Comparison of rectal swabs and fecal samples for the detection of Clostridioides difficile infections with a new in-house PCR assay.

The detection of Clostridioides difficile infections (CDI) relies on testing the stool of patients by toxin antigen detection or PCR methods. Although PCR and antigenic methods have significantly reduced the time to results, delays in stool collection can significantly add to the turnaround time. The use of rectal swabs to detect C. difficile could considerably reduce the time to diagnosis of CDI. We developed a new rapid PCR assay for the detection of C. difficile and evaluated this PCR assay on both stool and rectal swab specimens. We recruited a total of 623 patients suspected of C. difficile infection. Stool samples and rectal swabs were collected from each patient and tested by our PCR assay. Stool samples were also tested by the cell cytotoxicity neutralization assay (CCNA) as a reference. The PCR assay detected C. difficile in 60 stool specimens and 61 rectal swabs for the 64 patients whose stool samples were positive for C. difficile by CCNA. The PCR assay detected an additional 35 and 36 stool and rectal swab specimens positive for C. difficile, respectively, for sensitivity with stools and rectal swabs of 93.8% and 95.3%, specificity of 93.7% and 93.6%, positive predictive values of 63.2% and 62.9%, and negative predictive values of 99.2% and 99.4%. Detection of C. difficile using PCR on stools or rectal swabs yielded reliable and similar results. The use of PCR tests on rectal swabs could reduce turnaround time for CDI detection, thus improving CDI management and control of C. difficile transmission. IMPORTANCE: Clostridioides difficile infection (CDI) is the leading cause of healthcare-associated diarrhea, resulting in high morbidity, mortality, and economic burden. In clinical laboratories, CDI testing is currently performed on stool samples collected from patients with diarrhea. However, the diagnosis of CDI can be delayed by the time required to collect stool samples. Barriers to sample collection could be overcome by using a rectal swab instead of a stool sample. Our study showed that CDI can be identified rapidly and reliably by a new PCR assay developed in our laboratory on both stool and rectal swab specimens. The use of PCR tests on rectal swabs could reduce the time for the detection of CDI and improve the management of this infection. It should also provide a useful alternative for infection-control practitioners to better control the spread of C. difficile.

Internal control for nucleic acid testing based on the use of purified Bacillus atrophaeus subsp. globigii spores.

Commonly used internal controls (ICs) to monitor the efficiency of nucleic acid testing (NAT) assays do not allow verification of nucleic acid extraction efficiency. Since microbial cells are often difficult to lyse, it is important to ensure that nucleic acids are efficiently extracted from any target organism. For this purpose, we developed a cellular IC based on the use of nonpathogenic Bacillus spores. Purified Bacillus atrophaeus subsp. globigii (referred to hereafter as simply B. atrophaeus) spores were added to vaginal and anal samples, which were then subjected to rapid DNA extraction and subsequent PCR amplification. The proof of concept of this cellular IC was made through the use of both manual and automated DNA extraction methods, using vaginal or anal samples spiked with B. atrophaeus spores, combined with a multiplex real-time PCR assay for the specific detection of group B streptococci (GBS) and B. atrophaeus. The performance of the cellular IC was compared to that of a standard IC plasmid added to PCRs. Approximately 500 B. atrophaeus spores per PCR was found to be optimal since this did not interfere significantly with GBS detection for either DNA extraction method and yielded reproducible amplification and/or detection of B. atrophaeus genomic DNA serving as an IC template. Performance of the cellular IC was comparable to that of the standard IC. This novel IC system using nonpathogenic and hard-to-lyse B. atrophaeus spores allowed validation of both the DNA extraction procedure and the amplification and detection process. Use of a spore-based control also provides a universal control for microbial cell lysis.

Identification of methicillin-resistant Staphylococcus aureus carriage in less than 1 hour during a hospital surveillance program.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has spread worldwide and is responsible for significant morbidity, mortality, and health care costs. Control strategies to limit the emergence and spread of this organism rely on rapid and sensitive tests for detection of MRSA carriage. However, the standard surveillance culture method for detecting MRSA is labor intensive and time-consuming (2-3 days per procedure). There is thus a need for a rapid and accurate method to screen for MRSA carriage. METHODS: We recently developed an easy-to-use real-time polymerase chain reaction (PCR) assay suitable for specific detection of MRSA in nasal specimens in <1 h. We studied the efficacy of our new PCR assay in routine screening for nasal MRSA carriage during a hospital surveillance program. A total of 331 nasal specimens obtained from 162 patients at risk for colonization were tested by both the standard mannitol agar culture method and our PCR assay. RESULTS: The PCR assay detected MRSA in all 81 samples that were culture positive for MRSA. The PCR assay detected 4 additional MRSA-positive specimens, for a specificity of 98.4%, a positive predictive value of 95.3%, and a sensitivity and negative predictive value of 100%. CONCLUSIONS: This novel PCR assay allows reliable identification of MRSA carriers in <1 h. This test should facilitate the efficacy of MRSA surveillance programs.