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 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.

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.

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.

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 best serological test for confirming HTLV-1 and HTLV-2 infections, independently of being applied in HTLV-monoinfected or HTLV-coinfected individuals.

Performance of Commercially Available Serological Screening Tests for Human T-Cell Lymphotropic Virus Infection in Brazil.

Serological screening for human T-cell lymphotropic virus type 1 (HTLV-1) is usually performed using enzyme-linked immunosorbent assay (ELISA), particle agglutination, or chemiluminescence assay kits. Due to an antigen matrix improvement entailing the use of new HTLV antigens and changes in the format of HTLV screening tests, as well as newly introduced chemiluminescence assays (CLIAs), a systematic evaluation of the accuracy of currently available commercial tests is warranted. We aimed to assess the performance of commercially available screening tests for HTLV infection diagnosis. A diagnostic accuracy study was conducted on a panel of 397 plasma samples: 200 HTLV-negative plasma samples, 170 HTLV-positive plasma samples, and 27 plasma samples indeterminate by Western blotting (WB). WB-indeterminate samples (i.e., those yielding no specific bands for HTLV-1 and/or HTLV-2) were assessed by PCR, and the results were used to compare agreement among the commercially available ELISA screening tests. For performance analysis, WB-indeterminate samples were excluded, resulting in a final study panel of 370 samples. Three ELISA kits (Murex HTLV-1/2 [Murex], anti-HTLV-1/2 SYM Solution [SYM Solution], and Gold ELISA HTLV-1/2 [Gold ELISA]) and one CLIA kit (Architect rHTLV-1/2) were evaluated. All screening tests demonstrated 100% sensitivity. Concerning the HTLV-negative samples, the SYM Solution and Gold ELISA kits had specificity values of >99.5%, while the Architect rHTLV-1/2 test presented 98.1% specificity, followed by Murex, which had a specificity of 92.0%. Regarding the 27 samples with WB-indeterminate results, after PCR confirmation, all ELISA kits showed 100% sensitivity but low specificity. Accuracy findings were corroborated by the use of Cohen's kappa value, which evidenced slight and fair agreement between PCR analysis and ELISAs for HTLV infection diagnosis. Based on the data, we believe that all evaluated tests can be safely used for HTLV infection screening.

Making the invisible visible: searching for human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) in Brazilian patients with viral hepatitis B and C.

With this study, the authors hope to alert clinicians regarding the presence of human T-cell lymphotropic virus type 1 and 2 (HTLV-1/-2) infections in patients with viral hepatitis B and C in Brazil. HTLV-1/-2 were detected in 1.3% of hepatitis B virus (HBV)- and 5.3% of hepatitis C virus (HCV)-infected blood samples sent for laboratory viral load measurements. A partial association of human immunodeficiency virus (HIV)-1 and HTLV-1/-2 infection was detected in patients with HCV (HIV+, 27.3%), whereas this association was almost 100% in HBV-infected patients (HIV+, all except one). The high prevalence of HTLV-1/-2 infection among patients with hepatitis C was of concern, as HTLV-1/-2 could change the natural course of subsequent liver disease. The authors suggest including HTLV-1/-2 serology in the battery of tests used when following patients with viral hepatitis in Brazil, regardless of the HIV status.

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$11 415 per quality-adjusted life-year (QALY). 88% of all probabilistic sensitivity analysis simulations had ICER values lower than the Brazilian cost-effectiveness 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. FUNDING: None. TRANSLATIONS: For the Portuguese and Spanish translations of the abstract see Supplementary Materials section.

LTR point mutations in the Tax-responsive elements of HTLV-1 isolates from HIV/HTLV-1-coinfected patients.

BACKGROUND: In Virology Journal 2011, 8:535, Neto et al. described point mutations into Tax-responsive elements (TRE) of the LTR region of HTLV-1 isolates from asymptomatic carriers from Sao Paulo, Brazil, and hypothesized that the presence of the G232A mutation in the TRE-1 increase viral proliferation and consequently the proviral load (PvL), while the A184G mutation in the TRE-2 do not have such effect. FINDINGS: We performed the real-time PCR assay (pol) and sequenced LTR region of HTLV-1 isolates from 24 HIV/HTLV-1-coinfected patients without HTLV-1-associated diseases from the same geographic area. These sequences were classified as belonging to the transcontinental subgroup A of the Cosmopolitan subtype a. The frequency of G232A mutation (16/24, 66.7%) was high as much as 61.8% reported by Neto's in HTLV-1 asymptomatic carriers with high PvL. High frequency (13/24, 54.2%) of double mutations G232A and A184G was also detected in HIV/HTLV-1-coinfected patients. We did not quantify PvL, but comparative analyses of the cycle threshold (Ct) median values of the group of isolates presenting the mutated-types sequences (Ct 33.5, n = 16) versus the group of isolates with the wild-type sequences (Ct 32, n = 8) showed no statistical difference (p = 0.4220). CONCLUSION: The frequencies of mutated-type sequences in the TRE-1 and TRE-2 motifs were high in HIV/HTLV-1-coinfected patients from Sao Paulo, Brazil. If these LTR point mutations have predictive value for the development of HTLV-1-associated diseases or they correspond to the subtype of virus that circulate in this geographic area has to be determined.

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 HTLV-1/-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.

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, and HIV viral load (VL) in HIV single-infected and co-infected individuals has prognostic value. We analyzed such biomarkers in 129 blood samples of HIV-infected individuals matched 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-γ) and G6 (IL-6 and IL1-ß) and of chemokines in G1 (MIG, IP10, RANTES), G4 (MCP1), and G6 (MIP1-ß). 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-γ 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.

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 (INNO-LIA 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. Funding: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), and Instituto Adolfo Lutz.

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). This multi-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.

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

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.

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.

Comparative performances of seven quantitative Reverse-Transcription Polymerase Chain Reaction assays (RT-qPCR) for detecting SARS-CoV-2 infection in samples from individuals suspected of COVID-19 in São Paulo, Brazil.

Introduction: Brazil is the second largest country with COVID-19 positive cases worldwide. Due to the potent spread of the virus and the scarcity of kits and supplies, the Brazilian Ministry of Health has granted authorization for the use of kits available during this emergency, without an accurate evaluation of their performance. This study compared the performance and cost-effectiveness of seven molecular assays/kits available in São Paulo, Brazil, for SARS-CoV-2 diagnosis. Materials and methods: A total of 205 nasopharyngeal/oropharyngeal samples from suspected cases of COVID-19, were tested using the following assays: (i) GeneFinder COVID-19 plus RealAmp kit; (ii) 2019-nCoV RNA PCR-Fluorescence Probing, Da An Gene Co.; (iii) in-house RT-qPCR SARS-CoV-2 IAL; (iv) 2019-nCoV kit, IDT; (v) molecular SARS-CoV-2 (E) kit, Bio-Manguinhos; (vi) Allplex 2019-nCoV modified Assay, Seegene Inc, and (vii) Biomol one-step COVID-19 kit, IBMP. The criteria for determining a SARS-CoV-2 true positive result included the cycle threshold cut-off values, the characteristics of exponential/linear curves, the gene target diversity, and a positive result in at least two assays. Results: The overall sensitivity of the assays listed were GeneFinder 83.6%, Da An Gene 100.0%, IAL 90.4%, IDT 94.6%, Bio-Manguinhos 87.7%, Allplex 97.3%, and IBMP 87.7%. The minor sensitive gene target was RdRP. Although all assays had a Cohen's Kappa index ≥0.893, the best tests used multiplex assays identifying N-gene and/or E-gene targets. Conclusion: All assays tested accurate for diagnosis, but considering cost-effectiveness (cost, time consumption, number of samples tested, and performance), the in-house IAL assay was ideal for COVID-19 diagnosis in São Paulo, Brazil.

Blocking HTLV-1/2 silent transmission in Brazil: Current public health policies and proposal for additional strategies.

Human T-cell lymphotropic viruses 1 and 2 (HTLV-1/2) are relatively common in Brazil but remain silent and neglected infections. HTLV-1 is associated with a range of diseases with high morbidity and mortality. There is no curative treatment for this lifelong infection, so measures to prevent transmission are essential. This narrative review discusses HTLV-1/2 transmission routes and measures to prevent its continuous dissemination. The public health policies that are currently implemented in Brazil to avoid HTLV-1/2 transmission are addressed, and further strategies are proposed.

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 SARS-CoV-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).

Patterns of antibodies against latent and lytic antigens of human herpesvirus 8 in an endemic population and patients with Kaposi's sarcoma in Mozambique.

The patterns of antibodies against latent and lytic antigens of human herpesvirus 8 (HHV-8) were assessed using immunofluorescence assays of samples from 155 persons seropositive for HHV-8 seen at public health centers and 24 patients with Kaposi's sarcoma (KS) from Mozambique. Of the 155 persons without KS, 48 (31%) had antibodies against latent antigens only, 29 (18.7%) had antibodies against lytic antigens only, and 78 (50.3%) had antibodies against both types of antigen. The HHV-8 antibody titer tended to increase with age until age 40, after which it began to decrease. High titers of antibodies against latent and lytic antigens of HHV-8 were detected mostly in persons co-infected with HIV, and these increased titers could have a predictive value. All patients with KS except four patients who were seronegative for HHV-8 had elevated titers of HHV-8 antibodies, predominantly against latent antigens. The data suggest the potential for an increase in the development of KS in this endemic area for HHV-8.

Seroprevalence of human herpesvirus 8 infection in individuals from health care centers in Mozambique: potential for endemic and epidemic Kaposi's sarcoma.

Human herpesvirus 8 (HHV-8) infection is common in sub-Saharan Africa, but its prevalence in Mozambique is unknown. The seroprevalence of HHV-8 in a cohort of individuals seen at public health centers in Northern (n = 208), Central (n = 226), or Southern (n = 318) Mozambique was examined. All individuals were interviewed to obtain socioeconomic, demographic and clinical data and were tested for serum anti-HHV-8 antibodies using an immunofluorescence assay. The overall frequency of HHV-8 antibodies was 21.4% and, in spite of the diversity of epidemiological characteristics of the tested individuals, did not differ significantly among regions: 18.7%, 24.3% and 21.4% in the North, Center, and South, respectively (chi(2), 2.37; P = 0.305). The variables that were associated significantly with the presence of HHV-8 antibodies were gender, age, level of education, number of siblings and HIV serostatus, but these differed across the regions. In the North, although tested individuals lived under poor socioeconomic conditions, no association between HHV-8 infection and household variables was detected, with the exception of the number of siblings (P = 0.042). In the Central region, HHV-8 infection was associated with gender (P = 0.010), the number of household members (P = 0.031), and the place of attendance (P = 0.021). In the South, HHV-8 infection was associated with the number of siblings (P = 0.023) and HIV status (P = 0.002). The overall prevalence of HHV-8 seropositivity increased with age. These results demonstrate that Mozambique is another country in Africa with endemic HHV-8 infection, and, because of the AIDS epidemic, continued access to antiretroviral treatment is necessary to avert an outbreak of AIDS-Kaposi's sarcoma.

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.

Pooling of sera for human T-cell lymphotropic virus (HTLV) screening in a time of increasing health care expenditure and limited resources.

Identifying the true prevalence of human T-cell lymphotropic virus, mostly type 1 (HTLV-1), and the number of patients with HTLV-1-associated diseases, in addition to introducing HTLV-1/2 serology during the prenatal of pregnant women and in individuals infected with other viruses that share transmission routes with HTLV-1, are actions that could help to recognize the importance of this virus by WHO and national health organizations, and to control its transmission/dissemination. As Brazil is endemic to HTLV and there is an increase in health care expenditure, but resources are limited, any strategy that could reduce the cost of HTLV screening is needed and welcomed. This study aimed to determine whether the strategy of pooling sera for HTLV antibody determination is feasible and reduces the costs. Two enzyme immunoassays (EIA Murex HTLV-I+II, Diasorin, UK, and Gold ELISA HTLV-1+2, REM Ind. Com. Ltda., SP, Brazil), and serum samples that resulted in different levels of HTLV-1/2 antibodies by EIA and of which a volume allowed assay validation were employed for analysis. The diagnostic sensitivity and specificity and Cohen's Kappa value, as well as the accuracy and precision were analyzed. After validating the five-sample pool using the EIA Murex (Cohen's Kappa = 1.0), the technique was employed for individual cost comparison in 2,625 serum samples from populations at risk of HTLV infections (HBV, HCV, and HIV-infected individuals). The results from individual and pooled samples confirmed the diagnostic sensitivity (100%) and specificity (100%) of the pooling and a cost minimization varying from 60.7% to 73.6%. In conclusion, the results of this study suggest the use of pooling sera in sero-epidemiological surveillance studies and possibly in prenatal care screening programs in Brazil.

The reasons to include the serology of human T-lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) in the clinical follow-up of patients with viral hepatitis B and C in Brazil.

BACKGROUND: The WHO established targets for 2030 to globally reduce new viral hepatitis B and C infections by 90% and deaths by 65% and recommends searching for coinfections that increase the progression of chronic liver infections towards cirrhosis and hepatocellular carcinoma. AIMS AND METHODOLOGY: This study aimed to add information concerning the influence of human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) infections in hepatitis B and C, since in Brazil, these human retroviruses are endemic but neglected. Serum samples from 1,910 patients with hepatitis B and 1,315 with hepatitis C from São Paulo, southeast Brazil, that were previously tested and grouped for HIV and HTLV-1/-2 coinfections were analyzed for hepatitis B virus (HBV) and hepatitis C virus (HCV) loads measurements and subsequent clearance using data from laboratory records. KEY RESULTS: Briefly, the lowest HBV viral load (VL) was detected in HBV/HTLV-2 coinfected patients, regardless of whether they were infected with HIV (all comparisons p<0.05). In contrast, higher HCV VL was detected in HCV/HIV, HCV/HIV/HTLV-1/-2 coinfected patients (all p<0.05), and the lowest HCV VL was detected in HCV/HTLV-2 coinfected patients. Curiously, 61.1% of the patients with HBV/HTLV-2 coinfection had an undetectable HBV VL at the beginning of the study versus 21.4% in the patients with HBV/HTLV-1 coinfection. Although the percentages of undetectable HCV loads in HCV/HTLV-1 and HCV/HTLV-2 coinfected patients were quite similar, during follow-up, more HCV clearance was detected in patients with HCV/HTLV-2 coinfection [OR 2.65; 95% IC (1.17-5.99)]. MAJOR CONCLUSIONS: HTLV-2 positively impacts HBV and HCV viral loads and HCV clearance, while HIV and/or HTLV-1 negatively impacts HCV viral load. Thus, the search for HTLV-1/-2 in viral hepatitis B and C infected patients has virological prognostic value, which is a strong reason to suggest including HTLV serology in the follow-up of patients.