Population density profiles of nasopharyngeal carriage of 5 bacterial species in pre-school children measured using quantitative PCR offer potential insights into the dynamics of transmission

Abstract

Bacterial vaccines can reduce carriage rates. Colonization is usually a binary endpoint. Real time quantitative PCR (qPCR) can quantify bacterial DNA in mucosal samples over a wide range. Using culture and single-gene species-specific qPCRs for Streptococcus pneumoniae (lytA), Streptococcus pyogenes (ntpC), Moraxella catarrhalis (ompJ), Haemophilus influenzae (hdp) and Staphylococcus aureus (nuc) and standard curves against log-phase reference strain broth cultures we described frequency and peak density distributions of carriage in nasopharyngeal swabs from 161 healthy 2-4 y old children collected into STGG broth. In general, detection by qPCR and culture was consistent. Discordance mostly occurred at lower detection thresholds of both methods, although PCR assays for S. pyogenes and S. aureus were less sensitive. Density varied across 5-7 orders of magnitude for the 5 species with the abundant species skewed toward high values (modes: S. pneumoniae log3-4, M. catarrhalis & H. influenzae log4-5 CFU/ml broth). Wide ranges of bacterial DNA concentrations in healthy children carrying these bacteria could mean that different individuals at different times vary greatly in infectiousness. Understanding the host, microbial and environmental determinants of colonization density will permit more accurate prediction of vaccine effectiveness.

The Effects of Live Attenuated Influenza Vaccine on Nasopharyngeal Bacteria in Healthy 2 to 4 Year Olds. A Randomized Controlled Trial

“…Had cultures been done, doubtless some PCR-positive, culture-negative individuals would have been found. However, we have recently shown that individuals with discordant PCR and culture results tend to have low density colonization (16). Although DNA may be present in the absence of living organisms, it nevertheless represents a footprint of recent bacterial presence, which is what this study was designed to measure.…”

“…Nucleic acid was extracted from all samples using the Virus/Pathogen Mini Kit from QIAGEN (Valencia, CA), yielding 110-ml extracts from 300 ml of skim milk, tryptone, glucose, and glycerol broth. These extracts were stored at 280 8 C. Monoplex qPCR assays were used to detect S. pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, and S. aureus (16). The cycle threshold (Ct) value used to define detection was set at less than or equal to 35 cycles.…”

“…The cycle threshold (Ct) value used to define detection was set at less than or equal to 35 cycles. For density values, Ct values were converted into gene copies (GC)/ml using standard curves (16). qPCR assays for adenovirus, bocavirus, influenza (A and B), respiratory syncytial virus, rhinovirus, human metapneumovirus, parainfluenza (types 1-3), parechovirus, coronavirus (229E, OC43, and NL63), and enterovirus were performed using methods described previously (4,18).…”

“…Real-time quantitative PCR (qPCR) can enumerate bacterial species-specific gene copies and thus both viable and nonviable bacteria in biological samples over wide concentration ranges with less effort and expense than required with conventional quantitative plate cultures. Using qPCR, we recently described upper respiratory bacterial density ranges as wide as 6 orders of magnitude in healthy children (16). Such differences may result in wide variation in efficiency of onward transmission among carriers.…”

Specific PCR, bacterial culture, serology and pharyngeal sampling to enhance the aetiological diagnosis of cellulitis

“…PCR of a T4 bacteriophage internal process control confirmed successful DNA and inhibitor-free nucleic acid extracts. The methods and determination of the detection limit of the PCR method were as described by Thors et al (2015).…”