COVID-19 in hematopoietic stem cell transplant recipients during three years of the pandemic: a multicenter study in Brazil.

Hematopoietic stem cell transplant (HSCT) recipients are at -increased risk for severe COVID-19. The aim of this study was to evaluate the burden of COVID-19 in a cohort of HSCT recipients. This retrospective study evaluated a cohort of adult hospitalized HSCT recipients diagnosed with COVID-19 in two large hospitals in São Paulo, Brazil post-HSCT, from January 2020 to June 2022. The primary outcome was all-cause mortality. Of 49 cases, 63.2% were male with a median age of 47 years. Allogeneic-HSCT (51.2%) and autologous-HSCT (48.9%) patients were included. The median time from HSCT to COVID-19 diagnosis was 398 days (IQR: 1211-134), with 22 (44.8%) cases occurring within 12 months of transplantation. Most cases occurred during the first year of the pandemic, in non-vaccinated patients (n=35; 71.4%). Most patients developed severe (24.4%) or critical (40.8%) disease; 67.3% received some medication for COVID-19, primarily corticosteroids (53.0%). The probable invasive aspergillosis prevalence was 10.2%. All-cause mortality was 40.8%, 51.4% in non-vaccinated patients and 14.2% in patients who received at least one dose of the vaccine. In the multiple regression analyses, the variables mechanical ventilation (OR: 101.01; 95% CI: 8.205 - 1,242.93; p = 0.003) and chest CT involvement at diagnosis ≥50% (OR: 26.61; 95% CI: 1.06 - 664.26; p = 0.04) remained associated with all-cause mortality. Thus, HSCT recipients with COVID-19 experienced high mortality, highlighting the need for full vaccination and infection prevention measures.

Comparison of four different human papillomavirus genotyping methods in cervical samples: Addressing method-specific advantages and limitations.

Since human papillomavirus (HPV) is recognized as the causative agent of cervical cancer and associated with anogenital non-cervical and oropharyngeal cancers, the characterization of the HPV types circulating in different geographic regions is an important tool in screening and prevention. In this context, this study compared four methodologies for HPV detection and genotyping: real-time PCR (Cobas® HPV test), nested PCR followed by conventional Sanger sequencing, reverse hybridization (High + Low PapillomaStrip® kit) and next-generation sequencing (NGS) at an Illumina HiSeq2500 platform. Cervical samples from patients followed at the Family Health Strategy from Juiz de Fora, Minas Gerais, Brazil, were collected and subjected to the real-time PCR. Of those, 114 were included in this study according to the results obtained with the real-time PCR, considered herein as the gold standard method. For the 110 samples tested by at least one methodology in addition to real-time PCR, NGS showed the lowest concordance rates of HPV and high-risk HPV identification compared to the other three methods (67-75 %). Real-time PCR and Sanger sequencing showed the highest rates of concordance (97-100 %). All methods differed in their sensitivity and specificity. HPV genotyping contributes to individual risk stratification, therapeutic decisions, epidemiological studies and vaccine development, supporting approaches in prevention, healthcare and management of HPV infection.

COVID-19 in hematopoietic stem cell transplant recipients during three years of the pandemic: a multicenter study in Brazil

ABSTRACT Hematopoietic stem cell transplant (HSCT) recipients are at -increased risk for severe COVID-19. The aim of this study was to evaluate the burden of COVID-19 in a cohort of HSCT recipients. This retrospective study evaluated a cohort of adult hospitalized HSCT recipients diagnosed with COVID-19 in two large hospitals in São Paulo, Brazil post-HSCT, from January 2020 to June 2022. The primary outcome was all-cause mortality. Of 49 cases, 63.2% were male with a median age of 47 years. Allogeneic-HSCT (51.2%) and autologous-HSCT (48.9%) patients were included. The median time from HSCT to COVID-19 diagnosis was 398 days (IQR: 1211-134), with 22 (44.8%) cases occurring within 12 months of transplantation. Most cases occurred during the first year of the pandemic, in non-vaccinated patients (n=35; 71.4%). Most patients developed severe (24.4%) or critical (40.8%) disease; 67.3% received some medication for COVID-19, primarily corticosteroids (53.0%). The probable invasive aspergillosis prevalence was 10.2%. All-cause mortality was 40.8%, 51.4% in non-vaccinated patients and 14.2% in patients who received at least one dose of the vaccine. In the multiple regression analyses, the variables mechanical ventilation (OR: 101.01; 95% CI: 8.205 - 1,242.93; p = 0.003) and chest CT involvement at diagnosis ≥50% (OR: 26.61; 95% CI: 1.06 - 664.26; p = 0.04) remained associated with all-cause mortality. Thus, HSCT recipients with COVID-19 experienced high mortality, highlighting the need for full vaccination and infection prevention measures.

Toxoplasmosis outbreak in São Paulo, Brazil: Epidemiology and visual outcome

ABSTRACT Purpose: To describe a 2019 acute toxoplasmosis outbreak in the city of São Paulo, Brazil, and to evaluate the laboratory serological profile for toxoplasmosis for three consecutive years. The ophthalmological manifestations of the patients involved in the outbreak were also studied. Methods: A cross-sectional descriptive study of a toxoplasmosis outbreak in São Paulo, Brazil, between February and May 2019. Epidemiological data were described, as were the observed ocular manifestations. As part of this study the number of patients with positive IgM toxoplasmosis serology was obtained from a large laboratory network (DASA) for three consecutive years, including the year of the outbreak (2018, 2019, 2020). Results: Eighty-three individuals were identified in the outbreak and two clusters were studied. The clinical picture of at least 77% of the patients, the epidemiological analysis, and the short incubation period (5-8 days) suggested contamination by oocysts. Serological laboratory data analysis revealed an increase of positive toxoplasmosis IgM in 2019 of 73% compared to the previous year. Ophthalmological examination revealed that at least 4.8% of the patients developed toxoplasmic retinochoroiditis, none of whom had been treated during the acute systemic disease. Conclusion: Our findings indicate vegetable contamination as the possible source of this outbreak, a high prevalence of toxoplasmosis in São Paulo during the outbreak period, and a drop in the number of tests during the COVID-19 pandemic. Retinochoroiditis was observed in at least 4.8% of the cases. We confirm the need to implement effective means for the prevention, diagnosis, and treatment of the disease. This may involve raising awareness among the population of the importance of vegetable hygiene, and improved quality control of food and water.

Genital Human Papillomavirus Infection in women from Florianópolis - Santa Catarina, Brazil / Infecção genital pelo Papilomavírus Humano em mulheres de Florianópolis - Santa Catarina, Brasil

Introduction: Human Papillomavirus (HPV) infection is the most common sexually transmitted infection in women. About 80% of sexually active women will have contact with this virus at some age in their lives. Most infections will be transient, but when the infection becomes persistent, associated with high oncogenic risk HPV, there may be progression to cancer, especially cervical cancer. The best way to prevent HPV infection is through the use of vaccines. Objective: To assess which are the most prevalent types of HPV in the city of Florianópolis, Brazil and if the majority of the diagnosed types are contained in the HPV vaccines currently available on the market and in the public health sector. Methods: More than 14,727 HPV tests were evaluated for the diagnosis of genital HPV infection in women from Florianópolis. The prevalence of infection was evaluated according to age of the women. HPV detection was performed using molecular biology tests, such as hybrid capture (for diagnosis of the HPV group, high or low oncogenic risk) and PCR (viral genotyping) techniques. Results: The diagnosis of HPV infection was made for women between one and 102 years of age. The highest positivity of the exams was observed in women aged 20­25 years (51% of the exams). The most prevalent age group was 31­35 years old (23.5%), and the lowest was for women aged 70 and above (0.6%). High oncogenic risk HPV was detected in 94.1% of positive samples and was the most frequent in all age groups. Mixed infection (high- and low-risk HPV) was more prevalent in the 66­70 age group (25.6%). The most frequent genotypes were non-16/18 high oncogenic risk HPV (77% of positive cases). HPV 16 was found in 17.1% of positive cases, and HPV 18 in 6.5%. Conclusion: The most prevalent types of HPV in Florianópolis in the last 6 years are non-16/18 high oncogenic risk HPV types, viral types not covered by the current HPV vaccine available in the public health sector in Brazil.

Introdução: A infecção pelo Papilomavírus Humano (HPV)é a infecção sexualmente transmissível mais frequente na mulher. Cerca de 80% das mulheres sexualmente ativas irão entrar em contato com este vírus em algum momento da sua vida. A maioria das infecções será transitória, mas quando a infecção se torna persistente, associada aos HPV de alto risco oncogênico, poderá haver a progressão para o câncer, principalmente o câncer de colo de útero. A melhor forma de prevenção da contaminação pelo HPV é através da utilização das vacinas. Objetivo: Avaliar quais são os tipos de HPV mais prevalentes na cidade de Florianópolis, Brasil, e se a maioria dos tipos diagnosticados estão contidos nas vacinas contra o HPV atualmente disponíveis no mercado e no setor público de saúde. Métodos: Foram avaliados 14.727 exames para diagnóstico da infecção genital pelo HPV em mulheres de Florianópolis, de acordo com a idade das mulheres. A detecção do HPV foi realizada através dos exames de biologia molecular pelas técnicas de captura híbrida (para diagnóstico do grupo de HPV, alto ou baixo risco oncogênico) e PCR (genotipagem viral). Resultados: Foram avaliados exames para diagnóstico da infecção de mulheres entre um e 102 anos de idade. A maior positividade dos exames foi observada em mulheres dos 20­25 anos (51% dos exames). A faixa etária de maior prevalência foi dos 31­35 anos (23,5%), e a menor, após os 70 anos (0,6%). O HPV de alto risco oncogênico foi detectado em 94,1% dos casos positivos e foi o mais frequente em todas as faixas etárias. A infecção mista (HPV de alto e baixo risco) foi mais prevalente na faixa etária dos 66­70 anos (25,6%). Os genótipos mais frequentes foram os HPV de alto risco oncogênico não 16/18 (77% dos casos positivos). O HPV 16 foi encontrado em 17,1% dos casos positivos, e o HPV 18 em 6,5%. Conclusão: Os tipos de HPV mais prevalentes em Florianópolis nos últimos 6 anos são os HPV de alto risco oncogênico não 16/18, tipos virais não cobertos pela atual vacina contra o HPV disponível no setor público de saúde do Brasil.Palavras-chave: HPV. Tipos de HPV. Câncer de colo de útero. Cobertura vacinal.

Zika virus RNA detection in blood donors in São Paulo, Brazil

ABSTRACT Introduction: The Zika Virus (ZIKV) is a single-stranded RNA genome virus, belonging to the family Flaviviridae, genus Flavivirus. Outbreaks around the world have demonstrated that the presence of asymptomatic viremic blood donors provides an increase in the risk of transfusion transmission (TT) and nucleic acid test (NAT) screening has been proposed to ensure the blood safety. This study implemented an "in-house" method to detect ZIKV RNA in blood sample donations. Methods: Primary plasma tubes are submitted to nucleic acid extraction on an automated platform. After extraction, the NAT set-up is performed in the robotic pipettor, in which an amplification mixture containing primers and probes for ZIKV and Polio vaccine virus (PV) are added in duplex as an internal control. The real-time polymerase chain reaction is then performed in a thermocycler, using the protocol established by the supplier. Results: From May 2016 to May 2018, 3,369 samples were collected from 3,221 blood donors (confidence coefficient 95%), of which 31 were considered false positive (0.92%), as they did not confirm initial reactivity when repeated in duplicates and 14 (0.42%) had their results invalid due to repeat failure in the internal control, 4 (0.12%), due to insufficient sample volume and 2 (0.05%), due to automatic pipettor failures. No Zika RNA reactive sample was identified. Conclusion: The test showed feasible to be incorporated into the blood screening routine. Our data do not indicate the need to screen for ZIKV RNA in São Paulo during the evaluated period. However, a generic NAT system covering a group of flaviviruses which are circulating in the region, such as DENV and YFV, among others, could be a useful tool.

Genital infection by Human Papillomavirus (HPV) in women from Santa Catarina/Brazil / Infecção genital pelo Papilomavírus Humano (HPV) em mulheres de Santa Catarina/Brasil

Human Papillomavirus (HPV) infection is the most common sexually transmitted infection in women. About 80% of sexually active women will have contact with this virus at some point in their lives. Most infections will be transient, but when the infection becomes persistent and associated with a high oncogenic risk of Human Papillomavirus, there may be progression to cancer, especially cervical cancer. The best way to prevent Human Papillomavirus infection is through the use of vaccines, which have been available to the public in Brazil since 2014. Objective: This study aimed to assess the most prevalent types of Human Papillomavirus in the state of Santa Catarina, Brazil, and its mesoregions and if the majority of diagnosed types are contained in the Human Papillomavirus vaccines currently available on the market. Methods: A total of 20,000 Human Papillomavirus tests were evaluated for the diagnosis of genital Human Papillomavirus infection in women from the state of Santa Catarina, Brazil. The prevalence of infection was evaluated according to age and the city of origin of the exams. Human Papillomavirus detection was performed using molecular biology tests, such as hybrid capture (for diagnosis of the Human Papillomavirus group, high or low oncogenic risk) and polymerase chain reaction (viral genotyping) techniques. Results: The diagnosis of Human Papillomavirus infection was performed on women between 1 and 102 years of age. The age with the highest Human Papillomavirus positivity, as expected, was 20­25 years (45.6%) and the lowest after 70 years (7.1%). The highest Human Papillomavirus positivity of the exams was observed in the Serrana region of Santa Catarina state (58.9% of the exams). A high-oncogenic-risk Human Papillomavirus was detected in 93% of positive samples and was the most frequent in all age groups. Mixed infection (high- and low-risk Human Papillomavirus) was more prevalent in the 66­70 age group (29.3%) and in the Southern Region of Santa Catarina (26.4%). The most frequent genotypes in the state of Santa Catarina were non-16/18 high oncogenic risk Human Papillomavirus (76.9% of positive cases). Human Papillomavirus 16 was found in 17.1% of positive cases and Human Papillomavirus 18 in 6.6%. Conclusion: The most prevalent types of Human Papillomavirus in the state of Santa Catarina in the past 6 years are the non-16/18 high oncogenic risk Human Papillomavirus types, which are viral types not covered by the current Human Papillomavirus vaccines available in Brazil.

A infecção por Papilomavírus Humano é a infecção sexualmente transmissível mais frequente na mulher. Cerca de 80% das mulheres sexualmente ativas entrarão em contato com esse vírus em algum momento. A maioria das infecções será transitória, mas quando ela é persistente, associada aos Papilomavírus Humano de alto risco oncogênico, poderá progredir para câncer, principalmente de colo de útero. A melhor forma de se prevenir da contaminação pelo vírus é por meio de vacina, disponível no sistema público do Brasil desde 2014. Objetivo: Avaliar os tipos de Papilomavírus Humano mais prevalentes no estado de Santa Catarina e suas mesorregiões, e se a maioria dos tipos diagnosticados estão contidos nas vacinas contra o Papilomavírus Humano atualmente disponíveis no mercado. Métodos: Foram avaliados 20 mil exames para diagnóstico da infecção genital pelo Papilomavírus Humano em mulheres de todo o estado. A prevalência da infecção foi comparada de acordo com a idade e a procedência dos exames. A detecção do Papilomavírus Humano deu-se pelos exames de biologia molecular pelas técnicas de captura híbrida (para diagnóstico do grupo de Papilomavírus Humano, alto ou baixo riscos oncogênicos) e de PCR (genotipagem viral). Resultados: Foram avaliados exames para diagnóstico da infecção de mulheres entre um e 102 anos de idade. A faixa etária de maior positividade, como era de ser esperado, foi dos 20 aos 25 anos (45.6%) e a menor depois dos 70 anos (7.1%). A maior positividade dos exames foi observada na região Serrana do estado (58.9% dos exames). O Papilomavírus Humano de alto risco oncogênico foi detectado em 93% dos casos positivos e foi o mais frequente em todas as faixas etárias. A infecção mista (Papilomavírus Humano de alto e baixo riscos) foi mais prevalente na faixa etária dos 66 aos 70 anos (29.3%) e na região Sul Catarinense (26.4%). Os genótipos mais frequentes no estado foram os Papilomavírus Humano de alto risco oncogênico não 16/18 (76.9% dos casos positivos). O Papilomavírus Humano 16 foi encontrado em 17.1% dos casos positivos e o Papilomavírus Humano 18 em 6.6%. Conclusão:Os tipos de Papilomavírus Humano mais prevalentes no estado de Santa Catarina, nos últimos seis anos, são os Papilomavírus Humano de alto risco oncogênico não 16/18, tipos virais não cobertos pelas atuais vacinas contra o Papilomavírus Humano disponíveis no Brasil.

Human Papillomavirus (HPV) seroprevalence, cervical HPV prevalence, genotype distribution and cytological lesions in solid organ transplant recipients and immunocompetent women in Sao Paulo, Brazil.

INTRODUCTION: Solid organ transplant (SOT) recipients are at increased risk of Human Papillomavirus (HPV) persistent infection and disease. This study aimed to evaluate HPV seroprevalence, cervical HPV prevalence, genotype distribution, and frequency of HPV-related cervical lesions in SOT recipients in comparison to immunocompetent women. METHODS: Cross-sectional study including SOT and immunocompetent women aged 18 to 45 years who denied previous HPV-related lesions. Cervical samples were screened for HPV-DNA by a polymerase chain reaction (PCR)-based DNA microarray system (PapilloCheck®) and squamous intraepithelial lesions (SIL) by liquid-based cytology. A multiplexed pseudovirion-based serology assay (PsV-Luminex) was used to measure HPV serum antibodies. RESULTS: 125 SOT and 132 immunocompetent women were enrolled. Cervical samples were collected from 113 SOT and 127 immunocompetent women who had initiated sexual activity. HPV-DNA prevalence was higher in SOT than in immunocompetent women (29.6% vs. 20.2%, p = 0.112), but this difference was not statistically significant. High-risk (HR)-HPV was significantly more frequent in SOT than in immunocompetent women (19.4% vs. 7.9%, p = 0.014). Simultaneous infection with ≥2 HR-HPV types was found in 3.1% of SOT and 0.9% of immunocompetent women. HPV seropositivity for at least one HPV type was high in both groups: 63.8% of 105 SOT and 69.7% of 119 immunocompetent women (p = 0.524). Low-grade (LSIL) and high-grade SIL (HSIL) were significantly more frequent in SOT (9.7% and 5.3%, respectively) than in immunocompetent women (1.6% and 0.8%, respectively) (p = 0.001). CONCLUSIONS: These results may reflect the increased risk of HPV persistent infection and disease progression in SOT women due to chronic immunosuppression.

Performance evaluation of the fully automated molecular system Alinity m in a high-throughput central laboratory.

BACKGROUND: New partially or fully automated molecular diagnostic testing platforms are being developed to address the growing demand for fast, accurate, and cost-effective testing. OBJECTIVES: To evaluate the analytical and clinical performance of the Alinity m system compared to the Abbott RealTime m2000 assay system in a large central molecular laboratory. STUDY DESIGN: Alinity m HIV-1, HCV, and HBV assay precision, reproducibility, and sensitivity were assessed using commercial customized dilution panels. Clinical performance of the Alinity m and m2000 assay systems was compared using standard lab protocols and residual, de-identified patient specimens. A workflow analysis of 1,068 samples compared turnaround times (TATs) on five m2000 systems and one Alinity m system running Alinity m HIV-1, HCV, HBV, HR HPV, and STI assays. RESULTS: The Alinity m assay system demonstrated high detectability and precision at clinical decision points and excellent correlation with Abbott RealTime assay results. Processing TAT for 100 % of results was 117 min on Alinity m. Sample onboard TAT, from sample loading to 95 % of results, was 5:15 h for Alinity m and 7:30 h for m2000. 100 % of STAT samples were processed within 4 h on Alinity m. Total TAT for 100 % of results from all five assays was 80 h for m2000 versus 9 h for Alinity m. CONCLUSIONS: The Alinity m system produces assay results comparable to those of the Abbott RealTime m2000 system, but with significantly faster turnaround times due to continuous loading and the ability to run multiple assays simultaneously on the same sample.

High prevalence of anal high-risk HPV infection among transwomen: estimates from a Brazilian RDS study.

INTRODUCTION: As the leading sexually transmitted infection worldwide, human papillomavirus (HPV) may disproportionately affect transwomen. We aimed to estimate anal HPV prevalence, especially focusing on high-risk (hr)-HPV types and evaluate their associated factors among transwomen living in Rio de Janeiro, Brazil. METHODS: Transwomen enrolled in a respondent-driven sampling (RDS)-based survey conducted between August 2015 and January 2016 self-collected anal samples, which were promptly stored at minus 80°C. After DNA extraction, HPV detection and genotyping were performed using the PapilloCheck test. We estimated HPV prevalences and evaluated the correlates of anal hr-HPV infection using a regression logistic model. RESULTS: Out of 345 transwomen, 272 (78.8%) were included in this analysis (122 [44.9%] HIV-positive). No participant had ever received HPV vaccination. Among participants enrolled, 212 (77.9%) were positive for any anal HPV type and 165 (60.7%) for hr-HPV. Most common hr-HPV were as follows: HPV16 (17.6%), HPV68 (14.7%), HPV39 (14.3%), HPV56 (12.5%), HPV51 (11.4%) and HPV52 (11.0%). HIV-positive transwomen had three times the odds of having an hr-HPV compared to HIV-negative transwomen. Participants who had a current rectal Neisseria gonorrhoeae infection had 3.7 times the odds of being coinfected with hr-HPV. Among HIV-positive transwomen, neither antiretroviral therapy use, undetectable viral load, current and nadir CD4 counts were associated with anal hr-HPV infection. CONCLUSIONS: Brazilian transwomen in our study exhibit some of the highest population-specific rates of HPV and hr-HPV. There is an urgent need to elucidate the burden of HPV infection, prevalence of HPV-related diseases and access to and uptake of HPV vaccination among transwomen, especially from low- and middle-income settings.

Presence of HPV with overexpression of p16INK4a protein and EBV infection in penile cancer-A series of cases from Brazil Amazon.

BACKGROUND: In Brazil, penile cancer (PC) is not uncommon. The highest incidence of PC is in the North and Northeast of the country. In addition to phimosis, the Human Papillomavirus (HPV) and Epstein-Baar Virus (EBV) infections are also related as risk factors for PC. The overexpression of p16INK4a is a surrogate sensitive marker of HPV infection in PC. OBJECTIVES: To correlate p16INK4a overexpression and HPV infection status with EBV infection in a series of PC patients from the Amazon region. METHODS: Tumor tissues from 47 PC cases were analyzed for the presence of HPV and EBV DNA by PCR. All PC patients were diagnosed between 2013 and 2018 at a public reference cancer center hospital in Manaus, Amazonas-Brazil. HPV was genotyped using E7 HPV16/HPV18 type-specific real-time PCR and the PapilloCheck® HPV-Screening assay. p16INK4a expression was evaluated by immunohistochemistry using the automated Ventana® BenchMark Ultra. RESULTS: The mean age of patients at the time of diagnosis was 57.4 years ±SD 17.8 ranging from 20 to 90 years old. Most of the patients (64%) came from rural areas of the Amazonas State. Thirty patients had phimosis (64%). Among the patients with phimosis, 43% (13/30) underwent circumcision, three during childhood and 10 in adulthood. 60% of the patients were smokers or ex-smokers. HPV infection was observed in 45% (21/47) of cases. HPV16 was detected in 13 patients (61%). Other HPV types detected were HPV 6, 11, 42, 51, 53, 68 and 44/55. EBV infection was observed in 30% (14/47) of the patients with PC. Co-infection with HPV and EBV was observed in 28% (6/21) cases. p16INK4a was only investigated in 26 samples. The p16INK4a overexpression was observed exclusively in HPV 16 positive cases and four HPV negative cases. In the survival analysis, the follow-up time was 35.4 months/patient. The mortality rate during the follow up time was 38%. CONCLUSIONS: p16INK4a positivity presented a high correlation to HPV 16 DNA detection, reinforcing its use as a surrogate marker for HPV-driven cancers. Infection with EBV was quite frequent and its role in epithelial penile oncogenesis needs to be demonstrated.

Sequential measurement of Trypanosoma cruzi parasitic load in endomyocardial biopsies for early detection and follow-up of Chagas disease reactivation after heart transplantation.

BACKGROUND: Reactivation of Chagas disease after heart transplantation is characterized by proliferation and dissemination of Trypanosoma cruzi parasites to several organs. Reactivation affecting the allograft can simulate acute cellular rejection, from which it should be distinguished through the analysis of endomyocardial biopsies (EMB). METHODS: We evaluated retrospectively 100 EMB collected in the first year of follow-up from 13 heart-transplanted, chagasic patients who presented reactivation and were successfully treated. Additionally, 37 EMB from 8 patients who did not present reactivation constituted the control group. We reviewed histopathology and performed a real-time PCR-based assay in order to evaluate the T cruzi parasitic load of each EMB. RESULTS: The parasitic load of the EMB at the time of reactivation ranged from 22.80 to 190 000/106 cells (median: 1555). In 6 patients, none of the EMB obtained prior to reactivation amplified T cruzi DNA. On the other hand, 10 EMB from 7 patients, obtained 9-105 days before reactivation (median: 26 days), showed parasitic load ranging from 8.25 to 625/106 cells (median: 167.55). In all patients, the parasitic load increased at the time of reactivation, usually sharply. After initiation of treatment, all patients showed negative PCR or a dramatic reduction of the parasitic load in the following EMB. None of the EMB from the control group amplified T cruzi DNA. CONCLUSIONS: Sequential measurement of T cruzi parasitic load in EMB is useful for monitoring Chagas disease reactivation after heart transplantation. Its increase suggests imminent reactivation and its decrease after treatment indicates favorable evolution for cure of the episode of reactivation.

High-Risk HPV Testing in Primary Screening for Cervical Cancer in the Public Health System, São Paulo, Brazil.

Every year there are approximately 16,000 new cases of cervical cancer in Brazil. Novel screening technologies may reduce this number by expanding the population coverage but also by improving the detection rate of precursor lesions. We aimed to evaluate human papillomaviruses (HPV)-DNA testing in the context of routine cervical cancer screening in the public health system of the city of São Paulo, Brazil. Women participating in the primary screening program were invited to enroll. Liquid-based cytology samples were collected and cytology and Hr-HPV DNA testing were performed in parallel. Cytologists were blind to HPV results. Women older than 24 years with a positive high-risk HPV test and/or cytology class ≥ ASC-US were referred to colposcopy. From December 2014 to December 2016, 16,102 women joined the study. High-risk human papillomavirus (HR HPV) DNA prevalence was 14.9%, whereas cytologic abnormalities were found in 7.2% of the women. Per protocol, 1,592 Hr-HPV+ women, in addition to 72 patients with cytologic classification > low-grade squamous intraepithelial lesion (LSIL) were referred. A total of 80 cervical intraepithelial neoplasia (CIN2+) cases were diagnosed, 79 were Hr-HPV DNA+ and 18 had normal cytology. Hr-HPV DNA detected a significant number of patients with premalignant lesions missed by cytology and all 16 CIN3+ cases were Hr-HPV DNA+ HPV genotyping may be useful in the management of Hr-HPV+ women, reducing the burden of colposcopic referral for those harboring genotypes with a weaker association to CIN3+ Use of HPV-DNA testing was shown to be feasible and advantageous over current cytologic screening in the public health system.

Loss of Raf kinase inhibitor protein expression is associated with human papillomavirus 16 infection in anal tumors.

There has been an increase in the incidence of anal cancer in the past two decades, with squamous cell carcinoma (SCC) being the most frequent histological type identified. Among the risk factors, high-risk human papillomavirus (HPV) infection is the most pervasive. Raf kinase inhibitor protein (RKIP) is expressed in a number of normal human tissues and previous studies have demonstrated the prognostic value of the loss of RKIP expression in several gastrointestinal tumors. Therefore, the present study aimed to evaluate the clinical implications of RKIP expression in a series of neoplastic lesions of the anal canal. The resected tumors of 48 patients [8 high-grade intraepithelial lesions (HSILs), 14 adenocarcinomas and 26 squamous cell carcinomas (SCCs)] were immunohistochemically evaluated for RKIP expression, and the results were correlated with clinicopathological data. The results identified a decreased 5-year overall survival rate in patients with adenocarcinoma (40.8%) compared with patients with SCC (76.7%), and a decreased 5-year disease-free survival rate in patients at clinical stages III/IV (37.3 vs. 62.5 and 82.6% for clinical stages 0 and I/II, respectively). Low RKIP expression was revealed in 62.5% of HSILs, 88.5% of SCCs and 100.0% of the adenocarcinomas. High RKIP expression was associated with patient ethnicity (37.5% in non-Caucasians vs. 7.5% in Caucasians) and patient age (33.3% in younger patients vs. 0.0% in older patients). Finally, high RKIP expression was correlated with HPV16 infection status (40% in HPV- vs. 5.3% in HPV+ patients). A correlation was identified between high RKIP expression and lesions with a generally improved prognosis, such as those diagnosed in younger patients, in situ lesions and lesions of lower clinical grades; there was also a negative correlation between high RKIP expression and HPV16 positivity in patients.

Self-sampling coupled to the detection of HPV 16 and 18 E6 protein: A promising option for detection of cervical malignancies in remote areas.

OBJECTIVE: To evaluate both the performance and acceptability of a method coupling self-sampling with detection of cervical malignancy via elevated HPV 16 and 18 E6 oncoproteins (OncoE6™ Cervical Test) in remote areas in Brazil. METHODS: Women living in rural villages in proximity to Coari city, Amazonas, Brazil were invited to participate in a cervical cancer screening study. 412 subjects were enrolled; there were no refusals. In addition to E6 protein detection, DNA was extracted from the brushes and evaluated for HPV genotypes by PCR (PGMY09/11), followed by typing by the Papillocheck™ if positive. Subjects who were found to be positive for OncoE6 or HPV-DNA were referred for colposcopy. RESULTS: For 110 subjects (27%) this was the first cervical cancer exam. Overall the HPV-DNA prevalence was 19.1% (n = 79); 1.4% (n = 6) were positive by the OncoE6 Test. Fifty-six women attended the invitation for colposcopy where nine had an abnormal cervix and were subsequently biopsied. Histopathological analysis revealed 2 CIN3, 2 carcinomas and 5 CIN1. OncoE6 called two out of the three HPV 16 or 18 associated CIN3+ lesions. CONCLUSIONS: The findings suggest that self-administered sample collection in combination with OncoE6 Test is feasible in this population. This could enable expanded screening coverage while ensuring a high specificity which is imperative given the remote geographic location, since women bearing abnormal test results would necessitate travel and logistical burden to access colposcopy and treatment.

Influence of Prior Knowledge of Human Papillomavirus Status on the Performance of Cytology Screening.

OBJECTIVES: This study aimed to evaluate the influence of prior knowledge of human papillomavirus (HPV) status in cervical cytopathology readings. METHODS: Participants comprised 2,376 women who underwent parallel cytology and HPV-DNA testing. Smears were read twice by the same team, first with previous knowledge of HPV-DNA status. RESULTS: Overall, 239 (10.2%) smears had their cytology classification altered by the HPV-informed review. Cytology readings with prior knowledge of the HPV status revealed 10.5% of abnormal smears (atypical squamous cells of undetermined significance or higher), while without prior knowledge, this rate dropped to 7.6%. When HPV status was informed, a significant increase in all categories of altered smears was observed. Cytology with prior knowledge of HPV status detected more cervical intraepithelial neoplasia grade 2 or higher (CIN 2+) compared with blinded: 86.7% vs 60.0%. CONCLUSIONS: Our data indicate that cytology interpreted with prior knowledge of the HPV status provides higher sensitivity for CIN 2+ lesions while marginally reducing the overall specificity compared with HPV status blinded cytology.

Genomic analysis of head and neck cancer cases from two high incidence regions.

We investigated how somatic changes in HNSCC interact with environmental and host risk factors and whether they influence the risk of HNSCC occurrence and outcome. 180-paired samples diagnosed as HNSCC in two high incidence regions of Europe and South America underwent targeted sequencing (14 genes) and evaluation of copy number alterations (SCNAs). TP53, PIK3CA, NOTCH1, TP63 and CDKN2A were the most frequently mutated genes. Cases were characterized by a low copy number burden with recurrent focal amplification in 11q13.3 and deletion in 15q22. Cases with low SCNAs showed an improved overall survival. We found significant correlations with decreased overall survival between focal amplified regions 4p16, 10q22 and 22q11, and losses in 12p12, 15q14 and 15q22. The mutational landscape in our cases showed an association to both environmental exposures and clinical characteristics. We confirmed that somatic copy number alterations are an important predictor of HNSCC overall survival.

MAIT cells are activated in acute Dengue virus infection and after in vitro Zika virus infection.

Dengue virus (DENV) and Zika virus (ZIKV) are members of the Flaviviridae and are predominantly transmitted via mosquito bites. Both viruses are responsible for a growing number of infections in tropical and subtropical regions. DENV infection can cause lethargy with severe morbidity and dengue shock syndrome leading to death in some cases. ZIKV is now linked with Guillain-Barré syndrome and fetal malformations including microcephaly and developmental disorders (congenital Zika syndrome). The protective and pathogenic roles played by the immune response in these infections is unknown. Mucosal-associated invariant T (MAIT) cells are a population of innate T cells with potent anti-bacterial activity. MAIT cells have also been postulated to play a role in the immune response to viral infections. In this study, we evaluated MAIT cell frequency, phenotype, and function in samples from subjects with acute and convalescent DENV infection. We found that in acute DENV infection, MAIT cells had elevated co-expression of the activation markers CD38 and HLA-DR and had a poor IFNγ response following bacterial stimulation. Furthermore, we found that MAIT cells can produce IFNγ in response to in vitro infection with ZIKV. This MAIT cell response was independent of MR1, but dependent on IL-12 and IL-18. Our results suggest that MAIT cells may play an important role in the immune response to Flavivirus infections.

Hybrid capture as a tool for cervical lesions screening in HIV-infected women: insights from a Brazilian cohort

ABSTRACT Introduction: Cervical cancer remains an important burden for HIV-infected women in the era of combination antiretroviral therapy. Recommendations for cervical screening in these women diverge and may include high-risk HPV (HRHPV) testing. We aimed to evaluate the clinical usefulness of a single HRHPV testing for cervical screening of HIV-infected women. Methods: 723 HIV-infected women from a Brazilian prospective cohort were included between 1996 and 2012. Inclusion criteria were: normal cervical cytology at baseline and having a HRHPV-test at baseline. We calculated incidence rates of any squamous intraepithelial lesion (SIL) and high grade SIL+ (HSIL+) and negative predictive values (NPV) within 12 and 36 months. Hazard Ratios were obtained using Cox proportional hazards regression models. Results: Incidence rate for both outcomes was low (9.9 cases per 100 PY [95% CI 8.8-11.0] for any SIL and 1.3 cases per 100 PY [95% IC 0.9-1.8] for HSIL+). Women with a HRHPV positive status at baseline had 1.7-fold (95% CI 1.3-2.2) and 3.2-fold (95% CI 1.5-7.1) increased risk of presenting any SIL and HSIL+, respectively, during follow-up. Negative-HRHPV test presented high NPV for both periods and outcomes (any SIL: 92.4% [95% CI 89.7-94.6] for 12 months and 80.9% [95% CI 77.2-84.3] for 36 months; and HSIL+: 99.8% [95% CI 98.9-100.0] for 12 months and 99.0 [95% CI 97.6-99.7] for 36 months). Conclusions: Incidence of any and high grade cytological abnormality was significantly higher among HIV-infected women with positive-HRHPV test. A single negative-HRHPV test helped reassure follow-up free of cytological abnormalities through three years of follow-up in HIV-infected women with negative cytology.