Pedro Brotons

Molecular isothermal techniques for combating infectious diseases: towards low-cost point-of-care diagnostics.

Nucleic acid amplification techniques such as PCR have facilitated rapid and accurate diagnosis in central laboratories over the past years. PCR-based amplifications require high-precision instruments to perform thermal cycling reactions. Such equipment is bulky, expensive and complex to operate. Progressive advances in isothermal amplification chemistries, microfluidics and detectors miniaturisation are paving the way for the introduction and use of compact ‘sample in-results out’ diagnostic devices. However, this paradigm shift towards decentralised testing poses diverse technological, economic and organizational challenges both in industrialized and developing countries. This review describes the landscape of molecular isothermal diagnostic techniques for infectious diseases, their characteristics, current state of development, and available products, with a focus on new directions towards point-of-care applications.

Rapid and Easy Identification of Capsular Serotypes of Streptococcus pneumoniae by Use of Fragment Analysis by Automated Fluorescence-Based Capillary Electrophoresis

ABSTRACT The purpose of this study was to develop a high-throughput method for the identification of pneumococcal capsular types. Multiplex PCR combined with fragment analysis and automated fluorescent capillary electrophoresis (FAF-mPCR) was utilized. FAF-mPCR was composed of only 3 PCRs for the specific detection of serotypes 1, 2, 3, 4, 5, 6A/6B, 6C, 7F/7A, 7C/(7B/40), 8, 9V/9A, 9N/9L, 10A, 10F/(10C/33C), 11A/11D/11F, 12F/(12A/44/46), 13, 14, 15A/15F, 15B/15C, 16F, 17F, 18/(18A/18B/18C/18F), 19A, 19F, 20, 21, 22F/22A, 23A, 23B, 23F, 24/(24A/24B/24F), 31, 33F/(33A/37), 34, 35A/(35C/42), 35B, 35F/47F, 38/25F, and 39. In order to evaluate the assay, all invasive pneumococcal isolates (n = 394) characterized at Hospital Sant Joan de Déu, Barcelona, Spain, from July 2010 to July 2011 were included in this study. The Wallace coefficient was used to evaluate the overall agreement between two typing methods (Quellung reaction versus FAF-mPCR). A high concordance with Quellung was found: 97.2% (383/394) of samples. The Wallace coefficient was 0.981 (range, 0.965 to 0.997). Only 11 results were discordant with the Quellung reaction. However, latex reaction and Quellung results of the second reference laboratory agreed with FAF-mPCR for 9 of these 11 strains (82%). Therefore, we considered that only 2 of 394 strains (0.5%) were not properly characterized by the new assay. The automation of the process allowed the typing of 30 isolates in a few hours with a lower cost than that of the Quellung reaction. These results indicate that FAF-mPCR is a good method to determine the capsular serotype of Streptococcus pneumoniae.

High invasiveness of pneumococcal serotypes included in the new generation of conjugate vaccines

The implementation of the seven-valent pneumococcal conjugate vaccine, PCV7, has resulted in significant changes in the pneumococcal population being carried and causing disease. We aimed to determine the invasive disease potential of serotypes causing invasive paediatric disease in the era of conjugate vaccines in Catalonia, Spain, and their potential coverage by the 13-valent pneumococcal conjugate vaccine, PCV13. As a secondary objective, we evaluated whether implementation of PCV7 had resulted in significant changes in the invasive disease potential of the most frequent serotypes circulating in the area. Two pneumococcal collections obtained from children admitted to the University Hospital Sant Joan de Déu (Barcelona, Spain) between 2007 and 2011 were compared: a first set of 159 invasive disease isolates, and a second set of 209 nasopharyngeal isolates recovered from healthy children admitted for minor surgery. The most common invasive serotypes were 1 (24.5%, n = 39), 19A (21.2%, n = 34), 5 (8.8%, n = 14), 7F (8.8%, n = 14) and 3 (5%, n = 8). The most common serotypes in carriage were 19A (10%, n = 21), 6C (9%, n = 19), 23B (8.1%, n = 17), 6A (7.6%, n = 16) and 19F (6.2%, n = 13). A significantly higher propensity to cause invasive disease was observed for serotypes 1, 3, 5, 7F and 19A, all of which are included in PCV13. After false-discovery-rate correction, the results were robust for serotypes 1, 5, 7F and 19A. Non-PCV13 serotypes had a low invasive disease potential. Our data reinforce the need for continuous surveillance and should encourage efforts to introduce universal vaccination with PCV13 in children in our region.

Estimation of the invasive disease potential of Streptococcus pneumoniae in children by the use of direct capsular typing in clinical specimens

Traditionally, invasiveness indexes have been based on culture methods. We aimed to establish a new classification of the invasive disease potential of pneumococcal serotypes causing invasive pediatric disease in the era of conjugate vaccines in Catalonia, Spain, by adding capsular typing of Streptococcus pneumoniae in direct sample. Two samples of children attended at the University Hospital Sant Joan de Déu (Barcelona, Spain) between 2007 and 2011 were compared: a first sample of 358 children with invasive pneumococcal disease and a second sample of 402 pneumococcal nasopharyngeal carriers selected from 714 healthy children admitted for minor surgical procedures. The most common invasive serotypes were 1 (20.1 %, n = 72), 19A (13.9 %, n = 50), 3 (12.3 %, n = 44), and 7FA (7.5 %, n = 27), whereas the most common serotypes in carriage were 19A (8.7 %, n = 38), 10FC33C (7.8 %, n = 34), 6C (6.9 %, n = 30), and 19FBC (5.5 %, n = 24). We detected a rate of cocolonization of 26.4 % (n = 89) among the 336 samples serotyped in the carriers population. Serotypes 1, 3, and 7FA were significantly associated with high invasiveness. Serotypes 6C, 10FC33C, 23A, 35B, 19FBC, 21, 11AD, 15BC, 23B, 34, and 6A were significantly associated with low invasiveness. Our results proved that the use of molecular techniques in direct sample for both the detection and the capsular identification of Streptococcus pneumoniae is very useful to obtain a more accurate calculation of the invasiveness of the different pneumococcal serotypes.

Comparison of NxTAG Respiratory Pathogen Panel and Anyplex II RV16 Tests for Multiplex Detection of Respiratory Pathogens in Hospitalized Children

ABSTRACT Multiplex molecular techniques can detect a diversity of respiratory viruses and bacteria that cause childhood acute respiratory infection rapidly and conveniently. However, currently available techniques show high variation in performance. We sought to compare the diagnostic accuracy of the novel multiplex NxTAG respiratory pathogen panel (RPP) RUO test versus a routine multiplex Anyplex II RV16 assay in respiratory specimens collected from children <18 years of age hospitalized with nonspecific symptoms of acute lower respiratory infection. Parallel testing was performed on nasopharyngeal aspirates prospectively collected at referral Childrens Hospital Sant Joan de Déu (Barcelona, Spain) between June and November 2015. Agreement values between the two tests and kappa coefficients were assessed. Bidirectional sequencing was performed for the resolution of discordant results. A total of 319 samples were analyzed by both techniques. A total of 268 (84.0%) of them yielded concordant results. Positive percent agreement values ranged from 83.3 to 100%, while the negative percent agreement was more than 99% for all targets except for enterovirus/rhinovirus (EV/RV; 94.4%). Kappa coefficients ranged from 0.83 to 1.00. Discrepancy analysis confirmed 66.0% of NxTAG RPP RUO results. A total of 260 viruses were detected, with EV/RV (n = 105, 40.4%) being the most prevalent target. Viral coinfections were found in 44 (14.2%) samples. In addition, NxTAG RPP RUO detected single bacterial and mixed viral-bacterial infections in seven samples. NxTAG RPP RUO showed high positive and negative agreement with Anyplex II RV16 for main viruses that cause acute respiratory infections in children, coupled with an additional capability to detect some respiratory bacteria.

Cost of hospitalizing children with invasive pneumococcal pneumonia

BACKGROUND Although invasive pneumococcal pneumonia remains responsible for a significant number of child hospitalizations, specific data on hospital resource utilization and related costs are limited. OBJECTIVES To assess the cost of hospitalizing children with invasive pneumococcal pneumonia and identify the cost determinants of the disease. PATIENTS AND METHODS Economic evaluation based on an observational study of all children <18 years of age with culture-proved invasive pneumococcal pneumonia admitted to a referral hospital in Barcelona (Spain) during the period January 2001-December 2011. Analysis included demographic, microbiological, epidemiological and clinical variables. RESULTS A total of 135 children were included in the study (median age 3.3 years). PCV13 serotypes were detected in 132 (97.8%) cases. Median hospital cost was €4533 (€4078-5435, 95% CI). Median length of stay was 11.0 days (10.6-13.0 days, 95% CI). Variables significantly associated with increased cost in the multivariate analysis were complicated pneumonia (≥2 and 1 complication) versus non-complicated pneumonia (€4919 and €2822 vs. €1399), performance of surgery versus no surgery (€4849 vs. €1435), intensive care versus no intensive care (€6488 vs. €3862) and identification of non-PCV7 serotypes versus PCV7 serotypes (€4656 vs. €1470). CONCLUSION Invasive pneumococcal pneumonia in children makes substantial demands on hospital health care and financial resources that could be mitigated with universal PCV13 childhood immunization programmes and early management of complications.

Validation of a loop-mediated isothermal amplification assay for rapid diagnosis of pertussis infection in nasopharyngeal samples

Objective: To develop and validate a novel loop-mediated amplification (LAMP) assay for rapid diagnosis (<1 hour) of whooping cough in nasopharyngeal samples versus the gold standard: real-time PCR. Methods: The study included all nasopharyngeal samples (n = 213) collected from children with clinical suspicion of pertussis admitted to Children’s University Hospital Sant Joan de Déu (Barcelona, Spain) during July–December 2014. Fresh samples were routinely analyzed by real-time PCR and stored for retrospective LAMP analysis, following an easy 30 minute DNA extraction step by Chelex-100. Results: Performance results of the LAMP assay were: linearity, 105–101 CFU/ml; Limit of Detection, 2 CFU/ml; precision (mean CV), 7.38%; diagnostic sensitivity, 96.55%; diagnostic specificity, 99.46%; time to detection, 12–30 minutes. Conclusion: The new test was shown to be 2.5-fold faster than real-time PCR while maintaining similar levels of analytical and clinical performance. Therefore it could become a useful diagnostic tool for molecular point-of-care testing.

Performance of a rapid multi-analyte 2-photon excitation assay in children with acute respiratory infection

Abstract The purpose of this study is to evaluate the diagnostic performance of the novel 2-photon excitation-based mariPOC© Assay (ArcDia Laboratories, Turku, Finland) for antigen detection of respiratory viruses versus real-time polymerase chain reaction (PCR). The mariPOC Assay and 2 multiplex real-time PCR techniques were performed on nasopharyngeal samples from pediatric patients with suspicion of acute respiratory infection admitted to a childrens hospital in Spain during October 2011 to January 2013. A total of 233 samples were studied. Sensitivities and specificities (95% confidence interval) of the mariPOC Assay were for respiratory syncytial virus (RSV), 78.4% (69.7–85.6) and 99.2% (96.3–100.0); influenza virus (IFV) A, 66.7% (26.2–94.0) and 99.6% (97.9–100.0); IFV-B, 63.6% (33.6–87.2) and 100.0% (98.7–100.0); human metapneumovirus (hMPV), 60.0% (34.5–81.9) and 100.0% (98.6–100.0); adenovirus (ADV), 12.5% (0.6–48.0) and 100.0% (98.7–100.0), respectively. The mariPOC Assay is a highly specific method for simultaneous detection of 8 respiratory viruses but has sensitivities that range from moderately high for RSV to moderate for IFV and hMPV and low for ADV.

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.