Centro de Imunologia do Instituto Adolfo Lutz ao longo dos anos / Immunology Center of Instituto Adolfo Lutz over the years

O Instituto Adolfo Lutz (IAL) foi criado em 1940 como resultado da unificação dos Institutos Bacteriológico e Bromatológico, um moderno laboratório voltado ao controle de doenças, inaugurando uma nova fase de laboratórios de saúde pública no estado de São Paulo. Os primeiros testes sorológicos oferecidos à população foram executados pelas "antigas" Seções de Sorologia e de Imunologia. Essas seções destacam-se no desenvolvimento científico do IAL pela realização de pesquisas, produção científica e inovação tecnológica, seguramente, fundamentais para a saúde pública no decorrer dos anos. O Centro de Imunologia do IAL (CIM-IAL) foi criado em 2010, com a unificação das Seções de Sorologia e Imunologia, quando ocorreu a reorganização institucional. O CIM-IAL contribuiu para importantes avanços científicos na área da saúde, reforçando sua capacidade de desenvolver pesquisas, executar e monitorar o diagnóstico e a vigilância de diferentes agravos. Este manuscrito tem como objetivo apresentar os principais acontecimentos que ressaltam o papel fundamental na busca de soluções para os problemas de saúde pública, desde a época das Seções de Sorologia e Imunologia até tornar-se o Centro de Imunologia. Na elaboração deste trabalho foram utilizadas bibliografias contendo dados históricos, científicos e relatos de profissionais da área.

A new phase of Public Health Laboratories in the state of São Paulo occurred in 1940, with the unification of Instituto Bacteriológico and Bromatológico, creating the Instituto Adolfo Lutz (IAL), a modern laboratory focused on solving problems in this area. The first diagnostic tests offered to the population were carried out by the "old" Serology and Immunology Sections. It's worth highlighting the importance of these sections in the scientific development of the IAL by carrying out research, scientific production and technological innovation, which have certainly been fundamental to public health over the years. The Immunology Center (CIM) of IAL was created in 2010, when organizational adaptation took place with the junction of the Serology and Immunology Sections. The CIM-IAL has undergone important advances in the health area, reinforcing its capacity to develop research, carry out and monitor the diagnosis and surveillance of different diseases. This manuscript aims to present the main events that highlight the fundamental role in the search for solutions to public health problems, from the time of the Serology and Immunology Sections until it became the CIM. In the preparation, bibliographies were used based on historical and scientific data and reports from professionals in the field.

Sex, Age, and Risk Group Variations among Individuals Infected with HIV, HTLV-1, and HTLV-2: Review of Data Records (1983­2017) from a Public Health Laboratory in São Paulo, Brazil

The inaugural AIDS Program in Brazil was established in São Paulo in 1983, with the Instituto Adolfo Lutz appointed for laboratory assistance. Subsequently, research on HIV infections and HIV/HTLV (HIV/HTLV-1 and HIV/HTLV-2) co-infections was conducted. This narrative review focuses on studies from the Immunology Department (1983­2017) that significantly influenced AIDS diagnosis or provided epidemiological data such as prevalence rates, sex, age, and risk factors. Twelve studies, encompassing over 8000 individuals, are discussed. During 1983­1985, nearly all AIDS cases were attributed to homosexual/bisexual men aged 31 years old. Subsequently, heterosexual men and women emerged as risk groups owing to intravenous drug use (IDU) and/or unprotected sexual intercourse with AIDS patients or multiple partners per year. From 1985 onwards, vertical transmission led to child infections. HIV/HTLV co-infection rates decreased over time, initially associated with male IDU, and in the 2010s with female IDU, and individuals aged >40 years. Trends in HIV and HIV/HTLV co-infections among younger men and women (<30 years of age) were observed from 2015 to 2017. The changing characteristics and risk groups for HIV and HIV/HTLV co-infections over the years underscore the necessity for ongoing public policies to prevent retrovirus transmission, particularly among adolescents and young adults. (AU)

Desafios e contribuição do Laboratório de Imunologia Celular do Instituto Adolfo Lutz de São Paulo nos primeiros anos da epidemia de AIDS no Brasil / Challenges and contribution of the Cellular Immunology Laboratory of the Instituto Adolfo Lutz of São Paulo in the early years of the AIDS epidemic in Brazil

No ano em que se comemora 40 anos da implantação do primeiro Programa de AIDS em São Paulo, não poderia deixar de relatar os desafios e a contribuição concedida pelo Laboratório de Imunologia Celular do Instituto Adolfo Lutz de São Paulo, no período que antecedeu a identificação e o isolamento do HIV; portanto, quando não havia testes disponíveis no mercado nacional e internacional para o diagnóstico laboratorial sorológico e molecular desta infecção/doença. O perfil imunológico celular dos 111 primeiros casos suspeitos de AIDS de São Paulo foi avaliado pela contagem de linfócitos T e B, subpopulações de linfócitos T (T4 e T8), cálculo da razão T4/T8, e teste de proliferação de linfócitos (cultura de linfócitos) após estímulo com mitógenos e antígeno PPD. Os resultados obtidos mostraram um perfil imunológico celular característico dos casos de AIDS: leucopenia, linfopenia, diminuição do número de linfócitos T4 e aumento de T8, inversão da relação T4/T8, e resposta diminuída de proliferação de linfócitos frente à mitógenos e PPD, corroborando resultados laboratoriais dos casos de AIDS publicados pelo CDC de Atlanta, EUA. Posteriormente, o laboratório dedicou-se ao diagnóstico de infecção perinatal pelo HIV, padronizando a técnica de secreção induzida de anticorpos in vitro (IVIAP), capaz de detectar anticorpos anti-HIV produzidos por linfócitos B de crianças infectadas, diferenciando-os daqueles passivamente adquiridos da mãe durante a gestação. A IVIAP mostrou alta sensibilidade e especificidade, podendo ser usada no diagnóstico de infecção pelo HIV em crianças com mais de dois meses de idade.

In the year of the 40th anniversary of the implementation of the first AIDS Program in São Paulo, I could not miss the occasion to report the challenges and contribution of the Cellular Immunology Laboratory of the Instituto Adolfo Lutz (IAL) of São Paulo, in the phase that preceded the identification and isolation of HIV; therefore, when there were no laboratorial serological and molecular tests in national and international market for the diagnosis of this infection/disease. The cellular immunological profile of the first 111 AIDS suspected cases from São Paulo was evaluated by counting T and B lymphocyte, subpopulations of T lymphocytes (T4 and T8), T4/T8 ratio calculation, and lymphocytes proliferation analysis (lymphocytes culture) after stimulation with mitogens and PPD antigen. The obtained results disclosed an immunological profile characteristic of AIDS cases: leukopenia, lymphopenia, decrease in the number of T4 and increase in the number of T8 lymphocytes, inversion of the T4/T8 ratio, and reduced lymphocytes proliferation response to mitogens and PPD, corroborating laboratorial findings description of AIDS cases published by the CDC, Atlanta, USA. Subsequently, the laboratory conducted studies on the diagnosis of perinatal HIV infection, standardizing an in vitro induced antibody secretion (IVIAP) technique, capable of detecting anti-HIV antibodies produced by B lymphocytes from infected children, and differentiating them from those passively acquired from the mother during pregnancy. IVIAP showed high sensitivity and specificity and could be used in the diagnosis of HIV infection in children older than two months of age.

Performance of the tetra-primer PCR technique compared to PCR-RFLP in the search for rs12979860 (C/T) and rs8099917 (T/G) single nucleotide polymorphisms (SNPs) in the IFNL4 gene / Desempenho da técnica tetra-primer PCR em relação a PCR-RFLP na pesquisa de polimorfismos de nucleotídeos únicos (SNPs) rs12979860 (C/T) e rs8099917 (T/G) no gene IFNL4

Single nucleotide polymorphisms (SNPs, rs12979860 e rs8099917) in the Interferon Lambda 4 gene (IFNL4, formerly IFNL3and/or IL28B) has been associated with failure in the innate immune response, sustained virological response in hepatitis C, and HTLV-1-associated myelopathy (HAM) development. To search for these polymorphisms several methodologies can be employed, such as sequencing, real-time or quantitative polymerase chain reaction (qPCR), restriction fragment length polymorphism analysis in PCR products (PCR-RFLP), and tetra-primer PCR. The present study compared the performance of the tetra-primer PCR in relation to the PCR-RFLP, both optimized in the Research HTLV Laboratory of the Center of Immunology of Instituto Adolfo Lutz in São Paulo. One hundred DNA samples obtained from patients of STD/Aids Reference Centre in São Paulo, previously analyzed for IL28B SNPs by PCR-RFLP were selected for analysis, after confirming that they represent all IL28B SNPs patterns described in the literature. The results obtained showed concordance between the PCR-RFLP and the tetra-primer PCR SNPs results, and because of the low cost, easy to perform, and minor employment of biological specimen and reagents, the tetra-primer PCR is of choice to be used in routine. (AU)

Polimorfismos de nucleotídeos únicos (single nucleotide polymorphisms, SNPs rs12979860 e rs8099917) no gene que codifica o Interferon Lambda 4 (IFNL4, antigamente IFNL3 e/ou IL28B) têm sido associados às falhas na resposta imune inata e resposta virológica sustentada na hepatite C, e a mielopatia associada ao HTLV-1 (HTLV-1-associated myelopathy, HAM). A pesquisa destes polimorfismos pode empregar diversas metodologias: sequenciamento, reação em cadeia da polimerase em tempo real ou quantitativa (quantitative polymerase chain reaction, qPCR), análise de fragmentos de restrição enzimática em produtos de PCR (restriction fragment length polymorphism in PCR products, PCR-RFLP) e a tetra-primer PCR. Este estudo comparou o desempenho da tetra-primer PCR em relação a PCR-RFLP, ambas otimizadas no Laboratório de Pesquisa em HTLV do Centro de Imunologia do Instituto Adolfo Lutz de São Paulo. Foram selecionadas 100 amostras de DNA obtidas de pacientes do Centro de Referência e Treinamento em DST/Aids de São Paulo cujos SNPs na IL28B foram anteriormente determinados por PCR-RFLP e representaram todos os perfis descritos em literatura. Os resultados obtidos mostraram concordância entre elas, e pelo fato da tetra-primer PCR ter menor custo, ser de fácil execução, empregar menos tempo, insumos e material biológico, é a técnica de escolha para uso em rotina. (AU)

CCR5-∆32, CCR2-64I, SDF1-3'A, and IFNλ4 rs12979860 and rs8099917 gene polymorphisms in individuals with HIV-1, HIV/HTLV-1, and HIV/HTLV-2 in São Paulo, Brazil

Background. Chemokine and chemokine-receptor polymorphisms have been associated with protection against HIV infection and delayed progression to AIDS, whereas polymorphisms in IFNλ4 (formerly IL28B) have been associated with human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy (HAM) development. Evolutionary selection against ancestral genes differs among human populations, resulting in varying risks of acquiring and developing viral diseases. Methods. DNA samples from 434 patients infected with HIV-1 and/or co-infected with HTLV-1/-2, and samples from 74 HIV and HTLV non-infected individuals from São Paulo, Brazil, were divided into five groups: HIV-naïve, n=160; HIV-ART, n=180; HIV/HTLV-1, n=53; HIV/HTLV-2, n=41; and control, n=74. These samples were analyzed for CCR5-∆32deletion, CCR2-64I, SDF1-3'A, and IFNλ4 rs12979860 and rs8099917 single nucleotide polymorphisms using PCR and PCR-RFLP techniques. These polymorphisms' genotype and allele frequencies were calculated and compared among groups using logistic regression analysis. Results. All polymorphism profiles described in the literature were detected in this study. The wild-type genotype predominated in all genes analyzed except for IFNλ4 rs12979860. Statistical differences in allele frequencies among groups were detected in the CCR5 and CCR2 genes, with a high frequency of ∆32 in HIV-naïve vs. HIV-ART (OR 2.45, P=0.037) and a minus mutant allele A (CCR2-64I) in HIV-naïve vs. HIV/HTLV-1 (OR 1.90, P=0.048), HIV-ART vs. HIV/HTLV-1 (OR 2.62, P=0.003), and HIV/ART vs. HIV/HTLV-2 (OR 2.42, P=0.016). Conclusions. The polymorphism profiles detected in the study groups corroborate the profiles described in racial admixed populations. High CCR2-64I mutant allele frequencies were detected in HIV/HTLV-1/-2 co-infected individuals, and CCR5-∆32 showed predictive value for ART initiation. (AU)

Economic analysis of antenatal screening for human T-cell lymphotropic virus type 1 in Brazil: an open access cost-utility model

Background Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes severe diseases, such as aggressive cancer or progressive neurological disease. HTLV-1 affects mainly people in areas with low human development index and can be transmitted from mother to child, primarily through breastfeeding. Refraining from breastfeeding is an effective intervention to reduce the risk of infection in infants. However, HTLV-1 antenatal screening is not offered globally. According to WHO, the scarcity of cost-effectiveness studies is considered one of the major barriers to the implementation of policies to prevent HTLV-1 infection. Therefore, this study aimed to assess the cost-effectiveness of antenatal screening and postnatal interventions to prevent HTLV-1 mother-to-child transmission in Brazil and to develop an open-access, editable, mathematical model that can be used by other countries and regions to assess different scenarios. Methods In this cost-utility analysis, we constructed a decision tree and a Markov model to assess the cost-effectiveness of HTLV-1 antenatal screening and postnatal interventions (ie, avoidance of breastfeeding, by suppression of lactation with cabergoline, and provision of formula feed) to reduce transmission. For our model, we used data from Brazil and we took the perspective of the public health-care system to estimate costs. Findings The implementation of both screening and interventions would result in the prevention of 1039 infections in infants every year in Brazil with an incremental cost-effectiveness ratio (ICER) of US$11415 per quality-adjusted lifeyear (QALY). 88% of all probabilistic sensitivity analysis simulations had ICER values lower than the Brazilian costeffectiveness threshold ($18 107·74 per QALY). HTLV-1 prevalence in pregnant women, the risk of HTLV-1 transmission when breastfeeding lasts for 6 months or more, and the cost of screening tests were the variables with the largest effect on ICER. Interpretation HTLV-1 antenatal screening is cost-effective in Brazil. An open-access model was developed, and this tool could be used to assess the cost-effectiveness of such policy globally, favouring the implementation of interventions to prevent HTLV-1 mother-to-child transmission worldwide. (AU)

Development and Validation of Multiplex Quantitative Real-Time PCR Assays for Simultaneous Detection and Differentiation of HTLV-1 and HTLV-2, Using Different PCR Platforms and Reagent Brands

Brazil currently has the highest number of individuals infected with human T-lymphotropic virus 1- and 2- (HTLV-1 and HTLV-2) globally. At present, neither molecular protocols nor commercial assays are available for HTLV-1/-2 diagnosis or validated by the Brazilian Ministry of Health regulatory agency (ANVISA). We developed and validated two in-house multiplex quantitative real-time PCR for HTLV1/-2 (mqPCR_HTLV) assays, targeting the pol and tax genes, for the simultaneous identification of HTLV-1, HTLV-2, and the albumin reference gene. The robustness of the assays was evaluated on two platforms using seven commercial master mix formulations. The reactions employed double plasmids (pHTLV1-Alb and pHTLV2-Alb) for the standard curve's construction and for expressing the detection limit of the assays. They were able to detect 10 and 10 copies of HTLV-1 and 10 and 70 copies of HTLV-2 for the tax and pol targets, respectively. High efficiency was obtained using both the platforms and all the reagents evaluated and were successfully reproduced by other analysts. DNA samples from HTLV-1/-2-infected and non-infected patients and from HIV/HTLV-coinfected patients were evaluated to determine the feasibility of their use in routine diagnosis. The mqPCR_HTLV (pol and tax) assays demonstrated an overall specificity of 100% and a sensitivity of 97.4% when testing samples from patients without HIV infection, and sensitivities of 77.1% (pol) and 74.6% (tax) in samples from HIV/HTLV-coinfected patients. In addition, they resolved the issue of HTLV western blotting (WB) indeterminate and WB-untyped results in 45.5 and 66.7% of cases, respectively. The developed mqPCR_HTLV (pol and tax) assays indicated their feasibility for efficient and reliable HTLV diagnosis in various core facility laboratories under different conditions and supplies. (AU)

Biomarkers in a cohort of HIV-Infected Patients Single- or Co-Infected with HTLV-1, HTLV-2, and/or HCV: a Cross-Sectional, Observational Study

HIV, HTLV-1/-2, and HCV share routes of transmission, and such virus co-infections could account for worse outcomes of associated diseases. Measuring cytokines/chemokines, CD4 and CD8 T cells, andHIV viral load (VL) inHIV single-infected and co-infected individuals has prognostic value. We analyzed such biomarkers in 129 blood samples ofHIV-infected individualsmatched for age and sex and divided into six groups (G1 (69 HIV); G2 (9 HIV/HTLV-1); G3 (6 HIV/HTLV-2); G4 (11 HIV/HCV); G5 (19 HIV/HCV/HTLV-1); and G6 (15 HIV/HCV/HTLV-2)). Eight cytokines/chemokines from fifteen analytes could be compared. The highest levels of Th1 and pro-inflammatory cytokines were detected in G2 (IFN-) and G6 (IL-6 and IL1- ) and of chemokines in G1 (MIG, IP10, RANTES), G4 (MCP1), and G6 (MIP1- ). The highest CD4 cells number and the lowest HIV VL were identified in G3 and the opposite results in G2. Positive correlations between CD4 and CD8 cells counts and IL-6 levels were detected in G2 and G5 and of HIV VL and RANTES in G4. Negative correlations were detected between CD8 and IFN- in G4 and HIV VL and RANTES in G6. Despite the small number of the cohort analyzed, and although the cross-sectional study design does not allow firm conclusions, the homogeneity of the characteristics of HIV/HTLV-co-infected individuals regarding age, time and route of HIV acquisition, and criteria for introducing ART enable us to suggest a negative impact of HTLV-1 and a possible protective role of HTLV-2 in HIV infection progression in such patients. (AU)

HTLV-1 and HTLV-2 infections in patients with endemic mycoses in São Paulo, Brazil: a cross-sectional, observational study

Background Brazil is a country endemic for human T-lymphotropic virus 1 and 2 (HTLV-1 and HTLV-2), systemic mycoses such as paracoccidioidomycosis (PCM) and histoplasmosis (HP), and aspergillosis (AP). The prevalence of HTLV-1/-2 infections in individuals with endemic mycoses in Latin America is unknown; however, an association between HTLV-1 and severe PCM and HP has been observed in Peru. Addressing this knowledge gap, we searched for HTLV-1/-2 antibodies in serum samples sent to the Instituto Adolfo Lutz, São Paulo, Brazil, for systemic mycosis diagnosis. Methods We used 387 sera from a biorepository that had seropositive results for Paracoccidioides spp. (G1, n=212), Histoplasma capsulatum (G2, n=95), Aspergillus spp. (G3, n=61), and at least two of these fungi (G4, n=19). We searched for the presence of HTLV-1/-2 antibodies using commercial immunoassays: enzyme immunoassay (HTLV-I+II Murex, Diasorin), western blotting (HTLV Blot 2.4, MP Biomedicals), and line immunoassay (INNOLIA HTLV I/II, Fujirebio). Demographic characteristics were evaluated in each group. Findings Different regions in São Paulo were sampled. Most samples were from males (76.2%; p=0.001), except for G3, in which no sex bias was detected. Mean age differences were observed between groups: patients with PCM and HP had a similar mean age (42.8 and 42.0 years, respectively), while those with AP and co-fungal infection were older (55.1 and 52.8 years, respectively, (p<0.001). Noteworthy, males were older than females in G1 (p=0.005). Screening detected HTLV-1/2 antibodies in five samples (1.30%; 95% CI: 0.8−1.8%), with two borderline results. HTLV-1/2 was confirmed in two samples: 2/387 (0.52%; 0.063−1.85%): one HTLV-2, male, 42 years, from G1: 1/212 (0.47%; 0.012−2.60%), and one HTLV-1, male, 51 years, from G3: 1/61 (1.64%; 0.042−8.80%). Interpretation In the state of São Paulo, HTLV-1 and HTLV-2 seem to circulate in male patients with systemic mycoses, and since HTLV-1 could impact fungal disease severity, the identification of co-infection is important regardless of prevalence.(AU)

Multi-Epitope Protein as a Tool of Serological Diagnostic Development for HTLV-1 and HTLV-2 Infections

A multi-epitope protein expressed in a prokaryotic system, including epitopes of Env, Gag, and Tax proteins of both HTLV-1 and HTLV-2 was characterized for HTLV-1/2 serological screening. This tool can contribute to support the implementation of public policies to reduce HTLV-1/2 transmission in Brazil, the country with the highest absolute numbers of HTLV-1/2 infected individuals. The chimeric protein was tested in EIA using serum/plasma of HTLV-infected individuals and non-infected ones from four Brazilian states, including the North and Northeast regions (that present high prevalence of HTLV-1/2) and Southeast region (that presents intermediate prevalence rates) depicting different epidemiological context of HTLV-1/2 infection in our country. We enrolled samples from Pará (n = 114), Maranhão (n = 153), Minas Gerais (n = 225) and São Paulo (n = 59) states; they are from blood donors' candidates (Pará and Minas Gerais), pregnant women (Maranhão) and HIV+/high risk for sexually transmitted infection (STI; São Paulo). Among the HTLV-1/2 positive sera, there were co-infections with viral (HTLV-1 + HTLV-2, HIV, HCV, and HBV), bacterial (Treponema pallidum) and parasitic (Trypanosoma cruzi, Schistosma mansoni, Strongyloides stercoralis, Entamoeba coli, E. histolytica, and Endolimax nana) pathogens related to HTLV-1/2 co-morbidities that can contribute to inconclusive diagnostic results. Sera positive for HIV were included among the HTLV-1/2 negative samples. Considering both HTLV-1 and HTLV-2-infected samples from all states and different groups (blood donor candidates, pregnant women, and individuals with high risk for STI), mono or co-infected and HTLV-/HIV+, the test specificity ranged from 90.09 to 95.19% and the sensitivity from 82.41 to 92.36% with high accuracy (ROC AUC = 0.9552). Thismulti-epitope protein showed great potential to be used in serological screening of HTLV-1 and HTLV-2 in different platforms, even taking into account the great regional variation and different profile of HTLV-1 and HTLV-2 mono or co-infected individuals. (AU)

O Instituto Adolfo Lutz na pandemia COVID-19: Experiência no diagnóstico molecular e vigilância de SARS-CoV-2 / The Adolfo Lutz Institute in the COVID-19 pandemic: Experience in diagnosis molecular and surveillance of SARS-CoV-2

Este trabalho descreve os ensaios de rotina e as pesquisas realizadas no Instituto Adolfo Lutz (IAL), durante a pandemia de COVID-19, relacionadas ao diagnóstico molecular da Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). É apresentada a partícula viral, seu genoma e os ensaios e kits utilizados durante o período, contextualizando seu uso e apontando os de melhor desempenho e custo-efetividade. Ademais, destaca as pesquisas relacionadas à utilização de material biológico obtido de gargarejo, saliva, urina e tecidos. Por meio de sequenciamento de nova geração, descreve as variantes virais encontradas no estado de São Paulo e depositadas no Global Initiative on Sharing All Influenza Data (GISAID) ao longo do tempo, dando ênfase às variantes de interesse (variant of interest, VOI) e de atenção (variant of concern, VOC). Apresenta estudos sobre: técnica de sequenciamento SANGER da região S (spike) do genoma viral, útil na triagem de VOI e VOC; predição de estrutura terciária, estabilidade e flexibilidade da proteína, com ênfase à região de ligação do vírus ao seu receptor na célula hospedeira na variante P.1; e casos de possível "falha vacinal" relacionados à idade, tipo/local de trabalho e de residência, e a variante viral infectante. Além disso, menciona e discute os Boletins de Monitoramento de SARS-CoV-2, Vigilância Epigenômica, emitidos regularmente pela Instituição, onde é possível acompanhar o surgimento de variantes virais identificadas em cada um dos 17

This paper describes the routine trials and research carried out at the Instituto Adolfo Lutz (IAL), during the COVID-19 pandemic, related to the molecular diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The viral particle, its genome and the assays and kits used during the period are presented, contextualizing its use and pointing out those with the best performance and cost-effectiveness. Furthermore, highlights the researches related to the use of biological material obtained from throat wash, saliva, urine and tissues. Through next-generation sequencing, describes the viral variants found in the state of São Paulo and deposited in the Global Initiative on Sharing All Influenza Data (GISAID) over time, emphasizing the variants of interest (VOI) and of concern (VOC). It presents studies on: SANGER sequencing technique of the S (spike) region of the viral genome, useful in VOI and VOC screening; the tertiary structure, molecular stability, and flexibility of S protein, with emphasis on the receptor-biding region of the P.1 variant; cases of possible "vaccination failure" related to age, type/place of work and residence, and the infectious viral variant. In addition, mentions and discusses the SARSCoV-2 Monitoring Bulletins, Epigenomic Surveillance, regularly issued by the Institution, where it is possible to monitor the emergence of viral variants identified in each of the 17 Regional Departments of Health of the state of São Paulo. Finally, presents the list of people directly or indirectly involved in IAL's COVID-19 task force. (AU)

Specificity of HTLV screening tests and its impact on health care program costs: The perspective of antenatal screening in Brazil

Abstract INTRODUCTION: Brazil ranks first in the number of HTLV-1/-2-infected individuals worldwide. The high morbidity and mortality of HTLV-1-associated diseases, especially following infection in infancy, requires strong action to reduce vertical transmission. METHODS: To facilitate the appraisal of the implementation of the HTLV antenatal screening program by the Brazilian Ministry of Health, we determined the costs in distinct scenarios according to HTLV seroprevalence, specificity of the screening test, and type of confirmatory test. RESULTS: HTLV antenatal screening would cost R$ 55,777,012-R$ 77,082,123/year. Screening assays with high specificity reduce the need and cost of confirmatory assays by up to 25%. CONCLUSIONS: Careful selection of the screening assay is required to optimize the program.

SARS-CoV-2 variants in severely symptomatic and deceased persons who had been vaccinated against COVID-19 in São Paulo, Brazil

[ABSTRACT]. COVID-19 vaccination began in São Paulo, Brazil in January 2021, first targeting healthcare workers (HCWs) and the elderly, using the CoronaVac vaccine (Sinovac/Butantan) and subsequently the Oxford/AstraZeneca (ChAdOx1) vaccine (AstraZeneca/FIOCRUZ-RJ). Studies on such vaccines have shown efficacy in preventing severe cases and deaths, but there is a lack of information regarding their effectiveness. This manuscript presents data from the Instituto Adolfo Lutz (IAL), a public health laboratory located in São Paulo City that receives samples from 17 Regional Health Departments under the Secretary of Health of São Paulo, for SARS-CoV-2 genomic surveillance. Through May 15, 2021 IAL received 20 samples for analysis from COVID-19 vaccinated individuals who needed hospitalization and/or died from COVID-19. Next-generation sequencing was performed on an Ion Torrent S5 platform using the AmpliSeq™ SARS-CoV-2 kit. Almost all cases were vaccinated with CoronaVac and presented the gamma variant of concern (VOC). Cases of death were observed mostly in the elderly in nursing homes, and severe cases in younger frontline HCWs. This data confirmed that the SARS-CoV-2 gamma variant is highly transmissible, severe, and lethal for COVID-19 in these groups of individuals, thereby highlighting the importance of continuous vaccination and non-pharmacological prevention measures to avoid virus dissemination and the emergence of new VOCs.

[RESUMEN]. La vacunación contra la COVID-19 empezó en São Paulo (Brasil) en enero del 2021 con los trabajadores de atención de salud (personal de salud) y las personas mayores, empleando la vacuna de CoronaVac (Sinovac/Butantan) y posteriormente la vacuna de Oxford/AstraZeneca (ChAdOx1) (AstraZeneca/FIOCRUZ-RJ). Los estudios sobre estas vacunas han mostrado su eficacia en la prevención de los casos graves y las muertes, pero existe falta de información con respecto a su efectividad. En este artículo se presentan datos del Instituto Adolfo Lutz (IAL), un laboratorio de salud pública ubicado en la ciudad de São Paulo que recibe muestras de 17 departamentos regionales de salud bajo la Secretaría de Salud de São Paulo, relativos a la vigilancia genómica del SARS-CoV-2. Hasta el 15 de mayo del 2021, el IAL había recibido 20 muestras para su análisis de personas vacunadas contra la COVID-19 que necesitaron hospitalización o murieron a causa de esta enfermedad. Se realizó una secuenciación de nueva generación en una plataforma Ion Torrent S5 mediante el kit para el SARS-CoV-2 AmpliSeq™. Casi todos los pacientes se habían vacunado con CoronaVac y presentaban la variante de preocupación gamma. Se observaron muertes principalmente de personas mayores en residencias y casos graves en personal de salud más joven de primera línea. Estos datos confirmaron que la variante gamma del SARS-CoV-2 es sumamente transmisible, grave y letal para la COVID-19 entre estos grupos y destacan la importancia de continuar con la vacunación y las medidas preventivas no farmacológicas para evitar la propagación del virus y la aparición de nuevas variantes de preocupación.

[RESUMO]. A vacinação contra a COVID-19 começou em São Paulo, Brasil, em janeiro de 2021, primeiramente dirigida a profissionais da saúde e idosos, utilizando a vacina CoronaVac (Sinovac/Butantan), e posteriormente a vacina Oxford/AstraZeneca (ChAdOx1) (AstraZeneca/Fiocruz-RJ). Os estudos sobre tais vacinas revelaram eficácia na prevenção de casos graves e mortes, mas há falta de informação em relação à sua efetividade. Este manuscrito apresenta dados do Instituto Adolfo Lutz (IAL), um laboratório de saúde pública localizado no município de São Paulo, que recebe amostras de 17 Departamentos Regionais de Saúde da Secretaria Estadual de Saúde de São Paulo para vigilância genômica do SARS-CoV-2. Até 15 de maio de 2021, o IAL recebeu 20 amostras para análise de indivíduos vacinados contra a COVID-19 que necessitaram de hospitalização e/ou morreram por COVID-19. O sequenciamento de nova geração foi realizado em plataforma Torrente de íon S5, utilizando o kit AmpliSeq™ SARS-CoV-2. Quase todos os casos foram vacinados com CoronaVac e apresentaram a variante de preocupação (VOC) gama. Os óbitos foram observados principalmente nos idosos de casas de repouso, e os casos graves em profissionais de saúde mais jovens da linha de frente. Esses dados confirmaram que a variante SARS-CoV-2 gama é altamente transmissível, grave e letal para COVID-19 nesses grupos de indivíduos, destacando, assim, a importância da vacinação contínua e de medidas preventivas não farmacológicas para evitar a disseminação viral e o surgimento de novas VOC.

SARS-CoV-2 variants in severely symptomatic and deceased persons who had been vaccinated against COVID-19 in São Paulo, Brazil

COVID-19 vaccination began in São Paulo, Brazil in January 2021, first targeting healthcare workers (HCWs) and the elderly, using the CoronaVac vaccine (Sinovac/Butantan) and subsequently the Oxford/AstraZeneca (ChAdOx1) vaccine (AstraZeneca/FIOCRUZ-RJ). Studies on such vaccines have shown efficacy in preventing severe cases and deaths, but there is a lack of information regarding their effectiveness. This manuscript presents data from the Instituto Adolfo Lutz (IAL), a public health laboratory located in São Paulo City that receives samples from 17 Regional Health Departments under the Secretary of Health of São Paulo, for SARS-CoV-2 genomic surveillance. Through May 15, 2021 IAL received 20 samples for analysis from COVID-19 vaccinated individuals who needed hospitalization and/or died from COVID-19. Next-generation sequencing was performed on an Ion Torrent S5 platform using the AmpliSeq™ SARS-CoV-2 kit. Almost all cases were vaccinated with CoronaVac and presented the gamma variant of concern (VOC). Cases of death were observed mostly in the elderly in nursing homes, and severe cases in younger frontline HCWs. This data confirmed that the SARSCoV-2 gamma variant is highly transmissible, severe, and lethal for COVID-19 in these groups of individuals, thereby highlighting the importance of continuous vaccination and non-pharmacological prevention measures to avoid virus dissemination and the emergence of new VOCs. (AU)

La vacunación contra la COVID-19 empezó en São Paulo (Brasil) en enero del 2021 con los trabajadores de atención de salud (personal de salud) y las personas mayores, empleando la vacuna de CoronaVac (Sinovac/Butantan) y posteriormente la vacuna de Oxford/AstraZeneca (ChAdOx1) (AstraZeneca/FIOCRUZ-RJ). Los estudios sobre estas vacunas han mostrado su eficacia en la prevención de los casos graves y las muertes, pero existe falta de información con respecto a su efectividad. En este artículo se presentan datos del Instituto Adolfo Lutz (IAL), un laboratorio de salud pública ubicado en la ciudad de São Paulo que recibe muestras de 17 departamentos regionales de salud bajo la Secretaría de Salud de São Paulo, relativos a la vigilancia genómica del SARS-CoV-2. Hasta el 15 de mayo del 2021, el IAL había recibido 20 muestras para su análisis de personas vacunadas contra la COVID-19 que necesitaron hospitalización o murieron a causa de esta enfermedad. Se realizó una secuenciación de nueva generación en una plataforma Ion Torrent S5 mediante el kit para el SARS-CoV-2 AmpliSeq™. Casi todos los pacientes se habían vacunado con CoronaVac y presentaban la variante de preocupación gamma. Se observaron muertes principalmente de personas mayores en residencias y casos graves en personal de salud más joven de primera línea. Estos datos confirmaron que la variante gamma del SARS-CoV-2 es sumamente transmisible, grave y letal para la COVID-19 entre estos grupos y destacan la importancia de continuar con la vacunación y las medidas preventivas no farmacológicas para evitar la propagación del virus y la aparición de nuevas variantes de preocupación. (AU)

A vacinação contra a COVID-19 começou em São Paulo, Brasil, em janeiro de 2021, primeiramente dirigida a profissionais da saúde e idosos, utilizando a vacina CoronaVac (Sinovac/Butantan), e posteriormente a vacina Oxford/AstraZeneca (ChAdOx1) (AstraZeneca/Fiocruz-RJ). Os estudos sobre tais vacinas revelaram eficácia na prevenção de casos graves e mortes, mas há falta de informação em relação à sua efetividade. Este manuscrito apresenta dados do Instituto Adolfo Lutz (IAL), um laboratório de saúde pública localizado no município de São Paulo, que recebe amostras de 17 Departamentos Regionais de Saúde da Secretaria Estadual de Saúde de São Paulo para vigilância genômica do SARS-CoV-2. Até 15 de maio de 2021, o IAL recebeu 20 amostras para análise de indivíduos vacinados contra a COVID-19 que necessitaram de hospitalização e/ou morreram por COVID-19. O sequenciamento de nova geração foi realizado em plataforma Torrente de íon S5, utilizando o kit AmpliSeq™ SARS-CoV-2. Quase todos os casos foram vacinados com CoronaVac e apresentaram a variante de preocupação (VOC) gama. Os óbitos foram observados principalmente nos idosos de casas de repouso, e os casos graves em profissionais de saúde mais jovens da linha de frente. Esses dados confirmaram que a variante SARS-CoV-2 gama é altamente transmissível, grave e letal para COVID-19 nesses grupos de indivíduos, destacando, assim, a importância da vacinação contínua e de medidas preventivas não farmacológicas para evitar a disseminação viral e o surgimento de novas VOC. (AU)

Laboratory diagnosis of human T-lymphotropic virus in Brazil: assays, flowcharts, challenges, and perspectives

Abstract INTRODUCTION We present a data analysis and review of recent studies regarding the laboratory diagnosis of human T-lymphotropic virus 1 and 2 (HTLV-1/2) infections in Brazil. METHODS Target populations, available diagnostic serological assays (screening and complementary tests), molecular assays (in-house), causes of false-positive and false-negative results, and flowcharts were analyzed. RESULTS A table presents the target populations, two diagnostic flowcharts (depending on laboratory infrastructure and study population), and recent research that may improve how HTLV-1/2 is diagnosed in Brazil. CONCLUSIONS: Our results support the implementation of public policies to reduce HTLV-1/2 transmission and its associated diseases.

COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols

The gold standard for the laboratory diagnosis of COVID-19 is the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay, which searches for SARS­CoV-2 target genes in nasopharyngeal/oropharyngeal (NP/OP) samples, and its performance depends on the quantity and quality of the RNA input. This study compared the performance and cost-effectiveness of three different kits/reagents for RNA extraction used in COVID-19 diagnosis in Sao Paulo, Brazil. A total of 300 NP/OP samples belonging to suspected cases of COVID-19 stored in a biorepository were randomly selected, and RNA was extracted using (i) automated extraction (Loccus, Extracta Kit FAST), (ii) manual extraction (BioGene Kit, Bioclin, Quibasa), and (iii) quick extraction methods (Lucigen, Quick DNA Extract Kit). Next, the samples were tested using RT-qPCR for SARS-CoV-2 with the Allplex 2019-nCoV modified assay and the Charité-Berlin protocol. All assays/kits were used according to the manufacturer's instructions. For the Allplex kit, the sensitivity in detecting SARS-CoV-2 with previously extracted RNA by different procedures was 100.0% for Loccus, 100.0% for BioGene and 91.9% for Quick. Using the Charité-Berlin protocol, the sensitivities were 81.4% for Loccus, 81.2% for BioGene and 60.7% for Quick. The least sensitive target gene and the gene most affected by RNA extraction procedures was the RNA-dependent RNA polymerase gene (Charité-Berlin protocol). No false-positive SARS-CoV-2 results were detected using RNA obtained from any of the different protocols. In conclusion, Loccus and BioGene RNA extractions were efficient for RT-qPCR assays, and although the BioGene procedure is less expensive, Loccus is the best choice because it allows the rapid handling of hundreds or thousands of samples, a desirable feature during pandemics. Although less sensitive, the Quick extraction is useful during outbreaks coupled with the Allplex amplification kit for SARS-CoV-2 diagnosis (κ = 0.925).

Laboratory diagnosis of human T-lymphotropic virus in Brazil: assays, flowcharts, challenges, and perspectives

Introduction: We present a data analysis and review of recent studies regarding the laboratory diagnosis of human T-lymphotropic virus 1 and 2 (HTLV-1/2) infections in Brazil. Methods: Target populations, available diagnostic serological assays (screening and complementary tests), molecular assays (in-house), causes of false-positive and false-negative results, and flowcharts were analyzed. Results: A table presents the target populations, two diagnostic flowcharts (depending on laboratory infrastructure and study population), and recent research that may improve how HTLV-1/2 is diagnosed in Brazil. Conclusions: Our results support the implementation of public policies to reduce HTLV-1/2 transmission and its associated diseases.

Provirus Mutations of Human T-Lymphotropic Virus 1 and 2 (HTLV-1 and HTLV-2) in HIV-1-Coinfected Individuals

Provirus mutations of human T-lymphotropic virus 1 (HTLV-1), mostly the lack of the 5= long terminal repeat (LTR) genomic region, have been described and associated with severe adult T cell leukemia/lymphoma (ATLL), non-sense point mutations with low proviral load, and Western blotting indeterminate results. Until now, no information concerning provirus mutations of HTLV-2 and its consequences, as well as those of HTLV-1/2 in HIV-coinfected individuals, had been described. Therefore, we searched for these mutations in provirus samples of 44 HIV/HTLV-1- and 25 HIV/HTLV-2-coinfected individuals. Using protocols well established for amplification and sequencing of segments of the LTR, env, and tax regions, we searched for defective type 1 particles that retain LTRs and lack internal sequences and type 2 particles that lack the 5=LTR region. In addition, using as references the prototypes ATK (HTLV-1) and Mo (HTLV-2), we searched for point mutations in the LTR and synonyms and nonsynonymous mutations and non-sense mutations in env and tax regions. Defective HTLV-1 and HTLV-2 provirus type 1 or 2 was detected in 31.8% of HIV/HTLV-1- and 32.0% of HIV/HTLV-2-coinfected individuals. Synonymous and nonsynonymous mutations were identified mostly in HTLV-2 and associated with lower levels of specific antibodies. No non-sense mutations that resulted in premature termination of Env and Tax proteins were detected. On the contrary, mutation in the stop codon of Tax2a produced a long protein characteristic of the HTLV-2c subtype. The clinical significance of these mutations in coinfected individuals remains to be defined, but they confirmed the lower sensitivity of serological and molecular diagnostic tests in HIV/HTLV-1/2 coinfections. IMPORTANCE HTLV-1 and HTLV-2 are endemic to Brazil, and they have different effects in HIV/AIDS disease progression. HIV/HTLV-1 has been described as accelerating the progression to AIDS and death, while HIV/HTLV-2 slows the progression to AIDS. Provirus mutations of HTLV-1 were implicated in severe leukemia development and in problems in the diagnosis of HTLV-1; in contrast, provirus mutations of HTLV-2 had not been confirmed and associated with problems in HTLV-2 diagnosis or disease outcome. Nevertheless, data obtained here allowed us to recognize and understand the false-negative results in serologic and molecular tests applied for HTLV-1 and HTLV-2 diagnosis. Defective proviruses, as well as synonymous and nonsynonymous mutations, were associated with the diagnosis deficiencies. Additionally, since HIV-1 and HTLV-1 infect the same cells (CD4 positive), the production of HIV-1 pseudotypes with HTLV-1 envelope glycoprotein during HIV/HTLV-1 coinfection cannot be excluded. Defective provirus of HTLV-2 and Tax2c is speculated to influence progression to AIDS. (AU)

Surveillance of human retroviruses in blood samples from patients with hepatitis B and C in São Paulo, Brazil

Abstract INTRODUCTION Human retroviruses and the hepatitis B and C viruses (HBV and HCV, respectively) share routes of transmission; thus, coinfections occur and could alter subsequent disease outcomes. A preliminary study on human T-lymphotropic virus types 1 and 2 (HTLV-1/2) in serum samples from HBV- and HCV-infected individuals in São Paulo revealed 1.3% and 5.3% rates of coinfection, respectively. These percentages were of concern since they were detected in HTLV-endemic regions and in high-risk individuals in Brazil. The present study was conducted to extend and confirm these data. METHODS HTLV-1/2 and human immunodeficiency virus (HIV) infection status were identified in 1,984 sera for HBV and HCV viral load quantification - 1,290 samples from HBV-infected individuals (53.3% men, mean age: 47.1 years) and 694 samples from HCV-infected individuals (56.3% men, mean age: 50.1 years). HTLV-1/2 antibodies were detected by enzyme immunoassay, followed by western blotting and line immunoassay; HIV infection was detected by enzyme immunoassay. RESULTS HTLV-1/-2 infection was detected in 1.9% HBV-infected individuals (0.7% HTLV-1 and 1.2% HTLV-2) and in 4.0% (2.4% HTLV-1 and 1.6% HTLV-2) HCV-infected individuals; HIV infection was detected in 9.2% and 14.5%, respectively. Strong associations with HTLV and HIV, male sex, and older age were found in HBV/HTLV and HCV/HTLV-coinfected individuals (p<0.05). CONCLUSIONS HTLV-1 and HTLV-2 were confirmed to be prevalent in individuals with HBV and HCV in São Paulo; coinfected individuals deserve further clinical and laboratory investigation.

Line immunoassay for confirmation and discrimination of human t-cell lymphotropic virus infections in inconclusive western blot serum samples from Brazil

Difficulties in confirming and discriminating human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 infections by serological Western blot (WB) assays (HTLV Blot 2.4; MP Biomedicals) have been reported in Brazil, mainly in HIV/AIDS patients, with a large number of WB-indeterminate and WB-positive but HTLV-untypeable results. Nonetheless, a line immunoassay (LIA) (INNO-LIA HTLV-I/II; Fujirebio) provided enhanced specificity and sensitivity for confirming HTLV-1/2 infections. To add information concerning the improved ability of the LIA in relation to WB when applied to samples of individuals from different risk groups from Brazil, we performed the present study. Three groups were analyzed group 1 (G1), with 62 samples from HIV/AIDS patients from São Paulo, SP (48 WB indeterminate and 14 HTLV untypeable); group 2 (G2), with 24 samples from patients with hepatitis B or hepatitis C from São Paulo (21 WB indeterminate and 3 HTLV untypeable; 17 HIV seropositive); and group 3 (G3), with 25 samples from an HTLV outpatient clinic in Salvador, Bahia (16 WB indeterminate and 9 HTLV untypeable; all HIV seronegative). Overall, the LIA confirmed HTLV-1/2 infection (HTLV-1, HTLV-2, or HTLV) in 66.1% (G1), 83.3% (G2), and 76.0% (G3) of samples. Interestingly, the majority of WB-indeterminate results were confirmed by the LIA as being HTLV-2 positive in G1 and G2 but not in G3, in which the samples were defined as being HTLV-1 or HTLV positive. These results agree with the virus types that circulate in such patients of different regions in Brazil and emphasize that the LIA is the bes