Positive ZIKV real-time RT-PCR results were confirmed by repeat testing and by analysis with alternative methods, i.e. 2016, a man in his early 40s returning to Italy from a two-week stay in Haiti developed fever (38.5?C) and pruritic maculopapular rash on his trunk and arms that fully resolved after three days. The patient, who reported mosquito bites in Haiti, had an unremarkable past personal medical history. Laboratory analyses, performed at day 3 after symptom onset, showed blood cell count Mirk-IN-1 and liver function tests within the normal range. Testing for dengue, chikungunya and ZIKV infection, according to previously described methods , demonstrated the presence of ZIKV RNA in plasma and urine at 175 copies/mL and 25,600 copies/mL, respectively, and ZIKV-specific IgM but not IgG antibodies. Dengue virus (DENV) IgG antibodies were also detected by ELISA, but they represented cross-reacting antibodies induced by previous vaccination against yellow fever virus, as confirmed by virus neutralisation assays; DENV IgM, DENV NS1 antigen and chikungunya virus IgM and IgG were negative. Sequencing of the full ZIKV genome was obtained directly from a urine sample collected at diagnosis (GenBank “type”:”entrez-nucleotide”,”attrs”:”text”:”KX269878″,”term_id”:”1032810831″,”term_text”:”KX269878″KX269878), which demonstrated over Rabbit Polyclonal to CATD (L chain, Cleaved-Gly65) 99.6% nucleotide sequence identity with ZIKV strains circulating in Haiti (GenBank “type”:”entrez-nucleotide”,”attrs”:”text”:”KU509998″,”term_id”:”1016563098″,”term_text”:”KU509998″KU509998 and “type”:”entrez-nucleotide”,”attrs”:”text”:”KX051563″,”term_id”:”1016884664″,”term_text”:”KX051563″KX051563). Follow-up evaluation Based on these findings, a diagnosis of ZIKV infection was made. The patient was informed about the risk of sexual transmission of ZIKV and was advised to adopt safer sex practices. Further laboratory testing was performed at five days post onset of symptoms, which demonstrated the presence of ZIKV RNA also in saliva (58,700 copies/mL) and semen (175 copies/mL), while stool samples and a conjunctiva swab were negative. The patient did not report haematospermia or prostatitis. The patient was invited to participate in a follow-up evaluation of ZIKV RNA kinetics in various bodily fluids and of ZIKV-specific antibodies in serum. During follow-up, saliva and urine samples were collected daily, while blood Mirk-IN-1 and semen samples were collected at least weekly. Follow-up visits for clinical evaluation and counselling were performed weekly. Follow-up is still ongoing at the time of this report, with the latest evaluation performed on day 181 after symptom onset. During follow-up, laboratory testing (Figure) demonstrated that viral Mirk-IN-1 RNA was detectable in his plasma at low titre (ca 100 copies/mL) up to day 9 after symptom onset. Viral load in urine was higher than in blood (ca 25,000 copies/mL), but rapidly decreased to undetectable levels at two weeks after symptom onset. Shedding of ZIKV RNA in saliva persisted up to day 47, at a median load of 400 copies/mL (range: 80C3,300), after peak values of 20,000C50,000 copies/mL during the first week after symptom onset. It is noteworthy that ZIKV RNA shedding in semen was sustained and persistent, and still detectable at day 181 after symptom onset. In particular, after a peak of ca 50,000 copies/mL at day 14, viral RNA load in semen was stable in consecutive specimens, ranging from 1,000 to 10,000 copies/mL (Figure, panel A). Separation by centrifugation of cellular and plasma fractions showed that viral RNA was associated with the cellular component of semen, while undetectable in seminal plasma. Positive ZIKV real-time RT-PCR results were confirmed by repeat testing and by analysis with alternative methods, i.e. a LightMix Modular Zika Mirk-IN-1 Virus kit (Roche Diagnostics, Basel, Switzerland), broad-range pan-flavivirus RT-PCR followed by Sanger sequencing  and Sanger sequencing of the viral genome. Open in a separate window Figure Clinical and laboratory findings in a patient with Zika virus infection returning from Haiti to Italy, January 2016 OD: optical density; ZIKV: Zika virus;.