Triplex Direct Quantitative Polymerase Chain Reaction for the Identification of Streptococcus pneumoniae Serotypes.

The quantitative polymerase chain reaction (qPCR) method presented in this study allows the identification of pneumococcal capsular serotypes in cerebrospinal fluid without first performing DNA extraction. This testing approach, which saves time and resources, demonstrated similar sensitivity and a high level of agreement between cycle threshold values when it was compared side-by-side with the standard qPCR method with extracted DNA.

Triplex real-time PCR assay for the detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae directly from clinical specimens without extraction of DNA.

This study presents a triplex real-time PCR assay that allows for the direct detection of Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in one reaction without DNA extraction, with similar sensitivity and specificity to singleplex assays. This approach saves time, specimen volume and reagents while achieving a higher testing throughput.

Identification of P1 variants of Mycoplasma pneumoniae by use of high-resolution melt analysis.

Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia. Although two genetically distinct types of M. pneumoniae are known, variants of each also exist. We used a real-time PCR high-resolution melt genotyping assay to identify clinical variants which may provide greater insight into the genetic distribution of M. pneumoniae strains.

Comparison of laboratory diagnostic procedures for detection of Mycoplasma pneumoniae in community outbreaks.

BACKGROUND: Mycoplasma pneumoniae continues to be a significant cause of community-acquired pneumonia (CAP). A more definitive methodology for reliable detection of M. pneumoniae is needed to identify outbreaks and to prevent potentially fatal extrapulmonary complications. METHODS: We analyzed 2 outbreaks of CAP due to M. pneumoniae. Nasopharyngeal and/or oropharyngeal swab specimens and serum samples were obtained from persons with clinically defined cases, household contacts, and asymptomatic individuals. Real-time polymerase chain reaction (PCR) for M. pneumoniae was performed on all swab specimens, and the diagnostic utility was compared with that of 2 commercially available serologic test kits. RESULTS: For cases, 21% yielded positive results with real-time PCR, whereas 81% and 54% yielded positive results with the immunoglobulin M and immunoglobulin G/immunoglobulin M serologic tests, respectively. For noncases, 1.8% yielded positive results with real-time PCR, whereas 63% and 79% yielded serologically positive results with the immunoglobulin M and immunoglobulin G/immunoglobulin M kits, respectively. The sensitivity of real-time PCR decreased as the duration between symptom onset and sample collection increased, with a peak sensitivity of 48% at 0-21 days. A specificity of 43% for the immunoglobulin M antibody detection assay was observed for persons aged 10-18 years, but the sensitivity increased to 82% for persons aged 19 years. DISCUSSION: Thorough data analysis indicated that no single available test was reliable for the identification of an outbreak of CAP due to M. pneumoniae. A combination of testing methodologies proved to be the most reliable approach for identification of outbreaks of CAP due to M. pneumoniae, especially in the absence of other suspected respiratory pathogens.

Community outbreak of Mycoplasma pneumoniae infection: school-based cluster of neurologic disease associated with household transmission of respiratory illness.

BACKGROUND: We investigated an outbreak of severe neurologic disease and pneumonia that occurred among students at 4 schools in Rhode Island. METHODS: We identified cases of encephalitis, encephalomyelitis, and pneumonia that occurred among schoolchildren from 1 September 2006 through 9 February 2007, and we performed serologic tests, polymerase chain reaction (PCR) analysis, and culture for the detection of multiple pathogens in oropharyngeal and nasopharyngeal specimens. Students with positive results of M. pneumoniae IgM serologic testing and no alternative diagnosis were considered to be infected with M. pneumoniae. At school A, we used questionnaires to identify students and their household contacts who made visits to physicians for pneumonia and cough. We compared observed and expected rates of pneumonia. RESULTS: Rates of pneumonia among elementary students (122 cases/1000 student-years) were > 5-fold higher than expected. Three students had encephalitis or encephalomyelitis, and 76 had pneumonia. Of these 2 groups of students, 2 (66%) and 57 students (75%), respectively, had M. pneumoniae infection. M. pneumoniae was detected by PCR in 10 students with pneumonia; 5 of these specimens were cultured, and M. pneumoniae was isolated in 4. Of 202 households of students attending school A, 20 (10%) accounted for 61% of visits to physicians for pneumonia or cough. Of 19 household contacts of students with pneumonia, 8 (42%) developed pneumonia and 6 (32%) reported visits for cough. CONCLUSIONS: M. pneumoniae caused a community-wide outbreak of cough illness and pneumonia and was associated with the development of life-threatening neurologic disease. Although M. pneumoniae was detected in schools, its transmission in households amplified the outbreak. Interrupting household transmission should be a priority during future outbreaks.

Detection of macrolide resistance in Mycoplasma pneumoniae by real-time PCR and high-resolution melt analysis.

Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, which is often empirically treated with macrolides or azalides such as erythromycin or azithromycin. Recent studies have discovered the existence of macrolide-resistant strains within the population that have been mapped to mutations within the domain V region of the 23S rRNA gene. Currently, identification of these resistant strains relies on time-consuming and labor-intensive procedures such as restriction fragment length polymorphism, MIC studies, and sequence analysis. The current study reports two distinct real-time PCR assays that can detect the A2063G or A2064G base mutation (A2058G or A2059G by Escherichia coli numbering) conferring macrolide resistance. By subjecting the amplicon of the targeted domain V region of the 23S rRNA gene to a high-resolution melt curve analysis, macrolide-resistant strains can quickly be separated from susceptible strains. Utilizing this method, we screened 100 clinical isolates and found 5 strains to possess mutations conferring resistance. These findings were concordant with both sequencing and MIC data. This procedure was also used successfully to identify both susceptible and resistant genotypes in 23 patient specimens. These patient specimens tested positive for the presence of M. pneumoniae by a separate real-time PCR assay, although the bacteria could not be isolated by culture. This is the first report of a real-time PCR assay capable of detecting the dominant mutations that confer macrolide resistance on M. pneumoniae, and these assays may have utility in detecting resistant strains of other infectious agents. These assays may also allow for clinicians to select appropriate treatment options more rapidly and may provide a convenient method to conduct surveillance for genetic mutations conferring antibiotic resistance.

Analysis of eight commercial enzyme immunoassay tests for detection of antibodies to Mycoplasma pneumoniae in human serum.

Mycoplasma pneumoniae is an important etiologic agent of primary atypical pneumonia in children and adults. The diagnosis of M. pneumoniae infection is commonly confirmed through serologic testing. In this study, we used paired sera from 51 patients (all with confirmed M. pneumoniae infection and positive complement fixation [CF] titers) to compare the results of eight enzyme immunoassays (EIAs) available commercially in the United States. We compared two single-use EIAs and six plate-type EIAs. Results from acute-phase sera ranged from only 7 (14%) positive by ImmunoWELL (GenBio) immunoglobulin M (IgM) EIA to 23 (45%) positive by Zeus IgG EIA. When both the acute-phase and convalescent-phase serum samples were examined, positive results ranged from 20 (39%) by the ImmunoWELL (GenBio) IgM assay to 45 (88%) positive by the Remel IgG-IgM EIA. In this study, the single-use EIAs by Remel and Meridian were more reliable than were the plate-type EIAs. Among the plate-type EIAs, the Zeus and DiaSorin assays (which detect antibodies to protein antigens) were more sensitive than the ImmunoWELL assay (which detects antibodies to glycolipid antigens). In general, IgG EIAs on convalescent-phase sera were more concordant with one another than were IgM EIAs with one another. Scatter plot analysis of convalescent-phase sera showed that, as the CF titer dropped, the IgM assays identified fewer positive convalescent-phase sera. In contrast, the IgG assays provided fairly consistent positive results for convalescent-phase sera with CF titers of 64 and above. Results of individual tests and overall limitations of serodiagnostics for M. pneumoniae infections are discussed.