A low-cost culture- and DNA extraction-free method for the molecular detection of pneumococcal carriage in saliva.

Molecular methods have improved the sensitivity of the detection of pneumococcal carriage in saliva. However, they typically require sample culture enrichment and nucleic acid extraction prior to performing the detection assay and may limit scalability for extensive surveillance of pneumococcus, particularly in low-resource settings. We evaluated the performance of a DNA-extraction-free method for the detection of pneumococcus in saliva. We developed a streamlined qPCR-based protocol for the detection of pneumococcus, omitting culture enrichment and DNA extraction. Using saliva samples collected from children attending childcare centers (New Haven, CT, USA), we evaluated the detection of pneumococcus using saliva lysates as compared to purified DNA extracted from culture-enriched aliquots of the paired samples using qPCR targeting the pneumococcal piaB gene. Of the 759 saliva samples tested from 92 children [median age 3.65 years; IQR (2.46-4.78)], pneumococcus was detected in 358 (47.2%) saliva lysates prepared using the extraction-free protocol and in 369 (48.6%) DNA extracted from culture-enriched samples. We observed near-perfect agreement between the two protocols (Cohen's kappa: 0.92; 95% CI: 0.90-0.95). Despite a high correlation between CT values generated by the two methods (r = 0.93, P < 0.0001), the CT values generated from saliva lysates were higher (lower concentration) than those from culture-enriched samples (ΔCT = 6.69, P < 0.00001). The cost of detecting pneumococcus using saliva lysates was at least fivefold lower (US$2.53) compared to the cost of the culture-enriched method (range: US$13.60-US$19.46). For pneumococcal carriage surveillance in children, our findings suggest that a DNA extraction-free approach may offer a cost-effective alternative to the resource-intensive culture-enrichment method.IMPORTANCESurveillance for carriage of pneumococcus is a key component of evaluating the performance of pneumococcal vaccines and informing new vaccination strategies. To improve the scalability of pneumococcal carriage surveillance, we show that molecular detection of pneumococcus in saliva from children can be performed without culture enrichment and DNA extraction. Our findings show that using the extraction-free method can improve surveillance efforts for pneumococcal carriage in children, overcoming the resource-intensive hurdle that comes with the use of molecular methods, particularly in low-resource settings.

Development and evaluation of a real-time multienzyme isothermal rapid amplification assay for rapid detection of Streptococcus pneumoniae.

Streptococcus pneumoniae is a significant pathogen causing infectious diseases, including pneumonia, otitis media, septicemia, and meningitis. The introduction of multivalent vaccines has coincided with a remarkable decrease in the number of pneumococcal-related deaths. Despite this, pneumococcal infection remains a significant cause of death among children under 5 years old and adults aged 65 or older at a global level. Therefore, early detection of S. pneumoniae infection is crucial for prognosis of pneumococcal infection patients. In this study, we evaluated the utility of a real-time multienzyme isothermal rapid amplification (MIRA) assay for detecting S. pneumoniae and other non-S. pneumoniae bacterial species. A primer-probe set targeting the S. pneumoniae lytA gene was designed, followed by optimization of parameters for the MIRA assay. At the same time, we validated the real-time MIRA assay for detecting S. pneumoniae using 79 clinical isolates identified by VITEK MS. The results showed a detection sensitivity and specificity of 100%. These results demonstrate that the designed real-time MIRA assay is a promising, rapid, simple, and reliable method for detecting S. pneumoniae infection in resource-limited areas. It has great potential for application in detecting not only S. pneumoniae but also other non-S. pneumoniae bacterial species.

Qualification of a 21-valent pneumococcal urine antigen detection assay and development of clinical positivity cutoffs.

Aim: Serotype-specific assays detecting pneumococcal polysaccharides in bodily fluids are needed to understand the pneumococcal serotype distribution in non-bacteremic pneumonia.Methods: We developed a urine antigen detection assay and using urine samples from adult outpatients without pneumonia developed positivity cutoffs for both a previously published 15-valent and the new 21-valent assay. Clinical sensitivity was confirmed with samples from patients with invasive pneumococcal disease.Results: Total assay precision ranged from 7.6 to 17.8% coefficient of variation while accuracy ranged between 80 and 150% recovery, except for three serotypes where recoveries ranged from 32 to 60%. Clinical sensitivity was 86.4% and specificity was 96.5% across all 30 serotypes.Conclusion: The assay could potentially assess serotype-distribution in non-infected and infected participants with pneumococcal disease.

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Pneumococcal carriage and serotype distribution in children with nephrotic syndrome.

BACKGROUND: Patients with nephrotic syndrome (NS) are at a higher risk of developing invasive pneumococcal disease (IPD). Pneumococcal carriage studies are helpful tools for detecting potentially infectious serotypes and guiding immunization efforts. Pneumococcal nasopharyngeal colonization is common, and IPD can easily occur in an immunosuppressed state. Limited information is available regarding the frequency of pneumococcal carriage in individuals with NS. The aim of this study was to evaluate pneumococcal carriage and serotype distribution in children with NS. METHODS: Pneumococcal carriage was detected by real-time PCR assays from nasopharyngeal swab samples from 98 children with NS, and 100 healthy controls. Isolates were serotyped by real-time PCR. RESULTS: The pneumococcal carriage rate was 44.9% in children with NS. Regarding the recommendation about pneumococcal immunization in children with NS, the vaccination rate was low. Also, non-PCV13 serotypes have been detected in at least 25% of PCV13-vaccinated children. There is no statistically significant difference in total pneumococcal carriage rate, PCV13 serotype carriage rate, or non-PCV13 serotype carriage rate between children with NS and healthy controls (p > 0.05 for all). CONCLUSIONS: The pneumococcal carriage rate was similar between children with NS and healthy controls. However, because children with NS have an increased risk for IPD, the serotype distribution of children with NS can demonstrate the improved protection offered by new pneumococcal vaccines. Regular monitoring for IPD is crucial for assessing the evolving sero-epidemiology of pneumococcal infections and evaluating the effectiveness of vaccines for children with NS.

A rapid simultaneous antigen detection of Haemophilus influenzae and Streptococcus pneumoniae for predicting the prognosis of acute otitis media.

BACKGROUND: Rapid identification of causative bacteria in treatment of acute otitis media (AOM) is of paramount importance for appropriate antibiotic use. MATERIALS AND METHODS: This prospective observational study was conducted in 15 hospitals and clinics in Japan between 2018 and 2020. A new rapid antigen test kit (AOS-116), which simultaneously detects antigens for Streptococcus pneumoniae (Sp) and Haemophilus influenzae (Hi), was applied for middle ear fluids (MEFs) and nasopharyngeal secretions (NPSs) in patients with moderate to severe AOM. We investigated relationship between the results of rapid test, severity at initial visit, and clinical course. RESULTS: Regarding performance accuracy based on culture results, AOS-116 showed 1) high (>80%) sensitivity, specificity, and negative predictive value (NPV) in MEFs for both antigens, 2) high sensitivity, specificity, and positive predictive value (PPV) in NPSs for Hi antigen, and 3) high specificity, and PPV in NPSs for Sp antigen. Regarding predictive value of nasopharyngeal culture and antigen detection for causative middle ear pathogens, similar results were observed between AOS-116 and culture, which was characterized with high sensitivity and NPV for both pathogens. MEFs/NPSs positive for Hi antigen were significantly associated with eardrum findings, and severity. MEFs/NPSs positive for pneumococcal antigen were significantly associated with severity of otalgia, fever, and otorrhea. Among patients with prior antimicrobial treatment, improvement tended to be slower in cases positive for Hi than in cases negative. CONCLUSION: The rapid antigen detection test is useful as a decision-making tool for prescribing antimicrobial agents and may play an important role in promoting appropriate antimicrobial use.

Pneumococcal antibody response in children with recurrent respiratory tract infections: A descriptive study.

BACKGROUND: The pneumococcal antibody response after vaccination with unconjugated pneumococcal vaccine can be evaluated as part of the diagnostic work-up of children with recurrent respiratory tract infections to detect an underlying polysaccharide antibody deficiency. Little is known about the prevalence of polysaccharide antibody deficiency in this population and its therapeutic consequences. OBJECTIVES: This study aimed to investigate the prevalence of polysaccharide antibody deficiency in children with recurrent respiratory tract infections and to correlate polysaccharide responsiveness with clinical severity. In addition, we aimed to evaluate differences in the immunoglobulin (Ig)G2/IgG ratio, IgA level, and age in relation to the number of deficient serotype-specific antibody responses. METHODS: Polysaccharide antibody titers for pneumococcal serotypes 8, 9N, and 15B; clinical characteristics; and immunoglobulin levels of 103 children with recurrent respiratory tract infections were retrospectively assessed. American Academy of Allergy, Asthma, and Immunology guidelines were used for the interpretation of the polysaccharide antibody response. RESULTS: Overall, 28 children (27.2 %) were diagnosed with polysaccharide antibody deficiency. No correlation was found between the number of deficient serotype-specific antibody responses and clinical severity. The study participants with a normal response to all three serotypes had a higher IgG2/IgG ratio than those with one or more deficient responses (p < 0.003). No significant correlation between IgA levels and polysaccharide responsiveness was found. The median age of children with normal polysaccharide responsiveness for the three tested serotypes was higher than that of children with a deficient response to one or more serotypes (p < 0.0025). CONCLUSION: For a large group of children (18.4 %) with recurrent respiratory tract infections, an underlying mechanism for their susceptibility was defined thanks to diagnostic unconjugated pneumococcal polysaccharide vaccination. Further research is needed to formulate age-specific normal values for polysaccharide responsiveness and to investigate the usefulness of the IgG2/IgG ratio in determining the need for diagnostic unconjugated pneumococcal polysaccharide vaccination.

Enhancing pneumococcal bacteraemia diagnosis: A comparative assessment of culture-independent assays (MALDI-TOF-MS Sepsityper® module and a lateral flow inmunochromatography test).

INTRODUCTION: Pneumococcal bacteraemia is a major contributor to global morbidity and mortality. Traditional culture-based methods lack sensitivity and are time-consuming. This study aimed to assess the effectiveness of two culture-independent assays, the MALDI-TOF-MS Sepsityper® module and the lateral flow inmunochromatography test (LFICT) with the Standard F® Streptococcus pneumoniae, directly from positive blood culture (BC) bottles. METHODS: A prospective study was conducted from December 2021 to July 2022. For all BC positives for S. pneumoniae a double centrifugation protocol was implemented. The resulting pellet was subsequently processed using both techniques. RESULTS: The LFICT showed exceptional performance with 100% sensitivity and specificity, outperforming the MALDI-TOF-MS Sepsityper® module, which achieved 85.2% sensitivity and 100% specificity. Nevertheless, the combination of these assays offers a robust and comprehensive approach to diagnosis. CONCLUSIONS: The simultaneous use of both techniques offers a promising alternative that can be integrated into routine practices directly from BC samples.

Changes in vaccination uptake against pneumococcal disease, influenza, and coronavirus disease 2019 (COVID-19) before and after a Head and Neck cancer diagnosis.

BACKGROUND: Pneumonia is one of the main contributors to non-cancer mortality among patients with head and neck cancer (HNC). This study aimed to determine the vaccine uptake for pneumococcal polysaccharide and conjugate vaccines, quadrivalent influenza vaccines, and mRNA COVID-19 vaccines before and after an HNC diagnosis. Furthermore, the study investigated the timing of vaccination after a cancer diagnosis. MATERIALS & METHODS: This register based multicentre study included Danish patients ≥ 18y diagnosed with HNC between 2018 and 2021. The vaccine uptake was assessed by calculating cumulative incidence (CI), while the timing of vaccination after an HNC diagnosis was explored by calculating incidence rates of vaccination the first and second half year after a cancer diagnosis. RESULTS: The cumulative incidence of vaccine uptake for pneumococcal vaccines was estimated to be 8 % and 16 % one year before and after an HNC diagnosis, respectively. The CIs were 36 % and 38 % for quadrivalent influenza vaccines, respectively, whereas the CIs of vaccine uptake for mRNA COVID-19 vaccines were 60 % and 89 %. The IR of mRNA COVID-19 vaccinations the first half year after HNC diagnosis were 273 per 1000 person-months of follow-up (PMFU) and 111 per 1000 PMFU the second half year, respectively (IRR: 0.38, p < 0.001). Comparing the same periods, the IR of quadrivalent influenza vaccination was 28 per 1000 PMFU and 51 per 1000 PMFU (IRR: 1.95, 0 < 0.001). The IRs of pneumococcal vaccinations were 11 per 1000 PMFU and 14 per 1000 PMFU (IRR 1.28, p = 0.21). CONCLUSIONS: Although our study shows a significant increase in pneumococcal and COVID-19 vaccine uptake after HNC diagnosis, a gap remains in vaccine uptake before diagnosis, underscoring the need for increased awareness of vaccination options and recommendations. Our findings could serve as a reference for future recommendations.

Performance Characteristics of a Real-Time PCR Assay for Direct Detection of Streptococcus pneumoniae in Clinical Specimens.

Community-acquired pneumonia and complications, such as bacteremia and meningitis due to Streptococcus pneumoniae infection, still occur in at-risk populations, despite the availability of effective vaccines. Laboratory confirmation of S. pneumoniae remains challenging despite advances in blood culture techniques and the availability of nucleic acid-amplification tests. The goal of this study was to determine the performance characteristics of a molecular assay designed as a diagnostic test using primary clinical specimens for invasive pneumococcal disease. The molecular assay adapted for the Luminex Aries instrument targets an S. pneumoniae-specific gene (autolysin, lytA) in clinical specimens. Using real-time PCR MultiCode technology, four different clinical specimen types were evaluated. Specimen types included bronchoalveolar lavage, whole blood, cerebrospinal fluid, and urine to cover the various presentations and appropriate specimen types for invasive pneumococcal infections. The lower limit of detection in urine was 10 colony forming units (CFU)/mL, while in bronchoalveolar lavage, cerebrospinal fluid, and whole blood, it was 100 CFU/mL. Accuracy and specificity were both 100%, and all specimen types were stable for 8 days at 4°C. Finally, 38 clinical specimens were tested to further evaluate the assay. The performance characteristics met Clinical Laboratory Improvement Amendments standards for a clinical diagnostic assay, and the assay offers a sensitive and specific real-time PCR test for direct detection of S. pneumoniae in relevant clinical specimens.

Utility of quantitative loop mediated isothermal amplification (qLAMP) assay for the diagnosis of invasive pneumococcal disease (IPD).

PURPOSE: Quantitative LAMP (qLAMP) assay is one of the recent and emerging diagnostic tests for infectious diseases. Only a few studies exist comparing this assay with quantitative real-time PCR (qPCR) for the diagnosis of invasive pneumococcal disease (IPD). AIM: To compare the diagnostic performance of qLAMP assay with qPCR targeting autolysin gene for the diagnosis of invasive pneumococcal disease. METHODS: Ninety six blood samples and 73 CSF samples from patients clinically suspected with community acquired pneumonia and acute meningitis were tested by qPCR and qLAMP assays using previously published primers and protocols. The qPCR was considered as the gold standard test and the diagnostic performance was assessed by calculating sensitivity, specificity, positive and negative predictive values, and kappa coefficient for the level of agreement between the tests. Chi-squared/Fisher exact test was used to compare categorical variables (positive/negative). RESULTS: Thirty two blood samples and 22 CSF samples were positive by qPCR while 24 and 20 samples were positive by qLAMP assay respectively. The sensitivity of qLAMP assay was only 86.4% and 75% when tested on CSF and blood samples respectively. However, the qLAMP assay was in substantial to almost perfect agreement when compared with qPCR. The results were statistically significant in both sample types (P < 0.001). CONCLUSIONS: The performance of qLAMP assay can vary based on the specimen type. It has very high specificity and had substantial to almost perfect agreement, and thus may be an alternative to qPCR for the diagnosis of IPD.

Invasive disease caused simultaneously by two different serotypes of Streptococcus pneumoniae: a microbiological appreciation.

We report a clinical case of a child with an invasive pneumococcal disease caused by two different pneumococcal serotypes that belonged to different sequence types. She was a 15-month-old girl with pneumonia and pleural effusion in which S. pneumoniae colonies with different morphologies grew, one from the blood culture (characteristic greyish appearance) and the other from the pleural fluid (mucoid appearance). The isolate from blood was serotype 22 F (ST698/CC698/GPSC61), while the isolate from the pleural fluid was serotype 3 (ST180/CC180/GPSC12). The patient fully recovered after treatment with intravenous ampicillin followed by oral amoxicillin.

Invasive pneumococcal disease serotype 23B1 causing multifocal septic arthritis, myositis and retroperitoneal abscess.

We describe a case of a previously healthy unvaccinated man in his 70s who developed penicillin-susceptible bacteraemic invasive pneumococcal disease due to non-vaccine serotype 23B with the unusual manifestations of multifocal myositis, intramuscular abscesses, polyarticular septic arthritis and synovitis. Blood cultures drawn prior to antibiotic therapy and culture of iliopsoas collection were helpful in making the diagnosis. At follow-up, he had persistent hip pain attributed to avascular necrosis of the head of femur, a possible late complication of his pyomyositis.

Elbow septic arthritis in an infant: an unusual presentation of invasive pneumococcal disease.

Acute septic arthritis is a rare, potentially severe infection that requires immediate treatment to avoid long-term morbidity. Most common aetiological agents are commonly used for empirical treatment, but the choice of antibiotics may be influenced by other factors, such as the patient's age and the epidemiological context.We report an infant with elbow arthritis, whose treatment was changed after Streptococcus pneumoniae serotype 9N was isolated in the blood and synovial fluid. The child underwent arthrocentesis and received intravenous ampicillin followed by oral amoxicillin, with a favourable response and no sequelae at 1-year follow-up.We report an uncommon manifestation of invasive pneumococcal disease in a young immunised healthy infant caused by a non-vaccine serotype. The presence of S. pneumoniae should be considered in joint infections, especially in infants and those with a history of respiratory symptoms.

Multiplexed suspension array immunoassays for detection of antibodies to pneumococcal polysaccharide and conjugate vaccines.

Combination and polyvalent vaccines not only provide protection against several different pathogens at the same time but can also increase vaccine protection against pathogens that have closely related pathogenic strains or serotypes. Multiplexed serological testing is a preferred method for determining the efficacy of combination and polyvalent vaccines, as it reduces the need for conducting multiple individual assays to confirm immune responses and cross-reactivity, uses less sample, and can be faster, more reliable, and more cost-effective. Bead-based suspension array technologies, such as the Luminex® xMAP® Technology, are often used for development of multiplexed serological assays for various vaccine trials and for routine testing in clinical laboratories to determine immune status of vaccinated individuals. This article reviews publications describing the development and implementation of bead-based multiplexed serological assays for detection of immune responses to polyvalent polysaccharide and conjugate vaccines against Streptococcus pneumoniae. Many of these serological assays on the bead array platform have been further optimized and expanded over time and are still widely used today.

Saliva as an alternative sample type for detection of pneumococcal carriage in young children.

For children, the gold standard for the detection of pneumococcal carriage is conventional culture of a nasopharyngeal swab. Saliva, however, has a history as one of the most sensitive methods for surveillance of pneumococcal colonization and has recently been shown to improve carriage detection in older age groups. Here, we compared the sensitivity of paired nasopharyngeal and saliva samples from PCV7-vaccinated 24-month-old children for pneumococcal carriage detection using conventional and molecular detection methods. Nasopharyngeal and saliva samples were collected from 288 24-month-old children during the autumn/winter, 2012/2013. All samples were first processed by conventional diagnostic culture. Next, DNA extracted from all plate growth was tested by qPCR for the presence of the pneumococcal genes piaB and lytA and a subset of serotypes. By culture, 161/288 (60 %) nasopharyngeal swabs tested positive for pneumococcus, but detection was not possible from saliva due to abundant polymicrobial growth on culture plates. By qPCR, 155/288 (54 %) culture-enriched saliva samples and 187/288 (65 %) nasopharyngeal swabs tested positive. Altogether, 219/288 (76 %) infants tested positive for pneumococcus, with qPCR-based carriage detection of culture-enriched nasopharyngeal swabs detecting significantly more carriers compared to either conventional culture (P<0.001) or qPCR detection of saliva (P=0.002). However, 32/219 (15 %) carriers were only positive in saliva, contributing significantly to the overall number of carriers detected (P=0.002). While testing nasopharyngeal swabs by qPCR proved most sensitive for pneumococcal detection in infants, saliva sampling could be considered as complementary to provide additional information on carriage and serotypes that may not be detected in the nasopharynx and may be particularly useful in longitudinal studies, requiring repeated sampling of study participants.

A simple PCR assay for the identification of the novel Streptococcus pneumoniae serotype 7D.

The identification of the novel pneumococcal serotype 7D by Neufeld quellung reaction requires significant expertise. To circumvent this, we developed a simple serotype-specific PCR method to discriminate serotype 7D from the closely related serotypes 7C, 7B and 40. The established PCR was validated with the strain collection of the German National Reference Center for Streptococci (GNRCS). However, no isolate initially assigned as serotype 7B, 7C or 40 was identified as serotype 7D.

Purpura fulminans in a hyposplenic patient arising from pneumococcal sinusitis.

We report a previously healthy woman in her 50s who presented with sepsis, rapidly progressive purpuric rash and disseminated intravascular coagulation. She was diagnosed with acute infective purpura fulminans due to invasive pneumococcal infection likely secondary to sinusitis. Our case report discusses our initial diagnostic uncertainty and approach in investigating and treating such a critically unwell patient.

A novel immuno-molecular strategy for the detection of Streptococcus pneumoniae serotypes in human cerebrospinal and middle ear fluids.

Streptococcus pneumoniae is one of the most common microorganisms causing acute otitis media (AOM) in children. While bacterial culture of middle ear fluid (MEF) is the gold standard to detect the etiological organisms, several host and pathogen factors impact the survival of the organisms resulting in false negatives. To overcome this limitation, we have developed and validated an innovative multiplex immuno-molecular assay to screen and detect the S. pneumoniae 15-valent pneumococcal conjugate vaccine (PCV15; STs 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) vaccine serotypes in MEF. This novel in vitro approach involves two-step testing. First, the MEF specimens were tested for highly conserved pneumococcal genes, autolysin, lytA, and pneumolysin, ply using direct PCR to identify pneumococcus positive specimens. The pneumococcus positive specimens were screened for the presence of vaccine serotype specific pneumococcal polysaccharides using a 15-plex Pneumococcal Antigen Detection (PAD) assay, with specific capture and detection monoclonal antibodies. Due to the lack of availability of MEF samples, cerebrospinal fluid (CSF) was used as the surrogate matrix for the development and validation of the PCR-PAD assays. The PCR and PAD assays were separately evaluated for sensitivity and specificity. Subsequently, the PCR-PAD assays were cross-validated with human MEF samples (n = 39) which were culture confirmed to contain relevant bacterial strains. The combined PCR-PAD assays demonstrated high rate of agreement 94.9% (95% CI; 82.7, 99.4%) with historical Quellung serotype data of these MEF samples. This novel PCR-PAD assay demonstrates the feasibility of combining molecular and immunological assays to screen and identify PCV15 pneumococcal vaccine serotypes in AOM clinical samples.

High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States.

While the sensitivity of detection of pneumococcal carriage can be improved by testing respiratory tract samples with quantitative PCR (qPCR), concerns have been raised regarding the specificity of this approach. We therefore investigated the reliability of the widely used lytA qPCR assay when applied to saliva samples from older adults in relation to a more specific qPCR assay (piaB). During the autumn/winter seasons of 2018/2019 and 2019/2020, saliva was collected at multiple time points from 103 healthy adults aged 21 to 39 (n = 34) and >64 (n = 69) years (n = 344 total samples). Following culture enrichment, extracted DNA was tested using qPCR for piaB and lytA. By sequencing the variable region of rpsB (S2 typing), we identified the species of bacteria isolated from samples testing lytA-positive only. While 30 of 344 (8.7%) saliva samples (16.5% individuals) tested qPCR-positive for both piaB and lytA, 52 (15.1%) samples tested lytA-positive only. No samples tested piaB-positive only. Through extensive reculture attempts of the lytA-positive samples collected in 2018/2019, we isolated 23 strains (in 8 samples from 5 individuals) that were also qPCR-positive for only lytA. Sequencing determined that Streptococcus mitis and Streptococcus infantis were predominantly responsible for this lytA-positive qPCR signal. We identified a comparatively large proportion of samples generating positive signals with the widely used lytA qPCR and identified nonpneumococcal Streptococcus species responsible for this signal. This highlights the importance of testing for the presence of multiple gene targets in tandem for reliable and specific detection of pneumococcus in polymicrobial respiratory tract samples. IMPORTANCE Testing saliva samples with quantitative PCR (qPCR) improves the sensitivity of detection of pneumococcal carriage. The qPCR assay targeting lytA, the gene encoding the major pneumococcal autolysin, has become widely accepted for the identification of pneumococcus and is even considered the "gold standard" by many. However, when applying this approach to investigate the prevalence of pneumococcal carriage in adults in New Haven, CT, USA, we identified nonpneumococcal Streptococcus spp. that generate positive signals in this widely used assay. By testing also for piaB (encoding the iron acquisition ABC transporter lipoprotein, PiaB), our findings demonstrate the importance of testing for the presence of multiple gene targets in tandem for reliable molecular detection of pneumococcus in respiratory tract samples; targeting only lytA may lead to an overestimation of true carriage rates.