To assess the persistence of Zika virus in different body fluids of persons with confirmed infection, we conducted a multicenter prospective cohort study (the ZIKABRA Study). Laboratory confirmation of infection was based on real-time reverse transcription PCR (rRT-PCR) performed on samples (urine, blood, or both) from persons in whom a rash developed <48 hours after initial symptom onset. We also invited household contacts or sex partners to participate in the study and enrolled those with positive rRT-PCR results for Zika virus. Full details about the study protocol, including ethics approval, are described by Calvet et al. (7).

Figure

Figure. Detection of Zika virus RNA in human biological specimens from 8 patients, according to C_t and days after disease onset. Patient identification numbers above charts correspond to numbers in the Table....

We collected samples (saliva, blood, urine, vaginal, and rectal swabs) at specific intervals from patients identified as Zika virus positive after the screening visit (Figure). Vaginal and rectal swab samples were diluted in 1 mL of sterile Hank’s Balanced Salt Solution (ThermoFisher Scientific, https://www.thermofisher.com) and stored at −80°C until processing. After collection, specimens were kept refrigerated and transported within 2 hours to the laboratory, where they were maintained at −80°C. Zika virus detection was performed by rRT-PCR by processing 200 μL of each specimen for RNA extraction through an automated nucleic acid purification platform by using the Maxwell 16 Viral Total Nucleic Acid Purification Kit (Promega Corporation, https://www.promega.com) in a final volume of 70 μL. For rRT-PCR, we used the commercially available ZDC Kit from Instituto de Tecnologia em Imunobiológicos Biomanguinhos, approved by Agência Nacional de Vigilância Sanitária/ANVISA (registry no. 80142170032; https://www.bio.fiocruz.br). We considered positive those samples that displayed positive amplification in the ZDC Kit internal control reaction (which consists of an RNA virus–like particle individually added to each specimen before RNA extraction) and those samples in which the target amplification was detected within 38 amplification cycles, as previously described (8).

We report 10 Zika virus–infected patients from 2 locations in Brazil. Of these, 3 patients were identified in Recife, northeastern Brazil, and 7 were from Manaus, northern Brazil. The targeted Zika virus amplicons were found in the plasma of 9 patients and in the urine of 7 patients, all tested at screening visits. We also tested all plasma and urine samples for dengue and chikungunya virus by rRT-PCR; results were negative. Of the 10 Zika virus–infected patients, 9 were symptomatic and 1 (a Zika virus–positive household contact) was asymptomatic at enrollment but subsequently reported muscular weakness and irritability on day 5 and arthralgia on day 7 after Zika virus identification. Median patient age was 31.5 years; 7 patients were nonpregnant women, and 3 were men (Table). All patients were negative for HIV-1, hepatitis B and C, and syphilis.

For all patients, Zika virus RNA was detected >1 time in different body fluids. The longest interval for Zika virus–positive results by rRT-PCR in plasma and urine was 14 days after symptom onset; median cycle threshold value (C_t) was 34.92 in plasma (interquartile range [IQR] 33.57–36.78) and 31.57 in urine (IQR 30.34–34.92). The longest duration of persistence in saliva was 21 days. Zika virus RNA was detected in rectal swab samples from all patients (median C_t 34.36 [IQR 30.08–36.07]); positive results were obtained at >1 time for 4 patients. Vaginal swab sample results were positive for 2 patients (Figure). We attempted virus isolation in Vero E6 cells from all rectal swab specimens positive by rRT-PCR. After filtering samples through a 0.22-μm syringe filter, a rectal swab sample from patient 191002 (at 7 days after symptom onset) was positive in Vero E6 cells, inducing cytopathic effect and returning a C_t of 28.77 after 6 days of incubation at 37°C.

Our detection of Zika virus RNA in human rectal swab samples demonstrates the presence of virus RNA in the anorectal mucosa of naturally infected patients. Because the anorectal mucosa is a major entry site for HIV-1 and other sexually transmitted disease organisms (9), this finding may have implications for Zika virus transmission. Direct contact with infected mucosa could present a risk for virus transmission. Moreover, recovery of infectious Zika virus from anorectal mucosa samples may be associated with active virus replication in this body compartment, implicating its permissiveness to Zika virus infection.

Recently, 2 studies explored the mucosa as an experimental entry site for Zika virus. Macaques exposed to high doses of the virus by direct inoculation on palatine tonsils, nasal mucosa, and conjunctival mucosa became infected, whereas virus-naive animals exposed to saliva from Zika virus–infected macaques, containing a 20-fold lower virus concentration, remained uninfected (6). In mice, intra-anal inoculation resulted in viremia, replication in multiple organs, and Zika virus RNA detection in feces (5). However, it seems that high viral loads are required for successful experimental infection through mucosal tissues. In humans, a report of a man infected through anal intercourse implies that the anorectal mucosa is an entry site for Zika virus (10). Also, a rapidly progressive fatal case, with secondary nonsexual transmission to a close contact who reported having had no contact with blood or other body fluids except tears from the original patient, raised the hypothesis of Zika virus transmission through mucosa (11). Although mucosal tissues have been extensively explored as a potential site of Zika virus infection, the amount of infectious particles in these tissues remains unclear.

In patients with yellow fever, mucocutaneous bleeding with virus presence has been reported (12). However, no mucosal bleeding was observed in the patients we report, despite extensive medical examination. In addition, in 5 of 9 patients, the virus had already cleared from the blood at the time of anorectal detection, which supports the concept of local virus replication. Our finding of Zika virus in the anorectal mucosa of naturally infected persons may influence the recommendations for prevention of Zika virus transmission. We suggest the use of rectal swabbing, a noninvasive method, for diagnosing infection with Zika virus, among other emerging viruses (13).