Molecular epidemiology of paediatric invasive pneumococcal disease in Andalusia, Spain.

This study aimed to assess the impact of the introduction of pneumococcal conjugate vaccine 13 (PCV13) on the molecular epidemiology of invasive pneumococcal disease (IPD) in children from Andalusia. A population-based prospective surveillance study was conducted on IPD in children aged <14 years from Andalusia (2018-2020). Pneumococcal invasive isolates collected between 2006 and 2009 in the two largest tertiary hospitals in Andalusia were used as pre-PCV13 controls for comparison of serotype/genotype distribution. Overall IPD incidence rate was 3.55 cases per 100 000 in 2018; increased non-significantly to 4.20 cases per 100 000 in 2019 and declined in 2020 to 1.69 cases per 100 000 (incidence rate ratio 2020 vs. 2019: 0.40, 95% confidence interval (CI) 0.20-0.89, P = 0.01). Proportion of IPD cases due to PCV13 serotypes in 2018-2020 was 28% (P = 0.0001 for comparison with 2006-2009). Serotypes 24F (15%) and 11A (8.3%) were the most frequently identified non-PCV13 serotypes (NVT) in 2018-2020. Penicillin- and/or ampicillin-resistant clones mostly belonged to clonal complex 156 (serotype 14-ST156 and ST2944 and serotype 11A-ST6521). The proportion of IPD cases caused by PCV13 serotypes declined significantly after the initiation of the PCV13 vaccination programme in 2016. Certain NVT, such as serotypes 24F and 11A, warrant future monitoring in IPD owing to invasive potential and/or antibiotic resistance rates.

Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS. One hundred ninety strains (179 clinical isolates previously identified by sequencing and the 11 reference strains), cultured on solid medium, were blindy analyzed by the MALDI-TOF MS technology to validate the generated in-house reference database. A 100% correlation was obtained with both identification methods, gene sequencing and MALDI-TOF MS, and no discordant identification was obtained. The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.84%) and for the remaining 25 (13.16%) a genus level identification (score between 1.7 and 2.0) was obtained. The routine MALDI-TOF MS analysis with the new database, was then challenged with 200 Aspergillus clinical isolates grown on solid medium in a prospective evaluation. A species identification was obtained in 191 strains (95.5%), and only nine strains (4.5%) could not be identified at the species level. Among the 200 strains, A. tubingensis was the only cryptic species identified. We demonstrated the feasibility and usefulness of the new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species, grown either on solid or liquid media.

Clinical and epidemiological characterisation of lymphogranuloma venereum in southwest Spain, 2013-2015.

OBJECTIVES: Lymphogranuloma venereum (LGV) infections caused by Chlamydia trachomatis L serovars have emerged in 2003 in Europe among HIV-positive men having sex with men (MSM). Our aim was to evaluate LGV prevalence and predictors in a high-risk population attending to two STI clinics in the southwest of Spain between December 2013 and April 2015. METHODS: Screening of C. trachomatis using commercial kits was carried out, followed by real-time pmpH-PCR discriminating LGV strains, and finally ompA gene was sequenced for phylogenetic reconstruction. RESULTS: A total of 6398 samples were tested, of which, 594 (9.3%) were C. trachomatis-positive specimens and successfully typed by pmpH PCR. Five hundred and eighty-one samples contained non-LGV and 13 (2.2%; 95% CI 1.3% to 3.7%) samples had LGV. One hundred and sixty-six (27.9%; 95% CI 24.5% to 31.7%) CT-positive results were found in MSM. All C. trachomatis LGV types were found in rectal samples from MSM (13/166, 7.8%; 95% CI 4.5% to 13.0%). Of these, five (38.5%; 95% CI 17.7% to 64.5%) patients were asymptomatic and 11 (84.6%; 95% CI 57.8% to 95.7%; p<0.001) were also HIV positive. Successful treatment of LGV was achieved in all patients including 11/13 (84.6%) who received single-dose azithromycin. All of the L types were confirmed to be genotype L2b with ompA PCR and sequencing. CONCLUSIONS: This analysis shows that LGV infections are occurring in MSM in southwest Spain, where no data about LGV have been described before, reinforcing the need for screening and genotyping for LGV. LGV should be taken into account when considering treatment and management of rectal C. trachomatis infections, including in asymptomatic HIV-positive MSM. Larger studies on appropriate treatment for asymptomatic LGV infection are needed.

[The relevance of correct identification and interpretation of susceptibility testing of Aeromonas spp. bacteremia isolates]. / Influencia de la correcta identificación en la interpretación de las pruebas de sensibilidad en aislados de Aeromonas spp. productoras de bacteriemia.

OBJECTIVE: To assess the relevance of correct identification and interpretation of susceptibility testing of Aeromonas spp. bacteremia isolates using newly developed molecular methods in comparison to previous conventional methods. MATERIAL AND METHODS: The study included 22 patients with bacteremia due to Aeromonas hydrophila group, microbiologically characterized using the MicroScan system. Further identification to species level was performed by mass spectrometry, and confirmed by sequencing the rpoB gene. The MIC of imipenem, cefotaxime, piperacillin-tazobactam, ciprofloxacin and cotrimoxazole was studied using a commercial broth microdilution and antibiotic gradient strips with low and high inocula. Detection of carbapenemase production was performed using the modified Hodge test, and was confirmed by amplifying the cphA gene by PCR. RESULTS: A total of 9 (40.9%) isolates were identified as Aeromonas hydrophila, 8 (36.4%) as Aeromonas veronii, and the remaining 5 (22.7%) isolates as Aeromonas caviae. Resistance to beta-lactams according to both the commercial microdilution and MIC gradient strips methods was: 36%-50% to imipenem; 4%-56% to cefotaxime, and 27%-56% to piperacillin/tazobactam. The agreement between results generated by the automated system and the diffusion antibiotic gradient strip was, for all 3 species, 68% for imipenem, 50% to cefotaxime, and 46% to piperacillin/tazobactam. No resistance to cotrimoxazole and ciprofloxacin was found by either of the two methods, although 22.7% of the strains were resistant to nalidixic acid. CONCLUSIONS: It is essential to identify the isolates of Aeromonas spp. at the species level, due to the fact that beta-lactam resistance is species- and method-dependent. The high rate of resistance to beta-lactam and quinolones reduce their application as empiric treatments for invasive infection by Aeromonas ssp.

Molecular epidemiology and antimicrobial susceptibility of Campylobacter coli clinical isolates.

INTRODUCTION: Campylobacter spp. is a major cause of acute bacterial diarrhea in humans worldwide, and C. coli is responsible for 10% of the cases. MATERIALS AND METHODS: A study was made of the antimicrobial susceptibility using the E-test(®), and the clonal relationship using PCR-RFLP, of the flaA gene, as well as PFGE techniques on 43 C. coli clinical isolates. RESULTS: Only 49% and 2% of the isolates were susceptible to erythromycin and ciprofloxacin, respectively. Imipenem and clindamyicn, with 100% and 84% of the strains, respectively, being susceptible, were the most active antimicrobials. The PCR-RFLP of flaA gene technique grouped fourteen isolates into six clusters, while the PFGE technique grouped eleven isolates into five clusters. CONCLUSION: Ciprofloxacin and erythromycin are not suitable for the treatment of C. coli infections. Clindamycin could be considered as a therapeutic alternative in cases of enteritis, while imipenem is the best alternative for extra-intestinal infections. Both PFGE and PCR-RFLP can be useful to detect clones.

Molecular epidemiology of pneumococcal carriage in children from Seville, following implementation of the PCV13 immunization program in Andalusia, Spain.

INTRODUCTION: The 13-valent pneumococcal conjugate vaccine (PCV13) universal vaccination programme was introduced in December 2016 in Andalusia. METHODS: A cross-sectional study was conducted on the molecular epidemiology of pneumococcal nasopharyngeal colonization. A total of 397 healthy children were recruited from primary healthcare centres in Seville for the periods 1/4/2018 to 28/2/2020 and 1/11/2021 to 28/2/2022 (PCV13 period). Data from a previous carriage study conducted among healthy and sick children from 1/01/2006 to 30/06/2008 (PCV7 period), were used for comparison of serotype/genotype distributions and antibiotic resistance rates. RESULTS: Overall, 76 (19%) children were colonized with S. pneumoniae during the PCV13 period and there were information available from 154 isolates collected during the PCV7 period. Colonization with PCV13 serotypes declined significantly in the PCV13 period compared with historical controls (11% vs 38%, p = 0.0001), being serotypes 19F (8%), 3 (1%) and 6B (1%) the only circulating vaccine types. Serotypes 15B/C and 11A were the most frequently identified non-PCV13 serotypes during the PCV13 period (14% and 11%, respectively); the later one increased significantly between time periods (p = 0.04). Serotype 11A was exclusively associated in the PCV13 period with ampicillin-resistant variants of the Spain9V-ST156 clone (ST6521 and genetically related ST14698), not detected in the preceding period. CONCLUSIONS: There was a residual circulation of vaccine types following PCV13 introduction, apart from serotype 19F. Serotype 11A increased between PCV13 and PCV7 periods due to emergence and clonal expansion of ampicillin-resistant genotype ST6521.

Diagnóstico de síndrome de Gitelman en paciente con condrocalcinosis / Diagnosis of gitelman syndrome in patient with condrocalcinosis

RESUMEN Introducción: El síndrome de Gitelman es una tubulopatía caracterizada por alcalosis metabólica hipopotasémica, hipomagnesemia e hipocalciuria. Sus efectos musculoesqueléticos son comunes, pudiendo provocar desarrollo de condrocalcinosis. Caso clínico: Paciente con condrocalcinosis de larga data asociada a hipomagnesemia crónica en tratamiento con calcio y magnesio. Tras la suspensión del tratamiento debido a una intervención quirúrgica presentó debilidad generalizada, alcalosis metabólica, hipopotasemia, hipomagnesemia e hipocalciuria con diagnóstico final de síndrome de Gitelman. Tras la instauración de tratamiento, mejoró clínica y analíticamente manteniendo cifras iónicas estables. Discusión y conclusiones: Resulta fundamental un adecuado diagnóstico de este tipo de tubulopatías, ya que un tratamiento adecuado evita complicaciones asociadas.

ABSTRACT Introduction: Gitelman syndrome is a renal tubule disease that involves hypokalaemic metabolic alkalosis, hypomagnesaemia and hypocalciuria. The musculoskeletal effects of Gitelman syndrome are common, including the development of chondrocalcinosis. Clinical case: A female patient with long-standing chondrocalcinosis associated with chronic hypomagnesaemia on treatment with calcium and magnesium. After the suspension of the treatment due to surgery, she presented with a generalised weakness, metabolic alkalosis, hypokalaemia, hypomagnesaemia and hypocalciuria, with final diagnosis of Gitelman syndrome. After re-introducing the treatment, she improved clinically, with electrolytes remaining stable. Discussion and conclusions: A proper diagnosis of this type of tubular diseases is essential because an adequate treatment avoids associated complications.

Vacunación frente al virus de la hepatitis B en una unidad de hemodiálisis: comparativa entre 2 pautas de administración / Vaccination against hepatitis B virus in a hemodyalisis unit: comparative analysis between two different schemes

INTRODUCCIÓN: La vacunación frente al virus de la hepatitis B (VHB) es más efectiva en prediálisis pero en ocasiones el paciente llega a diálisis de forma no programada sin realizar vacunación completa previa. Pretendemos determinar el grado de respuesta a inmunización frente a VHB con diferentes esquemas en hemodiálisis. MATERIAL Y MÉTODOS: Estudiamos 30 pacientes en hemodiálisis administrando 2 esquemas de vacunación frente a VHB. Un grupo realiza el esquema de 3 dosis (0, 1, 6 meses) (PAUTA 3) y otro de 4 dosis (0, 1, 2, 6 meses) (PAUTA 4). RESULTADOS: El 56.7% hizo pauta 3 con respuesta del 26.7% y el 43.3% hizo pauta 4 con 23.3% de respuesta, sin diferencias significativas entre ambas (p=0.6). La tasa de AcHBs postvacunacional fue significativamente mayor con pauta 4 (p<0.05) así como en las tasas de anticuerpos mantenidas al año; a los 2 años las tasas de anticuerpos no fueron significativamente diferentes entre ambas pautas. En el resto de análisis estadístico, la vacunación realizada en pacientes con más tiempo en diálisis tuvo mayor respuesta con pauta 4 (p<0.05) aunque con respecto a los niveles de anticuerpos no hubo diferencias en los que respondieron con cada pauta. CONCLUSIONES: No existen diferencias en la respuesta inmunológica a vacunación frente VHB en hemodiálisis entre esquemas de 3 y 4 dosis. Hay mayor respuesta con el esquema de 4 dosis en la vacunación en pacientes con mayor tiempo en diálisis. Por ello, aconsejamos realizar la inmunoprofilaxis con el esquema de 3 dosis en pacientes incidentes en hemodiálisis que no han sido vacunados anteriormente

INTRODUCTION: Vaccination against hepatitis B virus (HBV) is more effective in pre-dialysis, but sometimes the patient has to start treatment in an unscheduled way, without full immunization coverage. We try to establish the immune response rate against HBV in hemodialysis with different vaccination schemes. METHODS: We studied 30 patients in hemodialysis program with 2 different vaccination schemes against HBV. One group performed a 3-doses scheme (0, 1 and 6 months) (PATTERN 3) and the other group performed a 4-doses scheme (0, 1, 2 and 6 months) (PATTERN 4). RESULTS: 56.7% performed pattern 3, with immune response of 26.7%. 43.3% performed pattern 4, with immune response of 23.3%, without significant differences between them (p=0.06). Anti-hepatitis B surface antibodies (anti-HBs) rate after vaccination was significantly higher with pattern 4 (p<0.05) as well as the antibodies counts maintained within 1 year. After 2 years, anti-HBs rates were not significantly different between both patterns. In other statical analysis, vaccination carried out in patients who have been for a long time in hemodialysis treatment, was more responsive with pattern 4 (p<0.05) although anti-HBs levels were similar in patients that had response with each pattern. CONCLUSIONS: There are no differences in immune response between 3-doses scheme and 4-doses scheme in HBV vaccination for hemodialysis patients. There is greater response with 4-doses scheme in hemodialysis patients who have not been vaccinated previously

[Detection of inducible resistance to clindamycin in cutaneous isolates of Staphylococcus spp. by phenotypic and genotypic methods]. / Detección de resistencia inducible a clindamicina en aislados cutáneos de Staphylococcus spp. por métodos fenotípicos y genotípicos.

INTRODUCTION: Resistance to macrolides, lincosamides and type B streptogramins (MLSB) in Staphylococcus isolates can be due to several mechanisms. The most important are an active efflux mechanism (MSB phenotype) and ribosomal target modification (MLSB phenotype); this latter mechanism confers resistance to all three groups of antimicrobials (MLSB resistance). Expression of MLSB resistance can be constitutive (cMLSB) or inducible (iMLSB). METHODS: A group of 117 erythromycin-resistant Staphylococcus spp. clinical isolates from cutaneous samples were selected from 536 recent clinical isolates of this microorganism. Resistance phenotypes were determined by the double disk diffusion test. Presence of the ermA, ermC, ermB and msrA genes was detected by real time PCR. RESULTS: The MSB phenotype was the most common, comprising 11.2% (7.2% in S. aureus and 23% in CoNS) of the erythromycin-resistant strains. The rate of iMLSB resistance was significantly higher, 7.4% (5.2% in S. aureus and 14% in CoNS), than the rate of cMLSB resistance, 3.2% (1.7% in S. aureus and 7.4% in CoNS). The msrA gene was present in all isolates with the MSB phenotype, and the ermC gene was the most common among clindamycin-resistant strains with the MLSB phenotype (constitutive or inducible). CONCLUSION: The good correlation between the phenotypic (disk-diffusion) and genotypic (real time PCR) methods used allows prediction of the mechanisms of erythromycin and clindamycin resistance, provides insight into the epidemiological differences in their distribution, and is an aid to selecting the most appropriate antimicrobial therapy.

[Detection of methicillin resistance and identification of Staphylococcus spp. from positive blood culture bottles using the mecA and nucA genes with the LightCycler System]. / Detección de la resistencia a meticilina e identificación de Staphylococcus spp. en hemocultivos positivos amplificando los genes mecA y nucA con el sistema LightCycler.

INTRODUCTION: The aim of this study was to evaluate the feasibility of detecting Staphylococcus aureus and coagulase-negative staphylococci (CoNS) and of identifying methicillin resistance directly in positive BACTEC blood culture bottles using the LightCycler system. METHODS: One hundred thirty-one positive blood culture bottles in which Gram-positive cocci in cluster were observed after Gram staining and 40 positive bottles with microorganisms other than staphylococci were studied. A molecular assay based on an automated DNA extraction protocol with a MagNA Pure LC instrument was used. Oligonucleotide primers and fluorescence-labeled hybridization probes were designed for amplification and sequence-specific detection of both a 408-pb fragment within the mecA gene and a 279-pb fragment within the S. aureus-specific nucA gene. RESULTS: All the bottles that yielded methicillin-resistant S. aureus (MRSA), methicillin-sensitive S. aureus (MSSA) or methicillin-resistant CoNS (MRCoNS) strains were correctly identified by the nucA and mecA PCR assays. One bottle that yielded a mixed culture of MSSA and MRCoNS gave positive results for both genes. In the 21 bottles with methicillin-susceptible CoNS (MSCoNS), nucA PCR were negative, but two of these bottles gave positive results for the mecA gene. The sensitivity and specificity of the nucA gene assay were 100%. The sensitivity and specificity of the PCR assay for detection of methicillin resistance with the mecA gene were 100% and 97.5%, respectively. CONCLUSION: This is a sensitive and highly specific method for identifying staphylococci in positive blood cultures, allowing discrimination between methicillin-susceptible and -resistant strains in less than 3 hours after Gram stain.