondlIn the current study, the overall agreement in detection of malaria parasite between the study subjects and expert readers was 71.4% (Kappa: 0.4), which is categorized as 'fair agreement'. The overall agreement in identification of malaria parasites was 51.1% (kappa: 0.04), which is categorized as 'slight agreement' based on WHO guideline ,13. OverIn present study, 40(66.7%) of participants were classified as 'In-training' and 5(8.3%) were at expert level; which was lower performance than findings from Hawassa where 17(23.6%) participants were ‘in-training” and the 18(25%) were at expert levels , and theP. falciparum slides. Performance in species identification was better on high-density unstained (70%) than on the low density (13.3%) unstained P. falciparum slides. The study clearly showed the microscopists had low experience in staining of malaria slide smears and had low capacity in malaria diagnosis, particularly in the case of low parasite density. The overall agreement in detection of malaria parasites on low density stained slides was 63.3% (Kappa: 0.26) which is lower than an agreement of 100% in high density stained slide. The overall agreement on low density unstained slides was 58.7% (Kappa: 0.17) and an agreement on unstained slide with high density was 88% (kappa: 0.76).There were a big difference in parasite detection and species identification in terms of parasite density, and staining of blood film. Performance in species identification was better on high density stained blood film slides (75%) than on the low density (25%) stained The current study brought the first evidence on performance of malaria microscopists in Addis Ababa and its surrounding areas. However, it has come with some limitations, as it could not include all malaria microscopists in the study sites; only those who were available during the time of data collection were recruited. The species of malaria parasites used for malaria blood film slide panel preparation were not characterized or molecularly typed by polymerase chain reaction, only identified by certified laboratory technologists.The present study showed poor performance of the study participants in malaria microscopy among the diagnostic laboratories at defense health facilities in Addis Ababa and its surrounding areas, Ethiopia. The general agreement between the study participants and expert microscopists in malaria parasite detection and species identification was very low, particularly in the cases of low parasite density and mixed infections. The misinterpretation in malaria diagnosis in turn equally results in misadministration of inappropriate treatment to the patients. This may direct to emergence of drug resistance, exposing patients to suffer from repeated diseases and death, economical loss and wastage of costly drugs. Therefore, to handle these challenges, further regular external quality assessments and trainings are crucial for career competency of professionals, particularly to those who are working in malaria non-endemic areas.S1 File(DOCX)Click here for additional data file.


Plasmodium falciparum infections in cord-blood samples. In addition, we examined the usefulness of P. falciparum Histidine Rich Protein2 (PfHRP2) as surrogate biomarker of infection and explored association between congenital malaria and clinical outcomes. A prevalence of congenital malaria by qPCR of 4% (16/400) was found, which increased to 10% among newborns from mothers infected at delivery. RDT and LM showed poor performances indicating limited utility for congenital malaria screening in cord blood. Because PfHRP2 detection in cord blood could be affected by transplacental passage of parasite antigens, PfHRP2 might not be used as a surrogate biomarker of congenital malaria infections. There was no evidence of a significant clinical impact of congenital malaria on infant’s health from birth to 59 days of life. Case control studies including long-term follow up may provide additional understanding on the relevance of neonatal malaria infections.Congenital malaria diagnosis is challenging due to frequently observed low parasite density infections, while their clinical relevance during early infancy is not well characterized. In Nanoro health district (Burkina Faso), we determined the prevalence of congenital malaria by real-time quantitative PCR and we assessed the performance of rapid diagnosis test (RDT) and light microscopy (LM) to detect  Plasmodium asexual stages in newborn’s cord or peripheral blood during the first week of life as a result of materno-fetal transfer of malaria parasites1. The reported prevalence of congenital malaria in sub-Saharan Africa ranges from 0% to 54.2% depending on geographic area, endemicity, study design and detection method (i.e. light microscopy -LM- or Polymerase Chain Reaction -PCR-)5.Congenital malaria is defined as the presence of 8. However, some newborns with congenital malaria can develop symptoms within days14, weeks or months after birth17. Even though consequences of congenital malaria may be life-threatening little is known about the impact of these infections on the infant’s health, especially during the neonatal period (from birth to 28 days)19. Targeted diagnosis of high risk newborns e.g. those born from infected mothers, could inform neonatal clinical care and guide prompt malaria treatment in clinically relevant cases.In malaria-endemic areas, symptomatic neonatal malaria infections are rare, suggesting spontaneous clearance of parasites detected at birth21, while a third study performed on hospitalized neonates during the first week of life reported a prevalence of 24.4%9. Routine diagnostic of clinical malaria in the country includes parasitological confirmation with rapid diagnostic test (RDT) and LM. Malaria parasite identification on Giemsa-stained blood smears is performed in district referral hospitals whereas the RDTs are the only diagnostic tool available for malaria at peripheral health centers. Although malaria RDTs have been implemented country-wide by the National Malaria Control Program (NMCP) since 2009, its performance for the detection of congenital malaria among neonates has not yet been evaluated.In Burkina Faso, three studies have investigated the prevalence of congenital malaria by LM: two independent cross-sectional studies reported prevalences of 1.4–2.8% in cord blood samplesPlasmodium falciparum infections in cord blood samples. In addition, we used an Enzyme-linked Immunosorbent Assay (ELISA) kit to examine whether histidine-rich protein II antigen (PfHRP2) levels are good surrogate biomarkers of congenital malaria infections. Finally, we explored the association between cord-blood malaria infections as detected by qPCR and clinical outcomes.In the present study, we determined the prevalence of congenital malaria, defined as cord-blood malaria infections as detected by quantitative PCR (qPCR) and we assessed the performance of a HRP2 RDT in plasma and LM to detect P = 0.317). Their overall mean birth weight was 3013.8 g and 11.3% of newborns had a low birth weight (<2500 g).Characteristics of study participants are presented in Table P < 0.001) and placental-blood infections . The prevalence of congenital malaria increased to 10.9% (14/128) among newborns whose mothers had peripheral infections at delivery and to 10.2% (14/137) among those born from mothers with placental-blood infections. Additionally, we performed histological examination of placental tissue from those mothers with cord-blood parasitaemia (Table P = 0.980), although we observed a trend of decreasing prevalence in newborns from primigravid to multigravid women. The overall parasite densities in cord-blood infections cases were low with a geometric mean of 5.5 parasites/µL (95% CI: 1.4–22).The prevalence of congenital malaria, as detected by qPCR on cord-blood samples, was 4% (16/400) Table . These ii.e. the probability of having a qPCR-confirmed congenital malaria infection, was 66.7% (95% CI: 12.5–98.2%) while negative predictive value (NPV), i.e. the likelihood of not having the infection among those who had a negative RDT, was 96.5% (95% CI: 94–98%). There was a limited agreement between RDT and qPCR with a Cohen’s kappa coefficient equal 0.2.HRP2 was detected in cord-blood in 3 (0.75%) out of the 400 plasma samples tested by RDT, while LM failed to detect any infection in the study population (Table PfHRP2 antigen was positive in 6 (1.5%) out of the 400 tested cord blood samples (Table P = 0.007).ELISA test for es Table , althouges Table . RDT andes Table . Indeed,PfHRP2 as a biomarker for detection of congenital P. falciparum malaria infection, we investigated whether neonatal PfHRP2 levels in plasma were associated with the parasite densities in cord blood. The same association was tested for maternal blood parasite densities. The results suggested that the optical density (OD) of the PfHRP2-ELISA test could be associated with parasite densities from both cord and maternal blood, although OD values were much strongly associated with parasite densities in the mother  than those in cord blood . Indeed, as shown in Fig. PfHRP2 ODs followed a consistent trend towards increasing parasite densities in the placenta and maternal peripheral blood, while cord antigen levels showed a markedly different profile when compared to parasite density levels in cord blood. The strong correlation between cord antigen detection and the maternal parasite burden was further confirmed by comparing maternal parasite densities among ELISA-positive and qPCR-positive cases. As shown in Table P = 0.001) than those with congenital malaria infection detected by qPCR (P < 0.05).In order to assess the appropriateness of using P = 0.264). One neonate with congenital malaria by qPCR, developed clinical malaria at day 36 of the follow-up. Msp1 and msp2 genotyping of the paired samples, i.e. at birth and day 36, confirmed a new infection at day 36 indicating that the cord-blood infection at birth was cleared spontaneously without developing into a clinical case. In total, 10.5% (42/400) of children experienced at least one fever episode during the follow-up period and only two of them had cord-blood malaria infection. Finally, there was no evidence of association between fever episodes during the first two months of life and cord-blood infections .We investigated whether congenital malaria infections as detected by qPCR in cord-blood samples were associated with low birth weight (LBW), occurrence of fever and risk of malaria during the first two months of life. The overall prevalence of LBW in the study population was 11.3% (45/400 newborns). Although 18.7% (3/16) of the newborns with cord-blood infections were born with LBW, the association was non-significant 9. Several studies have discussed the impact of seasonality, diagnostic method and study design on the estimated prevalence of congenital malaria3. Although our study recruitment period is comparable to previous reports in the country21, the qPCR technique we used, which targets Pfvar genes (≈60 copies/genome)22, is 10 times more sensitive than the standard 18S rRNA-based qPCR used in other studies24 and could explain at least partially variability observed between studies. On the other hand, the current study is ancillary to a larger trial investigating the effectiveness of strategies for malaria prevention in pregnancy25. Uptake of pregnancy preventive and control measures such as intermittent preventive treatment of malaria in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP), insecticide treated net, malaria diagnosis and treatment is expected to be higher in the study population than in the general population due to the trial effect. These preventive measures, which are known to be effective in preventing maternal and placental malaria infections30, may have reduced the risk of infection and transplacental transmission of malaria parasites potentially leading to underestimation of congenital malaria prevalence.In this study we found a 4% prevalence of congenital malaria as detected by qPCR in cord-blood samples from mothers delivering in the Nanoro health district of Burkina Faso. Previous studies from Burkina Faso using LM to detect congenital malaria infections have reported lower prevalence estimates of 2.8% in Ouagadougou33, found in all detected cord-blood infections and discordant levels of malaria parasitemia between cord and placental/maternal blood  are in agreement with malaria infections acquired antenatally24.Indeed, cord-blood infections as detected by PCR were more often detected among mothers with maternal peripheral and placental infections at delivery (10.9% and 10.2% respectively) than in the general study population (4%). Although we cannot exclude that a number of these cord blood infections are due to admixture with maternal or placental blood at delivery, evidences of a compromised placenta, which increases the risk of transplacental passageTherefore, due to the increased risk of congenital malaria found among newborns from infected women at delivery, this high risk group could be targeted for congenital malaria screening to allow for prompt and adequate neonatal clinical care. In this scenario, malaria diagnosis using umbilical cord-blood sample would be the best option to avoid invasive blood sample collection of the newborn. However, detection of congenital malaria infections in cord blood samples using field implemented diagnostic tools such RDT and LM remains a challenge.21. The observed differences can be explained by the lower cord blood parasite densities detected by qPCR in our study  compared with parasite densities detected by LM in the other studies . Lower parasite densities could also be explained by a higher uptake of pregnancy preventive measures in the present study. Thus, LM does not appear as an optimal tool to screen for congenital malaria infections in cord blood samples. Indeed, limited sensitivity of LM to detect congenital malaria infections compared to molecular methods has also been observed in studies conducted in other African countries34.LM is the standard method for malaria diagnosis in Burkina Faso and is available at the health district referral hospitals. In this study, LM did not detect any umbilical cord blood infections, contrasting with previous reports from Burkina FasoPfHRP2 is the RDT recommended by the NMCP in Burkina Faso for malaria diagnosis in peripheral health centers in rural areas where there are no laboratory facilities available. Here, we assessed whether the use of PfHRP2-RDTs could be extended to screen umbilical cord-blood samples for P. falciparum detection. SD-Bioline PfHRP2 RDT missed 87.5% of umbilical cord-blood infections detected by qPCR indicating that diagnostic performance to detect congenital malaria is suboptimal. Moreover, RDT test was only found to be positive when PfHRP2 levels were very high (RDT positives >13 vs qPCR/ELISA positives <2). Of note, we did not follow manufacturers’ instructions in the use of the PfHRP2 RDT as we tested stored cord plasma samples instead of whole blood, which may have decreased test sensitivity. However, we found a good agreement between RDT and ELISA tests taking into account that RDT uses lower plasma volumes than ELISA. This is in agreement with previous reports35 and indicates that results obtained here are reliable. In the other hand, although we cannot exclude that RDT- or ELISA-negative/PCR-positive samples are due to the presence of pfhrp2-deleted P. falciparum parasites36, parasite densities detected by qPCR in cord blood samples are low and often under the limit of detection of the used RDT. Nevertheless, future studies should investigate the prevalence of pfhrp2-deletion particularly in areas of high malaria transmission as in the study area.The SD-Bioline 38. In Nigeria, pLDH-Optimal® RDT did not detect any congenital malaria infection while prevalence by LM was 10.9%37 and, in Burundi two newborns were found positive by SD-Bioline PfHRP2 but none by CareStart pan-pLDH38. When samples from these two newborns were subsequently tested by qPCR, both resulted negative and were resolved as false RDT-positives, even though WHO RDT performance evaluation reported no false positive SD Bioline Pf-antigen results after testing 140 true-negative samples39.Since PfHRP2-based tests sensitivity depends on antigen concentration and this seems to be determined by the parasite burden41, we expected positive RDT cases among samples with high parasite density in umbilical cord blood. Surprisingly, congenital malaria infections presenting higher parasitemia  were all negative by RDT, while RDT-positive cord-blood samples presented 79.8, 5.3 and 0 parasites/µL. Of note, the RDT-positive/qPCR-negative cord sample corresponded to a mother with very high parasite burden at the time of delivery . This observation is in line with the findings reported in the study from Burundi38, where the two false-positive RDT results also corresponded to newborns from mothers with very high peripheral parasitemia . Indeed, a significant correlation between cord PfHRP2 levels and maternal parasite burden was further confirmed by our results. On the one hand, median placental and peripheral densities were found to be significantly higher among mother-child pairs with ELISA-positive result (P = 0.001) but not among those with a cord qPCR-positive result. In addition, cord PfHRP2 levels correlated strongly with maternal parasite densities than cord blood parasite densities. Unfortunately, we had no data available to investigate the direct correlation between cord blood and maternal PfHRP2 levels at delivery. All these evidences suggest that PfHRP2 in cord blood may be the result of the transplacental passage of maternal antigens rather than a reflection of concurrent malaria infection in the newborn. Hence, detection of congenital malaria infection based on the presence of PfHRP2 antigen might not be a reliable approach.Only two other studies have previously evaluated RDT performance to detect congenital malaria infections42. Likewise, transplacental transfer of circulating antigens has been also documented in other parasitic diseases such as lymphatic filariasis44. However, in the present study high maternal parasite densities do not always correlated with high PfHRP2 levels and positive RDT and/or ELISA results suggesting that transplacental passage of parasite antigens can be influenced by different conditions during pregnancy. In fact, factors that contribute to mother-to-child transmission of malaria parasites (or antigens) have not yet been well elucidated, although pre-existing level of malaria immunity, history of maternal infections and malaria-related placental changes and damage seems to play an important role48. In summary, our results indicate that PfHRP2 levels in cord blood samples are not a good surrogate biomarker of P. falciparum infections. Hence, even if high-sensitive PfHRP2-based RDTs are available on the market in the near future49, these would not render a useful tool for parasite presence detection in cord blood. Further investigations should include quantification of P. falciparum antigens levels such as (but not limited to) PfHRP2 in maternal peripheral, placental and umbilical cord blood samples.Transplacental passage of malaria antigens was previously reported in a study conducted in The Gambia that found GLURP antigen in 28.6% of tested cord sera samplesAlthough there was a trend towards lower birth weight among newborns with congenital malaria, we did not find a significant association between LBW and cord-blood infections as detected by qPCR. The lack of association could be related to the low number of LBW newborns observed in our study population. We did not observe any case of clinical congenital malaria  neither clinical neonatal malaria (parasites and symptoms from 1 to 4 weeks of life). One newborn with congenital malaria developed a clinical malaria infection 36 days after birth. However genotyping of paired samples concluded that the clinical episode at day 36 was due to a new infection.18. The importance of maternal derived antibodies in controlling infections early in infancy could be one of the main explanations for the clearance of infections and the lack of association with symptoms and adverse outcomes after birth50. In addition, as in all studies based on high-sensitive detection of parasite DNA or antigen, we are indirectly detecting parasite material rather than alive parasites. Therefore, we cannot exclude that some of the positive results correspond to non-viable parasites51.Overall, the lack of a significant clinical impact of congenital malaria as detected by PCR on infant’s health during the first 2 months of life in our study, may be due to the low number of congenital malaria infections. In the other hand, these results are concordant with other studies in high malaria transmission areas of sub-Saharan Africa, where symptomatic neonatal infections are rare suggesting that congenital malaria is infrequently associated with clinical disease52. However, the role of congenital malaria in the modulation of that immune response is unknown. Hence, a long-term effect of congenital malaria on newborns health living in high malaria endemic settings cannot be excluded and deserves further investigations. In the meantime, prenatal exposure to malaria parasites/antigens should be prevented through effective strategies for malaria control in pregnancy.Prenatal exposure to blood-stage malaria parasites/antigens may have profound long-term effects during infancy and childhood by priming the immune responses of the fetus and/or by inducing immune tolerancePfHRP2 RDT compared to qPCR suggests a limited utility of RDTs for congenital malaria screening in cord-blood samples in Burkina Faso. In addition, because PfHRP2 detection in cord blood could be influenced by high levels of parasite antigens in maternal blood through a transplacental passage, PfHRP2 might not be used as a surrogate of congenital malaria infection. There was no evidence of a significant clinical impact of congenital malaria infections on infant’s health from birth to 59 days of life. Case-control studies including long-term follow up may provide additional understanding on the relevance of neonatal malaria infections.In conclusion, congenital malaria infection is not uncommon in Nanoro health district and the estimate of its prevalence using a high sensitive qPCR could reach 10% among newborns from mothers presenting peripheral and/or placental infections at delivery. The overall poor performance of SD-Bioline 53.This study was conducted in 10 peripheral health centers of Nanoro Health District (NHD) in Burkina Faso. The NHD comprises approximately 153,000 inhabitants and is located 85 km west of Ouagadougou, the country’s capital. Malaria transmission in the region is seasonal and hyperendemic with the highest transmission period overlapping with the rainy season from July to October. The mean prevalence of malaria in the country is estimated to 62.2% with a mean entomological inoculation rate of 118.1 per person per year25. For the present study, 400 mother-child pairs were enrolled at the moment of delivery (from July 2014 to April 2015) after inform consent from the mothers was obtained and newborns were followed-up by passive case detection for 59 days to identify occurrence of fever or clinical malaria cases. Sample size was estimated assuming a minimum congenital malaria prevalence of 1.4% as reported in a previous study in Burkina Faso20. Ethical approvals were obtained from institutional ethics committees at Centre Muraz, Bobo Dioulasso, Burkina Faso (006–2014/CE-CM), Institute of Tropical Medicine, Antwerp, Belgium (953/14) and University Hospital in Antwerp (UZA), Belgium (14/26/277). All procedures followed were carried out in accordance with the Helsinki Declaration as revised in 2013.This study is ancillary to a larger multicentric trial assessing the effectiveness of malaria in pregnancy preventive strategies in Burkina Faso, Benin and The Gambia. Details of the main trial protocol have been described elsewhereP. falciparum diagnosis by qPCR. One plasma aliquot was prepared from cord blood for ELISA and RDT test performance and stored at −80 °C. Newborns visiting the health center with presence or history of fever during the previous 24 hours were screened for malaria infection with RDT and, if positive, treated with artesunate-amodiaquine or artemether-lumefantrine according to national guidelines. In addition, a capillary blood sample was collected from finger-prick for qPCR analysis and LM examination.At delivery, 200 µL of peripheral blood was obtained from the mother by finger-prick and placental blood was collected by pipette aspiration after incision of the maternal side of the placenta. A placental tissue section was collected from the maternal side and stored into 10% neutral buffer formalin. Cord blood was collected in heparin containing tubes by venipuncture of the umbilical vein and used to prepare thick blood smears for LM examination. Blood drops of all samples were spotted onto filter paper for posterior 54. Parasite density was expressed as the number of asexual parasites per μL of blood based on an assumed 8000 white blood cells per μL of blood. A slide was considered negative if no parasites were seen after examining 100 fields. Each slide was read by two independent experienced microscopists blinded to each other’s results and in case of discrepancy, the slide was read by a third experienced microscopists. After the completion of the reads an internal quality control was performed by a fourth experienced reader for 10% of slides. Slides of the quality control included all positive samples detected by qPCR. Histological examination of placental tissue collected at delivery was performed as described elsewhere33.Thick blood films stained with Giemsa (10%) were used for malaria detection by LM according to standard proceduresPfHRP2 was used. Umbilical cord plasma was thawed at room temperature, centrifuged at 500 g for 10 min and five microliters were transferred into the RDT sample well, to which four drops of assay diluent were added. The RDT result was read in 15–30 min.RDT SD-Bioline malaria antigen P.f® test  detecting PfHRP2 antigen was performed with Malaria Ag CELISATM kit  following manufacturer’s recommendations. Briefly, 100 µL of umbilical cord plasma were tested and optical density (OD) read at 450 nm with MultiskanTM FC Microplate Photometer (Thermo Scientific). All samples and controls were tested in duplicate. Positive and negative controls were provided by the manufacturer and OD values were normalized using a reference positive control in all plates. Samples with a mean OD value above the cut-off level (OD of negative control +0.2 unit) were considered positive for PfHRP2 antigen as recommended by the manufacturer. The antigen index values were determined by dividing the mean OD value of each sample by the cut-off value as previously described55.Detection of P. falciparum var gene acidic terminal sequence  as previously described22. The limit of detection in our laboratory was 0.1 parasite/μL using DNA extracted from blood spot on filter paper. Parasite densities were obtained by extrapolating cycle thresholds (Ct) from a standard curve prepared with titrated samples containing known numbers of infected erythrocytes diluted in whole blood . Samples with Ct value ≤39.7 were considered positive.For the molecular diagnosis by qPCR, filter papers with dried blood samples from maternal peripheral, placental and cord blood were punched and three circles of 5 mm in diameter was used for DNA extraction with QIAamp 96 DNA blood kit . Extracted DNA was eluted in 150μL of water. Five microliters of DNA were used for qPCR analysis targeting P. falciparum msp1 and msp2 genes was used to genotype parasite clones in a paired-sample corresponding to cord blood and peripheral blood of a newborn that experienced a malaria infection during the study follow-up. Family specific primers for msp1  and msp2 (FC27 and IC(3D7)) were used according to the genotyping procedures recommended by WHO56.Nested PCR targeting P values < 0.05 were considered statistically significant. The following definitions were applied: (a) Congenital malaria infection: cord-blood malaria infections as detected by quantitative PCR; (b) Maternal peripheral and placental infections: presence of parasite DNA detected by qPCR in the peripheral and placental blood at delivery; (c) Low birth weight: <2,500 g; (d) Fever: axillary temperature ≥37.5 °C; (e) Clinical malaria episode: presence of fever at the moment of screening or during the previous 24 h and positive test by RDT, LM or qPCR.Data were double entered into the study databases  and analyzed using Epi Info version 3.5.4 and STATA 12.0 . Normally distributed data was described by mean ± SD and non-normally distributed data was described by the median with interquartile range (IQR). Congenital malaria prevalences were calculated with 95% Confidence Interval (CI). Parasite densities and ELISA optical densities (ODs) were not normally distributed even after logarithm transformation. Thus, the difference in median values of parasite density and ODs between groups were analyzed using the non-parametric Mann-Whitney and Kruskal Wallis tests. Sensitivity and specificity were calculated with 95% CI to compare RDT and LM against qPCR.


Whilst there have been substantial reductions in malaria transmission over the past decade, in many countries in West and Central Africa the malaria burden remains high. Monitoring and evaluation of malaria transmission trends and intervention strategies are key elements for malaria control programmes. This study uses a time series of annual malaria indicator surveys to track the progress of malaria control in Bioko Island, Equatorial Guinea, over a 13 year period of intensive interventions. Malaria infection and haemoglobin were measured annually in children (1 to 14 years) in cross-sectional household surveys from 2004 to 2016 in 18 sentinel sites across the island. Trends in transmission patterns were assessed and the impact of the vector control interventions (net use and spray coverage) was evaluated.Between 2004 and 2016 approximately 106,500 individual tests for parasitaemia were conducted using rapid diagnostic tests. Although spray coverage remained relatively high (> 70%) over the time period, reported net usage was generally below 40%. Parasite prevalence reduced from 43.3 to 10.5% between 2004 and 2016. The prevalence of moderate to severe anaemia in children aged 1–5 years reduced from 14.9 to 1.6%. Impact in individual sites ranged from 57 to 100% reductions in parasite prevalence between 2004 and 2016. Sleeping under a net and living in a house that had been sprayed in the past 12 months were independently protective against infection , whilst recent travel to the mainland increased the odds of infection nearly fourfold (OR = 3.94 [95%CI 2.79–5.56]).Island-wide interventions have resulted in a substantial reduction in malaria transmission on Bioko Island. This unique time series of 13 consecutive annual malaria indicator surveys clearly demonstrates the long-term effectiveness of the sustained use of two vector control interventions, indoor residual spraying and LLINs, and the value of comprehensive and sustained surveillance. Despite considerable success in reducing the burden on the island, malaria is still endemic, with populations in some areas remaining at high risk of infection.The online version of this article (10.1186/s12936-018-2213-9) contains supplementary material, which is available to authorized users. Despite a large scale-up in access to long-lasting insecticide-treated nets (LLIN) and indoor residual spraying (IRS), malaria remains a major public health burden across much of sub-Saharan Africa. As intervention coverage increases across the continent, monitoring and evaluation of control programmes become paramount, although reliable indicators are often lacking and can be contradictory . SeveralIslands are considered ideal settings for effective malaria control due to the limited population migration and well-defined geographical borders. Several island nations have achieved substantial reductions in malaria transmission attributed to successful interventions –8, with Malaria indicator surveys (MIS) take place annually within sentinel sites across the island as part of an enhanced monitoring and evaluation programme to assess the progress of the BIMCP towards its ultimate goal of malaria elimination. Transmission trends over more than a decade of vector control were examined using the time series of 13 consecutive cross-sectional assessments of parasitaemia and anaemia prevalence.2), is 32 km from the coast of Cameroon. Its population, of approximately 250,000  are at risk of malaria year-round. Before the launch of the BIMCP in 2004, entomological inoculation rates (EIR) on the island were in excess of 750 infectious bites per person per year [Bioko, the largest island of Equatorial Guinea  was the principal intervention implemented by the BIMCP on a biannual basis beginning in 2004. The insecticide deltamethrin was used in the first year, followed by bendiocarb from 2005 to 2012, then deltamethrin again from 2013 to 2014 with a switch back to bendiocarb in 2015 and 2016. Since the switch back to bendiocarb in 2015, IRS has been targeted at particular communities rather than the whole island, as a strategy for enhancing the cost-effectiveness of vector control, taking into account variable community acceptance of the intervention after a decade of persistent spraying. Targeting was based on a risk score composed of community-level parasite prevalence, quality of housing (a measure of susceptibility to vector infiltration), risk of parasite importation as expressed by prevalence of reported travel to the mainland within the preceding 8 weeks, and previous IRS coverage (as a proxy for acceptability in the community).A first mass distribution of long-lasting insecticide-treated nets (LLIN) was conducted on Bioko in late 2007 and early 2008, which achieved initial reported coverage of an average of three nets per household . A seconMIS have been conducted annually in August/September on Bioko since 2004. This study utilizes data from 13 surveys from 2004 through to 2016. The survey protocol has stayed broadly similar throughout this time period with the exception of the age group targeted for sampling. Between 2004 and 2007, only children aged between 1 and 14 years were sampled, with the addition of head of households between 2008 and 2012, and the inclusion of everyone present in the household from 2013 onwards.Seventeen sentinel sites were monitored in 2004, increasing to 18 sites (with the addition of Punta Europa) from 2005 onwards  in the later surveys. Participants testing positive were referred to a survey nurse for treatment, according to national treatment guidelines (artestunate/amodiaquine), whilst participants with low haemoglobin levels were advised to seek care.Haemoglobin level was measured using Haemocues (HemoCue AB): mild anaemia was classified as a haemoglobin level less than 12 g/dl, 11.5 g/dl and 11 g/dl for individuals over 12 years, between 5 and 12 years and under 5 years, respectively. Moderate to severe anaemia was classified as a haemoglobin level below 8 g/dl for all age groups. Participants were tested for the presence of parasitaemia using a Pf/pan rapid diagnostic test (RDT) (ICT diagnostics) from 2004 to 2011 and Carestart2 tests. A logistic regression model, allowing for the survey design, was performed to estimate the mean effect of time  on infection prevalence.Due to the restricted age groups in the early surveys, only results from children aged 1–14 years are included in figures and analyses. Prevalence of infection by RDT and 95% confidence intervals were calculated for each year. The association between parasite prevalence and prevalence of moderate/severe anaemia (< 8 g/dl) in children aged 1–5 years was assessed using regression models. Differences in proportions were assessed using ChiMedian altitude of each sentinel site was calculated based on household global positioning system (GPS) information and sites were classified as high altitude if they were above the median altitude for all sites (108 m). Site-level spray coverage (based on reported spraying within sampled households) was estimated to investigate benefits of high IRS coverage at the community level. Households were classified as having universal coverage of nets if they reported having at least one net for every two people in the household. To investigate the impact of original malaria endemicity, the median baseline site prevalence (45%) was used to stratify sites into high or moderate prevalence (< 45%).Information about intervention coverage (net use and IRS within the last 12 months) was collected from 2006 onwards. Univariate logistic regression was used to look at the differential effects of intervention coverage on infection, comparing (1) living in a sprayed household and not using a net (2) reporting sleeping under a net and not living in a sprayed house (3) living in a sprayed house and sleeping under a net, to a baseline of no interventions. Variables that showed a crude association with RDT positivity were included in the model in a stepwise manner, including age group, original site prevalence, and site-level spray coverage. To investigate whether the switch in IRS strategy had an effect on risk factors, models were examined with and without the surveys from 2015 to 2016. A separate model including recent travel to the mainland was used for data from surveys from 2013 to 2016. For consistency, this model also only included children aged 1 to 14 years.Sampling probabilities of children aged between 1 and 14 years in each sentinel site were computed using data from a country-wide 2015 census. Responses were weighted by the inverse of sampling probabilities to compensate for the different numbers of children tested in each survey. All models controlled for year to account for temporal variations. Standard errors were adjusted to account for correlation of responses, with sentinel site specified as the primary sampling unit (PSU) when calculating an island-wide prevalence and household when calculating sentinel site level prevalence.In the thirteen surveys between 2004 and 2016, 35,146 household visits were conducted, ranging from 1043 to 4721 households per year  to 80% (in 2005). Spray coverage varied substantially between sites. Sites in Malabo and the North West consistently did not achieve spray coverage over 80%-in part due to a large proportion of refusals from homeowners. A low proportion of houses reported universal net coverage, ranging from 13% (in 2011) up to 57% and 42%, following the LLIN campaigns in 2007/08 and 2014/15, respectively. In the subsequent year, following the first distribution, universal coverage had already substantially reduced (21%) whilst coverage remained similar in the year following the second distribution (40%). The percentage of children aged 1–14 years sleeping under a net the night before the survey was below 55% in all survey years except in 2008 when it increased to 81%  to 10.5% (8.8–12.5) (p < 0.001)  in children aged 1 to 5 years dropped by nearly 90% between 2004 and 2016 (15–2%) . Site-level anaemia prevalence was strongly correlated with parasite prevalence  and were more likely to live in sprayed households . Net use and access was similar in all six sites. Sites with highest impact had median altitudes over 200 m whilst lowest impact sites all had median altitudes lower than 55 m. Two out of three sites with the highest impact were classified as moderate prevalence pre-intervention (Baney and Rebola), whilst only one of the least impact was classed as moderate prevalence .In 2016, 9 of the 18 sentinel sites had prevalences below 10%: this included all sites in the North East, South West and Highland regions of the island. No sites in the North West region  had parasite prevalences under 10% Fig. .There was no evidence for risk factors varying substantially between years. There was not strong evidence that living in a site with high spray coverage (> 80%) or in a site with higher altitude was associated with lower infection prevalence Table . The assThe influence of recent travel to the mainland was examined for surveys where data was collected (2013–2016). The proportion of participants reporting travel was relatively small, ranging from 6% in 2013 and 2014, up to 10% in 2016, but with wide variation between sites. Travellers were more likely to be women and to be aged between 15 and 50 years. The proportion of people travelling was highest in sites in Malabo, increasing from 9% in 2013 to 14% in 2016. Infection was higher in people reporting recent travel than those who had not travelled . Before adjusting for other risk factors, travel to the mainland increased the odds of infection in children aged 1–14 nearly 4-fold ). When incorporated with the other risk factors in the final model, travel to the mainland remained strongly associated with infection ).Monitoring and evaluation play a key role in malaria control campaigns and are essential for assessing the impact of interventions and guiding programmes in adapting operational approaches. The World Health Organization (WHO) Global Technical Strategy  emphasizThe BIMCP has been an overall success, achieving a reduction in parasitaemia of over 75% over 13 years. In the early years of implementation, there was heterogeneity of impact between sites. Differential impact of universal interventions have been documented in other studies , 21 and The main preventative intervention employed on Bioko is IRS. Whilst IRS can protect the sprayed household through a repellent effect (depending on the insecticide used), its main effect is through killing action resulting in community-wide protection when a sufficiently high spray coverage is reached . The WHOIn addition to IRS, two mass LLIN distributions took place; however, MIS which took place within 6 months of the distributions suggest that if high coverage was originally achieved, it had quickly reduced. This may be because some of the nets that were distributed were subsequently taken to the mainland where little malaria control is in place and malaria transmission is substantially higher. Studies are ongoing to establish why attrition of nets was so high. In households with universal coverage, the use of nets was high, indicating that the barrier to higher LLIN utilization is the lack of access to nets, not unwillingness to use them.Net use was shown to be protective against infection, with children reporting sleeping under a net the previous night (and not living in a sprayed house) having 30% lower odds of being infected. A treated net provides a physical barrier against biting mosquitoes and protection through reduction of the mosquito population by insecticidal action. Several sites saw steep falls in parasite prevalence between 2007 and 2008, which may have been due to increased net use. The proportion of people with access to nets post-2008 reduced to approximately 20%, but despite this, there was no sign of a rebound in malaria prevalence (or severe anaemia), suggesting other factors were also contributing to maintenance of the new lower equilibrium. The highest infection prevalence was consistently detected in children aged between 5 and 14 years old, who were also least likely to be using a net.IRS and LLIN are often used in combination rather than alone. As with previous studies in Equatorial Guinea , 26, andIn recent years, population movement to the mainland of the country increased, perhaps due to more transport options, or an overall increase in wealth. Travel to the mainland, where malaria is higher , has beeWhilst interventions have reduced the risk of infection, substantial variation in transmission over the study period remains unexplained. A factor which may have resulted in reduced transmission over time is an increase in the quality of housing, which has been shown in Bioko , and elskdr mutation in the Anopheles gambiae population [kdr, the 2004 pyrethroid spray round nevertheless resulted in a sharp reduction in sporozoite infectivity in the main vector after spraying, and there was no apparent association between kdr and phenotypic resistance. In 2013 deltamethrin spraying was re-introduced as part of a programme of insecticide resistance management through rotation [Although there has been a decrease in prevalence in most years of the project, 2013 stood out as an anomaly, when prevalence increased in most sites, following a particularly low prevalence recorded in 2012. This may have been influenced by the choice of insecticide used that year. In 2004, the insecticide used for spraying was switched from deltamethrin to bendiocarb following the detection of the pulation . Subsequrotation . Followirotation . The preDespite the heterogeneous impact of interventions in Bioko, transmission has declined substantially overall. Understanding the factors that have contributed to differential impact will help the BIMCP to prioritize strategies and to continue the downward trajectory in parasite rate. Indeed, several areas, which have now reached pre-elimination levels, are now implementing further strategies, such as reactive case detection, to attempt elimination of local transmission. This study has shown that vector control with IRS and LLINs remained the most important protective intervention on Bioko, whilst travel to the mainland constituted a substantial risk for malaria infection. The low proportion of houses with universal coverage of LLINs, and data suggesting that the age group most likely to be infected are least likely to be using a net (10–14 yr olds) is concerning. Strategies to ensure a higher proportion of the population have access to nets have been implemented, but the lack of LLINs on the mainland and the high rate of associated leakage of nets, may have undermined the ability to maintain high coverage levels. Controlling parasite importation from the mainland, and maintaining universal access to the core prevention, namely LLIN use, represent two clear challenges to further reducing malaria transmission on Bioko.Whilst the gains over the past 13 years have been impressive, the slow, but steady progress highlights the importance of sustaining malaria control interventions on a long-haul basis. Long-term commitment and surveillance are necessary to chart the downward trend which may not necessarily advance in an uninterrupted linear manner.


Increased carotid-femoral pulse wave velocity (PWV) has been associated with incident cardiovascular disease, independently of traditional risk factors. Cardiac autonomic dysfunction is a common complication of diabetes and has been associated with reduced aortic distensibility. However, the association of cardiac autonomic dysfunction with PWV is not known. In this study we examined the association between cardiac autonomic function and PWV in subjects with type 2 diabetes mellitus.n = 193) or abnormal (n = 97) PWV values using age-corrected values. Cardiac autonomic nervous system activity was determined by measurement of parameters of heart rate variability (HRV).A total of 290 patients with type 2 diabetes were examined. PWV was measured at the carotid-femoral segment with applanation tonometry. Central mean arterial blood pressure (MBP) was determined by the same apparatus. Participants were classified as having normal , log total power  and log high frequency power .Subjects with abnormal PWV were older, had higher arterial blood pressure and higher heart rate than those with normal PWV. Most of the values of HRV were significantly lower in subjects with abnormal than in those with normal PWV. Multivariate analysis, after controlling for various confounding factors, demonstrated that abnormal PWV was associated independently only with peripheral MBP [odds ratio (OR) 1.049, 95% confidence intervals (CI) 1.015–1.085, In subjects with type 2 diabetes, arterial blood pressure and impaired cardiac autonomic function is associated independently with abnormal PWV. Pulse wave velocity (PWV) is the gold-standard method for the assessment of arterial stiffness and is an independent predictor of cardiovascular morbidity and mortality . PreviouCardiac autonomic dysfunction is a common although underestimated chronic microvascular complication of diabetes . CardiacSeveral studies have reported that early in the course of type 1 diabetes impaired cardiac autonomic function and arterial stiffness are strongly associated , 9. MoreRecent guidelines emphasize that use of normal and abnormal PWV values according to age represent a critical step in the implementation of PWV as a clinical tool for identification of people at higher cardiovascular risk . It is kBased on the above literature data, the research hypothesis we examined in this study is that impaired cardiac autonomic function is associated with abnormal PWV in people with T2DM, when diabetes-related and classical risk factors for atherosclerosis are taken into consideration.2], heart failure of stage III and IV and acute illness or hypoglycemia in the previous 24 h. In addition, in order to minimize the confounding effect of medications on parameters of HRV, we excluded patients on antiarrhythmic drugs other than b-blockers or drugs with an effect on cardiac autonomic nervous system activity like antidepressants and antihistamines. The study was approved by the ethics committee of our hospital and was conducted according to the principles of the Declaration of Helsinki .Increasing evidence suggests that central blood pressure may be a more accurate indicator of end organ damage and cardiovascular risk than brachial blood pressure in specific groups of patients, including individuals with T2DM . A recenThe strength of our study is that it is the first to investigate the association between impaired cardiac autonomic function and abnormal PWV using age-corrected values. A limitation is, however, the cross-sectional design that does not allow determination of a causal relationship between PWV and cardiac autonomic function. Although a non-causal association cannot be ruled out, causality could only be determined if the question of which of the two events  appears first could be answered . AnotherThe present study has demonstrated that blood pressure and impaired cardiac autonomic function are the main determinants of abnormal PWV in people with T2DM, while the association between impaired cardiac autonomic function and arterial stiffness is not mediated by low BRS or increased heart rate. Our findings suggest that cardiac autonomic nervous system activity influences arterial stiffness and it should be monitored and reported in studies examining factors affecting PWV.