Ralph Huits

Kinetics of Zika virus persistence in semen

Objective Interim guidelines to prevent sexual transmission of Zika virus (ZIKV) do not recommend diagnostic testing, because the knowledge of viral kinetics in semen after acute infection is limited. We studied the presence and duration of ZIKV in semen after onset of symptoms. Methods A prospective observational cohort study (NCT 02733796) is ongoing to determine the presence, persistence and kinetics of ZIKV in semen of men with confirmed ZIKV infection after returning from areas with vector-borne transmission. ZIKV RT-PCR is performed in weekly semen samples until ZIKV is no longer detectable. The main endpoint is the proportion of ZIKV positive semen samples over time after acute ZIKV infection. We report preliminary findings after enrolment of the first 4 patients. Findings Semen of two out of four immunocompetent men with symptomatic ZIKV infection tested positive for ZIKV-RNA by RT-PCR. ZIKV levels in semen declined to undetectable after 68 and 56 days respectively, suggesting elimination of the virus. Conclusion Our findings indicate that presence and persistence of ZIKV in semen after acute infection appear common. Algorithms can be developed that incorporate ZIKV-specific molecular and antibody detection assays and subsequent testing for ZIKV in semen of selected men. We suggest that future guidelines to prevent sexual transmission of ZIKV, adopt diagnostic testing of both symptomatic and asymptomatic individuals returning from endemic areas.

Hyperferritinaemia in dengue virus infected patients is associated with immune activation and coagulation disturbances.

Background During a dengue outbreak on the Caribbean island Aruba, highly elevated levels of ferritin were detected in dengue virus infected patients. Ferritin is an acute-phase reactant and hyperferritinaemia is a hallmark of diseases caused by extensive immune activation, such as haemophagocytic lymphohistiocytosis. The aim of this study was to investigate whether hyperferritinaemia in dengue patients was associated with clinical markers of extensive immune activation and coagulation disturbances. Methodology/Principal Findings Levels of ferritin, standard laboratory markers, sIL-2R, IL-18 and coagulation and fibrinolytic markers were determined in samples from patients with uncomplicated dengue in Aruba. Levels of ferritin were significantly increased in dengue patients compared to patients with other febrile illnesses. Moreover, levels of ferritin associated significantly with the occurrence of viraemia. Hyperferritinaemia was also significantly associated with thrombocytopenia, elevated liver enzymes and coagulation disturbances. The results were validated in a cohort of dengue virus infected patients in Brazil. In this cohort levels of ferritin and cytokine profiles were determined. Increased levels of ferritin in dengue virus infected patients in Brazil were associated with disease severity and a pro-inflammatory cytokine profile. Conclusions/Significance Altogether, we provide evidence that ferritin can be used as a clinical marker to discriminate between dengue and other febrile illnesses. The occurrence of hyperferritinaemia in dengue virus infected patients is indicative for highly active disease resulting in immune activation and coagulation disturbances. Therefore, we recommend that patients with hyperferritinaemia are monitored carefully.

Epidemiology of Guillain–Barré Syndrome in Aruba

The epidemiology of Guillain-Barré syndrome (GBS) in tropical areas is different compared with developed countries. We investigated the epidemiology of GBS on the Caribbean island of Aruba. Data were collected retrospectively from all 36 patients hospitalized with GBS between 2003 and 2011 in Aruba. We observed a seasonal distribution of GBS cases with a peak in February. The incidence rate (IR) fluctuated heavily between individual years. The overall IR was 3.93/100,000, which is higher than that observed in developed countries. Serological studies indicated a possible relation of GBS cases with dengue virus infections. We also observed a relation between the annual number of dengue cases in Aruba and the number of GBS cases in the same year. We conclude that the epidemiology of GBS in tropical areas can be different from temperate climate regions and that dengue may be a trigger for developing GBS.

Zika Virus in Semen: A Prospective Cohort Study of Symptomatic Travellers Returning to Belgium/ Presence Du Virus Zika Dans le Sperme: Etude Prospective De Cohorte De Voyageurs Symptomatiques De Retour En Belgique/El Virus del Zika En El Semen: Un Estudio Prospectivo De Cohortes De Viajeros Sintomaticos Que Regresan a Belgica

Abstract Objective To prospectively monitor Zika viral loads in semen from Belgian travellers with confirmed Zika virus infection, who returned from the Americas during the 2016 Zika virus epidemic. Methods We recruited symptomatic travellers consulting our clinic and we confirmed infection with either reverse-transcriptase (RT) polymerase chain reaction (PCR) assay or virus neutralization test. The participants produced semen samples weekly, either at the clinic or at home. For the initial sample, the laboratory staff did a microscopy analysis if they received the sample within an hour of production. Using RT–PCR, we monitored Zika virus ribonucleic acid (RNA) loads in semen until we obtained two negative results. Findings We detected Zika virus RNA in nine of 15 participants’ semen, one of whom was vasectomized. The median time to loss of RNA detection in semen was 83 days after symptom onset (95% confidence interval, CI: 57−108). The longest duration of viral shedding in semen before obtaining the first negative RT–PCR result was 144 days after symptom onset. All of the 11 participants, for whom we microscopically analysed their semen, had presence of leukocytes, 10 showed haematospermia and six showed oligospermia. These abnormalities occurred irrespective of Zika virus detection in semen. Conclusion The majority of men in our study had detectable Zika virus RNA in their semen. We recommend that semen from Zika virus-infected men should be analysed with RT–PCR and that health professionals should advise infected men, even if they are vasectomized, about current recommendations for prevention of sexual transmission of the virus.

Chikungunya virus infection in Aruba: Diagnosis, clinical features and predictors of post-chikungunya chronic polyarthralgia

Background Chikungunya virus (CHIKV) emerged in Aruba for the first time in 2014. We studied the clinical presentation of acute CHIKV infection and the contribution of serologic and molecular assays to its diagnosis. In a cohort of confirmed CHIKV cases, we analysed the frequency, duration and predictors of post-chikungunya chronic polyarthralgia (pCHIK-CPA), defined as joint pains lasting longer than 6 weeks or longer than 1 year. Methodology Patient sera obtained within 10 days of symptom onset were tested for CHIKV, using an indirect immunofluorescence test for the detection of CHIKV-specific Immunoglobulin M (IgM) and post-hoc, by reverse-transcription polymerase chain reaction (RT-PCR). CHIKV was isolated from selected samples and genotyped. For confirmed CHIKV cases, clinical data from chart review were complemented by a Telephone survey, conducted 18–24 months after diagnosis. When joint pain was reported, the duration, presence of inflammatory signs, type and number of joints affected, were recorded. Joint involvement was scored according to the 2010 ‘American College of Rheumatology/ European League Against Rheumatism’ criteria for seronegative rheumatoid arthritis (ACR-score). Risk factors for pCHIK-CPA were identified by logistic regression. Principal findings Acute CHIKV infection was diagnosed in 269 of 498 sera, by detection of IgM (n = 105), by RT-PCR (n = 59), or by both methods (n = 105). Asian genotype was confirmed in 7 samples. Clinical data were complete for 171 of 248 (69.0%) patients, aged 15 years or older (median 49.4 [35.0–59.6]). The female-to-male ratio was 2.2. The main acute symptoms were arthralgia (94%), fever (85%), myalgia (85%), headache (73%) and rash (63%). In patients with arthralgia (n = 160), pCHIK-CPA longer than 6 weeks was reported by 44% and longer than 1 year by 26% of cases. Inflammatory signs, stiffness, edema and redness were frequent (71%, 39% and 21%, respectively). Joints involved were knees (66%), ankles (50%), fingers (52%), feet (46%), shoulders (36%), elbows (34%), wrists (35%), hips (31%), toes (28.1%) and spine (28.1%). Independent predictors of pCHIK-CPA longer than 1 year were female gender (OR 5.9, 95%-CI [2.1–19.6]); high ACR-score (7.4, [2.7–23.3]), and detection of CHIKV-RNA in serum beyond 7 days of symptom onset (6.4, [1.4–34.1]. Conclusions We identified 269 CHIKV patients after the first outbreak of Asian genotype CHIKV in Aruba in 2014–2015. RT-PCR yielded 59 (28%) additional CHIKV diagnoses compared to IgM antibody detection alone. Arthralgia, fever and skin rash were the dominant acute phase symptoms. pCHIK-CPA longer than 1 year affected 26% of cases and was predicted by female gender, high ACR-score and CHIKV-RNA detection beyond 7 days of symptom onset.

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.

Clinical Utility of the NS1 Antigen Rapid Diagnostic Test in the Management of Dengue in Returning Travelers with Fever

Abstract Background Rapid diagnostic test (RDT) detecting the nonstructural 1 (NS1) antigen is increasingly used for dengue diagnosis in endemic and nonendemic settings, but its clinical utility has not been studied in travel clinic practice. Methods From August 2012 to July 2016, travelers returning from the tropics with fever were evaluated in the Institute of Tropical Medicine (Antwerp, Belgium) with the routine use of NS1 antigen RDT that provided results within 1 hour. We determined the diagnostic performance, assessed the management of patients with a positive RDT result, and compared it with that of historical cases of dengue diagnosed from 2000 to 2006, when only antibody detection assays were available. Results Of 335 travelers evaluated for fever, 54 (16%) were diagnosed with dengue, including 1 severe case. Nonstructural 1 antigen RDT was performed in 308 patients. It was truly positive in 43 of 52 tested dengue cases and falsely positive in only 1 of the 256 nondengue cases; therefore, sensitivity was 82.7% (95% confidence interval [CI], 74.4%–93.0%) and specificity was 99.6% (95% CI, 98.8%–100%). Only 3 (7%) of the 43 febrile travelers “immediately” diagnosed by RDT were admitted, and only 2 (5%) were given empirical antibacterial treatment, without adverse outcome. Admission and antibiotic prescription rates were significantly higher in the historical cases (n = 43) diagnosed by antibody detection (33%, P = .006 and 26%, P = .014, respectively), although the frequency of severe dengue was similar. Conclusions In our practice, the diagnostic performance of NS1 antigen RDT substantially contributed in withholding unnecessary hospitalization and antibiotherapy in dengue patients.

A cross-sectional analysis of Zika virus infection in symptomatic and asymptomatic non-pregnant travellers: Experience of a European reference center during the outbreak in the Americas

BACKGROUND Zika virus (ZIKV) infection a concern to travellers because of potential sexual transmission and adverse pregnancy outcomes. OBJECTIVE To describe our experience in diagnosing ZIKV in travellers returning from endemic territories. METHOD Travellers were evaluated for ZIKV at our clinic in a 12-month period during the outbreak, using ZIKV-specific RT-PCR and anti-ZIKV Immunoglobulin M/G ELISA when symptomatic, and ELISA only for asymptomatic travellers, preferably from 20 days after the last exposure. All positive ELISA results were subject to confirmation by Virus Neutralization Testing. We estimated post-test probabilities of ZIKV in asymptomatic travellers. RESULTS Of 462 travellers, 227 reported symptoms and 235 did not. Asymptomatic travellers had similar baseline characteristics, but were younger (median age 31 vs. 33 years, p = 0.01) and had reproductive concerns more often (75.8% vs. 24.2%). ZIKV infection was confirmed in 49 cases: 46/227 (20.3%) were symptomatic and 3/235 (1.3%) asymptomatic. Rash (positive likelihood ratio (LRP) 5.6) and conjunctivitis (LRP 10.8) predicted ZIKV infection. The post-test probability of a negative ELISA-result at 20-25 days was below 0.1%. CONCLUSION ZIKV infection was frequent in symptomatic, but not in asymptomatic travellers. We consider negative ELISA results at 20-25 days after exposure a safe strategy to rule out ZIKV infection. Testing for ZIKV-specific antibodies within this timeframe could be particularly valuable in the management of returning travellers who wish to conceive.

Variation at position 350 in the Chikungunya virus 6K-E1 protein determines the sensitivity of detection in a rapid E1-antigen test

Chikungunya virus (CHIKV), a mosquito-borne pathogen, consists of three genotypes: East/Central/South African (ECSA), West African (WA), and Asian. Although a current rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity to ECSA-genotype viruses, it showed poor performance against the Asian-genotype virus that is spreading in the American continents. To understand the basis for the low performance of this IC test against Asian-genotype virus, we re-examined the anti-CHIKV monoclonal antibodies (mAbs) used in the assay for their interaction with E1-antigen of the three CHIKV genotypes. We found that the reactivity of one mAb for Asian-genotype virus was lower than that for ECSA virus. Comparison of E1 amino acid sequences revealed that the ECSA virus used to generate these mAbs possesses glutamic acid (E) at position 350, in contrast to WA and Asian, which possess aspartic acid (D) at this position. Site-directed mutagenesis confirmed that the mutation altered mAb reactivity, since E-to-D substitution at position 350 in ECSA reduced recognition by the mAb, while D-to-E substitution at this position in Asian and WA increased affinity for the mAb. Taken together, these results indicate that residue 350 of the CHIKV 6K-E1 is a key element affecting the performance of this IC assay.

Re: Lack of Zika virus antibody response in confirmed patients in non-endemic countries

Zika virus (ZIKV) has spread in the last 2 years throughout America and South-Eastern Asia causing a widespread epidemic [1]. Detection of ZIKV RNA in body fluids confirms ZIKV infection, however ZIKV antibody testing is much more complex due to possible cross-reactivity with closely related flaviviruses [2]. From December 2015 to February 2017, 401 patients from eight reference laboratories in the Czech Republic, Israel, Italy, the Netherlands, Romania, Slovenia, and the United Kingdom had been confirmed for ZIKV infection by detection of ZIKV RNA in body fluids [2–4] (Table 1). Of these 401 patients, 148 were negative for ZIKV directed against IgM and IgG in serum collected at the time of PCR-positivity as tested by ELISA (7 laboratories, Euroimmun, Lübeck, Germany) or IFA and ELISA (2 laboratories, Euroimmun, Lübeck, Germany). For 80 of these 148 seronegative confirmed patients a second, follow-up serum sample was available. Altogether, 5 of these 80 patients remained without seroconversion in consecutive samples (Table 2) for ZIKV antibodies tested by ELISA and virus neutralization (VNT) (Table 2). The acute samples of these 5 patients were re-extracted and retested from original material which confirmed the presence of ZIKV RNA. Material from patients 1 and 2 were sequenced [5]. Ideally, each of the samples from the 5 patients would also have been tested in at least one of the other laboratories, but because of insufficient clinical material, this wasn’t possible. Most importantly, none of the sero-negative patients had any indication of immune-deficiency. Two patients were pregnant. One explanation for the lack of detection of ZIKV IgM or IgG antibodies in 5 of our patients is low sensitivity of the assays. Indeed, a few studies have previously demonstrated low sensitivity of the Euroimmun NS1 ELISA [6–9]. However, since neutralization is widely accepted as the gold standard test for arboviral infections and unlike the NS1 ELISA, neutralization primarily recognizes antibodies against surface proteins, the probability that both tests failed to detect ZIKV antibodies is low. Another explanation is that production of ZIKV antibodies was suppressed in these cases maybe due to a previous flavivirus infection which might suppress ZIKV immune response including the production of neutralizing antibodies (original antigenic sin [10,11]). In conclusion, our results show absence of ZIKV specific antibodies using routine serological assays in 5 of 80 of convalescent sera from PCR confirmed ZIKV cases in returning travelers. This may suggest significant under-diagnosis of ZIKV infections when diagnosis relies on serology alone. This is especially of importance in cases where congenital Zika syndrome might be involved such as diagnosis of pregnant women or males with pregnant partners. As serum of pregnant women, whole blood and semen provide a longer window of detection for PCR [12–15], these samples should be tested by RT-PCR alongside serology. Relating the absence of detectable ZIKV immune responses to the absence/severity of clinical symptoms and previous flavivirus antigen exposure in larger cohort studies might provide insight into the groups at risk for such under-diagnosis.