Dengue virus detection in Lao PDR and Colombia: Comparative evaluation of PCR tests.

OBJECTIVES: Dengue virus (DENV) detection by polymerase chain reaction (PCR) facilitates diagnosis of dengue fever, which is the most frequent arboviral disease globally. Two studies were performed in countries with high dengue incidence, to assess the diagnostic performance of different PCR techniques. METHODS/RESULTS: Two hundred and seventy-nine acute phase blood samples from febrile patients were analyzed for DENV by the RealStar Dengue RT-PCR kit (Altona Diagnostics) as gold standard in comparison with the Tropical Fever Core multiplex PCR (Fast Track Diagnostics). In total, 102 samples collected in Savannakhet Province (Lao PDR, Southeast Asia) in 2013 and 35 samples from Valledupar (Colombia, South America) tested positive for DENV by RealStar RT-PCR. In comparison, the Tropical Fever Core multiplex PCR detected 65.0% (65/102) and 68.6% (24/35) of these samples as positive for DENV in Savannakhet and Valledupar, respectively. Diagnostic sensitivity of the multiplex PCR strongly correlated with viral load. A subset of DENV PCR-confirmed samples was additionally tested by BNITM in house Dengue Type RT-PCR in comparison with two commercial test kits (RealStar Dengue Type RT-PCR [Altona Diagnostics], Dengue differentiation PCR [Fast Track Diagnostics]). The leading dengue serotype in Savannakhet was DENV-3 (58% [29/50]), while DENV-1 (53.8% [14/26]) was the predominant serotype found in samples collected in Valledupar by BNITM-type PCR. However, three DENV serotypes were circulating in Valledupar and in Savannakhet. In 2015, additional studies found predominantly DENV-4 (71% [12/17]) in Savannakhet. CONCLUSIONS: Both studies emphasized that routine diagnostics in both regions will benefit from an expanded use of highly sensitive pan-dengue PCRs.

Differential replicative fitness of the four dengue virus serotypes circulating in Colombia in human liver Huh7 cells

ABSTRACT Dengue has been a significant public health problem in Colombia since the simultaneous circulation of the four dengue virus serotypes. The replicative fitness of dengue is a biological feature important for virus evolution and contributes to elucidating the behavior of virus populations and viral pathogenesis. However, it has not yet been studied in Colombian isolates. This study aimed to compare the replicative fitness of the four dengue virus serotypes and understand the association between the serotypes, their in vitro infection ability, and their replication in target cells. We used three isolates of each DENV serotype to infect Huh-7 cells at an MOI of 0.5. The percentage of infected cells was evaluated by flow cytometry, cell viability was evaluated by MTT assay, and the pathogenicity index was calculated as a ratio of both parameters. The replicative fitness was measured by the number of viral genome copies produced using quantitative PCR and the production of infectious viral progeny was measured by plaque assay. We showed that Huh-7 cells were susceptible to infection with all the different strain isolates. Nevertheless, the biological characteristics, such as infectious ability and cell viability, were strain-dependent. We also found different degrees of pathogenicity between strains of the four serotypes, representative of the heterogeneity displayed in the circulating population. When we analyzed the replicative fitness using the mean values obtained from RT-qPCR and plaque assay for the different strains, we found serotype-dependent behavior. The highest mean values of replicative fitness were obtained for DENV-1 (log 4.9 PFU/ml) and DENV-4 (log 5.28 PFU/ml), followed by DENV-2 (log 3.9 PFU/ml) and DENV-3 (log 4.31 PFU/ml). The internal heterogeneity of the replicative fitness within each serotype could explain the simultaneous circulation of the four DENV serotypes in Colombia.

Differential replicative fitness of the four dengue virus serotypes circulating in Colombia in human liver Huh7 cells.

Dengue has been a significant public health problem in Colombia since the simultaneous circulation of the four dengue virus serotypes. The replicative fitness of dengue is a biological feature important for virus evolution and contributes to elucidating the behavior of virus populations and viral pathogenesis. However, it has not yet been studied in Colombian isolates. This study aimed to compare the replicative fitness of the four dengue virus serotypes and understand the association between the serotypes, their in vitro infection ability, and their replication in target cells. We used three isolates of each DENV serotype to infect Huh-7 cells at an MOI of 0.5. The percentage of infected cells was evaluated by flow cytometry, cell viability was evaluated by MTT assay, and the pathogenicity index was calculated as a ratio of both parameters. The replicative fitness was measured by the number of viral genome copies produced using quantitative PCR and the production of infectious viral progeny was measured by plaque assay. We showed that Huh-7 cells were susceptible to infection with all the different strain isolates. Nevertheless, the biological characteristics, such as infectious ability and cell viability, were strain-dependent. We also found different degrees of pathogenicity between strains of the four serotypes, representative of the heterogeneity displayed in the circulating population. When we analyzed the replicative fitness using the mean values obtained from RT-qPCR and plaque assay for the different strains, we found serotype-dependent behavior. The highest mean values of replicative fitness were obtained for DENV-1 (log 4.9 PFU/ml) and DENV-4 (log 5.28 PFU/ml), followed by DENV-2 (log 3.9 PFU/ml) and DENV-3 (log 4.31 PFU/ml). The internal heterogeneity of the replicative fitness within each serotype could explain the simultaneous circulation of the four DENV serotypes in Colombia.

Dengue serotype circulation in natural populations of Aedes aegypti.

Ae. aegypti is the main vector of dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viruses. The transmission dynamics of these arboviruses, especially the arboviral circulation in the mosquito population during low and high transmission seasons in endemic areas are still poorly understood. We conducted an entomological survey to determine dengue infection rates in Ae. aegypti and Aedes albopictus. These collections were performed in 2012-2013 during a Rio de Janeiro epidemic, just before the introduction and spread of ZIKV and CHIKV in the city. MosquiTrap© and BG-Sentinel traps were installed in three fixed and seven itinerant neighborhoods each month over ten months. Mosquitoes were in supernatants pools tested and individually confirmed for DENV infection using RT-PCR. A total of 3053 Aedes mosquitos were captured and Ae. aegypti was much more frequent (92.9%) than Ae. albopictus (6.8%). Ae. aegypti females accounted for 71.8% of captured mosquitoes by MosquitTrap© and were the only species found naturally infected with DENV (infection rate=0.81%). Only one Ae. aegypti male, collected by BG-sentinel, was also tested positive for DENV. The peak of DENV-positive mosquitoes coincided the season of the highest incidence of human cases. The most common serotypes detected in mosquitoes were DENV-3 (24%) and DENV-1 (24%), followed by DENV-4 (20%), DENV-2 (8%) and DENV-1 plus DENV4 (4%), while 95% of laboratory-confirmed human infections in the period were due to DENV-4. These contrasting results suggest silent maintenance of DENV serotypes during the epidemics, reinforcing the importance of entomological and viral surveillance in endemic areas.

Serotipos de dengue en México durante 2009 y 2010 / Dengue serotypes in México during 2009-2010

Introducción. El dengue en México es un problema prioritario de salud pública. Desde el 2008 el Departamento para la Vigilancia Epidemiológica y Virológica del InDRE implementó un nuevo algoritmo de diagnóstico del dengue, que utiliza la Red de Laboratorios Estatales de Salud Pública, para favorecer la representatividad geográfica, la oportunidad, la sensibilidad y la especificidad de la información que se obtiene. Métodos. La identificación de serotipos se realizó a partir de muestras positivas a la proteína NS1 por ensayo inmunoenzimático (ELISA). Las técnicas que se utilizaron fueron: aislamiento viral, PCR punto final y, desde 2009, RT-PCR en tiempo real (qRT-PCR). Resultados. En 2009 se analizaron 6,336 muestras; en 2,944 de éstas (46.6%) se identificó el serotipo DENV-1 que predominó sobre el serotipo DENV-2; el serotipo DENV-3 sólo se identificó en dos casos en Guerrero y el serotipo DENV-4 en un caso en Chiapas. En 2010 se analizaron 2,013 muestras. Se identificó algún serotipo en 1,607 muestras (79.88%) y, nuevamente, el serotipo DENV-1 predominó en todo el país. En Chiapas se identificaron los serotipos DENV-1, 2 y 4 y en Jalisco los serotipos DENV-1 y 3. Además, se identificó la circulación del serotipo DENV-3 en Guerrero y apareció el serotipo DENV-4 en San Luis Potosí. Conclusiones. Por la selección de muestras para vigilancia virológica de dengue mediante la positividad a la proteína NS1 y por la introducción de la técnica de qRT-PCR se optimizó la identificación de serotipos circulantes. La alta endemia, los brotes en nuevas regiones, el predominio del serotipo DENV-1 por varios años y la introducción lenta de otros serotipos, principalmente DENV-3, pueden favorecer la aparición de formas clínicas graves de dengue. La vigilancia epidemiológica inteligente del dengue brindará información para un mejor entendimiento de la enfermedad y promoverá acciones para su control y prevención.

Background. Dengue is a public health priority in Mexico. Since 2008, the dengue diagnostic algorithm for epidemiological and virological surveillance has been improved at InDRE and the public health laboratory network (RLESP) to optimize geographic representation, opportunity, sensitivity and specificity of the produced information. Methods. Dengue serotype identification is based on ELISA NS1 positive samples. Methods used are viral isolation, endpoint PCR and, since August 2009, real-time PCR (qRT-PCR). Results. In 2009, 6,336 serum samples were analyzed and 2,944 (46.6%) were positive for serotype identification. DENV-1 was detected in greater proportion followed by DENV-2, and DENV-3 4 was only identified in two cases in Guerrero and DENV-4 in one case in Chiapas. In 2010, 2,013 serum samples were analyzed and 1,607 (78.8%) were positive for serotype identification. DENV-1 was predominant throughout the country. In Chiapas, DENV-1, 2 and 4 were identified and in Jalisco DENV-1 and 3. DENV-3 was identified in Guerrero again and DENV-4 was detected in San Luis Potosí. Conclusions. The selection samples through NS1 positive samples and the introduction of qRT-PCR optimized serotype identification. Hyperendemicity, outbreaks in new geographic areas, the predominant circulation of DENV-1 for several years and the slow reintroduction of the other serotypes, mainly DENV-3, could increase clinical cases of severe dengue. An ¡intelligentí epidemiological surveillance program would offer information for a better understanding of the disease and promote action for its control and prevention.