Diagnostic accuracy of a rapid E1-antigen test for chikungunya virus infection in a reference setting.

OBJECTIVES: Rapid diagnostic tests targeting virus-specific antigen could significantly enhance the diagnostic capacity for chikungunya virus infections. We evaluated the accuracy of an immunochromatographic antigen test for diagnosis of chikungunya in a reference laboratory for arboviruses. METHODS: An immunochromatographic rapid test that uses mouse monoclonal antibodies as a tracer against the E1-envelope protein of chikungunya (ARKRAY, Inc. Kyoto, Japan) was evaluated. Sensitivity was tested in sera from travellers with RT-PCR confirmed chikungunya virus infection (Eastern/Central/Southern African (ECSA) genotype) (n=9) and from patients diagnosed during the 2014-2015 chikungunya outbreak on Aruba (Asian genotype, n=30). Samples from patients with other febrile and non-febrile illnesses (n=26), sera spiked with Flavivirus and Alphavirus reference strains (n=13, including non-spiked serum), and samples containing other selected pathogens (n=20) were used to test specificity of the E1-antigen test. RESULTS: Sensitivity of the E1-antigen test was 8/9 (88.9%, 95% CI 56.5-98.0) for the ECSA genotype, but only 10/30 (33.3%, 95% CI 19.2-51.2) for the Asian genotype. Overall diagnostic specificity was 49/59 (83.1%, 95% CI 71.5-90.5). CONCLUSIONS: The E1-antigen test we evaluated had fair diagnostic sensitivity for ECSA genotype chikungunya, but low sensitivity for Asian genotype, and poor overall specificity. Antibodies that react across genotypes will be required for further development of a rapid test for chikungunya. Performance of new tests should be evaluated against different chikungunya genotypes.

Twelve years of dengue surveillance in Belgian travellers and significant increases in the number of cases in 2010 and 2013.

During 12 years of surveillance in Belgium, dengue virus (DENV) infection was diagnosed in 676 of 7771 (8.7%) returning travellers by the use of ELISA, RT-PCR, and/or antigen detection. Men and women were equally infected. The mean age of the patients was 36.78 years (range, 3-77 years). Most of the infections occurred after a stay in Asia (55.9%), followed by the Americas (31.8%), Africa (7.2%), Oceania (1.0%), and Europe (0.4%). The number of patients coming from Africa increased as of 2009, to reach a proportion of 17% in 2011. The most prevalent serotype was DENV-1, followed by DENV-2, DENV-3, and DENV-4. Two remarkable increases in dengue incidence were noticed in 2010 and 2013.

Recovery of dengue virus from urine samples by real-time RT-PCR.

Recently, reverse transcription polymerase chain reaction (RT-PCR) for dengue virus (DENV) has been reported to test positive in urine samples for a longer time frame than in serum. We evaluated two RNA extraction procedures from urine and investigated the stability of DENV RNA in urine and serum up to 1 year at different storage temperatures. In addition, 24 urine samples collected from patients with a recent infection were tested with DENV real-time RT-PCR and compared to the RT-PCR results on serum. Five patients with an acute DENV infection were followed up for 6 months by RT-PCR on urine. The automated extraction method with the MagNA Pure LC 2.0 device had a higher yield of DENV RNA compared to the manual QIAGEN method, explained by the higher volume used in the former method. DENV RNA in both serum and urine was stable at room temperature up to 1 month and at 4 °C and -20 °C for at least 1 year. The detection rate by RT-PCR on urine was 50 % (4/8) until day 7, 100 % (6/6) between 1 and 3 weeks after symptom onset, and 25 % (2/8) thereafter. Generally, DENV RNA concentrations are higher in serum than in urine up till day 7, switching to lower concentrations in serum thereafter. Peak concentrations in urine are reached around day 10, and RNA becomes undetectable 3 to 4 weeks following disease onset. This diagnostic tool is of added value in clinical settings by extending the period during which DENV infections are diagnosed by RT-PCR.

Chikungunya virus and West Nile virus infections imported into Belgium, 2007-2012.

Arboviral infections are emerging among tourists travelling to (sub)tropical regions. This study aims to describe the importation of chikungunya virus (CHIKV) and West Nile virus (WNV) into Belgium over a 6-year period from 2007 to 2012. Clinical samples were obtained from travellers presenting at the outpatient clinic of the Institute of Tropical Medicine (ITM), Antwerp, Belgium or submitted to the Central Laboratory for Clinical Biology of the ITM. Testing was performed by serology and/or by real-time reverse transcriptase-polymerase chain reaction. A total of 1288 returning travellers were investigated for CHIKV infection resulting in 34 confirmed and two probable diagnoses (2·80%). Out of 899 patients, four confirmed and one probable imported WNV infections were diagnosed (0·55%). No locally acquired cases have been registered in Belgium until now and the geographical origin of the imported infections reflects the global locations where the viruses are circulating.

Heat-shock protein 70 gene sequencing for Leishmania species typing in European tropical infectious disease clinics.

We describe Leishmania species determination on clinical samples on the basis of partial sequencing of the heat-shock protein 70 gene (hsp70), without the need for parasite isolation. The method is especially suited for use in non-endemic infectious disease clinics dealing with relatively few cases on an annual basis, for which no fast high throughput diagnostic tests are needed. We show that the results obtained from this gene are in nearly perfect agreement with those from multilocus enzyme electrophoresis, which is still considered by many clinicians and the World Health Organization (WHO) as the gold standard in Leishmania species typing. Currently, 203 sequences are available that cover the entire hsp70 gene region analysed here, originating from a total of 41 leishmaniasis endemic countries, and representing 15 species and sub-species causing human disease. We also provide a detailed laboratory protocol that includes a step-by-step procedure of the typing methodology, to facilitate implementation in diagnostic laboratories.

Validation of a four-primer real-time PCR as a diagnostic tool for single and mixed Plasmodium infections.

Although microscopy remains the reference standard for malaria diagnosis, molecular tools are attracting increasing interest. To improve the detection of mixed infections, we developed a four-primer real-time PCR with four Plasmodium species-specific forward primers, based on the pan-primer design with universal Plasmodium primers as described previously. After validation for analytical sensitivity, specificity and reproducibility, the four-primer PCR was evaluated on 351 blood samples from patients presenting at the outpatient clinic of the Institute of Tropical Medicine (Belgium). With the four-primer PCR, we identified 188 Plasmodium falciparum (Pf), 54 Plasmodium vivax (Pv), 52 Plasmodium ovale (Po) and 13 Plasmodium malariae (Pm) single infections, 27 mixed infections (14 Pf + Pm; 12 Pf + Po; one Pv + Pm) and 17 negative specimens. We found lower cycle threshold values than with the pan-primer PCR, with a mean difference of 2.23, a higher analytical sensitivity (in asexual parasites/µL: Pf/Pv, 0.02; Po, 0.004; Pm, 0.006) and 15 extra mixed infections. As compared with microscopy, 17 extra mixed infections were detected and Plasmodium species were identified in four microscopy-positive samples in which species identification was not possible. Additionally, the PCR corrected 13 species mismatches between Po and Pv, and in 11 cases detected Pf as a second species that was not identified by microscopy and in five of them was not detected by rapid diagnostic tests (RDTs). PCR confirmed the presence of Pf in 30/46 histidine-rich protein-2-positive samples that were microscopy-negative. We conclude that the presently developed four-primer real-time PCR is complementary to standard malaria diagnostic tests in clinical laboratories, with an added value for simultaneous identification of the four Plasmodium species and the detection of mixed infections.

Evaluation of the Immunoquick+4 malaria rapid diagnostic test in a non-endemic setting.

The aim of this retrospective study was to evaluate the Immunoquick+4 (BioSynex, Strasbourg, France), a three-band malaria rapid diagnostic test (MRDT) targeting histidine-rich protein-2 (HRP-2) and pan Plasmodium-specific parasite lactate dehydrogenase, in a non-endemic reference setting. Stored whole-blood samples (n = 613) from international travellers suspected of malaria were used, with microscopy corrected by polymerase chain reaction (PCR) as the reference method. Samples infected by P. falciparum (n = 323), P. vivax (n = 97), P. ovale (n = 73) and P. malariae (n = 25) were selected, as well as 95 malaria-negative samples. The overall sensitivities of the Immunoquick+4 for the diagnosis of P. falciparum, P. vivax, P. malariae and P. ovale were 88.9, 75.3, 56.0 and 19.2%, respectively. Sensitivity was significantly related to parasite density for P. falciparum (93.6% versus 71.4% at parasite densities >100/microl and 500/microl and

Chikungunya infection confirmed in a Belgian traveller returning from Phuket (Thailand).

Chikungunya infection has been increasingly reported in international travellers following its epidemic re-emergence in the Indian Ocean islands in 2006 and its spread to southern Asia thereafter. We describe the first case of chikungunya in a Belgian traveller returning from Phuket, Thailand and discuss the potential implications of chikungunya cases imported to European countries for patient management and public health.