Severe A(H1N1)pdm09 influenza acute encephalopathy outbreak in children in Tuscany, Italy, December 2023 to January 2024.

A severe outbreak of influenza A(H1N1pdm09) infection in seven children (median age: 52 months) occurred between December 2023 and January 2024 in Tuscany, Italy. Clinical presentation ranged from milder encephalopathy to acute necrotizing encephalopathy (ANE) with coma and multiorgan failure; one child died. This report raises awareness for clinicians to identify and treat early acute encephalopathy caused by H1N1 influenza and serves as a reminder of severe presentations of influenza in young children and the importance of vaccination.

Epidemiology and Natural History of Childhood-Acquired Chronic Hepatitis C: A Single-Center Long-Term Prospective Study.

OBJECTIVES: To prospectively describe the epidemiology and long-term outcome of childhood-acquired hepatitis C virus (HCV) infection in a large cohort of children followed at a single center. METHODS: All children with chronic HCV infection followed at the Liver Unit of our tertiary Hospital in Florence (Italy) from January 1, 1988, to September 30, 2021, were included in the analysis. RESULTS: The final sample consisted of 163 children (median age at enrollment 4 years, interquartile range (IQR): 10; median age at last follow-up 14 years, IQR: 7). The median duration of follow-up was 86 months (IQR: 112). One hundred twenty-five children were vertically infected and 26 acquired the infection horizontally. Twenty-six of the 125 children who were vertically infected (20.8%) underwent spontaneous clearance of HCV RNA at a median age of 4 years (IQR: 2), whereas all the others remained persistently viremic. One patient was diagnosed with cirrhosis; 2 presented clinically detectable extrahepatic manifestations (chronic urticaria). Thirty-two children (19.6%) received antiviral therapy: 8 out of 32 (25%) were treated with pegylated-interferon alfa-2b [sustained virological response (SVR) 24 weeks after the end of treatment in 7/8]; 24 out of 32 (75%) were treated with direct-acting antivirals (SVR 12 weeks after the end of treatment in 23/24). CONCLUSIONS: The present study describes the largest cohort of children with chronic HCV infection prospectively evaluated with a long follow-up at a single center. HCV infection in children is often a chronic infection that can be cured with modern antiviral therapy. Early treatment could prevent the development of advanced liver disease.

How home anterior self-collected nasal swab simplifies SARS-CoV-2 testing: new surveillance horizons in public health and beyond.

The sample collection procedure for SARS-CoV-2 has a strong impact on diagnostic capability, contact tracing approach, ultimately affecting the infection containment performance. This study demonstrates that self-collected nasal-swab has shown to be a valid and well tolerated procedure to SARS-CoV-2 surveillance in a healthcare system. More significantly, no performance adequacy difference was detected in self-administered swabs between healthcare worker (HCW) and non-HCW which allows to speculate that this procedure could be successfully extended to the entire population for mass screening.

Carriage rates and risk factors during an outbreak of invasive meningococcal disease due to Neisseria meningitidis serogroup C ST-11 (cc11) in Tuscany, Italy: a cross-sectional study.

BACKGROUND: During 2015-2016 an outbreak of invasive meningococcal disease due to N. meningitidis serogroup C ST-11 (cc11) occurred in Tuscany, Italy. The outbreak affected mainly the age group 20-30 years, men who have sex with men, and the area located between the cities of Firenze, Prato and Empoli, with discos and gay-venues associated-clusters. A cross-sectional-survey was conducted to assess the prevalence and risk factors for meningococcal-carriage, in order to address public health interventions. METHODS: A convenience sample of people aged 11-45 years provided oropharyngeal swab specimens and completed questionnaires on risk factors for meningococcal carriage during a 3 months study-period, conducted either in the outbreak-area and in a control-area not affected by the outbreak (cities of Grosseto and Siena). Isolates were tested by culture plus polymerase chain reaction. Serogroup C meningococcal isolates were further characterized using multilocus sequence typing. Univariate and multivariate analyses were performed to estimate adjusted odds ratios (AORs) for meningococcal carriage. RESULTS: A total of 2285 oropharyngeal samples were collected. Overall, meningococcal carriage prevalence was 4.8% (n = 110), with nonencapsulated meningococci most prevalent (2.3%; n = 52). Among encapsulated meningococci, serogroup B was the most prevalent (1.8%; n = 41), followed by serogroup Y (0.5%; n = 11) and serogroup C (0.2%; n = 4); one carrier of serogroup E and one of serogroup Z, were also found (0.04%). Three individuals from the city of Empoli were found to carry the outbreak strain, C:ST-11 (cc11); this city also had the highest serogroup C carriage prevalence (0.5%). At the multivariate analyses, risk factors for meningococcal carriage were: illicit-drugs consumption (AOR 6.30; p < 0.01), active smoking (AOR 2.78; p = 0.01), disco/clubs/parties attendance (AOR 2.06; p = 0.04), being aged 20-30 years (AOR 3.08; p < 0.01), and have had same-sex intercourses (AOR 6.69; p < 0.01). CONCLUSIONS: A low prevalence of meningococcal serogroup C carriage in an area affected by an outbreak due to the hypervirulent N. meningitidis serogroup C ST-11 (cc11) strain was found. The city of Empoli had the highest attack-rate during the outbreak and also the highest meningococcal serogroup C carriage-prevalence due to the outbreak-strain. Multivariate analyses underlined a convergence of risk factors, which partially confirmed those observed among meningococcal outbreak-cases, and that should be considered in targeted immunization campaigns.

Interconnected clusters of invasive meningococcal disease due to Neisseria meningitidis serogroup C ST-11 (cc11), involving bisexuals and men who have sex with men, with discos and gay-venues hotspots of transmission, Tuscany, Italy, 2015 to 2016.

In 2015 an increased incidence of invasive meningococcal disease due to serogroup-C (MenC) occurred in Tuscany, Italy. This led the Regional Health Authority of Tuscany to implement a reactive immunisation campaign and to launch an epidemiological field investigation aiming to address targeted immunisation interventions. In 2011-14, 10 MenC cases had been reported compared with 62 cases in 2015-16. The case fatality rate was 21% (n = 13) and 51 cases (82.3%) were confirmed as C:P1.5-1,10-8:F3-6:ST-11(cc11). Overall, 17 clusters were recognised. Six discos and four gay-venues were found to have a role as transmission-hotspots, having been attended by 20 and 14 cases in the 10 days before symptoms onset. Ten and three cases occurred, respectively, among men who have sex with men (MSM) and bisexual individuals, who were involved in 11 clusters. In addition, heterosexual cases (n = 5) attending gay-venues were also found. Secondary cases were not identified. Molecular typing indicated close relationship with MenC clusters recently described among gay, bisexual and other MSM in Europe and the United States, suggesting a possible international spread of the serogroup-C-variant P1.5-1,10-8:F3-6:ST-11(cc11) in this population-group; however, epidemiological links were not identified. In December 2016, a targeted vaccination campaign involving discos and lesbian, gay, bisexual, and transgender (LGBT) associations was implemented. During 2017, 10 cases of MenC occurred, compared with 32 and 30 cases reported in 2015 and 2016 respectively, suggesting the effectiveness of the reactive and targeted immunisation programmes.

Underestimation of Invasive Meningococcal Disease in Italy.

Knowing the incidence of invasive meningococcal disease (IMD) is essential for planning appropriate vaccination policies. However, IMD may be underestimated because of misdiagnosis or insufficiently sensitive laboratory methods. Using a national molecular surveillance register, we assessed the number of cases misdiagnosed and diagnoses obtained postmortem with real-time PCR (rPCR), and we compared sensitivity of rPCR versus culture-based testing. A total of 222 IMD cases were identified: 11 (42%) of 26 fatal cases had been misdiagnosed or undiagnosed and were reclassified as IMD after rPCR showed meningococcal DNA in all available specimens taken postmortem. Of the samples tested with both rPCR and culture, 58% were diagnosed by using rPCR alone. The underestimation factor associated with the use of culture alone was 3.28. In countries such as Italy, where rPCR is in limited use, IMD incidence may be largely underestimated; thus, assessments of benefits of meningococcal vaccination may be prone to error.

First Human Case of Meningitis and Sepsis in a Child Caused by Actinobacillus suis or Actinobacillus equuli.

We report the first human case of meningitis and sepsis caused in a child by Actinobacillus suis or A. equuli, a common opportunistic pathogen of swine or horses, respectively. Identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and real-time PCR assay. A previous visit to a farm was suspected as the source of infection.

Diagnosis of immunodeficiency caused by a purine nucleoside phosphorylase defect by using tandem mass spectrometry on dried blood spots.

BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency is a rare form of autosomal recessive combined primary immunodeficiency caused by a enzyme defect leading to the accumulation of inosine, 2'-deoxy-inosine (dIno), guanosine, and 2'-deoxy-guanosine (dGuo) in all cells, especially lymphocytes. Treatments are available and curative for PNP deficiency, but their efficacy depends on the early approach. PNP-combined immunodeficiency complies with the criteria for inclusion in a newborn screening program. OBJECTIVE: This study evaluate whether mass spectrometry can identify metabolite abnormalities in dried blood spots (DBSs) from affected patients, with the final goal of individuating the disease at birth during routine newborn screening. METHODS: DBS samples from 9 patients with genetically confirmed PNP-combined immunodeficiency, 10,000 DBS samples from healthy newborns, and 240 DBSs from healthy donors of different age ranges were examined. Inosine, dIno, guanosine, and dGuo were tested by using tandem mass spectrometry (TMS). T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) levels were evaluated by using quantitative RT-PCR only for the 2 patients (patients 8 and 9) whose neonatal DBSs were available. RESULTS: Mean levels of guanosine, inosine, dGuo, and dIno were 4.4, 133.3, 3.6, and 3.8 µmol/L, respectively, in affected patients. No indeterminate or false-positive results were found. In patient 8 TREC levels were borderline and KREC levels were abnormal; in patient 9 TRECs were undetectable, whereas KREC levels were normal. CONCLUSION: TMS is a valid method for diagnosis of PNP deficiency on DBSs of affected patients at a negligible cost. TMS identifies newborns with PNP deficiency, whereas TREC or KREC measurement alone can fail.

Tandem mass spectrometry, but not T-cell receptor excision circle analysis, identifies newborns with late-onset adenosine deaminase deficiency.

BACKGROUND: Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear. OBJECTIVE: We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life. METHODS: We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 2'-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency. RESULTS: The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25, and 19 µmol/L (normal value, <1.5 µmol/L) and 2'-deoxyadenosine levels of 0.7, 2.7, and 2.4 µmol/L (normal value, <0.07 µmol/L); the mean levels of adenosine and 2'-deoxyadenosine were respectively 12.0- and 27.6-fold higher than normal values. DBSs taken at birth from all 3 patients with delayed-onset ADA deficiency had normal TREC levels, but TRECs were undetectable in blood samples taken from the same patients at the time of diagnosis. CONCLUSION: Tandem-MS but not TREC quantification identifies newborns with delayed- or late-onset ADA deficiency.

Analysis of a Cohort of 165 Pediatric Patients with Human Bocavirus Infection and Comparison between Mono-Infection and Respiratory Co-Infections: A Retrospective Study.

INTRODUCTION: Human Bocavirus (HBoV) is mainly associated with respiratory tract infections. However, its role as respiratory pathogen is not fully understood for a high co-infection rate in symptomatic patients and a significant HBoV detection rate in asymptomatic subjects. This study aimed to describe a large cohort of children with HBoV infection and to compare HBoV mono-infection and co-infections. METHODS: We retrospectively reviewed data from 165 children admitted to Meyer Children's Hospital IRCCS from March 2022 to March 2023 with the diagnosis of HBoV infection, detected using Reverse Transcription qPCR from nasal swabs. Thereafter, we compared patients with HBoV mono-infection (Group A) and those with HBoV co-infections (Group B) in terms of disease severity, established by the length of stay (LOS), the requirement of Pediatric Intensive Care Unit (PICU), and advanced respiratory support (ARS). RESULTS: The median age was 1.5 years; 80% of patients presented with respiratory symptoms. The discharge rate from the emergency department (ED) within 24 h was 42.4%. Most cases (57.6%) were hospitalized, and 7.3% were admitted to PICU due to respiratory failure. Group A comprised 69 patients, and Group B 96 children (95% viral co-infections, 2% bacterial, 3% viral and bacterial). Group A and Group B were similar in hospitalization rate but differed significantly in LOS (median 3 vs. 5 days) and requirement of PICU admission (0 vs. 12 patients, p < 0.001). Patients with a respiratory disease history (17.5%) showed significantly longer LOS and more necessity of inhaled bronchodilator therapy. CONCLUSIONS: HBoV should be considered a relevant respiratory pathogen especially in viral co-infections. Patients with HBoV co-infections have a higher risk of necessitating advanced respiratory support with more PICU admission and longer LOS; a previous respiratory disease puts them at a higher risk of longer hospitalization.

Epidemiology of respiratory syncytial virus in hospitalized children over a 9-year period and preventive strategy impact.

Background: Respiratory Syncytial Virus (RSV) is the primary cause of respiratory infections and hospitalizations in young children globally, leading to substantial disease burden and mortality. The aim of the present study was to review and provide updates on how the SARS-CoV-2 pandemic have significantly influenced RSV epidemiology on hospitalized children due to RSV infection. A potential impact of the available preventive strategies on the same population were provided. Methods: All children aged 0-6 years hospitalized at Meyer Children's Hospital IRCCS for RSV infection from September 2014 to March 2023 were retrospectively recorded. Seasonal trends before and after SARS-CoV-2 pandemic, age distribution, ICU admission and co-infections, comorbidities and prematurity were retrieved. Predictions on the number of hospitalizations avoided by the deployment of different preventive strategies were provided. Results: A total of 1,262 children with RSV infection were included in the study. The 70% of them had less than 1 year-of-age at the moment of hospitalization and almost 50% less than 3 months. In the post-pandemic seasons, a 317% increase in the number of hospitalizations was recorded with a significant increase in older children compared to the pre-pandemic seasons. ICU support was required for 22% of children, the majority of whom were under 3 months of age. Almost 16% of hospitalized children were born preterm and only 27% of hospitalized children had prior comorbidities. The rate of comorbidities among RSV hospitalized children increased with age. Nirsevimab prophylaxis could have prevented more than 46% of hospitalizations in this cohort. A preventive strategy addressing also children aged 7 months to 6 years of age with co-existing comorbidities would increase that rate above 57%. Discussion: The identification of RSV hospitalization-related features is informing the decision-maker for the deployment of the wisest preventive approach on a population scale.

Expanded Newborn Screening for Inborn Errors of Immunity: The Experience of Tuscany.

BACKGROUND: Inborn errors of immunity (IEIs) include 485 inherited disorders characterized by an increased susceptibility to life-threatening infectious diseases, autoimmunity, and malignant diseases with a high mortality rate in the first years of life. Severe combined immunodeficiency is the most severe of the IEIs, and its detection should be a primary goal in a newborn screening (NBS) program. The term "actionable" has recently been used for all IEIs with outcomes that can be demonstrably improved through early specialized intervention. OBJECTIVE: To evaluate the results of the expanded NBS strategy for IEIs in Tuscany Region (Italy), based on T-cell receptor excision circle, kappa recombining excision circle, and tandem mass-based assays. METHODS: This is a retrospective study collecting data from all infants born in Tuscany from October 10, 2018, to October 10, 2022. Tandem mass assay to identify adenosine deaminase and purine nucleoside phosphorylase deficiency, together with T-cell receptor excision circle and kappa recombining excision circle molecular analysis, was conducted on dried blood spot from the newborns' Guthrie Cards. A new dried blood spot and evaluation by an immunologist were carried out when the results of the first test were outside the diagnostic cutoffs. RESULTS: A total of 94,319 newborns were evaluated. Referral rates for T-cell recombining excision circles (0.031%) and kappa recombining excision circles (0.074%) in this study are in line with the data available in literature. The results from the expanded NBS strategy revealed an incidence rate of 1 per 9431 affected newborns. CONCLUSIONS: This work represents the first description of a sustainable and real-life-based expanded NBS program for IEIs with a high diagnostic incidence facilitating prompt management of identified patients.

Four-Component Recombinant Protein-Based Vaccine Effectiveness Against Serogroup B Meningococcal Disease in Italy.

Importance: Population-based data on the 4-component recombinant protein-based (4CMenB) vaccine effectiveness and reduction in incidence rate ratios (IRRs) are continuously needed to assess vaccine performance in the prevention of serogroup B invasive meningococcal disease (IMD). Objective: To assess the effectiveness and reduction in IRRs associated with the 4CMenB vaccine in the pediatric population in 6 regions in Italy. Design, Setting, and Participants: This retrospective cohort screening study and case-control study included data from children aged younger than 6 years in 6 highly populated Italian regions from January 1, 2006, to January 1, 2020. Participants included children younger than 6 years diagnosed with serogroup B IMD without predisposing factors. Data were collected from regional surveillance and vaccination registries and were analyzed from September 2021 to January 2022. Exposures: Routine 4CMenB vaccination, per regional vaccination programs. Main Outcomes and Measures: The main outcome was the effectiveness of the 4CMenB vaccine in the prevention of serogroup B IMD in the population of children aged younger than 6 years in 6 Italian regions. The percentages of vaccine effectiveness (VE) were obtained through the concomitant use of a screening method and a case-control study. Secondary outcomes were the comparison of effectiveness results obtained using the 2 different computational methods, the description of serogroup B IMD incidence rates, and reduction in IRRs before and after 4CMenB introduction, as a proxy for vaccine impact. Results: The cohort screening study included a resident population of 587 561 children younger than 6 years in 3 regions with similar surveillance protocols, and the matched-case controls study assessed a resident population of 1 080 620 children younger than 6 years in 6 regions. Analyses found that 4CMenB VE in fully immunized children was 94.9% (95% CI, 83.1%-98.4%) using the screening method and 91.7% (95% CI, 24.4%-98.6%) using the case-control method. Overall reduction in IRR was 50%, reaching 70% in regions with early-start vaccination schedules. The case-control method involving 6 highly-populated Italian regions included 26 cases and 52 controls and found an estimated VE of 92.4% (95% CI, 67.6%-97.9%) in children old enough for the first vaccine dose and 95.6% (95% CI, 71.7%-99.1%) in fully immunized children. VE was more than 90% for partially immunized children. Even in regions where the first dose was administered at age 2 months, almost 20% of unvaccinated cases were among infants too young to receive the first 4CMenB dose. Conclusions and Relevance: This screening cohort study and matched case-controls study found high effectiveness of 4CMenB vaccination and greater reduction in IRR for early-start vaccination schedules in preventing invasive serogroup B meningococcal disease. The high proportion of children too young to be vaccinated among unvaccinated cases suggests that starting the vaccination even earlier may prevent more cases. Screening and case-control methods provided similar estimates of VE: either method may be used in different study settings, but concomitant use can provide more robust estimates.

Association between mannose-binding lectin gene polymorphisms and necrotizing enterocolitis in preterm infants.

OBJECTIVES: The aim of the present study was to evaluate whether polymorphisms of the mannose-binding lectin (MBL-2) gene and MBL serum levels on admission to neonatal intensive care unit are associated with necrotizing enterocolitis (NEC) in preterm infants and to verify MBL expression in NEC bowels. METHODS: In this retrospective cohort study, 107 neonates (41 with NEC and 66 controls) were included. MBL-2 genotyping for the promoter polymorphism -221 and for the exon 1 variant alleles at codons 52, 54, and 57 was performed. MBL levels were determined by enzyme-linked immunosorbent assay in 55 infants. Immunohistochemical staining for MBL expression was performed on bowel specimens. The main study outcome was severe NEC (Bell stages II/III). RESULTS: The -221 Y allele and the MBL-2 YY genotype were more frequent in neonates with severe NEC than in controls (P = 0.04 and P = 0.004, respectively). In the multivariate analysis, the MBL-2 YA/YA genotype was associated with NEC (odds ratio = 3.03, 95% confidence interval 1.13%-8.13%, P = 0.024). Neonates with NEC had MBL level on admission >400 ng/mL more frequently than controls (P = 0.043). Among neonates with severe NEC, the deceased neonates were carriers of high or intermediate producing MBL-2 genotypes (P = 0.035). Finally, MBL was highly expressed in intestinal tissue from infants with NEC. CONCLUSIONS: MBL-2 genotypes associated with high MBL serum levels represent a risk factor for NEC. This finding, together with the MBL expression in bowel tissue, supports a role for MBL in the pathogenesis of NEC.

Real-time polymerase chain reaction on filter paper spotted samples: a gateway to molecular diagnosis of invasive bacterial diseases for rural areas in low-income countries.

BACKGROUND: Bacterial culture is the gold standard for the diagnosis of invasive bacterial diseases (IBDs) but molecular methods are more specific and sensitive. Fresh liquid samples (FLSs) show patent limitations for shipping and storage. We aimed to evaluate the sensitivity and specificity of real-time polymerase chain reaction (PCR) performed on dried sample spots (DSSs) obtained from different biological fluids compared with real-time PCR or culture performed on FLSs. METHODS: FLSs positive for Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus, Bordetella pertussis and/or Pseudomonas aeruginosa were spotted on filter paper. Real-time PCR was performed on both FLSs and DSSs and results were compared. The stability of the DSS results over time was evaluated. RESULTS: Real-time PCR performed on 114 DSSs showed a specificity of 99.1% and a sensitivity of 91.2% for IBD diagnosis. A positive correlation was found between FLS cycle threshold (Ct) and DSS Ct (r=0.84; r2=0.71) with the Pearson statistical test and Bland-Altman analysis showing that 95% of the specimens were within agreeable limits. Although we observed a trend towards signal reduction over time in the DSSs, there was no statistical evidence of an increase in Ct values. Real-time PCR on DSSs was 2.2 times more sensitive than culture. CONCLUSIONS: Real-time PCR applied to DSSs may be a useful approach in different situations, such as IBD diagnosis, both for rural areas of low-income countries and family practitioners in various settings.

Molecular surveillance of pneumococcal carriage following completion of immunization with the 13-valent pneumococcal conjugate vaccine administered in a 3 + 1 schedule.

In a cross-sectional study, with the use of molecular methods, we aimed to gain insight into oropharyngeal pneumococcal colonization over time in 1212 Greek children recruited in general pediatric settings throughout the country; they were fully vaccinated with PCV13 (3 + 1 schedule). A single sample was obtained from each child at a time interval of 26 days to 70 months after administration of the 4th (booster) PCV13 dose; sampling time was divided into six time intervals. Carriage of Streptococcus pneumoniae was detected by real-time PCR targeting the lytA gene and isolates were serotyped by singleplex real-time PCR assays. Multiple control procedures to avoid false-positive results were applied. We showed an overall S. pneumoniae carriage rate of 48.6%. Serotyping identified typeable isolates in 82% of the total lytA-positive samples. Non-PCV13 serotypes represented 83.8% of total isolates when excluding serogroups with mixed PCV13 and non-PCV13 serotypes. In multivariate analysis daycare/school attendance emerged as the main contributing factor. Notably, serotypes 19A and 3 were the only two PCV13 serotypes the colonization rate of which increased over time (χ2 for trend P < 0.001 and P = 0.012, respectively). The application of the SP2020 gene on lytA-positive serotyped samples showed pneumococcal colonization in 97% of cases, and the overall colonization profile over time closely resembled that of the lytA gene. With the provisions of the methodological approach and age group of our study, the use of the oropharynx emerges as a reliable alternative to the nasopharynx in estimating pneumococcal carriage in epidemiological studies.