Fatal acute undifferentiated febrile illness among clinically suspected leptospirosis cases in Colombia, 2016-2019.

BACKGROUND: Acute undifferentiated febrile illness is a common challenge for clinicians, especially in tropical and subtropical countries. Incorrect or delayed diagnosis of febrile patients may result in medical complications or preventable deaths. Common causes of acute undifferentiated febrile illness in Colombia include leptospirosis, rickettsioses, dengue fever, malaria, chikungunya, and Zika virus infection. In this study, we described the acute undifferentiated febrile illness in postmortem patients reported as suspected cases of leptospirosis through the national leptospirosis surveillance in Colombia, 2016-2019. METHODOLOGY/PRINCIPAL FINDINGS: We retrospectively analyze human fresh and formalin-fixed tissue samples from fatal suspected leptospirosis cases reported by the Public Health Laboratories in Colombia. Leptospirosis confirmation was made by immunohistochemistry, real-time polymerase chain reaction (PCR) in the tissue samples. In some cases, the serum sample was used for confirmation by Microagglutination test (MAT). Simultaneously, tissue samples were tested by PCR for the most common viral (dengue, Zika, and chikungunya), bacterial (Brucella spp., and Rickettsia spp.), and parasitic (malaria). Fresh tissue samples from 92 fatal suspected leptospirosis cases were reported to the National Reference Laboratory from 22/32 departments in Colombia. We confirmed leptospirosis in 27% (25/92) of cases. Other pathogens identified by real-time PCR were Brucella spp. (10.9%), Rickettsia spp. (14.1%), and dengue (2.2%). Dengue (6.9%), hepatitis (3.5%), and Yellow Fever cases (2.2%) were detected by the pathology. All patients were negative for chikungunya and Plasmodium spp. Most cases were classified as undifferentiated febrile illnesses (45.7%; 42/92). CONCLUSIONS/SIGNIFICANCE: This study underscores the importance of early and accurate recognition of leptospirosis to prevent mortalities. Moreover, it draws attention to the existence of other febrile syndromes in Colombia, including rickettsiosis and brucellosis, that currently lack sufficient human surveillance and regular reporting. Expanding laboratory surveillance to include viruses such as Hantavirus, Mayaro virus, Oropouche virus, and West Nile virus is crucial.

Pregnancy, Birth, Infant, and Early Childhood Neurodevelopmental Outcomes among a Cohort of Women with Symptoms of Zika Virus Disease during Pregnancy in Three Surveillance Sites, Project Vigilancia de Embarazadas con Zika (VEZ), Colombia, 2016-2018.

Project Vigilancia de Embarazadas con Zika (VEZ), an intensified surveillance of pregnant women with symptoms of the Zika virus disease (ZVD) in Colombia, aimed to evaluate the relationship between symptoms of ZVD during pregnancy and adverse pregnancy, birth, and infant outcomes and early childhood neurodevelopmental outcomes. During May-November 2016, pregnant women in three Colombian cities who were reported with symptoms of ZVD to the national surveillance system, or with symptoms of ZVD visiting participating clinics, were enrolled in Project VEZ. Data from maternal and pediatric (up to two years of age) medical records were abstracted. Available maternal specimens were tested for the presence of the Zika virus ribonucleic acid and/or anti-Zika virus immunoglobulin antibodies. Of 1213 enrolled pregnant women with symptoms of ZVD, 1180 had a known pregnancy outcome. Results of the Zika virus laboratory testing were available for 569 (48.2%) pregnancies with a known pregnancy outcome though testing timing varied and was often distal to the timing of symptoms; 254 (21.5% of the whole cohort; 44.6% of those with testing results) were confirmed or presumptive positive for the Zika virus infection. Of pregnancies with a known outcome, 50 (4.2%) fetuses/infants had Zika-associated brain or eye defects, which included microcephaly at birth. Early childhood adverse neurodevelopmental outcomes were more common among those with Zika-associated birth defects than among those without and more common among those with laboratory evidence of a Zika virus infection compared with the full cohort. The proportion of fetuses/infants with any Zika-associated brain or eye defect was consistent with the proportion seen in other studies. Enhancements to Colombia's existing national surveillance enabled the assessment of adverse outcomes associated with ZVD in pregnancy.

Zika Virus Disease and Pregnancy Outcomes in Colombia.

BACKGROUND: In 2015 and 2016, Colombia had a widespread outbreak of Zika virus. Data from two national population-based surveillance systems for symptomatic Zika virus disease (ZVD) and birth defects provided complementary information on the effect of the Zika virus outbreak on pregnancies and infant outcomes. METHODS: We collected national surveillance data regarding cases of pregnant women with ZVD that were reported during the period from June 2015 through July 2016. The presence of Zika virus RNA was identified in a subgroup of these women on real-time reverse-transcriptase-polymerase-chain-reaction (rRT-PCR) assay. Brain or eye defects in infants and fetuses and other adverse pregnancy outcomes were identified among the women who had laboratory-confirmed ZVD and for whom data were available regarding pregnancy outcomes. We compared the nationwide prevalence of brain and eye defects during the outbreak with the prevalence both before and after the outbreak period. RESULTS: Of 18,117 pregnant women with ZVD, the presence of Zika virus was confirmed in 5926 (33%) on rRT-PCR. Of the 5673 pregnancies with laboratory-confirmed ZVD for which outcomes had been reported, 93 infants or fetuses (2%) had brain or eye defects. The incidence of brain or eye defects was higher among pregnancies in which the mother had an onset of ZVD symptoms in the first trimester than in those with an onset during the second or third trimester (3% vs. 1%). A total of 172 of 5673 pregnancies (3%) resulted in pregnancy loss; after the exclusion of pregnancies affected by birth defects, 409 of 5426 (8%) resulted in preterm birth and 333 of 5426 (6%) in low birth weight. The prevalence of brain or eye defects during the outbreak was 13 per 10,000 live births, as compared with a prevalence of 8 per 10,000 live births before the outbreak and 11 per 10,000 live births after the outbreak. CONCLUSIONS: In pregnant women with laboratory-confirmed ZVD, brain or eye defects in infants or fetuses were more common during the Zika virus outbreak than during the periods immediately before and after the outbreak. The frequency of such defects was increased among women with a symptom onset early in pregnancy. (Funded by the Colombian Instituto Nacional de Salud and the Centers for Disease Control and Prevention.).

Zika Virus Disease in Colombia - Preliminary Report.

BACKGROUND: Colombia began official surveillance for Zika virus disease (ZVD) in August 2015. In October 2015, an outbreak of ZVD was declared after laboratory-confirmed disease was identified in nine patients. METHODS: Using the national population-based surveillance system, we assessed patients with clinical symptoms of ZVD from August 9, 2015, to April 2, 2016. Laboratory test results and pregnancy outcomes were evaluated for a subgroup of pregnant women. Concurrently, we investigated reports of microcephaly for evidence of congenital ZVD. RESULTS: By April 2, 2016, there were 65,726 cases of ZVD reported in Colombia, of which 2485 (4%) were confirmed by means of reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay. The overall reported incidence of ZVD among female patients was twice that in male patients. A total of 11,944 pregnant women with ZVD were reported in Colombia, with 1484 (12%) of these cases confirmed on RT-PCR assay. In a subgroup of 1850 pregnant women, more than 90% of women who were reportedly infected during the third trimester had given birth, and no infants with apparent abnormalities, including microcephaly, have been identified. A majority of the women who contracted ZVD in the first or second trimester were still pregnant at the time of this report. Among the cases of microcephaly investigated from January 2016 through April 2016, four patients had laboratory evidence of congenital ZVD; all were born to asymptomatic mothers who were not included in the ZVD surveillance system. CONCLUSIONS: Preliminary surveillance data in Colombia suggest that maternal infection with the Zika virus during the third trimester of pregnancy is not linked to structural abnormalities in the fetus. However, the monitoring of the effect of ZVD on pregnant women in Colombia is ongoing. (Funded by Colombian Instituto Nacional de Salud and the Centers for Disease Control and Prevention.).

Complete Genome Sequence of a Colombian Zika Virus Strain Obtained from BALB/c Mouse Brain after Intraperitoneal Inoculation.

A Zika virus (ZIKV) strain was isolated from an acute febrile patient during the Zika epidemics in Colombia. The strain was intraperitoneally inoculated into BALB/c mice, and 7 days postinoculation, neurological manifestations and ZIKV infection in the brain were demonstrated. The reported genome sequence is highly related to strains circulating in the Americas.

Genomic epidemiology supports multiple introductions and cryptic transmission of Zika virus in Colombia.

BACKGROUND: Colombia was the second most affected country during the American Zika virus (ZIKV) epidemic, with over 109,000 reported cases. Despite the scale of the outbreak, limited genomic sequence data were available from Colombia. We sought to sequence additional samples and use genomic epidemiology to describe ZIKV dynamics in Colombia. METHODS: We sequenced ZIKV genomes directly from clinical diagnostic specimens and infected Aedes aegypti samples selected to cover the temporal and geographic breadth of the Colombian outbreak. We performed phylogeographic analysis of these genomes, along with other publicly-available ZIKV genomes from the Americas, to estimate the frequency and timing of ZIKV introductions to Colombia. RESULTS: We attempted PCR amplification on 184 samples; 19 samples amplified sufficiently to perform sequencing. Of these, 8 samples yielded sequences with at least 50% coverage. Our phylogeographic reconstruction indicates two separate introductions of ZIKV to Colombia, one of which was previously unrecognized. We find that ZIKV was first introduced to Colombia in February 2015 (95%CI: Jan 2015 - Apr 2015), corresponding to 5 to 8 months of cryptic ZIKV transmission prior to confirmation in September 2015. Despite the presence of multiple introductions, we find that the majority of Colombian ZIKV diversity descends from a single introduction. We find evidence for movement of ZIKV from Colombia into bordering countries, including Peru, Ecuador, Panama, and Venezuela. CONCLUSIONS: Similarly to genomic epidemiological studies of ZIKV dynamics in other countries, we find that ZIKV circulated cryptically in Colombia. More accurately dating when ZIKV was circulating refines our definition of the population at risk. Additionally, our finding that the majority of ZIKV transmission within Colombia was attributable to transmission between individuals, rather than repeated travel-related importations, indicates that improved detection and control might have succeeded in limiting the scale of the outbreak within Colombia.

Dengue, chikungunya and zika virus coinfection: results of the national surveillance during the zika epidemic in Colombia.

Our objective was to determine the frequency of zika (ZIKV), chikungunya (CHIKV) and dengue (DENV) virus coinfection and describe the mortality cases that occurred during the epidemiologic surveillance of the ZIKV epidemic in Colombia. We analysed all cases of suspected ZIKV infection that were reported to the National Institute of Health (October 2015-December 2016). DENV, CHIKV and ZIKV RNA were detected in serum or tissue samples using polymerase chain reaction assay. Medical records of the fatal cases were reviewed. We identified that 23 871 samples were processed. The frequency of viral agents was 439 (1.84%) for DENV, 257 (1.07%) for CHIKV and 10118 (42.38%) for ZIKV. Thirty-four (0.14%) cases of coinfection were identified. The CHIKV-ZIKV coinfection was present in 28 cases (82.3%), DENV-CHIKV in three (8.8%) and DENV-ZIKV in three (8.8%). Seven (20.6%) coinfection cases were fatal (two DENV-CHIKV cases and five CHIKV-ZIKV cases). Two cases were foetal deaths and the others were related to neurological syndrome and sepsis. In conclusion, the frequency of arbovirus coinfection during epidemic of ZIKV was low, and CHIKV-ZIKV coinfection was the most common. Mortality was high among coinfection patients. The role of each virus in the mortality cases of coinfection warrants further studies.

Clinical Indicators of Fatal Dengue in Two Endemic Areas of Colombia: A Hospital-Based Case-Control Study.

According to the World Health Organization, 98% of fatal dengue cases can be prevented; however, endemic countries such as Colombia have recorded higher case fatality rates during recent epidemics. We aimed to identify the predictors of mortality that allow risk stratification and timely intervention in patients with dengue. We conducted a hospital-based, case-control (1:2) study in two endemic areas of Colombia (2009-2015). Fatal cases were defined as having either 1) positive serological test (IgM or NS1), 2) positive virological test (RT-PCR or viral isolation), or 3) autopsy findings compatible with death from dengue. Controls (matched by state and year) were hospitalized nonfatal patients and had a positive serological or virological dengue test. Exposure data were extracted from medical records by trained staff. We used conditional logistic regression (adjusting for age, gender, disease's duration, and health-care provider) in the context of multiple imputation to estimate exposure to case-control associations. We evaluated 110 cases and 217 controls (mean age: 35.0 versus 18.9; disease's duration pre-admission: 4.9 versus 5.0 days). In multivariable analysis, retro-ocular pain (odds ratios [OR] = 0.23), nausea (OR = 0.29), and diarrhea (OR = 0.19) were less prevalent among fatal than nonfatal cases, whereas increased age (OR = 2.46 per 10 years), respiratory distress (OR = 16.3), impaired consciousness (OR = 15.9), jaundice (OR = 32.2), and increased heart rate (OR = 2.01 per 10 beats per minute) increased the likelihood of death (AUC: 0.97, 95% confidence interval: 0.96, 0.99). These results provide evidence that features of severe dengue are associated with higher mortality, which strengthens the recommendations related to triaging patients in dengue-endemic areas.

Zika Virus Disease in Children in Colombia, August 2015 to May 2016.

BACKGROUND: Children are considered a potentially vulnerable population for Zika virus infection. However, data on paediatric Zika virus infection are sparse. METHODS: We analysed data from Colombia's national surveillance system during the 2015-2016 Zika virus outbreak on patients meeting the clinical case definition of Zika virus disease (ZVD) among children aged 1 month to 18 years to estimate incidence by demographic characteristics and characterize the occurrence of selected complications. RESULTS: Between August 14, 2015, and May 28, 2016, there were 18 576 reported cases of postnatal ZVD among children aged 1 month to 18 years. Laboratory testing was prioritized for high-risk patients (infants, pregnant women, adults aged ≥65 years, and persons with serious co-morbidities); among 1655 that were tested by real-time reverse transcriptase polymerase chain reaction, 1207 (72.9%) were positive. The cumulative incidence of reported ZVD was 114.4 per 100 000. The incidence differed by sex, depending on age group; the largest difference was observed for 15-18 year olds, with females having a higher incidence than males (cumulative incidence ratio 2.5, 95% confidence interval 2.3, 2.7). At the time of report to the surveillance system, 631 patients (3.4%) were hospitalised and 96 (0.5%) had a report of an accompanying neurological diagnosis, including Guillain-Barré syndrome in 40 patients. CONCLUSIONS: Only a small proportion of reported paediatric ZVD cases in Colombia were hospitalized or had reported neurological conditions following ZVD. However, the potential for some serious outcomes demonstrates the importance of preventing Zika virus infection in children.

Severe Neurologic Disorders in 2 Fetuses with Zika Virus Infection, Colombia.

We report the results of pathologic examinations of 2 fetuses from women in Colombia with Zika virus infection during pregnancy that revealed severe central nervous system defects and potential associated abnormalities of the eye, spleen, and placenta. Amniotic fluid and tissues from multiple fetal organs tested positive for Zika virus.

Análisis filogenético del virus del chikungunya en Colombia: evidencia de selección purificadora en el gen E1 / Phylogenetic analysis of Chikungunya virus in Colombia: Evidence of purifying selection in the E1 gene

Introducción. El virus del chikungunya, perteneciente al género Alphavirus de la familia Togaviridae, es un virus ARN de 11,8 kb, de cadena sencilla y polaridad positiva, transmitido por Aedes spp . Se han identificado tres genotipos a nivel mundial: el de Asia, el del este-centro-sur de África ( East/Central/South African, ECSA) y el de África occidental ( West African, WA). La fiebre del chikungunya es una enfermedad febril aguda, acompañada principalmente de inflamación en las articulaciones y erupción cutánea. Después de su aparición en las Américas en el 2013, los primeros casos en Colombia ocurrieron en septiembre de 2014 y hasta junio del 2015 se habían notificado 399.932 casos. Objetivo. Identificar el genotipo o los genotipos responsables de la primera epidemia por el virus del chikungunya en Colombia y la variabilidad genética asociada a su dispersión en el territorio nacional. Materiales y métodos. Se seleccionaron muestras de suero de pacientes con síntomas indicativos de fiebre del chikungunya durante 2014 y 2015. Se hizo una transcripción inversa seguida de reacción en cadena de la polimerasa del gen E1, así como su secuenciación, análisis filogenético y análisis de evolución adaptativa. Resultados. Se demostró la presencia exclusiva del genotipo de Asia en Colombia. Se registró un promedio de 0,001 sustituciones de bases por sitio, una identidad de 99,7 a 99,9 % en los nucleótidos y de 99,9 % en los aminoácidos entre las secuencias colombianas y las secuencias de las Américas. Los análisis de evolución adaptativa indicaron una fuerte selección purificadora en el gen E1 . Conclusiones. Se determinó la circulación del genotipo de Asia del virus del chikungunya como la causa de la primera epidemia en Colombia. Es necesario continuar con la vigilancia de genotipos, con el fin de detectar posibles cambios en la epidemiología, la eficacia ( fitness ) viral y la patogenia del virus.

Introduction: Chikungunya virus (CHIKV) is a single-stranded positive sense RNA virus that belongs to the Alphavirus genus of the family Togaviridae. Its genome is 11.8 kb in length, and three genotypes have been identified worldwide: Asian, East/Central/South African (ECSA) and West African. Chikungunya fever is an acute febrile disease transmitted by Aedes spp . that usually presents with polyarthralgia and cutaneous eruption. Following introduction of the virus to the Americas in 2013, the first cases in Colombia occurred in September of 2014, and they reached a cumulative total of 399,932 cases by June of 2015. Objective: To identify the genotype or genotypes responsible for the current epidemic in Colombia and to describe the genetic variability of the virus in the country. Materials and methods: Serum samples from patients presenting with symptoms compatible with Chikungunya fever during 2014-2015 were selected for the study. RT-PCR products of the E1 gene from these samples were used for sequencing and subsequent phylogenetic and adaptive evolution analyses. Results: The study identified only the presence of the Asian genotype in Colombia. Comparing the Colombian sequences with other sequences from the Americas revealed an average of 0.001 base substitutions per site, with 99.7% and 99.9% nucleotide identity and 99.9% amino acid identity. The adaptive evolution analysis indicated that the E1 gene is under strong purifying selection. Conclusions: The first epidemic of Chikunguya fever in Colombia was caused by the circulation of the virus Asian genotype. Further genotypic surveillance of the virus in Colombia is required to detect possible changes in its epidemiology, fitness and pathogenicity.

Clinical and histopathological features of fatal cases with dengue and chikungunya virus co-infection in Colombia, 2014 to 2015.

We report clinical features and histopathological findings in fatal cases with dengue (DENV) and chikungunya (CHIKV) co-infection identified at the Colombian National Institute of Health between September 2014 and October 2015. Seven such cases were documented. Dengue serotype 2 virus was identified in six cases. All patients were adults and comorbidities were present in four. Fever, arthralgia or myalgia was present in all cases. The frequency of rash, haemorrhage, oedema, and gastrointestinal symptoms was variable. Laboratory findings such as thrombocytopenia, renal failure, and leukocyte count were also inconsistent between cases. Post-mortem tissue examination documented focal hepatocellular coagulative necrosis in three cases, incipient acute pericarditis in one and tubulointerstitial nephritis in one. This study provides evidence of mortality in patients with DENV and CHIKV co-infection. Fatal cases were characterised by variable clinical and laboratory features. Evaluation of histopathology of autopsy tissues provided evidence of the pathological consequences of the disease.

Phylogenetic analysis of Chikungunya virus in Colombia: Evidence of purifying selection in the E1 gene.

INTRODUCTION: Chikungunya virus (CHIKV) is a single-stranded positive sense RNA virus that belongs to the Alphavirus genus of the family Togaviridae. Its genome is 11.8 kb in length, and three genotypes have been identified worldwide: Asian, East/Central/South African (ECSA) and West African. Chikungunya fever is an acute febrile disease transmitted by Aedes spp. that usually presents with polyarthralgia and cutaneous eruption. Following introduction of the virus to the Americas in 2013, the first cases in Colombia occurred in September of 2014, and they reached a cumulative total of 399,932 cases by June of 2015.  OBJECTIVE: To identify the genotype or genotypes responsible for the current epidemic in Colombia and to describe the genetic variability of the virus in the country.  MATERIALS AND METHODS: Serum samples from patients presenting with symptoms compatible with Chikungunya fever during 2014-2015 were selected for the study. RT-PCR products of the E1 gene from these samples were used for sequencing and subsequent phylogenetic and adaptive evolution analyses.  RESULTS: The study identified only the presence of the Asian genotype in Colombia. Comparing the Colombian sequences with other sequences from the Americas revealed an average of 0.001 base substitutions per site, with 99.7% and 99.9% nucleotide identity and 99.9% amino acid identity. The adaptive evolution analysis indicated that the E1 gene is under strong purifying selection.  CONCLUSIONS: The first epidemic of Chikunguya fever in Colombia was caused by the circulation of the virus Asian genotype. Further genotypic surveillance of the virus in Colombia is required to detect possible changes in its epidemiology, fitness and pathogenicity.

Desarrollo de un método de transcripción inversa seguida de reacción en cadena de la polimerasa para la detección del virus de la fiebre amarilla / Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection

Introduction. Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. Objective. To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. Materials and methods. RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. Results. The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. Conclusion. This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.

Introducción. La fiebre amarilla se considera una enfermedad reemergente y endémica en regiones tropicales de África y Suramérica. Actualmente, no existen estuches estandarizados o comerciales disponibles para la detección del virus de la fiebre amarilla y, por lo tanto, el diagnóstico debe hacerse mediante técnicas de rutina que consumen mucho tiempo y algunas veces no garantizan la detección del virus o de sus proteínas. Además, la cocirculación con otros flavivirus, incluyendo el del dengue, hacen el diagnóstico más complicado. Objetivo. Desarrollar un ensayo específico de amplificación basado en transcripción inversa seguida de reacción en cadena de la polimerasa, con el fin de mejorar la detección y el diagnóstico de la fiebre amarilla, tanto a partir de suero como de tejido fresco. Materiales y métodos. Se diseñaron iniciadores específicos para amplificar un fragmento conservado del virus de la fiebre amarilla. Un segundo par de iniciadores se usó en una reacción de amplificación anidada para incrementar la sensibilidad. Se probaron 33 muestras clínicas con la técnica estandarizada. Resultados. El amplímero esperado se obtuvo en 25 de las 33 muestras analizadas usando este método y 2 más resultaron positivas después de la reacción anidada. Conclusión. Esta técnica mejorada garantiza la detección de todos los genotipos virales de fiebre amarilla y puede incrementar la sensibilidad del ensayo introduciendo una segunda etapa de amplificación, lo cual permite el diagnóstico diferencial con infección por dengue y otros flavivirus, lo cual es de gran importancia para la vigilancia y la toma de medidas epidemiológicas oportunas.

Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection.

INTRODUCTION: Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. OBJECTIVE: To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. MATERIALS AND METHODS: RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. RESULTS: The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. CONCLUSION: This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.