Sustained increase of paediatric invasive Streptococcus pyogenes infections dominated by M1UK and diverse emm12 isolates, Portugal, September 2022 to May 2023.

Since autumn 2022, observed numbers of paediatric invasive group A Streptococcus infections in Portugal (n = 89) were higher than in pre-COVID-19 seasons. Between September 2022 and May 2023, the dominant diagnoses were pneumonia (25/79), mostly with empyema (20/25), and sepsis (22/79). A number of cases required admission to intensive care (27/79) and surgery (35/79), and the case fatality rate was 5.1% (4/79). Genomic sequencing (n = 55) revealed multiple genetic lineages, dominated by the M1UK sublineage (26/55) and more diverse emm12 isolates (12/55).

Pediatric Complicated Pneumonia Caused by Streptococcus pneumoniae Serotype 3 in 13-Valent Pneumococcal Conjugate Vaccinees, Portugal, 2010-2015.

Despite use of 7-valent pneumococcal conjugate vaccine, incidence of pleural effusion and empyema (pediatric complicated pneumococcal pneumonia [PCPP]) is reportedly increasing globally. We cultured and performed PCR on 152 pleural fluid samples recovered from pediatric patients in Portugal during 2010-2015 to identify and serotype Streptococcus pneumoniae. We identified only 17 cases by culture, but molecular methods identified S. pneumoniae in 68% (92/135) of culture-negative samples. The most frequent serotypes were 3, 1, and 19A, together accounting for 62% (68/109) of cases. Nineteen cases attributable to 13-valent pneumococcal conjugate vaccine (PCV13) serotypes (mostly serotype 3) were detected among 22 children age-appropriately vaccinated with PCV13. The dominance of the additional serotypes included in PCV13 among PCPP cases in Portugal continues, even with PCV13 available on the private market (without reimbursement) since 2010 and with average annual coverage of 61% among age-eligible children. Our data suggest reduced effectiveness of PCV13 against serotype 3 PCPP.

Automatic determination of NET (neutrophil extracellular traps) coverage in fluorescent microscopy images.

MOTIVATION: Neutrophil extracellular traps (NETs) are believed to be essential in controlling several bacterial pathogens. Quantification of NETs in vitro is an important tool in studies aiming to clarify the biological and chemical factors contributing to NET production, stabilization and degradation. This estimation can be performed on the basis of fluorescent microscopy images using appropriate labelings. In this context, it is desirable to automate the analysis to eliminate both the tedious process of manual annotation and possible operator-specific biases. RESULTS: We propose a framework for the automated determination of NET content, based on visually annotated images which are used to train a supervised machine-learning method. We derive several methods in this framework. The best results are obtained by combining these into a single prediction. The overall Q(2) of the combined method is 93%. By having two experts label part of the image set, we were able to compare the performance of the algorithms to the human interoperator variability. We find that the two operators exhibited a very high correlation on their overall assessment of the NET coverage area in the images (R(2) is 97%), although there were consistent differences in labeling at pixel level (Q(2), which unlike R(2) does not correct for additive and multiplicative biases, was only 89%). AVAILABILITY AND IMPLEMENTATION: Open source software (under the MIT license) is available at https://github.com/luispedro/Coelho2015_NetsDetermination for both reproducibility and application to new data.

Changes in Streptococcus pyogenes causing invasive disease in Portugal: evidence for superantigen gene loss and acquisition.

The emergence of highly virulent and successful Streptococcus pyogenes (group A streptococci - GAS) clones has been attributed to the exchange of virulence factors by lateral gene transfer mechanisms, which strongly contribute to genomic diversity. We characterized a collection of 191 GAS isolates recovered from normally sterile sites in Portugal during 2006-2009 and compared them to invasive isolates obtained during 2000-2005. Antimicrobial resistance rates did not change significantly between the two periods and were generally low. In 2006-2009, emm1, emm89, emm3, and emm6 represented 60% of the isolates. The chromosomally encoded superantigen (SAg) genes speG and smeZ were present in the majority (>90%) of the isolates, while speJ was found in only 45%. The phage encoded SAgs varied greatly in prevalence (2-53%). The distribution of emm types, pulsed-field gel electrophoresis profiling (PFGE) clusters, and SAg profiles changed significantly between the periods, although there were no statistically supported changes in the prevalence of individual types. While the macrolide susceptible clone emm1-T1-ST28 remained dominant (28%), there was a significant decrease in clonal diversity as indicated by both PFGE profiling and emm typing. This was accompanied by intra-clonal divergence of SAg profiles, which was statistically confirmed for isolates representing emm1, emm28, and emm44. This diversification was associated with the loss and acquisition of SAg genes, carried by phages and of chromosomal origin. These data suggest an ongoing genomic diversification of GAS invasive isolates in Portugal that may contribute to the persistence of clones with improved fitness or virulence.

Group A streptococci clones associated with invasive infections and pharyngitis in Portugal present differences in emm types, superantigen gene content and antimicrobial resistance.

BACKGROUND: A few lineages of Group A streptococci (GAS) have been associated with a reemergence of severe invasive streptococcal disease in developed countries. However, the majority of the comparisons between invasive and non-invasive GAS isolates have been performed for collections of reduced genetic diversity or relied on limited typing information to distinguish clones. We characterized by several typing methods and compared a collection of 160 isolates recovered from normally sterile sites with 320 isolates associated with pharyngitis and recovered in the same time period in Portugal. RESULTS: Although most of the isolates belonged to clones that were equally prevalent in invasive infections and pharyngitis, we identified markers of invasiveness, namely the emm types 1 and 64, and the presence of the speA and speJ genes. In contrast, emm4, emm75, and the ssa and speL/M genes were significantly associated with pharyngitis. There was a strong agreement between the emm type, the superantigen (SAg) genes and the clusters defined by pulsed-field gel electrophoresis (PFGE) profiling. Therefore, combinations of particular emm types and SAg genes frequently co-occurred in the same PFGE cluster, but there was no synergistic or antagonistic interaction between them in determining invasiveness. Only macrolide-susceptible PFGE clones were significantly associated with invasive infections or pharyngitis, while the clones of resistant isolates sharing all other molecular properties analyzed were equally prevalent in the two groups of isolates. CONCLUSIONS: This study confirmed the importance of the widely disseminated emm1-T1-ST28 clone in invasive infections but also identified other clones linked to either invasive infections (emm64-ST164) or pharyngitis (emm4-T4-ST39), which may be more limited in their temporal and geographical spread. Clonal properties like some emm types or SAg genes were associated with disease presentation, highlighting the importance of bacterial genetic factors to the outcome of GAS infections, although other, yet unidentified factors may also play an important role.

Continued Vaccine Breakthrough Cases of Serotype 3 Complicated Pneumonia in Vaccinated Children, Portugal (2016-2019).

We previously reported that despite the use of pneumococcal conjugate vaccines (PCVs), vaccine serotypes remained important causes of pneumonia with pleural effusion and empyema (pediatric complicated pneumococcal pneumonia [PCPP]). We cultured and performed PCR on 174 pleural fluid samples recovered from pediatric patients in Portugal from 2016 to 2019 to identify and serotype Streptococcus pneumoniae. Most PCPP cases (n = 87/98) were identified by PCR only. Serotypes 3 (67%), 14, and 8 (5% each) were the most frequent. Vaccine breakthrough cases were seen among age-appropriately, 13-valent, PCV vaccinated children (median: 3 years, range: 17 months to 7 years), mostly with serotype 3 (n = 27) but also with serotypes 14 and 19A (n = 2 each). One breakthrough was seen with serotype 14 in an age-appropriately, 10-valent, PCV-vaccinated child and another with serotype 3 in a child to whom the 23-valent polysaccharide vaccine was administered. While the relative risk of serotype 1 PCPP decreased almost 10-fold from the period of 2010 to 2015 to the period of 2016 to 2019 (relative risk [RR] = 0.106), that of serotype 3 PCPP almost doubled (RR = 1.835). Our data highlight the importance of molecular diagnostics in identifying PCPP and document the continued importance of serotype 3 PCPP, even when PCV13 use with almost universal coverage could be expected to reduce exposure to this serotype. IMPORTANCE The use of conjugate vaccines against Streptococcus pneumoniae in children has led to substantial reductions in pneumococcal invasive disease. However, the reductions seen in each of the 13 serotypes currently included in the highest-valency vaccine approved for use in children (PCV13), were not the same. It is becoming clear that most vaccine breakthroughs worldwide involve serotype 3 and are frequently associated with complicated pneumonia cases, often with empyema or pleural effusion. Here, we show that despite almost universal PCV13 use, which would be expected to reduce vaccine serotype circulation and further reinforce vaccine direct protection, pneumococci and serotype 3 remain the major causes of pediatric complicated pneumonia. Molecular methods are essential to identify and serotype pneumococci in these cases, which frequently reflect vaccine breakthroughs. A broader use of molecular diagnostics will be essential to determine the role of this important serotype in the context of PCV13 use in different geographic regions.

Streptococcus pyogenes Causing Skin and Soft Tissue Infections Are Enriched in the Recently Emerged emm89 Clade 3 and Are Not Associated With Abrogation of CovRS.

Although skin and soft tissue infections (SSTI) are the most common focal infections associated with invasive disease caused by Streptococcus pyogenes (Lancefield Group A streptococci - GAS), there is scarce information on the characteristics of isolates recovered from SSTI in temperate-climate regions. In this study, 320 GAS isolated from SSTI in Portugal were characterized by multiple typing methods and tested for antimicrobial susceptibility and SpeB activity. The covRS and ropB genes of isolates with no detectable SpeB activity were sequenced. The antimicrobial susceptibility profile was similar to that of previously characterized isolates from invasive infections (iGAS), presenting a decreasing trend in macrolide resistance. However, the clonal composition of SSTI between 2005 and 2009 was significantly different from that of contemporary iGAS. Overall, iGAS were associated with emm1 and emm3, while SSTI were associated with emm89, the dominant emm type among SSTI (19%). Within emm89, SSTI were only significantly associated with isolates lacking the hasABC locus, suggesting that the recently emerged emm89 clade 3 may have an increased potential to cause SSTI. Reflecting these associations between emm type and disease presentation, there were also differences in the distribution of emm clusters, sequence types, and superantigen gene profiles between SSTI and iGAS. According to the predicted ability of each emm cluster to interact with host proteins, iGAS were associated with the ability to bind fibrinogen and albumin, whereas SSTI isolates were associated with the ability to bind C4BP, IgA, and IgG. SpeB activity was absent in 79 isolates (25%), in line with the proportion previously observed among iGAS. Null covS and ropB alleles (predicted to eliminate protein function) were detected in 10 (3%) and 12 (4%) isolates, corresponding to an underrepresentation of mutations impairing CovRS function in SSTI relative to iGAS. Overall, these results indicate that the isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites, supporting a role for mutations impairing CovRS activity specifically in invasive infection and suggesting that this role relies on a differential regulation of other virulence factors besides SpeB.

Congenital adrenal hyperplasia: focus on the molecular basis of 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by defects in one of several steroidogenic enzymes involved in the synthesis of cortisol from cholesterol in the adrenal glands. More than 90% of cases are caused by 21-hydroxylase deficiency, and the severity of the resulting clinical symptoms varies according to the level of 21-hydroxylase activity. 21-Hydroxylase deficiency is usually caused by mutations in the CYP21A2 gene, which is located on the RCCX module, a chromosomal region highly prone to genetic recombination events that can result in a wide variety of complex rearrangements, such as gene duplications, gross deletions and gene conversions of variable extensions. Molecular genotyping of CYP21A2 and the RCCX module has proved useful for a more accurate diagnosis of the disease, and prenatal diagnosis. This article summarises the clinical features of 21-hydroxylase deficiency, explains current understanding of the disease at the molecular level, and highlights recent developments, particularly in diagnosis.

Consequences of the variability of the CovRS and RopB regulators among Streptococcus pyogenes causing human infections.

To evaluate the importance of covRS and ropB mutations in invasive disease caused by Group A Streptococci (GAS), we determined the sequence of the covRS and ropB genes of 191 isolates from invasive infections and pharyngitis, comprising a diverse set of emm types and multilocus sequence types. The production of SpeB and the activity of NAD glycohydrolase (NADase) and streptolysin S (SLS) were evaluated. The results support the acquisition of null covS alleles (predicted to eliminate protein function), resulting in downregulation of SpeB and upregulation of NADase and SLS, as a mechanism possibly contributing to higher invasiveness. Among the isolates tested, this mechanism was found to be uncommon (10% of invasive isolates) and was not more prevalent among clones with enhanced invasiveness (including M1T1) but occurred in diverse genetic backgrounds. In lineages such as emm64, these changes did not result in upregulation of NADase and SLS, highlighting the diversity of regulatory pathways in GAS. Despite abrogating SpeB production, null alleles in ropB were not associated with invasive infection. The covRS and ropB genes are under stabilising selection and no expansion of isolates carrying null alleles has been observed, suggesting that the presence of these regulators is important for overall fitness.

Macrolide-resistant Streptococcus pyogenes: prevalence and treatment strategies.

Although penicillin remains the first-choice treatment for Streptococcus pyogenes infection, macrolides are important alternatives for allergic patients and lincosamides are recommended together with ß-lactams in invasive infections. S. pyogenes may exhibit macrolide resistance because of active efflux (mef genes) or target modification (erm genes), the latter conferring cross resistance to lincosamides and streptogramin B. Worldwide, resistance is restricted to a limited number of genetic lineages, despite resistance genes being encoded on mobile genetic elements. For reasons that are not completely clear, resistance and the associated phenotypes are highly variable across countries. Although resistance remains high in several countries, particularly in Asia, an overall decreasing trend of resistance has been noted in recent years, mostly in Europe. This decrease is not always accompanied by declines in macrolide consumption, suggesting significant roles of other factors in determining the dynamics of macrolide-resistant clones. Continued surveillance is needed to obtain further insights into the forces governing macrolide resistance in S. pyogenes.

Novel role of the antimicrobial peptide LL-37 in the protection of neutrophil extracellular traps against degradation by bacterial nucleases.

Neutrophil extracellular traps (NETs) have been described as a fundamental innate immune defence mechanism. They consist of a nuclear DNA backbone associated with different antimicrobial peptides (AMPs) which are able to engulf and kill pathogens. The AMP LL-37, a member of the cathelicidin family, is highly present in NETs. However, the function of LL-37 within NETs is still unknown because it loses its antimicrobial activity when bound to DNA in the NETs. Using immunofluorescence microscopy, we demonstrate that NETs treated with LL-37 are distinctly more resistant to S. aureus nuclease degradation than nontreated NETs. Biochemical assays utilising a random LL-37-fragment library indicated that the blocking effect of LL-37 on nuclease activity is based on the cationic character of the AMP, which facilitates the binding to neutrophil DNA, thus protecting it from degradation by the nuclease. In good correlation to these data, the cationic AMPs human beta defensin-3 and human neutrophil peptide-1 showed similar protection of neutrophil-derived DNA against nuclease degradation. In conclusion, this study demonstrates a novel role of AMPs in host immune defence: beside its direct antimicrobial activity against various pathogens, cationic AMPs can stabilise neutrophil-derived DNA or NETs against bacterial nuclease degradation.

The Viriato study: update on antimicrobial resistance of microbial pathogens responsible for community-acquired respiratory tract infections in Portugal.

The Viriato study is a prospective, multicentre laboratory-based surveillance study of antimicrobial susceptibility in which 30 microbiology laboratories throughout Portugal are asked to isolate, identify and submit to a central laboratory for testing Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis responsible for community-acquired lower respiratory tract infections and Streptococcus pyogenes from tonsillitis. To monitor changes in antimicrobial resistance patterns of these frequent respiratory pathogens. Susceptibility was determined by disk diffusion (Kirby-Bauer) or using Etest strips following the Clinical and Laboratory Standards Institute guidelines. From 1999 to 2007 over 13 900 isolates were analysed. Among S. pneumoniae penicillin non-susceptibility decreased from 25% in 1999 to 18% in 2007 (p = 0.002) but resistance to macrolides showed a steady increase, reaching 20% in the last 6 years. Resistance to amoxicillin and the quinolones remained stable and very low (1-2%) throughout the study period. Antimicrobial resistance among H. influenzae and M. catarrhalis remained stable. The most significant resistance was to ampicillin, of 10-12% and greater than 80%, respectively, as a result of the production of beta-lactamases. Macrolide resistance among S. pyogenes was stable during 1999-2003 (20-23%) but after 2003 there was a steady decline in resistance, which in 2007 reached 10%. The Viriato surveillance study showed that penicillin remains the most active antimicrobial agent against S. pyogenes causing tonsillitis, and amoxicillin-clavulanate and the quinolones are the most active in vitro simultaneously against S. pneumoniae, H. influenzae and M. catarrhalis responsible for community-acquired lower respiratory tract infections in Portugal.

CYP21A2 mutations in Portuguese patients with congenital adrenal hyperplasia: identification of two novel mutations and characterization of four different partial gene conversions.

More than 90% of congenital adrenal hyperplasia (CAH) cases are caused by 21-hydroxylase deficiency. In this study, the CYP21 gene was genotyped in 56 Portuguese unrelated patients with clinical symptoms of 21-hydroxylase deficiency, in a total of 112 independent alleles. CYP21A2 mutations were identified in 99.1% of the alleles. The most common point mutation was 1688G>T (25.9%). A previously unreported partial gene conversion, extending from exon 1 to 7, was found in 16.1% of the alleles, in most cases associated to the mutation 1688G>T in the other chromosome, and in patients with nonclassical CAH. Other three distinct partial gene conversions were also identified, with lower frequencies: one extends from exon 1 to 3 and the others from exons 3 to 7 and 3 to 8. Two novel mutations were identified in two salt-wasting patients: a putative splicing mutation, IVS2+5G>A, and the transition 2557C>T, that gives rise to the nonsense mutation R445X. Seven point mutations and a partial gene conversion were responsible for 88 of the studied disease causing alleles, and the overall concordance between genotype and phenotype was 92.9%. With this study the molecular basis of CAH was characterized, for the first time, in Portuguese patients, providing useful results for clinicians in terms of prediction of disease severity, genetic and prenatal counseling.