Manoel Sarno

Zika Virus Infection and Stillbirths: A Case of Hydrops Fetalis, Hydranencephaly and Fetal Demise.

Background The rapid spread of Zika virus in the Americas and current outbreak of microcephaly in Brazil has raised attention to the possible deleterious effects that the virus may have on fetuses. Methodology/Principal Findings We report a case of a 20-year-old pregnant woman who was referred to our service after a large Zika virus outbreak in the city of Salvador, Brazil with an ultrasound examination that showed intrauterine growth retardation of the fetus at the 18th gestational week. Ultrasound examinations in the 2nd and 3rd trimesters demonstrated severe microcephaly, hydranencephaly, intracranial calcifications and destructive lesions of posterior fossa, in addition to hydrothorax, ascites and subcutaneous edema. An induced labor was performed at the 32nd gestational week due to fetal demise and delivered a female fetus. ZIKV-specific real-time polymerase chain reaction amplification products were obtained from extracts of cerebral cortex, medulla oblongata and cerebrospinal and amniotic fluid, while extracts of heart, lung, liver, vitreous body of the eye and placenta did not yield detectable products. Conclusions/Significance This case report provides evidence that in addition to microcephaly, there may be a link between Zika virus infection and hydrops fetalis and fetal demise. Given the recent spread of the virus, systematic investigation of spontaneous abortions and stillbirths may be warranted to evaluate the risk that ZIKV infection imparts on these outcomes.

Emergence of Congenital Zika Syndrome: Viewpoint From the Front Lines

Zika, a mosquito-borne flavivirus discovered in Uganda in 1947, remained obscure until its emergence in Micronesia in 2007. Six years later, it arrived in French Polynesia and other islands in the South Pacific (1). The virus was first detected in Brazil in early 2015 and has now spread throughout South and Central America and the Caribbean (2). Infection often remains unrecognized because it either is asymptomatic (75% to 80%) or has a nonspecific presentation of rash and fever. The first suggestion that Zika virus causes more than a self-limited illness was during the French Polynesian outbreak, when incidence of Guillain-Barr syndrome increased 20-fold (3). Likewise, a cluster of cases of this syndrome was identified in Brazil after the introduction of Zika virus (4). From July to September 2015, several months after the introduction of Zika virus into northeastern Brazil, obstetricians noticed an increased number of fetuses with congenital malformations during ultrasound screening. By October, the number of newborns with microcephaly had increased significantly in this area, according to birth registry data from previous years. Microcephaly had now increased in other regions along with the spread of Zika virus. To date, more than 4000 cases have been reported (Figures 1 and 2). Figure 1. Distribution of incident cases of microcephaly among Brazilian newborns, according to epidemiologic week and geographic region from 15 November 2015 to 16 January 2016. From the Brazilian Ministry of Health. Figure 2. Cumulative cases of microcephaly according to federal state from 15 November 2015 to 16 January 2016. From the Brazilian Ministry of Health. That Zika virus is the cause of the large number of microcephaly cases identified during the epidemic remains presumptive (5). Brazilian researchers first noted the viruss potential association with microcephaly when they investigated a newborn with this condition, who died soon after birth and was found to have detectable virus in tissues. Subsequently, Zika virus RNA was detected in additional cases of fetuses and stillbirths with congenital malformations (68). To date, the strongest evidence of the correlation between Zika virus and microcephaly is a circumstantial link between the spatial and temporal patterns of these infections and the appearance of microcephaly. In addition, this condition was retrospectively identified in infants born during the 2013 outbreak in French Polynesia. Despite these observations, investigators have not determined a definitive association between Zika virus and microcephaly cases in the Brazilian outbreak, most of which have been live-born infants. Our investigation is still in progress; however, we have gained insight into the scope and severity of microcephaly due to presumed congenital Zika syndrome (CZS), as well as challenges in confirming this association. Microcephaly is characterized by severe manifestations, such as marked cerebral atrophy and ventriculomegaly, extensive intracranial calcifications, simplified gyral patterns, dysgenesis of the corpus collosum, and cerebellar hypoplasia (Figure 3). Furthermore, CZS manifestations that extend beyond the central nervous system have been observed, including auditory impairment as well as ocular manifestations (9), such as focal pigment mottling and chorioretinal atrophy, which are distinct from other congenital conditions. Similar ocular lesions have been anecdotally identified in normocephalic newborns, suggesting that the overall burden may not be restricted to microcephaly cases. Figure 3. Computed tomography, reconstructed in the coronal oblique plane, of a newborn with microcephaly. Craniofacial dysmorphism, subcortical and basal ganglia calcifications, simplified gyral pattern, ventriculomegaly, and dysgenesis of the corpus callosum are seen. Although the apparent increase in microcephaly supports the assertion that Zika virus causes a distinct congenital syndrome, diagnostic limitations suggest caution in assuming a causal relationship. We have detected Zika virus RNA in only a fraction of microcephaly cases. Increased case ascertainment of microcephaly due to other causes has probably occurred contemporaneously. The inability to detect Zika virus in newborns with microcephaly may reflect compartmentalization of virus in tissues not sampled at the time of birth. Alternatively, intrauterine infections may be self-limited and Zika virus often cleared by birth. Screening approaches are essential for pregnant women who reside in impoverished regions where Zika virus has been recently introduced and who do not have access to ultrasonography and amniocentesis. Although detection is hampered by the extensive antigenic cross-reactivity with dengue and other circulating flaviviruses, a serologic test for prior intrauterine exposure to the virus in newborns is critically needednot only for diagnosis in pregnant women and newborns but also to identify individuals who have been infected with Zika virus and are presumably immune to reinfection. A more accurate IgG assay is essential to stratify risk in women of childbearing age and to facilitate targeted prenatal screening. Molecular detection is unlikely to be available in many regions, and infections are asymptomatic. Potential explanations for the recent explosion of Zika virus in the Pacific islands and the Americas and its continued spread are unclear but include recent genetic/phenotypic virus changes before or coincident with its disbursement beyond Asia. This could involve selection for enhanced infection of mosquito vectors, such as Aedes aegypti. Such vector-adaptive selection of more transmissible chikungunya virus strains has occurred since 2005. Other possibilities include selection for higher levels of human viremia in the urban cycle, which could increase the efficiency of transmission as well as enhance fetal infection. A simpler explanation is that the outbreaks began when, by chance, the virus was introduced into naive populations at the right time and place for initiation of the humanmosquito cycle. If the virus is able to establish endemic or enzootic circulation (as has been suggested as occurring in Asia on the basis of seroprevalence data) stable herd immunity may prevent future epidemics, as well as CZS in Brazil. Further, genetic, pathogenesis, and vector infection studies with diverse virus strains combined with improved surveillance and better, more affordable diagnostics that can be deployed even in remote, resource-limited settings, are needed to evaluate these hypotheses. Unfortunately, the immediate prospects for controlling the magnitude and spread of the current Zika virus epidemic are not promising. Until a vaccine is available, mosquito control and education of at-risk populations to reduce contact with the vector are the only short-term approaches available. These methods have had limited success for dengue and chikungunya viruses. Although recent advances in flavivirus vaccines may guide relatively rapid development of a Zika vaccine, availability is still probably years away. Treatment with a monoclonal antibody could also be developed quickly on the basis of promising past results with flaviviruses. However, systematic investigations of pregnant women and newborns will still be needed to determine the risk for transplacental infection and development of severe congenital sequelae that can, in turn, guide effective diagnostic and prevention efforts.

High Zika Virus Seroprevalence in Salvador, Northeastern Brazil Limits the Potential for Further Outbreaks

ABSTRACT During 2015 to 2016, Brazil reported more Zika virus (ZIKV) cases than any other country, yet population exposure remains unknown. Serological studies of ZIKV are hampered by cross-reactive immune responses against heterologous viruses. We conducted serosurveys for ZIKV, dengue virus (DENV), and Chikungunya virus (CHIKV) in 633 individuals prospectively sampled during 2015 to 2016, including microcephaly and non-microcephaly pregnancies, HIV-infected patients, tuberculosis patients, and university staff in Salvador in northeastern Brazil using enzyme-linked immunosorbent assays (ELISAs) and plaque reduction neutralization tests. Sera sampled retrospectively during 2013 to 2015 from 277 HIV-infected patients were used to assess the spread of ZIKV over time. Individuals were georeferenced, and sociodemographic indicators were compared between ZIKV-positive and -negative areas and areas with and without microcephaly cases. Epidemiological key parameters were modeled in a Bayesian framework. ZIKV seroprevalence increased rapidly during 2015 to 2016, reaching 63.3% by 2016 (95% confidence interval [CI], 59.4 to 66.8%), comparable to the seroprevalence of DENV (75.7%; CI, 69.4 to 81.1%) and higher than that of CHIKV (7.4%; CI, 5.6 to 9.8%). Of 19 microcephaly pregnancies, 94.7% showed ZIKV IgG antibodies, compared to 69.3% of 257 non-microcephaly pregnancies (P = 0.017). Analyses of sociodemographic data revealed a higher ZIKV burden in low socioeconomic status (SES) areas. High seroprevalence, combined with case data dynamics allowed estimates of the basic reproduction number R0 of 2.1 (CI, 1.8 to 2.5) at the onset of the outbreak and an effective reproductive number Reff of <1 in subsequent years. Our data corroborate ZIKV-associated congenital disease and an association of low SES and ZIKV infection and suggest that population immunity caused cessation of the outbreak. Similar studies from other areas will be required to determine the fate of the American ZIKV outbreak. IMPORTANCE The ongoing American Zika virus (ZIKV) outbreak involves millions of cases and has a major impact on maternal and child health. Knowledge of infection rates is crucial to project future epidemic patterns and determine the absolute risk of microcephaly upon maternal ZIKV infection during pregnancy. For unknown reasons, the vast majority of ZIKV-associated microcephaly cases are concentrated in northeastern Brazil. We analyzed different subpopulations from Salvador, a Brazilian metropolis representing one of the most affected areas during the American ZIKV outbreak. We demonstrate rapid spread of ZIKV in Salvador, Brazil, and infection rates exceeding 60%. We provide evidence for the link between ZIKV and microcephaly, report that ZIKV predominantly affects geographic areas with low socioeconomic status, and show that population immunity likely caused cessation of the outbreak. Our results enable stakeholders to identify target populations for vaccination and for trials on vaccine efficacy and allow refocusing of research efforts and intervention strategies. IMPORTANCE The ongoing American Zika virus (ZIKV) outbreak involves millions of cases and has a major impact on maternal and child health. Knowledge of infection rates is crucial to project future epidemic patterns and determine the absolute risk of microcephaly upon maternal ZIKV infection during pregnancy. For unknown reasons, the vast majority of ZIKV-associated microcephaly cases are concentrated in northeastern Brazil. We analyzed different subpopulations from Salvador, a Brazilian metropolis representing one of the most affected areas during the American ZIKV outbreak. We demonstrate rapid spread of ZIKV in Salvador, Brazil, and infection rates exceeding 60%. We provide evidence for the link between ZIKV and microcephaly, report that ZIKV predominantly affects geographic areas with low socioeconomic status, and show that population immunity likely caused cessation of the outbreak. Our results enable stakeholders to identify target populations for vaccination and for trials on vaccine efficacy and allow refocusing of research efforts and intervention strategies.

Progressive lesions of Central Nervous System in microcephalic fetuses with suspected congenital Zika virus syndrome

To describe the pattern and progression of central nervous system (CNS) lesions in microcephalic fetuses with suspected Zika virus (ZIKV) infection.

Chromosomal abnormalities in couples with recurrent first trimester abortions.

PURPOSE To investigate the prevalence of chromosomal abnormalities in couples with two or more recurrent first trimester miscarriages of unknown cause. METHODS The study was conducted on 151 women and 94 partners who had an obstetrical history of two or more consecutive first trimester abortions (1-12 weeks of gestation). The controls were 100 healthy women without a history of pregnancy loss. Chromosomal analysis was performed on peripheral blood lymphocytes cultured for 72 hours, using Trypsin-Giemsa (GTG) banding. In all cases, at least 30 metaphases were analyzed and 2 karyotypes were prepared, using light microscopy. The statistical analysis was performed using the Student t-test for normally distributed data and the Mann-Whitney test for non-parametric data. The Kruskal-Wallis test or Analysis of Variance was used to compare the mean values between three or more groups. The software used was Statistical Package for the Social Sciences (SPSS), version 17.0. RESULTS The frequency of chromosomal abnormalities in women with recurrent miscarriages was 7.3%, including 4.7% with X-chromosome mosaicism, 2% with reciprocal translocations and 0.6% with Robertsonian translocations. A total of 2.1% of the partners of women with recurrent miscarriages had chromosomal abnormalities, including 1% with X-chromosome mosaicism and 1% with inversions. Among the controls, 1% had mosaicism. CONCLUSION An association between chromosomal abnormalities and recurrent miscarriage in the first trimester of pregnancy (OR=7.7; 95%CI 1.2--170.5) was observed in the present study. Etiologic identification of genetic factors represents important clinical information for genetic counseling and orientation of the couple about the risk for future pregnancies and decreases the number of investigations needed to elucidate the possible causes of miscarriages.

Evidence for Congenital Zika Virus Infection From Neutralizing Antibody Titers in Maternal Sera, Northeastern Brazil

ZIKV-specific neutralizing antibody titers were significantly higher in 28 mothers of children with microcephaly than in 122 controls from northeastern Brazil, suggesting an unusually strong immunological stimulus and potential utility of maternal antibody titers to corroborate congenital ZIKV infection.

Lymphocyte immunotherapy in the treatment of recurrent miscarriage: systematic review and meta-analysis

PurposeRecurrent miscarriage (RM) affects up to 2–3% of couples of reproductive age. There are several causes for this condition, including immunologic. The embryo is considered an allograft, subject to the rejection mechanisms of the maternal immune system. Immunotherapy involving immunization with lymphocytes is considered in cases of idiopathic RM. However, there is still no consensus regarding the efficacy and safety of this therapy.MethodsThis systematic review and meta-analysis evaluated the data available in the literature regarding the efficacy and safety of the use of immunotherapy with lymphocytes in couples with history of RM. Searches in PubMed/Medline, SCOPUS, and Cochrane Library databases were conducted, using the following keywords: “recurrent miscarriage,” “lymphocyte immunotherapy,” and “meta-analysis.” Statistical analyses were performed using Review Manager 5.3 (RevMan), version 5.3.ResultsSix published meta-analysis were retrieved; two found no improvements in the rate of live births after the use of immunization with lymphocytes in the treatment of RM, and four found a beneficial effect of the use of immunotherapy with lymphocytes in cases of RM, with significant improvements in the rate of live births.ConclusionData available in the literature supports the efficacy and safety of immunotherapy with lymphocytes in cases of RM without an identified cause.

Specific detection of dengue and Zika virus antibodies using envelope proteins with mutations in the conserved fusion loop

Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross-reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA-format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre-incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity. Emerging Microbes & Infections (2017) 6, e99; doi:10.1038/emi.2017.87; published online 8 November 2017

Zika virus infection, a new public health challenge

Brazil is an endemic area for Dengue virus (DENV) infections. Recently, the country was affected by an outbreak caused by Chikungunya virus, and in the last year, a third arbovirus (Zika virus – ZIKV), caused a large number of infections.1 ZIKV is transmitted by mosquitoes, and was first isolated from a rhesus macaque placed as sentinel during a study about yellow fever in the Zika Forest, Uganda, Africa in 1947.2 Since that time, sporadic viral isolations from humans and from a variety of mosquitoes Aedes sp have been reported in Africa and Asia. ZIKV, a positive-sense, single-stranded RNA virus, member of genus Flavivirus, family Flaviviridae, is another flavivirus of a public health importance. The virus has been circulated in Southeast Asia for at least the past 50 years, and a recent epidemic on Yap Island, Federated States of Micronesia, demonstrated that ZIKV is also capable of causing human disease and is expanding its geographic distribution.3 In Bahia, in the first months of 2015, patients at Santa Helena Hospital in Camacari, a city distant 50 km from Salvador, the third largest city in Brazil, were given a presumptive diagnosis of an acute viral illness by emergency department physicians. The illness was characterized by maculopapular rash, fever, myalgias/arthralgia, conjunctivitis, severe rash in arms and legs, and low grade fever. This atypic, dengue-like illness was investigated to confirm the presence of several arbovirus by conventional RT-PCR: West Nile, Mayaro virus, Sant Louis virus and DENV. Since all tests resulted negative, there was only one more possibility to investigate: ZIKV. On March 26 2015, Campos et al. identified ZIKV in sera of affected patients, demonstrating, for the first time, circulation of ZIKV in Brazil and Latin America.4 It was immediately informed to the national health authorities (Ministry of Health, Brazil) and the presence of ZIKV (autoctones cases) in Bahia was

Pitfalls in the prenatal diagnosis of mucolipidosis II alpha/beta: A case report

Mucolipidosis II alpha/beta is an autosomal recessive disorder caused by deficient activity of GlcNAc-1-phosphotransferase. We report the prenatal diagnosis of a fetus who was found to exhibit normal levels of lysosomal enzymes in the amniotic fluid but low levels in amniocytes, and who was found to be heterozygous for the most common GNPTAB mutation. As in some carriers of Mucolipidosis II biochemical abnormalities may hinder prenatal diagnosis, we suggest DNA analysis should be performed whenever possible.