Spectinomycin, gentamicin, and routine disc diffusion testing: An alternative for the treatment and monitoring of multidrug-resistant Neisseria gonorrhoeae?

INTRODUCTION: Neisseria gonorrhoeae is a major concern of public health due to its extraordinary capacity to develop and acquire resistance to different antimicrobials used to treat gonorrhoea. Limited treatment options and uncontrolled transmission have raised the need to assess the antimicrobial susceptibility profile of the isolates and to establish affordable alternatives for laboratory diagnosis. OBJECTIVES: This study aimed to (i) determine the susceptibility profile of 336 clinical isolates of N. gonorrhoeae to ceftriaxone, azithromycin, ciprofloxacin, spectinomycin and gentamicin by the gold standard agar dilution method; (ii) assess the agreement among agar dilution and disc diffusion results for ciprofloxacin, azithromycin, ceftriaxone, spectinomycin and gentamicin. RESULTS: All isolates were susceptible to ceftriaxone and spectinomycin. The levels of resistance to azithromycin and ciprofloxacin were 3.9% and 35.1%, respectively. Intermediate susceptibility to gentamicin was observed in 19.4% of isolates. There was 100% agreement between methods for spectinomycin and ceftriaxone, 99.7% for ciprofloxacin, and 85.7% for azithromycin. For gentamicin, there was 86.3% agreement between agar dilution and disc diffusion, resulting in intermediate susceptible by one method and susceptible by the other method, defined as minor errors. The discordance among agar dilution and disc diffusion results is acceptable for ciprofloxacin, ceftriaxone and spectinomycin as per CLSI M23-Ed4. CONCLUSIONS: Spectinomycin and gentamicin can be considered in some cases as options for the treatment of gonorrhoea in Brazil. Disc diffusion can be an alternative method in routine testing with comparable accuracy to agar dilution.

Analysis of Ki-67 expression in women with breast cancer: Comparative evaluation of two different methodologies by immunophenotyping.

The Ki-67 antigen is a nuclear protein with proven prognostic value in different neoplasms and recognizes the predictive value in breast cancer (BC). No consensus exists on the ideal cutoff point. In this study, Ki-67 expression was evaluated in samples of BC by flow cytometry (FC) and compared with immunohistochemical (IHC) examination. For this, the BC tissue samples were sectioned, macerated, filtered, and marked with anti-Ki-67 FITC and anti-CD45 V500 antibodies. We selected the neoplastic cells according to CD45 expression and size and internal complexity (FSC × SSC) using the Infinicity 1.7 software. Lymphocytes were negative control. We compared the results with IHC analyses carried out in parallel and independently. The expression of Ki-67 was evaluated in both methodologies through Bland-Altman analysis. Among the 44 samples analyzed, only three showed bias higher than the established confidence interval (mean bias 2.1%, p = 0.62), with no significant difference for the perfect mean bias (0%). Therefore, one can state that FC provides results equivalent to IHC analysis and possibly analyzes more cells simultaneously. The results obtained in this study show the absence of observational bias through software analysis in a larger number of tumor cell populations. We can conclude that FC may be a promising alternative method for investigating Ki-67 in solid tumours.

Genomic epidemiology of Neisseria gonorrhoeae elucidating the gonococcal antimicrobial resistance and lineages/sublineages across Brazil, 2015-16.

OBJECTIVES: Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance is imperative internationally, but only eight (22.9%) countries in the WHO Region of the Americas reported complete AMR data to the WHO Global Gonococcal Antimicrobial Surveillance Program (WHO GASP) in 2016. Genomic studies are ideal for enhanced understanding of gonococcal populations, including the spread of AMR strains. To elucidate the circulating gonococcal lineages/sublineages, including their AMR determinants, and the baseline genomic diversity among gonococcal strains in Brazil, we conducted WGS on 548 isolates obtained in 2015-16 across all five macroregions in Brazil. METHODS: A total of 548 gonococcal isolates cultured across Brazil in 2015-16 were genome sequenced. AMR was determined using agar dilution and/or Etest. Genome sequences of isolates from Argentina (n = 158) and the 2016 WHO reference strains (n = 14) were included in the analysis. RESULTS: We found 302, 68 and 214 different NG-MAST, MLST and NG-STAR STs, respectively. The phylogenomic analysis identified one main antimicrobial-susceptible lineage and one AMR lineage, which was divided into two sublineages with different AMR profiles. Determination of NG-STAR networks of clonal complexes was shown as a new and valuable molecular epidemiological analysis. Several novel mosaic mtrD (and mtrR and mtrE) variants associated with azithromycin resistance were identified. CONCLUSIONS: We describe the first genomic baseline data to support the Brazilian GASP. The high prevalence of resistance to ciprofloxacin, tetracycline and benzylpenicillin, and the high number of isolates with mosaic penA and azithromycin resistance mutations, should prompt continued and strengthened AMR surveillance, including WGS, of N. gonorrhoeae in Brazil.

Evaluation of seven rapid tests for syphilis available in Brazil using defibrinated plasma panels.

OBJECTIVES: In 2012, the WHO estimated that 6 million new cases of syphilis per year would occur worldwide, including 937 000 in Brazil. Early diagnosis and treatment of syphilis are essential to reduce morbidity and prevent transmission. The availability of rapid tests (RTs) for this diagnosis means that testing can be performed more quickly, as a point-of-care test, even in non-laboratory environments and requires only simple technical training to antibodies detection. The objective of this study was to evaluate the performance and operational aspects of seven commercially available RTs for syphilis in Brazil. METHODS: Seven rapid treponemal tests were evaluated for sensitivity, specificity, accuracy and Kappa value, according to a panel composed of 493 members. The operational performance of the assay was also determined for these tests. RESULTS: The seven RTs showed sensitivity ranging from 94.5% to 100% when compared with the reference tests and specificity of between 91.5% and 100%. All the RTs evaluated presented good operational performance, and only one failed to present the minimum specificity as defined by Brazil's Ministry of Health. CONCLUSION: All the tests presented good operational performance, and the professionals who performed them considered them to be easy to use and interpret. This evaluation is important for making informed choices of tests to be used in the Brazilian Unified Health System.

Frequência e genotipagem do papilomavírus humano em mulheres submetidas à citologia oncótica / Frequency and genotyping of human papillomavirus in women submitted to citology

Dentre os vírus de transmissão sexual, o papilomavírus humano (HPV) é o mais prevalente, podendo ser detectado em considerável número de mulheres sexualmente ativas. Ele é considerado o principal agente causador do câncer do colo do útero. Portanto, a identificação do HPV de alto risco pode auxiliar na prevenção de lesões do colo uterino. Objetivo: Avaliar a ocorrência de infecções pelo HPV, comparando diferentes metodologias, assim como alguns fatores de risco e seu potencial de associação no desenvolvimento do câncer do colo uterino em mulheres submetidas à citopatologia atendidas nos ambulatórios da Unidade de Saúde Familiar e Comunitária (USFC) da Universidade do Vale do Itajaí (UNIVALI). Métodos: Foram avaliadas 118 amostras de mulheres sexualmente ativas que buscaram atendimento para rastreio do câncer cervical na USFC e da UNIVALI. Todas as amostras foram submetidas à reação em cadeia da polimerase (PCR) e às citologias líquida e convencional. Entretanto, apenas 64 mulheres foram submetidas à metodologia de captura híbrida (CH2). Resultados: A prevalência do HPV foi de 43,22% pela técnica de PCR e de 25% pela CH2; na análise dos resultados observou-se associação do HPV com as seguintes variáveis: etnia (p<0,016), escolaridade (p<0,012), vírus da imunodeficiência humana (HIV) (p<0,008), preservativo (p<0,02),anticoncepcional (p<0,03), início da atividade sexual (p<0,07), citologia convencional (p<0,002) e citologia líquida (p<0,029). Conclusão: A ocorrência de infecção pelo HPV é elevada e o HPV de alto risco foi principalmente associado ao início precoce da atividade sexual

Among the sexually transmitted virus, the human papilloma virus (HPV) is the most prevalent and may be detected a considerable number o fsexually active women. He is considered the main agent of cervical cancer. Therefore, the high-risk HPV identification can aid in the prevention of cervical lesions. Objective: To evaluate the occurrence of HPV infections, comparing different methodologies, as well as some risk factors and their potential association in the development of cervical cancer in women submitted to cytopathology treated in ambulatory Unit Family and Community Health (USFC) ofthe University of Vale do Itajaí (UNIVALI). Methods: 118 samples were evaluated sexually active women who sought care for screening of cervical cancerin USFC and UNIVALI. All samples were subjected to polymerase chain reaction (PCR) and the liquid and conventional cytology. However, only 64 women were subjected to hybrid capture methodology (CH2). Results: The prevalence of HPV was 43.22% by PCR and 25% for CH2; analysis of the results was observed association between HPV and the following variables: ethnicity (p<0.016), scholarity (p<0.012), human immunodeficiency virus (HIV) (p<0.008),preservative (p<0.02), oral contraceptives (p<0.03), younger age at first sexual intercourse (p<0.07), conventional cytology (p<0.002) and liquid cytology(p<0.029). Conclusion: The incidence of HPV infection is high and the high-risk HPV was primarily associated with the younger age at first sexual intercourse.

Lower bifidobacteria counts in adult patients with celiac disease on a gluten-free diet.

CONTEXT: The ingestion of gluten is responsible for the symptoms of Celiac disease, but other environmental factors can also influence. Strains of the Bifidobacterium genus have been shown to afford protection against the inflammatory response and mucosal damage caused by gliadin peptides in vitro. OBJECTIVES: This study was designed to compare the concentration of fecal bifidobacteria and pH of patients with celiac disease on gluten-free diet and control subjects in order to identify if the imbalance on fecal microbiota still remain during the treatment of celiac disease and identify the necessity of dietary supplementation with pre- or probiotics. METHODS: It was analyzed the feces of 42 healthy subjects and 14 celiac patients. The bifidobacteria count in feces was done in selective medium BIM-25. Microscopic analysis of the colonies was performed by Gram stain. The identification of the genus Bifidobacterium was performed by determination of fructose-6-phosphate phosphoketolase. Fecal pH was measured using a pH meter. RESULTS: The concentration of bifidobacteria per gram of feces was significantly higher in healthy subjects (controls) (1.5 ± 0.63 x108 CFU/g) when compared to celiac patients (2.5 ± 1.5 x107 CFU/g). The fecal pH was not different between celiac patients (7.19 ± 0.521) and controls (7.18 ± 0.522). CONCLUSIONS: These results suggest that with lower levels of bifidobacteria, celiac patients have an imbalance in the intestinal microbiota, regardless of pH, even while on a gluten-free diet. This fact could favor the pathological process of the disorder.

Lower bifidobaceeria counts in adult patients with celiac disease on a gluten-free deet / Baixa contagem de bifidobactérias em pacientes adultos com doença celíaca, em dieta isenta de glúten

Context The ingestion of gluten is responsible for the symptoms of Celiac disease, but other environmental factors can also influence. Strains of the Bifidobacterium genus have been shown to afford protection against the inflammatory response and mucosal damage caused by gliadin peptides in vitro. Objectives This study was designed to compare the concentration of fecal bifidobacteria and pH of patients with celiac disease on gluten-free diet and control subjects in order to identify if the imbalance on fecal microbiota still remain during the treatment of celiac disease and identify the necessity of dietary supplementation with pre- or probiotics. Methods It was analyzed the feces of 42 healthy subjects and 14 celiac patients. The bifidobacteria count in feces was done in selective medium BIM-25. Microscopic analysis of the colonies was performed by Gram stain. The identification of the genus Bifidobacterium was performed by determination of fructose-6-phosphate phosphoketolase. Fecal pH was measured using a pH meter. Results The concentration of bifidobacteria per gram of feces was significantly higher in healthy subjects (controls) (1.5 ± 0.63 x108 CFU/g) when compared to celiac patients (2.5 ± 1.5 x107 CFU/g). The fecal pH was not different between celiac patients (7.19 ± 0.521) and controls (7.18 ± 0.522). Conclusions These results suggest that with lower levels of bifidobacteria, celiac patients have an imbalance in the intestinal microbiota, regardless of pH, even while on a gluten-free diet. This fact could favor the pathological process of the disorder. .

Contexto A ingestão do glúten é responsável pelos sintomas da doença celíaca, mas outros fatores ambientais também podem influenciar. Tem sido mostrado que as cepas do género Bifidobacterium proporcionam proteção contra a resposta inflamatória, lesão da mucosa causada por péptidos da gliadina in vitro. Objetivos Este estudo foi desenvolvido para comparar as concentrações de bifidobactérias e pH fecal de pacientes com doença celíaca em dieta isenta de glúten e indivíduos controles, a fim de identificar se o desequilíbrio na microbiota fecal ainda permanece durante o tratamento da doença celíaca e, identificar a necessidade de suplementação alimentar com pré ou probióticos. Métodos Foram analisadas as fezes de 42 indivíduos saudáveis e 14 pacientes com doença celíaca. A contagem de bifidobactérias nas fezes foi feita em meio seletivo BIM-25. A análise microscópica das colônias foi realizada por coloração de Gram. A identificação do género Bifidobacterium foi realizada por determinação de phosphoketolase frutose-6-fosfato. O pH fecal foi medido usando um medidor de pH. Resultados As concentrações de bifidobactérias por grama de fezes foi significativamente mais elevada nos indivíduos saudáveis (controles) (1,5 ± 0,63 x108 UFC/g), quando comparada com pacientes com doença celíaca (2,5 ± 1,5 x107 UFC/g). O pH fecal não foi diferente entre pacientes celíacos (7,19 ± 0,521) e controles (7,18 ± 0,522). Conclusões Estes resultados sugerem que, com concentrações inferiores de bifidobactérias, pacientes com doença celíaca tem um desequilíbrio na microbiota intestinal, independentemente do pH, mesmo durante uma dieta isenta de ...

External quality assurance with dried tube specimens (DTS) for point-of-care syphilis and HIV tests: experience in an indigenous populations screening programme in the Brazilian Amazon.

OBJECTIVES: The availability of point-of-care (POC) tests for infectious diseases has revolutionised the provision of healthcare for remote rural populations without access to laboratories. However, quality assurance for POC tests has been largely overlooked. We have evaluated the use and stability of dry tube specimens (DTS) for External Quality Assurance (EQA) for HIV and syphilis screening in remote indigenous populations in the Amazon region of Brazil. METHODS: All healthcare workers (HCWs) participating in the community-screening were trained. We used HIV and syphilis DTS panels developed by the reference laboratory, containing samples with negative and positive results at different antibody concentrations, for both infections. DTS panels were distributed to HCWs in the communities for reconstitution and testing using POC HIV and syphilis tests. The results of testing were sent to the reference laboratory for marking and remedial action taken where necessary. RESULTS: In total 268 HCWs tested 1607 samples for syphilis and 1608 samples for HIV. Results from HCWs showed a concordance rate of 90% for syphilis and 93% for HIV (κ coefficients of 0.74 and 0.78, respectively) with reference laboratories. Most false negatives were in samples of very low antibody concentration. DTS syphilis specimens produced the expected results after storage at 2-8°C or at 18-24°C for up to 3 weeks. CONCLUSIONS: The results show that POC tests for syphilis and HIV give valid results in environments where traditional tests do not work, but errors in the interpretation of POC test results were identified by the EQA programme using DTS. EQA using DTS can help to improve the quality of screening programmes using POC tests in remote regions.