Comparison of next generation technologies and bioinformatics pipelines for capsular typing of Streptococcus pneumoniae.

Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods. In silico serotyping from WGS has not yet been applied to long-read sequences produced by third-generation technologies. The objective of the study was to determine the capsular types of pneumococci causing invasive disease in Catalonia (Spain) using serological typing and WGS and to compare the performance of different bioinformatics pipelines using short- and long-read data from WGS. All invasive pneumococcal pediatric isolates collected in Hospital Sant Joan de Déu (Barcelona) from 2013 to 2019 were included. Isolates were assigned a capsular type by serological testing based on anticapsular antisera and by different WGS-based pipelines: Illumina sequencing followed by serotyping with PneumoCaT, SeroBA, and Pathogenwatch vs MinION-ONT sequencing coupled with serotyping by Pathogenwatch from pneumococcal assembled genomes. A total of 119 out of 121 pneumococcal isolates were available for sequencing. Twenty-nine different serotypes were identified by serological typing, with 24F (n = 17; 14.3%), 14 (n = 10; 8.4%), and 15B/C (n = 8; 6.7%) being the most common serotypes. WGS-based pipelines showed initial concordance with serological typing (>91% of accuracy). The main discrepant results were found at the serotype level within a serogroup: 6A/B, 6C/D, 9A/V, 11A/D, and 18B/C. Only one discrepancy at the serogroup level was observed: serotype 29 by serological testing and serotype 35B/D by all WGS-based pipelines. Thus, bioinformatics WGS-based pipelines, including those using third-generation sequencing, are useful for pneumococcal capsular assignment. Possible discrepancies between serological typing and WGS-based approaches should be considered in pneumococcal capsular-type surveillance studies.

Differential nasopharyngeal microbiota composition in children according to respiratory health status.

Acute respiratory infections (ARIs) constitute one of the leading causes of antibiotic administration, hospitalization and death among children <5 years old. The upper respiratory tract microbiota has been suggested to explain differential susceptibility to ARIs and modulate ARI severity. The aim of the present study was to investigate the relation of nasopharyngeal microbiota and other microbiological parameters with respiratory health and disease, and to assess nasopharyngeal microbiota diagnostic utility for discriminating between different respiratory health statuses. We conducted a prospective case-control study at Hospital Sant Joan de Deu (Barcelona, Spain) from 2014 to 2018. This study included three groups of children <18 years with gradual decrease of ARI severity: cases with invasive pneumococcal disease (IPD) (representative of lower respiratory tract infections and systemic infections), symptomatic controls with mild viral upper respiratory tract infections (URTI), and healthy/asymptomatic controls according to an approximate case-control ratio 1:2. Nasopharyngeal samples were collected from participants for detection, quantification and serotyping of pneumococcal DNA, viral DNA/RNA detection and 16S rRNA gene sequencing. Microbiological parameters were included on case-control classification models. A total of 140 subjects were recruited (IPD=27, URTI=48, healthy/asymptomatic control=65). Children's nasopharyngeal microbiota composition varied according to respiratory health status and infection severity. The IPD group was characterized by overrepresentation of Streptococcus pneumoniae, higher frequency of invasive pneumococcal serotypes, increased rate of viral infection and underrepresentation of potential protective bacterial species such as Dolosigranulum pigrum and Moraxella lincolnii. Microbiota-based classification models differentiated cases from controls with moderately high accuracy. These results demonstrate the close relationship existing between a child's nasopharyngeal microbiota and respiratory health, and provide initial evidence of the potential of microbiota-based diagnostics for differential diagnosis of severe ARIs using non-invasive samples.

Validation and implementation of a direct RT-qPCR method for rapid screening of SARS-CoV-2 infection by using non-invasive saliva samples.

OBJECTIVE: To validate and implement an optimized screening method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA combining use of self-collected raw saliva samples, single-step heat-treated virus inactivation and RNA extraction, and direct RT-qPCR. METHODS: This was a three-phase study conducted in Barcelona (Spain) during June to October, 2020. The three phases were (1) analytical validation against standard RT-qPCR in saliva samples; (2) diagnostic validation against standard RT-qPCR using paired saliva-nasopharyngeal samples obtained from asymptomatic teenagers and adults in a sports academy; and (3) pilot screening of asymptomatic health workers in a tertiary hospital. RESULTS: In phase 1, the detection yield of the new method was comparable to that of standard RT-qPCR. In phase 2, the diagnostic sensitivity and specificity values in 303 self-collected saliva samples were 95.7% (95% confidence interval 79.0-99.2%) and 100.0% (95% confidence interval 98.6-100.0%), respectively. In phase 3, only 17 (0.6%) of the saliva samples self-collected by 2709 participants without supervision were invalid. The rapid analytical workflow with the new method (up to 384 batched samples could be processed in less than 2 hours) yielded 24 (0.9%) positive results in the remaining 2692 saliva samples. Paired nasopharyngeal specimens were all positive by standard RT-qPCR. CONCLUSIONS: Direct RT-qPCR on self-collected raw saliva is a simple, rapid, and accurate method with potential to be scaled up for enhanced SARS-CoV-2 community-wide screening.

Nasopharyngeal bacterial load as a marker for rapid and easy diagnosis of invasive pneumococcal disease in children from Mozambique.

BACKGROUND: Current diagnostic methods for detection of Streptococcus pneumoniae in children with suspected invasive pneumococcal disease have limitations of accuracy, timeliness, and patient convenience. This study aimed to determine the performance of pneumococcal load quantified with a real-time polymerase-chain reaction in nasopharyngeal samples to diagnose invasive pneumococcal disease in children. METHODS: Matched case-control study of patients <5 years of age with invasive pneumococcal disease admitted to the Manhiça District Hospital (Mozambique) and asymptomatic controls recruited in different periods between 2006 and 2014. Cases were confirmed by a positive bacterial culture for S. pneumoniae in blood or cerebrospinal fluid. Nasopharyngeal aspirates were collected from cases and controls and pneumococcal density was quantified by lytA real-time polymerase-chain reaction. RESULTS: Thirty cases (median age 12.8 months) and sixty controls (median age 11.7 months) were enrolled and 70% of them were male. Nasopharyngeal pneumococcal carriage was high in both groups: 28/30 (93.3%) for cases vs. 53/60 (88.3%) for controls (p = 0.71). Mean nasopharyngeal pneumococcal load was identified as a marker for invasive pneumococcal disease (7.0 log10 copies/mL in cases vs. 5.8 log10 copies/mL in controls, p<0.001) and showed good discriminatory power (AUC-ROC: 82.1%, 95% CI 72.5%-91.8%). A colonization density of 6.5 log10 copies/mL was determined as the optimal cut-off value to distinguish cases from controls (sensitivity 75.0%, specificity 73.6%). CONCLUSION: Use of non-invasive nasopharyngeal aspirates coupled with rapid and accurate quantification of pneumococcal load by real-time polymerase chain reaction has the potential to become a useful surrogate marker for early diagnosis of invasive pneumococcal disease in children.