Evaluation of a multiplex-qPCR for paediatric pleural empyema-An observational study in hospitalised children.

Pleural empyema is a serious complication of pneumonia in children. Negative bacterial cultures commonly impede optimal antibiotic therapy. To improve bacterial identification, we developed a molecular assay and evaluated its performance compared with bacterial culture. Our multiplex-quantitative PCR to detect Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Haemophilus influenzae was assessed using bacterial genomic DNA and laboratory-prepared samples (n = 267). To evaluate clinical performance, we conducted the Molecular Assessment of Thoracic Empyema (MATE) observational study, enrolling children hospitalised with empyema. Pleural fluids were tested by bacterial culture and multiplex-qPCR, and performance determined using a study gold standard. We determined clinical sensitivity and time-to-organism-identification to assess the potential of the multiplex-qPCR to reduce the duration of empiric untargeted antibiotic therapy. Using spiked samples, the multiplex-qPCR demonstrated 213/215 (99.1%) sensitivity and 52/52 (100%) specificity for all organisms. During May 2019-March 2023, 100 children were enrolled in the MATE study; median age was 3.9 years (IQR 2-5.6). A bacterial pathogen was identified in 90/100 (90%) specimens by multiplex-qPCR, and 24/100 (24%) by bacterial culture (P <0.001). Multiplex-qPCR identified a bacterial cause in 68/76 (90%) culture-negative specimens. S. pneumoniae was the most common pathogen, identified in 67/100 (67%) specimens. We estimate our multiplex-qPCR would have reduced the duration of untargeted antibiotic therapy in 61% of cases by a median 20 days (IQR 17.5-23, range 1-55). Multiplex-qPCR significantly increased pathogen detection compared with culture and may allow for reducing the duration of untargeted antibiotic therapy.

Membranome-based identification of amino acid substitution in Haemophilus influenzae multidrug efflux pump HmrM for reduced chloramphenicol susceptibility.

A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.

Impact of the COVID-19 pandemic on Haemophilus influenzae infections in pediatric patients hospitalized with community acquired pneumonia.

The COVID-19 pandemic has altered the infection landscape for many pathogens. This retrospective study aimed to compare Haemophilus influenzae (H. influenzae) infections in pediatric CAP patients hospitalized before (2018-2019) and during (2020-2022) the COVID-19 pandemic. We analyzed the clinical epidemiology and antimicrobial resistance (AMR) patterns of H. influenzae from a tertiary hospital in southwest China. A total of 986 pediatric CAP patients with H. influenzae-associated infections were included. Compared to 2018, the positivity rate increased in 2019 but dropped significantly in 2020. Although it rose in the following 2 years, the rate in 2022 remained significantly lower than in 2019. Patients' age during the pandemic was significantly higher than in 2018 and 2019, while gender composition remained similar across both periods. Notably, there were significant changes in co-infections with several respiratory pathogens during the pandemic. Resistance rates of H. influenzae isolates to antibiotics varied, with the highest resistance observed for ampicillin (85.9%) and the lowest for cefotaxime (0.0%). Resistance profiles to various antibiotics underwent dramatic changes during the COVID-19 pandemic. Resistance to amoxicillin-clavulanate, cefaclor, cefuroxime, trimethoprim-sulfamethoxazole, and the proportion of multi-drug resistant (MDR) isolates significantly decreased. Additionally, MDR isolates, alongside isolates resistant to specific drugs, were notably prevalent in ampicillin-resistant and ß-lactamase-positive isolates. The number of pediatric CAP patients, H. influenzae infections, and isolates resistant to certain antibiotics exhibited seasonal patterns, peaking in the winter of 2018 and 2019. During the COVID-19 pandemic, sharp decreases were observed in February 2020, and there was no resurgence in December 2022. These findings indicate that the COVID-19 pandemic has significantly altered the infection spectrum of H. influenzae in pediatric CAP patients, as evidenced by shifts in positivity rate, demographic characteristics, respiratory co-infections, AMR patterns, and seasonal trends.

Prevalence of Haemophilus influenzae in the nasopharynx of children from regions with varying incidence of invasive H. influenzae serotype a disease: Canadian Immunization Research Network (CIRN) study.

Haemophilus influenzae serotype a (Hia) has recently emerged as an important cause of invasive disease in the North American Arctic and Sub-Arctic regions, mainly affecting young Indigenous children. In this study, we addressed the question of whether the prevalence of Hia and all H. influenzae in the nasopharynx differed between paediatric populations from regions with high versus low incidence of invasive Hia disease. Nasopharyngeal specimens from children with acute respiratory tract infections (ARTI) collected for routine diagnostic detection of respiratory viruses were analysed with molecular-genetic methods to identify and serotype H. influenzae. In Nunavut, a region with a high incidence of invasive Hia disease, all H. influenzae and particularly Hia were found in the nasopharynx of 60.6% and 3.0% children. In Southern Ontario (Hamilton region), where Hia invasive disease is rare, the frequencies of all H. influenzae and Hia detection were 38.5% and 0.6%, respectively. In both cohorts, non-typeable H. influenzae was prevalent (57.0% and 37.9%, respectively). Considering that Hia is an important cause of severe invasive disease in Nunavut children, 3% prevalence of Hia among children with ARTI can reflect continuing circulation of the pathogen in the Northern communities that may result in invasive disease outbreaks.

Epidemiological and bacterial profile of childhood meningitis in Tunisia.

The worldwide burden of disease of bacterial meningitis remains high, despite the decreasing incidence following introduction of routine vaccination campaigns.The aim of our study was to evaluate the epidemiological and bacteriological profile of paediatric bacterial meningitis (BM) in Tunisian children, during the period 2003-2019, following the implementation of Haemophilus influenzae type b (Hib) vaccine (April 2011) and before 10-valent pneumoccocal conjugate vaccine (PCV10) introduction to the childhood immunization program.All bacteriologically confirmed cases of BM admitted to children's hospital of Tunis were recorded (January 2003 to April 2019). Serogroups of Neisseria meningitidis (Nm) and serotypes of Streptococcus pneumoniae (Sp) and H. influenzae (Hi) and antibiotic resistance were determined using conventional and molecular methods.Among 388 cases, the most frequent species were Sp (51.3%), followed by Nm (27.5%) and Hi (16.8%). We observed a significant decrease in Hi BM rate during the conjugated Hib vaccine use period (P < 0.0001). The main pneumococcal serotypes were 14, 19F, 6B, 23F and 19A and the serotype coverage of PCV10, PCV13, PCV15 and PCV20 was 71.3 and 78.8%, 79.4 and 81.9% respectively. The most frequent Nm serogroup was B (83.1%). Most Hi strains were of serotype b (86.9%). High levels of resistance were found: Sp and Nm to penicillin (respectively 60.1 and 80%) and Hi to ampicillin (42.6%). All meningococcal and Hi isolates were susceptible to third-generation cephalosporins and 7.2% of pneumococcal strains had decreased susceptibility to these antibiotics.The Hib conjugate vaccine decreased the rate of BM. Sp dominated the aetiology of BM in children in Tunisia. Conjugate vaccines introducing decreases not only BM cases but also antimicrobial resistance.

Increase in invasive Haemophilus influenzae serotype A infections during the COVID-19 pandemic in New South Wales, Australia.

Haemophilus influenzae, a causative agent of severe invasive infections such as meningitis, sepsis and pneumonia, is classified into encapsulated or typeable (represented by serotypes A to F) and non-typeable varieties (NTHi) by the presence or absence of the polysaccharide capsule. Invasive disease caused by H. influenzae type B (HIB) can be prevented through vaccination which remains the main disease control intervention in many countries. This study examined the genomic diversity of circulating H. influenzae strains associated with invasive disease in New South Wales, Australia, before and during the COVID-19 pandemic. Ninety-six isolates representing 95 cases of invasive H. influenzae infections (iHi) diagnosed between January 2017 and September 2022 were typed and characterised using whole genome sequencing. These cases were caused by serotypes A (n=24), B (n=35), E (n=3), F (n=2) and NTHi (n=32). There was an apparent decline in the number of iHi infections during the COVID-19 pandemic, with a corresponding increase in the proportion of iHi cases caused by serotype A (HIA), which returned to pre-pandemic levels in 2022. Fifteen isolates associated with HIB or non-typeable iHi were resistant to ß-lactams due to a PBP3 mutation or carriage of blaTEM-1. Further, capsular gene duplication was observed in HIB isolates but was not found in HIA. These findings provide important baseline genomic data for ongoing iHi surveillance and control.

A novel biosensing strategy for identification of three important bacteria causing meningitis.

Bacterial meningitis is an acute infection which requires rapid diagnosis and treatment due to the high mortality and serious consequences of the disease. The purpose of this study was to design a homemade multiplex PCR and a novel fluorescence biosensor on chip (FBC) to detect three important agents of meningitis including Streptococcus pneumoniae (S. pneumoniae), Neisseria meningitidis (N. meningitidis), and Haemophilus influenzae (H. influenzae). The homemade multiplex PCR can diagnose three bacterial species simultaneously. Fabrication of FBC was carried out based on the deposition of lead nanoparticles on a quartz slide using the thermal evaporation method. Then, the SH-Cap Probe/Target ssDNA /FAM-Rep probe was loaded on lead film. The evaluation of the fluorescence reaction when the probes bind to the target ssDNA was assessed by a Cytation 5 Cell Imaging Multimode Reader Bio-Tek. The limit of detections (LOD) in homemade PCR and FBC to identify S. pneumoniae were 119 × 102 CFU/mL (0.27 ng/µL) and 380 CFU/mL (9 pg/µL), respectively. The LODs of homemade PCR and FBC for detection of N. meningitidis were 4.49 CFU/mL (1.1 pg/µL) and 13 × 103 CFU/mL (30 pg/µL), respectively. Our results confirmed the LODs of homemade PCR and FBC in detection of H. influenzae were 15.1 CFU/mL (30 fg/µL) and 41 × 102 CFU/mL (90 pg/ µL), respectively. Both techniques had appropriate sensitivity and specificity in detection of S. pneumoniae, N. meningitidis and H. influenzae.

Respiratory pathogen and clinical features of hospitalized patients in acute exacerbation of chronic obstructive pulmonary disease after COVID 19 pandemic.

Respiratory infections are common causes of acute exacerbation of chronic obstructive lung disease (AECOPD). We explored whether the pathogens causing AECOPD and clinical features changed from before to after the coronavirus disease 2019 (COVID-19) outbreak. We reviewed the medical records of patients hospitalized with AECOPD at four university hospitals between January 2017 and December 2018 and between January 2021 and December. We evaluated 1180 patients with AECOPD for whom medication histories were available. After the outbreak, the number of patients hospitalized with AECOPD was almost 44% lower compared with before the outbreak. Patients hospitalized with AECOPD after the outbreak were younger (75 vs. 77 years, p = 0.003) and more often stayed at home (96.6% vs. 88.6%, p < 0.001) than patients of AECOPD before the outbreak. Hospital stay was longer after the outbreak than before the outbreak (10 vs. 8 days. p < 0.001). After the COVID-19 outbreak, the identification rates of S. pneumoniae (15.3 vs. 6.2%, p < 0.001) and Hemophilus influenzae (6.4 vs. 2.4%, p = 0.002) decreased, whereas the identification rates of P. aeruginosa (9.4 vs. 13.7%, p = 0.023), Klebsiella pneumoniae (5.3 vs. 9.8%, p = 0.004), and methicillin-resistant Staphylococcus aureus (1.0 vs. 2.8%, p = 0.023) increased. After the outbreak, the identification rate of influenza A decreased (10.4 vs. 1.0%, p = 0.023). After the outbreak, the number of patients hospitalized with AECOPD was lower and the identification rates of community-transmitted pathogens tended to decrease, whereas the rates of pathogens capable of chronic colonization tended to increase. During the period of large-scale viral outbreaks that require quarantine, patients with AECOPD might be given more consideration for treatment against strains that can colonize chronic respiratory disease rather than community acquired pathogens.

Exploring the pediatric nasopharyngeal bacterial microbiota with culture-based MALDI-TOF mass spectrometry and targeted metagenomic sequencing.

The nasopharynx is an important reservoir of disease-associated and antimicrobial-resistant bacterial species. This proof-of-concept study assessed the utility of a combined culture, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and targeted metagenomic sequencing workflow for the study of the pediatric nasopharyngeal bacterial microbiota. Nasopharyngeal swabs and clinical metadata were collected from Cambodian children during a hospital outpatient visit and then biweekly for 12 weeks. Swabs were cultured on chocolate and blood-gentamicin agar, and all colony morphotypes were identified by MALDI-TOF MS. Metagenomic sequencing was done on a scrape of all colonies from a chocolate agar culture and processed using the mSWEEP pipeline. One hundred one children were enrolled, yielding 620 swabs. MALDI-TOF MS identified 106 bacterial species/40 genera: 20 species accounted for 88.5% (2,190/2,474) of isolates. Colonization by Moraxella catarrhalis (92.1% of children on ≥1 swab), Haemophilus influenzae (87.1%), and Streptococcus pneumoniae (83.2%) was particularly common. In S. pneumoniae-colonized children, a median of two serotypes [inter-quartile range (IQR) 1-2, range 1-4] was detected. For the 21 bacterial species included in the mSWEEP database and identifiable by MALDI-TOF, detection by culture + MALDI-TOF MS and culture + mSWEEP was highly concordant with a median species-level agreement of 96.9% (IQR 86.8%-98.8%). mSWEEP revealed highly dynamic lineage-level colonization patterns for S. pneumoniae which were quite different to those for S. aureus. A combined culture, MALDI-TOF MS, targeted metagenomic sequencing approach for the exploration of the young child nasopharyngeal microbiome was technically feasible, and each component yielded complementary data. IMPORTANCE: The human upper respiratory tract is an important source of disease-causing and antibiotic-resistant bacteria. However, understanding the interactions and stability of these bacterial populations is technically challenging. We used a combination of approaches to determine colonization patterns over a 3-month period in 101 Cambodian children. The combined approach was feasible to implement, and each component gave complementary data to enable a better understanding of the complex patterns of bacterial colonization.

Microbial aetiology of community-acquired pneumonia in hospitalised adults: A prospective study utilising comprehensive molecular testing.

OBJECTIVES: This study aimed to describe the microbial aetiology of community-acquired pneumonia (CAP) in adults admitted to a tertiary care hospital and assess the impact of syndromic polymerase chain reaction (PCR) panels on pathogen detection. METHODS: Conducted at Haukeland University Hospital, Norway, from September 2020 to April 2023, this prospective study enrolled adults with suspected CAP. We analysed lower respiratory tract samples using both standard-of-care tests and the BIOFIRE® FILMARRAY® Pneumonia Plus Panel (FAP plus). The added value of FAP Plus in enhancing the detection of clinically relevant pathogens, alongside standard-of-care diagnostics, was assessed. RESULTS: Of the 3238 patients screened, 640 met the inclusion criteria, with 384 confirmed to have CAP at discharge. In these patients, pathogens with proven or probable clinical significance were identified in 312 (81.3%) patients. Haemophilus influenzae was the most prevalent pathogen, found in 118 patients (30.7%), followed by SARS-CoV-2 in 74 (19.3%), and Streptococcus pneumoniae in 64 (16.7%). Respiratory viruses were detected in 186 (48.4%) patients. The use of FAP plus improved the pathogen detection rate from 62.8% with standard-of-care methods to 81.3%. CONCLUSIONS: Pathogens were identified in 81% of CAP patients, with Haemophilus influenzae and respiratory viruses being the most frequently detected pathogens. The addition of the FAP plus panel, markedly improved pathogen detection rates compared to standard-of-care diagnostics alone.

Characterization of Ceftriaxone-Resistant Haemophilus influenzae Among Korean Children.

BACKGROUND: Haemophilus influenzae is a frequently encountered pathogen responsible for respiratory tract infections in children. Following the detection of ceftriaxone-resistant H. influenzae at our institution, we aimed to investigate the resistance mechanisms of ceftriaxone in H. influenzae, with a particular focus on alterations in penicillin-binding protein 3 (PBP3) and ß-lactamase production. METHODS: Among H. influenzae isolates collected at Asan Medical Center Children's Hospital from March 2014 to April 2019, ceftriaxone-resistant strains by the disk-diffusion test were included. Ceftriaxone minimum inhibitory concentrations (MICs) were determined using the E-test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The presence of ß-lactamase was assessed through cefinase test and TEM-1/ROB-1 polymerase chain reaction (PCR). PBP3 alterations were explored via ftsI gene sequencing. RESULTS: Out of the 68 collected strains, 21 exhibited resistance to ceftriaxone in disk diffusion tests. Two strains were excluded due to failed subculture. Among 19 ceftriaxone-resistant H. influenzae isolates, eighteen were non-typeable H. influenzae, and twelve were positive for TEM-1 PCR. Isolates were classified into groups II (harboring only N526K, n = 3), III (N526K+S385T, n = 2), III+ (S385T+L389F+N526K, n = 11), and III-like+ (S385T+L389F+R517H, n = 3) according to the PBP3 alteration pattern. With a median ceftriaxone MIC of 0.190 mg/L (range, 0.008-0.750), the median ceftriaxone MIC was the highest in group III-like+ (0.250 mg/L), followed by groups III+ (0.190 mg/L), III (0.158 mg/L), and II (0.012 mg/L). All three strains belonging to group II, which did not harbor the S385T substitution, had ceftriaxone MICs of ≤ 0.125 mg/L. CONCLUSION: The emergence of ceftriaxone-resistant H. influenzae with ceftriaxone MIC values of up to 0.75 mg/L was observed even in children in South Korea, with most associated with S385T and L389F substitutions. The N526K mutation alone does not significantly impact ceftriaxone resistance. Further large-scale studies are essential to investigate changes in antibiotic resistance patterns and factors influencing antibiotic resistance in H. influenzae isolated from pediatric patients in Korea.

Recurrent community-acquired pneumococcal meningitis in adults with and without identified predisposing factors.

Bacterial meningitis is still a significant public health concern, with high morbidity and mortality rates. Despite this, it is still a rare event that requires the bacterial invasion of the meninges. However, some predisposing factors can trigger recurrent episodes of meningitis. This study is aimed at determining the clinical characteristics and the molecular epidemiology of episodes of recurrent community-acquired meningitis with and without predisposing factors. For this purpose, we performed a retrospective study of our laboratory database during the period of 2010 to 2020. Additionally, using molecular tools developed in our previous works, the epidemiology of the pathogens causing these episodes was analyzed using cerebrospinal fluid samples, especially in the absence of isolated strains. We observed a total of 1,779 meningitis cases and 230 were caused by Streptococcus pneumoniae. Of those, 16 were recurrent meningitis episodes (16/1,779; 0.9%) from seven patients. Pneumococcus was the main agent responsible in these recurrent episodes and only two episodes were caused by Haemophilus influenzae. The mean age of these patients was 20 years old and three had predisposing factors which could have led to contracting meningitis. The samples presented different pneumococcal serotypes. Most of them were non-vaccine-covered serotypes and antibiotic susceptible strains. Therefore, it was demonstrated how the practical employment of molecular tools, developed for research, when applied in the routine of diagnosis, can provide important information for epidemiological surveillance. Furthermore, it was shown how pneumococcus was the leading cause of recurrent community-acquired meningitis without predisposing factors, suggesting that pneumococcal vaccination may be necessary, even in those groups of individuals considered to be less susceptible.

An Investigation of Pediatric Case-patients With Invasive Haemophilus influenzae in Alaska, 2005-2011.

BACKGROUND: Haemophilus influenzae (Hi) can cause severe disease in children. This study aimed to identify risk factors related to invasive Hi disease in Alaska children and evaluate carriage in people around them. METHODS: From 2005 to 2011, we investigated episodes of invasive, typeable Hi disease in Alaska children <10 years old. Three age-matched control children were enrolled for each case-patient. We evaluated oropharyngeal Hi carriage in people in close contact with Hi case-patients (contacts) as well as control children and their household members. Individual and household risk factors for illness and carriage were evaluated using questionnaires and chart reviews. RESULTS: Thirty-eight of 44 (86%) children with invasive, typeable Hi disease were recruited: 20 Hi serotype a (53%), 13 serotype b (Hib) (34%) and 5 serotype f (13%). Children with the invasive Hi disease were more likely than controls to have underlying health problems (67% vs. 24%, P = 0.001), other carriers of any Hi in their household (61% vs. 15%, P < 0.001), and inadequate Hib vaccination (26% vs. 9%, P = 0.005). People who carried Hi were younger than noncarriers (mean 12.7 vs. 18.0 years, P = 0.008). The carriage was clustered within case-patient households, with carriage in 19% of household contacts, while only 6.3% of nonhousehold contacts and 5.5% of noncontacts carried the Hi serotype of interest ( P < 0.001). CONCLUSIONS: Factors associated with invasive Hi disease in children included underlying health problems, household carriage and inadequate Hib vaccination. The high level of carriage in case-patient households is important to consider when evaluating treatment and prophylaxis strategies.

Molecular characteristics and biofilm formation capacity of nontypeable Haemophilus influenza strains isolated from lower respiratory tract in children.

With the widespread introduction of the Hib conjugate vaccine, Nontypeable Haemophilus influenzae (NTHi) has emerged as the predominant strain globally. NTHi presents a significant challenge as a causative agent of chronic clinical infections due to its high rates of drug resistance and biofilm formation. While current research on NTHi biofilms in children has primarily focused on upper respiratory diseases, investigations into lower respiratory sources remain limited. In this study, we collected 54 clinical strains of lower respiratory tract origin from children. Molecular information and drug resistance features were obtained through whole gene sequencing and the disk diffusion method, respectively. Additionally, an in vitro biofilm model was established. All clinical strains were identified as NTHi and demonstrated the ability to form biofilms in vitro. Based on scanning electron microscopy and crystal violet staining, the strains were categorized into weak and strong biofilm-forming groups. We explored the correlation between biofilm formation ability and drug resistance patterns, as well as clinical characteristics. Stronger biofilm formation was associated with a longer cough duration and a higher proportion of abnormal lung imaging findings. Frequent intake of ß-lactam antibiotics might be associated with strong biofilm formation. While a complementary relationship between biofilm-forming capacity and drug resistance may exist, further comprehensive studies are warranted. This study confirms the in vitro biofilm formation of clinical NTHi strains and establishes correlations with clinical characteristics, offering valuable insights for combating NTHi infections.

High Prevalence of Non-typeable Haemophilus influenzae and Haemophilus haemolyticus Among Vaccinated Children with Community-Acquired Pneumonia in Vietnam.

Among 467 children under five hospitalized with community-acquired pneumonia, the prevalence of Haemophilus influenzae or Haemophilus haemolyticus was 60.8%, all cases were non-typable H. influenzae (NTHi) or H. haemolyticus. NTHi/H. haemolyticus PCR detection was associated with about twice the risk for severe disease. The results highlight the need for increased awareness and research efforts to investigate the role of NTHi/H. haemolyticus in severe CAP among children.

Identifying Children Likely to Benefit From Antibiotics for Acute Sinusitis: A Randomized Clinical Trial.

Importance: The large overlap between symptoms of acute sinusitis and viral upper respiratory tract infection suggests that certain subgroups of children being diagnosed with acute sinusitis, and subsequently treated with antibiotics, derive little benefit from antibiotic use. Objective: To assess if antibiotic therapy could be appropriately withheld in prespecified subgroups. Design, Setting, and Participants: Randomized clinical trial including 515 children aged 2 to 11 years diagnosed with acute sinusitis based on clinical criteria. The trial was conducted between February 2016 and April 2022 at primary care offices affiliated with 6 US institutions and was designed to evaluate whether symptom burden differed in subgroups defined by nasopharyngeal Streptococcus pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis on bacterial culture and by the presence of colored nasal discharge. Interventions: Oral amoxicillin (90 mg/kg/d) and clavulanate (6.4 mg/kg/d) (n = 254) or placebo (n = 256) for 10 days. Main Outcomes and Measures: The primary outcome was symptom burden based on daily symptom scores on a validated scale (range, 0-40) during the 10 days after diagnosis. Secondary outcomes included treatment failure, adverse events including clinically significant diarrhea, and resource use by families. Results: Most of the 510 included children were aged 2 to 5 years (64%), male (54%), White (52%), and not Hispanic (89%). The mean symptom scores were significantly lower in children in the amoxicillin and clavulanate group (9.04 [95% CI, 8.71 to 9.37]) compared with those in the placebo group (10.60 [95% CI, 10.27 to 10.93]) (between-group difference, -1.69 [95% CI, -2.07 to -1.31]). The length of time to symptom resolution was significantly lower for children in the antibiotic group (7.0 days) than in the placebo group (9.0 days) (P = .003). Children without nasopharyngeal pathogens detected did not benefit from antibiotic treatment as much as those with pathogens detected; the between-group difference in mean symptom scores was -0.88 (95% CI, -1.63 to -0.12) in those without pathogens detected compared with -1.95 (95% CI, -2.40 to -1.51) in those with pathogens detected. Efficacy did not differ significantly according to whether colored nasal discharge was present (the between-group difference was -1.62 [95% CI, -2.09 to -1.16] for colored nasal discharge vs -1.70 [95% CI, -2.38 to -1.03] for clear nasal discharge; P = .52 for the interaction between treatment group and the presence of colored nasal discharge). Conclusions: In children with acute sinusitis, antibiotic treatment had minimal benefit for those without nasopharyngeal bacterial pathogens on presentation, and its effects did not depend on the color of nasal discharge. Testing for specific bacteria on presentation may represent a strategy to reduce antibiotic use in this condition. Trial Registration: ClinicalTrials.gov Identifier: NCT02554383.

Sudden death of a child associated with invasive non-typeable Haemophilus influenzae infection with underlying IgG2 subclass deficiency.

Haemophilus influenzae can be divided into typeable and non-typeable strains. Although non-typeable Haemophilus influenzae (NTHi) is less likely to be a fatal bacterium, invasive NTHi infection has been reported to increase worldwide. This study presents a case of sudden death of a child with invasive NTHi infection and underlying immunoglobulin G2 (IgG2) deficiency. A two years seven months male child with a high fever was found unresponsive in bed, lying face down on a soft pillow. Later, the hospital declared the subject dead. An autopsy revealed that the only noteworthy finding was tissue congestion. The histopathological findings disclosed neutrophils within blood vessels of major organs. Meanwhile, the formation of the micro abscess was not visible, which indicated bacteremia. The bacterial blood culture was positive for Haemophilus Influenzae. Polymerase chain reaction assay revealed the absence of an entire capsule locus. The transmission electron microscopy showed that the colonies did not have polysaccharide capsules. Based on the above findings, the strain was identified as NTHi. Furthermore, the value of serum IgG2 was deficient, indicating the presence of IgG2 subclass deficiency. The subject eventually died from asphyxia by smothering due to a comorbid condition with a high fever brought on by NTHi-induced bacteremia and lying face down. IgG2 subclass deficiency contributed to the development of invasive NTHi infection. The invasive NTHi infection might present a risk of sudden death, particularly for immunocompromised children. As forensic pathologists and pediatricians may encounter such a problematic clinical condition, they should be aware of this.

Nasopharyngeal carriage and antibiotic susceptibility patterns of streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis and staphylococcus aureus among urban Ugandan children post-PCV10 introduction: a cross-sectional study.

In 2013, Uganda introduced the PCV10 pneumococcal vaccine and it is given to children at 6, 10 and 14 weeks after birth. Carriage prevalence studies post PCV10-introduction are necessary for monitoring the impact of vaccination and trends in antibiotic resistance. Here, we studied carriage/antibiotic resistance of Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus isolated from 194 children at the Mulago Assessment Centre clinic in Kampala-Uganda, 5 years post-PCV10 introduction. Almost all the children were vaccinated with PCV10 (98.5%, 191/194). The overall carriage prevalence (any species) was 62% (120/194), and it was associated with a history of antibiotics use (p=0.0159) and having respiratory symptoms (p=0.0003). The pneumococcus, H. influenzae, M. catarrhalis, and S. aureus carriage prevalence was 46% (90/194), 21% (40/194), 7% (14/194), and 6% (12/194), respectively. Species co-carriage occurred in 32 children (17%, 32/194), predominantly multidrug resistant pneumococcus + H. influenzae (23 children). Furthermore, pneumococci were highly resistant to cotrimoxazole (100%), erythromycin (76%), and tetracycline (52%), 42% being multidrug-resistant. Overall, we note an increase in antibiotic resistance post-PCV10 introduction, and microbial shifts i.e., a decrease in pneumococcus, M. catarrhalis and S. aureus carriage and an increase in H. influenzae carriage suggesting vaccine-associated perturbation of the respiratory ecology.

Whole-genome analysis of Haemophilus influenzae strains isolated from persons with cystic fibrosis.

Introduction. Haemophilus influenzae is a commensal of the respiratory tract that is frequently present in cystic fibrosis (CF) patients and may cause infection. Antibiotic resistance is well described for CF strains, and virulence factors have been proposed.Hypothesis/Gap. The genetic diversity of H. influenzae strains present in the lungs of persons with CF is largely unknown despite the fact that this organism is considered to be a pathogen in this condition. The aim was to establish the genetic diversity and susceptibility of H. influenzae strains from persons with CF, and to screen the whole genomes of these strains for the presence of antibiotic resistance determinants and proposed virulence factors.Methods. A total of 67 strains, recovered from respiratory samples from persons with CF from the UK (n=1), Poland (n=2), Spain (n=24) and the Netherlands (n=40), were subjected to whole-genome sequencing using Illumina technology and tested for antibiotic susceptibility. Forty-nine of these strains (one per different sequence type) were analysed for encoded virulence factors and resistance determinants.Results. The 67 strains represented 49 different sequence types. Susceptibility testing showed that all strains were susceptible to aztreonam, ciprofloxacin, imipenem and tetracycline. Susceptibility to ampicillin, ampicillin/sulbactam, amoxicillin/clavulanic acid, cefuroxime, cefixime, ceftriaxone, cefepime, meropenem, clarithromycin, co-trimoxazole and levofloxacin ranged from 70.2-98.5%. Only 6/49 strains (12.2%) harboured acquired resistance genes. Mutations associated with a ß-lactamase-negative ampicillin-resistant phenotype were present in four strains (8.2 %). The potential virulence factors, urease, haemoglobin- and haptoglobin-binding protein/carbamate kinase, and OmpP5 (OmpA), were encoded in more than half of the strains. The genes for HMW1, HMW2, H. influenzae adhesin, a IgA-specific serine endopeptidase autotransporter precursor, a TonB-dependent siderophore, an ABC-transporter ATP-binding protein, a methyltransferase, a BolA-family transcriptional regulator, glycosyltransferase Lic2B, a helix-turn-helix protein, an aspartate semialdehyde dehydrogenase and another glycosyltransferase were present in less than half of the strains.Conclusion. The H. influenzae strains showed limited levels of resistance, with the highest being against co-trimoxazole. Sequences encoding a carbamate kinase and a haemoglobin- and haemoglobin-haptoglobin-binding-like protein, a glycosyl transferase and an urease may aid the colonization of the CF lung. The adhesins and other identified putative virulence factors did not seem to be necessary for colonization.