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.

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.

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.

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.