Quantitative detection of Moraxella catarrhalis in nasopharyngeal secretions by real-time PCR

Abstract

The recognition of Moraxella catarrhalis as an important cause of respiratory tract infections has been protracted, mainly because it is a frequent commensal organism of the upper respiratory tract and the diagnostic sensitivity of blood or pleural fluid culture is low. Given that the amount of M. catarrhalis bacteria in the upper respiratory tract may change during infection, quantification of these bacteria in nasopharyngeal secretions (NPSs) by real-time PCR may offer a suitable diagnostic approach. Using primers and a fluorescent probe specific for the copB outer membrane protein gene, we detected DNA from serial dilutions of M. catarrhalis cells corresponding to 1 to 10(6) cells. Importantly, there was no difference in the amplification efficiency when the same DNA was mixed with DNA from NPSs devoid of M. catarrhalis. The specificity of the reaction was further confirmed by the lack of amplification of DNAs from other Moraxella species, nontypeable Haemophilus influenzae, H. influenzae type b, Streptococcus pneumoniae, Streptococcus oralis, Streptococcus pyogenes, Bordetella pertussis, Corynebacterium diphtheriae, and various Neisseria species. The assay applied to NPSs from 184 patients with respiratory tract infections performed with a sensitivity of 100% and a specificity of up to 98% compared to the culture results. The numbers of M. catarrhalis organisms detected by real-time PCR correlated with the numbers detected by semiquantitative culture. This real-time PCR assay targeting the copB outer membrane protein gene provided a sensitive and reliable means for the rapid detection and quantification of M. catarrhalis in NPSs; may serve as a tool to study changes in the amounts of M. catarrhalis during lower respiratory tract infections or following vaccination against S. pneumoniae, H. influenzae, or N. meningitidis; and may be applied to other clinical samples.

FIG. 1.

Sensitivity, detection range, and specificity of the real-time PCR assay for M. catarrhalis. The reproducibility of the assay was determined by testing a dilution series of M. catarrhalis, followed by DNA extraction, in three independent assays with independent DNA extractions.

FIG. 2.

Sensitivity and specificity of the real-time PCR assay for M. catarrhalis in clinical samples (NPSs) compared with culture results. The dashed line indicates the arbitrary cutoff C_T value. C_T values 30 or below were regarded as positive for M. catarrhalis. Horizontal bars indicate medians, which are also given as absolute values.

FIG. 3.

Comparison of M. catarrhalis quantification by real-time PCR with that by semiquantitative culture. The samples were inoculated onto sheep blood agar by fractionation with a calibrated wire loop (5 μl). Growth only in the first fraction was defined as a low level of growth (+), growth in the second fraction as well was defined as an intermediate level of growth (++), and growth in all three fractions was defined as abundant growth (+++).