Panorama do controle de qualidade em laboratórios clínicos e a experiência do Instituto Adolfo Lutz / Overview of quality control in clinical laboratories and the Adolfo Lutz Institute experience

No cenário dos laboratórios clínicos é de suma importância controlar e melhorar continuamente a qualidade dos serviços para obtenção de resultados confiáveis. Para alcançar essa condição, é imprescindível acompanhar os avanços tecnológicos. Uma das causas fundamentais tem sido a introdução e o monitoramento de indicadores de qualidade, tais como as especificações de desempenho analítico, principalmente a precisão e a exatidão. São componentes-chave desse processo a utilização do controle de qualidade interno (CQI) e a participação em programas de avaliação externa da qualidade (AEQ). Este relato de experiência se propõe a apresentar algumas tendências passadas e presentes da utilização do controle de qualidade nos processos de monitoramento da fase analítica em laboratórios clínicos. Destina-se também a compartilhar a experiência do Instituto Adolfo Lutz (IAL) no desenvolvimento e na implementação de programas de controle de qualidade em ensaios sorológicos, contextualizando seu propósito e benefício dos resultados. A qualidade em laboratórios clínicos torna-se cada vez mais relevante à medida que as evidências científicas destacam o seu papel importante no processo de tomada de decisão clínica e no monitoramento de pacientes. No desempenho de suas atividades, o Centro de Imunologia do IAL tem contribuído para a melhoria da qualidade dos resultados oferecidos à população pelos laboratórios da sub-rede do estado de São Paulo.

In the clinical laboratories setting, it is extremely important to continuously control and improve the quality of services to obtain reliable results. To achieve this condition, it is essential to keep up with technological advances. One of the fundamental causes has been the introduction and monitoring of quality indicators such as the analytical performance specifications in particular accuracy and precision. The key components of this process are the use of internal quality control (IQC) and participation in external quality assessment programs (EQA). This experience report intends to present some past and present trends in the use of quality control for monitoring analytical phase in clinical laboratories. It is also intended to share the experience of the Adolfo Lutz Institute (IAL) in the development and implementation of quality control programs in serological assays, which contextualizes their purpose and benefit of the results. The quality in clinical laboratories has become increasingly relevant as scientific evidence highlights the important role it plays in the clinical decision-making and patient monitoring process. In carrying out its activities, the Center of Immunology at IAL has contributed for improving the quality of results offered to the population by laboratories of the sub-network of São Paulo State.

Ocular manifestations of monkeypox: a case report / Manifestações oculares da monkeypox: um relato de caso

ABSTRACT Monkeypox disease is a viral zoonosis with symptoms similar to those seen in the past in smallpox (variola), although clinically less severe. Following the eradication of smallpox in 1980 and the subsequent cessation of smallpox vaccination, monkeypox has emerged as the most important orthopoxvirus from a public health standpoint. Monkeypox virus occurs primarily in central and western Africa, often in tropical forests, and has increasingly manifested in urban areas. Animal hosts include various rodents and nonhuman primates. We report the case of a patient with monkeypox disease who developed ocular complaints (eye discomfort and conjunctivitis) and had detectable conjunctival lesions on biomicroscopy and fluorescein testing. Its ophthalmological manifestations are still poorly known.

RESUMO Varíola do Macaco é uma zoonose viral com sintomas semelhantes aos observados no passado em pacientes com Varíola, embora seja clinicamente menos grave. Com a erradicação da varíola em 1980 e a subsequente cessação da vacinação contra a varíola, a varíola dos macacos emergiu como o ortopoxvírus mais importante em saúde pública. O vírus monkeypox ocorre principalmente na África central e ocidental, muitas vezes nas proximidades de florestas tropicais, e tem se manifestado cada vez mais em áreas urbanas. Os hospedeiros animais incluem uma variedade de roedores e primatas não humanos. O presente estudo relata o caso de um paciente com Monkeypox que evoluiu com queixa oftalmológica de desconforto ocular e conjuntivite e, à biomicroscopia e teste da fluoresceína, detecção de lesões conjuntivais. Alterações oftalmológicas da doença são, ainda, pouco conhecidas.

Ocular manifestations of monkeypox: a case report.

Monkeypox disease is a viral zoonosis with symptoms similar to those seen in the past in smallpox (variola), although clinically less severe. Following the eradication of smallpox in 1980 and the subsequent cessation of smallpox vaccination, monkeypox has emerged as the most important orthopoxvirus from a public health standpoint. Monkeypox virus occurs primarily in central and western Africa, often in tropical forests, and has increasingly manifested in urban areas. Animal hosts include various rodents and nonhuman primates. We report the case of a patient with monkeypox disease who developed ocular complaints (eye discomfort and conjunctivitis) and had detectable conjunctival lesions on biomicroscopy and fluorescein testing. Its ophthalmological manifestations are still poorly known.

Immunogenicity After a Heterologous BNT262b2 Versus Homologous Booster in Kidney Transplant Recipients Receiving 2 Doses of CoronaVac Vaccine: A Prospective Cohort Study.

BACKGROUND: Comparative studies of third heterologous doses following the CoronaVac vaccine against coronavirus disease 2019 (COVID-19) in kidney transplant recipients are lacking. METHODS: This prospective, single-center cohort study included kidney transplant recipients without previous COVID-19. Patients received a third heterologous (BNT162b2 mRNA) or homologous dose at least 4 wk after 2 doses of the CoronaVac vaccine. Immunoglobulin G antibody response and seroprevalence for neutralizing anti-severe acute respiratory syndrome coronavirus 2 antibodies immediately before and 28 d after third doses were compared between the groups. RESULTS: There were 307 patients in the heterologous group and 777 in the homologous group. Patients in the heterologous group were older (54 versus 50 y; P < 0.0001), with a longer time since transplant (11 versus 6 y; P < 0.0001). Immediately before the third dose, immunoglobulin G seroprevalence (36% versus 34%; P = 0.597) and antibody titers (246 versus 268 AU/mL; P = 0.279) were similar. After booster, seroconversion was higher in the heterologous group (49% versus 32%; P < 0.0001), resulting in a higher seroprevalence (67% versus 55%; P = 0.0003); however, 42% of all patients remained seronegative. Antibody titers after booster in seropositive patients were higher in the heterologous group (7771 versus 599 AU/mL; P < 0.0001). These results persisted after adjusting for confounding variables. Lastly, a similar proportion of patients became seropositive for neutralizing antibodies (98% versus 94%; P = 0.098). CONCLUSIONS: In kidney transplant recipients fully vaccinated with CoronaVac, a third dose with an mRNA vaccine produced a higher seroconversion rate and antibody titers than a third homologous dose. However, both boosters achieved equivalent seroprevalence for neutralizing antibodies. The high proportion of still seronegative patients indicates the need for alternative strategies of protection.

Prediction of SARS-CoV-2-positivity from million-scale complete blood counts using machine learning.

Background: The Complete Blood Count (CBC) is a commonly used low-cost test that measures white blood cells, red blood cells, and platelets in a person's blood. It is a useful tool to support medical decisions, as intrinsic variations of each analyte bring relevant insights regarding potential diseases. In this study, we aimed at developing machine learning models for COVID-19 diagnosis through CBCs, unlocking the predictive power of non-linear relationships between multiple blood analytes. Methods: We collected 809,254 CBCs and 1,088,385 RT-PCR tests for SARS-Cov-2, of which 21% (234,466) were positive, from 900,220 unique individuals. To properly screen COVID-19, we also collected 120,807 CBCs of 16,940 individuals who tested positive for other respiratory viruses. We proposed an ensemble procedure that combines machine learning models for different respiratory infections and analyzed the results in both the first and second waves of COVID-19 cases in Brazil. Results: We obtain a high-performance AUROC of 90 + % for validations in both scenarios. We show that models built solely of SARS-Cov-2 data are biased, performing poorly in the presence of infections due to other RNA respiratory viruses. Conclusions: We demonstrate the potential of a novel machine learning approach for COVID-19 diagnosis based on a CBC and show that aggregating information about other respiratory diseases was essential to guarantee robustness in the results. Given its versatile nature, low cost, and speed, we believe that our tool can be particularly useful in a variety of scenarios-both during the pandemic and after.

COVID-19 containment management strategies in a nursing home.

OBJECTIVE: To describe the first COVID-19 pandemic at Casa Ondina Lobo, a philanthropic nursing home in São Paulo city, and the containment measures against the pandemic that proved to be effective. METHODS: Several preventive measures were taken before and during the pandemic, with emphasis on universal testing by reverse transcription polymerase chain reaction for COVID-19. All residents and employees were tested twice in a D9 period. RESULTS: Among the 62 residents and 55 employees, in both testing, eight residents and nine employees tested positive for COVID-19. Of 22% of employees and 75% of residents evolved asymptomatic, emphasizing the importance of universal testing for the detection and isolation of these cases. A quarter of residents evolved without any symptoms, however, with COVID-19 signs, reinforcing the importance of monitoring vital signs. The second testing did not detect any new cases among residents, demonstrating the effectiveness of the containment measures, however, it found four new cases among employees. This emphasized their role in COVID-19 outbreaks in nursing homes. Only one patient died, a 12.5% lethality among those known to be infected and a 1.6% mortality in the total population of residents were seen. CONCLUSION: The adoption of appropriate containment measures enabled to contain an COVID-19 pandemic in studied nursing home. Universal reverse transcription polymerase chain reaction testing for COVID-19 has proved to be particularly important and effective.

Diagnóstico sorológico da infecção por HIV/aids no Brasil / Serological diagnosis of HIV/AIDS infection in Brazil

O teste imunoenzimático do tipo ELISA foi comercializado no Brasil logo após ser anunciado nos EUA e Europa, sendo imediatamente utilizado em vários laboratórios públicos e privados. Tecnologias mais recentes para a testagem de HIV, como a de quarta geração, que detecta anticorpos anti-HIV e o antígeno p24, e os testes baseados em ácido nucleico reduziram o intervalo entre a infecção e a detecção da doença. Esta breve revisão narrativa se propõe a apresentar os diferentes fluxogramas de testes para diagnóstico do HIV utilizados nacionalmente, desde os ensaios baseados apenas em anticorpos anti-HIV até os novos fluxogramas em que foram incluídos os testes moleculares. Até 1998, as autoridades sanitárias brasileiras ainda não haviam normatizado um algoritmo para a realização do diagnóstico da infecção pelo HIV. Desde então, diferentes algoritmos de testagem foram preconizados pelo Ministério da Saúde e seguidos pelos laboratórios. Considerando os diferentes cenários em que o diagnóstico do HIV tem sido realizado, há necessidade de avaliações frequentes dos ensaios, visto que a qualidade dos resultados pode ser influenciada por diversos fatores biológicos do hospedeiro e do agente.

COVID-19 containment management strategies in a nursing home

ABSTRACT Objective: To describe the first COVID-19 pandemic at Casa Ondina Lobo, a philanthropic nursing home in São Paulo city, and the containment measures against the pandemic that proved to be effective. Methods: Several preventive measures were taken before and during the pandemic, with emphasis on universal testing by reverse transcription polymerase chain reaction for COVID-19. All residents and employees were tested twice in a D9 period. Results: Among the 62 residents and 55 employees, in both testing, eight residents and nine employees tested positive for COVID-19. Of 22% of employees and 75% of residents evolved asymptomatic, emphasizing the importance of universal testing for the detection and isolation of these cases. A quarter of residents evolved without any symptoms, however, with COVID-19 signs, reinforcing the importance of monitoring vital signs. The second testing did not detect any new cases among residents, demonstrating the effectiveness of the containment measures, however, it found four new cases among employees. This emphasized their role in COVID-19 outbreaks in nursing homes. Only one patient died, a 12.5% lethality among those known to be infected and a 1.6% mortality in the total population of residents were seen. Conclusion: The adoption of appropriate containment measures enabled to contain an COVID-19 pandemic in studied nursing home. Universal reverse transcription polymerase chain reaction testing for COVID-19 has proved to be particularly important and effective.

Establishing a mass spectrometry-based system for rapid detection of SARS-CoV-2 in large clinical sample cohorts.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Here, we develop a high-throughput targeted proteomics assay to detect SARS-CoV-2 nucleoprotein peptides directly from nasopharyngeal and oropharyngeal swabs. A modified magnetic particle-based proteomics approach implemented on a robotic liquid handler enables fully automated preparation of 96 samples within 4 hours. A TFC-MS system allows multiplexed analysis of 4 samples within 10 min, enabling the processing of more than 500 samples per day. We validate this method qualitatively (Tier 3) and quantitatively (Tier 1) using 985 specimens previously analyzed by real-time RT-PCR, and detect up to 84% of the positive cases with up to 97% specificity. The presented strategy has high sample stability and should be considered as an option for SARS-CoV-2 testing in large populations.

Padronização de teste para quantificar anticorpos anti-poliovírus utilizando vírus vacinal / Standardization of a test for quantifying the antibodies against poliovirus using the vaccine virus

A poliomielite é uma doença endêmica no Afeganistão e no Paquistão, apesar dos esforços para ser erradicada, representando uma ameaça para outros países principalmente devido às viagens internacionais. A Organização Mundial da Saúde (OMS) tem como objetivo erradicar a poliomielite causada pelo poliovírus selvagem no mundo. O requisito essencial para a erradicação da poliomielite é a eliminação da cepa do poliovírus selvagem, que é empregada no teste padrão-ouro. Com o intuito de auxiliar na erradicação do poliovírus selvagem, o objetivo deste estudo foi modificar o teste padrão-ouro usando o poliovírus derivado da vacina oral atenuada. Foram testados 63 soros pelo ensaio de neutralização utilizando-se antígenos vacinais. A concordância do sorotipo 1 (k=0,74) foi considerada substancial, enquanto o sorotipo 2 (k=1,00) e sorotipo 3 (k= 0,95) foram consideradas quase perfeitas. A sensibilidade dos testes de soroneutralização utilizando os sorotipos 1, 2 e 3 foi de 94,83%, 100,00% e 100,00%, respectivamente. Em conclusão, o ensaio com antígenos vacinais pode ser usado como procedimento laboratorial seguro, especialmente em estudos de vigilância em larga escala. (AU)

Poliomyelitis is an endemic disease in Afghanistan and Pakistan in despite of the efforts to eradicate it, and it represents a threat to other countries mainly due to the international trips. The World Health Organization (WHO) aims at eradicating the polio disease worldwide. An essential requirement for eradicating the poliomyelitis is the elimination of the wild poliovirus strain, which is employed in the gold standard test. As a support for the eradication of wild poliovirus, the present study aimed at modifying the gold standard test by using poliovirus derived from the oral attenuated vaccine. Sixty-three sera samples were tested by neutralization assay using vaccine antigens. The degree of agreement of the serotype 1 (k=0.74) was considered substantial, while the serotype 2 (k=1.00) and 3 (k= 0.95) showed almost perfect agreement. The sensitivity of serotypes 1, 2 and 3 was 94.83%, 100.00% and 100.00%, respectively. In conclusion, the assay with the vaccine antigens can be used as a safe application, especially for large-scale surveillance studies. (AU)

Padronização de teste para quantificar anticorpos anti-poliovírus utilizando vírus vacinal / Standardization of a test for quantifying the antibodies against poliovirus using the vaccine virus

A poliomielite é uma doença endêmica no Afeganistão e no Paquistão, apesar dos esforços para ser erradicada, representando uma ameaça para outros países principalmente devido às viagens internacionais. A Organização Mundial da Saúde (OMS) tem como objetivo erradicar a poliomielite causada pelo poliovírus selvagem no mundo. O requisito essencial para a erradicação da poliomielite é a eliminação da cepa do poliovírus selvagem, que é empregada no teste padrão-ouro. Com o intuito de auxiliar na erradicação do poliovírus selvagem, o objetivo deste estudo foi modificar o teste padrão-ouro usando o poliovírus derivado da vacina oral atenuada. Foram testados 63 soros pelo ensaio de neutralização utilizando-se antígenos vacinais. A concordância do sorotipo 1 (k=0,74) foi considerada substancial, enquanto o sorotipo 2 (k=1,00) e sorotipo 3 (k= 0,95) foram consideradas quase perfeitas. A sensibilidade dos testes de soroneutralização utilizando os sorotipos 1, 2 e 3 foi de 94,83%, 100,00% e 100,00%, respectivamente. Em conclusão, o ensaio com antígenos vacinais pode ser usado como procedimento laboratorial seguro, especialmente em estudos de vigilância em larga escala.

Poliomyelitis is an endemic disease in Afghanistan and Pakistan in despite of the efforts to eradicate it, and it represents a threat to other countries mainly due to the international trips. The World Health Organization (WHO) aims at eradicating the polio disease worldwide. An essential requirement for eradicating the poliomyelitis is the elimination of the wild poliovirus strain, which is employed in the gold standard test. As a support for the eradication of wild poliovirus, the present study aimed at modifying the gold standard test by using poliovirus derived from the oral attenuated vaccine. Sixty-three sera samples were tested by neutralization assay using vaccine antigens. The degree of agreement of the serotype 1 (k=0.74) was considered substantial, while the serotype 2 (k=1.00) and 3 (k= 0.95) showed almost perfect agreement. The sensitivity of serotypes 1, 2 and 3 was 94.83%, 100.00% and 100.00%, respectively. In conclusion, the assay with the vaccine antigens can be used as a safe application, especially for large-scale surveillance studies.

Development and validation of a whole-cell ELISA for serologically diagnosing Helicobacter pylori infection in Brazilian children and adults: a diagnostic accuracy study.

BACKGROUND: Serological tests are practical, with low cost, but no noninvasive tests are available for diagnosing Helicobacter pylori (H. pylori) infection in Brazil. The aim here was to develop and validate enzyme-linked immunosorbent assay (ELISA) serological tests to detect anti-H. pylori immunoglobulin G antibodies, based on cultured strains from Brazilian patients. DESIGN AND SETTING: Cross-sectional, diagnostic accuracy study comparing a locally developed and validated ELISA and invasive tests among dyspeptic patients at two public hospitals in São Paulo, Brazil. METHODS: An ELISA test was prepared using whole-cell antigen from 56 strains. After genotypic characterization, it was standardized and optical density (OD) cutoffs were determined based on the serum antibody response of 100 H. pylori-negative samples, compared with 82 H. pylori-positive samples. Validation was performed on 174 symptomatic patients. RESULTS: The optimal OD cutoffs established (for monoclonal and polyclonal tests, respectively) were 0.167 and 0.164; overall ELISA sensitivity: 84.3%, 78.9%; specificity: 88.6%, 90.6%; positive predictive value (PPV): 75.4%, 80%; negative predictive value (NPV): 93.1%, 81.8%; accuracy: 87.3%, 86.2%; child and adolescent ELISA sensitivity: 74.2%, 81.8%; specificity: 90.8%, 86.7%; PPV: 66.6%, 84.3%; NPV: 95.8%, 84.8%; accuracy: 88.5%, 84.6; adult ELISA sensitivity: 84.4%, 75%; specificity: 86.9%, 93%; PPV: 81.8%, 78.3%; NPV: 88.9%, 91.8%; accuracy: 85.9%, 88.5%. CONCLUSION: The polyclonal serological test developed using local strains presented better diagnostic performance among children and adolescents, while the monoclonal test was better among adults. The results from both tests suggest that these in-house serological tests could be used to detect anti-H. pylori antibodies in our population, for screening purposes.

Rotavirus genotypes as etiological agents of diarrhoea in general populations of two geographic regions of Brazil.

Rotavirus is the main global cause of severe childhood diarrhoea among children. In 2006, Rotarix® (G1P[8]) was introduced into Brazil's National Immunization Program. The vaccine coverage rate was 84.4% in 2009. Evidences of increasing G2P[4] after 2006 opened up the discussion about the vaccine effectiveness to non-G1 strains. The aim of this study was to identify the circulating rotavirus genotypes in two Brazilian regions during 2009. A total of 223 positive samples by immunochromatography and latex agglutination assay from the Northeast (Bahia/Pernambuco States) and Southeast (São Paulo/Rio de Janeiro States) regions were included in the study. The samples were submitted to genotyping by nested-PCR according to VP7(G) and VP4(P) and 175 samples (78.5%) were able to be characterized. Considering the characterization of VP7, the G-types detected were G1, G2, and G4 in the Northeast, and G2, G3, G5, and G9 in the Southeast. Considering the characterization of VP4, the P-types detected were P[4], P[8], and P[6]/P[9] in the Northeast and the Southeast. The most frequent mixed types found were G2P[4]/G2P[NT](81.4%), G2P[6](5.2%), G1P[6](5.2%) in the Northeast, and G2P[4]/G2P[NT](78.8%), G2P[6](8.2%), G9P[8](4.7%) in the Southeast. Among immunized individuals whose age ranged from 0-4 years, the G2P[4]/G2P[NT] genotype was identified in 91,0% of cases, and among non-immunized individuals of the same age, the G2P[4]/G2P[NT] genotype was identified in 85.7% of the cases. In accordance with the high level of vaccine coverage, the data suggest that the circulation of G2P[4] in these regions had a considerable increase after the introduction of Rotarix®.

Detection of herpes simplex-1 and -2 and varicella zoster virus by quantitative real-time polymerase chain reaction in corneas from patients with bacterial keratitis.

OBJECTIVE:: Bacterial keratitis occurs worldwide, and despite recent developments, it remains a potentially blinding condition. This study assesses the presence of herpes simplex virus (HSV-1 and -2) and varicella zoster virus (VZV) by quantitative real-time polymerase chain reaction (qPCR) in corneal scrapings from patients with bacterial keratitis. METHODS:: A total of 65 patients with clinical diagnoses of infectious corneal ulcers prospectively underwent clinical eye examinations. Corneal scrapings were investigated by Gram staining, Giemsa staining, culture, and qPCR (the study group). Risk factors and epidemiological data were recorded. The control group comprising 25 eyes with typical herpes dendritic keratitis was also analyzed by qPCR. RESULTS:: From the study group (n=65), nine patients (13.8%) had negative smears, cultures, and qPCR findings. Fifty-six (86.2%) patients had positive cultures: 51 for bacteria, 4 for fungi, and 1 for amoebae. Of the patients who had positive bacterial cultures, qPCR identified 10 patients who were also positive for virus: one for VZV and nine for HSV-1. Of the 25 patients in the control group, 21 tested positive for HSV-1 by qPCR analysis. CONCLUSIONS:: Herpes may be present in patients with bacterial corneal ulcers, and qPCR may be useful in its detection.

High frequency of hepatitis E virus infection in swine from South Brazil and close similarity to human HEV isolates

Abstract Hepatitis E virus is responsible for acute and chronic liver infections worldwide. Swine hepatitis E virus has been isolated in Brazil, and a probable zoonotic transmission has been described, although data are still scarce. The aim of this study was to investigate the frequency of hepatitis E virus infection in pigs from a small-scale farm in the rural area of Paraná State, South Brazil. Fecal samples were collected from 170 pigs and screened for hepatitis E virus RNA using a duplex real-time RT-PCR targeting a highly conserved 70 nt long sequence within overlapping parts of ORF2 and ORF3 as well as a 113 nt sequence of ORF2. Positive samples with high viral loads were subjected to direct sequencing and phylogenetic analysis. hepatitis E virus RNA was detected in 34 (20.0%) of the 170 pigs following positive results in at least one set of screening real-time RT-PCR primers and probes. The swine hepatitis E virus strains clustered with the genotype hepatitis E virus-3b reference sequences in the phylogenetic analysis and showed close similarity to human hepatitis E virus isolates previously reported in Brazil.

Detection of herpes simplex-1 and -2 and varicella zoster virus by quantitative real-time polymerase chain reaction in corneas from patients with bacterial keratitis / Detecção de vírus herpes simplex tipo 1 e 2 e varicella zoster por reação em cadeia de polimerase em tempo real em córneas de pacientes com ceratites bacterianas

ABSTRACT Objective: Bacterial keratitis occurs worldwide, and despite recent developments, it remains a potentially blinding condition. This study assesses the presence of herpes simplex virus (HSV-1 and -2) and varicella zoster virus (VZV) by quantitative real-time polymerase chain reaction (qPCR) in corneal scrapings from patients with bacterial keratitis. Methods: A total of 65 patients with clinical diagnoses of infectious corneal ulcers prospectively underwent clinical eye examinations. Corneal scrapings were investigated by Gram staining, Giemsa staining, culture, and qPCR (the study group). Risk factors and epidemiological data were recorded. The control group comprising 25 eyes with typical herpes dendritic keratitis was also analyzed by qPCR. Results: From the study group (n=65), nine patients (13.8%) had negative smears, cultures, and qPCR findings. Fifty-six (86.2%) patients had positive cultures: 51 for bacteria, 4 for fungi, and 1 for amoebae. Of the patients who had positive bacterial cultures, qPCR identified 10 patients who were also positive for virus: one for VZV and nine for HSV-1. Of the 25 patients in the control group, 21 tested positive for HSV-1 by qPCR analysis. Conclusions: Herpes may be present in patients with bacterial corneal ulcers, and qPCR may be useful in its detection.

RESUMO Objetivo: Ceratites bacterianas ocorrem mundialmente e apesar dos novos desenvolvimentos permanece como uma condição que pode levar à cegueira. Avaliar a presença de herpes simples (-1 e -2) e vírus varicella zoster (VZV) por reação em cadeia quantitativa de polimerase em tempo real (qPCR) em raspados corneanos de pacientes com ceratite bacteriana. Métodos: Sessenta e cinco pacientes com ceratite infecciosa foram submetidos a raspados corneanos estudados para gram, Giemsa, cultura e qPCR (grupo de estudo). Foram avaliados fatores de risco e epidemiológicos. O grupo controle foi composto por 25 casos de úlcera dendrítica típica por herpes analisados por qPCR. Resultados: Do grupo de estudo (n=65), nove pacientes (13,8%) apresentaram cultura, qPCR e raspado negativos. Cinquenta e seis (86,2%) pacientes apresentaram cultura positiva, 51 para bacteria, 4 para fungo e 1 para ameba. A qPCR identificou 10 pacientes do grupo de cultura positiva para bactéria que também foram positivos para vírus, um VZV e 9 para HSV-1. Dos 25 pacientes que compunham o grupo controle, 21 apresentaram qPCR positivo para HSV-1. Conclusão: Herpes pode estar presente em pacientes com úlceras de córnea bacterianas e a qPCR pode ser útil na sua detecção.

High frequency of hepatitis E virus infection in swine from South Brazil and close similarity to human HEV isolates.

Hepatitis E virus is responsible for acute and chronic liver infections worldwide. Swine hepatitis E virus has been isolated in Brazil, and a probable zoonotic transmission has been described, although data are still scarce. The aim of this study was to investigate the frequency of hepatitis E virus infection in pigs from a small-scale farm in the rural area of Paraná State, South Brazil. Fecal samples were collected from 170 pigs and screened for hepatitis E virus RNA using a duplex real-time RT-PCR targeting a highly conserved 70nt long sequence within overlapping parts of ORF2 and ORF3 as well as a 113nt sequence of ORF2. Positive samples with high viral loads were subjected to direct sequencing and phylogenetic analysis. hepatitis E virus RNA was detected in 34 (20.0%) of the 170 pigs following positive results in at least one set of screening real-time RT-PCR primers and probes. The swine hepatitis E virus strains clustered with the genotype hepatitis E virus-3b reference sequences in the phylogenetic analysis and showed close similarity to human hepatitis E virus isolates previously reported in Brazil.

Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies.

Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2), which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV) protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

Simultaneous Quantification of the 8 Human Herpesviruses in Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Human herpesviruses may cause severe complications after allogeneic hematopoietic stem cell transplantation (HSCT). However, the impact of some of these infections on transplant outcomes is still unclear. A prospective survey on the incidence and clinical features of herpesviruses infections after HSCT has not yet been conducted in Brazilian patients, and the impact of these infections on HSCT outcome remains unclear. METHODS: We prospectively analyzed the incidence of infection of the eight human herpesviruses simultaneously in 1 045 peripheral blood samples from 98 allogeneic HSCT recipients. Samples were collected weekly starting at the time of transplant until day +100. All herpesviruses were screened and quantified in plasma by quantitative real-time polymerase chain reaction. Median follow up time was 24 months. RESULTS: The incidences of infection for each herpesvirus were as follows: cytomegalovirus (CMV), 44%; human herpesvirus [HHV] 6, 18%; HHV8, 6%; Epstein-Barr virus, 3%; herpes simplex virus 1, 3%; varicella zoster virus, 3%; HHV7, 2%; and herpes simplex virus 2, 1%. The CMV infection was significantly more frequent among adults and was associated with a higher risk of developing acute graft-versus-host disease. The HHV6 infection was significantly more frequent after umbilical cord blood transplant and was associated with an increased risk of platelet engraftment failure. There was no significant impact of these infections on the other transplant outcomes. CONCLUSIONS: Herpesviruses infections were uncommon after HSCT, except for CMV and HHV6, which, although relatively frequent, had no clinically relevant impact on the outcomes.

Serum lipidomic profiling as a useful tool for screening potential biomarkers of hepatitis B-related hepatocellular carcinoma by ultraperformance liquid chromatography-mass spectrometry.

BACKGROUND: Chronic hepatitis B (CHB) virus infection is a major cause of hepatocellular carcinoma (HCC), as late diagnosis is the main factor for the poor survival of patients. There is an urgent need for accurate biomarkers for early diagnosis of HCC. The aim of the study was to explore the serum lipidome profiles of hepatitis B-related HCC to identify potential diagnostic biomarkers. METHODS: An ultraperformance liquid chromatography mass spectrometry (UPLC-MS) lipidomic method was used to characterize serum profiles from HCC (n = 32), liver cirrhosis (LC) (n = 30), CHB (n = 25), and healthy subjects (n = 34). Patients were diagnosed by clinical laboratory and imaging evidence and all presented with CHB while healthy controls had normal liver function and no infectious diseases. RESULTS: The UPLC-MS-based serum lipidomic profile provided more accurate diagnosis for LC patients than conventional alpha-fetoprotein (AFP) detection. HCC patients were discriminated from LC with 78 % sensitivity and 64 % specificity. In comparison, AFP showed sensitivity and specificity of 38 % and 93 %, respectively. HCC was differentiated from CHB with 100 % sensitivity and specificity using the UPLC-MS approach. Identified lipids comprised glycerophosphocolines, glycerophosphoserines and glycerophosphoinositols. CONCLUSIONS: UPLC-MS lipid profiling proved to be an efficient and convenient tool for diagnosis and screening of HCC in a high-risk population.