Luiz Carlos Júnior Alcântara

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.

A case-control study of HTLV-infection among blood donors in Salvador, Bahia, Brazil - associated risk factors and trend towards declining prevalence

Previous data suggest that Salvador, the capital of the State of Bahia, a northeastern state of Brazil, has the highest prevalence of HTLV infection in blood donors among Brazilian cities. The aim of this case-control study was to identify the determinants of risk for HTLV infection among blood donors in the city of Salvador. Between January 2000 and December 2003, 504 blood donors with positive screening tests for HTLV infection (unconfirmed prevalence of 0.48%) were invited to participate in our study. A total of 154 had performed a Western Blot (WB) test, 139 were of which found to be positive (false positive screening rate 9.9%). Using a standardized questionnaire, a single interviewer obtained information on demographic, socio-economical and educational characteristics, as well as sexual behavior from 91 out of the 139 positive by WB and from 194 HTLV-negative blood donors. Prevalence of HTLV infection was 0.48%. Multivariate analysis revealed women (OR 3.79 [1.61-8.88], p=0.002), low family income* (OR 3.37 [1.17-9.66], p=0.02), self-reported history of sexual transmitted diseases (OR 6.15 [2.04-18.51], p=0.001), 2 or more sexual partners during life (OR 9.29 [2.16-39.94], p=0.0020) and inconsistent use of condoms (OR 4.73 [1.98-11.26], p=0.0004) as risk factors for HTLV infection. In accordance with previous published data, our results point to an association between low socio-economical level, poor education and unsafe sexual behavior with HTLV infection. We observed a lower prevalence of HLTV infection when compared to previous data.

Prevalência da infecção pelo vírus linfotrópico humano de células T e fatores de risco associados à soropositividade em doadores de sangue da cidade de Rio Branco, AC, Brasil (1998-2001)

Between December 1998 and March 2001, for each HTLV-I/II seropositive blood donor (ELISA, Abbott), two HTLV-I/II seronegative blood donors were also chosen. The blood samples were re-tested by ELISA (Murex), and those seropositives were also tested by Western Blot and PCR. Of the 11, 121 blood samples, 73 (0.66%) were positives (Abbott), but only 12 (0.11%) remained positives (Murex), whereas the 146 seronegatives were confirmed, even though the concordance index between these two ELISA tests was hopeless. The Western Blot confirmed the twelve blood samples as seropositives: 8 (0.07%) HTLV-I; two (0.02%) HTLV-II and two (0.02%) indeterminate, being by PCR, one HTLV-I and the other HTLV-II. Concluding, in this Western Amazon population the HTLV-I/II seroprevalence was too low, in spite of the greater prevalence of HTLV-II expected due to a great indigenous racial mixture.

HTLV-1 in pregnant women from the Southern Bahia, Brazil: a neglected condition despite the high prevalence

BackgroundAs the most frequent pathway of vertical transmission of HTLV-1 is breast-feeding, and considering the higher prevalence in women, it is very important to perform screening examinations for anti-HTLV-1 antibodies as part of routine prenatal care. So far, no studies of HTLV-1 seroprevalence in pregnant women in the Southern region of Bahia, Brazil, have been described.MethodsPregnant women were selected at the two regional reference centers for health care from Southern Bahia. A total of 2766 pregnant women attending the antenatal unit between November 2008 and May 2010 have been analyzed. An extra blood sample was drawn during their routine antenatal testing. A standardized questionnaire was applied and all positive plasma samples were tested by ELISA and were confirmed by Western Blot and PCR. Besides that, positive women were contacted and visited. The family members that were present during the visit were asked to be serologically screened to the virus. A prospective study was also carried out and newborns were followed up to two years for evaluation of vertical transmission.ResultsHTLV prevalence was 1.05% (CI 95%: 0.70-1.50). There was no association of HTLV-1 infection with age, education, income and ethnic differences. The association with marital status was borderline (OR = 7.99; 95% CI 1.07-59.3; p = 0.042). In addition, 43 family members of the HTLV-1 seropositive women have been analyzed and specific reactivity was observed in 32.56%, including two children from previous pregnancy.Conclusion: It is very important to emphasize that the lack of HTLV-1 screening in pregnant women can promote HTLV transmission especially in endemic areas. HTLV screening in this vulnerable population and the promotion of bottle-feeding for children of seropositive mothers could be important cost-effective methods to limit the vertical transmission. Besides that, our data reinforce the need to establish strategies of active surveillance in household and family contacts as important epidemiological surveillance actions for the early detection of virus infection and the prevention of transmission by sexual or and parenteral contact.

Sickle red cells as danger signals on proinflammatory gene expression, leukotriene B4 and interleukin-1 beta production in peripheral blood mononuclear cell

This study tested the hypothesis that sickle red blood cell (SS-RBC) induce Toll-like receptors (TLR) and Nod-like receptor family, pyrin domain containing 3 (NLRP3)- inflammasome expression in peripheral blood mononuclear cells (PBMC). TLR and NLRP3 inflammasome could contribute to the maintenance of the inflammatory status in sickle cell anemia (SCA) patients, since SS-RBC act as danger signals activating these pathways. In this study, first, we evaluated TLR (2, 4, 5 and 9), NLRP3, Caspase-1, interleukin (IL)-1β and IL-18 expression in PBMC freshly isolated from SCA patients (SS-PBMC) in comparison with PBMC from healthy individuals (AA-PBMC). In the second moment, we investigated whether SS-RBC could interfere with the expression of these molecules in PBMC from healthy donor, in the absence or presence of hydroxyurea (HU) in vitro. TLRs and NLRP3 inflammasome expression were investigated by qPCR. IL-1β, Leukotriene-B4 (LTB4) and nitrite production were measured in PBMC (from healthy donor) culture supernatants. TLR2, TLR4, TLR5, NLRP3 and IL-1β were highly expressed in SS-PBMC when compared to AA-PBMC. Additionally, SS-RBC induced TLR9, NLRP3, Caspase-1, IL-1β and IL-18 expression and induced IL-1β, LTB4 and nitrite production in PBMC cultures. HU did not prevent TLR and NLRP3 inflammasome expression, but increased TLR2 and IL-18 expression and reduced nitrite production. In conclusion, our data suggest that TLR and inflammasome complexes may be key inducers of inflammation in SCA patients, probably through SS-RBC; also, HU does not prevent NLRP3 inflammasome- and TLR-dependent inflammation, indicating the need to develop new therapeutic strategies to SCA patients that act with different mechanisms of those observed for HU.

The origin of HTLV-1 in the South Bahia by phylogenetic, mitochondrial DNA and β-globin analysis

In order to clarify the HTLV introduction and dispersion in the south of Bahia, we analyzed 29 samples from HTLV-1 seropositive women. Before the blood collection, all of them answered a standardized questionnaire. The DNA was extracted by QIAgen Kit. It was performed a nested-PCR to the LTR region of the HTLV. The sequences were submitted to the LASP HTLV-1 Automated Subtyping Tool. The phylogenetic analyses were generated using the neighbor-joining e maxima-verossimilhanca methods. The evolutionary model of Tamura-Nei with gamma distribution was selected as the best adaptive model by software Modeltest 3.7. The likelihood ratio was used to calculate the statistical support for the branches in trees. Bayesian tree were constructed to verify the posterior probability statistical parameter. To evaluate genetic ancestry of the population, it was analyzed mtDNA ancestry markers and β-globina haplotypes. From the total, 21 samples were successfully amplified and sequenced and they were classified as HTLV-1 aA (bootstrap support of 100%). The phylogeny analysis showed multiple introductions of the virus in Brazil. In addition, for the first time, Mozambique sequences were grouped with Brazilian and South Africa sequences, supporting the hypothesis that Africans infected with the virus have been brought from the southern regions of Africa. In relation to the genetic ancestry, the African ethnicity was predominantly found by mtDNA markers. In addition, the type benin was detected by β-globin analyses. These data corroborate to clarify the introduction and dispersion of this virus in Brazil, especially in the Bahia.

Interfamilial transmission of HTLV-1 in the South of Bahia, Brazil

The objective of this study was to investigate the prevalence of HTLV in the south of Bahia and to verify the interfamilial transmission of this virus. Initially, blood was collected from pregnant women in hospitals in southern Bahia.The HTLV-1 status was analyzed by ELISA and confirmed by Western Blot and PCR. The HTLV positive women were contacted and visited. In this opportunity, it was explained the results and collected samples from family members. They signed a consent informed and it was applied questionnaire. A total of 2766 pregnant women were analyzed. The prevalence was 1.05% (n=29) (CI95%:0,70-1,50). Besides, it was possible to follow 18 mothers (16 positives and 2 indeterminates) for 2 years. During the following, it was collected samples from 34 family members, including: partners (n=8), mothers (n=7), father (n=1), sisters (n=2), brothers (n=4), sons (n=5), daughters (n=4), others (n=3). Specific reactivity was observed in 11/34 (32,35%) individuals, of which two samples were from children (1 son and 1 daughter - 2,3 and 8 years old, respectively). The others members were: 3 mothers, 1 father, 3 partners, 2 sisters. In one case, we had three generation HTLV-1 positive: grandmother, mother and daughter. These results are very relevant because: (1) so far no studies of HTLV-1 seroprevalence in pregnant/puerperal women from the South region of Bahia had been described, (2) previous studies in areas endemic for the HTLV in Brazil found: 0.84% - Salvador, Bahia and 1.1% in Belo Horizonte, Minas Gerais, (3) evidence of important interfamilial transmission in this area.