Possible Association Between Zika Virus Infection and Microcephaly — Brazil, 2015

Febrile or Exanthematous Illness Associated with Zika, Dengue, and Chikungunya Viruses, Panama.

Zika virus in the dock

"What's called a difficult decision is a difficult decision because either way you go there are penalties."—Elia Kazan Infectious diseases potentially transmissible through blood transfusion continue to emerge or reemerge globally. Ten articles in this issue of TRANSFUSION focus on the most recent of these, Zika virus, the third major arbovirus within the past two decades to have been introduced to the Western Hemisphere. Zika virus follows the introduction of West Nile virus to New York City in 1999 and chikungunya virus to Saint Martin Island in 2013.1, 2 Factors contributing to their introduction and subsequent spread throughout the Western Hemisphere include causes well identified for other emerging diseases, such as increased travel and trade, urbanization, and population growth. These viruses have become transfusion safety threats despite their relatively short durations of viremia because of their high incidence of infection of the human population during outbreaks and because a proportion of those infected remain asymptomatic and donate blood or alternatively may donate blood before developing symptoms.3, 4 Despite certain similarities of these arbovirus infections, important differences in their biology, epidemiology, and clinical impact have distinct implications for transfusion medicine. Although the widely dispersed Culex species mosquitoes that transmit West Nile virus have permitted viral spread throughout much of the Americas, including all states of the contiguous United States, for unclear reasons large human outbreaks have occurred with variable seasonal intensity only in the United States and Canada.5 Humans develop insufficient viremia to efficiently infect mosquitoes and do not contribute to viral transmission, while many bird species produce high-level viremias and serve as reservoirs for West Nile virus.6 The relatively short life span and high turnover of avian reservoir species do not permit the development of long-standing herd immunity; thus, repeated outbreaks will continue indefinitely.5 Given this epidemiology, along with the propensity of West Nile virus to cause severe neuroinvasive disease, particularly among the elderly, blood donor screening has been proven to be of benefit to public health surveillance over a number of years.7 However, the future epidemiology and impact of chikungunya and Zika viruses are far less certain. Both viruses produce sufficient viremia in humans to efficiently infect Aedes aegypti mosquitoes, thus permitting a human-mosquito-human transmission cycle. The A. aegypti mosquito is ubiquitous in urban environments throughout the tropical and subtropical world, enabling both viruses to become established throughout the Western Hemisphere as a result of spread by human travel. However, A. aegypti is endemic only in the southernmost United States, with occasional seasonal introductions further north, thus limiting the geographic potential for autochthonous viral transmission.8, 9 This trend is similar to the observations with dengue, another arbovirus spread from human to human via A. aegypti mosquitoes, which has only caused transient focal outbreaks in southern Florida and Texas in recent decades, and that autochthonous chikungunya and Zika virus transmission has only been documented in these areas to date.10-13 While Aedes albopictus is a competent vector for dengue, chikungunya, and Zika viruses with wider distribution in the United States than A. aegypti, to date, only one case of autochthonous transmission of dengue in New York and none of chikungunya and Zika viruses has been convincingly shown to result from A. albopictus vectored transmission in the United States.14 Nevertheless, thousands of people with travel-associated chikungunya and Zika virus infection return to nearly all areas of the United States after travel to areas of ongoing transmission, thus presenting a potential risk of transfusion transmission in areas without autochthonous transmission.13 Concerns about transfusion transmission of chikungunya virus have been tempered by its usually short-lived illness without permanent sequelae and the likelihood that development of human herd immunity would greatly reduce transmission.3 The latter may have in fact occurred, with the halving of chikungunya cases reported to the Pan American Health Association each year since its introduction to the Western Hemisphere (http://www.paho.org/hq/index.php?option=com_topics&view=readall&cid=5927&Itemid=40931&lang=en).15 At first, Zika virus' introduction into the Western Hemisphere seemed to raise a level of concern similar to that for chikungunya virus. However, in the fall of 2015, several months after the recognition of mosquito-borne transmission of Zika virus in Brazil, investigators noted a sharp increase in the number of infants born with microcephaly, which prompted the World Health Organization to declare a Public Health Emergency of International Concern on February 1, 2016.16 Insufficient evidence existed at that time to make a causal link between maternal Zika virus infection and birth defects; considerable skepticism remained particularly since mosquito-borne viruses had never been linked definitively to human birth defects. Another confounding factor in February 2016 was reports of sexual transmission of Zika virus, particularly since sexual contact had never been associated with transmission of any other mosquito-borne virus.17 Accumulating case reports and a case series of infants with microcephaly began to strengthen the causal relationship between Zika virus and microcephaly and to define a specific phenotype among affected infants consisting of severe microcephaly, intracranial calcifications, redundant scalp skin, hypertonia/spasticity, clubfoot, and congenital joint contractures.18 These data, along with experimental data showing that Zika virus produced cell death and attenuated future growth of human neural progenitor cells, led the CDC to conclude in April 2016 that Zika virus caused microcephaly and other serious brain defects.19, 20 Subsequent animal models demonstrated that Zika virus is a teratogen, and a case-control study demonstrated a strong relationship between maternal infection and microcephaly.21-25 The full spectrum of adverse fetal outcomes and the risk associated with infection throughout pregnancy is currently unknown and remains an area of active investigation. What is now clear, however, is the potential for serious lifelong adverse impact to a child if Zika virus were to be transmitted by transfusion to a pregnant woman or her sexual partner. The articles focusing on Zika virus in this issue collectively describe a tremendous amount of knowledge gained over the relatively short time span of about a year. For a graphic illustration of the progression of Zika virus and progress in knowledge made regarding the virus in 2016, please see Oussayef and coworkers (https://www.cdc.gov/mmwr/volumes/65/wr/mm6552e1.htm?s_cid=mm6552e1_e).26 For further discussion, the articles in this issue are categorized into five themes. When the first case of Zika virus infection was reported in Puerto Rico by the CDC at the end of December 2015 (https://www.cdc.gov/media/releases/2015/s1231-zika.html), no commercial tests were available in the United States for its detection. However, work conducted about a decade prior on West Nile virus provided a directly relevant paradigm for the development of NAT for screening of the blood supply. In addition, NAT had previously been used for the detection of Zika virus in outbreak settings outside of the Western Hemisphere. Such prior work helped facilitate both test development and the implementation of screening. Several different noncommercial entities initiated the development of laboratory developed tests for diagnostic purposes, and two commercial sponsors experienced in NAT screening tests for blood engaged in the development of commercial assays to screen potential donors. The three articles in this issue relevant to the development of NAT screening of the blood supply are illustrative of the progress made in this field. Bielaire and colleagues27 describe the use of their Zika virus laboratory developed test that was used at the time of the 2013 to 2014 Zika virus outbreak in French Polynesia, which they had determined to have a limit of detection of approximately 100 copies/mL. For context, during the period when the samples screened were collected there was a 30-day deferral in place for symptoms of arbovirus infection at the French Polynesia blood bank in Tahiti. Given the relatively high asymptomatic infection rate with Zika virus, it is not too surprising that using minipools of 3 samples they found that 2.8% of donor samples were reactive for Zika virus RNA. No transfusion-transmitted cases of Zika virus were detected. With the development of more sensitive NAT assays and knowledge of the increased sensitivity provided by individual donor NAT described below, it is interesting to speculate that the actual percentage of reactive donor samples may have been higher than that described in French Polynesia, where it was estimated that approximately 11.5% of the population reported symptomatic infection. Stone and colleagues28 describe the evaluation of a 25-member panel of samples by 11 laboratories using 17 different assays. The comparison of the various assays provided the notable finding that enhanced sensitivity was associated with using a greater sample volume of plasma for the initial RNA extraction. Although not designed to compare the investigational commercial assays developed for use in screening the blood supply, the study found that both of these assays had 100% detection of standards at levels of down to 10 to 40 estimated copies/mL and LOD50 values of less than 5 copies/mL. Given that differences in the sensitivity of various assays are a well-recognized phenomenon with NAT, the development of a reference standard to harmonize results between different assays and laboratories was highly desirable. Once again emanating from global collaboration and using data provided by 21 different laboratories, Baylis and colleagues29 report the development of such a standard: IS 11468/16 for Zika virus RNA. The understanding of the critical variables for assay sensitivity, along with the availability of a reference standard, should greatly facilitate testing with appropriate sensitivity and allow comparison of results obtained across the globe. Although a publication has appeared describing the incidence of blood donations positive for Zika virus in Puerto Rico, to date there has not been a scientific publication regarding blood donations in the rest of the United States.30 The articles by Galel and colleagues31 and Williamson and colleagues32 represent the initial positive results from the commercial investigational blood screening tests of Roche Molecular Systems, Inc., and Hologic, Inc., respectively. The Roche test was the first to be implemented, and its use was allowed to proceed under an investigational new drug application in March 2016, just 3 months after the report of the first case in Puerto Rico.30, 31 The results of this study were highly informative regarding both the assay itself and the epidemiology of Zika virus in the United States outside of Puerto Rico. There were 23 initially reactive donations out of 358,786 samples tested using individual-donor NAT, and after follow-up testing, 14 of these were determined to represent true-positive donations. All of the positive donations identified were collected in Florida. Of note regarding the assay itself, using simulated minipools of 6 samples, only seven of these donations were identified. This finding is consistent with the Zika virus assay sensitivity characteristics described above. Ten of the 14 positive donations came from individuals who had risk factors for sexual transmission or who had traveled to areas with local transmission of Zika virus within the 90 days prior. However, three individuals had neither sexual nor travel risk factors identified. Roughly 6 months after the first case of Zika was reported in Puerto Rico, testing using the Hologic assay began in June 2016.32 The report by Williamson and colleagues provides complementary data to that above for regions of the United States outside of Florida and Puerto Rico. Of 466,834 donations screened with individual-donor NAT, five were found to be positive by supplemental testing. These donations were collected in Nevada, New York, Arizona, California, and Texas. One donor had donated platelets (PLTs) 1 week before the donation that was identified as being positive, and follow-up testing available did not suggest that Zika virus was transmitted to the recipient. Based on characteristics of the donors, the authors speculate that the individuals providing the donations were likely at the tail end of their RNA-positive period yet acknowledge that there is still uncertainty regarding how long transmission can occur after initial infection. The small number of positive donations identified with either of these assays in the United States outside of Puerto Rico stands in stark contrast to the average of 1% positive donations reported from Puerto Rico during the summer months of 2016. Pathogen reduction technology is currently approved in the United States for use with apheresis PLTs and plasma, and the ability of amotosalen and UV light pathogen inactivation technology to reduce Zika virus in plasma has been previously reported.33, 34 The article in this issue by Laughhunn and colleagues35 reporting on pathogen reduction of Zika virus in red blood cell (RBC) components is significant because of both its findings and its potential implications for safety of the blood supply in the future. In vitro use of an investigational technology for pathogen reduction of RBCs, amustaline (S-303) and glutathione, was associated with complete inactivation of more than 7.75 log genomic equivalents of Zika virus RNA using NAT and 5.99 log of infectivity relative to sham treatment using a cell culture assay. This work is significant in that it provides further evidence that after appropriate additional studies, pathogen reduction technologies may ultimately be effective in mitigating arboviral threats in addition to a variety of other pathogens in RBCs, PLTs, and plasma. Although inactivation of Zika, an enveloped virus, by solvent/detergent (S/D) treatment would be expected based on evidence obtained with other similar pathogens, formal documentation of this inactivation, as well as documentation of the effect of other viral clearance interventions, is welcome. The trio of articles by Blümel, Farcet, and Kühnel and their colleagues36-38 document that Zika is indeed inactivated by S/D treatment and by standard pasteurization technology (58 to 60°C for 2 hr for albumin). Indeed, Zika virus seems to be more sensitive to heat than other closely related viruses. In addition, nanofiltration with a pore size of 40 nm or less was found to remove all Zika virus infectious activity.36 All of these findings represent reassuring news regarding Zika virus and the safety of plasma derivatives. Given that Zika virus infection is frequently asymptomatic and that serious complications outside of pregnancy are uncommon, defining a population most at risk of complications from transfusion-transmitted Zika virus is a reasonable undertaking. Note that recent publications indicate the Zika virus is most likely to cause congenital malformations with infection during the first or early second trimesters.25, 39 Infection during that gestational period is estimated to be associated with rates of microcephaly of 11% to 13%.40, 41 The article by Murphy and colleagues42 reports on the number of women receiving blood transfusions in a large tertiary care hospital in Ottawa, Canada. They note that in their hospital only 0.04% of expectant mothers receive a transfusion during the first trimester. Although this information is a welcome addition to the literature, it must be interpreted with caution for two reasons. First, the data are not directly applicable to areas with higher rates of sickle cell disease and other hemoglobinopathies. Second, and perhaps of much greater relevance, is that the data must be interpreted in the context of the potential for sexual transmission of Zika virus.43 At this time, the potential for male-to-female sexual transmission is well documented, with presence of Zika virus RNA in semen reported for up to over 90 days after infection, and the rate of clearance has been reported to be variable.44 The mean duration during which semen is infectious is not yet known. Therefore, one can conclude that the issue is larger than just transfusing a pregnant woman—it is transfusing her male partner. Pending further data on the period of infectivity, an analysis is required that takes into consideration men receiving transfusions who might then have sexual contact in the next 90 days with women who are in the first or second trimester of pregnancy. Zika virus represents yet another in a series of emerging or reemerging threats to the blood supply.45, 46 Though a virus of known identity for several decades, the potential implications of the extensive Zika virus outbreak in the Western Hemisphere became more apparent with each month as the year 2016 progressed. Given the uncertainty regarding the spread of the epidemic and the broad range of the potential vector of Aedes mosquitos in the United States, a cautious approach led initially to implementation of donor deferrals for travel to areas with local Zika virus transmission and then to testing throughout the United States and its territories. As the articles in this issue illustrate, building on experience with prior emerging infectious diseases, much has been learned in the relatively brief period of a year both about the nature of the virus and its epidemiology. This knowledge is invaluable as we refine the response to this epidemic. However, in addition to uncertainty regarding whether Zika virus will spread further or become endemic in some areas, there is also much that remains unknown about the complications of infection itself. Clearly, universal screening of the blood supply was a significant undertaking in the United States, and concern regarding resource utilization is understandable.47 However, it is too early to tell whether or not such continued universal screening is necessary. This potential requirement should become clearer during the next year as we observe whether warmer months in the Northern Hemisphere are associated with a resurgence of spread of the virus. In the meantime, as noted by Galel and colleagues, in addition to helping to facilitate an adequate blood supply by removing regional deferrals and allowing collection to continue in places such as Puerto Rico and Florida, screening of the blood supply has had the benefit of leading to the prompt reporting of reactive donors, facilitating a rapid public health response to evaluate and address potential local transmission of Zika virus.31 With the combination of the rapidity of the Zika virus outbreak in the Western Hemisphere, the potential for adverse fetal and other outcomes, and the uncertainty involved, decision-making certainly was not easy. Indeed there are real costs associated with the course that was taken that must be balanced against the potential costs that could have been incurred with a different course of action and different potential outcomes. The articles in this issue fill important gaps in our knowledge as we continue to learn more about this arboviral pathogen. The authors have disclosed no conflicts of interest. The opinions expressed herein are those of the authors and do not represent those of the Centers for Disease Control and Prevention, the U.S. Food and Drug Administration, the Department of Health and Human Services, or the U.S. Government. Peter W. Marks, MD, PhD1 e-mail: [email protected] Lyle R. Petersen, MD, MPH2 1Center for Biologics Evaluation and Research U.S. Food and Drug Administration Silver Spring, MD 2Division of Vector-Borne Diseases National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention Atlanta, GA

Guillain–Barré syndrome associated with Zika virus infection in Honduras, 2016–2017

To the Editor: Isolated in 1947 from a rhesus monkey in Zika forest, Uganda, Zika virus (ZIKV) is a mosquito-borne flavivirus (1).For half a century, ZIKV was described only as causing sporadic human infections in Africa and Asia, which was mostly confirmed by serologic methods (2).In 2007, the first ZIKV outbreak reported outside Africa and Asia was retrospectively documented from biological samples of patients on Yap Island, Federated States of Micronesia, North Pacific, who had received an incorrect diagnosis of dengue virus (DENV) (3,4).We report here the early investigations that led to identification of ZIKV as the causative agent of an outbreak that started in October 2013 in French Polynesia.French Polynesia is a French overseas territory located in the South Pacific.The ≈270,000 inhabitants live on 67 islands distributed into 5 archipelagoes (Society, Marquesas, Tuamotu, Gambier, and Austral Islands).Surveillance for acute febrile illnesses is coordinated by the Department of Health with the contribution of a sentinel network of public and private practitioners, the main public hospital (Centre Hospitalier

Before it emerged as the suspected cause for the increased incidence of microcephaly observed in north-east Brazil in 2015, the vast majority of physicians were not familiar with Zika virus. But, that changed dramatically with the worldwide publicity given to this emerging public health threat. On February 1, 2016, the Director General of the World Health Organization (WHO) declared a Public Health Emergency of International Concern (PHEIC). This followed growing concerns about reports of Guillain-Barré syndrome appearing concomitantly with a Zika virus outbreak in French Polynesia from late 2013 to early 2014. Although Zika virus has not so far been proven to cause microcephaly or Guillain-Barré syndrome, scientific publications have started to emerge that have strengthened the aetiological connection between Zika virus and both maladies. The virus was first isolated in 1947 from a rhesus macaque captured in the Zika forest of Uganda from which it got its name 1. Zika virus is an arbovirus belonging to the Flaviviridae family and is transmitted by mosquitos. Unlike many pathogenic flaviviruses, like yellow fever virus, Japanese encephalitis virus, the four dengue viruses and West Nile virus, Zika virus had previously been considered to cause just a mild disease, with symptoms such as fever, rash, arthralgia, muscle pain and conjunctivitis 1, 2. In fact, more than 80% of those infected with Zika virus are asymptomatic. As Zika virus antibodies cross-react with dengue virus, the specific diagnosis of Zika virus infection relies on the detection of the virus nucleic acid in bodily fluids by reverse-transcriptase polymerase chain reaction or by detecting virus-specific immunoglobulin M antibodies. Until 2007, when an outbreak of the virus affected 70% of the population of the Micronesian islands of the Yap State, Zika virus had only been seen in Africa and South-East Asia. Six years later, a Zika virus epidemic occurred in French Polynesia, and it is estimated that more than 32 000 people were infected 3. The virus was probably introduced to the Americas in 2014, and the first report of autochthonous transmission in Brazil was as late as May 2015 1. Probably, the virus spread from Polynesia to the Americas by competitors from Pacific countries who took part in an international canoeing competition in Rio de Janeiro in August 2014 2. Zika virus has now spread to 31 countries and territories in the Americas, including Puerto Rico 4. The virus that infects human is carried by mosquitoes of the Aedes species, primarily by Aedes aegypti and probably also by Aedes albopictus 1. As the virus has been found in Puerto Rico, it is now considered to be only a matter of time before mosquito-born transmission of Zika virus will also be found in the United States. The virus can be found in urine and sperm and sexual transmission has been reported. Zika virus infections have also been found in patients in Europe and the United States who travelled to infected areas. Autochthonous transmission from such cases has been reported, probably due to sexual contact 4. As mentioned above, the reason for the recent public attention focused on Zika virus, and the PHEIC issued by the WHO, is its connection to Guillain-Barré syndrome and microcephaly. Guillain-Barré syndrome is an autoimmune postinfectious neurological disorder characterised by an ascending weakness starting in the distal legs 5, and in 20–30% of cases, it presents with rapid progressive paralysis and respiratory failure 5. The disease usually reaches its peak in two to 4 weeks, followed by a plateau phase of some months followed by a recovery phase. The mortality rate is 3–7%. It can be classified as an acute motor axonal neuropathy, where complement-fixing autoimmune antiganglioside antibodies attack nerve axon membranes, or an acute inflammatory demyelinating polyneuropathy. The former condition is usually the more serious 5. Guillain-Barré syndrome has been described after infections, particularly with campylobacter jejuni, but also with cytomegalovirus, Epstein–Barr virus, influenza A virus, hepatitis E virus, Mycoplasma pneumonia and Haemophilus influenzae 5. More recently, it has also been associated with arboviruses like chikungunia virus, West Nile virus, Japanese encephalitis virus and dengue virus 3. When the outbreak of Zika virus occurred in French Polynesia in 2013–2014, the diagnosis of Guillain-Barré syndrome increased from three to ten cases per year to 42 cases between November of 2013 and February of 2014 3, a 20-fold higher incidence than the previous four years 4. These 42 patients were affected by the acute motor axonal neuropathy type of the syndrome, with rapid progression but a short plateau phase that only lasted days. In an age- and sex-matched case-controlled study of these 42 patients, the risk of developing Guillain-Barré syndrome was calculated to be 0.24 per 1000 Zika virus infections 3. The study showed that 37 of the 42 patients had a history of viral syndrome for a median of six days prior to the onset of Guillain-Barré syndrome, and all of them (100%) had serological evidence of a recent Zika virus infection. This was compared with 54 (56%) of the 98 patients in the control group, who were recruited to the same hospital, but did not have febrile illness (p < 0.0001) 3. No other infection was diagnosed in the 42 patients, making it highly probable that Zika virus was the cause of Guillain-Barré syndrome in these patients. Although it has not yet been proven to be the causative agent, Zika virus infections have been reported to be linked to an increased incidence of Guillain-Barré syndrome in the state of Bahia in Brazil, Colombia, El Salvador, Suriname and Venezuela. In addition, cases of Guillain-Barré syndrome combined with Zika virus have been reported from Martinique, Puerto Rica and Panama 4. The other neurological disorder that has recently been linked to the Zika virus is microcephaly. During the four months from October 22, 2015 to February 27, 2016, no less than 5909 suspected cases of microcephaly were reported in Brazil, particularly in its northeast region, compared to a national average of 163 cases per year in the previous 15 years 4. Of these cases, 1687 have been investigated further and 641 (38%) were confirmed to fulfil the definition of microcephaly and/or central nervous system malformations associated with congenital infection 4, corresponding to at least a 40-fold increase in incidence. During the outbreak of Zika virus in French Polynesia, described above, 19 cases of congenital central nervous system malformations, including eight cases of microcephaly, were reported, compared to a national average of zero to two cases per year 4. Apart from associations with the timings and geographical locations of Zika virus outbreaks and these congenital central nervous system malformations, three reports published this year have identified Zika virus infection as the probable a cause of microcephaly 6, 7. In the first two reports, Zika virus ribonucleic acid was detected in the brain tissue, but not any other tissue, of a 32-week-old Slovenian foetus with microcephaly 6 and in a Finnish foetus with brain abnormalities terminated at 21 weeks of gestation 7. The two mothers had probably been infected in north-eastern Brazil and Central America, respectively, both at the end of the first trimester. Of note, ultrasonography may not show microcephaly at 20 weeks of gestation 6, 7. The third report was a study by Tang et al. 8 that showed that the Zika virus could infect and propagate in cultured cortical human neural progenitor cells derived from induced pluripotent stem cells, proving that the virus to be neurotropic. Unfortunately, there is probably little that can be done to halt the present Zika virus epidemic in the Americas. There is no antiviral intervention or vaccine available yet, which are both desperately needed, so are specific and easy to perform serological diagnostic tests for the detection of past Zika virus infection. All we can do at the moment is to try to control the mosquitos that carry the virus, discourage pregnant women from travelling to infected areas and raise awareness of the risk of transmission from sexual contact. In the meantime, readers may be interested in the frequently asked questions document on the Zika virus, which can be accessed from the home page of the Global Virus Network's website at: http://gvn.org/zika/.

BackgroundWe evaluated whether outbreaks of Zika virus (ZIKV) infection, newborn microcephaly, and Guillain-Barré syndrome (GBS) in Latin America may be detected through current surveillance systems, and how cases detected through surveillance may increase health care burden.MethodsWe estimated the sensitivity and specificity of surveillance case definitions using published data. We assumed a 10% ZIKV infection risk during a non-outbreak period and hypothetical increases in risk during an outbreak period. We used sensitivity and specificity estimates to correct for non-differential misclassification, and calculated a misclassification-corrected relative risk comparing both periods. To identify the smallest hypothetical increase in risk resulting in a detectable outbreak we compared the misclassification-corrected relative risk to the relative risk corresponding to the upper limit of the endemic channel (mean + 2 SD). We also estimated the proportion of false positive cases detected during the outbreak. We followed the same approach for microcephaly and GBS, but assumed the risk of ZIKV infection doubled during the outbreak, and ZIKV infection increased the risk of both diseases.ResultsZIKV infection outbreaks were not detectable through non-serological surveillance. Outbreaks were detectable through serologic surveillance if infection risk increased by at least 10%, but more than 50% of all cases were false positive. Outbreaks of severe microcephaly were detected if ZIKV infection increased prevalence of this condition by at least 24.0 times. When ZIKV infection did not increase the prevalence of severe microcephaly, 34.7 to 82.5% of all cases were false positive, depending on diagnostic accuracy. GBS outbreaks were detected if ZIKV infection increased the GBS risk by at least seven times. For optimal GBS diagnosis accuracy, the proportion of false positive cases ranged from 29 to 54% and from 45 to 56% depending on the incidence of GBS mimics.ConclusionsCurrent surveillance systems have a low probability of detecting outbreaks of ZIKV infection, severe microcephaly, and GBS, and could result in significant increases in health care burden, due to the detection of large numbers of false positive cases. In view of these limitations, Latin American countries should consider alternative options for surveillance.

ABSTRACT.Serological cross-reactivity has proved to be a challenge to diagnose Zika virus (ZIKV) infections in dengue virus (DENV) endemic countries. Confirmatory testing of ZIKV IgM positive results by plaque reduction neutralization tests (PRNTs) provides clarification in only a minority of cases because most individuals infected with ZIKV were previously exposed to DENV. The goal of this study was to evaluate the performance of a ZIKV/DENV DUO IgM antibody capture ELISA (MAC-ELISA) for discriminating between DENV and ZIKV infections in endemic regions. Our performance evaluation included acute and convalescent specimens from patients with real-time reverse transcription polymerase chain reaction (RT-PCR)-confirmed DENV or ZIKV from the Sentinel Enhanced Dengue Surveillance System in Ponce, Puerto Rico. The ZIKV/DENV DUO MAC-ELISA specificity was 100% for DENV (N = 127) and 98.4% for ZIKV (N = 275) when specimens were tested during the optimal testing window (days post-onset of illness [DPO] 6–120). The ZIKV/DENV DUO MAC-ELISA sensitivity of RT-PCR confirmed specimens reached 100% for DENV by DPO 6 and for ZIKV by DPO 9. Our new ZIKV/DENV DUO MAC-ELISA was also able to distinguish ZIKV and DENV regardless of previous DENV exposure. We conclude this novel serologic diagnostic assay can accurately discriminate ZIKV and DENV infections. This can potentially be useful considering that the more labor-intensive and expensive PRNT assay may not be an option for confirmatory diagnosis in areas that lack PRNT capacity, but experience circulation of both DENV and ZIKV.

Zika virus infection in humans is usually mild or asymptomatic. However, some babies born to women infected with Zika virus have severe neurological sequelae. An unusual cluster of cases of congenital microcephaly and other neurological disorders in the WHO Region of the Americas, led to the declaration of a public health emergency of international concern by the World Health Organization (WHO) on 1 February 2016. By 5 May 2016, reports of newborns or fetuses with microcephaly or other malformations – presumably associated with Zika virus infection – have been described in the following countries and territories: Brazil (1271 cases); Cabo Verde (3 cases); Colombia (7 cases); French Polynesia (8 cases); Martinique (2 cases) and Panama (4 cases). Additional cases were also reported in Slovenia and the United States of America, in which the mothers had histories of travel to Brazil during their pregnancies.1 Zika virus is an intensely neurotropic virus that particularly targets neural progenitor cells but also – to a lesser extent – neuronal cells in all stages of maturity. Viral cerebritis can disrupt cerebral embryogenesis and result in microcephaly and other neurological abnormalities.2 Zika virus has been isolated from the brains and cerebrospinal fluid of neonates born with congenital microcephaly and identified in the placental tissue of mothers who had had clinical symptoms consistent with Zika virus infection during their pregnancies.3–5 The spatiotemporal association of cases of microcephaly with the Zika virus outbreak and the evidence emerging from case reports and epidemiologic studies, has led to a strong scientific consensus that Zika virus is implicated in congenital abnormalities.6,7 Existing evidence and unpublished data shared with WHO highlight the wider range of congenital abnormalities probably associated with the acquisition of Zika virus infection in utero. In addition to microcephaly, other manifestations include craniofacial disproportion, spasticity, seizures, irritability and brainstem dysfunction including feeding difficulties, ocular abnormalities and findings on neuroimaging such as calcifications, cortical disorders and ventriculomegaly.3–6,8–10 Similar to other infections acquired in utero, cases range in severity; some babies have been reported to have neurological abnormalities with a normal head circumference. Preliminary data from Colombia and Panama also suggest that the genitourinary, cardiac and digestive systems can be affected (Pilar Ramon-Pardo, unpublished data). The range of abnormalities seen and the likely causal relationship with Zika virus infection suggest the presence of a new congenital syndrome. WHO has set in place a process for defining the spectrum of this syndrome. The process focuses on mapping and analysing the clinical manifestations encompassing the neurological, hearing, visual and other abnormalities, and neuroimaging findings. WHO will need good antenatal and postnatal histories and follow-up data, sound laboratory results, exclusion of other etiologies and analysis of imaging findings to properly delineate this syndrome. The scope of the syndrome will expand as further information and longer follow-up of affected children become available. The surveillance system that was established as part of the epidemic response to the outbreak initially called only for the reporting of microcephaly cases. This surveillance guidance has been expanded to include a spectrum of congenital malformations that could be associated with intrauterine Zika virus infection.11 Effective sharing of data is needed to define this syndrome. A few reports have described a wide range of abnormalities,3–6,8–10 but most data related to congenital manifestations of Zika infection remain unpublished. Global health organizations and research funders have committed to sharing data and results relevant to the Zika epidemic as openly as possible.12 Further analysis of data from cohorts of pregnant women with Zika virus infection are needed to understand all outcomes of Zika virus infection in pregnancy. Thirty-seven countries and territories in the Region of the Americas now report mosquito-borne transmission of Zika virus and risk of sexual transmission. With such spread, it is possible that many thousands of infants will incur moderate to severe neurological disabilities. Therefore, routine surveillance systems and research protocols need to include a larger population than simply children with microcephaly. The health system response, including psychosocial services for women, babies and affected families will need to be fully resourced. The Zika virus public health emergency is distinct because of its long-term health consequences and social impact. A coordinated approach to data sharing, surveillance and research is needed. WHO has thus started coordinating efforts to define the congenital Zika virus syndrome and issues an open invitation to all partners to join in this effort.

Zika virus (ZIKV) infection in pregnant women is a serious threat to the development and viability of the fetus. The primary mode of ZIKV transmission to humans is through mosquito bites, but sexual transmission has also been well documented in humans. However, little is known of the short- and long-term effects of ZIKV infection on the human male reproductive system. This study examines the effects of ZIKV infection on the male reproductive organs and semen and the immune response of the olive baboon (Papio anubis). Nine mature male baboons were infected with ZIKV (French Polynesian strain) subcutaneously. Six animals were euthanized at 41 days, while three animals were euthanized at 10 or 11 days postinfection (dpi). Viremia and clinical evidence of infection were present in all nine baboons. ZIKV RNA was present in the semen of five of nine baboons. ZIKV was present in the testes of two of three males euthanized at 10 or 11 dpi, but in none of six males at 41 dpi. Immunofluorescence of testes suggested the presence of ZIKV in sperm progenitor cells, macrophage penetration of seminiferous tubules, and increased tumor necrosis factor alpha (TNF-α), particularly in vascular walls. These data demonstrate that male olive baboons approximate the male human ZIKV response, including viremia, the adaptive immune response, and persistent ZIKV in semen. Although gross testicular pathology was not seen, the demonstrated breach of the testes-blood barrier and targeting of spermatogenic precursors suggest possible long-term implications in ZIKV-infected primates.IMPORTANCE Zika virus (ZIKV) is an emerging flavivirus spread through mosquitoes and sexual contact. ZIKV infection during pregnancy can lead to severe fetal outcomes, including miscarriage, fetal death, preterm birth, intrauterine growth restriction, and fetal microcephaly, collectively known as congenital Zika syndrome. Therefore, it is important to understand how this virus spreads, as well as the resulting pathogenesis in translational animal models that faithfully mimic ZIKV infection in humans. Such models will contribute to the future development of efficient therapeutics and prevention mechanisms. Through our previous work in olive baboons, we developed a nonhuman primate model that is permissive to ZIKV infection and transfers the virus vertically from mother to fetus, modeling human observations. The present study contributes to understanding of ZIKV infection in male baboon reproductive tissues and begins to elucidate how this may affect fertility, reproductive capacity, and sexual transmission of the virus.

In 2007, physicians on Yap Island reported an outbreak of illness characterized by rash, conjunctivitis, and arthralgia. Although serum from some patients had IgM antibody against dengue virus, the illness seemed clinically distinct from previously detected dengue. Subsequent testing with the use of consensus primers detected Zika virus RNA in the serum of the patients but no dengue virus or other arboviral RNA. No previous outbreaks and only 14 cases of Zika virus disease have been previously documented. We obtained serum samples from patients and interviewed patients for information on clinical signs and symptoms. Zika virus disease was confirmed by a finding of Zika virus RNA or a specific neutralizing antibody response to Zika virus in the serum. Patients with IgM antibody against Zika virus who had a potentially cross-reactive neutralizing-antibody response were classified as having probable Zika virus disease. We conducted a household survey to estimate the proportion of Yap residents with IgM antibody against Zika virus and to identify possible mosquito vectors of Zika virus. We identified 49 confirmed and 59 probable cases of Zika virus disease. The patients resided in 9 of the 10 municipalities on Yap. Rash, fever, arthralgia, and conjunctivitis were common symptoms. No hospitalizations, hemorrhagic manifestations, or deaths due to Zika virus were reported. We estimated that 73% (95% confidence interval, 68 to 77) of Yap residents 3 years of age or older had been recently infected with Zika virus. Aedes hensilli was the predominant mosquito species identified. This outbreak of Zika virus illness in Micronesia represents transmission of Zika virus outside Africa and Asia. Although most patients had mild illness, clinicians and public health officials should be aware of the risk of further expansion of Zika virus transmission.

Zika virus (ZIKV) and dengue virus (DENV) are co-endemic in many parts of the world, but the impact of ZIKV infection on subsequent DENV infection is not well understood. Here we show in rhesus macaques that the time elapsed after ZIKV infection affects the immune response to DENV infection. We show that previous ZIKV exposure increases the magnitude of the antibody and T cell responses against DENV. The time interval between ZIKV and subsequent DENV infection further affects the immune response. A mid-convalescent period of 10 months after ZIKV infection results in higher and more durable antibody and T cell responses to DENV infection than a short period of 2 months. In contrast, previous ZIKV infection does not affect DENV viremia or pro-inflammatory status. Collectively, we find no evidence of a detrimental effect of ZIKV immunity in a subsequent DENV infection. This supports the implementation of ZIKV vaccines that could also boost immunity against future DENV epidemics.

Zika virus is one Flavi virus, which is similar to dengue virus. At present, Zika virus infection occurs in some countries located in Central and South America. It is carried by Aedes aegypti mosquitoes, which are not living in Korea. However, Aedes albopictus has been spotted in Jeju Island and now this particular mosquito is known for carrying the virus, but at the current moment this mosquito is not carrying the Zika virus in Korea (1). Zika virus was initially discovered in rhesus monkeys in Zika forest, Tanzania in 1947 (2). After that, the virus sporadically spread in Asia and Africa. Aside from the two continents, the first report of the outbreak came from Yap Island in the Federated States of Micronesia. At that time, it was recorded that 14.6 per 10 million people were infected by the virus (3). From this time on, French Polynesia in the South Pacific reported the largest outbreak of Zika virus infection in 2013; a rise was recorded where 28,000 people (approximately 11% of the population) had been infected in 2013-2014 (4). On 1 February 2016, the World Health Organization declared a Public Health Emergency of International Concern to encourage prevention of international spreading of Zika virus infection. In Central and South America, including Brazil, about 1.5 million people were estimated to be infected by the virus in 2015. With the outbreak of Zika virus from 2015 to 16 January 2016, 3,893 babies with microcephaly or underdeveloped brains and skulls were born in Brazil and 49 babies died. Zika virus was confirmed in the amniotic fluid of pregnant women and the brain and body tissues of dead or stillborn children (5,6). However, it is unknown how Zika virus causes microcephaly when pregnant women are infected by the virus. The prevalence of Guillain-Barre syndrome also increases in endemic areas of Zika virus infection but relation of that syndrome and the infection is not proven (6,7). Nations where Zika virus is found recently are summarized in Table 1 (8). Table 1 Nations that Zika virus has been found from 2015. Zika virus is rarely transferred between people. It is assumed that the virus could spread through blood transfusion. When an outbreak of Zika virus was reported in French Polynesia between 2013 and 2014, 3% of blood donations tested positive by PCR (9). There was a report that Zika virus could be transmitted through sexual contact. When an outbreak happened in French Polynesia in 2013, semen samples of patients who were believed to be infected tested positive for Zika virus by PCR (10). Moreover, after a person travelled to Senegal, Zika virus was found in his semen and his wife was observed to have Zika virus infection symptoms after 4 days of having sexual contact (11). However, this is very rare. Further research will be needed to determine whether Zika virus could be transmitted by sexual contact. Although Zika virus was found in patients' urine or saliva, it is not confirmed transmittable through these kinds of body fluid (12, 13). Zika fever is officially specified as a group 4 legal infectious disease by Korean Center for Disease Control and Prevention (KCDC) on 29 January 2016 (14). All medical staffs were directed to report to KCDC when they find patients who are suspected to have Zika virus infection or those who are confirmed to have the Zika virus infection. KCDC recommends that pregnant women do not visit nations where Zika virus is reported to have occurred in the last 2 months. KCDC started to promote means of preventing Zika virus infection to travelers who will visit the identified countries. Since there is a possibility for Zika virus infection among travelers who visit high-risk countries, there is a need to monitor travelers coming from the countries identified to be at risk. Additionally, due to global warming, the ecosystem of mosquitoes in Korea has been changed. Monitoring the mosquitoes' habitat as well as research on the possibility of domestic occurrence should be continued. Many climate scientists and infectious disease specialists have said that environment destruction, industrial developments, and global warming are the major reasons for the spread of mosquito-borne infections. Since 1970, destruction of tropical forests due to development of large-scale resorts, plantations, and industries has been happening in Southeast Asia and South America. Because of the urbanization of tropical forests and global warming, mosquitoes' growth condition is at its peak. This causes the outbreak of dengue fever in Southeast Asia and Zika fever in South America. Environmental destruction leads to a potential disaster that could totally change our life. Zika virus infection is a new disease that threatens global public health, as WHO declared. It is the time to come up with a long-term perspective and systematic preparations to control this disease.

Zika virus (ZIKV) infection usually presents as a mild and self-limited illness, but it may be associated with severe outcomes. We describe a case of a 30-year-old man with systemic erythematous lupus and common variable immunodeficiency who became infected with both Zika (ZIKV) and Chikungunya (CHIKV) virus during the 2016 outbreak in Rio de Janeiro, Brazil. The patient presented with intense wrist and right ankle arthritis, and ZIKV RNA and virus particles were detected in synovial tissue, blood and urine, and CHIKV RNA in serum sample, at the time of the diagnosis. During the follow up, ZIKV RNA persisted for 275 days post symptoms onset. The patient evolved with severe arthralgia/arthritis and progressive deterioration of renal function. Fatal outcome occurred after 310 days post ZIKV and CHIKV co-infection onset. The results show the development of severe disease and fatal outcome of ZIKV infection in an immunosuppressed adult. The data suggests a correlation between immunodeficiency and prolonged ZIKV RNA shedding in both blood and urine with progressive disease. The results also indicate a possible role for arbovirus co-infections as risk factors for severe and fatal outcomes from ZIKV infection.

The frequency of Zika virus (ZIKV)-specific IgA and IgM and the cytokine expression profile of ZIKV-infected patients in hyperendemic areas remain unclear. This study investigated the rates of ZIKV non-structural protein 1 (NS1)-specific IgA and IgM and evaluated serum cytokine levels of ZIKV and Dengue virus (DENV) cases in Thailand to identify potential diagnostic biomarkers, elucidate the immunity against ZIKV and DENV, and investigate the association between cytokine levels and ZIKV symptoms. Low rates of positivity for ZIKV NS1-specific IgA and IgM were detected in our study. ZIKV NS1 IgA/M (11%, 11/101) in combination was more frequently detected than ZIKV NS1 IgM (2%, 2/101) or ZIKV NS1 IgA (4%, 4/96) alone, especially in acute ZIKV cases with previous DENV exposure (14%, 10/72). Cytokine analysis showed that both ZIKV and DENV infections induced polyfunctional immunity, and the latter triggered more prolonged responses. The existence of significant differences in IL-4 and IL-10 levels between acute ZIKV and acute DENV cases suggested that IL-4 (p = 0.0176) and IL-10 (p = 0.0003) may represent biomarkers for acute ZIKV and acute DENV infections, respectively. Analysis of the association between increased cytokine levels and ZIKV symptoms indicated that CXCL10 (p = 0.0029) was associated with exanthema, while IL-5 (p = 0.0496) was linked to headache. The detection of ZIKV NS1 IgA and IgM in combination may enhance the diagnosis of early ZIKV infection, particularly when levels of IgM or IgA alone are low or undetectable. IL-4 and IL-10 may serve as targets for the development of diagnostic tools to detect ZIKV and DENV infections early, respectively, in flavivirus-endemic regions.