Zika virus (ZIKV) is an RNA flavivirus with the first documented case of ZIKV infection reported in 1947 in the Zika Forest in Uganda. In 2013 an outbreak occurred in French Polynesia spreading widely throughout the world including South America (1).

While most infected people are asymptomatic, nearly 20-30% of patients may develop clinical symptoms, including fever, skin rash, arthralgia, arthritis, and conjunctivitis (1–5). Notably, neurological complications such as Guillain Barré Syndrome are life-threatening complications associated with acute infection (6). During the ZIKV outbreak in Brazil in 2015 (7), there were also numerous cases of Congenital Zika Syndrome, which is characterized by severe neurological conditions, particularly microcephaly (8).

Zika transmission typically occurs through the bite of the Aedes aegypti mosquito and through vertical transmission, though sexual transmission has been well documented. It primarily occurs from men to women with reports of infection in partners of travelers to ZIKV endemic areas (9–12). One important aspect of sexual transmission is the potential for ZIKV persistence in semen (13–15). The male genital tract provides a favorable microenvironment for viral persistence, particularly in the testis, where the blood-testis barrier confers immune privilege (15). Mathematical modeling suggests that 3-23% of Zika infections may occur through sexual transmission, even in areas with mosquito derived ZIKV infections (16–18).

Given the risk of Zika sexual transmission, guidelines have been developed for couples during reproductive planning. These recommendations include the use of condoms or abstaining from sexual activity for at least 3 months after traveling to an area with ongoing Zika virus circulation. In cases of confirmed infection or residence in a high-risk area, continuous condom use is advised for the duration of pregnancy (19, 20). In addition, ZIKV investigations in assisted fertilization clinics were implemented in endemic areas during the outbreak period (21), including testing of couples and semen donors.

The gold standard for diagnosing an ongoing ZIKV infection depends on the presence of viral RNA in blood or other body fluids (including urine, vaginal secretions, saliva, or semen) using RT-qPCR. However, serologic tests may be an alternative to molecular testing in certain situations, particularly in the later stages of infection or when there is a strong suspicion of Zika virus infection despite negative RT-PCR results (4, 19). In Brazil, only ZIKV serology tests (IgM and IgG) were incorporated into the infectious disease screening protocol for Cell and Germinative Tissue Banks, which includes testing for other infectious diseases such as HIV (Human Immunodeficiency Virus), Hepatitis B, and Hepatitis C (22).

It remains unknown whether immunoglobulin tests for ZIKV are sufficiently accurate to determine presence of prior infection and eligibility as a semen donor. The present study seeks to evaluate whether serologic tests are reliable for screening semen donors for ZIKV compared with virus screening using RT-qPCR.

A total of 36 samples from 23 healthy donors with no arboviral-like symptoms and a median age of 31 years (IQR: 26-39), were evaluated using RT-qPCR. Four donors provided more than one sample, each collected at distinct time points ( Figure 1 ). Ten donors were tested for both IgM and IgG antibodies against ZIKV. Among these, five tested positive and one was inconclusive for IgG anti-ZIKV. Two IgG-positive donors made four and five semen donations, respectively, during the study period. One of these donors was tested for IgG at three different time points during semen donation, yielding positive IgG results in all samples. All semen samples, whether from IgG-positive or IgG-negative donors, tested negative for ZIKV via RT-qPCR ( Table 2 ).

In this study, no positive results were observed for ZIKV through RT-qPCR in 8 semen samples from 5 individuals with IgG positive serology for ZIKV. Therefore, despite the small sample size, these findings provide additional evidence that IgG serologic testing for ZIKV may be unreliable to determine risk of sexual transmission of ZIKV, particularly within the immunological window. With confirmation of ZIKV sexual transmission through semen, protocols have been established for human reproduction clinics that rely on serologic testing for ZIKV (20, 21). In addition to the recommendations for travelers and couples of childbearing age, accurate testing is required to exclude infection and allow use of semen samples (19). Typically, candidates for assisted fertilization undergo serological testing, specifically IgM and IgG tests. RT-qPCR testing of the collected semen samples is not commonly performed, raising two important points. First, serological testing may result in false positive testing of ongoing ZIKV infection, particularly in areas with other endemic arboviruses (Dengue in particular) that may have demonstrate cross-reactivity serologically. Second, the duration and viability of ZIKV in samples from various body fluids may not correlate with serological test results.

A positive serological test for ZIKV can occur due to cross-reactivity with other flaviviruses, including Dengue virus (DENV) (23, 24). IgM typically becomes positive within two weeks after infection and persists for up to 12 weeks (about 3 months), while IgG antibodies can persist for an extended period (24). Plaque Reduction Neutralization Test (PRNT) detects IgM and IgG broad neutralizing action though may have significant cross-reactivity among viruses from same family. Both IgG and PRNT can remain positive for several years suggesting that IgM may be a better diagnostic alternative to assess risk during the ZIKV sexual transmissible window. However, many IgM tests are either highly specific with low sensitivity or highly sensitive with low specificity limiting their diagnostic accuracy. An alternative method to assess ZIKV infection in travelers that is viable would be the CDC Zika IgM MAC ELISA test (24). This test demonstrates excellent specificity and sensitivity for Zika virus diagnosis, without the risk of cross-reactivity with other arboviruses, as seen in traditional serology utilized in the current study. Nevertheless, it is a labor-intensive test requiring several days with a limited number of samples able to be tested simultaneously.

ZIKV may persist up to 6 months in the male reproductive tract and semen after serological conversion (14, 23), leading to prolonged shedding of viral particles in sperm. However, in a study by Paz-Bailey et al. (2017) (25), RT-qPCR positive results in sperm from ZIKV infected men reaches 55% in the first month, gradually decreasing thereafter with an average time for semen to test negative between 40 to 54 days (about 2 months), similar to the testing window of IgM serology. Moreover, in a study attempting to culture ZIKV in Vero cells from 78 ZIKV positive semen samples, only three samples had a positive culture. A higher likelihood of culture positivity occurred in samples collected closer to the acute stage of infection, with a higher viral load, and in individuals over 50 years of age, implying that sexual transmission might occur only during the first month (23). Additionally, further studies investigating the correlation between virus persistence in semen and IgM titers are required to evaluate the utility of IgM as a biomarker of ZIKV in semen.

The current study has several limitations, including a small sample size of semen samples analyzed, which were obtained through convenience sampling, and retrospective design. Moreover, positive IgM or IgG identified in our study by standard serology may be false positive for ZIKV given cross-reactivity with DENV and may limit interpretation of discordant serologic positive and negative RT-qPCR results. With a new outbreak of Zika infection in the future, a prospective study may determine the accuracy of different diagnostic tests (serologic PRNT and MAC-ELISA and nucleic acid testing) over a specified timeline during and after infection. This approach would aid in the development of diagnostic algorithms and evaluate their real-world reliability and cost-effectiveness for individuals of reproductive age and assisted reproduction clinics.

Several unresolved questions linger: Is it necessary to test semen samples using the RT-qPCR technique? Would serological testing alone be sufficient for a couple in which one partner has traveled to the ZIKV endemic zone? Does a positive RT-qPCR in the semen correspond to viable viruses with the potential for infection? An approach based on available data may include condom use for the first 30 days (about 4 and a half weeks) following possible ZIKV exposure. Following this period, if available, the next recommended test would be the ZIKV IgM MAC-ELISA, which can indicate a probable recent infection. If this test is positive, RT-qPCR testing of semen would be advised. Once the RT-qPCR results are negative, it would be considered safe for spontaneous or assisted contraception. For populations residing in endemic areas, where infection can be transmitted by mosquitoes at any time, routine precautions against mosquito bites must be maintained.