Constanza Martínez-Valdebenito

Person-to-person household and nosocomial transmission of andes hantavirus, Southern Chile, 2011.

Four persons became ill after exposure to a patient infected with the virus; 2 cases involved hospital transmission.

Rickettsia felis in Rhipicephalus sanguineus from Two Distant Chilean Cities

INTRODUCTION Rickettsia felis is an emerging agent considered a human threat; although its natural reservoir and agent of transmission is the cat flea, it has been also found in other vectors. R. felis has been identified in Chile in cat fleas and in one specimen of Rhipicephalus sanguineus collected in the Metropolitan Region. The objective of this study was to detect the presence of Rickettsia spp. in R. sanguineus from dogs of two different and distant geographical areas in Chile. MATERIAL AND METHODS We performed a domiciliary sampling in urban and rural localities of two distant areas of the country-the Metropolitan Region in the center and the northern city of Arica. A total of 460 households were visited; one dog per household was included in the study and ectoparasites were collected from them. RESULTS R. sanguineus was found in 50% of the 460 dogs. R. felis was identified by amplification and sequencing of gltA, ompA, and ompB genes in R. sanguineus from both regions, with predominance in Arica. DISCUSSION The presence of R. felis in R. sanguineus from two distant regions of Chile suggests that this rickettsial agent is well established in the country. Considering that no human spotted fever group infections have been recognized in the country, the results should alert clinicians about such possible cases. The role of R. sanguineus in the epidemiology and transmission of R. felis should be further investigated.

Development and Characterization of a Highly Specific and Sensitive SYBR Green Reverse Transcriptase PCR Assay for Detection of the 2009 Pandemic H1N1 Influenza Virus on the Basis of Sequence Signatures

ABSTRACT The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.

Association of Single-Nucleotide Polymorphisms in IL28B, but Not TNF-α, With Severity of Disease Caused by Andes Virus

BACKGROUND Andes virus (ANDV) is the sole etiologic agent of hantavirus cardiopulmonary syndrome (HCPS) in Chile, with a fatality rate of about 35%. Individual host factors affecting ANDV infection outcome are poorly understood. In this case-control genetic association analysis, we explored the link between single-nucleotide polymorphisms (SNPs) rs12979860, rs8099917 and rs1800629 and the clinical outcome of ANDV-induced disease. The SNPs rs12979860 and rs8099917 are known to play a role in the differential expression of the interleukin 28B gene (IL28B), whereas SNP rs1800629 is implicated in the expression of tumor necrosis factor α gene (TNF-α). METHODS A total of 238 samples from confirmed ANDV-infected patients collected between 2006 and 2014, and categorized according to the severity of the disease, were genotyped for SNPs rs12979860, rs8099917, and rs1800629. RESULTS Analysis of IL28B SNPs rs12979860 and rs8099917 revealed a link between homozygosity of the minor alleles (TT and GG, respectively), displaying a mild disease progression, whereas heterozygosity or homozygosity for the major alleles (CT/CC and TG/TT, respectively) in both IL28B SNPs is associated with severe disease. No association with the clinical outcome of HCPS was observed for TNF-α SNP rs1800629 (TNF -308G>A). CONCLUSIONS The IL28B SNPs rs12979860 and rs8099917, but not TNF-α SNP rs1800629, are associated with the clinical outcome of ANDV-induced disease, suggesting a possible link between IL28B expression and ANDV pathogenesis.

A rapid method for infectivity titration of Andes hantavirus using flow cytometry

The focus assay is currently the most commonly used technique for hantavirus titer determination. This method requires an incubation time of between 5 and 11 days to allow the appearance of foci after several rounds of viral infection. The following work presents a rapid Andes virus (ANDV) titration assay, based on viral nucleocapsid protein (N) detection in infected cells by flow cytometry. To this end, an anti-N monoclonal antibody was used that was developed and characterized previously. ANDV N could be detected as early as 6 h post-infection, while viral release was not observed until 24-48 h post-infection. Given that ANDV detection was performed during its first round of infection, a time reduction for titer determination was possible and provided results in only two days. The viral titer was calculated from the percentage of N positive cells and agreed with focus assay titers. Furthermore, the assay was applied to quantify the inhibition of ANDV cell entry by patient sera and by preventing endosome acidification. This novel hantavirus titration assay is a highly quantitative and sensitive tool that facilitates infectivity titration of virus stocks, rapid screening for antiviral drugs, and may be further used to detect and quantify infectious virus in human samples.

Fleas and ticks in carnivores from a domestic-wildlife interface: Implications for public health and wildlife

Abstract Fleas and ticks are parasites of wild and domestic mammals, and can be vectors of several pathogens. In rural areas, domestic carnivores such as the domestic dog (Canis lupus familiaris L.), may act as a “bridge” between natural areas and human settlements where ectoparasites can be used as a metric of such link. The aim of this study was to identify fleas, ticks, and Rickettsia spp., collected from domestic and wild carnivores in a natural reserve and surrounding human settlements in Central Chile, using morphological keys and molecular analysis. We surveyed 170 households from which 107 dogs and eight cats were sampled. From the natural reserve, we sampled two chilla foxes (Pseudalopex griseus Gray), two lesser grison (Galictis cuja Molina), three kodkods (Leopardus guigna Molina), and four dogs. From dogs, we collected Ctenocephalides felis Bouché, Ctenocephalides canis Curtis, Pulex irritans L., and Rhipicephalus sanguineus s.l. Latreille; C. felis was the most frequent ectoparasite. Cats were infested only by C. felis and Rh. sanguineus s.l. From wild carnivores, we obtained C. canis and P. irritans, the latter being most frequent. Molecular analysis of P. irritans detected 10 haplotypes and two main clades, which tended to separate fleas from wild and domestic hosts. Molecular analysis of ompA and ompB genes confirmed the presence of Rickettsia felis in fleas collected from owned dogs and cats, which could represent a potential risk factor of R. felis transmission in the area.

Molecular method for the detection of Andes hantavirus infection: validation for clinical diagnostics

Hantavirus cardiopulmonary syndrome is a severe disease caused by exposure to New World hantaviruses. Early diagnosis is difficult due to the lack of specific initial symptoms. Antihantavirus antibodies are usually negative until late in the febrile prodrome or the beginning of cardiopulmonary phase, while Andes hantavirus (ANDV) RNA genome can be detected before symptoms onset. We analyzed the effectiveness of quantitative reverse transcription polymerase chain reaction (RT-qPCR) as a diagnostic tool detecting ANDV-Sout genome in peripheral blood cells from 78 confirmed hantavirus patients and 166 negative controls. Our results indicate that RT-qPCR had a low detection limit (~10 copies), with a specificity of 100% and a sensitivity of 94.9%. This suggests the potential for establishing RT-qPCR as the assay of choice for early diagnosis, promoting early effective care of patients, and improving other important aspects of ANDV infection management, such as compliance of biosafety recommendations for health personnel in order to avoid nosocomial transmission.

Geographical distribution and phylogenetic analysis of Rhipicephalus sanguineus sensu lato in northern and central Chile

The presented study analyzed the presence and geographical distribution of the tropical and temperate lineages of Rhipicephalus sanguineus sensu lato in Chile. R. sanguineus s.l. ticks were collected from dogs at 14 sites in northern and central Chile for morphological and genetic analysis based on the 16S rDNA gene. Phylogenetic studies proved the existence of both, the tropical and the temperate lineages. The former was represented by a single haplotype and occurred in the far north; the latter included four haplotypes and was observed from the Tarapacá Region southwards. In four sites at latitudes from 20°S to 22°S, both lineages were found to coexist. Our study discovered for the first time the existence of the tropical lineage in Chile and demonstrated that distributions of the tropical and temperate lineages overlap, forming a transitional zone of approximately 200 km in northern coastal Chile.

Imported scrub typhus: first case in South America and review of the literature

BackgroundScrub typhus is a neglected vector-borne zoonosis causing life-threatening illnesses, endemic in the Asian-Pacific region and, as recently discovered, in southern Chile. Scrub typhus is rarely reported in travelers, most probably due to the lack of clinical experience and diagnostic tests in non-endemic countries. We report the first case of imported scrub typhus in South America.Case presentationA 62-year-old tourist from South Korea presented severely ill with fever, rash, and eschar in Santiago, Chile. Laboratory exams showed thrombocytopenia and elevated inflammation parameters, hepatic enzymes, and LDH. With the clinical suspicion of scrub typhus, empirical treatment with doxycycline was initiated and the patient recovered rapidly and without complications. The diagnosis was confirmed by IgM serology and by real-time PCR, which demonstrated infection with Orientia tsutsugamushi (Kawasaki clade).ConclusionsOnly due to the emerging clinical experience with endemic South American scrub typhus and the recent implementation of appropriate diagnostic techniques in Chile, were we able to firstly identify and adequately manage a severe case of imported scrub typhus in South America. Physicians attending febrile travelers need to be aware of this rickettsiosis, since it requires prompt treatment with doxycycline to avoid complications.

Parotiditis en Chile: caracterización clínica y molecular de dos casos en una población altamente inmunizada

Mumps virus usually produces a benign infection characterized by increased parotid volume which, prior to vaccination, mainly affected children and adolescents. After the introduction of measles, mumps and rubella (MMR) vaccine, mumps incidence decreased dramatically. This intervention also produced a change in its clinical presentation, moving to young adult patients, with an increased risk of complications. We report two clinical mumps cases in young adults with different clinical presentations. In both cases, serologic assays were assessed and, in one case, a polymerase chain reaction (PCR) was performed in order to confirm the diagnosis. The isolated virus was characterized and identifed as G genotype, the same genotype observed during outbreaks in United States and Europe, and different to the vaccinal strain. Mumps virus is currently circulating in Chile and it is important to be aware of possible outbreaks. Viral diagnosis can be difficult, particularly in populations with high vaccination coverage. Therefore, the access to etiologic study through PCR and serology becomes more relevant in order to optimize clinical management and secondary prevention measures.