Identifying the research, advocacy, policy and implementation needs for the prevention and management of respiratory syncytial virus lower respiratory tract infection in low- and middle-income countries

Introduction: The high burden of respiratory syncytial virus (RSV) infection in young children disproportionately occurs in low- and middle-income countries (LMICs). The PROUD (Preventing RespiratOry syncytial virUs in unDerdeveloped countries) Taskforce of 24 RSV worldwide experts assessed key needs for RSV prevention in LMICs, including vaccine and newer preventive measures. Methods: A global, survey-based study was undertaken in 2021. An online questionnaire was developed following three meetings of the Taskforce panellists wherein factors related to RSV infection, its prevention and management were identified using iterative questioning. Each factor was scored, by non-panellists interested in RSV, on a scale of zero (very-low-relevance) to 100 (very-high-relevance) within two scenarios: (1) Current and (2) Future expectations for RSV management. Results: Ninety questionnaires were completed: 70 by respondents (71.4% physicians; 27.1% researchers/scientists) from 16 LMICs and 20 from nine high-income (HI) countries (90.0% physicians; 5.0% researchers/scientists), as a reference group. Within LMICs, RSV awareness was perceived to be low, and management was not prioritised. Of the 100 factors scored, those related to improved diagnosis particularly access to affordable point-of-care diagnostics, disease burden data generation, clinical and general education, prompt access to new interventions, and engagement with policymakers/payers were identified of paramount importance. There was a strong need for clinical education and local data generation in the lowest economies, whereas upper-middle income countries were more closely aligned with HI countries in terms of current RSV service provision. Conclusion: Seven key actions for improving RSV prevention and management in LMICs are proposed

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

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. Children's Hospital, 1280 Main Street West, Room 3A-30, Hamilton, Ontario L9H 4K6. Telephone 905-521-2100 C ommunity-acquired pneumonia (CAP) commonly occurs in children. Pediatric hospitalization rates for CAP in the Western world are one to four per 1000 per year, with pneumonia accounting for up to 20% of all pediatric admissions in some settings (1). Streptococcus pneumoniae is the most common bacterial cause of pediatric CAP (2,3); the introduction of the conjugate pneumococcal vaccine in North America led to a substantial decrease in pneumonia incidence (4). Curiously, subsequent to this, rates of CAP complicated by origiNAL ArticLE This open-access article is distributed under the terms of the Creative Commons Attribution Non-Commercial License (CC BY-NC) (http:// creativecommons.org/licenses/by-nc/4.0/), which permits reuse, distribution and reproduction of the article, provided that the original work is properly cited and the reuse is restricted to noncommercial purposes. For commercial reuse, contact [email protected] parapneumonic effusion were observed to increase in Canada and other countries (5-7). The optimization of the antimicrobial management of CAP with parapneumonic effusion is important because children with this type of infection are often severely ill, requiring admission to hospital, and many require radiological or operative intervention. Choosing empirical antimicrobial therapy is difficult because there are numerous pyogenic bacteria in addition to S pneumoniae that cause complicated CAP. These include Staphylococcus aureus (both methicillin sensitive and methicillin resistant), group A streptococcus (Stretococcus pyogenes), Streptococcus anginosus group organisms, Haemophilus influenzae, anaerobes and others (8-11). Specific (ie, targeted) antibiotic therapy is not possible in the majority of cases because blood and pleural fluid cultures have been shown to be positive in only 17% to 35% of children (8-13). The low sensitivity of pleural fluid cultures in particular may be due to the fact that antibiotics are often started before pleural fluid specimens are obtained. A recent prospective study that enrolled children with complicated pneumonia presenting to the largest children's hospital in Canada (11) obtained a microbiological diagnosis using culture-based techniques in only 22 of 88 participants. The vast majority of these children were treated with multiple parenteral antimicrobials and more than one-half received vancomycin, yet only a single case of methicillin-resistant S aureus empyema was documented. Molecular techniques may be uniquely well suited to the microbiological diagnosis of complicated CAP because, in contrast to traditional culture-based methods, detection is not predicated on the growth of viable bacteria. However, molecular methods have their own limitations: false-positive results may result from inadequately controlled background contamination (14) or because of very low bacterial concentrations that are not clinically significant but detectable by the assay (15), among other reasons. The purpose of the present study was to determine whether the microbiological diagnosis of CAP with parapneumonic effusion in children could be improved using nonculture-dependent molecular testing of pleural fluids for bacterial pathogens. Although similar studies have described the results of molecular testing to define bacterial etiology of pediatric parapneumonic effusion in American and European populations METHODS Study population Children with pneumonia and parapneumonic effusion often receive thoracentesis and/or thoracostomy tube placement for both diagnostic and therapeutic purposes as part of routine care at the Children's Hospital of Eastern Ontario (CHEO, Ottawa, Ontario) and McMaster Children's Hospital (MCH, Hamilton, Ontario). A convenience sample of pleural fluids from children with a diagnosis of complicated pneumonia/ empyema/parapneumonic effusion were collected at CHEO (n=47) between January 2009 and March 2011, and at MCH (n=9) between December 2010 and March 2011. The infectious disease service at both hospitals had been involved with all study participants. Any pleural fluid from a patient without a diagnosis of complicated CAP (eg, from neonates with fetal hydrops) was not eligible for inclusion. All children with complicated CAP at both CHEO and MCH during the study period were treated empirically with broad-spectrum intravenous antimicrobials; antibacterials were continued if cultures were negative and rationalized if cultures were positive. Because the present study was retrospective in design, molecular testing results were not available to the treating clinicians. The present study was approved by the Research Ethics Boards of both CHEO and McMaster University (Hamilton, Ontario). Traditional culture-based sample processing As part of routine care, all pleural fluids were processed using standard microbiological methods. Aliquots were plated on sheep blood, chocolate and MacConkey agar under aerobic conditions at 37°C and monitored daily for five days. An aliquot was inoculated into thioglycollate broth and aliquots were plated on anaerobic agar media and kept for five days under strict anaerobic conditions; these were also checked daily after being left initially for 48 h. Bacteria were identified using standard laboratory methods. First, Gram stain, colony morphology and growth characteristics on culture media were examined. Based on these results, additional tests were performed. For example, optochin and bile solubility were performed for S pneumoniae identification; latex agglutination for group A antigen and bacitracin disk testing were performed for S pyogenes; and for streptococcal species other than S pyogenes and S pneumoniae, an API 20 Strep test (bioMérieux, USA) was performed. The majority of patients had blood drawn at the study sites and cultures processed using the BacT/Alert platform (bioMérieux, USA). Nucleic acid extraction Nucleic acids were extracted from 400 μL of pleural fluid samples. An extraction and amplification control organism that is not associated with human pulmonary infections (Bacillus atrophaeus, Steris Life Sciences, USA) was added before extraction (20). Samples underwent bead beating using 0.5 mm beads (ZR BashingBeads, Zymo Research, USA) to break down bacterial cell walls using the Disruptor Genie device (Scientific Industries Inc, USA). Samples were then extracted and purified using an automated NA extraction device (iPrep, Life Technologies, USA), with a final volume of 50 μL. Extraction time was approximately 45 min. Real-time polymerase chain reaction 5′exonuclease polymerase chain reaction (PCR) assays for the major bacterial CAP pathogens were used to test pleural fluid specimens. The PCR targets were the lytA gene for S pneumoniae (21), the hpd gene for H influenzae (21), the nuc gene for S aureus (22), the spy gene for S pyogenes (23), and the 16S ribosomal RNA gene of both Streptococcus intermedius and Streptococcus constellatus (two of the three species in the S anginosus group) (24). The limit of detection (LOD) for the assays was determined using serial dilutions of DNA extracted from reference strains of the organisms. The LOD was 100 organisms per PCR reaction for the S pyogenes, S aureus and H influenzae assays; 10 organisms per PCR reaction for the S pneumoniae assay; and one organism per PCR reaction for the S intermedius/S constellatus assay Uniplex PCR reaction assays for each target organism were prepared in 20 μL volumes in 96-well PCR plates using an automated liquid handler (Eppendorf 5070, Eppendorf Canada, Canada). A negative control (no template) was performed with each sample. PCR plates were covered with adhesive film (MicroAmp Optical Adhesive Film, Life Technologies Inc, USA) to prevent cross-contamination. PCR was performed using a 96-well fast-cycling block on a ViiA7 thermocyler (Life Technologies Inc, USA) using 40 cycles of two-temperature thermocyling (95°C × 3 s and 60°C × 30 s), taking approximately 40 min to complete. Statistical analysis Data were analyzed using STATA version 11.0 (Stata Corp, USA); 95% CIs for proportions were calculated. Differences between PCR diagnostic results and traditional culture-based results were compared using the McNemar exact test. RESULTS A total of 56 pleural fluids were analyzed; the majority of patients had received antimicrobials before pleural fluid sampling. Molecular methods detected a pathogen in 46 of 56 samples, yielding an overall positivity rate of 82% (95% CI 70% to 91%). Pneumococcus was detected in 35 of 56 samples (62% [95% CI 49% to 75%]). The second most common causative pathogen was group A streptococcus, detected in nine of 56 samples (16% [95% CI 8% to 28%]). Two of 56 samples (3.6% [95% CI 0.4% to 12%) were positive using the S intermedius/S constellatus assay. No pleural fluids were positive for S aureus. Cultures of blood and pleural fluids detected a pathogen in 14 of 56 patients (25% [95% CI 14% to 38%]) -less than one-third the rate of detection using PCR; this difference was statistically significant (P<0.0001). Cultures were positive for S pneumoniae in eight of 56 patients Every patient for whom an organism was grown in blood or pleural fluid had the same organism identified by pleural fluid PCR. Three pleural fluid samples had a positive Gram stain but were culturenegative. Two showed Gram-positive cocci in chains (one PCRpositive for group A streptococcus, one PCR-positive for S pneumoniae) and one showed Gram-negative cocci. This latter sample was PCR positive for S pneumoniae. No pleural fluids or blood cultures were positive for S aureus using culture or PCR. DISCUSSION In the present series, multiple-target uniplex real-time PCR of pleural fluids clearly outperformed traditional culture-based methods for the microbiological diagnosis of pneumonia with parapneumonic effusion. The benefit of molecular testing relative to culture was found to be greatest for the detection of S pneumoniae, the most common cause of bacterial pneumonia in children. Other investigators have also explored the use of PCR-based techniques for microbiological diagnosis of CAP complicated by parapneumonic effusion One approach to the detection of multiple pathogens has been broad-range 16S ribsomal RNA PCR. Pathogen detection in culturenegative cases has been shown to be 55% to 65% using this technique Microbiological diagnosis has practical value in that it can facilitate the optimization of antimicrobial therapy. The Infectious Disease Society of America recommends that "empiric therapy with a 3rd-generation parenteral cephalosporin should be prescribed…for infants and children with life-threatening infection, including those with empyema" (25). Our experience, and that of others The solution may be to improve diagnostics and thereby avoid both the under-and overtreatment risks inherent in empirical antimicrobial treatment algorithms. In our series, for example, had molecular testing been available in a timely manner, many patients (ie, those with group A streptococcus isolated from pleural fluids) may have been switched immediately to penicillin G without fear of decreased efficacy. Furthermore, in our region, high-level penicillin resistance (minimum inhibitory concentration ≥8 μ/mL) in S pneumoniae is very rare, suggesting that penicillin G or ampicillin could have been used successfully in most of our patients, consistent with Infectious Diseasees Society of America recommendations (25). Molecular methods to reliably determine penicillin susceptibility of pneumococci are not yet available; therefore, future studies should be performed to verify the safety of step-down therapy with ampicillin subsequent to the identification of S pneumoniae in populations in which rates of high-level penicillin resistance are low. There were limitations to the present study. Given that our study population was a convenience sample of pleural fluids, we cannot make inferences about the relative frequency of respiratory pathogens causing pediatric complicated pneumonia in the greater population. We also did not collect data on the immunocompetence of the study participants nor their vaccination records. Regardless, it is interesting that group A streptococcus was the second-most common cause of complicated pneumonia and that we did not find S aureus in any of our patients, despite the fact that participants were recruited during the 2009 pH1N1 epidemic. It may be that our PCR assay may have had low sensitivity for S aureus; however, unlike S pneumoniae, S aureus can typically be grown in blood or pleural fluid cultures from the majority of children with empyema There are barriers to the widespread implementation of molecular testing for the detection of bacterial pathogens. These include concerns about the costs of such testing, the requirement to train laboratory staff to perform these tests and the need for dedicated space to perform testing. However, in clinical situations in which patients have been given antibiotics before specimen collection, such as described in our study of parapneumonic effusion, molecular detection of bacteria was clearly superior to culture-based methods

Identification and management of Shigella infection in children with diarrhoea: a systematic review and meta-analysis

Summary: Background: Shigella infections are a leading cause of diarrhoeal death among children in low-income and middle-income countries. WHO guidelines reserve antibiotics for treating children with dysentery. Reliance on dysentery for identification and management of Shigella infection might miss an opportunity to reduce Shigella-associated morbidity and mortality. We aimed to systematically review and evaluate Shigella-associated and dysentery-associated mortality, the diagnostic value of dysentery for the identification of Shigella infection, and the efficacy of antibiotics for children with Shigella or dysentery, or both. Methods: We did three systematic reviews (for mortality, diagnostic value, and antibiotic treatment of Shigella and dysentery), and meta-analyses where appropriate, of studies in resource-limited settings. We searched MEDLINE, Embase, and LILACS database for studies published before Jan 1, 2017, in English, French, and Spanish. We included studies of human beings with diarrhoea and accepted all study-specific definitions of dysentery. For the mortality and diagnostic value searches, we excluded studies that did not include an effect estimate or data necessary to calculate this estimate. The search for treatment included only randomised controlled trials that were done after Jan 1, 1980, and assessed antibiotics in children (aged <18 years) with dysentery or laboratory-confirmed Shigella. We extracted or calculated odds ratios (ORs) and 95% CIs for relative mortality and did random-effects meta-analysis to arrive at pooled ORs. We calculated 95% CIs assuming a binomial distribution and did random-effects meta-regression of log-transformed sensitivity and specificity estimates for diagnostic value. We assessed the heterogeneity of papers included in these meta-analyses using the I2 statistic and evaluated publication bias using funnel plots. This review is registered with PROSPERO (CRD42017063896). Findings: 3649 papers were identified and 60 studies were included for analyses: 13 for mortality, 27 for diagnostic value, and 20 for treatment. Shigella infection was associated with mortality (pooled OR 2·8, 95% CI 1·6–4·8; p=0·000) whereas dysentery was not associated with mortality (1·3, 0·7–2·3; p=0·37). Between 1977 and 2016, dysentery identified 1·9–85·9% of confirmed Shigella infections, with sensitivity decreasing over time (p=0·04). Ten (50%) of 20 included antibiotic trials were among children with dysentery, none were placebo-controlled, and two (10%) evaluated antibiotics no longer recommended for acute infectious diarrhoea. Ciprofloxacin showed superior microbiological, but not clinical, effectiveness compared with pivmecillinam, and no superior microbiological and clinical effectiveness compared with gatifloxacin. Substantial heterogeneity was reported for meta-analyses of the Shigella-associated mortality studies (I2=78·3%) and dysentery-associated mortality studies (I2=73·2%). Too few mortality studies were identified to meaningfully test for publication bias. No evidence of publication bias was found in this analysis of studies of diagnostic value. Interpretation: Current WHO guidelines appear to manage dysentery effectively, but might miss opportunities to reduce mortality among children infected with Shigella who present without bloody stool. Further studies should quantify potential decreases in mortality and morbidity associated with antibiotic therapy for children with non-dysenteric Shigella infection. Funding: Bill & Melinda Gates Foundation and the Center for AIDS Research International Core

Glucose-6-phosphate dehydrogenase deficiency genotypes and allele frequencies in the Kavango and Zambezi regions of northern Namibia

Background: Namibia has made significant gains in the fight against malaria, with a target of elimination by 2023. We examined the genotype and allele frequencies of glucose-6-phosphate dehydrogenase (G6PD) deficiency to inform decisions on primaquine use, as we recently detected clusters of Plasmodium ovale curtisi in Kavango. Methods: A multistaged cross-sectional sampling method was used to enrol 212 children 2-9 y of age from schools and clinics in the Okavango and Zambezi regions of northern Namibia. Genotypes for the 202 G→A and 376 A→G mutations were assigned by polymerase chain reaction restriction fragment length polymorphism. Results: Of the 212 subjects enrolled, genotypes were available for 210, made up of 61 males and 149 females. G6PD-deficient males (hemizygotes) and females (homozygotes) constituted 3.27% (2/61) and 0.0% (0/149), respectively. Female heterozygotes (AA- and BA-) constituted 10.07% (15/149), while G6PD wild-type males (with A or B haplotype) and females (with AA, BB or AB haplotypes) consisted of 96.72% (59/61) and 89.93% (134/149), respectively. The A-, A and B allele frequencies were 0.0474, 0.3036 and 0.6490, respectively. Hardy-Weinberg equilibrium tests for female genotype frequencies did not show deviation (p=0.29). Conclusions: The frequency of G6PD deficiency alleles in males in the Kavango and Zambezi regions of northern Namibia constitute 3.27%, a first report to inform policy on primaquine role out

Correction: Rapid enteric testing to permit targeted antimicrobial therapy, with and without Lactobacillus reuteri probiotics, for paediatric acute diarrhoeal disease in Botswana: A pilot, randomized, factorial, controlled trial.

[This corrects the article DOI: 10.1371/journal.pone.0185177.]

Physician vaccination practices in mild to moderate inborn errors of immunity and retrospective review of vaccine completeness in IEI: results from the Canadian Immunization Research Network

Background and objectives Safety and effectiveness concerns may preclude physicians from recommending vaccination in mild/moderate inborn errors of immunity (IEI). This study describes attitudes and practices regarding vaccination among physicians who care for patients with mild/moderate B cell or mild/moderate combined immunodeficiencies (CID) and vaccination completeness among patients diagnosed with IEIs. Methods Canadian physicians caring for children with IEI were surveyed about attitudes and practices regarding vaccination in mild/moderate IEI. Following informed consent, immunization records of pediatric patients with IEI evaluated before 7 years of age were reviewed. Vaccine completeness was defined at age 2 years as 4 doses of diphtheria-tetanus-pertussis (DTaP), 3 doses pneumococcal conjugate (PCV), and 1 dose measles-mumps-rubella (MMR) vaccines. At 7 years 5 doses of DTP and 2 doses MMR were required. Results Forty-five physicians from 8 provinces completed the survey. Most recommended inactivated vaccines for B cell deficiency: (84% (38/45) and CID (73% (33/45). Fewer recommended live attenuated vaccines (B cell: 53% (24/45), CID 31% (14/45)). Of 96 patients with IEI recruited across 7 centers, vaccination completeness at age 2 was 25/43 (58%) for predominantly antibody, 3/13 (23%) for CID, 7/35 (20%) for CID with syndromic features, and 4/4 (100%) for innate/phagocyte defects. Completeness at age 7 was 15%, 17%, 5%, and 33%, respectively. Conclusion Most physicians surveyed recommended inactivated vaccines in children with mild to moderate IEI. Vaccine completeness for all IEI was low, particularly at age 7. Further studies should address the reasons for low vaccine uptake among children with IEI and whether those with mild-moderate IEI, where vaccination is recommended, eventually receive all indicated vaccines.Medicine, Faculty ofNon UBCPediatrics, Department ofReviewedFacultyResearche