In vitro susceptibility of eighteen clinical isolates of human monkeypox virus to tecovirimat

BACKGROUND In 2022, an outbreak of mpox that started in European countries spread worldwide through human-to-human transmission. Cases have been mostly mild, but severe clinical presentations have been reported. In these cases, tecovirimat has been the drug of choice to treat patients with aggravated disease. OBJECTIVES Here we investigated the tecovirimat susceptibility of 18 clinical isolates of monkeypox virus (MPXV) obtained from different regions of Brazil. METHODS Different concentrations of tecovirimat were added to cell monolayers infected with each MPXV isolate. After 72 hours, cells were fixed and stained for plaque visualization, counting, and measurement. The ortholog of F13L gene from each MPXV isolate was polymerase chain reaction (PCR)-amplified, sequenced, and the predicted protein sequences were analyzed. FINDINGS The eighteen MPXV isolates generated plaques of different sizes. Although all isolates were highly sensitive to the drug, two showed different response curves and IC50 values. However, the target protein of tecovirimat, F13 (VP37), was 100% conserved in all MPXV isolates and therefore does not explain the difference in sensitivity. MAIN CONCLUSIONS Our results support screening different MPXV isolates for tecovirimat susceptibility as an important tool to better use of the restricted number of tecovirimat doses available in low-income countries to treat patients with mpox.

COVID-19 diagnosis by RT-qPCR in alternative specimens

BACKGROUND The high demand for adequate material for the gold standard reverse transcription real-time polymerase chain reaction (RT-qPCR)-based diagnosis imposed by the Coronavirus disease 2019 (COVID-19) pandemic, combined with the inherent contamination risks for healthcare workers during nasopharyngeal swab (NP) sample collection and the discomfort it causes patients, brought the need to identify alternative specimens suitable for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVES The aim of this work was to compare saliva and gingival fluid swabs to NP swabs as specimens for RT-qPCR-based SARS-CoV-2 diagnosis. METHODS We compared gingival fluid swabs (n = 158) and saliva (n = 207) to the rayon-tipped NP swabs obtained from mild-symptomatic and asymptomatic subjects as specimens for RT-qPCR for SARS-CoV-2 detection. FINDINGS When compared to NP swabs, gingival fluid swabs had a concordance rate of 15.4% among positive samples, zero among inconclusive, and 100% among negative ones. For saliva samples, the concordance rate was 67.6% among positive samples, 42.9% among inconclusive, and 96.8% among negative ones. However, the concordance rate between saliva and NP swabs was higher (96.9%) within samples with lower cycle threshold (Ct) values (Ct > 10 ≤ 25). MAIN CONCLUSIONS Our data suggests that whereas gingival fluid swabs are not substitutes for NP swabs, saliva might be considered whenever NP swabs are not available or recommended.

Conventional and liquid-based cytology (Liqui-PREP™) anal findings in male HIV anoreceptive patients in a referral University hospital in Rio de Janeiro, Brazil / Achados de citologia convencional e de citologia em base líquida (Liqui-PREP®) anal em pacientes masculinos HIV soropositivos anorreceptivos, em um hospital universitário de referência do Rio de Janeiro, Brasil

Introduction: The anal lesions seem to have a natural history that closely resembles cervical lesions, with signs that precede the invasion. Cytological changes of anal epithelium induced by HPV can be detected through cytology, as it is considered an effective screening method. Objective: To identify the frequency of atypical epithelial conventional cytology results by comparing anal samples through Liqui-PREPTM technology in HIV-positive men. Methods: Cross-sectional descriptive and analytical study of 33 men who have sex with men (MSM), HIV-positive and anoreceptive attended at the Gaffrèe and Guinle University Hospital (HUGG), Rio de Janeiro, from June to July, 2016. Collection of anal samples for the conventional cytology and Liqui-PREPTM cytology was carried out. For significance of findings, Fisher exact test with 95% confidence interval was used and cytological Kappa index was employed for concordance between the two cytological methods. Results: The age ranged from 23 to 60 years (mean=39.06). The CD4 cell count was between 200 to 500/mm3 on 16 (48.5%) and 13 (39.4%), and 50% was diagnosed with HIV for more than 6 years. In conventional cytology one case was considered unsatisfactory (3%). Among the cases considered satisfactory, 9 (28.1%) were diagnosed with ASC-US; 4 (12.5%) LSIL; 2 (6.3%) ASC-H, and 2 (6.3%) HSIL. Through Liqui-PREPTM method, 7 cases were considered unsatisfactory (21.2%). Among the satisfactory cases, 7 showed ASC-US (26.9%); 4 (15.4%) ASC-H; 2 (7.7%) LSIL; and 2 (7.7%) HSIL. The difference of unsatisfactory cases between both methods, although higher for Liqui-PREPTM, was not statistically significant (p=0.054). The correlation was moderate (0503; p<0.006 [0.1765­0.8298]). Conclusion: The cytologic atypia is common among MSM HIV (+), and the anal conventional cytology and liquid by Liqui-PREPTM cytology are equivalent, although they are more unsatisfactory in the latter technique.

Introdução: As lesões anais parecem ter uma história natural, que se assemelha às de lesões de colo uterino, com sinais que precedem a invasão. As alterações citológicas do epitélio anal induzidas pelo HPV podem ser detectadas por citologia, um método de rastreio considerado efetivo. Objetivo: Identificar a frequência de atipias epiteliais nos resultados da citologia convencional comparando amostras anais pela tecnologia Liqui-PREP® em homens HIV positivos. Métodos: Estudo transversal, descritivo e analítico de 33 homens que fazem sexo com homens (HSH), HIV positivos e anorreceptivos atendidos no Hospital Universitário Gaffrèe e Guinle (HUGG), Rio de Janeiro, no período de junho a julho de 2016. Os pacientes foram submetidos à coleta de amostras anais para citologia convencional e citologia Liqui-PREP®. Para significância de achados, foi usado o teste exato de Fisher com intervalo de confiança de 95%, e para concordância entre os dois métodos citológicos, foi utilizado o índice de Kappa. Resultados: A idade variou de 23 a 60 anos (média=39,06). A contagem de células CD4 foi entre 200 e 500/mm3 para 16 (48,5%) e 13 (39,4%) dos casos analisados, e 50% tinham o diagnóstico de HIV há mais de seis anos. Na citologia convencional, um caso foi considerado insatisfatório (3%). Entre os casos considerados satisfatórios, 9 (28,1%) foram diagnosticados como células escamosas atípicas de significado indeterminado possivelmente não neoplásicas (ASC-US); 4 (12,5%) como lesão intraepitelial de baixo grau (LSIL); 2 (6,3%) como células escamosas atípicas não sendo possível excluir lesão intraepitelial de alto grau (ASC-H) e 2 (6,3%) como lesão intraepitelial de alto grau (HSIL). Pelo método Liqui-PREP®, 7 casos foram considerados insatisfatórios (21,2%). Entre os casos satisfatórios, 7 como ASC-US (26,9%); 4 (15,4%) como ASC-H; 2 (7,7%) como LSIL e 2 (7,7%) como HSIL. A diferença de insatisfatório entre os métodos, embora maior para Liqui-PREP®, não foi estatisticamente significativa (p=0,054). A concordância foi moderada (0,503; p<0,006 [0,1765­0,8298]). Conclusão: É frequente a atipia citológica entre HSH HIV (+), e as citologias anal convencional e em meio líquido pela técnica Liqui-PREPTM se equivalem, embora sejam mais insatisfatórias na técnica citológica Liqui-PREP®.

Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients

Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1) antibodies (Abs) represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Western blotting (WB) for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF) and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF) indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I) and gag (p24) proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.

Diagnóstico laboratorial da mielopatia associada ao HTLV-I: métodos para análise do líquido cefalorraquidiano / Laboratorial diagnosis of HTLV-I associated myelopathy: methods for the cerebrospinal fluid analysis

O vírus linfotrópico de células T humanas do tipo I (HTLV-I) pode causar uma doença neurológica inflamatória, crônica e incapacitante, que acomete a medula espinhal, denominada mielopatia associada ao HTLV-I/paraparesia espástica tropical (PET/MAH). A verificação de anticorpos da classe G (IgG) anti-HTLV-I no soro e no líquido cefalorraquidiano (LCR) representa importante parâmetro para o diagnóstico laboratorial da PET/MAH. OBJETIVO: Avaliação crítica dos métodos utilizados para verificação da presença e da produção intratecal de anticorpos totais e anti-HTLV-I no LCR para o diagnóstico de PET/MAH. MÉTODO: Realizou-se uma revisão sistemática de artigos da literatura médica, usando-se palavras-chave da língua inglesa como cerebrospinal fluid, intrathecal synthesis of antibodies, HTLV-I, HAM/TSP. As bases de dados utilizadas incluíram Pubmed, Literatura Latino-Americana e do Caribe em Ciências da Saúde (Lilacs), MEDlars onLINE (Medline) e Cochrane Library. RESULTADO: Foram selecionados 14 artigos: cinco relacionados com a presença do anticorpo IgG específico no LCR; nove sobre síntese intratecal de anticorpos totais (IgG ou IgG/IgA/IgM) e específicos anti-HTLV-I (IgG ou IgM). DISCUSSÃO: O estudo isolado da presença de anticorpo IgG anti-HTLV-I no LCR não discrimina a fração produzida no sistema nervoso central (SNC), possui baixa especificidade (40 por cento) para o diagnóstico de PET/MAH. A demonstração da síntese intratecal de anticorpos IgG anti-HTLV-I possui maior relevância por suas elevadas especificidade (89 por cento) e sensibilidade (83 por cento). Entre os métodos para a avaliação da síntese intratecal de anticorpo específico, destaca-se o índice de IgG anti-HTLV-I, segundo Reiber e Felgenhauer(18), o qual se baseia no teste do ensaio imunossorvente ligado à enzima (ELISA), com análise simultânea do LCR e do soro. Outros estudos utilizam pequenas amostragens e não demonstram sensibilidade e especificidade no teste do LCR...

The human T-cell lymphotropic virus type I (HTLV-I) may cause HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP), an incapacitating chronic inflammatory disease of the spinal cord. The detection of IgG anti-HTLV-I antibodies in the serum and cerebrospinal fluid (CSF) has been an important parameter for the laboratorial diagnosis of HAM/TSP. OBJECTIVE: critical evaluation of the methods applied to detect the presence and intrathecal production of total antibodies and anti-HTLV-I in the CSF for the diagnosis of HAM/TSP. METHODS: We performed a systematic review of medical articles by using the key words: "cerebrospinal fluid, intrathecal synthesis of antibodies, HTLV-I associated myelopathy, HTLV-I, HAM/TSP". The used databases included: PubMed, Lilacs, Medline and Cochrane Library. RESULTS: A total of 14 articles were selected: five studies were related to the presence of specific IgG antibody in the CSF and nine studied the intrathecal synthesis of total antibodies (IgG or IgG/IgA/IgM) and specific anti-HTLV-I (IgG or IgM). DISCUSSION: The isolated study of the presence of IgG antibody anti-HTLV-I in the CSF does not show the fraction produced in the central nervous system, which represents low specificity (40 percent) for the diagnosis of HAM/TSP. The demonstration of the intrathecal synthesis of IgG anti-HTLV-I antibodies is more relevant due to its high specificity (89 percent) and sensibility (83 percent). According to Reiber & Felgenhauer (1987), the index IgG anti-HTLV-I, which is based on ELISA test with simultaneous CSF and serum analysis, stands out from the other methods applied to evaluate the intrathecal synthesis of specific antibody. Other studies use small samples and do not demonstrate the sensibility and specificity of the test in the CSF. Only one study shows statistical analysis. CONCLUSION: The immunological diagnosis of the CSF in HAM/TSP requires the standardization of methods, which should be based...