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

Jan E. Conn Wadsworth Center, New York State Department of Health, Albany, New York;
Department of Biomedical Sciences, School of Public Health, State University of New York-Albany, Albany, New York;

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Sara A. Bickersmith Wadsworth Center, New York State Department of Health, Albany, New York;

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Marlon P. Saavedra Amazonian International Center of Excellence for Malaria Research, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru;

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Juliana A. Morales Amazonian International Center of Excellence for Malaria Research, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru;

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Freddy Alava Ministry of Health, Iquitos, Peru;

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Gloria A. Diaz Rodriguez Laboratorio de Salud Pública-Gerencia Regional de Salud de Loreto, GERESA, Iquitos, Peru;

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Clara R. del Aguila Morante Laboratorio de Salud Pública-Gerencia Regional de Salud de Loreto, GERESA, Iquitos, Peru;

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Carlos G. Tong Laboratorio de Salud Pública-Gerencia Regional de Salud de Loreto, GERESA, Iquitos, Peru;

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Carlos Alvarez-Antonio Gerencia Regional de Salud de Loreto, GERESA, Iquitos, Peru;

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Jesus M. Daza Huanahui Red de Salud Datem del Marañon – Gerencia Regional de Salud de Loreto, GERESA, Iquitos, Peru;

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Joseph M. Vinetz Amazonian International Center of Excellence for Malaria Research, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru;
Laboratorio de Malaria: Parásitos y Vectores, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru;
Section of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut;
VA Connecticut Healthcare System, West Haven, Connecticut;
Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru

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Dionicia Gamboa Amazonian International Center of Excellence for Malaria Research, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru;
Laboratorio de Malaria: Parásitos y Vectores, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru;
Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru

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ABSTRACT.

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.

INTRODUCTION

In Peru, many malaria cases (∼84% of 26,621 in 2022)1 originate in the hypoendemic region of Loreto Department.2,3 One indicator of the health burden due to malaria is the measure of the economic burden of productivity loss, calculated using the disability-adjusted life year and the gross domestic product. A recent study based on Peruvian data determined that whereas the economic burden of productivity loss for Loreto from 2001 to 2019 was several times that of Peru overall, in 2019 alone it was estimated to be 30 times higher, a stark reminder of the disproportionate effect of health disparities in this region.4 Locally and globally, malaria is heterogeneous owing to differences in landscape, vector distribution and ecology, and human behavior, among other factors,5,6 and in Loreto many riverine villages are transmission hotspots.7 Malaria endemic regions in several parts of Amazonian Peru have been identified and investigated, yet the province of Datem del Marañon remains understudied.

Aside from well-known annual seasonal mosquito abundance and malaria cycles (high during the rainy season and low during the dry season), malaria case numbers in Amazonian Peru have a history of fluctuation.8 Despite a marked reduction in cases associated with the major control initiative “Control de la Malaria en las Zonas Fronterizas de la Región Andina: Un Enfoque Comunitario – PAMAFRO (2006–2011), by 2012 case levels began to increase,9 reaching epidemic proportions in many communities by 2017.10 Notably, case numbers since 2017 declined by an estimated 50–60%,11,12 attributed mainly to the Malaria Zero Program (MZP), put into place in 2017 by the Peruvian government.4,13 The MZP control activities have included free antimalarials, test-and-treat strategies, larviciding, and pyrethroid spray. Nevertheless, during epidemiological weeks 1–31 in Peru in 2022, the number of cases reported was 15,811, a major increase from 2021 data, when 9,698 cases were reported during the same period.1

Within Loreto, the principal malaria vector is Nyssorhynchus darlingi, a remarkably adaptable species that invaded Iquitos, Peru in the 1990 s1416 and continues to dominate malaria transmission throughout much of the Amazon region of northern South America.17,18 However, prior to the reinvasion of Ny. darlingi into Loreto in the 1990s, Nyssorhynchus benarrochi was considered to be the main vector, especially in some areas of western Loreto and eastern Peru.1921 Based on distribution and molecular taxonomy, it is probable that the species previously known as Ny. benarrochi in Peru is actually Nyssorhynchus benarrochi B,22,23 a member of a species complex that comprises Nyssorhynchus benarrochi s.s., Ny. benarrochi B, Nyssorhynchus benarrochi G1, and Nyssorhynchus benarrochi G2.24 The current known distribution of Ny. benarrochi B, aside from Peru, includes southern Colombia,25 Amazonian Ecuador,23 and both western (Acre state) and eastern (Para state) Amazonian Brazil.24,26 We hypothesized a role for Ny. benarrochi B in malaria transmission in Datem del Marañon based in part on reported high anthropophily and infection with Plasmodium in eastern Peru and southern Colombia,21,27 although there have also been instances where Ny. benarrochi is abundant and highly anthropophilic but not detected as infected.28

Knowledge of biting behavior is an essential part of understanding transmission risk, and such information provides insights that can be used to improve surveillance and intervention.29,30 Rarely, if ever, have peak biting time and location been investigated in Ny. benarrochi B, and it is not known whether or to what extent its biting activity is influenced by environmental variables including indoor residual spray, long-lasting insecticidal bed net use, or human sociodemographics. Intensity of malaria transmission can be estimated by use of the entomological inoculation rate (EIR). In the Amazon Basin, the main technique to calculate Plasmodium infectivity of mosquitoes remains ELISA,31 although molecular methods such as nested polymerase chain reaction (PCR)32 and real-time PCR of the small subunit of the 18S ribosomal RNA (rRNA) have become more common.33,34 The EIR is useful for evaluation and comparison of the effectiveness of vector interventions across landscapes and during malaria elimination efforts. It is also considered to be one of the best metrics for measuring malaria transmission that can be incorporated into the development of model-predicted maps to accurately pinpoint locations of malaria transmission risk, particularly in low-transmission settings during malaria elimination efforts, and where asymptomatic infections are not detected by more traditional surveillance systems.35

Datem del Marañon is one of eight provinces that together comprise Loreto Department. Although large (46,000 km2), it is sparsely populated, with an estimated one inhabitant/km2. From 2000 to 2017, high transmission of Plasmodium falciparum and Plasmodium vivax characterized Datem del Marañon, along with other northwestern provinces in Loreto.36 Perhaps because of its remoteness and low population, Datem del Marañon has not been the focus of any systematic vector biology studies. The objective of this study was to identify the malaria vectors in this region of Datem del Marañon in Amazonian Peru and to calculate the entomological indices to estimate the risk of local malaria transmission.

MATERIALS AND METHODS

Study region.

Loreto Department is characterized by a distinctive rainy season (November–May) and a dry season (June–October), although rainfall (cumulative average of 2,500 mm) occurs throughout the year. The human population of 883,510 is distributed among cities and large and small villages.35 Datem del Marañon province is mainly a tropical rainforest climate according to the Köppen climate classification.37 The major rivers are the Marañon, Pastaza, Huasaga, and Morona (Figure 1). The capital, San Lorenzo de Loreto, situated on the northern banks of the Marañon River has a population of 8,216 (Peruvian census 2017) and is accessible by river or small aircraft.38 Most inhabitants are engaged in swidden-fallow agroforestry, cultivation of crops in the floodplains and exposed riverbeds, extraction of forest products, fishing, and hunting.39 Crops include rice, cowpea, plantain, manioc, and maize.40 In the six main study villages, the annual parasite indice (API; the number of confirmed new malaria cases registered in a specific year per 1,000 individuals under surveillance) ranged from 18 in the largest village of Ullpayacu (population 1,716) to 2,398 in the village of Hortencia Cocha with 83 inhabitants (Table 1).

Figure 1.
Figure 1.

Map of mosquito collection localities within the Datem del Marañon province, Loreto Department, Amazonian Peru.

Citation: The American Journal of Tropical Medicine and Hygiene 109, 2; 10.4269/ajtmh.23-0058

Table 1

Details of collection localities within the province of Datem del Marañon where mosquito collections occurred, human population numbers, annual human malaria case estimates, and API

Locality District Latitude Longitude Population 2018 Cases API 2019 Cases API 2020 Cases API
Pf Pv Pf Pv Pf Pv
Washientza Andoas −3.064560 −76.757360 604 227 502 1,207 180 394 950 132 280 682
Loboyacu Andoas −3.670834 −76.355690 301 23 80 332 6 13 63 7 9 53
Hortencia Cocha Andoas −4.088269 −76.618550 83 75 124 2,398 80 98 2,145 87 43 1,566
Nueva Yarina Pastaza −4.211420 −76.937980 122 24 230 2,082 22 127 1,221 42 70 918
Ullpayacu Pastaza −4.644330 −76.598210 1,716 5 93 57 2 29 18 10 25 20
Trueno Cocha Pastaza −4.658039 −76.461150 253 2 70 285 0 39 154 9 59 269
Totals 356 1,099 290 700 287 486

API = annual parasite index; Pf = Plasmodium falciparum; Pv = Plasmodium vivax.

Mosquito sampling.

Mosquitoes were collected from three localities within the Pastaza district (Ullpayacu, Trueno Cocha, and Nueva Yarina) and three within the Andoas district (Hortencia Cocha, Loboyacu, and Washientza) of Datem del Marañon province (Figure 1, Table 1). Collections were conducted in 2019 indoors and outdoors (peridomestic, within approximately 10 m of the main house entrance) using human landing catch (HLC) by two persons/locality for 12 hours from 18:00 to 06:00, for two nights/locality as follows: Nueva Yarina and Hortencia Cocha, August 3–5; Washientza and Ullpayacu, August 5–7; and Loboyacu and Truena Cocha, August 7–9.

Mosquito collecting including HLC is conducted as part of the routine work of field personnel at Laboratorio de Salud Pública-Gerencia Regional de Salud de Loreto, GERESA, Peru, and, as such, is considered a safety management issue. All field personnel are trained in the safe and responsible collection of mosquitoes and other vectors that might transmit pathogens.

We also examined two samples of anophelines collected during 4-hour collections (18:00–22:00) by HLC indoors and outdoors from Puerto Rubina on April 10, 2021 and Triunfo on April 25, 2021 (Figure 1), confirmed the Anophelinae species identification, and tested these specimens for Plasmodium. These secondary collections were not used to calculate any entomological indices.

Testing for Plasmodium.

A total of 7,827 mosquitoes, using heads and thoraces, in pools of one to eight mosquitoes based on capture time, species (Ny. benarrochi B or Ny. darlingi), and specific locality were tested at the Universidad Peruana Cayetano Heredia in Lima, Peru, for detection of Plasmodium infection with an ELISA as in Saavedra et al.41 To calculate the infection rate (IR) for each Anophelinae species (IR = # mosquitoes infected with Plasmodium/# mosquitoes of the same species tested) and the EIR = HBR × IR, each of the positive pools was conservatively estimated to include one positive mosquito. The human biting rate (HBR) was calculated as the mean number of mosquitoes collected by HLC per person per night.

At the Vector Biology and Population Genetics Laboratory at the Wadsworth Center, New York State Department of Health in Albany, New York, genomic DNA was extracted from each individual abdomen (Qiagen DNeasy Blood & Tissue Kit, Germantown, MD) of the 33 specimens that comprised the positive (N = 6) and possible positive (N = 3) pools for ELISA and was tested for Plasmodium species infection using 18S rRNA, in duplicate, with a triplex real-time PCR.33 The possible positives were below the optical density (OD) but close to the average of twice the OD of the negative controls, although the real-time PCR tests of abdomens were negative. We used only the six positive ELISA results to calculate the entomological indices. Most vectors in malaria endemic Peru have relatively low infectivity rates; consequently, abdomens may contain very low titers of Plasmodium. Despite the higher sensitivity of real-time PCR, it is not uncommon to find a discrepancy between the two results, that is, more positive or possible positive pools from ELISA versus real-time PCR.

Morphological and molecular species identification.

All captured mosquitoes were identified initially using regional morphological keys42,43 at the Laboratorio de Salud Publica-Gerencia Regional de Salud de Loreto, DIRESA in Iquitos, Peru. The 2021 mosquito samples from Puerto Rubina and Triunfo, as well as individual Ny. benarrochi B and Ny. darlingi from the positive ELISA pools, were identified molecularly for species confirmation using a PCR of the ribosomal internal transcribed spacer 2 (ITS2) region, followed by a double-digest restriction fragment length polymorphism (RFLP).44 The cytochrome c oxidase subunit I (COI) barcode region was amplified45 for all mosquito samples that could not be identified by the ITS2-PCR-RFLP patterns and sent for Sanger sequencing in the forward direction only at the Advanced Genomic Technologies Core (Wadsworth Center). Raw sequences were cleaned, edited, and checked for stop codons and pseudogenes using the platform Geneious Prime.46 Sequences were queried for species match in the Barcode of Life Data System (www.barcodinglife.org) or GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Following GenBank protocol, 16 sequences of Anopheles benarrochi B, one Anopheles darlingi, three Anopheles tadei, and one Anopheles mattogrossensis (21 total) from this study have been deposited under accession numbers OP964656–OP964676.

RESULTS

Vector biology.

A total of 7,844 Anophelinae mosquitoes were captured across the six main collection localities. Species detected were Ny. benarrochi B, Ny. darlingi, Nyssorhynchus triannulatus s.l., and An. mattogrossensis. The most abundant species captured was Ny. benarrochi B (N = 7,550) in all but one locality (Washientza), where Ny. darlingi was the most abundant, albeit at low numbers (Table 2, Figure 2). The samples captured in 2021 in Puerto Rubina were all Ny. darlingi (N = 18); in contrast, of the 19 mosquitoes collected in Triunfo, nine were An. mattogrossensis, four were Ny. darlingi, three were Nyssorhynchus tadei, a recently named species in the Oswaldoi-Konderi complex,47 and two were Ny. benarrochi B. One could not be identified (degraded DNA).

Table 2

Number of mosquitoes collected by species, indoors and outdoors, per locality in the province of Datem del Marañon, HBR, IR, and EIR

Locality Species Indoor N Outdoor N Total N HBR (± SE) # Infected IR EIR
Pv Pf
Washientza Ny. benarrochi B 2 0 2 0.5 (0.5) 0.0000 0.000
Ny. darlingi 52 54 106 26.5 (8.5) 1 0.0094 0.250
An. mattogrossensis 0 0 0 0 (0) N/A N/A
Ny. triannulatus s.l. 0 0 0 0 (0) N/A N/A
Total 54 54 108
Loboyacu Ny. benarrochi B 921 1,450 2,371 592.8 (308.3) 1 0.0004 0.250
Ny. darlingi 0 0 0 0 (0) N/A N/A
An. mattogrossensis 0 2 2 0.5 (0.5) 0.0000 0.000
Ny. triannulatus s.l. 0 0 0 0 (0) N/A N/A
Total 921 1,452 2,373
Hortencia Cocha Ny. benarrochi B 1,046 1,272 2,318 579.5 (19) 0.0000 0.000
Ny. darlingi 20 25 45 11.3 (5.3) 1 1 0.0444 0.500
An. mattogrossensis 0 4 4 1 (0) 0.0000 0.000
Ny. triannulatus s.l. 0 0 0 0 (0) N/A N/A
Total 1,066 1,301 2,367
Nueva Yarina Ny. benarrochi B 803 1,137 1,940 485 (199) 0.0000 0.000
Ny. darlingi 35 93 128 32 (6.5) 2 0.0156 0.500
An. mattogrossensis 0 2 2 0.5 (0) 0.0000 0.000
Ny. triannulatus s.l. 0 0 0 0 (0) N/A N/A
Total 838 1,232 2,070
Ullpayacu Ny. benarrochi B 22 295 317 79.3 (67.3) 0.0000 0.000
Ny. darlingi 0 0 0 0 (0) N/A N/A
An. mattogrossensis 0 1 1 0.3 (0.3) 0.0000 0.000
Ny. triannulatus s.l. 0 0 0 0 (0) N/A N/A
Total 22 296 318
Trueno Cocha Ny. benarrochi B 115 487 602 150.5 (34.5) 0.0000 0.000
Ny. darlingi 1 1 2 0.5 (0.5) 0.0000 0.000
An. mattogrossensis 0 3 3 0.8 (0.3) 0.0000 0.000
Ny. triannulatus s.l. 1 0 1 0.3 (0.3) 0.0000 0.000
Total 117 491 608

An. = Anopheles; EIR = the number of infective bites per person per 12-hour night; HBR = the average bites per person per night (b/p/n) calculated from a mean of two nights/12 hours per night per locality; Indoor N = number of mosquitoes captured inside a house; IR = infection rate; Ny. = Nyssorhynchus; Outdoor N = number of mosquitoes captured in the peridomestic environment (within 10 m of a main house entry); Pf = Plasmodium falciparum; Pv = Plasmodium vivax; N/A = not applicable.

Figure 2.
Figure 2.

Total number (counts) of Nyssorhynchus benarrochi B and Nyssorhynchus darlingi captured indoors (in) or outdoors (out) in Datem del Marañon, Peru, in 2019 by locality.

Citation: The American Journal of Tropical Medicine and Hygiene 109, 2; 10.4269/ajtmh.23-0058

Among the six main collection communities, 61.5% of all Anophelinae were captured outdoors (4,826/7,844). By community, most (five/six) had more outdoor specimens captured; however, in Washientza, the outdoor and indoor collection sizes were the same (N = 54) and all the samples were Ny. darlingi (Figure 2). Focusing exclusively on the biting pattern of Ny. benarocchi B because of its high abundance, the peak (average proportion/hour) occurred between 18:00 and 20:00, with a small peak detected at 01:00 only in Nueva Yarina (Figure 3). The highest HBRs for Ny. benarrochi B were in Loboyacu (592.8 bites/person/night) and Hortencia Cocha (579.5 b/p/n), whereas the highest HBR for Ny. darlingi was 32 b/p/n in Nueva Yarina (Table 2).

Figure 3.
Figure 3.

Average proportion of Nyssorhynchus benarrochi B collected per hour for all localities except Washientza, where Ny. benarrochi B was not collected.

Citation: The American Journal of Tropical Medicine and Hygiene 109, 2; 10.4269/ajtmh.23-0058

Plasmodium infection.

Six ELISA pools of mosquitoes were confirmed positive for P. falciparum or P. vivax, and calculations were based on the assumption of one infective mosquito per positive pool (Table 2; Supplemental Table 1). The real-time PCR assay confirmed four of six infected individuals from the positive ELISA pools. Of the four species identified, only Ny. darlingi and Ny. benarrochi B were infected with Plasmodium. Infection rates, calculated per mosquito species, varied among localities; that is, IRs for Ny. darlingi were 0.94% (1/106) Washientza, 4.44% (2/45) Hortencia Cocha, and 1.56% (2/128) Nueva Yarina. Infection rates for Ny. benarrochi B were 0.04% (1/2,371) Loboyacu and 0 for the other localities (Table 2). Time of the collection of the six infected mosquitoes ranged across the night (Supplemental Table 1), and most (five/six) were collected outdoors. The infected specimen collected indoors was Ny. darlingi in Nueva Yarina (21:00–22:00), with P. falciparum.

Nueva Yarina and Hortencia Cocha had the highest EIR/night (0.5) where Ny. darlingi was infected (two P. falciparum [Pf] in Nueva Yarina; one Pf and one P. vivax [Pv] in Hortencia Cocha). In Loboyacu, we calculated an EIR of 0.25/night where Ny. benarrochi B was infected with Pv and in Washientza, the same EIR (0.25/night) where Ny. darlingi was infected with Pv. No infected mosquitoes were detected in either the largest village, Ullpayacu, or in Trueno Cocha (Table 2).

Human malaria case incidence.

Overall, from 2018 to 2020, fewer cases of both P. falciparum and P. vivax were registered in the six riverine villages by 2020 (Table 1). However, in four villages, Hortencia Cocha, Nueva Yarina, Ullpayacu, and Trueno Cocha, P. falciparum cases increased from 2018 to 2020 at the same time as P. vivax cases decreased (except in Trueno Cocha). The overall proportion of P. falciparum from 2018 to 2020 was 29%, with P. vivax responsible for ∼71% (Table 1).

Data management.

All anopheline individuals sequenced for the DNA COI barcode region (N = 21) have been deposited in GenBank. All sample results will be deposited in VectorBase48 pending publication.

DISCUSSION

Entomological inoculation rate results greater than 0/night in four/six of the study villages in Datem del Marañon study were higher than generally reported for the dry season in Loreto (∼June–October), when both mosquito abundance and malaria cases trend lower.7,49 On the other hand, spatial heterogeneity among villages combined with occupation-related travel can lead to high EIR rates for Ny. darlingi even during the dry season.50 A striking result from this study is that in these riverine villages in Datem del Marañon both Ny. darlingi and Ny. benarrochi B were infected with Plasmodium, indicating that both are involved in local transmission. These data also support the role of Ny. darlingi as a primary vector in Loreto because even at low numbers and a relatively low biting rate (especially in contrast to the overwhelming abundance and high biting rate of Ny. benarrochi B in five/six villages), the EIR values for both species were in the same range.

Previous reports in Loreto have demonstrated that Ny. benarrochi (presumed to be Ny. benarrochi B) is both anthropophilic and abundant19,20 and can be infected.21 Importantly, we note that in these Datem del Marañon villages, the peak biting time of Ny. benarrochi B is early evening (∼18:00–20:00), a time when studies in many malaria endemic regions have shown that inhabitants are active, unprotected by bed nets, and vulnerable to transmission.41,51,52 Furthermore, most (five/six) infected mosquitoes were collected outdoors and were biting throughout the night. Such behavior patterns were recorded in Loreto previously for Ny. darlingi,53,54 although this species appears to revert to indoor biting when insecticide pressure is reduced.55 Biting behavior of Ny. darlingi is extremely plastic and depends on a wide array of microgeographic environmental conditions such as house location, forest cover, previous and current insecticide use, and human behavior.56,57

The current study, together with data from Colombia and Brazil,24,26,27 indicates that the role of Ny. benarrochi B in malaria transmission remains locally and regionally relevant, and additional studies on behavior and ecology are warranted. One limitation of this study is that collections were undertaken only in August during the dry season, generally the time of year in Amazonian Peru when both mosquito abundance and malaria cases are lower. Another limitation is that only two 12-hour collections were conducted per village for the main study. This collection is not representative of the common transmission pattern in Amazonian Peru, which is seasonal, high during the rainy season and low during the dry season.41 On the other hand, these data provide an entomological snapshot of Plasmodium infectivity in multiple villages in a region that has been neglected.

A study that analyzed 697,916 malaria cases in Peru between 2000 and 2017 determined that the highest mean API of P. falciparum occurred in Datem del Marañon (M = 26.2; SD = 25.9), whereas the highest API for P. vivax (M = 50.3, SD = 35.5) was in Loreto province.36 Despite the much smaller sample size in our study from six villages in Datem del Marañon (2018–2020), our findings also demonstrate an elevated proportion of P. falciparum of 28.99% (933/3,218) of the malaria cases (Table 1), in contrast to an average 25% of malaria cases in Peru.11,12 It is also noteworthy that subsequently, in 2021–2022, the district of Andoas was categorized as very high risk for P. vivax and P. falciparum malaria transmission (in Peru, an API > 50), with Pastaza in the same category for P. vivax but slightly lower for P. falciparum (high risk, API 10.00–49.99).1

Although we detected only three specimens of Ny. tadei from one village (Triunfo), it is the first confirmed report of this species in Datem del Marañon. This species belongs to the broadly distributed Amazonian Oswaldoi-Konderi complex (Nyssorhynchus oswaldoi s.s., Nyssorhynchus oswaldoi A, Nyssorhynchus oswaldoi B, Nyssorhynchus konderi, and Ny. tadei) (Saraiva and Scarpassa47 and references therein). However, Ny. tadei (as Nyssorhynchus sp. nr. konderi) is known from Loreto, Peru,41,58,59 and is hypothesized to be allopatric in Loreto to the north and Madre de Dios to the south.58 In contrast, only a single specimen of Ny. konderi has been confirmed with molecular markers from the village of Salvador on the Napo River northwest of Iquitos, Loreto.59 Several earlier studies reported the presence of Nyssorhynchus oswaldoi s.l. in Peru, where it has been considered an effective malaria vector.19,60,61

In conclusion, in the malaria endemic province of Datem del Marañon, Amazonian Peru, Ny. benarrochi B was infected by P. vivax in Loboyacu and Ny. darlingi was infected by both P. falciparum and P. vivax in several villages. Although the risk to local human inhabitants of being bitten is highest during the early evening, risk of transmission was found to be throughout the night, mainly but not exclusively outdoors. As work toward malaria eradication in Peru moves forward with programs such as MZP, we recommend that remote and relatively understudied parts of Peru be a particular focus of attention and intervention.

Supplemental Materials

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ACKNOWLEDGMENTS

We thank all the residents and the local authorities of Ullpayacu, Trueno Cocha, Nueva Yarina, Hortencia Cocha, Loboyacu, Washientza, Triunfo, and Puerto Rubina for their support. We acknowledge the entomology technicians from Laboratorio Referencial Regional de Salud Pública de Loreto and the personnel (especially J. Campos, P. Rojas, and W. Orellana) at the health facility in Datem del Marañon for all the assistance during field collections. This publication has been possible thanks to the authorization and permits (no. 0424-2012-AG-DGFFS-DGEFFS) from Dirección de Gestión Forestal y de Fauna Silvestre y la Dirección General Forestal y de Fauna Silvestre del Ministerio de Agricultura de la República del Perú. Sample DNA was sequenced at the Advanced Genomic Technologies Core Wadsworth Center, Albany, NY.

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Author Notes

Address correspondence to Jan E. Conn, Griffin Laboratory, Wadsworth Center, New York State Department of Health, 5668 State Farm Road, Slingerlands, NY 12159, E-mail: jan.conn@health.ny.gov or Dionicia Gamboa, Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Lima 31, Peru, E-mail: dionigamboa@yahoo.com

Financial support: This research was supported by the NIH, U.S. International Centers of Excellence in Malaria Research (ICEMR) program, grant no. U19 AI089681 to J. M. V.

Authors’ addresses: Jan E. Conn, Wadsworth Center, New York State Department of Health, Albany, NY, and Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY, E-mail: jan.conn@health.ny.gov. Sara A. Bickersmith, Wadsworth Center, New York State Department of Health, Albany, NY, E-mail: sara.bickersmith@health.ny.gov. Marlon P. Saavedra and Juliana A. Morales, Amazonian International Center of Excellence for Malaria Research, Laboratorios de Investigación y Desarollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru, E-mails: marlon.saavedra.r@upch.pe and jmoralesmonje@gmail.com. Freddy Alava, Ministry of Health, Iquitos, Perú, E-mail: ffalare@hotmail.com. Gloria A. Diaz Rodriguez, Clara R. del Aguila Morante, and Carlos G. Tong, Laboratorio de Salud Pública-Gerencia Regional de Salud de Loreto, GERESA, Iquitos, Peru, E-mails: gloriaadiaz@yahoo.com, cdelaguilamorante@gmail.com, and ctong32@gmail.com. Carlos Alvarez-Antonio, Gerencia Regional de Salud de Loreto, Iquitos, GERESA, Peru, E-mail: calvarez@diresaloreto.gob.pe. Jesus M. Daza Huanahui, Red de Salud Datem del Marañon – Gerencia Regional de Salud de Loreto, Iquitos, GERESA, Peru, E-mail: jesusdaza1986@gmail.com. Joseph M. Vinetz, Amazonian International Center of Excellence for Malaria Research, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru, Laboratorio de Malaria: Parásitos y Vectores, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru, Section of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, VA Connecticut Healthcare System, West Haven, CT, and Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru, E-mail: joseph.vinetz@yale.edu. Dionicia Gamboa, Amazonian International Center of Excellence for Malaria Research, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru, Laboratorio de Malaria: Parásitos y Vectores, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru, and Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru, E-mail: dionigamboa@yahoo.com.

  • Figure 1.

    Map of mosquito collection localities within the Datem del Marañon province, Loreto Department, Amazonian Peru.

  • Figure 2.

    Total number (counts) of Nyssorhynchus benarrochi B and Nyssorhynchus darlingi captured indoors (in) or outdoors (out) in Datem del Marañon, Peru, in 2019 by locality.

  • Figure 3.

    Average proportion of Nyssorhynchus benarrochi B collected per hour for all localities except Washientza, where Ny. benarrochi B was not collected.

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    MINSA , 2022. Ministerio de Salud del Perú: Boletín epidemiológico del Perú SE 31-2022. Lima, Perú: Centro Nacional de Epidemiología, Prevención y Control de Enfermedades. Available at: https://www.dge.gob.pe/portalnuevo/publicaciones/boletines-epidemiologicos/. Accessed May 26, 2023.

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    Branch O, Casapia WM, Gamboa DV, Hernandez JN, Alava FF, Roncal N, Alvarez E, Perez EJ, Gotuzzo E, 2005. Clustered local transmission and asymptomatic Plasmodium falciparum and Plasmodium vivax malaria infections in a recently emerged, hypoendemic Peruvian Amazon community. Malar J 4: 27.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 3.

    Rosas-Aguirre A et al., 2016. Epidemiology of Plasmodium vivax malaria in Peru. Am J Trop Med Hyg 95: 133144.

  • 4.

    Sanchez-Castro EE, Cahuana GM, García-Ríos CJ, Guerra-Duarte C, Chauca P, Tapia-Limonchi R, Chenet SM, Soria B, Chavez-Olortegui C, Tejedo JR, 2022. Health and economic burden due to malaria in Peru over 30 years (1990–2019): findings from the global burden of diseases study 2019. Lancet Reg Health Am 15: 100347.

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  • 5.

    Clements ACA, Reid HL, Kelly GC, Hay SI, 2013. Further shrinking the malaria map: how can geospatial science help to achieve malaria elimination? Lancet Infect Dis 13: 709718.

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    Antiporta DA, Rosas-Aguirre A, Chang J, Llanos-Cuentas A, Lescano AG, 2020. Malaria eradication. Lancet 395: e67.

  • 7.

    Soto-Calle V et al., 2017. Spatio-temporal analysis of malaria incidence in the Peruvian Amazon region between 2002 and 2013. Sci Rep 7: 40350.

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    Griffing SM, Gamboa D, Udhayakumar V, 2013. The history of the 20th century malaria control in Peru. Malar J 12: 303.

  • 9.

    Rosas-Aguirre A et al., 2015. Hotspots of malaria transmission in the Peruvian Amazon: rapid assessment through a parasitological and serological survey. PLoS One 10: e0137458.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10.

    Recht J, Siqueira AM, Monteiro WM, Herrera SM, Herrera S, Lacerda MVG, 2017. Malaria in Brazil, Colombia, Peru and Venezuela: current challenges in malaria control and elimination. Malar J 16: 273.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11.

    WHO , 2022. World Malaria Report 2022. Geneva, Switzerland: World Health Organization, 293.

  • 12.

    MINSA , 2020. Ministerio de Salud del Perú: Centro Nacional de Epidemiológica, Prevención y Control de Enfermedades Hasta la SE 44 - 2020: Centro Nacional de Epidemiología, Prevención y Control de Enfermedades. Available at: https://www.dge.gob.pe/portal/docs/vigilancia/sala/2020/SE44/mmaterna.pdf. Accessed May 26, 2023.

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    • Export Citation
  • 13.

    Torres K, Alava F, Soto-Calle V, Llanos-Cuentas A, Rodriguez H, Llacsahuanga L, Gamboa D, Vinetz J, 2020. Malaria situation in the Peruvian Amazon during the COVID-19 pandemic. Am J Trop Med Hyg 103: 17731776.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14.

    Fernández R, Carbajal F, Quintana J, Chauca H, Watts DM, 1996. Presencia del A. (N) darlingi (Diptera: Culicidae), en alrededores de la ciudad de Iquitos, Loreto-Peru. Boletín de la Soc Peruana de Enfermedades Infecciosas y Trop 5: 1020.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15.

    Vittor AY, Gilman RH, Tielsch J, Glass G, Shields T, Lozano WS, Pinedo-Cancino V, Patz JA, 2006. The effect of deforestation on the human-biting rate of Anopheles darlingi, the primary vector of falciparum malaria in the Peruvian Amazon. Am J Trop Med Hyg 74: 311.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16.

    Lainhart W, Bickersmith S, Nadler K, Moreno M, Saavedra M, Chu VM, Ribolla PE, Vinetz JM, Conn JE, 2015. Evidence for temporal population replacement and the signature of ecological adaptation in a major neotropical malaria vector in Amazonian Peru. Malar J 14: 375.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17.

    Sinka ME et al., 2012. A global map of dominant malaria vectors. Parasit Vectors 5: 69.

  • 18.

    Carlos BC, Rona LDP, Christophides GK, Souza-Neto JA, 2019. A comprehensive analysis of malaria transmission in Brazil. Pathog Glob Health 113: 113.

  • 19.

    Calderón G, Fernández R, Valle J, 1995. Especies de la fauna anofelina, su distribucion y algunas consideraciones sobre su abundancia e infectividad en el Peru. Rev Peru Epidemiol 8: 523.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 20.

    Schoeler GB, Flores-Mendoza C, Fernandez R, Davila JR, Zyzak M, 2003. Geographical distribution of Anopheles darlingi in the Amazon basin region of Peru. J Am Mosq Control Assoc 19: 286296.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21.

    Flores-Mendoza C, Fernandez R, Escobedo-Vargas KS, Vela-Perez Q, Schoeler GB, 2004. Natural Plasmodium infections in Anopheles darlingi and Anopheles benarrochi (Diptera: Culicidae) from eastern Peru. J Med Entomol 41: 489494.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22.

    Conn JE et al., 2013. Molecular taxonomy of Anopheles (Nyssorhynchus) benarrochi (Diptera: Culicidae) and malaria epidemiology in southern Amazonian Peru. Am J Trop Med Hyg 88: 319324.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23.

    Morales Viteri D et al., 2021. New records of Anopheles benarrochi B (Diptera: Culicidae) in malaria hotspots in the Amazon regions of Ecuador and Peru. J Med Entomol. 58: 12341240.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 24.

    Bourke BP, Conn JE, de Oliveira TMP, Chaves LSM, Bergo ES, Laporta GZ, Sallum MAM, 2018. Exploring malaria vector diversity on the Amazon frontier. Malar J 17: 342.

  • 25.

    Ruiz F, Quinones ML, Erazo HF, Calle DA, Alzate JF, Linton YM, 2005. Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from southern Colombia. Mem Inst Oswaldo Cruz 100: 155160.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26.

    Oliveira TMP, Laporta GZ, Bergo ES, Chaves LSM, Antunes JLF, Bickersmith SA, Conn JE, Massad E, Sallum MAM, 2021. Vector role and human biting activity of Anophelinae mosquitoes in different landscapes in the Brazilian Amazon. Parasit Vectors 14: 236.

    • PubMed
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