Multiplex Assay Detects Atypical Bacterial Respiratory Pathogens

The multiplex polymerase chain reaction (PCR) assay was compared with singleplex assays to determine the presence of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella species and the human ribonuclease P (RNase P) gene.

Scientists at the US Centers for Disease Control and Prevention (CDC; Atlanta, GA, USA), used four distinct hydrolysis probes to detect three of the leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a dilution series of each specific target and 197 clinical specimens. In total, 177 nasopharyngeal and/or oropharyngeal swabs were tested by all four singleplex assays in addition to the multiplex assay. Twenty clinical specimens including lung tissue, bronchial lavage, sputa, and spleen tissue were also tested, in triplicate, with the singleplex assay designed specifically to detect Legionella species (Pan-Leg), as well as the multiplex assay.

A comparison to previously validated singleplex real-time PCR assays for each agent was also performed and specificity testing demonstrated no cross-reactivity. The analytical sensitivity for specific pathogen targets in both the singleplex and multiplex was identical at 50 fg, while efficiencies ranged from 82% to 97% for the singleplex assays and from 90% to 100% for the multiplex assay. This multiplex assay provides an overall improvement in the diagnostic capability for these agents by demonstrating a sensitive, high-throughput and rapid method. This procedure may allow for a practical and efficient means to test respiratory clinical specimens for atypical pneumonia agents in health care settings and facilitate an appropriate public health response to outbreaks.

The authors concluded that the use of this assay in surveillance studies may provide greater insight into the etiology and prevalence of atypical bacterial pneumonias among a population. The assay will also facilitate the rapid identification of the causative agent for community acquired pneumonia outbreaks. Widespread availability of this and similar assays may lead to a more effective public health response and expedite appropriate treatment of patients. The study was published in May 2011 in the journal Diagnostic Microbiology and Infectious Disease.

Related Links:
US Centers for Disease Control and Prevention