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Laboratory findings in Zika infection: The experience of a reference centre in North-West Italy
Journal of Clinical Virology 101 (2018) 18–22
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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Laboratory findings in Zika infection: The experience of a reference centre in North-West Italy
T
⁎
Elisa Burdinoa, , Maria Grazia Miliaa, Tiziano Allicea, Gabriella Gregoria, Tina Ruggieroa, Guido Callerib, Filippo Lipanic, Anna Lucchinic, Giulietta Venturid, Giovanni Di Perric, Valeria Ghisettia a
Laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, Turin, Italy Unit A of Infectious and Tropical Diseases, Amedeo di Savoia Hospital, Turin, Italy c Department of Infectious Diseases, Amedeo di Savoia Hospital, Turin, Italy d National Reference Laboratory for Arboviruses, Department of Infectious, Parasitic and Immune- Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy b
A R T I C L E I N F O
A B S T R A C T
Keywords: Zika virus Travelers Real time RT-PCR Blood Urine
Background: Zika virus (ZIKV) remains a public health concern due to its association with fetal malformation and neurologic disease. Objective: To report a reference centre experience on ZIKA virus (ZIKV) infection in travelers from epidemic countries from January 1 to September, 30, 2016 in Italy North-West (a geographic area covering 4.424 million inhabitants, corresponding to almost 73% of Italy North-West area). Study design: One hundred and twelve febrile travelers were studied to rule out a tropical fever [e.g. malaria, dengue (DENV), chikungunya (CHIKV), West Nile (WNV) and ZIKV]. Molecular tests for detecting ZIKV RNA were applied on serum or urine as well as IgG and IgM specific serology. Results: ZIKV was the most frequent “tropical infection (11.6%) with 12 infected travelers and one sexual partner of an infected traveler. At the time of the diagnosis, ZIKV RNA was detected in the blood from 9 patients (69%) within 7 days from symptom onset; afterwards, the virus was detected only in urine (5 patients) and ZIKV IgM was reactive in 9 patients (69%). Travelers with ZIKV infection tested negative for DENV, CHIKV, WNV and malaria and completely recovered. Other infections identified in travelers were DENV (5 patients, 4.5%), CHIKV (1, 0.9%), malaria (Plasmodium vivax, 1, 0.9%), measles (1, 0.9%) and tuberculosis (1, 0.9%). Conclusions: The etiologic diagnosis of a febrile illness in travelers where ZIKV is endemic is highly desirable as they are sentinel of a challenging epidemiology including the risk of autochthonous transmission in non endemic countries where the competent or carrier vector is present.
1. Background Zika virus (ZIKV), a mosquito-borne member of the Flaviviridae family, is the focus of a great public health concern due to its association with fetal malformation and neurologic disease. While ZIKV was previously limited to sporadic cases in Africa and Asia, but in the year 2015 it rapidly spread to Brazil and throughout the Americas and Caribbean causing 0.5–1.5 million human infections [1–5]. Moreover, in 2016, due to the Olympic Games in Brazil, an increasing number of travelers from the Americas have been observed during the period of vector activity (May–November). Neither an effective treatment nor a vaccine
is available; therefore, prevention, surveillance and accurate diagnosis are the primary public health response. Since 2007, 31 of the 70 countries and territories reporting evidence of ZIKV transmission (67 from the year 2015 onwards) reported also the association of the disease with microcephaly and other CNS malformations [6]. Although a decline of Zika infection has been recently reported by the last WHO estimates, vigilance needs to remain high [7] and increasing reports of imported ZIKV cases have been observed throughout Europe, United States and Asia [6,8–11]. From a public health perspective the etiologic diagnosis of febrile travelers from countries where ZIKV is epidemic, is highly desirable and
Abbreviations: ZIKV, Zika virus; PCR, Polymerase Chain Reaction; RT-PCR, Reverse Transcription PCR; rtRT-PCR, real time RT-PCR; PRNT, Plaque Reduction Neutralization Tests; PNRT80, PRNT ≥ 80% of plaque reduction; GEN, Genesig Advanced Kit; ALT, Altona Diagnostics RealStar Zika virus RT-PCR Kit 1.0; RU, relative units; ELISA, Enzyme-Linked Immunosorbent Assay; CV, coefficient of variation; Ct, cycle threshold ⁎ Corresponding author at: Laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, Department of Infectious Diseases, Corso Svizzera 164, 10149 Torino, Italy. E-mail address: [email protected] (E. Burdino). https://doi.org/10.1016/j.jcv.2018.01.010 Received 15 May 2017; Received in revised form 11 January 2018; Accepted 20 January 2018 1386-6532/ © 2018 Elsevier B.V. All rights reserved.
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3.2. Diagnostic tests for ZIKV infection
efforts in this direction are highly recommended, as travelers can be a sentinel of rapidly challenging epidemiology including the risk of autochthonous transmission in non epidemic countries where the competent or carrier vector is present. Currently, the diagnosis of ZIKV infection relies on the detection of the virus by Reverse Transcription Polymerase Chain Reaction (RTPCR)-based tests especially useful during the acute phase of the disease (that is within 7 days from symptom onset) and the identification of specific antibodies. Accurate detection of ZIKV in biological samples can be highly demanding due to the low viral load and the short viraemic period, implying the risk of false-negative results in the acute phase. To increase the diagnostic sensitivity in this stage of the disease, testing other specimens than plasma/serum, such as whole blood [12], urine and more recently saliva [13] is now highly recommended as viral shedding is longer in these compartments [8,14]. As observed by Gourinat [15] and Bingham [16] urine might be the preferred specimen to identify acute/recent ZIKV disease because viral RNA can be detected at higher levels for longer periods of time than in serum; moreover, urine has the advantage of a much easier sample to collect than blood. ZIKV serology should be considered in those patients with a high suspicion of ZIKV infection in whom ZIKV RNA is negative by RT-PCR and whose symptoms last for more than one week. Serology has substantial limitations [17], mainly due to the high cross-reactivity among flavivirus antibodies, but recently, ELISA tests based on ZIKV NS1-antigen have been developed for a more specific identification of ZIKV antibodies [18]. Final evaluation of serologic results needs to be confirmed by the detection of neutralizing antibody that remains the gold standard, although this test is restricted to reference laboratories due to its high complexity [19].
One hundred eighty-five specimens were processed (130 serum and 55 urine) for ZIKV molecular testing and/or ZIKV serology. For the detection of ZIKV RNA in serum and urine, a CE-marked real time (rt) RT-PCR assays (RealStar Zika virus RT-PC Kit 1.0, Altona Diagnostics, Germany) was used, while quantification of ZIKV RNA in positive samples was performed with the Genesig Advanced Kit (Primerdesign Ltd, United Kingdom) using a plasmid as standard curve from 2 × 105 to 2 copies/μl after ZIKV RNA extraction from 600 ml of serum and urine using the semi-automated system NucliSENS easyMAG (Biomérieux, France), according to manufacturer’s protocols. Performances of the two molecular tests was done with the Asian reference strain ZIKV H/PF/2013 kindly provided by the Istituto Superiore di Sanità (ISS), Rome. All patients underwent ZIKV IgG and IgM antibody detection with a commercial ELISA assay (Euroimmun AG, Germany) against ZIKV NS1 protein [18]; confirmation with Plaque Reduction Neutralization Tests (PRNT) was performed at the ISS, as previously described [22], in those patients in whom serology was positive but ZIKV RNA was not detectable in serum/urine (therefore classified as probable cases according to the Italian Ministry of Health and WHO case definition for ZIKV virus disease [21]). Briefly, the assay was prepared in six-well tissue culture plates with sub confluent VERO cell monolayers and the ZIKV H/PF/2013 strain of the Asian genotype (kindly provided by Dr. Isabelle Leparc-Goffart of the French National Reference Center on Arboviruses in Marseille). Patient sera were diluted 1:10 in serum-free maintenance medium. Equal volumes (100 μl) of ZIKV dilution containing approximately 80 Plaque Forming Units (PFU) and serum dilutions, were mixed and incubated overnight at 4 °C. Subsequently, VERO cells plates were infected with 200 μl/well of virus-serum mixtures in duplicate, incubated at 37 °C (5% CO2) for 4 days, and stained with 1.5% crystal violet. A titration of ZIKV with three dilutions in duplicate was performed in each assay as control and neutralizing antibody titers were calculated as the reciprocal of the serum dilution that gave an 80% reduction of the number of plaques (PRNT80), compared to the virus control. PRNT80 ≥ 10 were considered positive. Diagnosis of DENV, CHIKV and WNV infection was ruled out with rapid tests against to NS1 DENV antigen, DENV IgM and IgG (Panbio ALERE, I) and indirect immunofluorescence for both CHIKV and WNV IgG and IgM (Euroimmun AG, Lubek, Germany). A multiplex rtRT-PCR was applied to detect DENV and CHIKV RNA in serum (Dengue/Chik, FastTrack Diagnostics assay) and WNV RNA was identified with the West Nile Virus ELITe MGB® Kit (ELITechGroup, Italy).
2. Objective To report ZIKV infection in febrile travelers returning from epidemic areas (Venezuela, Dominican Republic, Puerto Rico, Mexico, Costa Rica), who referred at the regional Centre for Infectious Diseases, Amedeo di Savoia Hospital, Turin, Italy (a geographic area characterized by the widespread presence of the ZIKV vector Aedes albopictus and serving 4.424 million inhabitants), between January 1 and September 30, 2016 [19,20]. Ours represents a significant cohort of ZIKV imported infection in North-West Italy within the national surveillance system that since January 2014 has been enhanced for ZIKV (Ministero della Salute. Sorveglianza dei casi umani di chikungunya, dengue, West Nile Disease ed altre arbovirosi e valutazione del rischio di trasmissione in Italia 2015. 0020115-16/06/2015-DGPRE-COD_UO-P. 2014:1–135) and accounts for approximately 60 notified ZIKA cases in the year 2016. (http://www.iss.it/binary/arbo/cont/Casi_confermati_Chikungunya_ Den_Zika.pdf; data not-published).
4. Results According to WHO case definition, ZIKV infection was identified in 13 out of 112 febrile (11.6%) patients (Table 1): 12 were travelers (ID#1-8 and ID#10-13), one was the sexual partner of an imported case (ID#9). Twelve patients (92%) reported maculopapular rash (with itching in half of them); 9 (69%) arthralgia/myalgia, 4 (30.7%) headache and/or retrorbital pain, 3 presented with conjunctivitis (23%), malaise and fatigue (23%) and 1 with diarrhea (7.7%). Leukopenia (< 4000 WBC/μl) was present in 4 patients, while impaired liver function in two. Paracetamol and steroids for uncontrollable itching were administered and all patients completely recovered within 1 week without hospitalization. A case most likely of sexual transmission of ZIKV was suspected in the female partner (ID#9) of a male traveler (ID#5) returning home from the Dominican Republic with ZIKV infection. The partner (patients ID#9) had no history of travelling abroad and of mosquito bites, but reported a sexual intercourse with travel ID#5 after his returning home and falling ill. The female partner had fever, a maculopapular itchy rash and ZIKV RNA was detected in urine, but not in serum. Other body fluids from the two partners were not available for testing, therefore, a
3. Study design 3.1. Patients and samples Febrile travelers (N = 112) returning home from ZIKV epidemic areas reporting symptoms and signs such as fever, maculopapular and itchy rash and/or arthralgia/myalgia who referred to the outpatient Clinic of Travel Medicine at the regional reference Centre for Infectious Diseases, Amedeo di Savoia Hospital, Turin, from January, 1, to September, 30, 2016 were studied. A suspicion of ZIKV infection was posed. Patients underwent hematological, biochemical, microbiologic, serologic and virological examinations to rule out a tropical fever [e.g. malaria, dengue (DENV), chikungunya (CHIKV), West Nile (WNV) and ZIKV fever] and the Italian Ministry of Health and WHO case definition for ZIKV, CHIKV, DENV and WNV disease were followed [21].
19
20
29 Male / > 15 days Jan 9 Jan 6 Jan 11
Yes Yes Yes No No Yes Yes No Yes
Age (yrs) Sex Pregnancy Stay duration Date of returning to Italy Date of symptom onset Date of sampling
Symptom Fever Maculopapular rash Itching Conjunctivitis Headache Retroorbital pain Arthralgia/Myalgia Diarrhea Malaise/Fatigue
Paracetamol Recovery (4 days)
ALT x 4
Normal
Paracetamol Recovery (1 week)
Normal
Normal
Normal
Yes Yes No No No No Yes No No
31 Female No > 15 days Jan 9 Jan 4 Jan 11
Venezuela
ID# 2
Steroid Recovery (2 days)
Normal
Normal
3880
Yes Yes Yes Yes No No Yes No No
Dominican Republic 40 Female No 30 days Apr 8 Apr 11 Apr 17
ID# 3
NA = Not available; ALT = alanine aminotransferase. a Sexually transmitted, sexual partner of patient ID#5. b Patients ID#11 and ID#12, brother and sister. c Indicated as normal (within reference value) or x upper reference value.
Treatment Outcome
Thrombocytopenia (PLTS Count < 140,000 μl) Liver functionc
3500
Venezuela
Travelling from
Laboratory test (reference) Leukopenia (WB Count < 4,000 μl)
ID# 1
Characteristic
No therapy Recovery
Normal
Normal
Normal
Yes Yes Yes Yes No No Yes Yes No
Dominican Republic 30 Male / 15 days May 26 May 30 Jun 3
ID# 4
No therapy Recovery
NA
NA
NA
Yes Yes No No No No No No No
Dominican Republic 20 Male / 30 days July 11 July 6 July 15
ID# 5
No therapy Recovery
Normal
Normal
Normal
Yes Yes No No No No No No Yes
29 Male / 15 days July 4 July 11 July 15
Puerto Rico
ID# 6
Table 1 Clinical and epidemiological characteristics of the 13 patients with confirmed Zika infection (January–September 2016).
No therapy Recovery
NA
NA
NA
Yes Yes No No No No Yes No No
Dominican Republic 21 Female No 30 days July 14 July 7 July 18
ID# 7
No therapy Recovery
NA
NA
NA
Yes Yes No No No No Yes No No
Dominican Republic 18 Male / 30 days July 14 July 4 July 18
ID# 8
No therapy Recovery
NA
NA
NA
Yes Yes Yes No Yes No Yes No No
Sexual transmissiona 19 Female No 15 days July 25 July 17 July 28
ID# 9a
No therapy Recovery
NA
NA
Normal
Yes Yes Yes No No Yes Yes No No
No therapy Recovery (1 week)
Normal
123,000
3610
Yes Yes Yes Yes Yes Yes Yes No Yes
21 Male / 15 days August 29 August 26 August 29
Costa Rica
Mexico 28 Male / 4 months August 23 August 19 August 29
ID# 11b
ID# 10
No therapy Recovery (1 week)
Normal
Normal
2320
Yes No No No No No No No No
23 Female No 15 days August 29 August 27 August 29
Costa Rica
ID# 12b
No therapy Recovery
ALT x 2
Normal
Normal
Yes Yes No No No No No No No
Dominican Republic 32 Male / 15 days August 18 August 22 August 26
ID# 13
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Journal of Clinical Virology 101 (2018) 18–22
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Table 2 Laboratory findings of ZIKV infection for the 13 patients. Patient ID
Days from symptoms onset
ZIKV ELISA IgMa
ZIKV ELISA IgGa
ZIKV RNA in serum Real-time PCRb
ZIKV RNA in urine Real-time PCRb
#1
5c 56 7c 55 6 19 28 4 13 9 4 9 14c 11 10 3 7 21 2 7 21 4c 25
Positive Negative Negative Negative Negative Negative Negative Positive Positive Positive Positive Positive Positive Positive Positive Negative Positive Negative Negative Positive Negative Positive Negative
Negative Positive Positive Positive Positive Positive Positive Negative Positive Positive Negative Positive Positive Negative Positive Negative Negative Positive Negative Positive Positive Negative Positive
Negative NA Negative NA Negative NA NA Positive (40 cps/ml) NA Negative Negative Negative Negative Negative Positive (n.q.) Positive (157 cps/ml) Negative Negative Positive (20510 cps/ml) Negative Negative NA NA
NA NA NA NA Positive (5913 cps/ml) Positive (310 cps/ml) NA NA NA Positive (1321 cps/ml) Positive (41338 cps/ml) Positive (117 cps/ml) Negative Positive (13608 cps/ml) Positive (619 cps/ml) Positive (3252 cps/ml) Negative Negative Positive (3501 cps/ml) NA Positive (n.q.) NA NA
#2 #3
#4 #5 #6 #7 #8 #9 #10 #11
#12
#13
PCR = Polymerase Chain Reaction; ELISA = Enzyme-Linked Immunosorbent Assay; NA = Not available; cps = copies; n.q. = not quantifiable. a Commercial ELISA assays (Euroimmun AG, G). Reference values: positive ≥ 22 RU/ml, negative ≤ 16 RU/ml). b RealStar Zika virus RT-PC Kit 1.0 (Altona Diagnostics, G) and Genesig Advanced Kit (Primerdesign™ Ltd, UK). See text for details on the protocol used. c Confirmatory Plaque Reduction Neutralization Test (PRNT80) performed at the Istituto Superiore di Sanità, Rome, Italy, (positive result for titers ≥ 1:10).
Analytical sensitivity of molecular tests applied for ZIKV RNA detection in this study was assessed with known concentrations of the Asian reference strain ZIKV H/PF/2013 kindly provided by the ISS. The detection rate was 100% at 1425, 142 and 14 copies/mL for both the two rtRT-PCR assays. Test specificity was probed against 10 plasma samples positive for DENV RNA and supernatants from DENV and CHIKV infected cell cultures (Quality Control Molecular Diagnostics, QCMD 2016 ZIKV External Quality Pilot Study) that tested negative for ZIKV RNA with both the assays.
confirmed match between the viruses isolated from the two patients could not be obtained. The last ZIKV infection was reported on August 2016. None of the women with ZIKV infection were pregnant. Table 2 shows ZIKV findings in the 13 infected patients. As expected, in patients referring at the outpatient clinic within 7 days from symptoms onset (N = 7, Table 2, ID#1, 3, 4, 6, 11–13), molecular tests detected the virus in the majority of them(83%, in serum and/or urine; in one patient, ID#13, molecular test were not performed due to the lack of sample); serology for IgM was positive in only 57% of them (the presence of IgG for ZIKV was found in one patient). In those patients with ZIKV infection referring later than 7 days from symptom onset (N = 6, Table 2, ID#2, 5, 7–10), the diagnosis of infection was made by molecular tests in 66.7% of them and by serology in 83% of them (Fisher’s exact test for IgG reactivity within and after 7 days from symptoms onset: p = 0.0291; no statistically significant difference for both IgM and molecular tests). At the time of diagnosis, molecular tests identified ZIKV RNA in 9 infected patients (69%), in the serum of 4 (45%, ID#4, 10–12) and in the urine of 8 (89%, ID#3, 5–7, 9–12) of them, Median viral load was lower in serum than in urine (2.7 ± 1.4 log10 copies/mL vs. 3.4 ± 0.8) and urine shedding was detectable up to 21 days after symptom onset. IgM reactivity was found in 9 patients [6 ZIKV RNA positive in serum (1 patient, ID#4) or urine (5 patients, ID#5, 6, 7, 9 and 10)]; ZIKV IgG were detected in 6 patients (4 of them with urine viral shedding, patients ID#3, 5, 7 and 10). In those patients in whom the virus was absent in both serum and urine (ID#1, 2, 8 and 13) confirmation of ZIKV antibodies was obtained by a PRNT80 titer ≥ 1:10. In 5 patients who could be monitored over time (ID#1, 4, 11–13), IgM seroconversion from negative to positive was observed in 3 of them (within 7 days from symptom onset), while ZIKV IgG became detectable from day 7 to day 21–25. Patients with ZIKV infection were negative for DENV, CHIKV, WNV and malaria and they completely recovered without hospital admission. Other infectious diseases in the traveler study-group were DENV (5 patients, 4.5%), CHIKV (1, 0.9%), malaria (Plasmodium vivax, 1, 0.9%), measles (1, 0.9%) and tuberculosis (1, 0.9%).
5. Discussion Highly sensitive molecular testing for ZIKV RNA detection in clinical samples is a key factor for the diagnosis of ZIKV infection and this also applies to ZIKV congenital disease and the safety of blood transfusion in epidemic areas. During the acute phase of the disease, ZIKV infection is generally characterized by low virus concentration in clinical specimens, irrespective of the sample type. Thus, it can be expected that a significant proportion of patients may not be diagnosed if molecular tests are insufficiently sensitive. At present, the availability of real time PCR assays provides more accurate and reproducible results with higher sensitivity and specificity than conventional PCR, significantly simplifying and accelerating the process of ZIKV detection in biological samples and in the framework of a global response to the outbreak specific quality controls are extremely helpful. A number of clinical reports [8,10,14–16] suggest that ZIKV can be found in plasma or serum and saliva a few days after the infection, and in the urine of the majority of patients approximately 1 week after symptom onset. Thus, urine testing is certainly very helpful to the diagnosis of ZIKV infection in travelers. Recent data suggests a longer recovery of the virus in whole blood than in plasma/serum [12] and that the median time for RNA loss in serum is 14 days (95th percentile: 54 days), while 8 days in urine (95th percentile: 39 days) [23]. In addition to the above mentioned critical issues, quantification of ZIKV viral load is important in investigating the risk of transmission through other different specimens considering that most likely the sexual transmission of the virus occurs as ZIKV can be detected in semen for 21
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kindly providing Zika virus strain H/PF/2013.
several weeks [24]. According to WHO case definition [21], we identified ZIKV infection by molecular testing in 9 patients (69%), while serology and PRNT confirmation allowed the identification of other 4. In our experience, urine was very helpful for ZIKV RNA detection (8/13 positive patients); in this compart viral shedding lasted for 21–25 days after symptoms onset, that is longer than in serum (about 3–4 days) and the viral load was generally higher in urine than in serum. The combination of nucleic acid testing of more than one type of specimen per patient is certainly an added value to the diagnosis of ZIKV infection. In this study, for ZIKV RNA detection we applied two commercially available real-time–PCR (e.g. Altona and Genesig) assays, whose performances were assessed with serial dilution of known concentrations of an Asian ZIKV reference strain. The analytical sensitivity of both assays was very high (100% detection rate at 14 copies/mL) and in line with the majority of published rtRT-PCR protocols for ZIKV RNA [14]. ZIKV genotype inclusivity could not be tested being the primer and probe design covered by company confidentiality. Quality controls at known concentration of ZIKV virus and external quality control programs for the standardization of molecular assays play a key role for the most appropriate diagnosis of ZIKV infection, patient management and outbreak control. In our case-series of febrile travelers the prevalence of ZIKV was higher than that of DENV or other tropical diseases, but ZIKV casefinding should also focus on sexual partners as sexual transmission of the virus has been documented and this can be a possible source of additional vector-borne spread in non-epidemic area where competent and carrier vectors are present. The paucity of the knowledge of ZIKV persistence and dynamics in semen is a challenge for the prevention of the sexual transmission of ZIKV and further studies are warrant in this direction. Together this data emphasize how important is the implementation of vector control measures around ZIKV cases. In conclusion, a prompt etiologic diagnosis of febrile diseases in travelers is highly desirable as they are sentinel of a challenging epidemiology and require a careful surveillance for their implications in subsequent autochthonous transmission of the disease.
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Remoli, et al., Experimental studies of susceptibility of Italian Aedes albopictus to Zika virus, Euro Surveill. 21 (2016) 1–4, http://dx.doi.org/10.2807/1560-7917.ES.2016.21.18.30223. [20] E. Burdino, M.G. Milia, G. Sergi, G. Gregori, T. Allice, M.L. Cazzato, et al., Diagnosis of dengue fever in North West Italy in travelers from endemic areas: a retrospective study, J. Clin. Virol. Off. Publ. Pan Am. Soc. Clin. Virol. 51 (2011) 259–263, http://dx.doi.org/10. 1016/j.jcv.2011.05.011. [21] World Health Organization, Zika Virus Disease–Interim Case Definitions–12 February 2016, (2016) file:///E:/Zika virus/WHO_ZIKV_Case definition 12 Feb 2016.pdf. [22] C. Fortuna, M.E. Remoli, C. Rizzo, E. Benedetti, C. Fiorentini, A. Bella, Arbovirus Working Group, et al., Imported arboviral infections in Italy, July 2014-October 2015: a National Reference Laboratory report, BMC Infect. Dis. 17 (2017) 216, http://dx.doi.org/10.1186/ s12879-017-2320-1 Erratum in: BMC Infect Dis. (2017), 17:335. [23] G. Paz-Bailey, E.S. Rosenberg, K. Doyle, J. Munoz-Jordan, G.A. Santiago, L. Klein, et al., Persistence of Zika virus in body fluids–preliminary report, N. Engl. J. Med. (2017) Feb 14. https://doi.org/10.1056/NEJMoa1613108. [24] G.E. van, C. Reusken, S. Pas, C. GeurtsvanKessel, R. Mögling, J. van Kampen, T. Langerak, M. Koopmans, A. van der Eijk, Longitudinal follow-up of Zika virus RNA in semen of a traveller returning from Barbados to the Netherlands with Zika virus disease, March 2016, Euro Surveill. 21 (2016) 30251, http://dx.doi.org/10.2807/1560-7917.ES.2016. 21.23.30251.
Conflicts of interest None of the authors has a conflict of interest to declare. Author contribution Acquisition of clinical data and sample analysis (EB, TA, GG,TR, GC, FL, AL), validation of real time PCR assays for ZIKA virus (MGM), neutralization assays for ZIKA antibody confirmation (GV), drafting the manuscript (EB, VG), critical revision of the manuscript (VG and GDP). Funding None. Tests were done for clinical purpose to rule out the clinical suspicion of ZIKV infection. Ethical approval Not required. Acknowledgements We are grateful to Giulio Pisani, MD, from the Centro Nazionale per la Ricerca e valutazione dei prodotti immunobiologici (CRIVIB), Istituto Superiore di Sanità, Rome and to Dr. Isabelle Leparc-Goffart of the French National Reference Center on Arboviruses in Marseille for
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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Laboratory findings in Zika infection: The experience of a reference centre in North-West Italy
T
⁎
Elisa Burdinoa, , Maria Grazia Miliaa, Tiziano Allicea, Gabriella Gregoria, Tina Ruggieroa, Guido Callerib, Filippo Lipanic, Anna Lucchinic, Giulietta Venturid, Giovanni Di Perric, Valeria Ghisettia a
Laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, Turin, Italy Unit A of Infectious and Tropical Diseases, Amedeo di Savoia Hospital, Turin, Italy c Department of Infectious Diseases, Amedeo di Savoia Hospital, Turin, Italy d National Reference Laboratory for Arboviruses, Department of Infectious, Parasitic and Immune- Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy b
A R T I C L E I N F O
A B S T R A C T
Keywords: Zika virus Travelers Real time RT-PCR Blood Urine
Background: Zika virus (ZIKV) remains a public health concern due to its association with fetal malformation and neurologic disease. Objective: To report a reference centre experience on ZIKA virus (ZIKV) infection in travelers from epidemic countries from January 1 to September, 30, 2016 in Italy North-West (a geographic area covering 4.424 million inhabitants, corresponding to almost 73% of Italy North-West area). Study design: One hundred and twelve febrile travelers were studied to rule out a tropical fever [e.g. malaria, dengue (DENV), chikungunya (CHIKV), West Nile (WNV) and ZIKV]. Molecular tests for detecting ZIKV RNA were applied on serum or urine as well as IgG and IgM specific serology. Results: ZIKV was the most frequent “tropical infection (11.6%) with 12 infected travelers and one sexual partner of an infected traveler. At the time of the diagnosis, ZIKV RNA was detected in the blood from 9 patients (69%) within 7 days from symptom onset; afterwards, the virus was detected only in urine (5 patients) and ZIKV IgM was reactive in 9 patients (69%). Travelers with ZIKV infection tested negative for DENV, CHIKV, WNV and malaria and completely recovered. Other infections identified in travelers were DENV (5 patients, 4.5%), CHIKV (1, 0.9%), malaria (Plasmodium vivax, 1, 0.9%), measles (1, 0.9%) and tuberculosis (1, 0.9%). Conclusions: The etiologic diagnosis of a febrile illness in travelers where ZIKV is endemic is highly desirable as they are sentinel of a challenging epidemiology including the risk of autochthonous transmission in non endemic countries where the competent or carrier vector is present.
1. Background Zika virus (ZIKV), a mosquito-borne member of the Flaviviridae family, is the focus of a great public health concern due to its association with fetal malformation and neurologic disease. While ZIKV was previously limited to sporadic cases in Africa and Asia, but in the year 2015 it rapidly spread to Brazil and throughout the Americas and Caribbean causing 0.5–1.5 million human infections [1–5]. Moreover, in 2016, due to the Olympic Games in Brazil, an increasing number of travelers from the Americas have been observed during the period of vector activity (May–November). Neither an effective treatment nor a vaccine
is available; therefore, prevention, surveillance and accurate diagnosis are the primary public health response. Since 2007, 31 of the 70 countries and territories reporting evidence of ZIKV transmission (67 from the year 2015 onwards) reported also the association of the disease with microcephaly and other CNS malformations [6]. Although a decline of Zika infection has been recently reported by the last WHO estimates, vigilance needs to remain high [7] and increasing reports of imported ZIKV cases have been observed throughout Europe, United States and Asia [6,8–11]. From a public health perspective the etiologic diagnosis of febrile travelers from countries where ZIKV is epidemic, is highly desirable and
Abbreviations: ZIKV, Zika virus; PCR, Polymerase Chain Reaction; RT-PCR, Reverse Transcription PCR; rtRT-PCR, real time RT-PCR; PRNT, Plaque Reduction Neutralization Tests; PNRT80, PRNT ≥ 80% of plaque reduction; GEN, Genesig Advanced Kit; ALT, Altona Diagnostics RealStar Zika virus RT-PCR Kit 1.0; RU, relative units; ELISA, Enzyme-Linked Immunosorbent Assay; CV, coefficient of variation; Ct, cycle threshold ⁎ Corresponding author at: Laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, Department of Infectious Diseases, Corso Svizzera 164, 10149 Torino, Italy. E-mail address: [email protected] (E. Burdino). https://doi.org/10.1016/j.jcv.2018.01.010 Received 15 May 2017; Received in revised form 11 January 2018; Accepted 20 January 2018 1386-6532/ © 2018 Elsevier B.V. All rights reserved.
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3.2. Diagnostic tests for ZIKV infection
efforts in this direction are highly recommended, as travelers can be a sentinel of rapidly challenging epidemiology including the risk of autochthonous transmission in non epidemic countries where the competent or carrier vector is present. Currently, the diagnosis of ZIKV infection relies on the detection of the virus by Reverse Transcription Polymerase Chain Reaction (RTPCR)-based tests especially useful during the acute phase of the disease (that is within 7 days from symptom onset) and the identification of specific antibodies. Accurate detection of ZIKV in biological samples can be highly demanding due to the low viral load and the short viraemic period, implying the risk of false-negative results in the acute phase. To increase the diagnostic sensitivity in this stage of the disease, testing other specimens than plasma/serum, such as whole blood [12], urine and more recently saliva [13] is now highly recommended as viral shedding is longer in these compartments [8,14]. As observed by Gourinat [15] and Bingham [16] urine might be the preferred specimen to identify acute/recent ZIKV disease because viral RNA can be detected at higher levels for longer periods of time than in serum; moreover, urine has the advantage of a much easier sample to collect than blood. ZIKV serology should be considered in those patients with a high suspicion of ZIKV infection in whom ZIKV RNA is negative by RT-PCR and whose symptoms last for more than one week. Serology has substantial limitations [17], mainly due to the high cross-reactivity among flavivirus antibodies, but recently, ELISA tests based on ZIKV NS1-antigen have been developed for a more specific identification of ZIKV antibodies [18]. Final evaluation of serologic results needs to be confirmed by the detection of neutralizing antibody that remains the gold standard, although this test is restricted to reference laboratories due to its high complexity [19].
One hundred eighty-five specimens were processed (130 serum and 55 urine) for ZIKV molecular testing and/or ZIKV serology. For the detection of ZIKV RNA in serum and urine, a CE-marked real time (rt) RT-PCR assays (RealStar Zika virus RT-PC Kit 1.0, Altona Diagnostics, Germany) was used, while quantification of ZIKV RNA in positive samples was performed with the Genesig Advanced Kit (Primerdesign Ltd, United Kingdom) using a plasmid as standard curve from 2 × 105 to 2 copies/μl after ZIKV RNA extraction from 600 ml of serum and urine using the semi-automated system NucliSENS easyMAG (Biomérieux, France), according to manufacturer’s protocols. Performances of the two molecular tests was done with the Asian reference strain ZIKV H/PF/2013 kindly provided by the Istituto Superiore di Sanità (ISS), Rome. All patients underwent ZIKV IgG and IgM antibody detection with a commercial ELISA assay (Euroimmun AG, Germany) against ZIKV NS1 protein [18]; confirmation with Plaque Reduction Neutralization Tests (PRNT) was performed at the ISS, as previously described [22], in those patients in whom serology was positive but ZIKV RNA was not detectable in serum/urine (therefore classified as probable cases according to the Italian Ministry of Health and WHO case definition for ZIKV virus disease [21]). Briefly, the assay was prepared in six-well tissue culture plates with sub confluent VERO cell monolayers and the ZIKV H/PF/2013 strain of the Asian genotype (kindly provided by Dr. Isabelle Leparc-Goffart of the French National Reference Center on Arboviruses in Marseille). Patient sera were diluted 1:10 in serum-free maintenance medium. Equal volumes (100 μl) of ZIKV dilution containing approximately 80 Plaque Forming Units (PFU) and serum dilutions, were mixed and incubated overnight at 4 °C. Subsequently, VERO cells plates were infected with 200 μl/well of virus-serum mixtures in duplicate, incubated at 37 °C (5% CO2) for 4 days, and stained with 1.5% crystal violet. A titration of ZIKV with three dilutions in duplicate was performed in each assay as control and neutralizing antibody titers were calculated as the reciprocal of the serum dilution that gave an 80% reduction of the number of plaques (PRNT80), compared to the virus control. PRNT80 ≥ 10 were considered positive. Diagnosis of DENV, CHIKV and WNV infection was ruled out with rapid tests against to NS1 DENV antigen, DENV IgM and IgG (Panbio ALERE, I) and indirect immunofluorescence for both CHIKV and WNV IgG and IgM (Euroimmun AG, Lubek, Germany). A multiplex rtRT-PCR was applied to detect DENV and CHIKV RNA in serum (Dengue/Chik, FastTrack Diagnostics assay) and WNV RNA was identified with the West Nile Virus ELITe MGB® Kit (ELITechGroup, Italy).
2. Objective To report ZIKV infection in febrile travelers returning from epidemic areas (Venezuela, Dominican Republic, Puerto Rico, Mexico, Costa Rica), who referred at the regional Centre for Infectious Diseases, Amedeo di Savoia Hospital, Turin, Italy (a geographic area characterized by the widespread presence of the ZIKV vector Aedes albopictus and serving 4.424 million inhabitants), between January 1 and September 30, 2016 [19,20]. Ours represents a significant cohort of ZIKV imported infection in North-West Italy within the national surveillance system that since January 2014 has been enhanced for ZIKV (Ministero della Salute. Sorveglianza dei casi umani di chikungunya, dengue, West Nile Disease ed altre arbovirosi e valutazione del rischio di trasmissione in Italia 2015. 0020115-16/06/2015-DGPRE-COD_UO-P. 2014:1–135) and accounts for approximately 60 notified ZIKA cases in the year 2016. (http://www.iss.it/binary/arbo/cont/Casi_confermati_Chikungunya_ Den_Zika.pdf; data not-published).
4. Results According to WHO case definition, ZIKV infection was identified in 13 out of 112 febrile (11.6%) patients (Table 1): 12 were travelers (ID#1-8 and ID#10-13), one was the sexual partner of an imported case (ID#9). Twelve patients (92%) reported maculopapular rash (with itching in half of them); 9 (69%) arthralgia/myalgia, 4 (30.7%) headache and/or retrorbital pain, 3 presented with conjunctivitis (23%), malaise and fatigue (23%) and 1 with diarrhea (7.7%). Leukopenia (< 4000 WBC/μl) was present in 4 patients, while impaired liver function in two. Paracetamol and steroids for uncontrollable itching were administered and all patients completely recovered within 1 week without hospitalization. A case most likely of sexual transmission of ZIKV was suspected in the female partner (ID#9) of a male traveler (ID#5) returning home from the Dominican Republic with ZIKV infection. The partner (patients ID#9) had no history of travelling abroad and of mosquito bites, but reported a sexual intercourse with travel ID#5 after his returning home and falling ill. The female partner had fever, a maculopapular itchy rash and ZIKV RNA was detected in urine, but not in serum. Other body fluids from the two partners were not available for testing, therefore, a
3. Study design 3.1. Patients and samples Febrile travelers (N = 112) returning home from ZIKV epidemic areas reporting symptoms and signs such as fever, maculopapular and itchy rash and/or arthralgia/myalgia who referred to the outpatient Clinic of Travel Medicine at the regional reference Centre for Infectious Diseases, Amedeo di Savoia Hospital, Turin, from January, 1, to September, 30, 2016 were studied. A suspicion of ZIKV infection was posed. Patients underwent hematological, biochemical, microbiologic, serologic and virological examinations to rule out a tropical fever [e.g. malaria, dengue (DENV), chikungunya (CHIKV), West Nile (WNV) and ZIKV fever] and the Italian Ministry of Health and WHO case definition for ZIKV, CHIKV, DENV and WNV disease were followed [21].
19
20
29 Male / > 15 days Jan 9 Jan 6 Jan 11
Yes Yes Yes No No Yes Yes No Yes
Age (yrs) Sex Pregnancy Stay duration Date of returning to Italy Date of symptom onset Date of sampling
Symptom Fever Maculopapular rash Itching Conjunctivitis Headache Retroorbital pain Arthralgia/Myalgia Diarrhea Malaise/Fatigue
Paracetamol Recovery (4 days)
ALT x 4
Normal
Paracetamol Recovery (1 week)
Normal
Normal
Normal
Yes Yes No No No No Yes No No
31 Female No > 15 days Jan 9 Jan 4 Jan 11
Venezuela
ID# 2
Steroid Recovery (2 days)
Normal
Normal
3880
Yes Yes Yes Yes No No Yes No No
Dominican Republic 40 Female No 30 days Apr 8 Apr 11 Apr 17
ID# 3
NA = Not available; ALT = alanine aminotransferase. a Sexually transmitted, sexual partner of patient ID#5. b Patients ID#11 and ID#12, brother and sister. c Indicated as normal (within reference value) or x upper reference value.
Treatment Outcome
Thrombocytopenia (PLTS Count < 140,000 μl) Liver functionc
3500
Venezuela
Travelling from
Laboratory test (reference) Leukopenia (WB Count < 4,000 μl)
ID# 1
Characteristic
No therapy Recovery
Normal
Normal
Normal
Yes Yes Yes Yes No No Yes Yes No
Dominican Republic 30 Male / 15 days May 26 May 30 Jun 3
ID# 4
No therapy Recovery
NA
NA
NA
Yes Yes No No No No No No No
Dominican Republic 20 Male / 30 days July 11 July 6 July 15
ID# 5
No therapy Recovery
Normal
Normal
Normal
Yes Yes No No No No No No Yes
29 Male / 15 days July 4 July 11 July 15
Puerto Rico
ID# 6
Table 1 Clinical and epidemiological characteristics of the 13 patients with confirmed Zika infection (January–September 2016).
No therapy Recovery
NA
NA
NA
Yes Yes No No No No Yes No No
Dominican Republic 21 Female No 30 days July 14 July 7 July 18
ID# 7
No therapy Recovery
NA
NA
NA
Yes Yes No No No No Yes No No
Dominican Republic 18 Male / 30 days July 14 July 4 July 18
ID# 8
No therapy Recovery
NA
NA
NA
Yes Yes Yes No Yes No Yes No No
Sexual transmissiona 19 Female No 15 days July 25 July 17 July 28
ID# 9a
No therapy Recovery
NA
NA
Normal
Yes Yes Yes No No Yes Yes No No
No therapy Recovery (1 week)
Normal
123,000
3610
Yes Yes Yes Yes Yes Yes Yes No Yes
21 Male / 15 days August 29 August 26 August 29
Costa Rica
Mexico 28 Male / 4 months August 23 August 19 August 29
ID# 11b
ID# 10
No therapy Recovery (1 week)
Normal
Normal
2320
Yes No No No No No No No No
23 Female No 15 days August 29 August 27 August 29
Costa Rica
ID# 12b
No therapy Recovery
ALT x 2
Normal
Normal
Yes Yes No No No No No No No
Dominican Republic 32 Male / 15 days August 18 August 22 August 26
ID# 13
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Table 2 Laboratory findings of ZIKV infection for the 13 patients. Patient ID
Days from symptoms onset
ZIKV ELISA IgMa
ZIKV ELISA IgGa
ZIKV RNA in serum Real-time PCRb
ZIKV RNA in urine Real-time PCRb
#1
5c 56 7c 55 6 19 28 4 13 9 4 9 14c 11 10 3 7 21 2 7 21 4c 25
Positive Negative Negative Negative Negative Negative Negative Positive Positive Positive Positive Positive Positive Positive Positive Negative Positive Negative Negative Positive Negative Positive Negative
Negative Positive Positive Positive Positive Positive Positive Negative Positive Positive Negative Positive Positive Negative Positive Negative Negative Positive Negative Positive Positive Negative Positive
Negative NA Negative NA Negative NA NA Positive (40 cps/ml) NA Negative Negative Negative Negative Negative Positive (n.q.) Positive (157 cps/ml) Negative Negative Positive (20510 cps/ml) Negative Negative NA NA
NA NA NA NA Positive (5913 cps/ml) Positive (310 cps/ml) NA NA NA Positive (1321 cps/ml) Positive (41338 cps/ml) Positive (117 cps/ml) Negative Positive (13608 cps/ml) Positive (619 cps/ml) Positive (3252 cps/ml) Negative Negative Positive (3501 cps/ml) NA Positive (n.q.) NA NA
#2 #3
#4 #5 #6 #7 #8 #9 #10 #11
#12
#13
PCR = Polymerase Chain Reaction; ELISA = Enzyme-Linked Immunosorbent Assay; NA = Not available; cps = copies; n.q. = not quantifiable. a Commercial ELISA assays (Euroimmun AG, G). Reference values: positive ≥ 22 RU/ml, negative ≤ 16 RU/ml). b RealStar Zika virus RT-PC Kit 1.0 (Altona Diagnostics, G) and Genesig Advanced Kit (Primerdesign™ Ltd, UK). See text for details on the protocol used. c Confirmatory Plaque Reduction Neutralization Test (PRNT80) performed at the Istituto Superiore di Sanità, Rome, Italy, (positive result for titers ≥ 1:10).
Analytical sensitivity of molecular tests applied for ZIKV RNA detection in this study was assessed with known concentrations of the Asian reference strain ZIKV H/PF/2013 kindly provided by the ISS. The detection rate was 100% at 1425, 142 and 14 copies/mL for both the two rtRT-PCR assays. Test specificity was probed against 10 plasma samples positive for DENV RNA and supernatants from DENV and CHIKV infected cell cultures (Quality Control Molecular Diagnostics, QCMD 2016 ZIKV External Quality Pilot Study) that tested negative for ZIKV RNA with both the assays.
confirmed match between the viruses isolated from the two patients could not be obtained. The last ZIKV infection was reported on August 2016. None of the women with ZIKV infection were pregnant. Table 2 shows ZIKV findings in the 13 infected patients. As expected, in patients referring at the outpatient clinic within 7 days from symptoms onset (N = 7, Table 2, ID#1, 3, 4, 6, 11–13), molecular tests detected the virus in the majority of them(83%, in serum and/or urine; in one patient, ID#13, molecular test were not performed due to the lack of sample); serology for IgM was positive in only 57% of them (the presence of IgG for ZIKV was found in one patient). In those patients with ZIKV infection referring later than 7 days from symptom onset (N = 6, Table 2, ID#2, 5, 7–10), the diagnosis of infection was made by molecular tests in 66.7% of them and by serology in 83% of them (Fisher’s exact test for IgG reactivity within and after 7 days from symptoms onset: p = 0.0291; no statistically significant difference for both IgM and molecular tests). At the time of diagnosis, molecular tests identified ZIKV RNA in 9 infected patients (69%), in the serum of 4 (45%, ID#4, 10–12) and in the urine of 8 (89%, ID#3, 5–7, 9–12) of them, Median viral load was lower in serum than in urine (2.7 ± 1.4 log10 copies/mL vs. 3.4 ± 0.8) and urine shedding was detectable up to 21 days after symptom onset. IgM reactivity was found in 9 patients [6 ZIKV RNA positive in serum (1 patient, ID#4) or urine (5 patients, ID#5, 6, 7, 9 and 10)]; ZIKV IgG were detected in 6 patients (4 of them with urine viral shedding, patients ID#3, 5, 7 and 10). In those patients in whom the virus was absent in both serum and urine (ID#1, 2, 8 and 13) confirmation of ZIKV antibodies was obtained by a PRNT80 titer ≥ 1:10. In 5 patients who could be monitored over time (ID#1, 4, 11–13), IgM seroconversion from negative to positive was observed in 3 of them (within 7 days from symptom onset), while ZIKV IgG became detectable from day 7 to day 21–25. Patients with ZIKV infection were negative for DENV, CHIKV, WNV and malaria and they completely recovered without hospital admission. Other infectious diseases in the traveler study-group were DENV (5 patients, 4.5%), CHIKV (1, 0.9%), malaria (Plasmodium vivax, 1, 0.9%), measles (1, 0.9%) and tuberculosis (1, 0.9%).
5. Discussion Highly sensitive molecular testing for ZIKV RNA detection in clinical samples is a key factor for the diagnosis of ZIKV infection and this also applies to ZIKV congenital disease and the safety of blood transfusion in epidemic areas. During the acute phase of the disease, ZIKV infection is generally characterized by low virus concentration in clinical specimens, irrespective of the sample type. Thus, it can be expected that a significant proportion of patients may not be diagnosed if molecular tests are insufficiently sensitive. At present, the availability of real time PCR assays provides more accurate and reproducible results with higher sensitivity and specificity than conventional PCR, significantly simplifying and accelerating the process of ZIKV detection in biological samples and in the framework of a global response to the outbreak specific quality controls are extremely helpful. A number of clinical reports [8,10,14–16] suggest that ZIKV can be found in plasma or serum and saliva a few days after the infection, and in the urine of the majority of patients approximately 1 week after symptom onset. Thus, urine testing is certainly very helpful to the diagnosis of ZIKV infection in travelers. Recent data suggests a longer recovery of the virus in whole blood than in plasma/serum [12] and that the median time for RNA loss in serum is 14 days (95th percentile: 54 days), while 8 days in urine (95th percentile: 39 days) [23]. In addition to the above mentioned critical issues, quantification of ZIKV viral load is important in investigating the risk of transmission through other different specimens considering that most likely the sexual transmission of the virus occurs as ZIKV can be detected in semen for 21
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kindly providing Zika virus strain H/PF/2013.
several weeks [24]. According to WHO case definition [21], we identified ZIKV infection by molecular testing in 9 patients (69%), while serology and PRNT confirmation allowed the identification of other 4. In our experience, urine was very helpful for ZIKV RNA detection (8/13 positive patients); in this compart viral shedding lasted for 21–25 days after symptoms onset, that is longer than in serum (about 3–4 days) and the viral load was generally higher in urine than in serum. The combination of nucleic acid testing of more than one type of specimen per patient is certainly an added value to the diagnosis of ZIKV infection. In this study, for ZIKV RNA detection we applied two commercially available real-time–PCR (e.g. Altona and Genesig) assays, whose performances were assessed with serial dilution of known concentrations of an Asian ZIKV reference strain. The analytical sensitivity of both assays was very high (100% detection rate at 14 copies/mL) and in line with the majority of published rtRT-PCR protocols for ZIKV RNA [14]. ZIKV genotype inclusivity could not be tested being the primer and probe design covered by company confidentiality. Quality controls at known concentration of ZIKV virus and external quality control programs for the standardization of molecular assays play a key role for the most appropriate diagnosis of ZIKV infection, patient management and outbreak control. In our case-series of febrile travelers the prevalence of ZIKV was higher than that of DENV or other tropical diseases, but ZIKV casefinding should also focus on sexual partners as sexual transmission of the virus has been documented and this can be a possible source of additional vector-borne spread in non-epidemic area where competent and carrier vectors are present. The paucity of the knowledge of ZIKV persistence and dynamics in semen is a challenge for the prevention of the sexual transmission of ZIKV and further studies are warrant in this direction. Together this data emphasize how important is the implementation of vector control measures around ZIKV cases. In conclusion, a prompt etiologic diagnosis of febrile diseases in travelers is highly desirable as they are sentinel of a challenging epidemiology and require a careful surveillance for their implications in subsequent autochthonous transmission of the disease.
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Conflicts of interest None of the authors has a conflict of interest to declare. Author contribution Acquisition of clinical data and sample analysis (EB, TA, GG,TR, GC, FL, AL), validation of real time PCR assays for ZIKA virus (MGM), neutralization assays for ZIKA antibody confirmation (GV), drafting the manuscript (EB, VG), critical revision of the manuscript (VG and GDP). Funding None. Tests were done for clinical purpose to rule out the clinical suspicion of ZIKV infection. Ethical approval Not required. Acknowledgements We are grateful to Giulio Pisani, MD, from the Centro Nazionale per la Ricerca e valutazione dei prodotti immunobiologici (CRIVIB), Istituto Superiore di Sanità, Rome and to Dr. Isabelle Leparc-Goffart of the French National Reference Center on Arboviruses in Marseille for
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