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.

Molecular epidemiology of dengue virus type 3 in Northern South America: 2000--2005.

A phylogenetic approach was used to identify genetic variants of DENV-3 subtype III that may have emerged during or after its expansion throughout South America. We sequenced the capsid, premembrane/membrane and envelope genes from 22 DENV-3 strains isolated from Venezuela, Bolivia, Ecuador and Peru between 2000 and 2005. Phylogenetic analysis showed that the isolates sequenced in this study formed three clades within subtype III: one with the isolates from Venezuela, one with the Bolivian isolates and one with the isolates from Ecuador and Peru.

Full-genome amplification and sequencing of Zika viruses using a targeted amplification approach.

We have developed methods for full-genome sequencing of Zika viruses (ZIKVs) based on a targeted amplification approach. We used alignments of publicly available complete genome data to design a primer set that selectively amplifies ZIKVs. The approach includes amplification strategies for templates present at both high- and low-copy number, and PCR cycling conditions that have been normalized across genome fragments in order to streamline laboratory handling. Abundant templates can be amplified using a strategy that uses 6 overlapping amplicons to cover the complete viral genome, whereas scarce templates can be amplified using a strategy that uses 11 overlapping amplicons of smaller size. The workflow is sequencing platform agnostic, and thus, can be used in low resource settings where access to traditional Sanger sequencing is the only option available. Given the scarcity of tools for ZIKV, this approach should facilitate epidemiological surveillance and other studies that require the generation of complete viral genomic information quickly and cost-effectively.

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.

Hantavirus pulmonary syndrome in Central Bolivia: relationships between reservoir hosts, habitats, and viral genotypes.

In August 2002, two cases of hantavirus pulmonary syndrome (HPS) were confirmed in Mineros and Concepcion, within the Santa Cruz Department of Bolivia. Extensive alteration of the native ecosystem, from dense forest to pasture or sugarcane, had occurred in both regions. An ecologic assessment of reservoir species associated with the human disease identified a single hantavirus antibody-positive Oligoryzomys microtis from Mineros and three hantavirus antibody-positive Calomys callosus from Concepcion. In Mineros, the virus from the O. microtis was 90% similar to sequences published for Rio Mamore virus. Viral nucleotide sequences from two C. callosus were 87-88% similar to the sequence of Laguna Negra virus. The viral sequence from the C. callosus was 99% identical to viral sequences obtained from the HPS patient in this area, implicating C. callosus as the host and Laguna Negra virus as the agent responsible for the HPS case near Concepcion.

Influenza-like illness sentinel surveillance in one hospital in Medellin, Colombia. 2007-2012.

BACKGROUND: The city of Medellin in Colombia has almost no documentation of the causes of acute respiratory infections (ARIs). As part of an ongoing collaboration, we conducted an epidemiologic surveillance for influenza and other respiratory viruses. It described the influenza strains that were circulating in the region along with their distribution over time, and performing molecular characterization to some of those strains. This will contribute to the knowledge of local and national epidemiology. OBJECTIVES: To analyze viral etiologic agents associated with influenza like illness (ILI) in participants reporting to one General hospital in Medelllin, Colombia. RESULTS: From January 2007 to December 2012, a total of 2039 participants were enrolled. Among them, 1120 (54.9%) were male and 1364 (69%) were under the age of five. Only 124 (6%) were older than the age of 15. From all 2039 participants, 1040 samples were diagnosed by either isolation or RT-PCR. One or more respiratory viruses were found in 737 (36%) participants. Of those, 426 (57.8%) got influenza A or B. Adenoviral and parainfluenza infections represented 19.1% and 14.9% of viral infections, respectively. Influenza A was detected almost throughout the whole year except for the first quarter of 2010, right after the 2009 influenza A pandemic. Influenza B was detected in 2008, 2010, and 2012 with no pattern detected. During 2008 and 2010, both types circulated in about the same proportion. Unusually, in many months of 2012, the proportion of influenza B infections was higher than influenza A (ranging between 30% and 42%). The higher proportion of adenovirus was mainly detected in the last quarter of years 2007 and 2010. Adenoviral cases are more frequent in participants under the age of four. CONCLUSIONS: The phylogenetic analysis of influenza viruses shows that only in the case of influenza A/H1N1, the circulating strains totally coincide with the vaccine strains each year.

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.

Detection of Rickettsia parkeri from within Piura, Peru, and the first reported presence of Candidatus Rickettsia andeanae in the tick Rhipicephalus sanguineus.

Domestic farm animals (n=145) were sampled for the presence of ectoparasites in northwestern Peru during March, 2008. Ninety domestic animals (62%) were positive for the presence of an ectoparasite(s) and produced a total collection of the following: 728 ticks [Amblyomma maculatum, Anocentor nitens, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus, and Otobius megnini], 12 lice (Haematopinus suis), and 3 fleas (Ctenocephalides felis). A Rickettsia genus-specific qPCR assay was performed on nucleic acid preparations of the collected ectoparasites that resulted in 5% (37/743, 35 ticks and 2 fleas) of the ectoparasites positive for the presence of Rickettsia. DNA from the positive individual ticks was tested with 2 other qPCR assays for the presence of the ompB gene in Candidatus Rickettsia andeanae or Rickettsia parkeri. Candidatus R. andeanae was found in 25 A. maculatum ticks and in two Rh. sanguineus ticks, whereas R. parkeri was detected in 6 A. maculatum ticks. Two A. maculatum were co-infected with both Candidatus R. andeanae and R. parkeri. Rickettsia felis was detected in 2 fleas, Ctenocephalides felis, by multilocus sequence typing of the 17-kD antigen and ompA genes. These findings expand the geographic range of R. parkeri to include Peru as well as expand the natural arthropod vector of Candidatus R. andeanae to include Rhipicephalus sanguineus.

Hantavirus pulmonary syndrome in Santa Cruz, Bolivia: outbreak investigation and antibody prevalence study.

We report the results of an investigation of a small outbreak of hantavirus pulmonary syndrome in 2002 in the Department of Santa Cruz, Bolivia, where the disease had not previously been reported. Two cases were initially reported. The first case was a physician infected with Laguna Negra virus during a weekend visit to his ranch. Four other persons living on the ranch were IgM antibody-positive, two of whom were symptomatic for mild hantavirus pulmonary syndrome. The second case was a migrant sugarcane worker. Although no sample remained to determine the specific infecting hantavirus, a virus 90% homologous with Río Mamoré virus was previously found in small-eared pygmy rice rats (Oligoryzomys microtis) trapped in the area. An antibody prevalence study conducted in the region as part of the outbreak investigation showed 45 (9.1%) of 494 persons to be IgG positive, illustrating that hantavirus infection is common in Santa Cruz Department. Precipitation in the months preceding the outbreak was particularly heavy in comparison to other years, suggesting a possible climatic or ecological influence on rodent populations and risk of hantavirus transmission to humans. Hantavirus infection appears to be common in the Santa Cruz Department, but more comprehensive surveillance and field studies are needed to fully understand the epidemiology and risk to humans.

Genetic characterization of Venezuelan equine encephalitis virus from Bolivia, Ecuador and Peru: identification of a new subtype ID lineage.

Venezuelan equine encephalitis virus (VEEV) has been responsible for hundreds of thousands of human and equine cases of severe disease in the Americas. A passive surveillance study was conducted in Peru, Bolivia and Ecuador to determine the arboviral etiology of febrile illness. Patients with suspected viral-associated, acute, undifferentiated febrile illness of <7 days duration were enrolled in the study and blood samples were obtained from each patient and assayed by virus isolation. Demographic and clinical information from each patient was also obtained at the time of voluntary enrollment. In 2005-2007, cases of Venezuelan equine encephalitis (VEE) were diagnosed for the first time in residents of Bolivia; the patients did not report traveling, suggesting endemic circulation of VEEV in Bolivia. In 2001 and 2003, VEE cases were also identified in Ecuador. Since 1993, VEEV has been continuously isolated from patients in Loreto, Peru, and more recently (2005), in Madre de Dios, Peru. We performed phylogenetic analyses with VEEV from Bolivia, Ecuador and Peru and compared their relationships to strains from other parts of South America. We found that VEEV subtype ID Panama/Peru genotype is the predominant one circulating in Peru. We also demonstrated that VEEV subtype ID strains circulating in Ecuador belong to the Colombia/Venezuela genotype and VEEV from Madre de Dios, Peru and Cochabamba, Bolivia belong to a new ID genotype. In summary, we identified a new major lineage of enzootic VEEV subtype ID, information that could aid in the understanding of the emergence and evolution of VEEV in South America.

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.

Effect of dengue-1 antibodies on American dengue-2 viral infection and dengue haemorrhagic fever.

In Iquitos, Peru, no cases of dengue haemorrhagic fever have been recorded in individuals infected with dengue-1 virus followed by American genotype dengue-2 (American dengue-2) virus. We assayed serum samples collected in Iquitos that tested positive for antibodies of monotype dengue-1 and monotype dengue-2 using a plaque reduction neutralisation test to determine their ability to neutralise the infectivity of two dengue-1 viruses, two American dengue-2 viruses, and two Asian dengue-2 viruses. Sera positive for the dengue-1 antibody neutralised dengue-1 viruses and American dengue-2 viruses much more effectively than Asian dengue-2 viruses. Neutralisation of American dengue-2 virus by sera positive for dengue-1 antibodies may account for the absence of dengue haemorrhagic fever in individuals infected with dengue-1 in 1990-91 followed by American dengue-2 virus in 1995 in Iquitos, Peru.