A Pediatric Investigators Collaborative Network on Infections in Children (PICNIC) multi-centre Canadian descriptive analysis of Haemophilus influenzae bacteremia in children: Emerging serotypes.

Background: There has been dramatic reduction in Haemophilus influenzae serotype b (Hib) since introduction of Hib vaccines, but children still experience serious invasive Haemophilus influenzae (Hi) disease caused by various serotype and non-typeable bacteria. The object of this study was to describe the serotype distribution and clinical spectrum of Hi bacteremia in children admitted to Canadian hospitals. Methods: All children with Hi bacteremia admitted 2013 through 2017 to 10 centres across Canada were included. Demographic, clinical, treatment and outcome data were collected. Results: Haemophilus influenzae bacteremia occurred in 118 children of median age 12 months (inter-quartile range: 7-48 months). Forty-three (36%) isolates were non-typeable (NTHi) and 8 were not typed. Of the 67 typeable (THi), Hia (H. influenzae serotype a) (n=36, 54%), Hif (serotype f) (n=19, 26%) and Hib (serotype b) (n=9, 13%) dominated. The THi was more likely than NTHi bacteremia to present as meningitis (p<0.001), particularly serotype a (p=0.04) and less likely to present as pneumonia (p<0.001). Complicated disease (defined as intensive care unit admission, need for surgery, long-term sequelae or death) occurred in 31 (26%) cases and were more likely to have meningitis (p<0.001) than were those with uncomplicated disease. Conclusion: In the era of efficacious conjugate Hib vaccines, NTHi, Hia and Hif have emerged as the leading causes of invasive Hi in Canadian children, with Hia being most likely to result in meningitis and complicated disease. A vaccine for all NTHi and THi would be ideal, but knowledge of the current disease burden from circulating strains will inform prioritization of vaccine targets.

Screening for Group A Streptococcal Disease via Solid-State Nanopore Detection of PCR Amplicons.

Single-molecule detection methods are becoming increasingly important for diagnostic applications. Practical early detection of disease requires sensitivity down to the level of single copies of the targeted biomarkers. Of the candidate technologies that can address this need, solid-state nanopores show great promise as digital sensors for single-molecule detection. Here, we present work detailing the use of solid-state nanopores as downstream sensors for a polymerase chain reaction (PCR)-based assay targeting group A streptococcus (strep A), which can be readily extended to detect any pathogen that can be identified with a short nucleic acid sequence. We demonstrate that with some simple modifications to the standard PCR reaction mixture, nanopores can be used to reliably identify strep A in clinical samples. We also discuss methodological best practices for both adapting PCR-based assays to solid-state nanopore readout and analytical approaches by which to decide on sample status.

Temporal Dynamics and Evolution of SARS-CoV-2 Demonstrate the Necessity of Ongoing Viral Genome Sequencing in Ontario, Canada.

Genome-wide variation in SARS-CoV-2 reveals evolution and transmission dynamics which are critical considerations for disease control and prevention decisions. Here, we review estimates of the genome-wide viral mutation rates, summarize current COVID-19 case load in the province of Ontario, Canada (5 January 2021), and analyze published SARS-CoV-2 genomes from Ontario (collected prior to 24 November 2020) to test for more infectious genetic variants or lineages. The reported mutation rate (∼10-6 nucleotide [nt]-1 cycle-1) for SARS-CoV-2 is typical for coronaviruses. Analysis of published SARS-CoV-2 genomes revealed that the G614 spike protein mutation has dominated infections in Ontario and that SARS-CoV-2 lineages present in Ontario have not differed significantly in their rate of spread. These results suggest that the SARS-CoV-2 population circulating in Ontario has not changed significantly to date. However, ongoing genome monitoring is essential for identification of new variants and lineages that may contribute to increased viral transmission.

Short-Course Antimicrobial Therapy for Pediatric Community-Acquired Pneumonia: The SAFER Randomized Clinical Trial.

Importance: Community-acquired pneumonia (CAP) is a common occurrence in childhood; consequently, evidence-based recommendations for its treatment are required. Objective: To determine whether 5 days of high-dose amoxicillin for CAP was associated with noninferior rates of clinical cure compared with 10 days of high-dose amoxicillin. Design, Setting, and Participants: The SAFER (Short-Course Antimicrobial Therapy for Pediatric Respiratory Infections) study was a 2-center, parallel-group, noninferiority randomized clinical trial consisting of a single-center pilot study from December 1, 2012, to March 31, 2014, and the follow-up main study from August 1, 2016, to December 31, 2019 at the emergency departments of McMaster Children's Hospital and the Children's Hospital of Eastern Ontario. Research staff, participants, and outcome assessors were blinded to treatment allocation. Eligible children were aged 6 months to 10 years and had fever within 48 hours, respiratory symptoms, chest radiography findings consistent with pneumonia as per the emergency department physician, and a primary diagnosis of pneumonia. Children were excluded if they required hospitalization, had comorbidities that would predispose them to severe disease and/or pneumonia of unusual origin, or had previous ß-lactam antibiotic therapy. Data were analyzed from March 1 to July 8, 2020. Interventions: Five days of high-dose amoxicillin therapy followed by 5 days of placebo (intervention group) vs 5 days of high-dose amoxicillin followed by a different formulation of 5 days of high-dose amoxicillin (control group). Main Outcomes and Measures: Clinical cure at 14 to 21 days. Results: Among the 281 participants, the median age was 2.6 (interquartile range, 1.6-4.9) years (160 boys [57.7%] of 279 with sex listed). Clinical cure was observed in 101 of 114 children (88.6%) in the intervention group and in 99 of 109 (90.8%) in the control group in per-protocol analysis (risk difference, -0.016; 97.5% confidence limit, -0.087). Clinical cure at 14 to 21 days was observed in 108 of 126 (85.7%) in the intervention group and in 106 of 126 (84.1%) in the control group in the intention-to-treat analysis (risk difference, 0.023; 97.5% confidence limit, -0.061). Conclusions and Relevance: Short-course antibiotic therapy appeared to be comparable to standard care for the treatment of previously healthy children with CAP not requiring hospitalization. Clinical practice guidelines should consider recommending 5 days of amoxicillin for pediatric pneumonia management in accordance with antimicrobial stewardship principles. Trial Registration: ClinicalTrials.gov Identifier: NCT02380352.

Paediatric Investigators Collaborative Network on Infections in Canada (PICNIC) study of the current landscape of invasive meningococcal disease in children.

BACKGROUND: Immunizations have led to a decrease in the incidence of invasive meningococcal disease (IMD) in Canada, but this infection still leads to significant morbidity and mortality. OBJECTIVES: The purpose of this study was to determine the burden of illness and management of IMD in paediatric hospitals. METHODS: Data were collected on all cases of IMD in eight paediatric hospitals from 2013 to 2017. RESULTS: There were 17 cases of IMD. Three of eight hospitals had no cases. Just over half of the cases were serogroup B (n=9); a quarter (n=4) were serogroup W; less than a quarter (n=3) were serogroup Y; and one was unknown. Two infected children were not started on antibiotics until day one and day five after the initial blood culture was collected, but had uneventful recoveries. Six cases required admission to intensive care units; two died. Six cases had probable or proven meningitis. Thrombocytopenia was documented in seven cases. All cases had elevated C-reactive protein levels. Seven children received more than seven days of antibiotics; of these seven, only two had complications that justified prolonged therapy (subdural empyema and septic knee). Six cases had a central line placed. CONCLUSION: IMD is now rare in Canadian children, but about one-third of the cases in our study required treatment in the intensive care unit and two died. Clinicians appear to not always be aware that a five to seven-day course is adequate for uncomplicated cases of bacteremia or meningitis.

First case report of infective endocarditis associated with Microbacterium maritypicum.

Microbacterium species are gram positive coryneforms generally considered as a contaminant when identified in gram stain of blood culture, especially when time-to-positivity is longer than 48 h. We encountered a case of infective endocarditis associated with Microbacterium maritypicum bacteremia, which became positive after 48 h of incubation in three out of four bottles. The antimicrobial management is controversial as vancomycin is generally assumed to cover most gram positive bacilli, but our susceptibility result demonstrated minimum inhibitory concentration of 4 µg/mL of vancomycin, indicating non-susceptibility. To the best of our knowledge, this is the first case report of infective endocarditis associated with Microbacterium maritypicum.

Pragmatic evaluation of a midstream urine collection technique for infants in the emergency department.

OBJECTIVES: Our objective was to examine the performance characteristics of a bladder stimulation technique for urine collection among infants presenting to the emergency department (ED). METHODS: This prospective cohort study enrolled a convenience sample of infants aged ≤ 90 days requiring urine testing in the ED. Infants were excluded if critically ill, moderately to severely dehydrated, or having significant feeding issues. Bladder stimulation consisted of finger tapping on the lower abdomen with or without lower back massage while holding the child upright. The primary outcome was successful midstream urine collection within 5 minutes of stimulation. Secondary outcomes included sample contamination, bladder stimulation time for successful urine collection, and perceived patient distress on a 100-mm visual analog scale (VAS). RESULTS: We enrolled 151 infants and included 147 in the analysis. Median age was 53 days (interquartile range [IQR] 27-68 days). Midstream urine sample collection using bladder stimulation was successful in 78 infants (53.1%; 95% confidence interval [CI] 45-60.9). Thirty-nine samples (50%) were contaminated. Most contaminated samples (n = 31; 79.5%) were reported as "no significant growth" or "growth of 3 or more organisms". Median bladder stimulation time required for midstream urine collection was 45 seconds (IQR 20-120 seconds). Mean VAS for infant distress was 22 mm (standard deviation 23 mm). CONCLUSIONS: The success rate of this bladder stimulation technique was lower than previously reported. The contamination rate was high, however most contaminated specimens were easily identified and had no clinical impact.

A comparison of the Quidel Solana GAS assay, the Luminex Aries Group A Strep assay and the Focus Diagnostics Simplexa Group A Strep Direct assay for detection of Group A Streptococcus in throat swab specimens.

We compared three commercially available group A Streptococcus (GAS) nucleic acid amplification tests, the Quidel Solana GAS assay, the Luminex Aries Group A Strep assay and the Focus Diagnostics Simplexa Group A Strep Direct assay, with GAS bacterial culture. A true positive result was defined as one positive by culture or positive by ≥2/3 molecular methods. Two hundred eighty-seven throat swabs (207 children, 80 adults) were collected. The sensitivity of culture was 84.8% (95% CI 77.7-90.3%) with a specificity of 100% (95% CI 97.5-100%). The Solana assay sensitivity was 94.2% (95% CI 88.9-97.5%) with a specificity of 98.7% (95% CI 95.2-99.8%). Simplexa assay sensitivity was 99.3% (95% CI 96.0-99.9%) with a specificity of 95.3% (95% CI 90.6-98.1%). Aries assay sensitivity was 96.4% (95% CI 91.8-98.8%) with a specificity of 98.0% (95% CI 94.2-99.6%). In summary, all the molecular methods evaluated showed high sensitivity and specificity and were more sensitive than culture.

A comparison of the Quidel Solana HSV 1 + 2/VZV Assay, the Focus Diagnostics Simplexa HSV 1 & 2 Direct Assay and the Luminex Aries HSV 1&2 Assay for detection of herpes simplex virus 1 and 2 from swab specimens.

BACKGROUND: Molecular methods enable more rapid and sensitive detection of herpes simplex virus (HSV) than viral culture. OBJECTIVE: Three commercial molecular methods, all of which detect both HSV-1 and HSV-2, were compared to viral culture for the detection of HSV from swab specimens. STUDY DESIGN: Pediatric and adult patient viral swab specimens were cultured for HSV. Residual swab fluid was frozen at -80 °C until tested with the 3 molecular methods: the Quidel Solana HSV 1 + 2/VZV Assay, the Focus Diagnostics Simplexa HSV 1 & 2 Direct Assay and the Luminex Aries HSV 1&2 Assay. A true positive was defined as positive by culture or positive by ≥ 2/3 molecular methods. RESULTS: 177 specimens were studied. The sensitivity of culture was 81.3% (61/75, 95% CI 70.7-89.4%) and specificity was 100% (102/102, 95% CI 96.4-100%). The sensitivities of both the Solana and Simplexa were 100% (75/75, 95% CI 95.2-100%) and specificities were also both 100% (102/102, 95% CI 96.4-100%). The Aries had a sensitivity of 98.7% (74/75, 95% CI 92.8-99.97%) and specificity 99.0% (101/102, 95% CI 94.7-99.98%). All three molecular methods were significantly more sensitive than culture (p ≤ 0.0005 for Solana and Simplexa and p ≤ 0.0012 for Aries). CONCLUSION: All the molecular methods studied provided a significantly higher sensitivity than culture. In addition, the molecular methods took 1-2 hours to perform compared to a mean of 2.1 days for culture results. Use of any of the three molecular methods could lead to improved patient care.

Direct molecular detection of amoxicillin-susceptible E. coli in urine samples from children with suspected urinary tract infection: A potential tool to improve antibiotic stewardship and patient care.

Background: Rapid detection of amoxicillin-susceptible Escherichia coli (ASEC) urinary tract infections (UTIs) could have a significant impact on patient care and improve antibiotic stewardship. This is especially true for infants and children, for whom antibiotic choices are more limited than for adults. Methods: A real-time polymerase chain reaction (PCR) uniplex panel for detection of ASEC using PCR assays for E. coli and five resistance genes (bla TEM, bla SHV, bla OXA, bla CTX-M, and bla CMY) and an internal control was designed. PCR was then performed directly on pediatric urine samples using an inhibitor-resistant DNA polymerase. The main outcome measure was the performance of the PCR panel (sensitivity, specificity, positive predictive value [PPV], negative predictive value [NPV], accuracy) for the detection of ASEC. ASEC samples were defined as those that were E. coli PCR positive and PCR negative for all five resistance genes. PCR results were compared with the reference standard for culture and susceptibility testing. Results: Two hundred and six urine samples with pyuria (>10 white blood cells/high power field) were tested with the PCR panel. Two samples showed PCR inhibition (1%). For ASEC detection, the PCR panel showed a sensitivity of 91.53% (95% CI 81.32% to 97.19%), specificity of 98.21% (95% CI 90.45% to 99.95%), PPV of 98.18% (95% CI 88.54% to 99.74%), NPV of 91.67% (95% CI 82.61% to 96.22%), and accuracy of 94.78% (95% CI 88.99% to 98.06%). Conclusions: This PCR method could potentially enable amoxicillin or ampicillin to be used in a greater proportion of children with E. coli UTIs, improving antibiotic stewardship.

Historique: La détection rapide des infections urinaires à Escherichia coli susceptibles à l'amoxicilline (ECSA) peut avoir des effets importants sur les soins aux patients et améliorer la gérance des antibiotiques. C'est particulièrement vrai chez les nourrissons et les enfants, pour qui les choix d'antibiotiques sont plus limités que pour les adultes. Méthodologie: Les chercheurs ont fait appel à un panel uniplex d'amplification en chaîne par polymérase (PCR) pour déceler l'ECSA au moyen d'épreuves PCR d'E. coli et de cinq gènes de résistance (bla TEM, bla SHV, bla OXA, bla CTX-M et bla CMY) et ont conçu un contrôle interne. Ils ont ensuite effectué la PCR directement sur les échantillons d'urine pédiatrique à l'aide d'une polymérase d'ADN résistante aux inhibiteurs. La principale mesure de résultat était l'exécution du panel de PCR (sensibilité, spécificité, valeur prédictive positive [VPP], valeur prédictive négative [VPN], précision) pour déceler l'ECSA. Les échantillons d'ECSA étaient définis comme ceux dont la PCR était positive à l'E. coli et négative aux cinq gènes de résistance. Les chercheurs ont comparé les résultats de la PCR aux normes de référence des tests de culture et susceptibilité. Résultats: Les chercheurs ont testé 206 échantillons d'urine pyurique (>10 globules blancs/champ à fort grossissement) avec le panel de PCR. Deux échantillons ont révélé une inhibition de la PCR (1 %). Pour déceler l'ECSA, le panel de PCR a révélé une sensibilité de 91,53 % (IC à 95 %, 81,32 % à 97,19 %), une spécificité de 98,21 % (IC à 95 %, 90,45 % à 99,95 %), une VPP de 98,18 % (IC à 95 %, 88,54 % à 99,74 %), une VPN de 91,67 % (IC à 95 %, 82,61 % à 96,22 %) et une précision de 94,78 % (IC à 95 %, 88,99 % à 98,06 %). Conclusions: Cette méthode de PCR pourrait permettre de prescrire de l'amoxicilline ou de l'ampicilline à une plus grande proportion d'enfants ayant une infection urinaire à E. coli, ce qui améliorera la gérance des antibiotiques.

A comparison of the Allplex™ bacterial and viral assays to conventional methods for detection of gastroenteritis agents.

OBJECTIVE: Molecular methods to detect diarrheal pathogens are increasingly being used in place of conventional methods. We compared a new multiplex real-time PCR assay for detection of both bacterial and viral gastroenteritis agents, the Allplex™ Gastrointestinal Panel Assays (AGPA), to conventional methods (stool culture for bacterial pathogens and electron microscopy (EM) for viral pathogens). RESULTS: Gastrointestinal viruses, in particular norovirus genogroup II viruses, were detected by the AGPA in a high number of specimens that were negative by EM. For bacterial pathogens, the AGPA was able to detect the organisms grown in culture with high sensitivity and additionally detected several types of E. coli, such as enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and non-O157 Shiga toxin-producing E. coli (STEC), that could not be detected with conventional culture methods. Overall, the AGPA had a > 2-fold higher detection rate than the conventional methods, with 24/135 (17.8%) samples positive by conventional methods and 60/135 (44.4%) by AGPA. Thus, diarrhea pathogen detection rates increased substantially with the use of the AGPA as compared to conventional methods.

Short-course antimicrobial therapy for paediatric respiratory infections (SAFER): study protocol for a randomized controlled trial.

BACKGROUND: Community-acquired pneumonia (CAP) is commonly diagnosed in children. The Infectious Disease Society of America guidelines recommend 10 days of high-dose amoxicillin for the treatment of non-severe CAP but 5-day "short course" therapy may be just as effective. Randomized trials in adults have already demonstrated non-inferiority of 5-day short-course treatment for adults hospitalized with severe CAP and for adults with mild CAP treated as outpatients. Minimizing exposure to antimicrobials is desirable to avoid harms including diarrhoea, rashes, severe allergic reactions, increased circulating antimicrobial resistance, and microbiome disruption. METHODS: The objective of this multicentre, randomized, non-inferiority, controlled trial is to investigate whether 5 days of high-dose amoxicillin is associated with lower rates of clinical cure 14-21 days later as compared to 10 days of high-dose amoxicillin, the reference standard. Recruitment and enrolment will occur in the emergency departments of McMaster Children's Hospital and the Children's Hospital of Eastern Ontario. All children in the study will receive 5 days of amoxicillin after which point they will receive either 5 days of a different formulation of amoxicillin or a placebo. Assuming a clinical failure rate of 5% in the reference arm, a non-inferiority margin of 7.5%, one-sided alpha set at 0.025 and power of 0.80, 270 participants will be required. Participants from a previous feasibility study (n = 60) will be rolled over into the current study. We will be performing multiplex respiratory virus molecular testing, quantification of nasopharyngeal pneumococcal genomic loads, salivary inflammatory marker testing, and faecal microbiome profiling on participants. DISCUSSION: This is a pragmatic study seeking to provide high-quality evidence for front-line physicians evaluating children presenting with mild CAP in North American emergency departments in the post-13-valent pneumococcal, conjugate vaccine era. High-quality evidence supporting the non-inferiority of short-course therapy for non-severe paediatric CAP should be generated prior to making changes to established guidelines. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02380352 . Registered on 2 March 2015.

Higher atypical enteropathogenic Escherichia coli (a-EPEC) bacterial loads in children with diarrhea are associated with PCR detection of the EHEC factor for adherence 1/lymphocyte inhibitory factor A (efa1/lifa) gene.

BACKGROUND: Typical enteropathogenic Escherichia coli (t-EPEC) are known to cause diarrhea in children but it is uncertain whether atypical EPEC (a-EPEC) do, since a-EPEC lack the bundle-forming pilus (bfp) gene that encodes a key adherence factor in t-EPEC. In culture-based studies of a-EPEC, the presence of another adherence factor, called EHEC factor for adherence/lymphocyte activation inhibitor (efa1/lifA), was strongly associated with diarrhea. Since a-EPEC culture is not feasible in clinical laboratories, we designed an efa1/lifA quantitative PCR assay and examined whether the presence of efa1/lifA was associated with higher a-EPEC bacterial loads in pediatric diarrheal stool samples. METHODS: Fecal samples from children with diarrhea were tested by qPCR for EPEC (presence of eae gene) and for shiga toxin genes to exclude enterohemorrhagic E. coli, which also contain the eae gene. EPEC containing samples were then tested for the bundle-forming pilus gene found in t-EPEC and efa1/lifA. The eae gene quantity in efa1/lifA-positive and negative samples was compared. RESULTS: Thirty-nine of 320 (12%) fecal samples tested positive for EPEC and 38/39 (97%) contained a-EPEC. The efa1/lifA gene was detected in 16/38 (42%) a-EPEC samples. The median eae concentration for efa1/lifA positive samples was significantly higher than for efa1/lifA negative samples (median 16,745 vs. 1183 copies/µL, respectively, p = 0.006). CONCLUSIONS: Atypical enteropathogenic E. coli-positive diarrheal stool samples containing the efa1/lifA gene had significantly higher bacterial loads than samples lacking this gene. This supports the idea that efa1/lifA contributes to diarrheal pathogenesis and suggests that, in EPEC-positive samples, efa/lifA may be a useful additional molecular biomarker.

Direct molecular detection of a broad range of bacterial and viral organisms and Streptococcus pneumoniae vaccine serotypes in children with otitis media with effusion.

BACKGROUND: Otitis media with effusion (OME) causes significant morbidity in children, but the causes of OME and methods for prevention are unclear. To look for potential infectious etiologies, we performed a pilot study using multiple-target real-time polymerase chain reaction (qPCR) for 27 infectious agents, including nine bacterial organisms and 18 respiratory viruses in middle ear fluids (MEFs) from children with OME. QPCR was also performed for the 13 Streptococcus pneumoniae serotypes contained in the current vaccine. RESULTS: Forty-eight MEF samples were obtained and qPCR detected bacterial nucleic acid (NA) in 39/48 (81%) and viral NA in 7/48 (15%). Alloiococcus otitidis and S. pneumoniae were both detected in 15/48 (31%) MEFs, followed by M. catarrhalis in 14/48 (29%), H. influenzae in 5/48 (10%) and M. pneumoniae in 4/48 (8%). Rhinoviruses were most common virus type detected, found in 4/48 (8%) MEFs. Serotypes included in the current 13-serotype vaccine were detected in only 3/15 (20%) S. pneumoniae qPCR-positive MEFs. CONCLUSIONS: Bacteria may play an important role in OME, since over 80% of MEFs contained bacterial NA. Further research into the role of A. otitidis in OME will be helpful. Serotypes of S. pneumoniae not included in the current 13-serotype vaccine may be involved in OME. Larger studies of OME S. pneumoniae serotypes are needed to help determine which additional serotypes should be included in future vaccine formulations in order to try to prevent OME.

Polymerase chain reaction detection of Kingella kingae in children with culture-negative septic arthritis in eastern Ontario.

BACKGROUND: The bacterium Kingella kingae may be an under-recognized cause of septic arthritis in Canadian children because it is difficult to grow in culture and best detected using molecular methods. OBJECTIVES: To determine whether K kingae is present in culture-negative joint fluid specimens from children in eastern Ontario using polymerase chain reaction (PCR) detection methods. METHODS: K kingae PCR testing was performed using residual bacterial culture-negative joint fluid collected from 2010 to 2013 at a children's hospital in Ottawa, Ontario. The clinical features of children with infections caused by K kingae were compared with those of children with infections caused by the 'typical' septic arthritis bacteria, Staphylococcus aureus and Streptococcus pyogenes. RESULTS: A total of 50 joint fluid specimens were submitted over the study period. Ten were culture-positive, eight for S aureus and two for S pyogenes. Residual joint fluid was available for 27 of the 40 culture-negative specimens and K kingae was detected using PCR in seven (25.93%) of these samples. Children with K kingae were significantly younger (median age 1.7 versus 11.3 years; P=0.01) and had lower C-reactive protein levels (median 23.8 mg/L versus 117.6. mg/L; P=0.01) than those infected with other bacteria. CONCLUSIONS: K kingae was frequently detected using PCR in culture-negative joint fluid specimens from children in eastern Ontario. K kingae PCR testing of culture-negative joint samples in children appears to be warranted.

HISTORIQUE: La bactérie Kingella kingae peut être une cause sousdiagnostiquée d'arthrite septique chez les enfants canadiens, car elle est difficile à développer en culture et qu'elle est plus facile à déceler à l'aide de méthodes moléculaires. OBJECTIFS: Déterminer si le K kingae est présent dans des prélèvements de liquide articulaire à bactériologie négative provenant d'enfants de l'est de l'Ontario, à l'aide des méthodes de détection par amplification en chaîne par polymérase (PCR). MÉTHODOLOGIE: Les chercheurs ont réalisé la PCR du K kingae dans le liquide articulaire à bactériologie négative résiduel prélevé entre 2010 et 2013 dans un hôpital pour enfants d'Ottawa, en Ontario. Ils ont comparé les caractéristiques cliniques des enfants atteints d'une infection causée par le K kingae à celles d'enfants infectés par les bactéries habituellement responsables de l'arthrite septique, soit le Staphylococcus aureus et le Streptococcus pyogenes. RÉSULTATS: Au total, 50 prélèvements de liquide articulaire ont été soumis pendant la période de l'étude. Dix étaient à bactériologie positive, soit huit au S aureus et deux au S pyogenes. Il y avait du liquide articulaire résiduel pour 27 des 40 prélèvements à bactériologie négative, et le K kingae a été décelé par PCR dans sept d'entre eux (25,93 %). Les enfants infectés par le K kingae étaient beaucoup plus jeunes (âge médian de 1,7 an plutôt que de 11,3 ans; P=0,01) et avaient un taux de protéine C-réactive plus faible (médiane de 23,8 mg/L plutôt que de 117,6 mg/L; P=0,01) que ceux infectés par d'autres bactéries. CONCLUSIONS: Le K kingae était souvent décelé par PCR dans les prélèvements de liquide articulaire à bactériologie négative réalisés chez des enfants de l'est de l'Ontario. Il semble justifié d'effectuer une PCR du K kingae dans les prélèvements articulaires à bactériologie négative réalisés chez les enfants.

Detection of respiratory viruses and bacteria in children using a twenty-two target reverse-transcription real-time PCR (RT-qPCR) panel.

BACKGROUND: Rapid detection of the wide range of viruses and bacteria that cause respiratory infection in children is important for patient care and antibiotic stewardship. We therefore designed and evaluated a ready-to-use 22 target respiratory infection reverse-transcription real-time polymerase chain reaction (RT-qPCR) panel to determine if this would improve detection of these agents at our pediatric hospital. METHODS: RT-qPCR assays for twenty-two target organisms were dried-down in individual wells of 96 well plates and saved at room temperature. Targets included 18 respiratory viruses and 4 bacteria. After automated nucleic acid extraction of nasopharyngeal aspirate (NPA) samples, rapid qPCR was performed. RT-qPCR results were compared with those obtained by the testing methods used at our hospital laboratories. RESULTS: One hundred fifty-nine pediatric NPA samples were tested with the RT-qPCR panel. One or more respiratory pathogens were detected in 132/159 (83%) samples. This was significantly higher than the detection rate of standard methods (94/159, 59%) (P<0.001). This difference was mainly due to improved RT-qPCR detection of rhinoviruses, parainfluenza viruses, bocavirus, and coronaviruses. The panel internal control assay performance remained stable at room temperature storage over a two-month testing period. CONCLUSION: The RT-qPCR panel was able to identify pathogens in a high proportion of respiratory samples. The panel detected more positive specimens than the methods in use at our hospital. The pre-made panel format was easy to use and rapid, with results available in approximately 90 minutes. We now plan to determine if use of this panel improves patient care and antibiotic stewardship.

Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in diarrhoeic patients in the Qikiqtani Region, Nunavut, Canada.

BACKGROUND: Although the prevalences of infection with the protozoan parasites Cryptosporidium spp. and Giardia duodenalis in humans appear to be relatively high in the Canadian North, their transmission patterns are poorly understood. OBJECTIVE: To determine the detection rate and the molecular characteristics of Cryptosporidium spp. and Giardia duodenalis in diarrhoeic patients in the Qikiqtani (Baffin Island) Region of Nunavut, Canada, in order to better understand the burden of illness and the potential mechanisms of transmission. STUDY DESIGN/METHODS: Diarrhoeal stool specimens (n=108) submitted to the Qikiqtani General Hospital for clinical testing were also tested for the presence of Cryptosporidium spp. and Giardia duodenalis using epifluorescence microscopy and polymerase chain reaction (PCR). DNA sequencing and restriction fragment length polymorphism (RFLP) analyses were performed on PCR-positive specimens to determine the species, genotypes and sub-genotypes of the parasites. RESULTS: Cryptosporidium was detected in 15.7% of the diarrhoeic patients, while Giardia was detected in 4.6%. DNA sequencing of a fragment of the small subunit rRNA gene indicated that all of the Cryptosporidium amplicons had a 100% homology to C. parvum, and a gp60 assay showed that all aligned with C. parvum sub-genotype IIa. Microsatellite analysis revealed 3 cases of sub-genotype IIaA15G2R1, 2 of IIaA15G1R and 1 case each of sub-genotypes IIaA16G1R1 and IIaA15R1. For Giardia, results based on the amplification of both the 16S rRNA gene and the gdh gene were generally in agreement, and both DNA sequencing and RFLP demonstrated the presence of the G. duodenalis Assemblage B genotype. CONCLUSIONS: Both C. parvum and G. duodenalis Assemblage B were present in human diarrhoeal stool specimens from Nunavut, which was suggestive of zoonotic transmission, although human-to-human transmission cannot be ruled out. To fully understand the public health significance of the different Cryptosporidium and Giardia species and genotypes in diarrhoeic patients, it will be imperative to establish the extent of genetic diversity within these parasites through comprehensive studies of the molecular epidemiology of cryptosporidiosis and giardiasis in the Nunavut region.

Acute effects of viral respiratory tract infections on sputum bacterial density during CF pulmonary exacerbations.

BACKGROUND: Airway proliferation of Pseudomonas aeruginosa bacteria is thought to trigger CF exacerbations and may be affected by the presence of viral infections. METHODS: A 2-year prospective study was conducted on 35 adults with CF. P. aeruginosa sputum density was analyzed during stable, exacerbation and post exacerbation assessments. Upon exacerbation, samples were sent for PCR detection of respiratory viruses and the sputum density of P. aeruginosa in patients with a viral infection versus those without was compared. RESULTS: Twenty-two patients experienced 30 exacerbations during the study period; 50% were associated with a viral infection. There was no change in sputum density of P. aeruginosa from the stable to exacerbation state when measured by quantitative culture or by PCR. Virus-associated exacerbations did not result in significant increases in P. aeruginosa sputum density compared to non-viral exacerbations. CONCLUSION: Sputum density of P. aeruginosa was not increased at the time of CF exacerbation and was not influenced by the presence of viral infection.

Real-time polymerase chain reaction for microbiological diagnosis of parapneumonic effusions in Canadian children.

BACKGROUND: Community-acquired pneumonia (CAP) complicated by parapneumonic effusion/empyema is an infectious syndrome commonly encountered by physicians caring for children in Canada. OBJECTIVE: To investigate the incremental benefit of novel molecular testing for the microbiological diagnosis of pediatric CAP complicated by parapneumonic effusion/empyema in Canada. METHODS: A convenience sample of pleural fluid from 56 children who had been admitted to hospital in Ontario with CAP complicated by parapneumonic effusion between 2009 and 2011 was examined. Multiple uniplex real-time polymerase chain reaction (PCR) testing was performed on these pleural fluids and compared with traditional culture-based testing of blood and pleural fluid samples. RESULTS: Molecular methods detected a pathogen in 82% of cases, whereas traditional cultures of blood and pleural fluids detected a pathogen in only 25%. The majority of parapneumonic effusions were associated with pneumococcal infection; Streptococcus pneumoniae was detected in 62% of the samples using molecular methods but in only 14% of samples using culture-based methods. Streptococcus pyogenes, detected in 16% of samples using PCR, was the second most common pathogen found. No patients were found to have empyema caused by Staphylococcus aureus. DISCUSSION: The results showed that multiple uniplex real-time PCR performed substantially better than traditional culture methods for microbiological diagnosis of CAP complicated by effusion/ empyema. S pneumoniae and S pyogenes were found to be responsible for the majority of infections. The approach detected pathogens in a similar proportion of pleural fluid samples as previously reported nested PCR assays; furthermore, the real-time closed-well approach also minimized the risk of nonspecificity due to cross-contamination relative to nested PCR. CONCLUSIONS: Real-time PCR for the detection of bacterial DNA in pleural fluids has the potential to better define the microbiological cause of pediatric CAP. This approach could help clinicians provide targeted antimicrobial therapy.

HISTORIQUE: La pneumonie d'origine non nosocomiale (PONN) compliquée par un épanchement parapneumonique ou un empyème est un syndrome infectieux qu'observent souvent les médecins qui soignent des enfants au Canada. OBJECTIF: Examiner les avantages incrémentiels de nouveaux tests moléculaires pour poser un diagnostic microbiologique de PONN pédiatrique compliquée par un épanchement parapneumonique ou un empyème au Canada. MÉTHODOLOGIE: Les chercheurs ont examiné un échantillon de commodité de liquide pleural prélevé chez 56 enfants hospitalisés en Ontario à cause d'une PONN compliquée par un épanchement parapneumonique entre 2009 et 2011. Ils ont effectué de multiples tests de réaction en chaîne de la polymérase (PCR) uniplexe en temps réel sur ce liquide pleural et les ont comparés aux examens classiques des cultures de sang et de liquide pleural. RÉSULTATS: Les méthodes moléculaires ont permis de déceler un pathogène dans 82 % des cas, tandis que les cultures classiques de sang et de liquide pleural n'ont permis d'en déceler que dans 25 % des cas. La majorité des épanchements parapneumoniques s'associait à une infection pneumococcique. En effet, les chercheurs ont décelé un Streptococcus pneumoniae dans 62 % des échantillons au moyen des méthodes moléculaires, mais seulement dans 14 % des échantillons au moyen des méthodes de culture. Le Streptococcus pyogenes, décelé dans 16 % des échantillons par PCR, était le deuxième pathogène en importance à avoir été décelé. Aucun patient n'avait d'empyème causé par le Staphylococcus aureus. EXPOSÉ: Les résultats ont démontré que de multiples tests de PCR uniplexe en temps réel donnaient des résultats beaucoup plus précis que les cultures classiques pour poser un diagnostic microbiologique de PONN compliquée par un épanchement ou un empyème. Le S pneumoniae et le S pyogenes étaient responsables de la majorité des infections. Cette méthode permet de déceler des pathogène dans une proportion similaire d'échantillons de liquide pleural que les PCR nichées déclarées antérieurement. De plus, la technique en temps réel par système fermé réduisait le risque de non-spécificité attribuable à la contamination croisée observée en cas de PCR nichée. CONCLUSIONS: La PRC en temps réel pour déceler l'ADN bactérien dans le liquide pleural définit peut-être mieux la cause microbiologique de la PONN pédiatrique. Cette approche pourrait aider les cliniciens à proposer un traitement antimicrobien ciblé.

Direct Streptococcus pneumoniae real-time PCR serotyping from pediatric parapneumonic effusions.

BACKGROUND: To determine the serotypes of Streptococcus pneumoniae responsible for pneumonia complicated by parapneumonic effusion in children, we performed real-time PCR based pneumococcal "serotyping" directly on parapneumonic fluid samples. METHODS: Specimens were collected at two children's hospitals in Ontario, Canada from 2009 to 2011. Samples in which S. pneumoniae was detected by PCR were tested with serotype-specific 5'exonuclease PCR assays for the 13 serotypes contained in the 13-serotype pneumococcal vaccine. RESULTS: Thirty-five S. pneumoniae PCR-positive pleural samples were studied. Pneumococcal serotyping PCR assays were positive for 34 of 35 (97%). Serotype 3 was detected most frequently, in 19/35 (54%), followed by serotype 19A in 9/35 (26%), serotype 7 F/A in 4/35 (11%), serotype 1 in 1/35 (3%), and serotype 6A also in 1/35 (3%). CONCLUSIONS: PCR testing demonstrated that the vast majority (97%) of S. pneumoniae parapneumonic effusions were caused by serotypes present in the 13-serotype vaccine that were not present in the original 7 serotype vaccine. This suggests that use of the 13-serotype vaccine could potentially prevent many S. pneumoniae pneumonias complicated by parapneumonic effusion in our region, provided serotype replacement does not occur.