Effect of spatiotemporal variables on abundance, biting activity and parity of Nyssorhynchus darlingi (Diptera: Culicidae) in peri-Iquitos, Peru.

BACKGROUND: In malaria endemic regions of the Peruvian Amazon, rainfall together with river level and breeding site availability drive fluctuating vector mosquito abundance and human malaria cases, leading to temporal heterogeneity. The main variables influencing spatial transmission include location of communities, mosquito behaviour, land use/land cover, and human ecology/behaviour. The main objective was to evaluate seasonal and microgeographic biting behaviour of the malaria vector Nyssorhynchus (or Anopheles) darlingi in Amazonian Peru and to investigate effects of seasonality on malaria transmission. METHODS: We captured mosquitoes from 18:00 to 06:00 h using Human Landing Catch in two riverine (Lupuna, Santa Emilia) and two highway (El Triunfo, Nuevo Horizonte) communities indoors and outdoors from 8 houses per community, during the dry and rainy seasons from February 2016 to January 2017. We then estimated parity rate, daily survival and age of a portion of each collection of Ny. darlingi. All collected specimens of Ny. darlingi were tested for the presence of Plasmodium vivax or Plasmodium falciparum sporozoites using real-time PCR targeting the small subunit of the 18S rRNA. RESULTS: Abundance of Ny. darlingi varied across village, season, and biting behaviour (indoor vs outdoor), and was highly significant between rainy and dry seasons (p < 0.0001). Biting patterns differed, although not significantly, and persisted regardless of season, with peaks in highway communities at ~ 20:00 h in contrast to biting throughout the night (i.e., 18:00-06:00) in riverine communities. Of 3721 Ny. darlingi tested for Plasmodium, 23 (0.62%) were infected. We detected Plasmodium-infected Ny. darlingi in both community types and most (20/23) were captured outdoors during the rainy season; 17/23 before midnight. Seventeen Ny. darlingi were infected with P. vivax, and 6 with P. falciparum. No infected Ny. darlingi were captured during the dry season. Significantly higher rates of parity were detected in Ny. darlingi during the rainy season (average 64.69%) versus the dry season (average 36.91%) and by community, Lupuna, a riverine village, had the highest proportion of parous to nulliparous females during the rainy season. CONCLUSIONS: These data add a seasonal dimension to malaria transmission in peri-Iquitos, providing more evidence that, at least locally, the greatest risk of malaria transmission is outdoors during the rainy season mainly before midnight, irrespective of whether the community was located adjacent to the highway or along the river.

Mutations Linked to Insecticide Resistance Not Detected in the Ace-1 or VGSC Genes in Nyssorhynchus darlingi from Multiple Localities in Amazonian Brazil and Peru.

Indoor residual spray (IRS), mainly employing pyrethroid insecticides, is the most common intervention for preventing malaria transmission in many regions of Latin America; the use of long-lasting insecticidal nets (LLINs) has been more limited. Knockdown resistance (kdr) is a well-characterized target-site resistance mechanism associated with pyrethroid and DDT resistance. Most mutations detected in acetylcholinesterase-1 (Ace-1) and voltage-gated sodium channel (VGSC) genes are non-synonymous, resulting in a change in amino acid, leading to the non-binding of the insecticide. In the present study, we analyzed target-site resistance in Nyssorhynchus darlingi, the primary malaria vector in the Amazon, in multiple malaria endemic localities. We screened 988 wild-caught specimens of Ny. darlingi from three localities in Amazonian Peru and four in Amazonian Brazil. Collections were conducted between 2014 and 2021. The criteria were Amazonian localities with a recent history as malaria hotspots, primary transmission by Ny. darlingi, and the use of both IRS and LLINs as interventions. Fragments of Ace-1 (456 bp) and VGSC (228 bp) were amplified, sequenced, and aligned with Ny. darlingi sequences available in GenBank. We detected only synonymous mutations in the frequently reported Ace-1 codon 280 known to confer resistance to organophosphates and carbamates, but detected three non-synonymous mutations in other regions of the gene. Similarly, no mutations linked to insecticide resistance were detected in the frequently reported codon (995) at the S6 segment of domain II of VGSC. The lack of genotypic detection of insecticide resistance mutations by sequencing the Ace-1 and VGSC genes from multiple Ny. darlingi populations in Brazil and Peru could be associated with low-intensity resistance, or possibly the main resistance mechanism is metabolic.

Natural Infection of Nyssorhynchus darlingi and Nyssorhynchus benarrochi B with Plasmodium during the Dry Season in the Understudied Low-Transmission Setting of Datem del Marañon Province, Amazonian Peru.

The persistence of malaria hotspots in Datem del Marañon Province, Peru, prompted vector control units at the Ministry of Health, Loreto Department, to collaborate with the Amazonian International Center of Excellence for Malaria Research to identify the main vectors in several riverine villages that had annual parasite indices > 15 in 2018-2019. Anophelinae were collected indoors and outdoors for two 12-hour nights/community during the dry season in 2019 using human landing catch. We identified four species: Nyssorhynchus benarrochi B, Nyssorhynchus darlingi, Nyssorhynchus triannulatus, and Anopheles mattogrossensis. The most abundant, Ny. benarrochi B, accounted for 96.3% of the total (7,550/7,844), of which 61.5% were captured outdoors (4,641/7,550). Six mosquitoes, one Ny. benarrochi B and five Ny. darlingi, were infected by Plasmodium falciparum or Plasmodium vivax. Human biting rates ranged from 0.5 to 592.8 bites per person per hour for Ny. benarrochi B and from 0.5 to 32.0 for Ny. darlingi, with entomological inoculation rates as high as 0.50 infective bites per night for Ny. darlingi and 0.25 for Ny. benarrochi B. These data demonstrate the risk of malaria transmission by both species even during the dry season in villages in multiple watersheds in Datem del Marañon province.

Dengue and COVID-19 Co-Circulation in the Peruvian Amazon: A Population-Based Study.

The COVID-19 pandemic affected the main Amazon cities dramatically, with Iquitos City reporting the highest seroprevalence of anti-SARS-CoV-2 antibodies during the first COVID-19 wave worldwide. This phenomenon raised many questions about the possibility of a co-circulation of dengue and COVID-19 and its consequences. We carried out a population-based cohort study in Iquitos, Peru. We obtained a venous blood sample from a subset of 326 adults from the Iquitos COVID-19 cohort (August 13-18, 2020) to estimate the seroprevalence of anti-dengue virus (DENV) and anti-SARS-CoV-2 antibodies. We tested each serum sample for anti-DENV IgG (serotypes 1, 2, 3, and 4) and SARS-CoV-2 antibodies anti-spike IgG and IgM by ELISA. We estimated an anti-SARS-CoV-2 seroprevalence of 78.0% (95% CI, 73.0-82.0) and an anti-DENV seroprevalence of 88.0% (95% CI, 84.0-91.6), signifying a high seroprevalence of both diseases during the first wave of COVID-19 transmission in the city. The San Juan District had a lower anti-DENV antibody seroprevalence than the Belen District (prevalence ratio, 0.90; 95% CI, 0.82-0.98). However, we did not observe these differences in anti-SARS-CoV-2 antibody seroprevalence. Iquitos City presented one of the highest seroprevalence rates of anti-DENV and anti-SARS-CoV-2 antibodies worldwide, but with no correlation between their antibody levels.

Seroprevalence of anti-SARS-CoV-2 antibodies in Iquitos, Peru in July and August, 2020: a population-based study.

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.

Integrating Parasitological and Entomological Observations to Understand Malaria Transmission in Riverine Villages in the Peruvian Amazon.

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.

Malaria Situation in the Peruvian Amazon during the COVID-19 Pandemic.

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.

Guaroa Virus and Plasmodium vivax Co-Infections, Peruvian Amazon.

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.

Higher risk of malaria transmission outdoors than indoors by Nyssorhynchus darlingi in riverine communities in the Peruvian Amazon.

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.

Malaria vector species in Amazonian Peru co-occur in larval habitats but have distinct larval microbial communities.

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.

High-accuracy detection of malaria vector larval habitats using drone-based multispectral imagery.

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.

Nyssorhynchus dunhami: bionomics and natural infection by Plasmodium falciparum and P. vivax in the Peruvian Amazon.

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.

Automated microscopy for routine malaria diagnosis: a field comparison on Giemsa-stained blood films in Peru.

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.

Decreasing proportion of Anopheles darlingi biting outdoors between long-lasting insecticidal net distributions in peri-Iquitos, Amazonian Peru.

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.

Nyssorhynchus dunhami: bionomics and natural infection by Plasmodium falciparum and P. vivax in the Peruvian Amazon

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.

Loop-mediated isothermal DNA amplification for asymptomatic malaria detection in challenging field settings: Technical performance and pilot implementation in the Peruvian Amazon.

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.

Micro-epidemiology and spatial heterogeneity of P. vivax parasitaemia in riverine communities of the Peruvian Amazon: A multilevel analysis.

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.

Hotspots of Malaria Transmission in the Peruvian Amazon: Rapid Assessment through a Parasitological and Serological Survey.

BACKGROUND: With low and markedly seasonal malaria transmission, increasingly sensitive tools for better stratifying the risk of infection and targeting control interventions are needed. A cross-sectional survey to characterize the current malaria transmission patterns, identify hotspots, and detect recent changes using parasitological and serological measures was conducted in three sites of the Peruvian Amazon. MATERIAL AND METHODS: After full census of the study population, 651 participants were interviewed, clinically examined and had a blood sample taken for the detection of malaria parasites (microscopy and PCR) and antibodies against P. vivax (PvMSP119, PvAMA1) and P. falciparum (PfGLURP, PfAMA1) antigens by ELISA. Risk factors for malaria infection (positive PCR) and malaria exposure (seropositivity) were assessed by multivariate survey logistic regression models. Age-specific seroprevalence was analyzed using a reversible catalytic conversion model based on maximum likelihood for generating seroconversion rates (SCR, λ). SaTScan was used to detect spatial clusters of serology-positive individuals within each site. RESULTS: The overall parasite prevalence by PCR was low, i.e. 3.9% for P. vivax and 6.7% for P. falciparum, while the seroprevalence was substantially higher, 33.6% for P. vivax and 22.0% for P. falciparum, with major differences between study sites. Age and location (site) were significantly associated with P. vivax exposure; while location, age and outdoor occupation were associated with P. falciparum exposure. P. falciparum seroprevalence curves showed a stable transmission throughout time, while for P. vivax transmission was better described by a model with two SCRs. The spatial analysis identified well-defined clusters of P. falciparum seropositive individuals in two sites, while it detected only a very small cluster of P. vivax exposure. CONCLUSION: The use of a single parasitological and serological malaria survey has proven to be an efficient and accurate method to characterize the species specific heterogeneity in malaria transmission at micro-geographical level as well as to identify recent changes in transmission.

Implications for changes in Anopheles darlingi biting behaviour in three communities in the peri-Iquitos region of Amazonian Peru.

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.

Clustered local transmission and asymptomatic Plasmodium falciparum and Plasmodium vivax malaria infections in a recently emerged, hypoendemic Peruvian Amazon community.

BACKGROUND: There is a low incidence of malaria in Iquitos, Peru, suburbs detected by passive case-detection. This low incidence might be attributable to infections clustered in some households/regions and/or undetected asymptomatic infections. METHODS: Passive case-detection (PCD) during the malaria season (February-July) and an active case-detection (ACD) community-wide survey (March) surveyed 1,907 persons. Each month, April-July, 100-metre at-risk zones were defined by location of Plasmodium falciparum infections in the previous month. Longitudinal ACD and PCD (ACP+PCD) occurred within at-risk zones, where 137 houses (573 persons) were randomly selected as sentinels, each with one month of weekly active sampling. Entomological captures were conducted in the sentinel houses. RESULTS: The PCD incidence was 0.03 P. falciparum and 0.22 Plasmodium vivax infections/person/malaria-season. However, the ACD+PCD prevalence was 0.13 and 0.39, respectively. One explanation for this 4.33 and 1.77-fold increase, respectively, was infection clustering within at-risk zones and contiguous households. Clustering makes PCD, generalized to the entire population, artificially low. Another attributable-factor was that only 41% and 24% of the P. falciparum and P. vivax infections were associated with fever and 80% of the asymptomatic infections had low-density or absent parasitaemias the following week. After accounting for asymptomatic infections, a 2.6-fold increase in ACD+PCD versus PCD was attributable to clustered transmission in at-risk zones. CONCLUSION: Even in low transmission, there are frequent highly-clustered asymptomatic infections, making PCD an inadequate measure of incidence. These findings support a strategy of concentrating ACD and insecticide campaigns in houses adjacent to houses were malaria was detected one month prior.