Samuel Bernal

The aim of this multicentre project (seven hospitals across the Spanish National Health Service) was to study the phenotypic and genotypic susceptibility of C. trachomatis to the main antimicrobials used (macrolides, doxycycline, and quinolones) in isolates from patients with clinical treatment failure in whom reinfection had been ruled out. During 2018–2019, 73 clinical isolates were selected. Sixty-nine clinical specimens were inoculated onto confluent McCoy cell monolayers for phenotypic susceptibility testing. The minimum inhibitory concentration for azithromycin and doxycycline was defined as the lowest concentration associated with an at least 95% reduction in inclusion-forming units after one passage in the presence of the antibiotic compared to the initial inoculum for each strain (control). Sequencing analysis was performed for the genotypic detection of resistance to macrolides, analysing mutations in the 23S rRNA gene (at positions 2057, 2058, 2059, and 2611), and quinolones, analysing a fragment of the gyrA gene, and searching for the G248T mutation (Ser83->Ile). For tetracyclines, in-house RT-PCR was used to test for the tet(C) gene. The phenotypic susceptibility testing was successful for 10 isolates. All the isolates had minimum inhibitory concentrations for azithromycin ≤ 0.125 mg/L and for doxycycline ≤ 0.064 mg/L and were considered sensitive. Of the 73 strains studied, no mutations were found at positions T2611C or G248T of the gyrA gene. We successfully sequenced 66 isolates. No macrolide resistance-associated mutations were found at positions 2057, 2058, 2059, or T2611C. None of the isolates carried the tet(C) gene. We found no evidence for genomic resistance in this large, clinically relevant dataset.

Chlamydia trachomatis infection is an important public health problem. Our objective was to assess the dynamics of the transmission of this infection, analysing the distribution of circulating ompA genotypes and multilocus sequence types of C. trachomatis in Spain as a function of clinical and epidemiological variables. During 2018 and 2019, we genetically characterized C. trachomatis in tertiary hospitals in six areas in Spain (Asturias, Barcelona, Gipuzkoa, Mallorca, Seville and Zaragoza), with a catchment population of 3.050 million people. Genotypes and sequence types were obtained using polymerase chain reaction techniques that amplify a fragment of the ompA gene, and five highly variable genes (hctB, CT058, CT144, CT172 and pbpB), respectively. Amplicons were sequenced and phylogenetic analysis was conducted. We obtained genotypes in 636/698 cases (91.1%). Overall and by area, genotype E was the most common (35%). Stratifying by sex, genotypes D and G were more common among men, and genotypes F and I among women (p < 0.05). Genotypes D, G and J were more common in men who have sex with men (MSM) than in men who have sex with women (MSW), in whom the most common genotypes were E and F. The diversity index was higher in sequence typing (0.981) than in genotyping (0.791), and the most common sequence types were ST52 and ST108 in MSM, and ST30, ST148, ST276 and ST327 in MSW. Differences in genotype distribution between geographical areas were attributable to differences in population characteristics. The transmission dynamics varied with sexual behaviour: the predominant genotypes and most frequent sequence types found in MSM were different to those detected in MSW and women.

One of the most important threats to public health is the appearance of multidrug-resistant pathogenic bacteria, since they are the cause of a high number of deaths worldwide. Consequently, the preparation of new effective antibacterial agents that do not generate antimicrobial resistance is urgently required. We report on the synthesis of new linear cationic antibacterial polytriazoles that could be a potential source of new antibacterial compounds. These polymers were prepared by thermal- or copper-catalyzed click reactions of azide and alkyne functions. The antibacterial activity of these materials can be modulated by varying the size or nature of their side chains, as this alters the hydrophilic/hydrophobic balance. Antibacterial activity was tested against pathogens of the ESKAPE group. The P3TD polymer, which has butylated side chains, was found to have the highest bactericidal activity. The toxicity of selected polytriazoles was investigated using human red blood cells and a human gingival fibroblast cell line. The propensity of prepared polytriazoles to induce resistance in certain bacteria was studied. Some of them were found to not produce resistance in methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa. The interaction of these polytriazoles with the Escherichia coli membrane produces both depolarization and disruption of the membrane.

Importance: The actual demand on SARS-CoV-2 diagnosis is a current challenge for clinical laboratories. Sample pooling may help to ameliorate workload in clinical laboratories. Objective: to evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of CoVID-19, by using different commercial platforms for nucleic acid extraction and amplification. Design and settings: observational, prospective, multicentre study across 9 Spanish clinical microbiology laboratories including SARS-CoV-2 RNA testing performed in April 2020, during the first three days after acceptance to participate. Participants and Methods: 3519 naso-oro-pharyngeal samples received at the participating laboratories were processed individually and in pools (351 pools) according to the existing methodology in each of the centres. Results: We found that 253 pools (2519 samples) were negative, and 99 pools (990 samples) were positive; with 241 positive samples (6.85%), our pooling strategy would have saved 2167 PCR tests. For 29 pools (made out of 290 samples) we found discordant results when compared to their correspondent individual samples: in 24/29 pools (30 samples), minor discordances were found; for five pools (5 samples), we found major discordances. Sensitivity, specificity, positive and negative predictive values for pooling were 97.93%, 100%, 100% and 99.85% respectively; accuracy was 99.86% and kappa concordant coefficient was 0.988. As a result of the sample dilution effect of pooling, a loss of 2-3 Cts was observed for E, N or RdRP genes. Conclusion: we show a high efficiency of pooling strategies for SARS-CoV-2 RNA testing, across different RNA extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity, and positive and negative predictive values. We believe that our results may help clinical laboratories to respond to the actual demand and clinical need on SARS-CoV-2 testing, especially for the screening of low prevalence populations. Key points: Question: May clinical laboratories implement sample pooling as an efficient and safe strategy for SARS-COV-2 RT-PCR screening? Findings: Sensitivity, specificity, positive and negative predictive values for pooling were 97.93%, 100%, 100% and 99.85% respectively; accuracy was 99.86% and kappa concordant coefficient was 0.988. Meaning: Sample pooling can be used safely at clinical laboratories, especially for the screening of low prevalence populations.

Background: The early identification of the Chlamydia trachomatis variants that cause lymphogranuloma venereum (LGV) is very important to establish an adequate antibiotic treatment. This identification should be made with molecular techniques that are easy to perform and accessible to most microbiology laboratories. The objective of this study was to evaluate 2 real-time polymerase chain reaction (PCR)-based assay (VIASURE Haemophilus ducreyi + C. trachomatis (LGV) real-time PCR detection kit and the Allplex Genital ulcer Assay) for the detection of LGV in rectal samples. Materials and Methods: Prospective study on positive rectal samples for C. trachomatis. All samples were processed in parallel by both tests. As a molecular reference method and to solve possible discrepancies between both assays, a PCR-based restriction fragment length polymorphism analysis of the major outer membrane protein gene (omp1) was used. Results: In total, we detected 157 positive rectal samples for C. trachomatis, of which 36 were identified as LGV by PCR-based restriction fragment length polymorphism analysis. The positive percent agreement, negative percent agreement, and overall percent agreement were 88.9%, 100%, and 97.3%, respectively, for the Allplex Genital ulcer assay and 91.6%, 100%, and 97.1%, respectively, for the VIASURE assay. In the direct comparison between the Seegene assay and the VIASURE assay, we obtained a kappa concordance index of 0.98 between both tests. Conclusions: According to the results obtained, both tests could be used for the detection of LGV in rectal samples.