Lack of Evidence of Sylvatic Transmission of Dengue Viruses in the Amazon Rainforest Near Iquitos, Peru.

Dengue viruses (DENV) are currently responsible for more human morbidity and mortality than any other known arbovirus, and all four DENV are known to exist in sylvatic cycles that might allow these viruses to persist if the urban (Aedes aegypti) cycle could be controlled. To determine whether DENV were being maintained in a sylvatic cycle in a forested area about 14 km southwest of Iquitos, Peru, a city in which all 4 serotypes of DENV circulate, we placed 20 DENV seronegative Aotus monkeys in cages either in the canopy or near ground level for a total of 125.6 months. Despite capturing >66,000 mosquitoes in traps that collected some of the mosquitoes attracted to these monkeys, blood samples obtained once a month from each animal were tested and found to be negative by an enzyme-linked immunosorbent assay for IgM and IgG antibodies to dengue, yellow fever, Venezuelan equine encephalitis, Oropouche, and Mayaro viruses. Although all four DENV serotypes were endemic in nearby Iquitos, the findings of this study did not support a DENV sylvatic maintenance and transmission cycle in a selected area of the Amazon rainforest in northeastern Peru.

Identification and Genomic Analysis of a Novel Group C Orthobunyavirus Isolated from a Mosquito Captured near Iquitos, Peru.

Group C orthobunyaviruses are single-stranded RNA viruses found in both South and North America. Until very recently, and despite their status as important vector-borne human pathogens, no Group C whole genome sequences containing all three segments were available in public databases. Here we report a Group C orthobunyavirus, named El Huayo virus, isolated from a pool of Culex portesi mosquitoes captured near Iquitos, Peru. Although initial metagenomic analysis yielded only a handful of reads belonging to the genus Orthobunyavirus, single contig assemblies were generated for L, M, and S segments totaling over 200,000 reads (~0.5% of sample). Given the moderately high viremia in hamsters (>107 plaque-forming units/ml) and the propensity for Cx. portesi to feed on rodents, it is possible that El Huayo virus is maintained in nature in a Culex portesi/rodent cycle. El Huayo virus was found to be most similar to Peruvian Caraparu virus isolates and constitutes a novel subclade within Group C.

Taming a tiger in the city: comparison of motorized backpack applications and source reduction against the Asian tiger mosquito, Aedes albopictus.

We evaluated 2 strategies to manage Aedes albopictus: 1) motorized backpack applications and 2) source reduction (coupled with hand-applied applications of larvicide). Backpack applications used a water-dispersible granular formulation (VectoBac WDG) of Bacillus thuringiensis var. israelensis (Bti), whereas source reduction used granular formulations of the insect growth regulator methoprene (Altosid) combined with a monomolecular film surfactant (Agnique). Six subplots (total 8.02 ha) were selected for backpack applications, source reduction, and control groups. The experiments were blind with applications conducted randomly and independently. Efficacy was determined through placement of bioassay cups with larvae within experimental plots 1 day before treatment. Backpack applications resulted in 76% (+/- 8.2% SE) and source reduction resulted in 92% (+/- 4.1% SE) larval mortality. Backpack applications required 50 times less labor than source reduction (0.25 versus 0.005 ha/h). The cost of backpack applications, including labor, was $159.88/ha, compared with $659.65/ha for source reduction. Although overall efficacy was slightly lower, motorized backpack applications of Bti were more efficient and cost-effective than source reduction methods to control Ae. albopictus in urban settings at the community level.

Point-source and area-wide field studies of pyriproxyfen autodissemination against urban container-inhabiting mosquitoes.

Autodissemination of insecticides is a novel strategy for mosquito management. We tested if contaminated Aedes albopictus (Skuse) mosquitoes from a small area treated with commercial formulations (79gm a.i. pyriproxyfen/ha) using conventional techniques, would disseminate pyriproxyfen over a wider area. Pyriproxyfen showed LC50=0.012 ppb for Ae. albopictus. Direct treatment and autodissemination efficacy was measured as a pupal mortality by conducting Ae. albopictus larval bioassay. A tire pile (n=100 tires) treated by backpack sprayer as a point-source treatment showed higher pupal mortality in 2010 (60.8% for week 0-6) than in 2011 (38.3% for week 0-6). The sentinel containers placed for autodissemination in four compass directions out to 200-400m from the tire pile showed 15.8% pupal mortality (week 1-6) in the first year, and 1.4% pupal mortality in the second year. No significant difference was detected among the distances and direction for pupal mortality. In area-wide treatments, vegetation was sprayed in checkerboard pattern (3.7% of 105ha) using backpack sprayer in 2010 and in strips (24.8% of 94ha) using truck-mounted ultra-low volume (ULV) sprayer in 2011. In both years, the area-wide direct treatment efficacy was lower (30.3% during 2010 and 5.3% in 2011) than point-source treatments. Autodissemination in area-wide plots was higher in 2010 (10.3%) than 2011 (2.9%). However, area-wide treatments were ineffective on field populations of Ae. albopictus as monitored by using BGS traps. We found accumulation of pyriproxyfen in the week 6 autodissemination containers in both experiments. The differences in autodissemination in 2010 and 2011 can be attributed to higher rainfall in the second year that may have eroded the pyriproxyfen from treatment surfaces and sentinel containers. Our study shows that ULV surface treatments of conventional formulation do not work for autodissemination. The effectiveness of pyriproxyfen in autodissemination may be improved by developing specific formulations to treat vegetation and tires that can load high doses on mosquitoes.

Species Diversity, Seasonal, and Spatial Distribution of Mosquitoes (Diptera: Culicidae) Captured in Aotus Monkey-Baited Traps in a Forested Site Near Iquitos, Peru.

This study was conducted to determine the relative abundance, diversity, seasonal, and vertical distributions of potential mosquito vectors in the Amazon Basin, Peru. A total of 66,097 mosquitoes (50 mosquito species from 12 genera) were collected from May 2001 through March 2002 at a forested site near Iquitos, Peru. Mosquitoes were collected using Aotus nancymae Hershkovitz monkey-baited CDC light traps set for 12-h day and night periods at varying heights (e.g., ground and canopy) in the forest. Of the 12 genera, three accounted for 75% of all mosquitoes collected: Culex (33%), Aedes (23%), and Psorophora (18%). The most prevalent species collected were Aedes serratus (Theobald), Culex pedroi Sirivanakarn & Belkin, Psorophora albigenu (Peryassu), and a combination of Mansonia indubitans Dyar & Shannon and Mansonia titillans (Walker), which accounted for 56% of all mosquitoes captured. In general, mosquitoes were collected more often at night and on the ground. Exceptions include Coquillettidia venezuelensis (Theobald), which were collected in relatively even numbers at both day and night and most Mansonia and some species of Anopheles, which were collected more often in the canopy. Total mosquito populations had two peaks, June-July (Ma. indubitans/titillans and Cq. venezuelensis) and December-January (Ps. albigenu, Cx. pedroi, and Ae. serratus). Observations of the eight most collected mosquitoes indicated that behavioral shifts were not observed between collection months. These data provide a better understanding of the species diversity, population density, and seasonal distribution of potential mosquito vectors within the Amazon Basin region and allow for the development of appropriate vector and disease prevention strategies.

Larval mosquito habitat utilization and community dynamics of Aedes albopictus and Aedes japonicus (Diptera: Culicidae).

Aedes albopictus (Skuse) and Ae. japonicus (Theobald) are important container-inhabiting mosquitoes that transmit disease agents, outcompete native species, and continue to expand their range in the United States. Both species deposit eggs in natural and artificial containers and thrive in peridomestic environments. The goal of our study was to examine the types and characteristics of containers that are most productive for these species in the northeastern United States. In total, 306 containers were sampled in urban, suburban, and rural areas of New Jersey. Multiple biotic and abiotic factors were recorded in an attempt to identify variables associated with the productivity of each species. Based on pupal abundance and density of container types, results showed that tires, trash cans, and planter dishes were the most important containers for Ae. albopictus, while planter dishes were the most important containers for Ae. japonicus. Container color (black and gray), material (rubber), and type (tires) were correlated with species presence for Ae. albopictus and Ae. japonicus. These factors may play a role in the selection of oviposition sites by female mosquitoes or in the survival of their progeny. Differences in species composition and abundance were detected between areas classified as urban, suburban, and rural. In urban and suburban areas, Ae. albopictus was more abundant in container habitats than Ae. japonicus; however, Ae. japonicus was more abundant in rural areas, and when water temperatures were below 14 degrees C. Our results suggest many variables can influence the presence of Ae. albopictus and Ae. japonicus in container habitats in northeastern United States.

Human spotted fever rickettsial infections.

Serum specimens from patients at 4 sites in Peru were tested for evidence of spotted fever group rickettsial infection. Results showed that 30 (18%) of 170 patients had spotted fever group rickettsial infections, which likely caused their illnesses. These findings document laboratory-confirmed spotted fever from diverse areas of Peru.

Phylogenetic analysis of a novel molecular isolate of spotted fever group Rickettsiae from northern Peru: Candidatus Rickettsia andeanae.

Phylogenetic analysis of five rickettsial genes (17-kDa gene, gltA, ompB, ompA, and sca4) from two molecular isolates of Candidatus Rickettsia andeanae from two ticks (Amblyomma maculatum and Ixodes boliviensis) collected from two domestic horses living in two separate locations in northern Peru (Coletas and Naranjo) was conducted to more clearly characterize this recently reported novel spotted fever group (SFG) rickettsia. Following nested polymerase chain reaction (PCR) amplification of the 17-kDa gene, gltA, ompB, ompA, and sca4, amplicons were purified, sequenced, and compared to those downloaded from GenBank. Phylogenetic analyses of the Candidatus Rickettsia andeanae sequences generated from 17-kDa gene (483 bp), gltA (1185 bp), ompA (1598 bp), ompB (4839 bp), and sca4 (2634 bp) demonstrated that they aligned strongly with those of SFG rickettsiae. Moreover, the sequences of these five genes most closely aligned with the following rickettsiae: ompA: Rickettsia sp RpA4 (98.03%), R. sp DnS28 (97.90%), and R. rhipicephali and R. massiliae (97.11%); ompB: R. aeschlimannii (97.22%), R. rhipicephali (97.20%), and R. sp Bar 29 (97.10%); and sca4: R. massiliae (97.8%), R. rhipicephali, and R. slovaca (97.7%). These results from the additional phylogenetic analyses of Candidatus Rickettsia andeanae confirm its inclusion within, and distance and uniqueness from, other known SFG rickettsiae.

Presencia de Anopheles (Nyssorhynchus) benarrochi en áreas de la selva con transmisión malárica / Presence of Anopheles (Nyssorhynchus) benarrochi in Amazona areas where malaria transmision occurs

Objetivos: Demostrar la presencia de An. (Nys. ) benarrochi en áreas de transmisión malárica, donde es frecuentemente identificado como An. evansae. Materiales y métodos: Se colectaron mosquitos en San José (Pucallpa), Muniches y Andoas (Alto Amazonas), localidades con transmisión de malaria. Con estos mosquitos se efectuaron crías biológicas en laboratorio con la finalidad de obtener material de estudio como son exuvias de larvas, pupas, adultos hembras y machos. Se efectuaron montajes de exuvias de larvas y pupas asociados con sus respectivos adultos, disecciones de las genitalias de machos y biometría de los adultos machos y hembras. Resultados: Además de las verificaciones efectuadas en el campo, basadas en el estudio de inmaduros y adultos, las crías biológicas efectuadas confirmaron la presencia de An. benarrochi en las localidades mencionadas. Asimismo, confirmaron también la existencia de gran variabilidad de los caracteres morfológicos externos, que pueden inducir al error en las identificaciones. Conclusiones: En las áreas maláricas estudiadas, la especie identificada como An. (Nys.) evansae corresponde a An. (Nys. ) benarrochi, demostrado con nuestros estudios. Dada la variabilidad de caracteres que dificultan la identificación de las especies afines, es necesario realizar estudios completos con la finalidad de conocer la fauna local de anofelinos.

Objective: Determine the presence of An. (Nys.) benarrochi in those malaria transmission areas where it is often identified as An. evansae. Material and methods: Collections were conducted in San José (Pucallpa), Muniches and Andoas (Alto Amazonas), localities where malaria transmission occurs. Mosquitoes were reared in the laboratory where larval and pupal exuviae were preserved along with corresponding adults specimens, dissection of male genitalia and biometry of male and female adult mosquitoes was also conducted. Results: The presence of An. benarrochi in the above localities was confirmed with both field collected adults and laboratory-reared specimens. Considerable morphological variability was noted in the laboratory-reared specimens, which has likely contributed to previous incorrect identifications. Conclusions: Based on these studies, the species identified as An. (Nys.) evansae correspond to An. (Nys.) benarrochi, in these malaria endemic areas. Morphological variability makes identification of similar species difficult, thus it is necessary to conduct thorough studies to know local fauna of anophelines.

Characterization of spotted fever group rickettsiae in flea and tick specimens from northern Peru.

Evidence of spotted fever group (SFG) rickettsiae was obtained from flea pools and individual ticks collected at three sites in northwestern Peru within the focus of an outbreak of febrile disease in humans attributed, in part, to SFG rickettsia infections. Molecular identification of the etiologic agents from these samples was determined after partial sequencing of the 17-kDa common antigen gene (htrA) as well as pairwise nucleotide sequence homology with one or more of the following genes: gltA, ompA, and ompB. Amplification and sequencing of portions of the htrA and ompA genes in pooled samples (2 of 59) taken from fleas identified the pathogen Rickettsia felis. Four tick samples yielded molecular evidence of SFG rickettsiae. Fragments of the ompA (540-bp) and ompB (2,484-bp) genes were amplified from a single Amblyomma maculatum tick (tick 124) and an Ixodes boliviensis tick (tick 163). The phylogenetic relationships between the rickettsiae in these samples and other rickettsiae were determined after comparison of their ompB sequences by the neighbor-joining method. The dendrograms generated showed that the isolates exhibited close homology (97%) to R. aeschlimannii and R. rhipicephali. Significant bootstrap values supported clustering adjacent to this nodule of the SFG rickettsiae. While the agents identified in the flea and tick samples have not been linked to human cases in the area, these results demonstrate for the first time that at least two SFG rickettsia agents were circulating in northern Peru at the time of the outbreak. Furthermore, molecular analysis of sequences derived from the two separate species of hard ticks identified a possibly novel member of the SFG rickettsiae.

Natural Plasmodium infections in Anopheles darlingi and Anopheles benarrochi (Diptera: Culicidae) from eastern Peru.

Malaria, both Plasmodium falciparum (Welch) and Plasmodium vivax (Grassi & Feletti), has reemerged as a significant public health disease issue in Peru, especially in forested areas in the eastern part of the country. The spread of Anopheles darlingi Root, the principal South American malaria vector, into new areas of Peru is thought to be a factor in this resurgence. However, epidemiological evidence suggests that in malaria endemic areas of eastern Peru where An. darlingi does not occur, other species are involved in malaria transmission. The objective of this study was to analyze Anopheles species collected from 11 provinces within four departments in eastern Peru during 2001 and 2002 for infections with P. falciparum and P. vivax. More than 84,000 Anopheles mosquitoes representing 13 species were tested by enzyme-linked immunosorbent assay for the presence of Plasmodium circumsporozoite (CS) proteins. Of these, only An. darlingi and Anopheles benarrochi Gabaldón, Cova García & López were found positive. In total, 14 (0.98%) of 1,432 pools of An. darlingi were positive for Plasmodium species; specifically 10 (0.70%) pools were positive for P. falciparum, two (0.14%) were positive for P. vivax VK210, and two (0.14%) were positive for P. vivax VK247 proteins. Nine (0.14%) of 6,323 pools of An. benarrochi were positive for Plasmodium; five (0.08%) of 6,323 pools were positive for P. falciparum, two (0.03%) were positive for P. vivax VK247, one (0.02%) was positive for mixed P. vivax VK210/VK247 infections, and one (0.02%) was positive for mixed P. falciparum and P. vivax VK210 CS-proteins. Although infection rates in An. benarrochi were significantly lower (0.14%) than rates found for An. darlingi (0.98%), our data suggest that An. benarrochi may play a role in transmitting and maintaining Plasmodium species in various malaria endemic areas of eastern Peru.

Evidence of rickettsial and leptospira infections in Andean northern Peru.

Between May and October 2002, a cluster of acute febrile illnesses occurred in the subtropical Andean foothills of Peru. Serologic evidence in villages where disease had been documented showed that the prevalence of IgM antibody to Leptospira ranged from 6% to 52%, that of IgM antibody to spotted fever group (SFG) rickettsia ranged from 10% to 19%, and that of IgM antibody to Coxiella burnetii from 1% to 15%. Measurement of IgG antibodies for SFG rickettsiae suggested that this disease was endemic. In contrast, IgG antibodies against C. burnetii were largely absent. In humans, microagglutination tests identified pathogenic variants of Leptospira. The presence of an SFG rickettsial infection was confirmed in four febrile patients following polymerase chain reaction and sequencing of the conserved 17-kD common antigen gene (htrA). Collectively, these analyses indicated that Rickettsia sp., C. burnetii, and Leptospira sp. were circulating in the region during the time of disease outbreak and implicate the involvement of an as yet undetermined SFG rickettsia in northwestern Peru.

Evaluation of surveillance devices for monitoring Aedes aegypti in an urban area of northeastern Peru.

In this study, we assessed the efficacy of the American Biophysics Corporation Standard Professional (ABC-PRO) light trap, the Omni-Directional Fay-Prince trap (with and without CO2), and the Centers for Disease Control and Prevention Wilton trap as a means of evaluating populations of adult Aedes aegypti in an urban area of northeastern Peru. Efficacies of collections from each of the trap types were compared to backpack-aspirator collections and human-landing collections. Collections were conducted twice daily, 3 days per week, for 27 wk from July 2001 to July 2002. Backpack-aspirator collections yielded significantly more mosquitoes (1,764) than any of the other collecting methods with a mean of 21.80 mosquitoes collected per sampling period. This method was less specific for Ae. aegypti than were human-landing collections because only 28.3% of mosquitoes collected with backpack aspirators were Ae. aegypti. Human-landing collections yielded only 23% (554/2,411) of the total mosquitoes collected. However, more than 80% (445/554) of the mosquitoes collected by this method were Ae. aegypti. None of the trapping devices evaluated collected mosquitoes, specifically Ae. aegypti, as effectively as backpack-aspirator or human-landing collections. The ABC-PRO trap, which was the most effective device in collecting mosquitoes, particularly Ae. aegypti, collected less than 2% of the total mosquitoes (mean of 0.12 mosquitoes/sampling period), and less than 3% of total Ae. aegypti (mean of 0.11 Ae. aegypti/sampling period). We conclude that none of the trap devices evaluated in this study is an acceptable alternative to backpack-aspirator or human-landing collections for monitoring populations of adult Ae. aegypti in Peru.

Geographical distribution of Anopheles darlingi in the Amazon Basin region of Peru.

Malaria has reemerged as a significant public health disease threat in Peru, especially within the Amazon Basin region. This resurgence of human cases caused by infection with Plasmodium falciparum and Plasmodium vivax is thought to be associated with the spread of Anopheles darlingi, the principal South American malaria vector, into new areas of the Amazon Basin. However, comprehensive studies of the distribution for this species have not been conducted in Peru for several years, nor are historical accounts accurate enough to determine if An. darlingi was actually present and not collected or misidentified. Therefore, the objective of this study is to define the distribution of An. darlingi as well as obtain data on distribution and abundance of other Anopheles species in this region. Mosquitoes were collected during 2001 in the Departments of Loreto and Ucayali, the two largest Amazonian Departments of Peru. A total of 60,585 specimens representing 12 species of the subgenera Nyssorhynchus and Anopheles were collected at 82 (88.2%) of 93 collecting sites. The majority of mosquitoes obtained were identified as An. benarrochi, comprising 70.7% of mosquitoes collected, followed by An. darlingi (24.0%), Anopheles mattogrosensis (2.4%), and Anopheles triannulatus (1.5%). Anopheles darlingi was collected from 48.8% of sites, indicating that this species is established throughout central Loreto, including further west in the Amazon Basin than previously reported. These data suggest that this species is now found in areas of the Amazon Basin region where it has not been previously reported.