First confirmation of imported dengue virus serotype 2 complete genome in urine from a Chinese traveler returning from India
© Ma et al.; licensee BioMed Central Ltd. 2014
Received: 17 December 2013
Accepted: 19 March 2014
Published: 25 March 2014
Dengue virus (DENV) is a mosquito-borne virus that has four serotypes. Collection of serum from patients is time- and labor- consuming, and presents a high injury risk for infants and children. The genomic and serological diagnosis of imported dengue fever from a urine sample was used as a non-invasive diagnostic method in this study. A serum sample was collected on disease day 5, and a serum and urine sample were collected on disease day 8 and 18. The results of serological tests for DENV IgM revealed that the serum samples were positive for DENV. The results of RT-qPCR assay revealed that the serum sample collected on day 5 was DENV-positive; however, the serum sample collected on day 8 and 18 were negative for DENV. The urine sample collected on day 8 and 18 were DENV-positive. We also sequenced the complete DENV genome (10723 bp) from the urine sample (GenBank KF479233). The results of phylogenetic and epidemiological analysis indicated strong confirmation that the strain was located within the DENV-2 group with a 100% bootstrap value. In this report, we (1) provided the first evidence of a DENV infection that was imported from India to a non-endemic city of China, (2) investigated the DENV genome detection having a longer timeframe for positive detection in urine sample compared to previous studies, (3) provided the sequence results for the complete DENV-2 genome from a concentrated urine sample (4) discussed how virus-typing results could be used to manage the risk of sero-specific and re-infected travel-associated dengue fever.
Dengue virus (DENV) has four different serotypes and any one of related types will cause dengue infection with the main transmission of mosquitoes . DENV infection with one serotype may drive the development of dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) after re-infection with another serotype, which has a high mortality rate (1–2.5%) . To prevent re-infection, it is important to detect the primary DENV serotype infection in travelers returning from highly endemic areas. The laboratory DENV diagnoses include viral isolation, viral antigen detection, antibody detection and genomic test. The combination of serological tests and RT-qPCR is an approach that is often to confirm DENV infection . Laboratory diagnostic detections in health examination for international travelers usually need patients’ serum as detecting samples. However, collection of serum sample is time- and labor-consuming, presents a high injury risk for infants and children, and depends on patients’ compliance. Several studies have reported that DENV genome detection in urine is an alternative non-invasive diagnostic method, and the timeframes for positive detection in urine are longer than they are in serum [4–6]. Considering the severe DENV secondary infection, the DENV long-last timeframe in urine sample provides a monitoring chance rather than the serological test to detect positive DENV infection with the identification of specific serotypes. This case study reported a longer timeframe for positive detection of dengue virus in urine samples than previous studies have reported. In addition to the difficulties of patients’ serum collection, the weakness of serological test is a high risk of the cross-reaction with other flaviviruses such as Yellow Fever Virus (YFV) . The complete DENV genome provides a strong molecular evidence to determine dengue virus and its serotype. Here, this case report first represents the diagnosis and serotyping of a case of imported dengue fever from the urine sample and provide the full genome sequence of this DENV-2 at a point of entry in North China.
The patient had no previous medical history of dengue fever or renal disease, and had not received Japanese encephalitis or yellow fever vaccines. He visited Chennai, India, for 5 days, from Dec 18 to Dec 22 2013, and arrived in Qinhuangdao on Dec 23. The patient recalled that he had been bitten by mosquitoes sometime between Dec 19 and Dec 22. If disease day is defined as the time of the fever onset, he developed fever (37.5–39.5°C), arthralgia, retro-orbital pain, waist pain, and abdominal pain on Dec 27 (disease day 0). His clinical examination data were: fever (39.2°C); face exanthema; white blood cell count, 3.8 × 109/L (reference range, 4–10 × 109/L); platelet count, 31 × 109/L (reference range, 100–300 × 109/L); and urine protein +++. He had no previous history of renal disease, blood urea nitrogen, creatinine, β2-microglobulin levels, and urinalysis results were within normal limits from Dec 27, 2013 to Jan 14, 2013. No apparent renal dysfunction was present. By Jan 4, 2013 (day 8), his headache, waist pain, and abdominal pain subsided, and no rashes or hemorrhage were present. There was no bleeding or bruising under the skin, and no pachyemia, or hepatomegaly.
Nucleotide sequence of primers and probe used in the RT-qPCR assay*
TaqMan MGB probe
Results of real time RT-PCR and serum IgM tests
Real time RT-PCR
Serum antibodies (IgM)
NS1 antigen colloidal gold test
The viral RNA was reverse transcribed using the reverse primer with AMV reverse transcriptase (Promega Corporation, Madison, WI, USA). The reverse transcription reaction was carried out at 42°C for 1 h. Further conventional PCR amplification covering the full genome with 16 sets of DENV specific primers succeeded in cDNA template. Amplified products were detected using agarose gel electrophoresis and sequence identification. The sequencing was performed using an ABI PRISM 3730 DNA Sequencer. Overlapping sequencing fragments were assembled to generate a contiguous full-genome sequence (10723 bp) using Lasergene (DNASTAR, Madison, WI, USA). The complete DENV genome was submitted to GenBank (accession number KF479233).
The assembled sequence isolated from the patient’s urine was aligned with NCBI BLASTn default parameter against the non-redundant nucleotide database (nr/nt). An E-value of 0 was used as the cutoff. The cutoff of the query coverage and the identity were >99% and >95% above, respectively. The result indicated that all query hits were dengue virus type 2, but the hits were variant strains from different South Asian countries.
Besides the early clinical diagnosis at the onset of symptoms, our results indicated that urine sample analysis was a noninvasive diagnostic approach, and the timeframes for positive detection in urine were longer than that in serum . Serum samples can have a high detection rate (>50%) in the first week after the onset of infection. However, the last positive detection of virus in serum samples was no later than disease day 11. Urine sample detection rates can be >50% between disease day 6 to 16, with the last positive detection of virus on day 16 . In this case, our data were consistent with previous studies that reported a longer timeframe of positive detection (Table 2). The full DENV-2 genome was sequenced from a urine sample on disease day 18, which was the longest timeframe reported for DENV positive detections. Based on the results of previous studies, the application of DENV detection from urine samples has an important future role for a fast, accurate and noninvasive diagnosis [4, 6, 14].
Data on the geographical and phylogenetic relatedness of DENV strains provide further evidence on between-country transmission of imported strains. Phylogenetic analysis provided strong evidence to confirm that the sample was associated with the DENV type 2 India group. The most similar strain to the KF479233 strain was isolated in Singapore (bootstrap values, 100%), and indicated that imported from India . The second closest strain to the KF479233 strain was isolated in India in 2009 (JN568258.1); it has been confirmed as an imported case in Australian residents returning from India . DENV-2 was emerged as the prominent serotype outbreaks in Chennai, Tami Nadu, from the 1970s to the 2000s (Figure 1) . The high endemic DENV outbreaks were mostly observed during the rainy season (Oct to Dec) in Chennai, which coincided with the time of travel for the patient in this report . The phylogenetic and epidemiological analysis confirmed that the KF479233 serotype was the DENV-2 imported from India.
DENV infection can develope into two types of clinical illness, dengue fever and more severe DHF or DSS. The first single serotype infection will result in a lifelong immunity to the infecting serotype. During a heterotypic DENV secondary infection, the cross-reactivity will enhance DENV replication and may cause severe DHF or DSS. Although secondary infection by any of the DENV serotypes may cause a severe symptom, not all infections present the same risk for severe development. Early studies of DHF in Thailand suggested that secondary DENV-2 infection was 5–7 times more frequent compared with secondary DENV-1, DENV-3 or DENV-4 infection . As the co-circulation of multiple DENV serotypes in endemic dengue areas, in cases of international travelers, there are more chances of exposure to a cross-reacting transmitted DENV infection. The identification of DENV serotypes present in a primary infection will provide a clear description of the clinical diagnosis for clinicians, and play an important role as an evidence supports for understanding of the mechanism of DHF/DSS and the research of cross-reactivity of different serotype antibodies in a secondary infection. As the recent research and studies reported, there is still no vaccine against DENV and no medications to treat a dengue infection. The prevention will be the most important step, and it means avoiding mosquito bites if re-traveling to an dengue endemic area. The dengue serotype-specific risk needs to monitor against re-infection on the pre-traveling advice for international travelers who will stay in dengue high risk regions in the future traveling.
In this report, we (1) provided the first evidence of a DENV infection that was imported from India to a non-endemic city of China, (2) investigated the DENV genome detection having a longer timeframe for positive detection in urine sample compared with previous studies, (3) provided the sequence results for the complete DENV-2 genome from a concentrated urine sample, and identified DENV-2 by comparing it to isolates from nearby geographic areas, (4) discussed medical advice on the risk of sero-specific and re-infected travel-associated dengue fever based on the virus-typing results.
Written informed consent was obtained from the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
We would like to thank Dr. Kongxin Hu for his critically reviewing and technical support. This work was supported by the grant from Development of Imported Infectious Disease Detected Methods (2013ZX10004101-006), and CAIQ Research Fund: The Evaluation of Imported Non-invasive Hemorrhagic Fever Diagnosis and Surveillance (2014JK008).
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