Abstract
We report the first two cases of imported Zika virus (ZIKV) infection in Romanian patients returning from areas with ongoing outbreaks and challenges for laboratory diagnostic; first one with a classical pattern of acute flaviviral infection and the second one with an interesting pattern of a secondary flaviviral (ZIKV) infection in a yellow fever-vaccinated child living abroad in an endemic area.
Introduction
Z
We describe the first two cases of Zika infection in Romanian patients returning from areas with ongoing outbreaks and the challenges for laboratory diagnostic.
Case Reports
Patient 1
On July 5, 2016, a 27-year-old Romanian woman presented to the “Dr. Victor Babes” Hospital of Infectious and Tropical Diseases, Bucharest, Romania, returning from a holiday in Martinique (June 26–July 2). She reported mosquito bites in Martinique. On July 3, she developed a diffused erythematous rash on the trunk, arms, and legs, accompanied by fever, arthralgia, headache, and asthenia. The intensity of the clinical picture was mild with fever remission in 24 h and exanthema disappearance at day 7. The articular manifestations persisted till day 5.
Laboratory tests on day 3 after symptom onset showed leucopenia (2977/mm3) with neutropenia (1526/mm3) and mild anemia (Hb 11.45 g/dL). Blood cell count became normal after 6 days. ZIKV infection was confirmed based on the molecular detection of ZIKV genome in acute serum and seroconversion for ZIKV-specific IgM and IgG (Table 1).
Molecular tests: 1Dengue Real-TM Genotype (Sacace Biotechnologies, Como, Italy); 2aZika Virus Real-time RT-PCR in house TaqMan assay using ZIKV 1107-FAM probe, and ZIKV 1086 and ZIKV 1162c primers, after Lanciotti et al. (2008); 2bZika virus Real-TM (Sacace Biotechnologies, Como, Italy); 3West Nile Virus Real-TM (Sacace Biotechnologies, Como, Italy).
Serological tests
ELISA tests: 4a,4bAnti-Dengue Virus ELISA IgM and IgG (Euroimmun, Lübeck, Germany); 4cDengue Virus NS1 ELISA (Euroimmun, Lübeck, Germany); 5a,5bAnti-Zika Virus ELISA IgM and IgG (Euroimmun, Lübeck, Germany); 6a,6bAnti-West Nile Virus ELISA IgM and IgG (Euroimmun, Lübeck, Germany).
Interpretation for ELISA: For IgM and IgG ELISA: Antibodies index value: Negative <0.8; Borderline 0.8–1.1; Positive >1.1. For NS1 Dengue antigen: Negative <0.8; Borderline 0.8–1.1; Positive >1.1.
Indirect Immunofluorescence Antibodies: 7Anti-Yellow fever IIFT—IgG (Euroimmun, Lübeck, Germany); 8 Rickettsia conorii IFA IgG (Vircell, Granada, Spain).
Bold letters for positive or borderline results.
Ct, cycle threshold; Neg, negative; NS, no sample; NT, not tested; Pos, positive.
Patient 2
On July 15, 2016, a 10-year-old child presented to our hospital on the third day after arriving in Bucharest; the child lived in French Guyana for the last 2 years where he received the yellow fever (YF) vaccination. He left French Guyana on 6th of July and traveled by car through southern France, Italy, Slovenia, Serbia, and Croatia until 12th of July. The parents reported that the child had been suspected with Zika infection on April in French Guyana, but only on clinical symptoms. After arriving in Bucharest, on 13th July, he developed mild fever (37.6°), dysphagia, and micropapular rash on trunk, arms, and legs, which disappeared after a few hours and reappeared after 48 h. He addressed to hospital with mild dysphagia and erythematous maculopapular rash on the trunk, arms, and legs, painless mobile bilateral adenopathy, hyperemic pharynx. Laboratory tests performed were normal.
ZIKV infection was confirmed based on the genome detection in urine using two different Real-time PCR assays (Table 1). Serological tests for ZIKV, dengue (DENV), yellow fever virus (YFV), and West Nile virus (WNV) show a significant increase in YFV-specific IgG antibodies and a seroconversion for IgG against WNV and DENV. Enzyme immunoassay tests detecting IgM and IgG against NS1 nonstructural antigen of ZIKV showed only borderline IgM antibodies in the convalescent serum, but no seroconversion of IgG specific to NS1 antigen of ZIKV was detected (Table 1).
Discussion
These are the first cases in Romania diagnosed with ZIKV infection. In case 1, the pattern of ZIKV genome detection and of IgM and IgG antibodies dynamics against ZIKV, with IgM documenting acute infection, was typical for a flavivirus naive individual. In case 2, a vaccinated for YF individual, the serological testing against ZIKV and the other flaviviruses tested (WNV, DENV, YFV) revealed a different dynamics, which may be attributable to the vaccination status of this patient. Indeed the difficulty of serological diagnostic in a case of ZIKV infection after YF vaccination was already reported (Filipe et al. 1973). The mainstays of the routine diagnosis in ZIKV infection are the detection of viral nucleic acid by RT-PCR and the detection of IgM by enzyme-linked immunosorbent assay (ELISA) (Petersen et al. 2016). The confirmatory tests for the diagnostic of ZIKV infection in case 2 was the detection of ZIKV genome in urine by both an in-house and a commercial real-time RT-PCR test. Molecular testing positive for urine and negative for serum, early postonset of ZIKV infection, was not uncommon (Leparc-Goffart, personal communication). A suspicion of previous ZIKV infection, based on clinical symptoms and epidemiological context only, was not confirmed by our laboratory results. We excluded the possibility of a persistent ZIKV infection with urine virus shedding because the serological pattern was suggestive for an acute secondary flavivirus infection. Cross-reactivity among flaviviruses is a major challenge for diagnosis, and because antibodies to nonstructural protein 1 (NS1) were largely ZIKV-specific, they were used to develop a serological diagnostic tool (Stettler et al. 2016), the ELISA commercial tests we used being based on NS1 antigen for both IgM and IgG ZIKV-specific detection.
Secondary flavivirus-infected persons show high degree of serologic cross-reactivity with other flaviviruses, and a higher titer against other flaviviruses than the virus causing the current infection may be interpreted as a demonstration of the “original antigenic sin” phenomenon already observed among flaviviruses (Petersen et al. 2016). In cases of Zika infections as secondary flaviviral infections, the seroneutralization test performed poorly in identifying the virus causing the actual infection. Indeed, previous reports showed that if ZIKV is the secondary flavivirus infection, the individual may not have the highest neutralizing titer to ZIKV compared with other flaviviruses, but against the flavivirus causing the primary infection, cross-reactivity to other heterologous flavivirus antigens being also observed (Lanciotti et al. 2008, Rabe et al. 2016). The case–patient showed seroconversion for IgG targeting envelope antigens of YF, DENV, and WNV, but not for the NS1 antigen of ZIKV. A borderline level of IgM antibodies to NS1 antigen of ZIKV was detected in the convalescent serum only, a pattern which is frequently found in secondary infections.
The NS1 antigen-based ELISA showed previously high specificity for ZIKV primary infections (Huzly et al. 2016), and no cross-reactivity to sera from acute DENV and tick-borne encephalitis virus acute infections, or from persons vaccinated for tick borne encephalitis or YF, but the dynamics of antibody response to NS1 antigen of ZIKV infection as a secondary infection post-YF vaccination was yet not reported.
Rapid diagnosis of disease in returning travelers, testing all asymptomatic pregnant women returning from endemic areas, and the control of local mosquito population should be considered, taking into account that potential vector species (A. albopictus) are established (Prioteasa et al. 2015) in urban areas in southern Romania.
Footnotes
Author Disclosure Statement
No competing financial interests exist.