Persistent Oropouche virus viremia in two travellers returned to Italy from Cuba, July 2024

Abstract

Oropouche virus (OROV) disease is mainly transmitted by biting midges (Culicoides paraensis) and mosquitos (Culex quinquefasciatus and Aedes serratus). OROV infection is currently endemic in tropical areas of Central and South America. An unprecedented outbreak is ongoing since December 2023 in the Amazon region and surrounding territories. In May 2024, the Pan American Health Organization (PAHO) declared a new wave of OROV cases outside the Amazon area and the first confirmed OROV outbreak was reported in Cuba.1 Moreover, in August 2024, the first fatal outcomes were reported in two Brazilian women with no comorbidities, due to haemorrhagic manifestations.2 Concurrently, from late May 2024, more than 50 imported cases were reported in non-endemic areas including European countries, USA and Canada.1 Five imported cases from Cuba or Brazil have been reported in Italy between March and August 2024.3 We observed and conducted a microbiological follow-up in two cases of OROV infection imported from Cuba. Clinical samples were sent for diagnostic purpose to the Infectious and Tropical Diseases Department of the IRCCS Sacro Cuore Don Calabria Hospital, Negrar (Verona). A real-time polymerase chain reaction (RT-PCR, PAHO)4 targeting the S segment was performed using the iScript Reverse Transcription Supermix (BioRad Hercules, CA, USA). OROV-specific IgG and IgM were detected by indirect immunofluorescence assay using homemade slides prepared with Vero-E6 cells infected with an OROV isolate (strain OROV-IRCCS-SCDC_1/2024). Further details on microbiological investigation are reported in Supplementary Materials. A Cuban 59-year-old woman, living in Italy for more than 10 years, with no remarkable clinical history (despite a previous episode of haemorrhagic dengue 15 years before), travelled to Moron, central Cuba, between 26 May and 1 July 2024. Starting from 15 June, she reported a sudden-onset fever (with maximum temperature 38°C) that persisted for about 3 days, retro-orbital pain, headache, arthromyalgia and diarrhoea. While these symptoms were persisting, 9 days after symptoms onset (SO), she reported appearance of a diffuse maculopapular rash on upper and lower limbs, sparing palms and soles. She was admitted to a Cuban health facility, reporting a positive Dengue serological test. Back to Italy, she presented on 3 July 2024 (20 days after SO), at the Emergency Department of Careggi University Hospital (CUH) due to a worsening headache. Laboratory findings were in range (Supplementary Table 1), so she was discharged and referred to our Infectious and Tropical Diseases (ITD) outpatient clinic, where she was evaluated on 8 July 2024. At the time of our evaluation, headache was completely resolved, and no pathological findings were reported at the physical examination. After excluding Dengue (DENV), Zika (ZIKV) and Chikungunya (CHIKV) viruses infection (Figure 1), specific OROV IgM and IgG resulted positive (≥1:20 and 1:80, respectively). RT-PCR for OROV-RNA was positive on a whole-blood sample but negative on serum and urine samples. Follow-up samples at 10 weeks after SO detected a persistent positivity for OROV-RNA on whole-blood samples and an increase in antibody titres, whereas OROV-RNA was not detected on whole blood 110 days after SO (Figure 1). The patient is currently in good clinical state. Main microbiological investigations conducted for Case 1 (left) and Case 2 (right). N/A = not available; Ct = cycle threshold value; *Days after symptoms onset; #Positive ≥1:20; °Test performed at the Department of Infectious/Tropical Diseases and Microbiology of the IRCCS Sacro Cuore-Don Calabria Hospital (Negrar, Verona, Italy); §Test performed at the Microbiology and Virology Unit, Careggi University Hospital (Florence, Italy). Positive results are reported in bold. An Italian 31-year-old woman with no significant previous medical history travelled to Cuba from 3 July to 22 July 2024. She reported diarrhoea with no blood or mucous in the stools starting from 5 July, which resolved in about 24 hours. On 8 July, she again reported more intense diarrhoea, accompanied by fever (with maximum temperature 38°C), retroorbital pain, headache and a maculopapular rash, more evident on her back and lower limbs. Symptoms lasted for about 2 weeks, then gradually disappeared. During her stay in Cuba, due to dehydration and persistent fever, she was hospitalized a week after SO, receiving intravenous fluids and antipyretics. She was discharged after 2 days with a suspected diagnosis of dengue fever. About 3 weeks after SO, she was evaluated at the CUH ITD outpatient clinic, presenting in good clinical condition with full blood count within the normal limits (Supplementary Table 2). After DENV, CHIKV, ZIKV and Yellow Fever virus infection (Figure 1) were ruled out, OROV infection was suspected. RT-PCR for OROV-RNA resulted negative on urine, plasma and serum samples collected at 3, 4, 7 and 12 weeks after SO (the patient was asymptomatic at all timepoints), whereas whole-blood RT-PCR tested persistently positive. OROV-specific IgG and IgM were positive at 3 weeks (IgM 1:80, IgG 1:320), 4 weeks (IgM 1:160, IgG ≥1:640), 7 weeks (IgM 1:40, IgG 1:640) and 12 weeks (IgM 1:40, IgG 1:2560) (Figure 1). The patient is currently in good clinical state. Beta-Human Chorionic Gonadotropin (Beta-HCG) dosage was negative, and she was precautionary advised to avoid pregnancy up to the viremia clearance. Both cases presented with persistently positive viremia up to 10 and 12 weeks after SO, respectively. OROV-RNA was detectable in whole-blood samples, but not on serum, urine or plasma, showing similar behaviour with ZIKV.5 It can be assumed that persistent viral detection in whole blood could be favoured by the presence of a virological reservoir. For ZIKV, persistent viremia in pregnant women could be caused by persistent viral replication in the foetus or placenta, which acts as viral reservoirs.6 Regarding OROV, the virus seems to be able to replicate and persist in low titers in human T-cells and monocytes, possibly through an immunological escape strategy.7 Of note, due to the Ct values close to the detection limit of the RT-PCR test4 in the patients’ samples described here, it is not possible to establish whether the molecular signal represents viable virus or viral remnants, as shown for other RNA viruses,8 since it is generally known that sensitivity of virus isolation is lower than that of molecular detection. The presence of a virological reservoir in OROV-infected patients needs further investigation to be confirmed; however, our findings suggest that the whole-blood molecular testing for OROV could raise diagnostic sensitivity and prolong the diagnostic period, compared to urine, serum or plasma. Another alarming similarity between OROV and ZIKV infection is the potential of vertical transmission, which is currently being investigated by health authorities, since specific IgM and positive RT-PCR for OROV have been detected in samples of newborns with microcephaly or died foetuses.9 The epidemiological implications of persistent viremia in OROV-infected patients need further investigation, raising concern regarding organ transplantation and blood donation safety, and timing for safe conception after infection or exposure in an endemic area. In the meantime, the Italian Ministry of Health set screening measures for OROV in blood and organ donors who visited endemic areas in the 28 days preceding the donation and issued specific precautions for prevention and screening of OROV for pregnant woman.10 Finally, other Culicoides, Culex and Aedes species are widely present in most European countries, and their competence as potential OROV vectors needs to be urgently defined. L.Z., F.G. and C.C. conceived and designed the work. N.C., M.G.C., G.M.R., S.P., R.P.M. and S.A. contributed to the laboratory analysis. A.B. and G.F. contributed to samples collection and patients’ enrollment. A.B., G.F. and C.C. drafted the paper. A.B., M.S., F.G., G.M.R., C.C. and L.Z. supervised the research work. All authors have read and agreed to the current version of the manuscript. Anna Barbiero (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Writing—original draft [equal], Writing—review & editing [equal]. Giuseppe Formica Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Writing—original draft [equal], Writing—review & editing [equal]), Rebeca Passarelli Mantovani (Data curation [equal], Formal analysis [equal], Investigation [equal], Writing—review & editing [equal]), Silvia Accordini (Data curation [equal], Formal analysis [equal], Investigation [equal], Writing—review & editing [equal]), Federico Gobbi (Conceptualization [equal], Methodology [equal], Supervision [equal], Writing—review & editing [equal]), Michele Spinicci (Conceptualization [equal], Methodology [equal], Supervision [equal], Writing—review & editing [equal]), Maria Grazia Colao (Investigation [equal], Writing—review & editing [equal]), Simona Pollini (Investigation [equal], Writing—review & editing [equal]), Nunziata Ciccone (Investigation [equal], Writing—review & editing [equal]), Gian Maria Rossolini (Investigation [equal], Supervision [equal], Writing—review & editing [equal]), Alessandro Bartoloni (Supervision [equal], Writing—review & editing [equal]), Lorenzo Zammarchi (Conceptualization [equal], Data curation [equal], Investigation [equal], Methodology [equal], Supervision [equal], Writing—original draft [equal], Writing—review & editing [equal]), Concetta Castilletti (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Supervision [equal], Writing—original draft [equal], Writing—review & editing [equal]). This work was also supported by the Italian Ministry of Health ‘Fondi Ricerca Corrente’ to IRCCS Sacro Cuore Don Calabria Hospital—L1P3. The authors declare no conflicts of interest. Data is available from the authors upon reasonable request.

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