Serologic Analysis of Returned Travelers with Fever, Sweden

We studied 1,432 febrile travelers from Sweden who had returned from malaria-endemic areas during March 2005–March 2008. In 383 patients, paired serum samples were blindly analyzed for influenza and 7 other agents. For 21% of 115 patients with fever of unknown origin, serologic analysis showed that influenza was the major cause

Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections

Background: Dengue is a mosquito-borne viral disease that has become more prevalent in the last few decades. Most patients are viremic when they present with symptoms, and early diagnosis of dengue is important in preventing severe clinical complications associated with this disease and also represents a key factor in differential diagnosis. Here, we designed and validated a hydrolysis-probe-based one-step real-time RT-PCR assay that targets the genomes of dengue virus serotypes 1-4. Methodology/Principal Findings: The primers and probe used in our RT-PCR assay were designed to target the 39 untranslated region of all complete genome sequences of dengue virus available in GenBank (n=3,305). Performance of the assay was evaluated using in vitro transcribed RNA, laboratory-adapted virus strains, external control panels, and clinical specimens. The linear dynamic range was found to be 10(4)-10(11) GCE/mL, and the detection limit was between 6.0x10(2) and 1.1x10(3) GCE/mL depending on target sequence. The assay did not cross-react with human RNA, nor did it produce false-positive results for other human pathogenic flaviviruses or clinically important etiological agents of febrile illnesses. We used clinical serum samples obtained from returning travelers with dengue-compatible symptomatology (n = 163) to evaluate the diagnostic relevance of our assay, and laboratory diagnosis performed by the RT-PCR assay had 100% positive agreement with diagnosis performed by NS1 antigen detection. In a retrospective evaluation including 60 archived serum samples collected from confirmed dengue cases 1-9 days after disease onset, the RT-PCR assay detected viral RNA up to 9 days after appearance of symptoms. Conclusions/Significance: The validation of the RT-PCR assay presented here indicates that this technique can be a reliable diagnostic tool, and hence we suggest that it be introduced as the method of choice during the first 5 days of dengue symptoms

DENV RT-PCR assay specificity.

<p>DENV RT-PCR assay specificity.</p

Overview of the primers and probe in the DENV RT-PCR assay.

<p>The reverse primers DENV_R1–3 (A) and DENV_R4 (B) were specifically designed to target DENV serotypes 1–3 and serotype 4, respectively. Vertical bars and percentages show the fraction of sequences with nucleotides deviating from the consensus of DENV serotypes 1–3 (A) and serotype 4 (B). Percentages below 1 are not shown. Numbers indicate genomic positions.</p

Results of the DENV RT-PCR assay performed on serum samples obtained 1 to 9 days after onset of symptoms.

<p>(A) Archived serum samples that had been collected from 60 patients on days 1–3 (n = 5), 4 (n = 10), 5 (n = 7), 6 (n = 11), 7 (n = 12), 8 (n = 8), and 9 (n = 7) after disease onset were tested by the DENV RT-PCR assay, an NS1 antigen detection test, IgM capture ELISA, and IFA detecting DENV-specific IgG antibodies. The criteria for a positive result in each of these analyses are explained in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003416#s2" target="_blank">Methods</a>. The curves show the percent of samples testing positive in the individual assays for the specified days after disease onset. (B) Viral load in samples collected on days 1–9 after onset of symptoms. Each dot represents the mean of results for duplicate samples from a single patient. GCE = genome copy equivalents.</p

Flow-chart showing the laboratory test results for samples from returning travelers with dengue-compatible symptomatology.

<p>Serum samples collected during January and February 2014 were tested consecutively by the newly developed DENV RT-PCR method, an NS1 antigen detection test, IgM capture ELISA, and/or an in-house IFA detecting DENV-specific IgG antibodies. The criteria for a positive result in each individual assay are explained in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003416#s2" target="_blank">Methods</a>. The results of the laboratory analysis of the first sample arriving at the Public Health Agency of Sweden are shown. w/o = without.</p

Dynamic range and limit of detection of the DENV RT-PCR assay.

<p>(A) The linear dynamic range of the DENV RT-PCR assay was determined by testing triplicates of 10-fold serially diluted in vitro transcribed RNA. The sequences of the transcript RNA (RNA[DENV_R1–3] and RNA[DENV_R4]) were matched with the two reverse primers DENV_R1–3 and DENV_R4, respectively. Each dot represents the mean Cq-value from three replicates, the error bars indicate the 95% confidence interval, and the lines illustrate the result of the lin-log regression analysis. (B and C) Limit of detection was determined by assaying eight replicates of twofold serially diluted RNA transcripts in three separate experiments, and the results of testing are shown for RNA[DENV_R1–3] (B) and RNA[DENV_R4] (C). Horizontal lines indicate mean values, boxes denote the 25th to 75th percentiles and whiskers the 5–95% percentiles, and dots represent outliers. The number of positives per total number of replicates tested is given above each box. Limit of detection was defined as the last dilution in which transcript RNA was detected in all 24 replicates. GCE = genome copy equivalents.</p

Characteristics of the primers and probe targeting the 3′ UTR of DENV serotypes 1–4.

<p>Characteristics of the primers and probe targeting the 3′ UTR of DENV serotypes 1–4.</p