Validation of an expanded, in-house library and an optimized preparation method for the identification of fungal isolates using MALDI-TOF mass spectrometry.

The goal of this study was to validate an optimized sample preparation method for filamentous fungal isolates coupled with the use of an in-house library for the identification of moulds using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in a multicenter context. For that purpose, three Spanish microbiology laboratories participated in the identification of 97 fungal isolates using MALDI-TOF MS coupled with the Filamentous Fungi library 3.0 (Bruker Daltonics) and an in-house library containing 314 unique fungal references. The isolates analyzed belonged to 25 species from the genus Aspergillus, Fusarium, Scedosporium/Lomentospora, the Mucorales order and the Dermatophytes group. MALDI-TOF MS identification was carried out from hyphae resuspended in water and ethanol. After a high-speed centrifugation step, the supernatant was discarded and the pellet submitted to a standard protein extraction step. The protein extract was analyzed with the MBT Smart MALDI Biotyper system (Bruker Daltonics). The rate of accurate, species-level identification obtained ranged between 84.5% and 94.8% and the score values were 1.8 for 72.2-94.9% of the cases. Two laboratories failed to identify only one isolate of Syncephalastrum sp. and Trichophyton rubrum, respectively and three isolates could not be identified in the third center (F. proliferatum, n = 1; T.interdigitale, n = 2). In conclusion, the availability of an effective sample preparation method and an extended database allowed high rates of correct identification of fungal species using MALDI-TOF MS. Some species, such as Trichophyton spp. are still difficult to identify. Although further improvements are still required, the developed methodology allowed the reliable identification of most fungal species.

MALDI-TOF mass spectrometry has been improved as a diagnostic method for the rapid and reliable identification of filamentous fungi by means of the creation of an expanded database containing reference protein spectra of the most clinically impacting fungal species.

Genomic features of predominant non-PCV13 serotypes responsible for adult invasive pneumococcal disease in Spain.

BACKGROUND: Although pneumococcal conjugate vaccines (PCVs) effectively prevent invasive pneumococcal disease (IPD), serotype replacement has occurred. OBJECTIVES: We studied the pangenome, antibiotic resistance mechanisms and presence of mobile elements in predominant non-PCV13 serotypes causing adult IPD after PCV13 vaccine introduction in Spain. METHODS: We conducted a multicentre study comparing three periods in six Spanish hospitals and analysed through whole genome sequencing representative strains collected in the pre-PCV13, early-PCV13 and late-PCV13 periods. RESULTS: Among 2197 cases of adult IPD identified, 110 pneumococci expressing non-PCV13 capsules were sequenced. Seven predominant serotypes accounted for 42.6% of IPD episodes in the late-PCV13 period: serotypes 8 (14.4%), 12F (7.5%), 9N (5.2%), 11A (4.1%), 22F (3.9%), 24F (3.9%) and 16F (3.6%). All predominant non-PCV13 serotypes were highly clonal, comprising one or two clonal complexes (CC). In general, CC538, CC4048, CC3016F, CC43322F and CC669N, related to predominant non-PCV13 serotypes, were antibiotic susceptible. CC15611A was associated with resistance to co-trimoxazole, penicillin and amoxicillin. CC23024F was non-susceptible to penicillin and resistant to erythromycin, clindamycin, and tetracycline. Six composite transposon structures of the Tn5252-family were found in CC23024F, CC98912F and CC3016F carrying different combinations of erm(B), tet(M), and cat. Pangenome analysis revealed differences in accessory genomes among the different CC, with most variety in CC3016F (23.9%) and more conservation in CC15611A (8.5%). CONCLUSIONS: We identified highly clonal predominant serotypes responsible for IPD in adults. The detection of not only conjugative elements carrying resistance determinants but also clones previously associated with vaccine serotypes (CC15611A and CC23024F) highlights the importance of the accessory genome.

The Evolution and Distribution of Pneumococcal Serotypes in Adults Hospitalized With Community-Acquired Pneumonia in Spain Using a Serotype-Specific Urinary Antigen Detection Test: The CAPA Study, 2011-2018.

BACKGROUND: Spain introduced the 13-valent pneumococcal conjugate vaccine (PCV13) in the childhood National Immunization Program in 2015-2016 with coverage of 3 doses of 94.8% in 2018. We assessed the evolution of all pneumococcal, PCV13 vaccine type (VT), and experimental PCV20-VT (PCV13 + serotypes 8, 10A, 11A, 12F, 15B, 22F, 33F) hospitalized community-acquired pneumonia (CAP) in adults in Spain from 2011-2018. METHODS: A prospective observational study of immunocompetent adults (≥18 years) admitted to 4 Spanish hospitals with chest X-ray-confirmed CAP between November 2011 and November 2018. Microbiological confirmation was obtained using the Pfizer serotype-specific urinary antigen detection tests (UAD1/UAD2), BinaxNow test for urine, and conventional cultures of blood, pleural fluid, and high-quality sputum. RESULTS: Of 3107 adults hospitalized with CAP, 1943 were ≥65 years. Underlying conditions were present in 87% (n = 2704) of the participants. Among all patients, 895 (28.8%) had pneumococcal CAP and 439 (14.1%) had PCV13-VT CAP, decreasing from 17.9% (n = 77) to 13.2% (n = 68) from 2011-2012 to 2017-2018 (P = .049). PCV20-VT CAP occurred in 243 (23.8%) of those included in 2016-2018. The most identified serotypes were 3 and 8. Serotype 3 accounted for 6.9% (n = 215) of CAP cases, remaining stable during the study period, and was associated with disease severity. CONCLUSIONS: PCV13-VT caused a substantial proportion of CAP in Spanish immunocompetent adults 8 years after introduction of childhood PCV13 immunization. Improving direct PCV13 coverage of targeted adult populations could further reduce PCV13-VT burden, a benefit that could be increased further if PCV20 is licensed and implemented.

Assessment of the Optochin Susceptibility Test to Differentiate Streptococcus pneumoniae from other Viridans Group Streptococci.

BACKGROUND: Streptococcus pneumoniae identification has traditionally been based on two biochemical tests, susceptibility of pneumococci to optochin and solubility in bile-salt solution. Due to slowness and sometimes difficulty in interpretation, the bile solubility test has fallen into disuse. The main objective of this work was to assess the current effectiveness of the optochin susceptibility test in pneumococcal identification in clinical practice. MATERIALS AND METHODS: Overall 126 viridans group streptococci consecutively isolated from respiratory samples were analyzed using the optochin susceptibility test by picking one colony from the culture. Sixty-two were initially considered optochin susceptible, and 64 were considered optochin resistant and analyzed with the bile solubility test. If a discrepancy between the tests was observed (i.e., whether an isolate was optochin susceptible and bile insoluble or optochin resistant and bile soluble), then the optochin susceptibility test was repeated, adjusting the inoculum to a McFarland standard of 0.5. Species were identified by sequencing the lytA and recA genes. RESULTS: Twelve discrepancies were initially observed. The result of the repeated optochin test showed that the initial optochin test of 4 isolates had been wrongly interpreted. Of the remaining 8 discrepancies, 2 optochin-resistant bile-soluble isolates were identified by gene sequencing as S. pneumoniae, and of the 6 optochin-susceptible bile-nonsoluble isolates, 3 were identified as Streptococcus mitis and 3 as Streptococcus pseudopneumoniae. CONCLUSIONS: The optochin test correctly identified 90.5% of all recent viridans group streptococci clinical isolates which include both optochin susceptible (62/126 = 49.2%) and optochin resistant (64/126 = 50.8%) strains. Of the group of optochin susceptible viridans, 87.5% were correctly identified, and 93.5% of the optochin resistant group were correctly identified. However, this technique does not correctly differentiate between S. pneumoniae from other viridans group streptococci in the clinical setting. Additional testing is needed for that identification.

Comparison of Four Methods for Streptococcus pneumoniae Capsular Typing.

BACKGROUND: Pneumococcal capsular-type identification is essential for monitoring the epidemiology of pneumococcal infections and for establishing the effectiveness of pneumococcal vaccines. The objective of this work was to compare the accuracy of four methods of Streptococcus pneumoniae capsular typing. METHODS: A prospective blind study was carried out at Donostia University Hospital (northern Spain) to determine the capsular types of 50 pneumococcal clinical isolates using four techniques: a) S. PneumoStripTM: a reverse-hybridization strip-based commercial assay that detects 76 pneumococcal serotypes: 42 individually and 34 in pairs. b) FAF-mPCR: a single-step multiplex-PCR (polymerase chain reaction) assay combined with fragment analysis using automated fluorescent capillary electrophoresis, which can differentiate 92 serotypes in a single tube: 31 individually, 28 in pairs, and 33 in groups of 3 to 5 serotypes. c) PCRSeqTyping: which enables the detection of 91 serotypes after sequencing the regions of the cpsB gene in two steps: 59 directly and the remaining 32 serotypes in a second step. d) The Quellung reaction. RESULTS: The S. PneumoStripTM, FAF-mPCR and PCRSeqTyping identified the serotypes of all the 50 clinical isolates. With the Quellung reaction 46/50 (92%) isolates were correctly serotyped. The quickest technique was the S. PneumoStripTM, followed by the single-step multiplex PCR assay and PCRSeqTyping. The Quellung reaction was the slowest technique. CONCLUSIONS: The S. PneumoStripTM, PCRSeqTyping, and FAF-mPCR were very accurate techniques for pneumococcal serotyping, with S. PneumoStripTM obtaining results more rapidly. The combination of any of these S. pneumoniae molecular typing techniques and the Quellung reaction as confirmation reference method is a highly precise and fast strategy for the serotyping of high number of pneumococcal clinical isolates.

Epidemiological and clinical characteristics of Streptococcus tigurinus endocarditis.

BACKGROUND: Streptococcus tigurinus was recently described as a new streptococcal species within the viridans group streptococci (VGS). The objectives of the present work were to analyse the clinical and microbiological characteristics of S. tigurinus isolated from patients with bacteraemias, to determine the prevalence of S. tigurinus among VGS endocarditis in Spain, and to compare the clinical characteristics and outcomes of endocarditis caused by S. tigurinus and other VGS. METHODS: Retrospective nationwide study, performed between 2008 and 2016 in 9 Spanish hospitals from 7 different provinces comprising 237 cases of infective endocarditis. Streptococcal isolates were identified by sequencing fragments of their 16S rRNA, sodA and groEL genes. Clinical data of patients with streptococcal endocarditis were prospectively collected according to a pre-established protocol. RESULTS: Patients with endocarditis represented 7/9 (77.8%) and 26/86 (30.2%) of the bacteraemias caused by S. tigurinus and other VGS, respectively (p < 0.001), in two of the hospital participants. Among patients with streptococcal endocarditis, 12 different Streptococcus species were recognized being S. oralis, S. tigurinus and S. mitis the three more common. No relevant statistical differences were observed in the clinical characteristics and outcomes of endocarditis caused by the different VGS species. CONCLUSIONS: In this multicenter study performed in Spain, S. tigurinus showed a higher predilection for the endocardial endothelium as compared to other VGS. However, clinical characteristics and outcomes of endocarditis caused by S. tigurinus did not significantly differ from endocarditis caused by other oral streptococci.

Measuring Bacterial Glycosyl Hydrolase Activity with a Soluble Capture Probe by Mass Spectrometry.

A solution-phase enzymatic assay has been developed to track bacterial glycosyl hydrolase activity by surface-assisted MALDI-TOF mass spectrometry. Lactose was equipped with an azide-functionalized linker and was supplemented to bacterial cultures as an artificial substrate for bacterial ß-galactosidase enzyme. The azide linked glycoside probe was then covalently captured on an alkyne-functionalized indium tin oxide sample plate via a bio-orthogonal copper-catalyzed azide alkyne cycloaddition (CuAAC). The noncovalent immobilization of the alkyne capture tag via hydrophobic interactions on the ITO-sample plate allowed the analysis of the probe conjugate by surface-based mass spectrometry. The ratio of digested to nondigested lactose probe was then employed as a measure for bacterial hydrolase activity, which correlated well with bacterial growth measured by optical density. In addition, we established in a proof of concept experiment that the setup was well suited to identify antibiotic susceptibility of bacterial strains with a performance comparable to current state-of-the-art methods. While the proof of concept version is limited to the identification of a single enzyme activity, we envisage that the use of multiple substrate probes in a multiplexed version will allow the quantification of various glycosyl hydrolase activities with clinical relevance in a single experiment.

Primary Cutaneous Nocardia brasiliensis in a Spanish Child.

BACKGROUND: We report a case of a primary cutaneous nocardiosis by autochthonous Nocardia brasiliensis in a Spanish immunocompetent 9-year-old boy. METHODS: N. brasiliensis caused cellulitis showing the patient recovery after drainage and treatment with trimethoprim-sulfamethoxazole. Nocardia grew in pure culture and its identification was confirmed by sequencing (16S rRNA) and by MALDI-TOF MS (Bruker, Daltonics, Germany). CONCLUSIONS: In Spain although N. brasiliensis cutaneous infections in children are very infrequent should not be ruled out when an insect bite, stuck with a pine needle or an animal scratch has existed and the wound evolution is torpid.

Autochthonous Nocardia cerradoensis Infection in Humans, Spain, 2011 and 2014.

Nocardia cerradoensis was first isolated in 2003 in the El Cerrado region of Brazil; since then, only 2 human infections, in France and Spain, have been reported. We describe 3 autochthonous cases in residents of Spain during 2011 and 2014. Together these cases support the idea of an emerging global pathogenic microorganism.

Single-Step Multiplex PCR Assay for Determining 92 Pneumococcal Serotypes.

For pneumococcal disease surveillance, simple and cost-effective methods capable of determining all serotypes are needed. Combining a single-tube multiplex PCR with fluorescently labeled primers followed by amplicon analysis using automated fluorescent capillary electrophoresis, each serotype of 92 reference isolates and 297 recently collected clinical isolates was successfully determined.

Nocardia donostiensis sp. nov., isolated from human respiratory specimens.

Three human clinical isolates (X1654, X1655, and W9944) were recovered from the sputum and bronchial washings of two patients with pulmonary infections. The 16S rRNA gene sequence analysis of the isolates showed that they share 100 % sequence similarity with each other and belong to the genus Nocardia. Close phylogenetic neighbours are Nocardia brevicatena ATCC 15333(T) (98.6 %) and Nocardia paucivorans ATCC BAA-278T (98.4 %). The in silico DNA-DNA relatedness between the isolates ranges from 96.8 to 100 % suggesting that they belong to the same genomic species. The DNA-DNA relatedness between X1654 and N. brevicatena ATCC 15333(T) is 13.3 ± 2.3 % and N. paucivorans ATCC BAA-278T is 18.95 ± 1.1 % suggesting that they do not belong to the same genomic species. Believed to represent a novel species, these isolates were further characterised to establish their taxonomic standing within the genus. Chemotaxonomic data for isolate X1654 are consistent with those described for the genus Nocardia: this isolate produced saturated and unsaturated fatty acids, tuberculostearic acid (15.9 %), the major menaquinone was MK-8 (H4cyclic), mycolic acid chain lengths ranged from 38 to 58 carbons, produced meso-diaminopimelic acid with arabinose, glucose, and galactose as the whole cell sugars. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylinositol mannosides. The DNA G+C content is 66.7 mol  %. Based on the combination of phenotypic, chemotaxonomic, and genotypic data for X1654, X1655, and W9944, we conclude that these isolates represent a novel species within the genus Nocardia for which we propose the name Nocardia donostiensis sp. nov. with X1654(T) (=DSM 46814(T) = CECT 8839(T)) as the type strain.

MALDI-TOF MS as a tick identification tool in a tertiary hospital in Spain.

In Spain, as in other countries, the spectrum of tick-borne diseases and their number have increased in recent years. The tick identification, at species level, can be challenging outside research centers although this information is very usufull for decisions making. The performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in tick identification of specimens collected from patients have been seldomly reported. The aim of the present study was to desing a protein-extraction protocol and build a tick-legs reference spectra. This protocol was then validated using specimens from both patients and non-patient sources. Nine species of ticks that usually bites humans in Spain were included: Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis punctata, Hyalomma lusitanicum, Hyalomma marginatum, Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus pusillus and Rhipicephalus sanguineus sensu lato. Other less-frequent biting species were also included: Haemaphysalis inermis, Haemaphysalis concinna, Hyalomma scupense, Ixodes frontalis, Ixodes hexagonus, and Argas sp. specimens were identified by PCR and sequencing of a fragment of the 16S rRNA gene of ticks. In the tests performed with non-patient collected specimens, a 100% correlation was observed between molecular methods and MS, while in the tests performed with ticks collected from patients a 92.59% correlation was observed. Misidentification was observed only in two of I. ricinus nymphs (identified as Ctenocephalides felis). Therefore, mass- spectrometry can be confidently used as a tick identification tool in a hospital setting for the rapid identification of tick vectors.

Impact of the progressive uptake of pneumococcal conjugate vaccines on the epidemiology and antimicrobial resistance of invasive pneumococcal disease in Gipuzkoa, northern Spain, 1998-2022.

Objectives: To analyze the impact of pneumococcal conjugate vaccines (PCVs) on the incidence of invasive pneumococcal diseases (IPDs) and pneumococcal antibiotic resistance in Gipuzkoa, northern Spain for a 25 years period. Methods: All cases of IPD confirmed by culture between 1998 and 2022 in a population of around 427,416 people were included. Pneumococci were serotyped and antimicrobial susceptibility was assessed by the EUCAST guidelines. Results: Overall, 1,516 S. pneumoniae isolates were collected. Annual IPD incidence rates (per 100,000 people) declined from 19.9 in 1998-2001 to 11.5 in 2017-19 (42.2% reduction), especially in vaccinated children (from 46.7 to 24.9) and non-vaccinated older adult individuals (from 48.0 to 23.6). After PCV13 introduction, the decrease in the incidence of infections caused by PCV13 serotypes was balanced by the increase in the incidence of non-PCV13 serotypes. In the pandemic year of 2020, IPD incidence was the lowest: 2.81. The annual incidence rates of penicillin-resistant isolates also decreased, from 4.91 in 1998-2001 to 1.49 in 2017-19 and 0.70 in 2020. Since 2017, serotypes 14, 19A, and 11A have been the most common penicillin-resistant types. The incidence of erythromycin-resistant strains declined, from 3.65 to 1.73 and 0.70 in the same years. Conclusion: PCV use was associated with declines in the incidence of IPD and the spread of non-vaccine serotypes, that balanced the beneficial effect off PCV13, some of them showing high rates of antibiotic resistance.

Lung microbiome on admission in critically ill patients with acute bacterial and viral pneumonia.

Composition of pulmonary microbiome of patients with severe pneumonia is poorly known. The aim of this work was to analyse the lung microbiome of patients admitted to the intensive care unit  (ICU) with severe community acquired pneumonia (CAP) between 2019 and 2021 in comparison with a control group of 6 patients undergoing digestive surgery. As a second objective, the diagnostic capabilities of metagenomics was also studied in a small group of selected patients. The lung microbiome of patients with viral (5 with Influenza A and 8 with SARS-CoV-2) pneumonia at admission showed a similar diversity as the control group (p = 0.140 and p = 0.213 respectively). Contrarily, the group of 12 patients with pneumococcal pneumonia showed a significant lower Simpson´s index (p = 0.002). In the control group (n = 6) Proteobacteria (36.6%), Firmicutes (24.2%) and Actinobacteria (23.0%) were the predominant phyla. In SARS-CoV-2 patients (n = 8), there was a predominance of Proteobacteria (mean 41.6%) (Moraxella and Pelomonas at the genus level), Actinobacteria (24.6%) (Microbacterium) and Firmicutes (22.8%) mainly Streptococcus, Staphylococcus and Veillonella. In patients with Influenza A pneumonia (n = 5) there was a predominance of Firmicutes (35.1%) mainly Streptococcus followed by Proteobacteria (29.2%) (Moraxella, Acinetobacter and Pelomonas). In the group of pneumococcal pneumonia (n = 12) two phyla predominated: Firmicutes (53.1%) (Streptococcus) and Proteobacteria (36.5%) (Haemophilus). In the 7 patients with non-pneumococcal bacterial pneumonia Haemophilus influenzae (n = 2), Legionella pneumophila (n = 2), Klebsiella pneumoniae, Streptococcus pyogenes and Leptospira were detected by metagenomics, confirming the diagnosis done using conventional microbiological techniques. The diversity of the respiratory microbiome in patients with severe viral pneumonia at ICU admission was similar to that of the control group. Contrarily, patients with pneumococcal pneumonia showed a lower grade of diversity. At initial stages of SARS-CoV-2 infection, no important alterations in the pulmonary microbiome were observed. The analysis of bacterial microbiome showed promising results as a diagnostic tool.

Pneumococcal Serotypes Associated with Community-Acquired Pneumonia Hospitalizations in Adults in Spain, 2016-2020: The CAPA Study.

Newer higher valency pneumococcal conjugate vaccines (PCVs) have the potential to reduce the adult community-acquired pneumonia (CAP) burden. We describe the evolution and distribution of adult community-acquired pneumonia (CAP) serotypes in Spain, focusing on serotypes contained in the 20-valent PCV (PCV20). This was a prospective, observational study of chest X-ray (CXR)-confirmed CAP in immunocompetent adults hospitalized in one of four Spanish hospitals between November 2016 and November 2020. Pneumococci were isolated from cultures and detected in urine using BinaxNow® and Pfizer serotype-specific urinary antigen tests UAD1 and UAD2. We included 1948 adults hospitalized with CXR-CAP. The median age was 69.0 years (IQR: 24 years). At least one comorbidity was present in 84.8% (n = 1653) of patients. At admission, 76.1% of patients had complicated pneumonia. Pneumococcus was identified in 34.9% (n = 680) of study participants. The PCV20 vaccine-type CAP occurred in 23.9% (n = 465) of all patients, 68.4% (n = 465) of patients with pneumococcal CAP, and 82.2% (83/101) of patients who had pneumococcus identified by culture. Serotypes 8 (n = 153; 7.9% of all CAP) and 3 (n = 152; 7.8% of all CAP) were the most frequently identified. Pneumococcus is a common cause of hospitalized CAP among Spanish adults and serotypes contained in PCV20 caused the majority of pneumococcal CAP.

Decline and rise of the antimicrobial susceptibility of Streptococcus pneumoniae isolated from middle ear fluid in children: influence of changes in circulating serotypes.

Changes in the antimicrobial susceptibility of Streptococcus pneumoniae causing otitis media were studied in 916 isolates from children <5 years old between 1999 and 2010 in a region of northern Spain. The rate of antimicrobial resistance decreased between the period before the introduction of the heptavalent pneumococcal conjugate vaccine (from 1999 to 2001) and the period from 2005 to 2007. However, in 2008 to 2010, resistance rates increased again due to the spread of serotype 19A, especially the multidrug-resistant ST320 and ST276 clones.

Dynamics of pneumococcal nasopharyngeal carriage in healthy children attending a day care center in northern Spain. Influence of detection techniques on the results.

BACKGROUND: Pneumococcal nasopharyngeal carriage precedes invasive infection and is the source for dissemination of the disease. Differences in sampling methodology, isolation or identification techniques, as well as the period (pre -or post-vaccination) when the study was performed, can influence the reported rates of colonization and the distribution of serotypes carried. OBJECTIVES: To evaluate the prevalence and dynamics of pneumococcal nasopharyngeal colonization in healthy children aged 6-34 months attending a day care center with a high level of hygiene and no overcrowding. The study was performed 3-4 years after the 7-valent pneumococcal vaccine was introduced, using multiple methodologies to detect and characterize the isolates. METHODS: Over 12 months, 25 children were sampled three times, 53 children twice and 27 children once. Three Streptococcus pneumoniae typing techniques were used: Quellung, Pneumotest-Latex-kit and multiplex-polymerase chain reaction (PCR). The similarity of isolates of the same serotype was established by pulsed field gel electrophoresis (PFGE) and occasionally the multilocus sequence type (ST) was also determined. RESULTS: Overall pneumococcal carriage and multiple colonization rates were 89.5% (94/105) and 39%, respectively. Among 218 pneumococci detected, 21 different serotypes and 13 non-typeable isolates were found. The most prevalent serotypes were 19A, 16F and 15B. Serotypes 15B, 19A and 21 were mainly found as single carriage; in contrast serotypes 6B, 11A and 20, as well as infrequent serotypes, were isolated mainly as part of multiple carriage. Most 19A isolates were ST193 but most serotypes showed high genetic heterogeneity. Changes in the pneumococci colonizing each child were frequent and the same serotype detected on two occasions frequently showed a different genotype. By multiplex-PCR, 100% of pneumococci could be detected and 94% could be serotyped versus 80.3% by the Quellung reaction and Pneumotest-Latex in combination (p < 0.001). CONCLUSIONS: Rates of S. pneumoniae carriage and multiple colonization were very high. Prevalent serotypes differed from those found in similar studies in the pre-vaccination period. In the same child, clearance of a pneumococcal strain and acquisition of a new one was frequent in a short period of time. The most effective technique for detecting pneumococcal nasopharyngeal carriers was multiplex-PCR.

Considerations on diagnosis and surveillance measures of PTEN hamartoma tumor syndrome: clinical and genetic study in a series of Spanish patients.

BACKGROUND: The limited knowledge about the PTEN hamartoma tumor syndrome (PHTS) makes its diagnosis a challenging task. We aimed to define the clinical and genetic characteristics of this syndrome in the Spanish population and to identify new genes potentially associated with the disease. RESULTS: We reviewed the clinical data collected through a specific questionnaire in a series of 145 Spanish patients with a phenotypic features compatible with PHTS and performed molecular characterization through several approaches including next generation sequencing and whole exome sequencing (WES). Macrocephaly, mucocutaneous lesions, gastrointestinal polyposis and obesity are prevalent phenotypic features in PHTS and help predict the presence of a PTEN germline variant in our population. We also find that PHTS patients are at risk to develop cancer in childhood or adolescence. Furthermore, we observe a high frequency of variants in exon 1 of PTEN, which are associated with renal cancer and overexpression of KLLN and PTEN. Moreover, WES revealed variants in genes like NEDD4 that merit further research. CONCLUSIONS: This study expands previously reported findings in other PHTS population studies and makes new contributions regarding clinical and molecular aspects of PHTS, which are useful for translation to the clinic and for new research lines.

Antimicrobial susceptibilities and serotypes of Streptococcus pneumoniae isolates from elderly patients with pneumonia and acute exacerbation of chronic obstructive pulmonary disease.

In the elderly, Streptococcus pneumoniae is the most common cause of pneumonia and one of the most frequently isolated pathogens in cases of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). This study was conducted to compare the pneumococcal isolates obtained during episodes of AECOPD and pneumonia in patients of ≥65 years old and to analyze whether in patients with AECOPD and pneumonia within a short interval, the same isolate caused both episodes. This laboratory-based study was performed between 2005 and 2008. Pneumococcal isolates from episodes of pneumonia (n = 401) and AECOPD (n = 398), matched one-to-one by date of isolation, were characterized. The serotypes and genotypes of other pneumococcal isolates causing pneumonia and AECOPD in the same patient were compared. In patients with pneumonia, COPD as an underlying disease was not associated with more-drug-resistant pneumococci. In contrast, isolates causing AECOPD showed higher rates of resistance than those causing pneumonia. Serotypes 1, 3, and 7F were more frequent in pneumonia. The same pneumococcus was involved in 25.7% (9/35 patients) of patients with two consecutive AECOPD episodes but in only 6.3% (2/32 patients) of COPD patients with pneumonia and exacerbation (Fisher's exact test; P = 0.047). Less invasive serotypes were isolated more often in AECOPD and were more resistant to antimicrobials. The presence of a specific pneumococcal serotype in AECOPD does not predict the etiology of subsequent pneumonia.

Genomic Virulence Features of Two Novel Species Nocardia barduliensis sp. nov. and Nocardia gipuzkoensis sp. nov., Isolated from Patients with Chronic Pulmonary Diseases.

Strains 335427T and 234509T, isolated from two 76-year-old patients with chronic pulmonary diseases, were the subject of polyphasic taxonomic studies and comparative genomic analyses for virulence factors. The 16 rRNA gene sequence similarity between strains 335427T and 234509T and their closest phylogenetic neighbors Nocardia asiatica NBRC 100129T and Nocardia abscessus NBRC 100374T were 99.5% and 100%, respectively. Digital DNA-DNA hybridization values between the aforementioned studied strains were well below the 70% threshold for assigning prokaryotic strains to a novel species. Strains 335427T and 234509T have genome sizes of 8.49 Mpb and 8.07 Mpb, respectively, with G + C content of 68.5%. Isolate 335427T has C16:0, C18:1 ω9c, C18:0 and C18:0 10 methyl as major fatty acids (>15%) and mycolic acids formed of 52-54 carbon atoms. However, only C18:1 ω9c was detected for isolate 234509T, which had mycolic acids with 44-56 carbon. Based on phenotypic and genetic data, strains 335427T (DSM 109819T = CECT 9924T) and 234509T (DSM 111366T = CECT 30129T) merit recognition as novel species, which are named Nocardia barduliensis sp. nov. and Nocardia gipuzkoensis sp. nov., respectively. All the strains studied had homologous VF-associated genes to those described in M. tuberculosis, including experimentally verified virulence genes in humans related to tuberculosis. The narGHIJ (nitrate reduction pathway) and gvpAFGOJLMK (gas vesicles) genetic maps of strains 335427T, 234509T, NBRC 100129T and NBRC 100374T showed the same syntenic block and raise the question of whether their functions are interlinked during the infection of the human host. However, further research is required to decipher the role of the gas vesicle in the pathogenicity mechanism of Nocardia spp.