Detecção de Mycoplasma genitalium, Neisseria gonorrhoeae, Chlamydia trachomatis e Trichomonas vaginalis em amostras detectadas e não detectadas para HPV provenientes de mulheres vivendo com HIV no Brasil

TCC (graduação) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Farmácia.As infecções sexualmente transmissíveis (ISTs) podem ser causadas por mais de 30 tipos de microrganismos, entre eles Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG), Vírus da Imunodeficiência Humana (HIV) e Papilomavírus Humano (HPV). HPV, envolvido com casos de câncer cervical, facilita a aquisição e transmissão do HIV, e HIV, por sua vez, contribui com a persistência do HPV no organismo. CT, NG, MG e TV são responsáveis por causar desde infecções assintomáticas até potenciais complicações se não diagnosticados e tratados, além disso aumentam o risco de aquisição e/ou transmissão de HIV, podendo interagir também em diferentes níveis com HPV. MG naturalmente apresenta resistência aos antimicrobianos betalactâmicos, mas também possuem padrões de resistência a outros antimicrobianos utilizados no tratamento da infecção, sendo de suma importância estimar a resistência a fluoroquinolonas e macrolídeos por meio da detecção dos polimorfismos de nucleotídeos únicos (SNPs), nos genes parC, gyrA e na subunidade 23S do RNA ribossomal (RNAr) respectivamente. Atualmente, a forma mais sensível para diagnosticar e compreender grande parte das ISTs é por meio dos testes de amplificação de ácidos nucleicos, especialmente a reação em cadeia da polimerase (PCR), que foi utilizada neste estudo. Ao todo, 355 amostras de conteúdo vaginal provenientes de mulheres vivendo com HIV (MVHV) foram submetidas a PCR em tempo real para detecção de CT, NG, MG e TV e, das amostras com MG detectado, uma Nested PCR foi realizada para amplificar genes associados à resistência aos antimicrobianos macrolídeos e fluoroquinolonas, seguida por sequenciamento tipo Sanger para pesquisar SNPs nos genes alvos. Utilizando o software Statistical Package for Social Science (SPSS) para a análise estatística, foi visto que CT, NG e TV apresentaram alta prevalência (10,1%, 7,9% e 13,0%, respectivamente), MG uma prevalência de 2,8%. Coinfecções também foram detectadas, dessas CT e MG com 1,4% de prevalência, MG e TV com 0,6%, NG, MG e CT com 0,3%, NG e TV com 1,1% e CT e TV com 2,5%. Ademais, uma comparação de resultados entre dois testes moleculares foi avaliada, sendo eles Allplex CT/NG/MG/TV Assay (Seegene®) e RealTime CT/NG Assay (Abbott®), o grau de concordância obtido pelo coeficiente Kappa foi 0,736, considerado muito bom. Infecção por NG foi associada significativamente com HPV de baixo risco. Não foram encontradas SNPs em posições que predizem a resistência para MG nos alvos 23S do RNAr, gyrA e parC nas 10 amostras detectadas. As prevalências altas de CT, NG e TV podem estar associadas pela coinfecção HIV-HPV e à possível imunossupressão das participantes. A prevalência de MG foi condizente com a literatura assim como os dados de coinfecções entre estes patógenos. Por se tratar do primeiro dado, até o momento, do perfil de resistência de MG aos antimicrobianos no Brasil em MVHIV. Resultados nacionais para MVHIV corroboram para a o cenário epidemiológico brasileiro e podem auxiliar gestores públicos a embasar decisões diagnósticas e de tratamento das ISTs.Sexually transmitted infections (STIs) can be caused by more than 30 microorganisms, including Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG), Human Immunodeficiency Virus (HIV), and Human Papillomavirus (HPV). HPV, involved in cases of cervical cancer, facilitates HIV acquisition and transmission, whilst HIV contributes to the persistence of HPV infection. CT, NG, MG, and TV are responsible for causing asymptomatic infections but if not diagnosed and treated can cause complications; in addition, they can increase the risk of acquiring and/or transmitting HIV, and may also interact at different levels with HPV. MG is naturally resistant to beta-lactam antimicrobials but can also be resistant to other antimicrobials used in treatment, and it is of great importance to estimate MG resistance to fluoroquinolones and macrolides through the detection of single nucleotide polymorphisms (SNPs) in parC, gyrA, and 23S ribosomal RNA (RNAr) subunit genes respectively. Currently, the most sensitive way to diagnose and understand most STIs is through nucleic acid amplification tests, especially the polymerase chain reaction (PCR), which was used in this study. In all, 355 vaginal samples from women living with HIV (MVHV) were subjected to real-time PCR for CT, NG, MG, and TV detection, and from MG detected samples, a Nested PCR was performed to amplify genes associated with macrolide and fluoroquinolone resistance, followed by Sanger sequencing to search for SNPs in target genes. Using the Statistical Package for Social Sciences (SPSS) software for statistical analysis, it was observed that CT, NG, and TV had a high prevalence (10.1%, 7.9%, and 13.0%, respectively), MG had a 2.8% prevalence. Coinfections were also detected, such as CT and MG with 1.4% prevalence, MG and TV with 0.6%, NG, MG and CT with 0.3%, NG and TV with 1.1 % and CT and TV with 2.5%. In addition, a comparison between the results of two molecular tests was perfomed, them being Allplex CT/NG/MG/TV Assay (Seegene®) and RealTime CT/NG Assay (Abbott®), being the agreement degree obtained via Kappa coefficient was 0.736, considered very good. Infection by NG was significantly associated with low-risk HPV. No SNPs were found in positions that predict MG resistance in the 23S rRNA, gyrA, and parC targets in the 10 detected samples. The high prevalence of CT, NG, and TV may be associated with the HIV-HPV coinfection and the imunosupression of the participants. MG prevalence was consistent with the literature, as well as data on co-infections between these pathogens. As it is the first data so far on the profile of MG resistance to antimicrobials in MVHIV living in Brazil. National results for MVHIV corroborate the Brazilian epidemiological scenario and can help governmental institutions to support diagnostic and treatment decisions for STIs

In vitro selection of Neisseria gonorrhoeae unveils novel mutations associated with extended-spectrum cephalosporin resistance

The emergence of Neisseria gonorrhoeae strains resistant to extended-spectrum cephalosporins (ESCs) is a worldwide concern because this class of antibiotics represents the last empirical treatment option for gonorrhea. The abusive use of antimicrobials may be an essential factor for the emergence of ESC resistance in N. gonorrhoeae. Cephalosporin resistance mechanisms have not been fully clarified. In this study, we mapped mutations in the genome of N. gonorrhoeae isolates after resistance induction with cefixime and explored related metabolic pathways. Six clinical isolates with different antimicrobial susceptibility profiles and genotypes and two gonococcal reference strains (WHO F and WHO Y) were induced with increasing concentrations of cefixime. Antimicrobial susceptibility testing was performed against six antimicrobial agents before and after induction. Clinical isolates were whole-genome sequenced before and after induction, whereas reference strains were sequenced after induction only. Cefixime resistance induction was completed after 138 subcultures. Several metabolic pathways were affected by resistance induction. Five isolates showed SNPs in PBP2. The isolates M111 and M128 (ST1407 with mosaic penA-34.001) acquired one and four novel missense mutations in PBP2, respectively. These isolates exhibited the highest minimum inhibitory concentration (MIC) for cefixime among all clinical isolates. Mutations in genes contributing to ESC resistance and in other genes were also observed. Interestingly, M107 and M110 (ST338) showed no mutations in key determinants of ESC resistance despite having a 127-fold increase in the MIC of cefixime. These findings point to the existence of different mechanisms of acquisition of ESC resistance induced by cefixime exposure. Furthermore, the results reinforce the importance of the gonococcal antimicrobial resistance surveillance program in Brazil, given the changes in treatment protocols made in 2017 and the nationwide prevalence of sequence types that can develop resistance to ESC

Emergence of Two Distinct SARS-CoV-2 Gamma Variants and the Rapid Spread of P.1-like-II SARS-CoV-2 during the Second Wave of COVID-19 in Santa Catarina, Southern Brazil

The western mesoregion of the state of Santa Catarina (SC), Southern Brazil, was heavily affected as a whole by the COVID-19 pandemic in early 2021. This study aimed to evaluate the dynamics of the SARS-CoV-2 virus spreading patterns in the SC state from March 2020 to April 2021 using genomic surveillance. During this period, there were 23 distinct variants, including Beta and Gamma, among which the Gamma and related lineages were predominant in the second pandemic wave within SC. A regionalization of P.1-like-II in the Western SC region was observed, concomitant to the increase in cases, mortality, and the case fatality rate (CFR) index. This is the first evidence of the regionalization of the SARS-CoV-2 transmission in SC and it highlights the importance of tracking the variants, dispersion, and impact of SARS-CoV-2 on the public health systems