NATHALIA CAROLINE SANTIAGO E SOUZA
Projetos de Pesquisa
Unidades Organizacionais
Instituto do Câncer do Estado de São Paulo, Hospital das Clínicas, Faculdade de Medicina
LIM/52 - Laboratório de Virologia, Hospital das Clínicas, Faculdade de Medicina
LIM/52 - Laboratório de Virologia, Hospital das Clínicas, Faculdade de Medicina
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- DETECTION OF HUMAN ANTI-ZIKA VIRUS IgG BY ELISA USING AN ANTIGEN FROM in vitro INFECTED VERO CELLS: PRELIMINARY RESULTS(2016) SUMITA, Laura Masami; RODRIGUES, Jaqueline Polizeli; FERREIRA, Noely Evangelista; FELIX, Alvina Clara; SOUZA, Nathalia Caroline Santiago; MACHADO, Clarisse Martins; ANDRADE JUNIOR, Heitor Franco deZika virus (ZKV) infection is a huge public health problem in Brazil because of the increased incidence of microcephaly in neonates from infected mothers. Detection of specific IgG antibodies in maternal serum samples constitutes an important approach for diagnosing ZKV infection and evaluating its relationship with neonatal microcephaly. However, as there is no serological test produced in Brazil to detect IgM and IgG antibodies against ZKV, we sought to examine specific IgG in serum samples from patients or suspected mothers to detect previous infection and to test for specificity with regard to flaviviral infections occurring in the same area. Brazilian Zika virus native antigens were obtained from infected Vero cell layers or free virions in the culture medium and then used in ELISA. We tested sera from eight ZKV RNA-diagnosed infected patients (ZKVR), seven neonates with microcephaly and their mothers after delivery (MM), 140 dengue virus IgM-positive (DM) and IgG (DG)-positive patients, and 100 yellow fever (YF)-vaccinated patients. According to the ELISA, ZKVR samples were mostly positive (7/8), and all the MM serum samples were positive for ZKV IgG (7/7). In contrast, cross-reactions for dengue or yellow fever-vaccinated patients were observed, including DM (48/95), DG (10/45) or YF (3/100) serum samples; however, these cross-reactions exhibited low antigen avidity so that 6 M urea largely removed this cross-reactivity, with only a few cross-reacting samples remaining (8/140). ELISA based on extracted virions was much more specific, with all ZKVR (8/8) and MM sera being positive for ZKV IgG (7/7) and only borderline cross-reactivity found for DM (6/95), DG (3/45) or YF (4/100)-vaccinated serum samples. This technique (ELISA) can identify specific IgG in ZKV-infected patients and may be helpful in diagnosing congenital infetions after maternal RNA virus clearance or in epidemiological studies.
- Cross reactivity of commercial anti-dengue immunoassays in patients with acute Zika virus infection(2017) FELIX, Alvina Clara; SOUZA, Nathalia C. Santiago; FIGUEIREDO, Walter M.; COSTA, Angela A.; INENAMI, Marta; SILVA, Rosangela M. G. da; LEVI, Jose Eduardo; PANNUTI, Claudio Sergio; ROMANO, Camila MaltaSeveral countries have local transmission of multiple arboviruses, in particular, dengue and Zika viruses, which have recently spread through many American countries. Cross reactivity among Flaviviruses is high and present a challenge for accurate identification of the infecting agent. Thus, we evaluated the level of cross reactivity of anti-dengue IgM/G Enzyme-Linked Immunosorbent Assays (ELISA) from three manufacturers against 122 serum samples obtained at two time-points from 61 patients with non-dengue confirmed Zika virus infection. All anti-dengue ELISAs cross reacted with serum from patients with acute Zika infection at some level and a worrisome number of seroconversion for dengue IgG and IgM was observed. These findings may impact the interpretation of currently standard criteria for dengue diagnosis in endemic regions.
- Predictors of mortality in patients with yellow fever: an observational cohort study(2019) KALLAS, Esper G.; ZANELLA, Luiz Gonzaga F. A. B. D'Elia; V, Carlos Henrique Moreira; BUCCHERI, Renata; DINIZ, Gabriela B. F.; CASTINEIRAS, Anna Carla P.; COSTA, Priscilla R.; DIAS, Juliana Z. C.; MARMORATO, Mariana P.; SONG, Alice T. W.; MAESTRI, Alvino; BORGES, Igor C.; JOELSONS, Daniel; CERQUEIRA, Natalia B.; SOUZA, Nathalia C. Santiago e; CLARO, Ingra Morales; SABINO, Ester C.; LEVI, Jose Eduardo; I, Vivian Avelino-Silva; HO, Yeh-LiBackground Yellow fever virus infection results in death in around 30% of symptomatic individuals. The aim of this study was to identify predictors of death measured at hospital admission in a cohort of patients admitted to hospital during the 2018 outbreak of yellow fever in the outskirts of Sao Paulo city, Brazil. Methods In this observational cohort study, we enrolled patients with yellow fever virus from two hospitals in Sao Paolo-the Hospital das Clinicas, University of Sao Paulo and the Infectious Diseases Institute ""Emilio Ribas"". Patients older than 18 years admitted to hospital with fever or myalgia, headache, arthralgia, oedema, rash, or conjunctivitis were consecutively screened for inclusion in the present study. Consenting patients were included if they had travelled to geographical areas in which yellow fever virus cases had been previously confirmed. Yellow fever infection was confirmed by real-time PCR in blood collected at admission or tissues at autopsy. We sequenced the complete genomes of yellow fever virus from infected individuals and evaluated demographic, clinical, and laboratory findings at admission and investigated whether any of these measurements correlated with patient outcome (death). Findings Between Jan 11, 2018, and May 10, 2018, 118 patients with suspected yellow fever were admitted to Hospital das Clinicas, and 113 patients with suspected yellow fever were admitted to Infectious Diseases Institute ""Emilio Ribas"". 95 patients with suspected yellow fever were included in the study, and 136 patients were excluded. Three (3%) of 95 patients with suspected yellow fever who were included in the study were excluded because they received a different diagnosis, and 16 patients with undetectable yellow fever virus RNA were excluded. Therefore, 76 patients with confirmed yellow fever virus infection, based on detectable yellow fever virus RNA in blood (74 patients) or yellow fever virus confirmed only at the autopsy report (two patients), were included in our analysis. 27 (36%) of 76 patients died during the 60 day period after hospital admission. We generated 14 complete yellow fever virus genomes from the first 15 viral load-detectable samples. The genomes belonged to a single monophyletic clade of the South America I genotype, sub-genotype E. Older age, male sex, higher leukocyte and neutrophil counts, higher alanine aminotransferase, aspartate transaminase (AST), bilirubin, and creatinine, prolonged prothrombin time, and higher yellow fever virus RNA plasma viral load were associated with higher mortality. In a multivariate regression model, older age, elevated neutrophil count, increased AST, and higher viral load remained independently associated with death. All 11 (100%) patients with neutrophil counts of 4000 cells per mL or greater and viral loads of 5.1 log(10) copies/mL or greater died (95% CI 72-100), compared with only three (11%) of 27 (95% CI 2-29) among patients with neutrophil counts of less than 4000 cells per mL and viral loads of less than 5.1 log(10) copies/mL. Interpretation We identified clinical and laboratory predictors of mortality at hospital admission that could aid in the care of patients with yellow fever virus. Identification of these prognostic markers in patients could help clinicians prioritise admission to the intensive care unit, as patients often deteriorate rapidly. Moreover, resource allocation could be improved to prioritise key laboratory examinations that might be more useful in determining whether a patient could have a better outcome. Our findings support the important role of the virus in disease pathogenesis, suggesting that an effective antiviral could alter the clinical course for patients with the most severe forms of yellow fever.
- External Quality Assessment for Zika Virus Molecular Diagnostic Testing, Brazil(2018) FISCHER, Carlo; PEDROSO, Celia; MENDRONE JR., Alfredo; FILIPPIS, Ana Maria Bispo de; VALLINOTO, Antonio Carlos Rosario; RIBEIRO, Bergmann Morais; DURIGON, Edison Luiz; MARQUES JR., Ernesto T. A.; CAMPOS, Gubio S.; VIANA, Isabelle F. T.; LEVI, Jose Eduardo; SCARPELLI, Luciano Cesar; NOGUEIRA, Mauricio Lacerda; BASTOS, Michele de Souza; SOUZA, Nathalia C. Santiago; KHOURI, Ricardo; LIRA, Sanny M. Costa; KOMNINAKIS, Shirley Vasconcelos; BARONTI, Cecile; CHARREL, Remi N.; KUEMMERER, Beate M.; DROSTEN, Christian; BRITES, Carlos; LAMBALLERIE, Xavier de; NIEDRIG, Matthias; NETTO, Eduardo Martins; DREXLER, Jan FelixWe conducted an external quality assessment of Zika virus molecular diagnostic tests in Brazil using a new Zika virus standard. Of 15 laboratories, 73% showed limited sensitivity and specificity. Viral load estimates varied significantly. Continuous quality assurance is required for adequate estimates of Zika virus-associated disease and determination of patient care.
- Evaluation of serological cross-reactivity between yellow fever and other flaviviruses(2019) SOUZA, Nathalia Caroline Santiago e; FELIX, Alvina Clara; PAULA, Anderson Vicente de; LEVI, Jose Eduardo; PANNUTI, Claudio Sergio; ROMANO, Camila MaltaObjectives: This study was performed to determine whether neutralizing antibodies against yellow fever virus (YFV) generated by YFV vaccine could interfere in the specificity of dengue virus (DENV) and Zika virus (ZIKV) IgG ELISA tests. Methods: Seventy-nine pairs of serum samples (pre- and post-vaccination), collected during the years 1997-1998 from children with no history of yellow fever disease who had been vaccinated against YFV, were tested. The seroconversion post-vaccination was evaluated through plaque reduction neutralization test (PRNT), and four different commercial ELISA kits were used for the detection of DENV and ZIKV IgG antibodies. Results: A cross-reactivity rate of 3.9% with DENV IgG antibodies was found only with the Dengue Virus IgG Dx Select kit (Focus Diagnostics). Conclusions: As several countries have local transmission of multiple arboviruses, the absence of cross-reactivity or minimum cross-reactivity of YFV neutralizing antibodies with DENV and ZIKV antigens is a relevant finding, since the interpretation of sero-epidemiological investigations would be seriously impacted in many regions where YFV vaccination is mandatory. (C) 2019 The Authors.
- A cohort study to assess the incidence of dengue, Brazil, 2014-2018(2020) LUNA, Expedito J. A.; FIGUEIREDO, Gerusa M.; LEVI, Jose E.; CAMPOS, Sergio R. S. L. C.; FELIX, Alvina Clara; SOUZA, Nathalia Santiago E; FIGUEIREDO, Walter M.; COSTA, Angela A.; CARDOSO, Maria R. A.; PANNUTI, Claudio S.The present cohort study was set up with the aim of determining the incidence of dengue among children and adolescents, from 2 to 16 years of age, living in Araraquara, South-Eastern Brazil, a city classified as a mid-level endemicity location for dengue. Enrollment took place from September 2014 to March 2015. Baseline socio-demographic data were collected, and a blood sample from the participant was drawn, for dengue serology. Families were contacted weekly for fever surveillance. If the child developed fever, a nurse visited the household to collect a blood sample. PCR, NS1 and IgM were used for dengue diagnosis. Parents or legal guardians of participating children provided a written informed consent. 3,514 children and adolescents were enrolled in the cohort. Dengue baseline seroprevalence was 12.2% (95%CI: 11.1 - 13.3). The incidence density of symptomatic dengue was 8.94 per 100 person/years in the first year of follow-up, 0.58 in the second, and 0.19 in the fourth. No cases were confirmed in the third year. Incidence was associated with age, sex, baseline seroprevalence and with living in a house as opposed to an apartment. This study provides relevant information on the epidemiology of dengue in mid-level transmission settings that may be useful to policymakers in the evaluation of control strategies.
- Plaque Reduction Neutralization Test (PRNT) in the Congenital Zika Syndrome: Positivity and Associations with Laboratory, Clinical, and Imaging Characteristics(2020) RIBEIRO, Marizelia R. C.; KHOURI, Ricardo; SOUSA, Patricia S.; BRANCO, Maria R. F. C.; BATISTA, Rosangela F. L.; COSTA, Elaine P. F.; ALVES, Maria T. S. S. B.; AMARAL, Glaucio A.; BORGES, Marcella C. R.; TAKAHASI, Eliana H. M.; GOMES, Lillian N.; MENDES, Ana K. T.; CAVALCANTE, Tamires B.; COSTA, Luciana C.; FELIX, Alvina C.; SOUZA, Nathalia C. S.; SILVA, Antonio A. M.The short duration of viremia, low blood viral load, inaccessibility to timely specific diagnostic tests, and cross-reactions with other flaviviruses have hindered laboratory confirmation of Congenital Zika Syndrome (CZS). This study analyzes the positivity of the plaque reduction neutralization test (PRNT) in children with clinical or imaging characteristics of CZS and its association with laboratory, clinical, and imaging characteristics. The 94 clinical cases of CZS submitted to the ZIKV PRNT90 test were followed from 2016 to 2018. The mean age of children at PRNT90 collection was 22 +/- 6 months Standard Deviation. The ZIKV PRNT90 was positive (titer >= 10) in 40 (42.5%) children. ZIKV PRNT90 positivity was associated with severe microcephaly in newborns (p = 0.016), lower head circumference z-score at birth (p = 0.043) and 24 months of age (p = 0.031), and severe reduction of the cerebral parenchyma volume (p = 0.021), expressing greater disease severity. Negative PRNT90 in children with characteristic signs of CZS may be due to false-negative results, indicating that the diagnosis of CZS should be primarily syndromic.
- Zika virus infection among symptomatic patients from two healthcare centers in Sao Paulo State, Brazil: prevalence, clinical characteristics, viral detection in body fluids and serodynamics(2019) TOZETTO-MENDOZA, Tania Regina; AVELINO-SILVA, Vivian Iida; FONSECA, Silvia; CLARO, Ingra Morales; PAULA, Anderson Vicente de; LEVIN, Anna Sara; SABINO, Ester Cerdeira; MENDES-CORREA, Maria Cassia; FIGUEIREDO, Walter Manso; FELIX, Alvina Clara; SOUZA, Nathalia C. Santiago; COSTA, Angela Aparecida; INENAMI, Maria; SILVA, Rosangela M. Gasparetto da; LEVI, Jose Eduardo; ROMANO, Camila Malta; PARANHOS-BACCALA, Glaucia; SEGURADO, Aluisio Cotrim; MAYAUD, PhilippeZika virus (ZIKV) clinical presentation and frequency/duration of shedding need further clarification. Symptomatic ZIKV-infected individuals identified in two hospitals in Sao Paulo State, Brazil, were investigated regarding clinical characteristics, shedding in body fluids, and serodynamics. Ninety-four of 235 symptomatic patients (Site A: 58%; Site B: 16%) had Real-Time PCR-confirmed ZIKV infection; fever, headache and gastrointestinal symptoms were less frequent, and rash was more frequent compared to ZIKV-negative patients. Real-Time PCR in serum had worse performance compared to plasma, while urine had the highest sensitivity. Shedding in genital fluids and saliva was rare. IgM positivity was the highest <14 days after the symptoms onset (86%), decreasing >28 days (24%); IgG positivity increased >14 days (96%) remaining positive in 94% of patients >28 days. ZIKV prevalence varied importantly in two neighboring cities during the same transmission season. Urine Real-Time PCR can improve diagnostic sensitivity; serum testing is less useful. Accurate serological tests are needed to improve diagnosis and surveillance.
- High-level of viral genomic diversity in cervical cancers: A Brazilian study on human papillomavirus type 16(2015) OLIVEIRA, Cristina Mendes de; BRAVO, Ignacio G.; SOUZA, Nathalia Caroline Santiago e; GENTA, Maria Luiza Nogueira Dias; FREGNANI, Jose Humberto Tavares Guerreiro; TACLA, Maricy; CARVALHO, Jesus Paula; LONGATTO-FILHO, Adhemar; LEVI, Jose EduardoInvasive cervical cancer (ICC) is the third most frequent cancer among women worldwide and is associated with persistent infection by carcinogenic human papillomaviruses (HPVs). The combination of large populations of viral progeny and decades of sustained infection may allow for the generation of intra-patient diversity, in spite of the assumedly low mutation rates of PVs. While the natural history of chronic HPVs infections has been comprehensively described, within-host viral diversity remains largely unexplored. In this study we have applied next generation sequencing to the analysis of intra-host genetic diversity in ten ICC and one condyloma cases associated to single HPV16 infection. We retrieved from all cases near full-length genomic sequences. All samples analyzed contained polymorphic sites, ranging from 3 to 125 polymorphic positions per genome, and the median probability of a viral genome picked at random to be identical to the consensus sequence in the lesion was only 40%. We have also identified two independent putative duplication events in two samples, spanning the L2 and the L1 gene, respectively. Finally, we have identified with good support a chimera of human and viral DNA. We propose that viral diversity generated during HPVs chronic infection may be fueled by innate and adaptive immune pressures. Further research will be needed to understand the dynamics of viral DNA variability, differentially in benign and malignant lesions, as well as in tissues with differential intensity of immune surveillance. Finally, the impact of intralesion viral diversity on the long-term oncogenic potential may deserve closer attention.
- Modulated Zika virus NS1 conjugate offers advantages for accurate detection of Zika virus specific antibody in double antigen binding and Ig capture enzyme immunoassays(2019) TEDDER, Richard S.; DICKS, Steve; IJAZ, Samreen; SOUZA, Nathalia Caroline Santiago de; PAULA, Anderson Vincente de; LEVY, Flavia; MEDIALDEA-CARRERA, Raquel; LEVI, Jose Eduardo; PANNUTI, Claudio S.; SEQUEIRA, Patricia Carvalho de; BROWN, David W. G.; LUMB, Ines UshiroThe accurate diagnosis and seroprevalence investigations of Zika virus (ZKV) infections remain complex due to cross reactivity with other flaviviruses. Two assay formats, both using labelled Zika virus NS1 antigen as a revealing agent (a double antigen binding assay, DABA, and an immunoglobulin Ig capture assay, G capture) were initially developed and compared with the indirect EuroimmunZ assay for the detection of anti-Zika antibody. Of 147 pre-Zika period serum samples, 39 (27%) were reactive in the EuroimmunZ or the DABA assays, 28 sera concordantly so. Such false reactivity was influenced by the serotype of Dengue virus (DV) to which individuals had been exposed to. Thus, of sera from patients undergoing secondary Dengue virus infection of known serotype, 91%, 45% and 28% of Dengue virus serotype 2, 3 and 4 respectively were reactive in one or more of the three assays. A novel method of quenching false sero-reactivity was therefore developed for the DABA and G capture assays. Initial addition of a single homologous Dengue virus serotype 3 NS1Ag quench significantly ablated false reactivities in the pre-Zika period sera. An equipotent quadrivalent quench comprising homologous Dengue virus serotypes 1 to 4 NS1Ag was shown to be optimum yet retained sensitivity for the detection of specific anti-Zika antibody. Comparing DABA and G capture assays using quenched and unquenched conjugates in comparison with EuroimmunZ early in the course of PCR-confirmed infection indicated that a significant component of the apparent early anti-ZIKA antibody response is likely to be due to a Zika virus-driven anamnestic anti-Dengue virus response. The increased specificity provided by homologous antigen quenching is likely to provide a significant improvement in sero-diagnostics and to be of clinical value.