Cristina Domingo

West Nile virus in Spain : Report of the first diagnosed case (in Spain) in a human with aseptic meningitis

We report the first case of illness caused by West Nile virus (WNV) so far diagnosed in Spain. A 21-y-old male presented with clinical and biological signs compatible with viral meningitis. Acute and convalescent serum samples showed IgM and IgG positivity for WNV. These results were confirmed by microneutralization assays.

Use of a Short Fragment of the C-Terminal E Gene for Detection and Characterization of Two New Lineages of Dengue Virus 1 in India

ABSTRACT Here we propose the use of a 216-nucleotide fragment located in the carboxyl terminus of the E gene (E-COOH) and a pairwise-based comparison method for genotyping of dengue virus 1 (DENV-1) strains. We have applied this method to the detection and characterization of DENV-1 in serum samples from travelers returning from the tropics. The results obtained with the typing system correlate with the results obtained by comparison of the sequences of the entire E gene of the strains. The approach demonstrates utility in plotting the distribution and circulation of different genotypes of DENV-1 and also suggests the presence of two new clades of Indian strains. The integration of the method with an online database and a typing characterization tool enhances its strength. Additionally, the analysis of the complete E gene of DENV-1 strains suggested the occurrence of a nondescribed recombination event in the China GD23-95 strain. We propose the use of this methodology as a tool for real-time epidemiological surveillance of dengue virus infections and their pathogenesis.

Immune Response during Adverse Events after 17D-Derived Yellow Fever Vaccination in Europe

BACKGROUND In 1999-2000, reports of fatalities after vaccination with 17D-derived yellow fever vaccine (YEL) focused mainly on cases of YEL-associated adverse events (YEL-AEs) and YEL-associated viscerotropic disease (YEL-AVD). Here, we investigated 6 recent European cases to provide insight regarding immune response involvement and to identify potential risk factors. METHODS Clinical, microbiological, molecular biological, and immunological assays were performed on serum from 6 patients with YEL-AEs, including 5 with YEL-AVD and 1 with YEL-associated neurotropic disease (YEL-AND). RESULTS The levels of 3 liver enzymes associated with infection were clearly increased in all patients with YEL-AVD, but no elevations were observed in the patient with YEL-AND. In the patients with severe YEL-AVD, platelet counts were markedly reduced (< 100,000 cells/microL). The only patient with fatal YEL-AVD exhibited a cytokine profile comparable to that seen in YF: high levels of interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, monokine induced by interferon-gamma, and growth-related oncogene (GRO). The other patients with YEL-AVD exhibited similar but less severe cytokine profiles. The patient with YEL-AND exhibited a cytokine profile similar to that found in vaccinees without YEL-AEs: elevated levels of RANTES and low levels of GRO, MCP-1, transforming growth factor-beta1, and tumor necrosis factor-beta. CONCLUSIONS On the basis of these results, we conclude that elevations in cytokine levels and reductions in platelet counts are suitable surrogate markers for patients likely to experience severe adverse reactions to YEL.

Human West Nile Virus Infection, Catalonia, Spain

To the Editor: West Nile virus (WNV) is a mosquitoborne flavivirus that is widespread in Africa, the Middle East, Asia, and southern Europe, where it causes outbreaks and sporadic cases of the disease. It has become an emergent disease in North America, where it was detected for the first time in 1999 and became epidemic shortly thereafter (1). Although WNV was initially considered to have a minor health effect in the Mediterranean basin, human and equine outbreaks reported in the last decade in different countries (2–5) have made WNV infections a public health concern. The epidemiology of WNV in Europe differs from that in America and has only been associated with nonrecurrent, sporadic outbreaks. The reasons for this difference are controversial; it may be due to environmental factors, reservoirs, or even mosquito vectors. In Spain, neither equine nor human WNV cases have been reported. However, some human serosurveys that used hemagglutination inhibition suggested that WNV or closely related flaviviruses circulated during the 1970s in the Ebro delta and areas in Spain (6,7). The Ebro delta, a wetland in Catalonia, in the northeast of Spain, is a stopping-off point for birds migrating between regions of Africa and Europe where different WNV vectors and reservoirs have been identified. The delta could be considered a high-risk area for WNV and other arthropodborne virus infections. To evaluate WNV seroprevalence in the human population of the Ebro delta, a survey was conducted in 2001. After obtaining informed consent, 992 serum samples were obtained from inhabitants of the area. The population studied was representative of the whole area and was stratified by sex and age. Anti-WNV immunoglobulin G (IgG) antibodies were determined by using an in-house indirect enzyme-linked immunosorbent assay (ELISA), as previously described (8). Results were classified as the sample absorbance/positive control absorbance ratio. Samples showing ratio values >0.2 were tested for WNV IgG and IgM by using an indirect and a μ-chain capture ELISA, respectively (Focus Technologies, Cypress, CA, USA), and an in-house microneutralization test. For the microneutralization test, samples were tested in duplicate and assayed twice. Twofold dilutions (25 μL) of the samples (1:16–1:256 dilutions) were assayed by using 100 TCID50 (50% tissue culture infectious dose) of West Nile Eg-101 reference strain in 96-well tissue culture plates with Vero cells and after 7 days of incubation at 37°C and 5% CO2. Thirty-eight samples showed IgG ratios >0.2 by the in-house ELISA. Of these, 12 showed WNV IgG, and 1 was positive for WNV IgM and IgG, according to the Focus assays. Two samples showed positive neutralizing activity, with titers of 32 and 256. The highest titer was shown by the sample that yielded positive levels of both IgM and IgG in the ELISA, which suggests recent WNV infection. Anti-WNV IgG was more often detected in participants in the 20- to 29-year age group (odds ratio [OR] 4.23, 95% confidence interval [CI] 1.04–16.02, p = 0.03) and in persons who reported frequent mosquito bites (OR 8.62, 95% CI 0.44–169, p = 0.08). IgG-positive persons were equally divided by sex. No significant differences were found between antibody-positive or antibody-negative persons with respect to their profession, place of occupation, current residence, time in current residence, outdoor activities, use of insecticides and repellents, or symptoms related to WNV infection. No symptoms related to WNV infection were reported by the IgM/IgG-positive participant, who was 31 years of age, was born in the area, worked outdoors, and was frequently bitten by mosquitoes. He also reported travel to Cuba 1 year earlier, but he had not been vaccinated against flavivirus, and serologic test results for dengue were negative. The other IgG- and neutralizing antibody–positive participant was 45 years of age and was born and works in the area. He had never traveled abroad or been vaccinated against flavivirus. He reported a 4-day fever of unknown origin during the summer 1 or 2 years before the study. He often fishes in the areas and is frequently bitten by mosquitoes. In conclusion, the study found evidence of recent WNV infections in humans living in the Ebro delta, where previous flavivirus circulation has been suggested by Lozano and Filipe (6). IgG-positive results not confirmed by neutralization could be due to cross-reactive antibodies induced by other flavivirus infections or vaccinations (9,10). The probable WNV infection described was asymptomatic, as occurs in ≈80% of cases. Other WNV infections in the area may have remained undetected, including neuroinvasive cases. Intensified research and surveillance in this area will help determine and refine thresholds for public health interventions.

Detection of yellow fever 17D genome in urine

Yellow fever (YF) remains an important public health problem in regions where the disease is endemic, with a dramatic upsurge in the number of cases in recent years. So far, extensive YF epizooties occurred in South America in 2008, and during the past year YF outbreaks arose in Cameroon, Democratic

2nd International External Quality Control Assessment for the Molecular Diagnosis of Dengue Infections

Background Currently dengue viruses (DENV) pose an increasing threat to over 2.5 billion people in over 100 tropical and sub-tropical countries worldwide. International air travel is facilitating rapid global movement of DENV, increasing the risk of severe dengue epidemics by introducing different serotypes. Accurate diagnosis is critical for early initiation of preventive measures. Different reverse transcriptase PCR (RT-PCR) methods are available, which should be evaluated and standardized. Epidemiological and laboratory-based surveillance is required to monitor and guide dengue prevention and control programmes, i.e., by mosquito control or possible vaccination (as soon as an effective and safe vaccine becomes available). Objective The purpose of the external quality assurance (EQA) study described is to assess the efficiency and accuracy of dengue molecular diagnosis methods applied by expert laboratories. Study Design A panel of 12 human plasma samples was distributed and tested for DENV-specific RNA. The panel comprised 9 samples spiked with different DENV serotypes (DENV-1 to DENV-4), including 10-fold dilution series of DENV-1 and DENV-3. Two specificity controls consisted of a sample with a pool of 4 other flaviviruses and a sample with chikungunya virus. A negative control sample was also included. Results Thirty-seven laboratories (from Europe, Middle East Asia, Asia, the Americas/Caribbean, and Africa) participated in this EQA study, and reports including 46 sets of results were returned. Performance among laboratories varied according to methodologies used. Only 5 (10.9%) data sets met all criteria with optimal performance, and 4 (8.7%) with acceptable performance, while 37 (80.4%) reported results showed the need for improvement regarding accomplishment of dengue molecular diagnosis. Failures were mainly due to lack of sensitivity and the presence of false positives. Conclusions The EQA provides information on each laboratorys efficacy of RT-PCR techniques for dengue diagnosis and indicates for most laboratories an urgent need to improve sensitivity and specificity.

West Nile virus past infections in the general population of Southern Spain

OBJECTIVE To analyze the prevalence of past and recent infections by West Nile virus (WNV) and the risk factors associated with WNV exposure in a representative population from southern Spain. METHODS Sample size was established for an estimated prevalence of past WNV infections of 5 +/- 2.5% in 504 subjects. A pre-stratification was performed according to age distribution and place of residence. After random telephone solicitation and acquisition of informed consent, a serum sample was collected and an epidemiologic survey performed on all participating subjects. Samples were tested with ELISA-IgG and MAC-ELISA to detect specific IgG and IgM antibodies; results were confirmed by the plaque reduction neutralization test (PRNT). Multivariate analysis using a forward stepwise logistic regression model was performed to assess potential risk factors associated with WNV exposure. RESULTS Prevalence of past WNV infections confirmed by PRNT in the 504 participants was 0.6%, affecting mainly older persons (mean age 65 +/- 23 vs. 34 +/- 22 years; P = 0.018), those living in rural areas (5.4% vs. 0% in urban areas; P = 0.01), and individuals with risk professions (prevalence 2.8% vs. 0%; P = 0.048). None of the five recent infections detected by MAC-ELISA was confirmed by PRNT. CONCLUSIONS These results strongly suggest past circulation and exposure of the human population to WNV in southern Spain.

Phylogenetic history demonstrates two different lineages of dengue type 1 virus in Colombia.

BackgroundDengue Fever is one of the most important viral re-emergent diseases affecting about 50 million people around the world especially in tropical and sub-tropical countries. In Colombia, the virus was first detected in the earliest 70s when the disease became a major public health concern. Since then, all four serotypes of the virus have been reported. Although most of the huge outbreaks reported in this country have involved dengue virus serotype 1 (DENV-1), there are not studies about its origin, genetic diversity and distribution.ResultsWe used 224 bp corresponding to the carboxyl terminus of envelope (E) gene from 74 Colombian isolates in order to reconstruct phylogenetic relationships and to estimate time divergences. Analyzed DENV-1 Colombian isolates belonged to the formerly defined genotype V. Only one virus isolate was clasified in the genotype I, likely representing a sole introduction that did not spread. The oldest strains were closely related to those detected for the first time in America in 1977 from the Caribbean and were detected for two years until their disappearance about six years later. Around 1987, a split up generated 2 lineages that have been evolving separately, although not major aminoacid changes in the analyzed region were found.ConclusionDENV-1 has been circulating since 1978 in Colombia. Yet, the phylogenetic relationships between strains isolated along the covered period of time suggests that viral strains detected in some years, although belonging to the same genotype V, have different recent origins corresponding to multiple re-introduction events of viral strains that were circulating in neighbor countries. Viral strains used in the present study did not form a monophyletic group, which is evidence of a polyphyletic origin. We report the rapid spread patterns and high evolution rate of the different DENV-1 lineages.

Adverse events following yellow fever preventive vaccination campaigns in eight African countries from 2007 to 2010

BACKGROUND Serious, but rare adverse events following immunization (AEFI) have been reported with yellow fever (YF) 17D vaccine, including severe allergic reactions, YF vaccine-associated neurologic disease (YEL-AND) and YF vaccine-associated viscerotropic disease (YEL-AVD). The frequency with which YEL-AND and YEL-AVD occur in YF endemic countries is mostly unknown. METHODS From 2007 to 2010, eight African countries - Benin, Cameroon, Guinea, Liberia, Mali, Senegal, Sierra Leone, and Togo- implemented large-scale YF preventive vaccination campaigns. Each country established vaccine pharmacovigilance systems that included standard case definitions, procedures to collect and transport biological specimens, and National Expert Committees to review data and classify cases. Staff in all countries received training and laboratory capacity expanded. RESULTS In total, just over 38 million people were vaccinated against YF and 3116 AEFIs were reported of which 164 (5%) were classified as serious. Of these, 22 (13%) were classified as YF vaccine reactions, including 11 (50%) hypersensitivity reactions, six (27%) suspected YEL-AND, and five (23%) suspected YEL-AVD. The incidence per 100,000 vaccine doses administered was 8.2 for all reported AEFIs, 0.43 for any serious AEFI, 0.058 for YF vaccine related AEFIs, 0.029 for hypersensitivity reactions, 0.016 for YEL-AND, and 0.013 for YEL-AVD. Our findings were limited by operational challenges, including difficulties in obtaining recommended biological specimens leading to incomplete laboratory evaluation, unknown case ascertainment, and variable levels of staff training and experience. CONCLUSIONS Despite limitations, active case-finding in the eight different countries did not find an incidence of YF vaccine associated AEFIs that was higher than previous reports. These data reinforce the safety profile of YF vaccine and support the continued use of attenuated YF vaccine during preventive mass vaccination campaigns in YF endemic areas.

Advanced yellow fever virus genome detection in point-of-care facilities and reference laboratories

ABSTRACT Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.