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Summary: Background: Detection of anti-SARS-CoV-2 antibodies among people at risk of infection is crucial for understanding both the past transmission of COVID-19 and vulnerability of the population to continuing transmission and, when done serially, the intensity of ongoing transmission over an interval in a community. We aimed to estimate the seroprevalence of COVID-19 in a representative population-based cohort in Iquitos, one of the regions with the highest mortality rates from COVID-19 in Peru, where a devastating number of cases occurred in March, 2020. Methods: We did a population-based study of SARS-CoV-2 transmission in Iquitos at two timepoints: July 13–18, 2020 (baseline), and Aug 13–18, 2020 (1-month follow-up). We obtained a geographically stratified representative sample of the city population using the 2017 census data, which was updated on Jan 20, 2020. We included people who were inhabitants of Iquitos since COVID-19 was identified in Peru (March 6, 2020) or earlier. We excluded people living in institutions, people receiving any pharmacological treatment for COVID-19, people with any contraindication for phlebotomy, and health workers or individuals living with an active health worker. We tested each participant for IgG and IgM anti-SARS-CoV-2 antibodies using the COVID-19 IgG/IgM Rapid Test (Zhejiang Orient Gene Biotech, China). We used survey analysis methods to estimate seroprevalence accounting for the sampling design effect and test performance characteristics. Findings: We identified 726 eligible individuals and enrolled a total of 716 participants (99%), distributed across 40 strata (four districts, two sexes, and five age groups). We excluded ten individuals who: did not have consent from a parent or legal representative (n=3), had moved to Iquitos after March 6, 2020 (n=3), were in transit (n=2), or had respiratory symptoms (n=1). After adjusting for the study sampling effects and sensitivity and specificity of the test, we estimated a seroprevalence of 70% (95% CI 67–73) at baseline and 66% (95% CI 62–70) at 1 month of follow-up, with a test-retest positivity of 65% (95% CI 61–68), and an incidence of new exposures of 2% (95% CI 1–3). We observed significant differences in the seroprevalence between age groups, with participants aged 18–29 years having lower seroprevalence than those aged younger than 12 years (prevalence ratio 0·85 [95% CI 0·73–0·98]; p=0·029). Interpretation: After the first epidemic peak, Iquitos had one of the highest rates of seroprevalence of anti-SARS-CoV-2 antibodies worldwide. Nevertheless, the city experienced a second wave starting in January, 2021, probably due to the emergence of the SARS-CoV-2 P1 variant, which has shown higher transmissibility and reinfection rates. Funding: Dirección Regional de Salud de Loreto (DIRESA), Loreto, Peru. Translation: For the Spanish translation of the abstract see Supplementary Materials section.

Background. Remote rural riverine villages account for most of the reported malaria cases in the Peruvian Amazon. As transmission decreases due to intensive standard control efforts, malaria strategies in these villages will need to be more focused and adapted to local epidemiology. Methods. By integrating parasitological, entomological, and environmental observations between January 2016 and June 2017, we provided an in-depth characterization of malaria transmission dynamics in 4 riverine villages of the Mazan district, Loreto department. Results. Despite variation across villages, malaria prevalence by polymerase chain reaction in March 2016 was high (>25% in 3 villages), caused by Plasmodium vivax mainly and composed of mostly submicroscopic infections. Housing without complete walls was the main malaria risk factor, while households close to forest edges were more commonly identified as spatial clusters of malaria prevalence. Villages in the basin of the Mazan River had a higher density of adult Anopheles darlingi mosquitoes, and retained higher prevalence and incidence rates compared to villages in the basin of the Napo River despite test-and-treat interventions. Conclusions. High heterogeneity in malaria transmission was found across and within riverine villages, resulting from interactions between the microgeographic landscape driving diverse conditions for vector development, housing structure, and human behavior. This work was supported by the Consejo Nacional de Ciencia, Tecnologia e Innovacion Tecnologica (grant number 008-2014-FONDECYT); the Academie de Recherche et d'Enseignement Superieur-Commission de la Cooperation au Developpement of Belgium (grant number ARES-CCD, PRD-Peru 2014-2019 to N. S., A. L. C., and A. R. A.); World Health Organization Special Programme for Research and Training in Tropical Diseases (grant number 201460655 to D. G.); and National Institutes of Health, National Institute of Allergy and Infectious Diseases (grant numbers U19AI089681 to J. M. V. and R01AI110112 to J. E. C.). A. R. A. is a Postdoctoral Researcher of the Fonds de la Recherche Scientifique (FNRS, Belgium).

During April–June 2014 in a malaria-endemic rural community close to the city of Iquitos in Peru, we detected evidence of Guaroa virus (GROV) infection in 14 febrile persons, of whom 6 also had evidence of Plasmodium vivax malaria. Cases were discovered through a long-term febrile illness surveillance network at local participating health facilities. GROV cases were identified by using a combination of seroconversion and virus isolation, and malaria was diagnosed by thick smear and PCR. GROV mono-infections manifested as nonspecific febrile illness and were clinically indistinguishable from GROV and P. vivax co-infections. This cluster of cases highlights the potential for GROV transmission in the rural Peruvian Amazon, particularly in areas where malaria is endemic. Further study of similar areas of the Amazon may provide insights into the extent of GROV transmission in the Amazon basin.

Background Microscopic examination of Giemsa-stained blood films remains a major form of diagnosis in malaria case management, and is a reference standard for research. However, as with other visualization-based diagnoses, accuracy depends on individual technician performance, making standardization difficult and reliability poor. Automated image recognition based on machine-learning, utilizing convolutional neural networks, offers potential to overcome these drawbacks. A prototype digital microscope device employing an algorithm based on machine-learning, the Autoscope, was assessed for its potential in malaria microscopy. Autoscope was tested in the Iquitos region of Peru in 2016 at two peripheral health facilities, with routine microscopy and PCR as reference standards. The main outcome measures include sensitivity and specificity of diagnosis of malaria from Giemsa-stained blood films, using PCR as reference. Methods A cross-sectional, observational trial was conducted at two peripheral primary health facilities in Peru. 700 participants were enrolled with the criteria: (1) age between 5 and 75 years, (2) history of fever in the last 3 days or elevated temperature on admission, (3) informed consent. The main outcome measures included sensitivity and specificity of diagnosis of malaria from Giemsa-stained blood films, using PCR as reference. Results At the San Juan clinic, sensitivity of Autoscope for diagnosing malaria was 72% (95% CI 64–80%), and specificity was 85% (95% CI 79–90%). Microscopy performance was similar to Autoscope, with sensitivity 68% (95% CI 59–76%) and specificity 100% (95% CI 98–100%). At San Juan, 85% of prepared slides had a minimum of 600 WBCs imaged, thus meeting Autoscope’s design assumptions. At the second clinic, Santa Clara, the sensitivity of Autoscope was 52% (95% CI 44–60%) and specificity was 70% (95% CI 64–76%). Microscopy performance at Santa Clara was 42% (95% CI 34–51) and specificity was 97% (95% CI 94–99). Only 39% of slides from Santa Clara met Autoscope’s design assumptions regarding WBCs imaged. Conclusions Autoscope’s diagnostic performance was on par with routine microscopy when slides had adequate blood volume to meet its design assumptions, as represented by results from the San Juan clinic. Autoscope’s diagnostic performance was poorer than routine microscopy on slides from the Santa Clara clinic, which generated slides with lower blood volumes. Results of the study reflect both the potential for artificial intelligence to perform tasks currently conducted by highly-trained experts, and the challenges of replicating the adaptiveness of human thought processes.

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 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.

The Peruvian Ministry of Health reports a near absence of malaria cases in the Amazon region during the COVID-19 pandemic. However, the rapid increase in SARS-CoV-2 infections has overwhelmed the Peruvian health system, leading to national panic and closure of public medical facilities, casting doubt on how accurately malaria cases’ numbers reflect reality. In the Amazon region of Loreto, where malaria cases are concentrated, COVID-19 has led to near-complete closure of the primary healthcare system, and diagnosis and treatment of acute febrile illnesses, including malaria, has plummeted. Here, we describe the potential association of COVID-19 with a markedly reduced number of reported malaria cases due to the reduced control activities carried out by the Peruvian Malaria Zero Program, which could lead to malaria resurgence and an excess of morbidity and mortality.

Background Malaria remains an important public health problem in Peru where incidence has been increasing since 2011. Of over 55,000 cases reported in 2017, Plasmodium vivax was the predominant species (76%), with P. falciparum responsible for the remaining 24%. Nyssorhynchus darlingi (previously Anopheles darlingi) is the main vector in Amazonian Peru, where hyperendemic Plasmodium transmission pockets have been found. Mazán district has pronounced spatial heterogeneity of P. vivax malaria. However, little is known about behavior, ecology or seasonal dynamics of Ny. darlingi in Mazán. This study aimed to gather baseline information about bionomics of malaria vectors and transmission risk factors in a hyperendemic malaria area of Amazonian Peru. Methods To assess vector biology metrics, five surveys (two in the dry and three in the rainy season), including collection of sociodemographic information, were conducted in four communities in 2016–2017 on the Napo (Urco Miraño, URC; Salvador, SAL) and Mazán Rivers (Visto Bueno, VIB; Libertad, LIB). Human-biting rate (HBR), entomological inoculation rate (EIR) and human blood index (HBI) were measured to test the hypothesis of differences in entomological indices of Ny. darlingi between watersheds. A generalized linear mixed effect model (GLMM) was constructed to model the relationship between household risk factors and the EIR. Results Nyssorhynchus darlingi comprised 95% of 7117 Anophelinae collected and its abundance was significantly higher along the Mazán River. The highest EIRs (3.03–4.54) were detected in March and June in URC, LIB and VIB, and significantly more Ny. darlingi were infected outdoors than indoors. Multivariate analysis indicated that the EIR was >12 times higher in URC compared with SAL. The HBI ranged from 0.42–0.75; humans were the most common blood source, followed by Galliformes and cows. There were dramatic differences in peak biting time and malaria incidence with similar bednet coverage in the villages. Conclusions Nyssorhynchus darlingi is the predominant contributor to malaria transmission in the Mazán District, Peru. Malaria risk in these villages is higher in the peridomestic area, with pronounced heterogeneities between and within villages on the Mazán and the Napo Rivers. Spatiotemporal identification and quantification of the prevailing malaria transmission would provide new evidence to orient specific control measures for vulnerable or at high risk populations. Electronic supplementary material The online version of this article (10.1186/s13071-019-3619-0) contains supplementary material, which is available to authorized users.

In Amazonian Peru, the primary malaria vector, Nyssorhynchus darlingi (formerly Anopheles darlingi), is difficult to target using standard vector control methods because it mainly feeds and rests outdoors. Larval source management could be a useful supplementary intervention, but to determine its feasibility, more detailed studies on the larval ecology of Ny. darlingi are essential. We conducted a multi-level study of the larval ecology of Anophelinae mosquitoes in the peri-Iquitos region of Amazonian Peru, examining the environmental characteristics of the larval habitats of four species, comparing the larval microbiota among species and habitats, and placing Ny. darlingi larval habitats in the context of spatial heterogeneity in human malaria transmission. We collected Ny. darlingi, Nyssorhynchus rangeli (formerly Anopheles rangeli), Nyssorhynchus triannulatus s.l. (formerly Anopheles triannulatus s.l.), and Nyssorhynchus sp. nr. konderi (formerly Anopheles sp. nr. konderi) from natural and artificial water bodies throughout the rainy and dry seasons. We found that, consistent with previous studies in this region and in Brazil, the presence of Ny. darlingi was significantly associated with water bodies in landscapes with more recent deforestation and lower light intensity. Nyssorhynchus darlingi presence was also significantly associated with a lower vegetation index, other Anophelinae species, and emergent vegetation. Though they were collected in the same water bodies, the microbial communities of Ny. darlingi larvae were distinct from those of Ny. rangeli and Ny. triannulatus s.l., providing evidence either for a species-specific larval microbiome or for segregation of these species in distinct microhabitats within each water body. We demonstrated that houses with more reported malaria cases were located closer to Ny. darlingi larval habitats; thus, targeted control of these sites could help ameliorate malaria risk. The co-occurrence of Ny. darlingi larvae in water bodies with other putative malaria vectors increases the potential impact of larval source management in this region., Author summary The standard methods used to combat mosquitoes that transmit malaria, long-lasting insecticide treated nets and indoor residual spraying, target mosquitoes that bite people indoors and rest indoors after biting. In Amazonian Peru, the major malaria vector, Nyssorhynchus darlingi (formerly Anopheles darlingi), is known to bite and rest mostly outdoors and to feed on animals as well as humans. Therefore, additional methods are needed to control this species, such as targeting immature mosquitoes in water bodies using chemical or biological larvicides or environmental modification. To determine whether this is feasible, more ecological information about Ny. darlingi larval habitats in this region is needed. In this study, we found that Ny. darlingi were more likely to be collected from water bodies in more deforested areas, and in the presence of other species of mosquitoes that transmit malaria. We characterized the bacteria detected in three species of mosquito larvae, and found that the species of mosquito, and not the water body, determined which bacteria were identified. As we found that houses with more malaria cases were located closer to Ny. darlingi larval habitats, management of larval habitats may be an effective method to reduce the risk of malaria in this region.

BACKGROUND: Nyssorhynchus dunhami, a member of the Nuneztovari Complex, has been collected in Brazil, Colombia, and Peru and described as zoophilic. Although to date Ny. dunhami has not been documented to be naturally infected by Plasmodium, it is frequently misidentified as other Oswaldoi subgroup species that are local or regional malaria vectors. OBJECTIVES: The current study seeks to verify the morphological identification of Nuneztovari Complex species collected in the peri-Iquitos region of Amazonian Peru, to determine their Plasmodium infection status, and to describe ecological characteristics of their larval habitats. METHODS: We collected Ny. nuneztovari s.l. adults in 2011-2012, and Ny. nuneztovari s.l. larvae and adults in 2016-2017. When possible, samples were identified molecularly using cytochrome c oxidase subunit I (COI) barcode sequencing. Adult Ny. nuneztovari s.l. from 2011-2012 were tested for Plasmodium using real-time PCR. Environmental characteristics associated with Ny. nuneztovari s.l. larvae-positive water bodies were evaluated. FINDINGS: We collected 590 Ny. nuneztovari s.l. adults and 116 larvae from eight villages in peri-Iquitos. Of these, 191 adults and 111 larvae were identified by COI sequencing; all were Ny. dunhami. Three Ny. dunhami were infected with P. falciparum, and one with P. vivax, all collected from one village on one night. Ny. dunhami larvae were collected from natural and artificial water bodies, and their presence was positively associated with other Anophelinae larvae and amphibians, and negatively associated with people living within 250m. MAIN CONCLUSIONS: Of Nuneztovari Complex species, we identified only Ny. dunhami across multiple years in eight peri-Iquitos localities. This study is, to our knowledge, the first report of natural infection of molecularly identified Ny. dunhami with Plasmodium. We advocate the use of molecular identification methods in this region to monitor Ny. dunhami and other putative secondary malaria vectors to more precisely evaluate their importance in malaria transmission.

Background In Loreto Department, Peru, a successful 2005–2010 malaria control programme (known as PAMAFRO) included massive distribution of long-lasting insecticidal nets (LLINs). Additional local distribution of LLINs occurred in individual villages, but not between 2012 and 2015. A 2011–2012 study of the primary regional malaria vector Anopheles darlingi detected a trend of increased exophagy compared with pre-PAMAFRO behaviour. For the present study, An. darlingi were collected in three villages in Loreto in 2013–2015 to test two hypotheses: (1) that between LLIN distributions, An. darlingi reverted to pre-intervention biting behaviour; and, (2) that there are separate sub-populations of An. darlingi in Loreto with distinct biting behaviour. Results In 2013–2015 An. darlingi were collected by human landing catch during the rainy and dry seasons in the villages of Lupuna and Cahuide. The abundance of An. darlingi varied substantially across years, villages and time periods, and there was a twofold decrease in the ratio of exophagic:endophagic An. darlingi over the study period. Unexpectedly, there was evidence of a rainy season population decline in An. darlingi. Plasmodium-infected An. darlingi were detected indoors and outdoors throughout the night, and the monthly An. darlingi human biting rate was correlated with the number of malaria cases. Using nextRAD genotyping-by-sequencing, 162 exophagic and endophagic An. darlingi collected at different times during the night were genotyped at 1021 loci. Based on model-based and non-model-based analyses, all genotyped An. darlingi belonged to a homogeneous population, with no evidence for genetic differentiation by biting location or time. Conclusions This study identified a decreasing proportion of exophagic An. darlingi in two villages in the years between LLIN distributions. As there was no evidence for genetic differentiation between endophagic and exophagic An. darlingi, this shift in biting behaviour may be the result of behavioural plasticity in An. darlingi, which shifted towards increased exophagy due to repellence by insecticides used to impregnate LLINs and subsequently reverted to increased endophagy as the nets aged. This study highlights the need to target vector control interventions to the biting behaviour of local vectors, which, like malaria risk, shows high temporal and spatial heterogeneity. Electronic supplementary material The online version of this article (10.1186/s12936-018-2234-4) contains supplementary material, which is available to authorized users.

Interest in larval source management (LSM) as an adjunct intervention to control and eliminate malaria transmission has recently increased mainly because long-lasting insecticidal nets (LLINs) and indoor residual spray (IRS) are ineffective against exophagic and exophilic mosquitoes. In Amazonian Peru, the identification of the most productive, positive water bodies would increase the impact of targeted mosquito control on aquatic life stages. The present study explores the use of unmanned aerial vehicles (drones) for identifying Nyssorhynchus darlingi (formerly Anopheles darlingi) breeding sites with high-resolution imagery (~0.02m/pixel) and their multispectral profile in Amazonian Peru. Our results show that high-resolution multispectral imagery can discriminate a profile of water bodies where Ny. darlingi is most likely to breed (overall accuracy 86.73%- 96.98%) with a moderate differentiation of spectral bands. This work provides proof-of-concept of the use of high-resolution images to detect malaria vector breeding sites in Amazonian Peru and such innovative methodology could be crucial for LSM malaria integrated interventions., Author summary The most efficient malaria vector in the Latin American region is Nyssorhynchus darlingi (formerly Anopheles darlingi). In Amazonian Peru, where malaria is endemic, Ny. darlingi feeds both indoors and outdoors (endophagy, exophagy), depending on the local environment, and rests outdoors (exophily). LLINs and IRS, the most common tools employed for vector control, target endophagic and endophilic mosquitoes. Thus, they are only partially effective against Ny. darlingi. Control of the aquatic stages of vector mosquitoes, larval source management (LSM), targets the most productive breeding sites nearest to human habitation. In four riverine communities, we used drones with high-resolution imagery as a key initial step to analyze water bodies within the estimated flight range of Ny. darlingi, ~ 1 km. We found distinctive spectral profiles for water bodies that were positive versus negative for Ny. darlingi. The methodology and analysis reported here provide the basis for testing whether LSM can be combined successfully with LLINs and IRS to contribute to the elimination of transmission in malaria hotspots in the Amazon.

BACKGROUND: Malaria transmission in the peri-Iquitos region of Amazonian Peru has been designated as seasonal and hypo-endemic with recently described hyper-endemic hotspots. Despite relatively recent distribution of long-lasting insecticidal bed nets (LLINs), malaria in Amazonian Peru persists and increased substantially in 2014 compared to previous years. Anopheles darlingi, identified as the main malaria vector, is known for its variable behaviour depending on locality and environment. METHODS: To evaluate vector biology metrics in relation to seasonality and malaria transmission, mosquito collections were carried out in three localities in the peri-Iquitos region, Loreto, Peru in 2011-2012. Human landing catch (HLC) collection method, Shannon (SHA) and CDC trap types were compared for effectiveness in a neotropical setting. Abundance, human biting rate and entomological inoculation rate (EIR) were measured to provide an updated view of transmission patterns post-LLIN distribution. RESULTS: HLC collected significantly more anopheline mosquitoes than SHA and CDC light traps. Anopheles darlingi was the most prevalent species in all three villages (84% overall). Biting patterns varied depending on trap type, season and village. EIR varied temporally (monthly) and spatially and the highest (2.52) occurred during the 2012 malaria outbreak in Cahuide. Unexpectedly there was a high infection rate (1.47 and 1.75) outside the normal malaria transmission season, coincident with a second local outbreak in Cahuide. The first identification of Anopheles dunhami and Anopheles oswaldoi C in Peru, using molecular markers, is also reported in this study. CONCLUSION: These data underscore the importance of HLC as the most meaningful collection method for measuring vector biology indices in this region. The highest monthly EIR provides additional evidence of seasonal transmission in riverine localities correlated with high river levels, and An. darlingi as the only contributor to transmission. The trend of an increase in outdoor-biting together with early-evening infected mosquitoes may undermine the effectiveness of LLINs as a primary malaria intervention.

Background Loop-mediated isothermal DNA amplification (LAMP) methodology offers an opportunity for point-of-care (POC) molecular detection of asymptomatic malaria infections. However, there is still little evidence on the feasibility of implementing this technique for population screenings in isolated field settings. Methods Overall, we recruited 1167 individuals from terrestrial (‘road’) and hydric (‘riverine’) communities of the Peruvian Amazon for a cross-sectional survey to detect asymptomatic malaria infections. The technical performance of LAMP was evaluated in a subgroup of 503 samples, using real-time Polymerase Chain Reaction (qPCR) as reference standard. The operational feasibility of introducing LAMP testing in the mobile screening campaigns was assessed based on field-suitability parameters, along with a pilot POC-LAMP assay in a riverine community without laboratory infrastructure. Results LAMP had a sensitivity of 91.8% (87.7–94.9) and specificity of 91.9% (87.8–95.0), and the overall accuracy was significantly better among samples collected during road screenings than riverine communities (p≤0.004). LAMP-based diagnostic strategy was successfully implemented within the field-team logistics and the POC-LAMP pilot in the riverine community allowed for a reduction in the turnaround time for case management, from 12–24 hours to less than 5 hours. Specimens with haemolytic appearance were regularly observed in riverine screenings and could help explaining the hindered performance/interpretation of the LAMP reaction in these communities. Conclusions LAMP-based molecular malaria diagnosis can be deployed outside of reference laboratories, providing similar performance as qPCR. However, scale-up in remote field settings such as riverine communities needs to consider a number of logistical challenges (e.g. environmental conditions, labour-intensiveness in large population screenings) that can influence its optimal implementation.