Nasopharyngeal microbiota in children is associated with severe asthma exacerbations.

BACKGROUND: The respiratory microbiome has been associated with the etiology and disease course of asthma. OBJECTIVE: We sought to assess the nasopharyngeal microbiota in children with a severe asthma exacerbation and their associations with medication, air quality, and viral infection. METHODS: A cross-sectional study was performed among children aged 2 to 18 years admitted to the medium care unit (MCU; n = 84) or intensive care unit (ICU; n = 78) with an asthma exacerbation. For case-control analyses, we matched all cases aged 2 to 6 years (n = 87) to controls in a 1:2 ratio. Controls were participants of either a prospective case-control study or a longitudinal birth cohort (n = 182). The nasopharyngeal microbiota was characterized by 16S-rRNA-gene sequencing. RESULTS: Cases showed higher Shannon diversity index (ICU and MCU combined; P = .002) and a distinct microbial community composition when compared with controls (permutational multivariate ANOVA R2 = 1.9%; P < .001). We observed significantly higher abundance of Staphylococcus and "oral" taxa, including Neisseria, Veillonella, and Streptococcus spp. and a lower abundance of Dolosigranulum pigrum, Corynebacterium, and Moraxella spp. (MaAsLin2; q < 0.25) in cases versus controls. Furthermore, Neisseria abundance was associated with more severe disease (ICU vs MCU MaAslin2, P = .03; q = 0.30). Neisseria spp. abundance was also related with fine particulate matter exposure, whereas Haemophilus and Streptococcus abundances were related with recent inhaled corticosteroid use. We observed no correlations with viral infection. CONCLUSIONS: Our results demonstrate that children admitted with asthma exacerbations harbor a microbiome characterized by overgrowth of Staphylococcus and "oral" microbes and an underrepresentation of beneficial niche-appropriate commensals. Several of these associations may be explained by (environmental or medical) exposures, although cause-consequence relationships remain unclear and require further investigations.

Clinical and In Vitro Evidence Favoring Immunoglobulin Treatment of a Chronic Norovirus Infection in a Patient With Common Variable Immunodeficiency.

BACKGROUND: Immunocompromised individuals can become chronically infected with norovirus, but effective antiviral therapies are not yet available. METHODS: Treatments with nitazoxanide, ribavirin, interferon alpha-2a, and nasoduodenally administered immunoglobulins were evaluated sequentially in an immunocompromised patient chronically infected with norovirus. In support, these components were also applied to measure norovirus inhibition in intestinal enteroid cultures in vitro. Viral RNA levels were determined in fecal and plasma samples during each treatment and viral genomes were sequenced. RESULTS: None of the antivirals resulted in a reduction of viral RNA levels in feces or plasma. However, during ribavirin treatment, there was an increased accumulation of virus genome mutations. In vitro, an effect of interferon alpha-2a on virus replication was observed and a genetically related strain was neutralized effectively in vitro using immunoglobulins and post-norovirus-infection antiserum. In agreement, after administration of immunoglobulins, the patient cleared the infection. CONCLUSIONS: Intestinal enteroid cultures provide a relevant system to evaluate antivirals and the neutralizing potential of immunoglobulins. We successfully treated a chronically infected patient with immunoglobulins, despite varying results reported by others. This case study provides in-depth, multifaceted exploration of norovirus treatment that can be used as a guidance for further research towards norovirus treatments.

Prevalence and Impact of Fatigue in Children with Primary Immunodeficiency Disorders: a Quantitative Single-Center Study.

Although fatigue is a common symptom in adult patients with primary immunodeficiencies (PID), data in pediatric patients are limited. The goal of this study is to estimate the prevalence and impact of fatigue in children with PID as reported by patients, parents, and health-care providers. A retrospective single-center observational study was performed. Prevalence of fatigue was measured by reviewing medical charts of 54 children in our department who are on immunoglobulin replacement therapy. Both prevalence and impact were also measured by the PedsQL-Multidimensional Fatigue Scale (MFS) in 27 patients and 32 of their parents. This is an age-appropriate questionnaire for self-report of fatigue symptoms in patients aged 5-18 years and for parent proxy reports for patients aged 2-18 years. General, cognitive, and sleep-rest fatigue was measured, and a total fatigue score was calculated. Means, standard deviation and Z scores were calculated using age-specific reference values. Intraclass correlation coefficients (ICC) were calculated for comparison of scores provided by parents vs children's self-reported scores. Both chart review data and PedsQL-MFS showed fatigue rates of 65%. Pediatric PID patients of all ages had significantly lower scores on all subscales and total score of the PedsQL-MFS compared to healthy children, indicating greater perceived symptoms of fatigue. General fatigue was the most affected subscale in PID patients, suggesting that fatigue in these patients is mainly physical. Seventy-four percent of PID patients had a Z score lower than - 1 on the general fatigue subscale indicating severe fatigue. Child-parent concordance varied between 0.24 and 0.93. Our results show the feasibility of the PedsQL-MFS survey to evaluate the prevalence and severity of fatigue in children with PID and underscore the importance of this issue in our patient care.

Extended clinical and immunological phenotype and transplant outcome in CD27 and CD70 deficiency.

Biallelic mutations in the genes encoding CD27 or its ligand CD70 underlie inborn errors of immunity (IEIs) characterized predominantly by Epstein-Barr virus (EBV)-associated immune dysregulation, such as chronic viremia, severe infectious mononucleosis, hemophagocytic lymphohistiocytosis (HLH), lymphoproliferation, and malignancy. A comprehensive understanding of the natural history, immune characteristics, and transplant outcomes has remained elusive. Here, in a multi-institutional global collaboration, we collected the clinical information of 49 patients from 29 families (CD27, n = 33; CD70, n = 16), including 24 previously unreported individuals and identified a total of 16 distinct mutations in CD27, and 8 in CD70, respectively. The majority of patients (90%) were EBV+ at diagnosis, but only ∼30% presented with infectious mononucleosis. Lymphoproliferation and lymphoma were the main clinical manifestations (70% and 43%, respectively), and 9 of the CD27-deficient patients developed HLH. Twenty-one patients (43%) developed autoinflammatory features including uveitis, arthritis, and periodic fever. Detailed immunological characterization revealed aberrant generation of memory B and T cells, including a paucity of EBV-specific T cells, and impaired effector function of CD8+ T cells, thereby providing mechanistic insight into cellular defects underpinning the clinical features of disrupted CD27/CD70 signaling. Nineteen patients underwent allogeneic hematopoietic stem cell transplantation (HSCT) prior to adulthood predominantly because of lymphoma, with 95% survival without disease recurrence. Our data highlight the marked predisposition to lymphoma of both CD27- and CD70-deficient patients. The excellent outcome after HSCT supports the timely implementation of this treatment modality particularly in patients presenting with malignant transformation to lymphoma.

SARS-CoV-2 outbreaks in secondary school settings in the Netherlands during fall 2020; silent circulation.

BACKGROUND: In fall 2020 when schools in the Netherlands operated under a limited set of COVID-19 measures, we conducted outbreaks studies in four secondary schools to gain insight in the level of school transmission and the role of SARS-CoV-2 transmission via air and surfaces. METHODS: Outbreak studies were performed between 11 November and 15 December 2020 when the wild-type variant of SARS-CoV-2 was dominant. Clusters of SARS-CoV-2 infections within schools were identified through a prospective school surveillance study. All school contacts of cluster cases, irrespective of symptoms, were invited for PCR testing twice within 48 h and 4-7 days later. Combined NTS and saliva samples were collected at each time point along with data on recent exposure and symptoms. Surface and active air samples were collected in the school environment. All samples were PCR-tested and sequenced when possible. RESULTS: Out of 263 sampled school contacts, 24 tested SARS-CoV-2 positive (secondary attack rate 9.1%), of which 62% remained asymptomatic and 42% had a weakly positive test result. Phylogenetic analysis on 12 subjects from 2 schools indicated a cluster of 8 and 2 secondary cases, respectively, but also other distinct strains within outbreaks. Of 51 collected air and 53 surface samples, none were SARS-CoV-2 positive. CONCLUSION: Our study confirmed within school SARS-CoV-2 transmission and substantial silent circulation, but also multiple introductions in some cases. Absence of air or surface contamination suggests environmental contamination is not widespread during school outbreaks.

Case numbers of acute hepatitis of unknown aetiology among children in 24 countries up to 18 April 2022 compared to the previous 5 years.

An increase of acute hepatitis of unknown aetiology has been reported among children in multiple countries worldwide. With a rapid online survey among hospitals in and outside of Europe, we describe case numbers recorded from 1 January to 18 April 2022 vs the previous 5 years. Of 24 countries that responded, we identified 5/17 European and 1/7 non-European countries with an elevation in probable cases of unexplained acute hepatitis, and severe cases were elevated in five European countries.

Performance of Zika Assays in the Context of Toxoplasma gondii, Parvovirus B19, Rubella Virus, and Cytomegalovirus (TORCH) Diagnostic Assays.

Infections during pregnancy that may cause congenital abnormalities have been recognized for decades, but their diagnosis is challenging. This was again illustrated with the emergence of Zika virus (ZIKV), highlighting the inherent difficulties in estimating the extent of pre- and postnatal ZIKV complications because of the difficulties in establishing definitive diagnoses. We reviewed the epidemiology, infection kinetics, and diagnostic methods used for Toxoplasma gondii, parvovirus B19, rubella virus, and cytomegalovirus (TORCH) infections and compared the results with current knowledge of ZIKV diagnostic assays to provide a basis for the inclusion of ZIKV in the TORCH complex evaluations. Similarities between TORCH pathogens and ZIKV support inclusion of ZIKV as an emerging TORCH infection. Our review evaluates the diagnostic performance of various TORCH diagnostic assays for maternal screening, fetal screening, and neonatal screening. We show that the sensitivity, specificity, and positive and negative predictive value of TORCH complex pathogens are widely variable, stressing the importance of confirmatory testing and the need for novel techniques for earlier and accurate diagnosis of maternal and congenital infections. In this context it is also important to acknowledge different needs and access to care for different geographic and resource settings.

Viral Kinetics and Resistance Development in Children Treated with Neuraminidase Inhibitors: The Influenza Resistance Information Study (IRIS).

BACKGROUND: We studied the effect of age, baseline viral load, vaccination status, antiviral therapy, and emergence of drug resistance on viral shedding in children infected with influenza A or B virus. METHODS: Samples from children (aged ≤13 years) enrolled during the 7 years of the prospective Influenza Resistance Information Study were analyzed using polymerase chain reaction to determine the influenza virus (sub-)type, viral load, and resistance mutations. Disease severity was assessed; clinical symptoms were recorded. The association of age with viral load and viral clearance was examined by determining the area under the curve for viral RNA shedding using logistic regression and Kaplan-Meier analyses. RESULTS: A total of 2131 children infected with influenza (683, A/H1N1pdm09; 825, A/H3N2; 623, influenza B) were investigated. Age did not affect the mean baseline viral load. Children aged 1-5 years had prolonged viral RNA shedding (±1-2 days) compared with older children and up to 1.2-fold higher total viral burden. Besides, in older age (odds ratio [OR], 1.08; confidence interval [CI], 1.05-1.12), prior vaccination status (OR, 1.72; CI, 1.22-2.43) and antiviral treatment (OR, 1.74; CI, 1.43-2.12) increased the rate of viral clearance. Resistance mutations were detected in 49 children infected with influenza A virus (34, A/H1N1pdm09; 15, A/H3N2) treated with oseltamivir, most of whom were aged <5 years (n = 39). CONCLUSIONS: Children aged 1-5 years had a higher total viral burden with prolonged virus shedding and had an increased risk of acquiring resistance mutations following antiviral treatment. CLINICAL TRIALS REGISTRATION: NCT00884117.

The Role of B Cells in Carriage and Clearance of Mycoplasma pneumoniae From the Respiratory Tract of Mice.

Background: Carriage of Mycoplasma pneumoniae (Mp) in the nasopharynx is considered a prerequisite for pulmonary infection. It is interesting to note that Mp carriage is also detected after infection. Although B cells are known to be involved in pulmonary Mp clearance, their role in Mp carriage is unknown. Methods: In this study, we show in a mouse model that Mp persists in the nose after pulmonary infection, similar to humans. Results: Infection of mice enhanced Mp-specific immunoglobulin (Ig) M and IgG levels in serum and bronchoalveolar lavage fluid. However, nasal washes only contained elevated Mp-specific IgA. These differences in Ig compartmentalization correlated with differences in Mp-specific B cell responses between nose- and lung-draining lymphoid tissues. Moreover, transferred Mp-specific serum Igs had no effect on nasal carriage in B cell-deficient µMT mice, whereas this enabled µMT mice to clear pulmonary Mp infection. Conclusions: We report the first evidence that humoral immunity is limited in clearing Mp from the upper respiratory tract.

Ebola Virus Inactivation by Detergents Is Annulled in Serum.

Background: Treatment of blood samples from hemorrhagic fever virus (HFV)-infected patients with 0.1% detergents has been recommended for virus inactivation and subsequent safe laboratory testing. However, data on virus inactivation by this procedure are lacking. Here we show the effect of this procedure on diagnostic test results and infectious Ebola virus (EBOV) titers. Methods: Serum and whole-blood samples were treated with 0.1% or 1% sodium dodecyl sulfate (SDS) or 0.1% Triton X-100 and assayed for clinical chemistry and malaria antigen detection. Infectious EBOV titers were determined in SDS-treated plasma and whole blood from EBOV-infected nonhuman primates (NHPs). Infectious titers of EBOV or herpes simplex virus type 1 (HSV-1) in detergents-treated cell culture medium containing various serum concentrations were determined. Results: Laboratory test results were not affected by 0.1% detergent treatment of blood samples, in contrast with 1% SDS treatment. However, 0.1% detergent treatment did not inactivate EBOV in blood samples from infected NHPs. Experiments with cell culture medium showed that virus inactivation by detergents is annulled at physiological serum concentrations. Conclusions: Treatment of blood samples with 0.1% SDS or Triton X-100 does not inactivate EBOV. Inactivation protocols for HFV should be validated with serum and whole blood.

Antigenic Maps of Influenza A(H3N2) Produced With Human Antisera Obtained After Primary Infection.

BACKGROUND: Antigenic characterization of influenza viruses is typically based on hemagglutination inhibition (HI) assay data for viral isolates tested against strain-specific postinfection ferret antisera. Here, similar virus characterizations were performed using serological data from humans with primary influenza A(H3N2) infection. METHODS: We screened sera collected between 1995 and 2011 from children between 9 and 24 months of age for influenza virus antibodies, performed HI tests for the positive sera against 23 influenza viruses isolated between 1989 and 2011, and measured HI titers of antisera against influenza A(H3N2) from 24 ferrets against the same panel of viruses. RESULTS: Of the 17 positive human sera, 6 had a high response, showing HI patterns that would be expected from primary infection antisera, while 11 sera had lower, more dispersed patterns of reactivity that are not easily explained. The antigenic map based on the high-response human HI data was similar to the map created using ferret data. CONCLUSIONS: Although the overall structure of the ferret and human antigenic maps is similar, local differences in virus positions indicate that the human and ferret immune system might see antigenic properties of viruses differently. Further studies are needed to establish the degree of similarity between serological patterns in ferret and human data.

Identification of amino acid substitutions supporting antigenic change of influenza A(H1N1)pdm09 viruses.

UNLABELLED: The majority of currently circulating influenza A(H1N1) viruses are antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as population immunity increases. Amino acid substitutions in the hemagglutinin protein can result in escape from neutralizing antibodies, affect viral fitness, and change receptor preference. In this study, we constructed mutants with substitutions in the hemagglutinin of A/Netherlands/602/09 in an attenuated backbone to explore amino acid changes that may contribute to emergence of antigenic variants in the human population. Our analysis revealed that single substitutions affecting the loop that consists of amino acid positions 151 to 159 located adjacent to the receptor binding site caused escape from ferret and human antibodies elicited after primary A(H1N1)pdm09 virus infection. The majority of these substitutions resulted in similar or increased replication efficiency in vitro compared to that of the virus carrying the wild-type hemagglutinin and did not result in a change of receptor preference. However, none of the substitutions was sufficient for escape from the antibodies in sera from individuals that experienced both seasonal and pandemic A(H1N1) virus infections. These results suggest that antibodies directed against epitopes on seasonal A(H1N1) viruses contribute to neutralization of A(H1N1)pdm09 antigenic variants, thereby limiting the number of possible substitutions that could lead to escape from population immunity. IMPORTANCE: Influenza A viruses can cause significant morbidity and mortality in humans. Amino acid substitutions in the hemagglutinin protein can result in escape from antibody-mediated neutralization. This allows the virus to reinfect individuals that have acquired immunity to previously circulating strains through infection or vaccination. To date, the vast majority of A(H1N1)pdm09 strains remain antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as a result of increasing population immunity. We show that single amino acid substitutions near the receptor binding site were sufficient to escape from antibodies specific for A(H1N1)pdm09 viruses but not from antibodies elicited in response to infections with seasonal A(H1N1) and A(H1N1)pdm09 viruses. This study identified substitutions in A(H1N1)pdm09 viruses that support escape from population immunity but also suggested that the number of potential escape variants is limited by previous exposure to seasonal A(H1N1) viruses.

Determinants of Ethnic Differences in Cytomegalovirus, Epstein-Barr Virus, and Herpes Simplex Virus Type 1 Seroprevalence in Childhood.

OBJECTIVE: To identify whether there are ethnic differences in cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus type 1 (HSV-1) seroprevalence rates in children at 6 years of age, and when present, to evaluate how these differences can be explained by sociodemographic and environmental factors. STUDY DESIGN: This study was embedded within a multi-ethnic population-based prospective cohort study. Serum IgG levels against CMV, EBV, and HSV-1 were measured by enzyme-linked immunosorbent assay in 4464 children (median age 6.0 years). Information on demographics and characteristics were assessed by questionnaires. Herpesvirus seroprevalences between Surinamese-Creole, Surinamese-Hindustani, Turkish, Moroccan, Cape Verdean Antillean, and Native Dutch children were compared. RESULTS: Non-Western ethnicity was an independent risk factor for CMV (aOR, 2.16; 95% CI 1.81-2.57), EBV (1.76; 1.48-2.09), and HSV-1 seropositivity (1.52; 1.39-1.66). Among the ethnic groups, CMV seroprevalences ranged between 29% and 65%, EBV between 43% and 69%, and HSV-1 between 13% and 39%. Low family net household income, low maternal educational level, crowding, and lifestyle factors explained up to 48% of the ethnic differences in HSV-1 seroprevalences, and up to 39% of the ethnic differences in EBV seroprevalences. These factors did not explain ethnic differences in CMV seroprevalences. CONCLUSIONS: Socioeconomic position and factors related to lifestyle explain only a part of the large ethnic differences in EBV and HSV-1 seroprevalences, whereas they do not explain ethnic differences in CMV seroprevalences in childhood.

Herpesvirus Infections and Transglutaminase Type 2 Antibody Positivity in Childhood: The Generation R Study.

OBJECTIVES: Persistent viral infections have been implicated in the etiology of autoimmune diseases in adulthood, but it is not known whether herpesviruses are associated with the development of celiac disease autoimmunity in childhood. We assessed whether herpesvirus infections are associated with transglutaminase type 2 antibody (TG2A) concentrations in children at 6 years of age. METHODS: The present study was embedded within a population-based prospective cohort study. Serum immunoglobulin G levels against Epstein-Barr virus, cytomegalovirus (CMV), and herpes simplex virus type 1 were measured by enzyme-linked immunosorbent assay , and TG2A concentrations with fluorescence enzyme immunoassay in 4420 children at 6 years of age. Children were categorized based on TG2A concentrations into negative (<7 U/mL), positive (≥7-70 U/mL), and strongly positive (≥70 U/mL), that is, 10 times upper limit normal. RESULTS: Fifty-nine children (1.3%) were TG2A positive, and of these 31 (53%) had concentrations 70 U/mL or more. Children with TG2A concentrations 70 U/mL or more were less often infected with CMV (adjusted odds ratio (aOR) 0.38, 95% CI 0.14-0.98, P = 0.04) and with any of the 3 viruses (aOR 0.38, 95% CI 0.18-0.78, P < 0.01) than children with TG2A negative concentrations. In addition, children with TG2A concentrations 70 U/mL or more were less often infected with 2 or more viruses than children with TG2A negative concentrations (aOR 0.15, 95% CI 0.03-0.65, P = 0.01). CONCLUSIONS: Both CMV single infection and combined CMV, Epstein-Barr virus and/or herpes simplex virus type 1 infections are inversely associated with strongly TG2A positivity. This may indicate a protective effect of herpesvirus infections in the pathogenesis of celiac disease autoimmunity.

Severe acute respiratory infection caused by swine influenza virus in a child necessitating extracorporeal membrane oxygenation (ECMO), the Netherlands, October 2016.

In October 2016, a severe infection with swine influenza A(H1N1) virus of the Eurasian avian lineage occurred in a child with a previous history of eczema in the Netherlands, following contact to pigs. The patient's condition deteriorated rapidly and required life support through extracorporeal membrane oxygenation. After start of oseltamivir treatment and removal of mucus plugs, the patient fully recovered. Monitoring of more than 80 close unprotected contacts revealed no secondary cases.

Prolonged influenza virus shedding and emergence of antiviral resistance in immunocompromised patients and ferrets.

Immunocompromised individuals tend to suffer from influenza longer with more serious complications than otherwise healthy patients. Little is known about the impact of prolonged infection and the efficacy of antiviral therapy in these patients. Among all 189 influenza A virus infected immunocompromised patients admitted to ErasmusMC, 71 were hospitalized, since the start of the 2009 H1N1 pandemic. We identified 11 (15%) cases with prolonged 2009 pandemic virus replication (longer than 14 days), despite antiviral therapy. In 5 out of these 11 (45%) cases oseltamivir resistant H275Y viruses emerged. Given the inherent difficulties in studying antiviral efficacy in immunocompromised patients, we have infected immunocompromised ferrets with either wild-type, or oseltamivir-resistant (H275Y) 2009 pandemic virus. All ferrets showed prolonged virus shedding. In wild-type virus infected animals treated with oseltamivir, H275Y resistant variants emerged within a week after infection. Unexpectedly, oseltamivir therapy still proved to be partially protective in animals infected with resistant virus. Immunocompromised ferrets offer an attractive alternative to study efficacy of novel antiviral therapies.

Experiences of adolescents and youth with HIV testing and linkage to care through the Red Carpet Program (RCP) in Kenya.

Adolescents and youth living with HIV (AYLHIV) experience worse health outcomes compared to adults. We aimed to understand the experiences of AYLHIV in care in the youth-focused Red-Carpet program in Kenya to assess the quality of service provision and identify programmatic areas for optimization. We conducted focus group discussions among 39 AYLHIV (15-24 years) and structured analysis into four thematic areas. Within the HIV testing theme, participants cited fear of positive results, confidentiality and stigma concerns, and suggested engaging the community and youth in HIV testing opportunities. Within the HIV treatment adherence theme, participants cited forgetfulness, stigma, adverse side effects, lack of family support, and treatment illiteracy as barriers to adherence. Most participants reported positive experiences with healthcare providers and peer support. In terms of the HIV status disclosure theme, AYLHIV cited concerns about their future capacity to conceive children and start families and discussed challenges with understanding HIV health implications and sharing their status with friends and partners. Youth voices informing service implementation are essential in strengthening our capacity to optimize the support for AYLHIV within the community, at schools and healthcare facilities.

Carriage of Mycoplasma pneumoniae in the upper respiratory tract of symptomatic and asymptomatic children: an observational study.

BACKGROUND: Mycoplasma pneumoniae is thought to be a common cause of respiratory tract infections (RTIs) in children. The diagnosis of M. pneumoniae RTIs currently relies on serological methods and/or the detection of bacterial DNA in the upper respiratory tract (URT). It is conceivable, however, that these diagnostic methods also yield positive results if M. pneumoniae is carried asymptomatically in the URT. Positive results from these tests may therefore not always be indicative of a symptomatic infection. The existence of asymptomatic carriage of M. pneumoniae has not been established. We hypothesized that asymptomatic carriage in children exists and investigated whether colonization and symptomatic infection could be differentiated by current diagnostic methods. METHODS AND FINDINGS: This study was conducted at the Erasmus MC-Sophia Children's Hospital and the after-hours General Practitioners Cooperative in Rotterdam, The Netherlands. Asymptomatic children (n = 405) and children with RTI symptoms (n = 321) aged 3 mo to 16 y were enrolled in a cross-sectional study from July 1, 2008, to November 30, 2011. Clinical data, pharyngeal and nasopharyngeal specimens, and serum samples were collected. The primary objective was to differentiate between colonization and symptomatic infection with M. pneumoniae by current diagnostic methods, especially real-time PCR. M. pneumoniae DNA was detected in 21.2% (95% CI 17.2%-25.2%) of the asymptomatic children and in 16.2% (95% CI 12.2%-20.2%) of the symptomatic children (p = 0.11). Neither serology nor quantitative PCR nor culture differentiated asymptomatic carriage from infection. A total of 202 children were tested for the presence of other bacterial and viral pathogens. Two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Finally, longitudinal sampling showed persistence of M. pneumoniae in the URT for up to 4 mo. Fifteen of the 21 asymptomatic children with M. pneumoniae and 19 of the 22 symptomatic children with M. pneumoniae in this longitudinal follow-up tested negative after 1 mo. CONCLUSIONS: Although our study has limitations, such as a single study site and limited sample size, our data indicate that the presence of M. pneumoniae in the URT is common in asymptomatic children. The current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection.