Simultaneous detection and ribotyping of Clostridioides difficile, and toxin gene detection directly on fecal samples

Background Clostridioides difficile is the most common cause of nosocomial diarrhea. Ribotyping of cultured strains by a PCR-based test is used to study potential transmission between patients. We aimed to develop a rapid test that can be applied directly on fecal samples for simultaneous detection and ribotyping of C. difficile, as well as detection of toxin genes. Methods We developed a highly specific and sensitive primer set for simultaneous detection and ribotyping of C. difficile directly on total fecal DNA. Toxin genes were detected with primers adapted from Persson et al. (Clin Microbiol Infect 14(11):1057–1064). Our study set comprised 130 fecal samples: 65 samples with positive qPCR for C. difficile toxin A/B genes and 65 C. difficile qPCR negative samples. PCR products were analyzed by capillary gel electrophoresis. Results Ribosomal DNA fragment peak profiles and toxin genes were detected in all 65 C. difficile positive fecal samples and in none of the 65 C. difficile negative samples. The 65 samples were assigned to 27 ribotypes by the Dutch reference laboratory. Our peak profiles corresponded to these ribotypes, except for two samples. During a C. difficile outbreak, patients were correctly allocated to the outbreak-cluster based on the results of direct fecal ribotyping, before C. difficile isolates were cultured and conventionally typed. Conclusion C. difficile ribotyping directly on fecal DNA is feasible, with sensitivity and specificity comparable to that of diagnostic toxin gene qPCR and with ribotype assignment similar to that obtained by conventional typing on DNA from cultured isolates. This supports simultaneous diagnosis and typing to recognize an outbreak.

Presença de DNA da Leishmania (V.) braziliensis na Mucosa Nasal Clinicamente Saudável em Pacientes com Leishmaniose Cutânea

Considerando que a lesão da mucosa ocorre tardiamente após a infecção por leishmaniose cutanea (LC), avaliamos se pacientes com doença cutânea por L. V. braziliensi sem atividade podem carrear DNA do parasita na mucosa nasal aparentemente sadia. Realizamos um estudo de corte transversal, com exame otorrinolaringológico de todos os casos de LC (n-153) atendidos em uma área endêmica na Bahia- Brasil, em um período de 2 anos. Amostras de swab nasal foram coletadas antes do tratamento para avaliação de DNA do parasita. Após 3 meses, esses indivíduos foram reavaliados para identificar o status da doença. O DNA do parasita foi detectado em 7,8% (12/153) de pacientes com LC sem lesão aparente em mucosa nasal. O DNA estava presente em pacientes com fatores de risco conhecidos para desenvolvimento de lesão de mucosa, como lesões cutâneas em maior número (mediana de 1,5 vs 1,0, p=0,044) e maiores (mediana 2,7 vs 1,6cm, p=0,013). Além disso, esses indivíduos com parasita na mucosa evoluíram mais frequentemente para formas atípicas da LC [45,5% vs 11,5%; p=0,009], e necessitaram de mais ciclos de tratamento para atingir a cura clínica da lesão cutânea (mediana de 2 vs 1, p<0,05). Os resultados sugerem tropismo precoce do parasita para a mucosa nasal e um fenótipo clínico dos casos de LC com DNA de parasita detectável na mucosa. Estudos futuros poderão avaliar se a presença do parasita na mucosa servirá como marcador prognóstico de risco de desenvolver leishmaniose mucocutânea. Publicado na revista Journal of Microbiology and Infection, em Janeiro de 2019. Canário A, et al. Clin Microbiol Infect. 2019 Jan 4. pii: S1198-743X(18)30838-3. Doi: 10.1016/j.cmi.2018.12.02.

In Vitro Activity of Imipenem/Relebactam and Ceftolozane/Tazobactam Against Clinical Isolates of Gram-negative Bacilli With Difficult-to-Treat Resistance and Multidrug-resistant Phenotypes—Study for Monitoring Antimicrobial Resistance Trends, United States 2015–2017

Background Multidrug-resistant (MDR) bacteria are frequently defined using the criteria established by Magiorakos et al [Clin Microbiol Infect 2012;18:268–81]. Difficult-to-treat resistance (DTR) [Kadri et al, Clin Infect Dis 2018;67:1803–14] is a novel approach to defining resistance in gram-negative bacilli focusing on treatment-limiting resistance to first-line agents (all β-lactams and fluoroquinolones). Methods Clinical and Laboratory Standards Institute–defined broth microdilution minimum inhibitory concentrations (MICs) were determined for imipenem/relebactam, ceftolozane/tazobactam, and comparators against respiratory, intraabdominal, and urinary isolates of Enterobacterales (n = 10 516) and Pseudomonas aeruginosa (n = 2732) collected in 26 US hospitals in 2015–2017. Results Among all Enterobacterales, 1.0% of isolates were DTR and 15.6% were MDR; 8.4% of P. aeruginosa isolates were DTR and 32.4% were MDR. MDR rates for Enterobacterales and DTR and MDR rates for P. aeruginosa were significantly higher (P &lt; .05) in isolates collected in intensive care units (ICUs) than in non-ICUs and in respiratory tract isolates than in intraabdominal or urinary tract isolates. In addition, 82.4% of DTR and 92.1% of MDR Enterobacterales and 62.2% of DTR and 82.2% of MDR P. aeruginosa were imipenem/relebactam-susceptible, and 1.5% of DTR and 65.8% of MDR Enterobacterales and 67.5% of DTR and 84.0% of MDR P. aeruginosa were ceftolozane/tazobactam-susceptible. Conclusions MDR phenotypes defined using the Magiorakos criteria may overcall treatment-limiting resistance in gram-negative bacilli. In the US, DTR Enterobacterales were infrequent, while MDR Enterobacterales isolates and DTR and MDR P. aeruginosa were common. Imipenem/relebactam (Enterobacterales, P. aeruginosa) and ceftolozane/tazobactam (P. aeruginosa) retained in vitro activity against most DTR and MDR isolates.

24 Comparison of the tolerability of posaconazole versus voriconazole in children with cystic fibrosis

BackgroundTriazole antifungals (itraconazole and voriconazole), are commonly used for treating isolates of Aspergillus, or in combination with corticosteroids for the empiric treatment of Allergic Bronchopulmonary Aspergillosis (ABPA) in children with cystic fibrosis (CF). Posaconazole is a newer triazole that is as effective, but better tolerated than voriconazole and itraconazole in immunocompromised patients1 though there is no published use in CF and it is not licensed in children<18 years old. It is used as a 3rd line agent for Aspergillus or ABPA in our institution.AimOur aim was to evaluate why posaconazole was needed in some children, and to assess its tolerability. Given the difficulty in reaching therapeutic drug levels of triazoles in children with CF2 we also reviewed posaconazole blood levels.MethodA retrospective case note review of all children with CF who had received voriconazole or posaconazole from April 2014 to May 2015 in a tertiary paediatric CF centre with a clinic population of 350 children. Children were identified from pharmacy records and clinical data was collected from case notes and computerised laboratory records. We compared reported adverse effects for both drugs, and for the posaconazole group documented reason for use and blood levels (therapeutic >0.7 mg/L).ResultsVoriconazole was used in 10 children with a median age of 13.5 years (range 12–16), for median 8 weeks. Adverse effects were experienced in 5/10 (50%) of children (photosensitivity – 4; hallucinations and nausea – 1), and 2 children had raised liver functions tests (LFTs). Posaconazole was used in 7 children with a median age of 14 years (range 13–16) for median 37 weeks. No adverse effects were reported but LFTs were raised in 1 child. Posaconazole was commenced in 6/7 (85%) children due to severe photosensitivity with voriconazole. Of these, posaconazole was indicated for concurrent Scedosporium isolates in 2 children, and therapeutic failure with itraconazole in 4 children. Posaconazole levels were consistently therapeutic in 5/7 (71%) children (range 0.7–2.47 mg/L). Levels in 1 child fell to <0.2 mg/L following the introduction of an interacting drug (rifampicin), and a level of 2.47 mg/L was associated with raised LFTs in another, resulting in discontinuation of posaconazole.ConclusionIn this small cohort, posaconazole was better tolerated than voriconazole for the treatment of Aspergillus or ABPA in children with CF, due mainly to the lack of photosensitivity associated with its use. Posaconazole levels attained from our patients indicate that therapeutic levels can be readily obtained in this patient population. Larger studies are needed to support these conclusions.ReferencesDoring M, Blume O, Haufe S, et al. Comparison of itraconazole, voriconazole, and posaconazole as oral antifungal prophylaxis in paediatric patients following allogeneic hematopoietic stem cell transplantation. Eur J Clin Microbiol Infect Dis2014;33(4):629–38.Bentley S, Gupta A, Balfour-Lynn IM. Subtherapeutic itraconazole and voriconazole levels in children with cystic fibrosis. J Cyst Fibros2013;12:418–9.