Simultaneous determination of HCV genotype and NS5B resistance associated substitutions using dried serum spots from São Paulo state, Brazil.

Hepatitis C virus (HCV) is responsible for more than 180 million infections worldwide, and about 80 % of infections are reported in Low and Middle-income countries (LMICs). Therapy is based on the administration of interferon (INF), ribavirin (RBV) or more recently Direct-Acting Antivirals (DAAs). However, amino acid substitutions associated with resistance (RAS) have been extensively described and can contribute to treatment failure, and diagnosis of RAS requires considerable infrastructure, not always locally available. Dried serum spots (DSS) sampling is an alternative specimen collection method, which embeds drops of serum onto filter paper to be transported by posting to a centralized laboratory. Here, we assessed feasibility of genotypic analysis of HCV from DSS in a cohort of 80 patients from São Paulo state Brazil. HCV RNA was detected on DSS specimens in 83 % of samples of HCV infected patients. HCV genotypes 1a, 1b, 2a, 2c and 3a were determined using the sequence of the palm domain of NS5B region, and RAS C316N/Y, Q309R and V321I were identified in HCV 1b samples. Concerning therapy outcome, 75 % of the patients who used INF +RBV as a previous protocol of treatment did not respond to DAAs, and 25 % were end-of-treatment responders. It suggests that therapy with INF plus RBV may contribute for non-response to a second therapeutic protocol with DAAs. One patient that presented RAS (V321I) was classified as non-responder, and combination of RAS C316N and Q309R does not necessarily imply in resistance to treatment in this cohort of patients. Data presented herein highlights the relevance of studying circulating variants for a better understanding of HCV variability and resistance to the therapy. Furthermore, the feasibility of carrying out genotyping and RAS phenotyping analysis by using DSS card for the potential of informing future treatment interventions could be relevant to overcome the limitations of processing samples in several location worldwide, especially in LMICs.

Electrochemical Biosensor for Sensitive Detection of Hepatitis B in Human Plasma.

In this work, we report the construction of a novel electrochemical device for molecular diagnosis of hepatitis B virus in the blood plasma of infected patients, using graphite electrodes functionalized with poly(4-aminophenol) and sensitized with a specific DNA probe. The recognition of genomic DNA was evaluated by electrochemical techniques (DPV and EIS) and scanning electron microscopy. The genosensor was efficient in detecting genomic DNA with a linear range from 1.176 to 4.825 µg mL-1 and detection limit of 35.69 ng mL-1 (4.63 IU ml-1 or 25.93 copies.ml-1), which is better than the 10.00 IU ml-1 limit of reference method, real-time PCR, used in point of care. EIS analysis shows that the genosensor resistance increased exponentially with the concentration of the genomic DNA target. This novel platform has advantages to its applicability in real samples, such as good sensitivity, selectivity, low sample volume, and fast assay time (36 min), thus interesting for application in the diagnosis of hepatitis B virus in blood plasma. Also, the ease of synthesis of the low-cost polymer by electrosynthesis directly on the electrode surface allows the translation of the platform to portable devices.

Hepatitis B in the Northwestern region of Sao Paulo State: genotypes and resistance mutations.

In Brazil, few studies on the molecular aspects of hepatitis B virus (HBV) infection have been conducted in the interior regions of Sao Paulo State. This study aimed to identify HBV genotypes and evaluate strains with resistance mutations for nucleoside analogues in the Administrative Region (AR) of the municipality of Sao Jose do Rio Preto. We performed nested PCRs of 127 samples from the Health Care Services of the AR to amplify, sequence and analyze fragments of the HBV DNA, in order to identify genotypes and resistance mutations. The HBV S/Pol regions of 126 samples were successfully amplified and sequenced. Five different genotypes were found, and the main ones were A, D and F; a greater number of samples contained the subgenotypes A1 (n = 51; 40.5%), D3 (n = 36; 28.6%), A2 (n = 14; 11.1%) and F2a (n = 9; 7.1%). Resistance mutations (rtM204V/I/S) associated or not with compensatory mutations (rtL180M, rtV173L) were identified in 13.9% (5/36) of patients undergoing viral treatment and 1.1% (1/90) of naïve patients. The diversity of genotypes/subgenotypes found is probably due to the intense migration occurring in the region. These data can complement epidemiological and clinical surveillance, and can be used for a more effective management of chronic HBV patients.

SARS-CoV-2 reinfection in a healthcare professional in inner Sao Paulo during the first wave of COVID-19 in Brazil.

Coronavirus Disease 2019 pandemic remains a threat to public health. We report 2 cases of Coronavirus Disease 2019 infection in the same healthcare professional in Brazil. Genomic analysis identified that primoinfection was caused by the endemic lineage B.1.1.33 while reinfection by the lineage B.1.1.44, a lineage with an additional V1176F mutation in S protein.

Detecção de arboviroses em gestantes na região noroeste do Estado de São Paulo / Detection of arboviroses in pregnant women in the northwest region of the state of São Paulo

As infecções ocasionadas pelos vírus da Dengue (DENV), Zika (ZIKV) e Chikungunya (CHIKV) em gestantes são de grande preocupação pelos possíveis danos causados pelos mesmos às mães e fetos. O ZIKV está relacionado à microcefalia e outras anomalias cerebrais graves em neonatos e a infecção por CHIKV em gestantes no período intraparto pode levar à transmissão vertical, com possibilidade de agravamento no quadro do neonato. E, apesar de ainda não haver relatos de ocorrência de malformações congênitas associadas à infecção por DENV em gestantes, as mesmas são consideradas um grupo de risco, pois apresentam maiores chances de evolução para formas graves ou óbito e aumento no risco de partos prematuros decorrente da infecção materna perinatal. Neste estudo, foi realizada uma análise retrospectiva dos resultados envolvendo os vírus DENV, ZIKV e CHIKV para determinar a taxa de positividade destas arboviroses em gestantes no município de São José do Rio Preto-SP, nos anos de 2018 e 2019. Para isso, foram coletados resultados de PCR em tempo real (RT-PCR) para DENV, ZIKV e CHIKV de amostras de soro e urina obtidas de 557 gestantes com histórico de febre, bem como 93 amostras de recém-nascidos (RN). Na análise dos resultados foi verificado que o sorotipo-2 de Dengue (DENV-2) foi detectado em 106/557 correspondendo a 19% das amostras, o sorotipo-1 (DENV-1) foi detectado em apenas uma amostra e o ZIKV foi detectado em duas amostras. CHIKV não foi detectado. Não foi detectado arbovírus nas amostras de RN testadas pela técnica de RT-PCR. Quanto à idade, 40% das gestantes pertenciam à faixa etária de 25 a 32 anos, seguidas pelas faixas de 33 a 40 anos e 17 a 24 anos com percentuais de 31 e 29%, respectivamente. No período, uma gestante que estava na 13ª semana de gestação foi a óbito por DENV-2. Os resultados obtidos evidenciam a importância do diagnóstico precoce das arboviroses neste grupo, viabilizando a assistência adequada às gestantes. Nesse sentido, o monitoramento da circulação simultânea de arboviroses responsáveis por causarem complicações em gestantes e infecções congênitas deve continuar em áreas endêmicas como a de São José do Rio Preto, visando um diagnóstico materno precoce e manejo adequado de gestantes testadas positivas verificando a presença de sinais de alerta e de dengue grave.

Infections caused by Dengue (DENV), Zika (ZIKV) and Chikungunya (CHIKV) viruses in pregnant women represent great concern because of the possible damage that can be caused by these viruses to both mothers and fetuses. ZIKV is related to microcephaly and other severe brain abnormalities in neonates, while CHIKV infection in pregnant women in the intrapartum period can lead to vertical transmission, with the possibility of worsening in the neonate. And although there are no reports of congenital malformations associated with DENV infection in pregnant women, they are also considered of risk group, since they have greater probability to progress to severe forms or even death. In addition, there is an increased risk of premature childbirth. In this study, a retrospective result analysis involving DENV, ZIKV and CHIKV viruses was carried out to determine the positivity rate of those arboviruses in pregnant women in the city of São José do Rio Preto-SP, in the years 2018 and 2019. For this purpose, real-time PCR results (RT-PCR) were collected for DENV, ZIKV and CHIKV from serum and urine samples obtained from 557 pregnant women with a history of fever, as well as samples from 93 newborns (NB). Dengue serotype-2 (DENV-2) was detected in 106/557, which corresponds to 19% of the samples; dengue serotype-1 (DENV-1) was detected in only one sample, and the ZIKV was detected in two samples. CHIKV was not detected. Arboviruses were not detected in the NB samples tested by the RT-PCR technique. In relation to age groups, 40% of pregnant women were between 25 to 32 years old, followed by the groups of 33 to 40 years old, and 17 to 24 years old, with 31% and 29%, respectively. In the period, a pregnant woman who was in the 13th week of pregnancy died due to DENV-2. The results obtained emphasize the importance of the early diagnosis of arboviruses in this group, thus enabling adequate assistance to pregnant women. In this sense, the monitoring of arboviruses circulation responsible for causing complications and congenital infections in pregnant women should continue in endemic areas such as São José do Rio Preto, aiming at an early maternal diagnosis and adequate management of the patients who tested positive, checking for the presence of any alert signs and severe dengue.

SARS-CoV-2 reinfection in a healthcare professional in inner Sao Paulo during the first wave of COVID-19 in Brazil

Coronavirus Disease 2019 pandemic remains a threat to public health. We report 2 cases of Coronavirus Disease 2019 infection in the same healthcare professional in Brazil. Genomic analysis identified that primoinfection was caused by the endemic lineage B.1.1.33 while reinfection by the lineage B.1.1.44, a lineage with an additional V1176F mutation in S protein.

Cumprimento do algoritmo para o diagnóstico do HIV em crianças de 1 a 18 meses nascidas de mães HIV positivas / Complying with the HIV test algorithm for diagnosingchildren aged from 1 to 18 months born to HIV–positivemothers

The evolution of the epidemic of HIV in Brazil has especially affected women, and the controlof mother-to-child transmission of HIV (MTCT) has been a new challenge to be faced. Thisstudy assessed the compliance with the algorithm recommended by the Ministry of Health(MH) for diagnosing the children born to HIV-positive mothers, followed-up in the RegionalCentral Laboratory of the Instituto Adolfo Lutz of São José do Rio Preto-X, and to evaluate theoccurrence of mother-to-child transmission. This cross-sectional descriptive study collecteddata on the CD4+ T-cell count and viral load System for Controlling Laboratory Examinations(SISCEL) and System for Hospital Management (SIGH) from January 2009 to December 2014.In this period, 265 children were registered, being 141 (53.2%) male and 124 (46.8%) female; andonly 217 (81.9%) complied with the algorithm recommended by the MH. The mean prevalenceof MTCT was 5.9%. Despite the efforts exerted by the MH, the HIV algorithm noncompliancestill occurs, and the vertical transmission rate is higher than 2%. These data reinforce the need tointensify the surveillance, to improve the information and the follow-up of pregnant women, andto rescue those responsible who do not strictly comply with the algorithm proposed by the MH.

A epidemia do HIV no Brasil tem afetado de maneira especial as mulheres e trouxe, comoum novo desafio a ser enfrentado, o controle da transmissão vertical (TV) do HIV. O objetivodeste estudo foi de avaliar o cumprimento ao algoritmo preconizado pelo Ministério da Saúde(MS) no diagnóstico de crianças nascidas de mães HIV positivas, acompanhadas no Centro deLaboratório Regional Instituto Adolfo Lutz de São José do Rio Preto-X, e avaliar a ocorrência deTV. Foram coletados dados do Sistema de Informação e Gestão Hospitalar (SIGH) e do Sistemade Controle de Exames Laboratoriais da Rede Nacional de Contagem de Linfócitos T CD4+ eCarga Viral (SISCEL) no período de janeiro de 2009 a dezembro de 2014. Foram cadastradas265 crianças, 141 (53,2%) eram do sexo masculino e 124 (46,8%) feminino. Destas, 217 (81,9%)completaram o algoritmo preconizado pelo MS. A média da prevalência de TV foi de 5,9%.Apesar dos esforços do MS, há descumprimento no algoritmo e a taxa de TV é maior que ameta de 2%. Os dados reforçam a necessidade de intensificar a vigilância, melhorar a informaçãoe acompanhamento de gestantes e resgate dos responsáveis que não cumprem o algoritmo do MS.

Complying with the HIV test algorithm for diagnosing children aged from 1 to 18 months born to HIV–positive mothers / Cumprimento do algoritmo para o diagnóstico do HIV em crianças de 1 a 18 meses nascidas de mães HIV positivas

The evolution of the epidemic of HIV in Brazil has especially affected women, and the control of mother-to-child transmission of HIV (MTCT) has been a new challenge to be faced. This study assessed the compliance with the algorithm recommended by the Ministry of Health (MH) for diagnosing the children born to HIV-positive mothers, followed-up in the Regional Central Laboratory of the Instituto Adolfo Lutz of São José do Rio Preto-X, and to evaluate the occurrence of mother-to-child transmission. This cross-sectional descriptive study collected data on the CD4+ T-cell count and viral load System for Controlling Laboratory Examinations (SISCEL) and System for Hospital Management (SIGH) from January 2009 to December 2014. In this period, 265 children were registered, being 141 (53.2%) male and 124 (46.8%) female; and only 217 (81.9%) complied with the algorithm recommended by the MH. The mean prevalence of MTCT was 5.9%. Despite the efforts exerted by the MH, the HIV algorithm noncompliance still occurs, and the vertical transmission rate is higher than 2%. These data reinforce the need to intensify the surveillance, to improve the information and the follow-up of pregnant women, and to rescue those responsible who do not strictly comply with the algorithm proposed by the MH.

A epidemia do HIV no Brasil tem afetado de maneira especial as mulheres e trouxe, como um novo desafio a ser enfrentado, o controle da transmissão vertical (TV) do HIV. O objetivo deste estudo foi de avaliar o cumprimento ao algoritmo preconizado pelo Ministério da Saúde(MS) no diagnóstico de crianças nascidas de mães HIV positivas, acompanhadas no Centro de Laboratório Regional Instituto Adolfo Lutz de São José do Rio Preto-X, e avaliar a ocorrência de TV. Foram coletados dados do Sistema de Informação e Gestão Hospitalar (SIGH) e do Sistema de Controle de Exames Laboratoriais da Rede Nacional de Contagem de Linfócitos T CD4+ e Carga Viral (SISCEL) no período de janeiro de 2009 a dezembro de 2014. Foram cadastradas265 crianças, 141 (53,2%) eram do sexo masculino e 124 (46,8%) feminino. Destas, 217 (81,9%) completaram o algoritmo preconizado pelo MS. A média da prevalência de TV foi de 5,9%. Apesar dos esforços do MS, há descumprimento no algoritmo e a taxa de TV é maior que a meta de 2%. Os dados reforçam a necessidade de intensificar a vigilância, melhorar a informação e acompanhamento de gestantes e resgate dos responsáveis que não cumprem o algoritmo do MS.

Identification of fungal diseases at necropsy.

The purpose of the Death Verification Service is to elucidate the causes of deaths that occur without medical assistance and of ill-defined deaths. In recent decades, the epidemiological reality of fungal infections has changed due to the rise in opportunistic infections chiefly in immunocompromised patients. A study of fungal diseases in autopsies performed in the Death Verification Service of the Medicine School in São José do Rio Preto between January 2000 and December 2009 was made. Sixty-seven cases of fungal disease, most involving men (70%), were found in 4824 autopsies. Cryptococcosis was the most prevalent (45%), followed by paracoccidioidomycosis, candidiasis, histoplasmosis, aspergillosis and mucormycosis. Associations between AIDS (n=14) and fungal diseases were identified for cryptococcosis (36%), candidiasis (28.5%) and histoplasmosis (28.5%). Pneumonia, AIDS and fungal diseases were evident in 26% of the cases, with the most prevalent etiologies being Cryptococcus neoformans (55.5%) and Histoplasma capsulatum (22%). Pneumonia alone occurred in 43% of cases, with cryptococcosis (53%) and paracoccidioidomycosis (33%) being the main infectious agents. Diabetes mellitus was associated with candidiasis in two cases and aspergillosis in one. One case of renal transplantation linked to paracoccidioidomycosis and one case of bone marrow aplasia with mucormycosis were reported. Despite the reduction in the number of autopsies over recent decades, these findings suggest that this procedure is useful to provide additional data on the etiology, underlying disease and specific risk factors, essential for quality control and to improve treatment protocols.

[Onychomycosis: clinical, epidemiological and mycological study in the municipality of São José do Rio Preto]. / Onicomicose: estudo clínico, epidemiológico e micológico no município de São José do Rio Preto.

This was a clinical-epidemiological and mycological study on 184 patients at the university hospital: 200 samples, 142 positive samples, 98 yeasts and 68 filamentous fungi. Candida parapsilosis (47%) and Trichophyton rubrum (38%) were prevalent. They were 100% sensitive to ketoconazole and 99% sensitive to amphotericin B. The highest prevalences were among women (80%), adults (62%) and toes (84%).

Onicomicose: estudo clínico, epidemiológico e micológico no município de São José do Rio Preto / Onychomycosis: clinical, epidemiological and mycological study in the municipality of São José do Rio Preto

Estudo clínico-epidemiológico e micológico em 184 pacientes de Hospital Escola: 200 amostras, 142 positivas, 98 leveduras e 68 fungos filamentosos. Candida parapsilosis (47 por cento) e Trichophyton rubrum (38 por cento) foram prevalentes. Ao cetoconazol, 100 por cento de sensibilidade e a anfotericina B, 99 por cento. Prevalência em mulheres (80 por cento), adultos (62 por cento) em pododáctilos (84 por cento).

This was a clinical-epidemiological and mycological study on 184 patients at the university hospital: 200 samples, 142 positive samples, 98 yeasts and 68 filamentous fungi. Candida parapsilosis (47 percent) and Trichophyton rubrum (38 percent) were prevalent. They were 100 percent sensitive to ketoconazole and 99 percent sensitive to amphotericin B. The highest prevalences were among women (80 percent), adults (62 percent) and toes (84 percent).

Genetic relatedness among clinical strains of Stenotrophomonas maltophilia in tertiary care hospital settings in São Paulo State, Brazil

Stenotrophomonas maltophilia is a Gram-negative bacillus, which is becoming widely recognized as an important nosocomial pathogen. The main objective of this study was to evaluate the genetic relatedness, by random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of 86 clinical isolates of S. maltophilia (colonization 22, infection 64) obtained from 79 hospitalized patients, from different geographic regions of São Paulo State. The genotypic analysis performed by RAPD and PFGE was used in 24 isolates for genetic identity confirmation. The results were congruent between the two methods but it was not possible to link genetic profiles with the studied variables, clinical state and geographic area, probably due to the great variability among the strains. The analyses by PFGE confirmed identity in 5 pairs of microorganisms and RAPD, in this study, showed to be a useful tool for investigation of diversity leading the identification of 85 genetic profiles. The genetic diversity shown may be due to re-infection by different strains or co-infection by multiple strains which suggests multiple entry sources of the bacterium in the hospital setting or of acquisition by patient. In this setting, colonization, infection and re-infection occur with unknown frequency, raising the need for the establishment of specific control measures.

Stenotrophomonas maltophilia é um bacilo Gram-negativo, conhecido como importante patógeno nosocomial. O principal objetivo desse estudo foi avaliar a relação genética, através da análise randômica do polimorfismo de DNA (RAPD) e eletroforese em gel de campo pulsado (PFGE), de 86 isolados clínicos de S. maltophilia (22 de colonização, 64 de infecção) obtidos de 79 pacientes hospitalizados em diferentes regiões geográficas do estado de São Paulo. A análise genotípica foi realizada através da técnica RAPD e o PFGE foi usado em 24 isolados para confirmar a identidade genética dos mesmos. Os resultados foram coerentes entre os dois métodos, mas não foi possível correlacionar um perfil genético com as variáveis estudadas, estado clínico e área geográfica, provavelmente pela ampla variabilidade entre as linhagens. A análise por PFGE confirmou a identidade genética em 5 pares de microrganismos e o RAPD, neste estudo, mostrou ser uma ferramenta útil para investigação da diversidade, possibilitando identificar 85 perfis genéticos. A diversidade genética observada através do RAPD pode ser devido à re-infecção por diferentes linhagens ou co-infecção por linhagens distintas, sugerindo múltiplas fontes de entrada da bactéria no hospital ou de aquisição pelo paciente. Nesse ambiente, a colonização, infecção e re-infecção ocorrem com freqüência, o que leva à necessidade do estabelecimento de medidas de controle específicas.