Development, verification, and validation of an RT-qPCR-based protocol for Yellow Fever diagnosis.

INTRODUCTION: Yellow fever (YF) is a public health threat with frequent outbreaks in tropical and subtropical areas, despite the existence of a safe and effective vaccine. The diagnosis of acute infection of the etiologic agent relies mainly on real-time reverse transcription-polymerase chain reaction (RT-qPCR)-based assays. OBJECTIVES: The aim of this study was to evaluate and compare this novel protocol for yellow fever virus (YFV) diagnosis against assays developed in-house by reference laboratories for arboviruses. METHODS: We developed a novel molecular protocol for the detection of YFV that includes an Internal Control to validate the reaction and an External Control to monitor the RNA extraction efficiency. RESULTS AND DISCUSSION: Our assay detects one viral genome per reaction and displays no cross-reactions with dengue (1-4), Zika, or Chikungunya viruses. This novel assay yielded 95% of agreement with the reference method recommended by the Pan American Health Organization when analyzing 204 clinical samples and cultured viruses, these samples were analyzed in 3 different diagnosis centers for arboviruses in Brazil. The data suggest the use of the proposed multiplex assay protocol to do routine tests in a clinical laboratory. This product adds higher specificity and sensitivity in addition to reduced cost per test due to hands-on time and reagent spending.

Sentinel hospital surveillance for rotavirus in latin american and Caribbean countries.

The burden of rotavirus disease in the Latin American region has been poorly understood despite the promise of effective vaccines. We describe here the implementation and results of a rotavirus surveillance network in the Latin American and Caribbean region. From 2005 through 2007, stool specimens and epidemiologic information were gathered from children <5 years of age who were hospitalized for acute diarrhea (3 looser-than-normal stools within <24 h) lasting <14 days with use of a standardized generic protocol. Stool samples were tested for rotavirus, and a proportion of detected strains were typed. The proportion of samples positive for rotavirus was applied to World Health Organization diarrhea-related mortality estimates to calculate rotavirus-associated mortality. In 2007, the network comprised 54 sites in 11 countries. During 2006-2007, specimens were collected from 19,817 children; 8141 of these specimens were positive for rotavirus. The median percentage of positive specimens in the country was 31.5% (range, 24%-47%). The risk of death from rotavirus diarrhea by age 5 years was 1 of 2874. Strong rotavirus winter seasonality was apparent, even in tropical Central America. Globally common strains (P[8] G1, P[8] G9, and P[4] G2) accounted for >75% of strains, although unusual strains, including G12, were detected at low levels. As rotavirus vaccines continue to be introduced in Latin America, maintenance of surveillance will provide robust pre-introduction data and a platform for estimating vaccine effectiveness and other measures of impact.