Isadora Siqueira

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

Proteins with bacterial immunoglobulin‐like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface‐expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig‐like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino‐terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy‐terminal non‐repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High‐pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis.

Establishment and cryptic transmission of Zika virus in Brazil and the Americas

Transmission of Zika virus (ZIKV) in the Americas was first confirmed in May 2015 in northeast Brazil. Brazil has had the highest number of reported ZIKV cases worldwide (more than 200,000 by 24 December 2016) and the most cases associated with microcephaly and other birth defects (2,366 confirmed by 31 December 2016). Since the initial detection of ZIKV in Brazil, more than 45 countries in the Americas have reported local ZIKV transmission, with 24 of these reporting severe ZIKV-associated disease. However, the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of this information for interpreting observed trends in reported microcephaly. Here we address this issue by generating 54 complete or partial ZIKV genomes, mostly from Brazil, and reporting data generated by a mobile genomics laboratory that travelled across northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Analyses of viral genomes with ecological and epidemiological data yield an estimate that ZIKV was present in northeast Brazil by February 2014 and is likely to have disseminated from there, nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates for the international spread of ZIKV from Brazil indicate the duration of pre-detection cryptic transmission in recipient regions. The role of northeast Brazil in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the basic reproduction number of the virus.

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.

Multi-centric prospective evaluation of rk39 rapid test and direct agglutination test for the diagnosis of visceral leishmaniasis in Brazil.

The diagnosis of visceral leishmaniasis (VL) is still a major problem in Brazil and several other countries where the disease is endemic. The use of an easy-to-use and interpret, sensitive, and specific method that requires no complex infrastructure or specialized professionals, such as direct agglutination test (DAT) and the rK39-based rapid immunochromatographic test may enhance the diagnosis of disease. This study evaluated the performance of a rapid test (DiaMed- IT-LEISH®) and the DAT for the diagnosis of VL in 213 parasitologically confirmed cases and 119 controls with clinical suspicion of VL and confirmation of another etiology. The sensitivities and specificities of the rapid test were 93% and 97%, respectively and those of the DAT were 90% and 96%, respectively. The positive predictive values of the rapid test and the DAT were 98% and 97%, respectively and the negative predictive values were 89% and 84%, respectively. The Kappa index showed agreement between both methods classified as substantial (0.77). This study showed that the DAT and the rapid test can be used to diagnose VL in Brazil, following a pilot study for implementation of the rapid test in the health services.

Validação do teste imunocromatográfico rápido IT-LEISH® para o diagnóstico da leishmaniose visceral humana

The rapid immunochromatographic test IT-LEISH® (DiaMed IT-LEISH) was validated for the diagnosis of visceral leishmaniasis (VL) in four endemic areas of Brazil. The performance of the IT-LEISH® was compared with that of the indirect fluorescent antibody test, and that of enzyme-linked immunosorbent assay, using soluble antigen of Leishmania chagasi and the recombinant K39 (rK39). The study group was composed by 332 patients with clinical suspicion of VL: 213 cases confirmed by parasitological tests; and 119 with confirmation of another etiology. The sensitivity of the test IT-LEISH® was of 93% and the specificity of 97%. Immunofluorescent antibody test, ELISA L. chagasi and ELISA rK39 showed sensitivity of 88%, 92%, and 97%, and specificity of 81%, 77%, and 84%, respectively. The results confirm the validity of the test IT-LEISH® for the diagnosis of the VL in Brazil.

Chromobacterium violaceum in Siblings, Brazil

Chromobacterium violaceum, a saprophyte bacterium found commonly in soil and water in tropical and subtropical climates, is a rare cause of severe, often fatal, human disease. We report 1 confirmed and 2 suspected cases of C. violaceum septicemia, with 2 fatalities, in siblings after recreational exposure in northeastern Brazil.

Zika virus complete genome from Salvador, Bahia, Brazil.

In May 2015 the first autochthonous Zika virus infection was reported in Brazil. Rapid and urgent measures are needed to contain the ongoing outbreak. Here we report the full-length ZIKV coding sequence from Bahia. Genetic analysis of outbreak sequences will be essential for characterizing the diversity of circulating strains, identifying hotspots of virus transmission and guiding public health control. Rapid and urgent measures are needed to contain the ongoing outbreak.

Transient hearing loss in adults associated with Zika virus infection

In 2015, during the outbreak of Zika virus (ZIKV) in Brazil, we identified 3 cases of acute hearing loss after exanthematous illness. Serology yielded finding compatible with ZIKV as the cause of a confirmed (n = 1) and a probable (n = 2) flavivirus infection, indicating an association between ZIKV infection and transient hearing loss.

Neurological symptoms and signs in HTLV-1 patients with overactive bladder syndrome

OBJECTIVE To compare neurological symptoms and signs in HTLV-1 asymptomatic carriers and HTLV-1 patients with overactive bladder (OB) syndrome. METHODS We studied 102 HTLV-1 positive individuals without HAM/TSP (HTLV-1 associated myelopathy/tropical spastic paraparesis) divided into two groups according to the presence or absence of OB syndrome. Clinical interview, neurological exam and proviral load was performed in all patients. RESULTS AND CONCLUSIONS Individuals with OB were more commonly female (84.3 vs. 60.8% of asymptomatics, p=0.01). The prevalence of neurological complaints was higher in OB group, especially hand or foot numbness and arm or leg weakness. There was no difference between the groups in neurological strength and reflexes. Weakness complaint remained strongly associated with OB in multivariate logistic regression analysis adjusting for sex and age [adjusted odds ratio and 95%CI 3.59 (1.45-8.88) in arms and 6.68 (2.63-16.93) in legs]. Proviral load was also different between the two groups with higher level on OB individuals.

Epidemic establishment and cryptic transmission of Zika virus in Brazil and the Americas

Zika virus (ZIKV) transmission in the Americas was first confirmed in May 2015 in Northeast Brazil1. Brazil has the highest number of reported ZIKV cases worldwide (>200,000 by 24 Dec 20162) as well as the greatest number of cases associated with microcephaly and other birth defects (2,366 confirmed cases by 31 Dec 20162). Following the initial detection of ZIKV in Brazil, 47 countries and territories in the Americas have reported local ZIKV transmission, with 22 of these reporting ZIKV-associated severe disease3. Yet the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of such information for interpreting past trends in reported microcephaly. To address this we generated 53 complete or partial ZIKV genomes, mostly from Brazil, including data generated by the ZiBRA project – a mobile genomics lab that travelled across Northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Joint analyses of viral genomes with ecological and epidemiological data estimate that the ZIKV epidemic first became established in NE Brazil by March 2014 and likely disseminated from there, both nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates of the international spread of ZIKV from Brazil coincide with periods of high vector suitability in recipient regions and indicate the duration of pre-detection cryptic transmission in those regions. NE Brazil’s role in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the virus’ basic reproduction number. One Sentence Summary Virus genomes reveal the establishment of Zika virus in Northeast Brazil and the Americas, and provide an appropriate timeframe for baseline (pre-Zika) microcephaly in different regions.