Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand

Background: AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. Objective: To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. Study design: A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011–2013. Results and conclusion: The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods

Polytopic vaccination with a live-attenuated dengue vaccine enhances B-cell and T-cell activation, but not neutralizing antibodies

Dengue, caused by dengue viruses (DENVs), is the most common arboviral disease of humans. Several dengue vaccine candidates are at different stages of clinical development and one has been licensed. Inoculation with live-attenuated DENV constructs is an approach that has been used by vaccine developers. Unfortunately, the simultaneous injection of all four attenuated DENV serotypes (DENV1-4) into a single injection site (monotopic vaccination) has been postulated to result in interference in the replication of some serotypes in favor of others, an important obstacle in obtaining a balanced immune response against all serotypes. Here, we demonstrate the virus replicative and immunostimulatory effects of polytopic monovalent dengue vaccination (PV) in which, each of the four components of the tetravalent vaccine is simultaneously delivered to four different sites versus the more traditional monotopic tetravalent vaccination (MV) in a non-human primate (NHP) model. With the exception of DENV-2, there was no significant difference in detectable viral RNA levels between PV and MV inoculation. Interestingly, longer periods of detection and higher viral RNA levels were seen in the lymph nodes of NHPs inoculated PV compared to MV. Induction of lymph node dendritic cell maturation and of blood T- and B-cell activation showed different kinetics in PV inoculated NHPs compared to MV. The MV inoculated group showed earlier maturation of dendritic cells and activation of B and T cells compared to PV inoculated NHPs. A similar kinetic difference was also observed in the cytokine response: MV induced earlier cytokine responses compared to PV. However, similar levels of DENV neutralizing antibodies were observed in PV and MV NHPs. These findings indicate that cellular immune response after vaccination may be affected by the location of inoculation. Design of vaccine delivery may need to take into account the effects of locations of vaccine delivery of multiples serotype live viral vaccine on the induction of immune response

Polytopic vaccination with a live-attenuated dengue vaccine enhances B-cell and T-cell activation, but not neutralizing antibodies

Dengue, caused by dengue viruses (DENVs), is the most common arboviral disease of humans. Several dengue vaccine candidates are at different stages of clinical development and one has been licensed. Inoculation with live-attenuated DENV constructs is an approach that has been used by vaccine developers. Unfortunately, the simultaneous injection of all four attenuated DENV serotypes (DENV1-4) into a single injection site (monotopic vaccination) has been postulated to result in interference in the replication of some serotypes in favor of others, an important obstacle in obtaining a balanced immune response against all serotypes. Here, we demonstrate the virus replicative and immunostimulatory effects of polytopic monovalent dengue vaccination (PV) in which, each of the four components of the tetravalent vaccine is simultaneously delivered to four different sites versus the more traditional monotopic tetravalent vaccination (MV) in a non-human primate (NHP) model. With the exception of DENV-2, there was no significant difference in detectable viral RNA levels between PV and MV inoculation. Interestingly, longer periods of detection and higher viral RNA levels were seen in the lymph nodes of NHPs inoculated PV compared to MV. Induction of lymph node dendritic cell maturation and of blood T- and B-cell activation showed different kinetics in PV inoculated NHPs compared to MV. The MV inoculated group showed earlier maturation of dendritic cells and activation of B and T cells compared to PV inoculated NHPs. A similar kinetic difference was also observed in the cytokine response: MV induced earlier cytokine responses compared to PV. However, similar levels of DENV neutralizing antibodies were observed in PV and MV NHPs. These findings indicate that cellular immune response after vaccination may be affected by the location of inoculation. Design of vaccine delivery may need to take into account the effects of locations of vaccine delivery of multiples serotype live viral vaccine on the induction of immune response

Data from: Clinical and laboratory predictors of influenza infection among individuals with influenza-like illness presenting to an urban Thai hospital over a five-year period

Early diagnosis of influenza infection maximizes the effectiveness of antiviral medicines. Here, we assess the ability for clinical characteristics and rapid influenza tests to predict PCR-confirmed influenza infection in a sentinel, cross-sectional study for influenza-like illness (ILI) in Thailand. Participants meeting criteria for acute ILI (fever > 38°C and cough or sore throat) were recruited from inpatient and outpatient departments in Bangkok, Thailand, from 2009-2014. The primary endpoint for the study was the occurrence of virologically-confirmed influenza infection (based upon detection of viral RNA by RT-PCR) among individuals presenting for care with ILI. Nasal and throat swabs were tested by rapid influenza test (QuickVue) and by RT-PCR. Vaccine effectiveness (VE) was calculated using the case test-negative method. Classification and Regression Tree (CART) analysis was used to predict influenza RT-PCR positivity based upon symptoms reported. We enrolled 4572 individuals with ILI; 32.7% had detectable influenza RNA by RT-PCR. Influenza cases were attributable to influenza B (38.6%), A(H1N1)pdm09 (35.1%), and A(H3N2) (26.3%) viruses. VE was highest against influenza A(H1N1)pdm09 virus and among adults. The most important symptoms for predicting influenza PCR-positivity among patients with ILI were cough, runny nose, chills, and body aches. The accuracy of the CART predictive model was 72.8%, with an NPV of 78.1% and a PPV of 59.7%. During epidemic periods, PPV improved to 68.5%. The PPV of the QuickVue assay relative to RT-PCR was 93.0% overall, with peak performance during epidemic periods and in the absence of oseltamivir treatment. Clinical criteria demonstrated poor predictive capability outside of epidemic periods while rapid tests were reasonably accurate and may provide an acceptable alternative to RT-PCR testing in resource-limited areas

Predictors of clinical severity and receipt of antimicrobial medications among those with PCR-confirmed influenza infection.

<p>Predictors of clinical severity and receipt of antimicrobial medications among those with PCR-confirmed influenza infection.</p

CART analysis to predict influenza RT-PCR positivity on the basis of clinical symptoms.

<p>CART analysis to predict influenza RT-PCR positivity on the basis of clinical symptoms.</p

Clinical and laboratory predictors of influenza infection among individuals with influenza-like illness presenting to an urban Thai hospital over a five-year period

<div><p>Early diagnosis of influenza infection maximizes the effectiveness of antiviral medicines. Here, we assess the ability for clinical characteristics and rapid influenza tests to predict PCR-confirmed influenza infection in a sentinel, cross-sectional study for influenza-like illness (ILI) in Thailand. Participants meeting criteria for acute ILI (fever > 38°C and cough or sore throat) were recruited from inpatient and outpatient departments in Bangkok, Thailand, from 2009–2014. The primary endpoint for the study was the occurrence of virologically-confirmed influenza infection (based upon detection of viral RNA by RT-PCR) among individuals presenting for care with ILI. Nasal and throat swabs were tested by rapid influenza test (QuickVue) and by RT-PCR. Vaccine effectiveness (VE) was calculated using the case test-negative method. Classification and Regression Tree (CART) analysis was used to predict influenza RT-PCR positivity based upon symptoms reported. We enrolled 4572 individuals with ILI; 32.7% had detectable influenza RNA by RT-PCR. Influenza cases were attributable to influenza B (38.6%), A(H1N1)pdm09 (35.1%), and A(H3N2) (26.3%) viruses. VE was highest against influenza A(H1N1)pdm09 virus and among adults. The most important symptoms for predicting influenza PCR-positivity among patients with ILI were cough, runny nose, chills, and body aches. The accuracy of the CART predictive model was 72.8%, with an NPV of 78.1% and a PPV of 59.7%. During epidemic periods, PPV improved to 68.5%. The PPV of the QuickVue assay relative to RT-PCR was 93.0% overall, with peak performance during epidemic periods and in the absence of oseltamivir treatment. Clinical criteria demonstrated poor predictive capability outside of epidemic periods while rapid tests were reasonably accurate and may provide an acceptable alternative to RT-PCR testing in resource-limited areas.</p></div

QuickView test performance, as compared to RT-PCR (“gold standard”).

<p>Lower limit of 95% confidence interval for each proportion is shown in parentheses.</p

Receiver operating characteristic (ROC) curves for (Fig 3A) CART analysis and (Fig 3B) QuickVue, compared to RT-PCR for identifying influenza infection among individuals with ILI.

<p>Receiver operating characteristic (ROC) curves for (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193050#pone.0193050.g003" target="_blank">Fig 3A</a>) CART analysis and (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193050#pone.0193050.g003" target="_blank">Fig 3B</a>) QuickVue, compared to RT-PCR for identifying influenza infection among individuals with ILI.</p