Molecular Detection of Culture-Confirmed Bacterial Bloodstream Infections with Limited Enrichment Time

Abstract

bConventional blood culturing using automated instrumentation with phenotypic identification requires a significant amount of time to generate results. This study investigated the speed and accuracy of results generated using PCR and pyrosequencing compared to the time required to obtain Gram stain results and final culture identification for cases of culture-confirmed bloodstream infections. Research and physician-ordered blood cultures were drawn concurrently. Aliquots of the incubating research blood culture fluid were removed hourly between 5 and 8 h, at 24 h, and again at 5 days. DNA was extracted from these 6 time point aliquots and analyzed by PCR and pyrosequencing for bacterial rRNA gene targets. These results were then compared to those of the physician-ordered blood culture. PCR and pyrosequencing accurately identified 92% of all culture-confirmed cases after a mean enrichment time of 5.8 ؎ 2.9 h. When the time needed to complete sample processing was included for PCR and pyrosequencing protocols, the molecular approach yielded results in 11.8 ؎ 2.9 h compared to means of 27.9 ؎ 13.6 h to obtain the Gram stain results and 81.6 ؎ 24.0 h to generate the final culture-based identification. The molecular approach enabled accurate detection of most bacteria present in incubating blood culture bottles on average about 16 h sooner than Gram stain results became available and approximately 3 days sooner than the phenotypic identification was entered in the Laboratory Information System. If implemented, this more rapid molecular approach could minimize the number of doses of unnecessary or ineffective antibiotics administered to patients.

In the era of the 24 h laboratory, does communicating Gram stain results from blood cultures flagging positive outside of conventional working hours alter patient management?

“…In this context, improved rapid diagnostic techniques such as direct matrix-assisted laser desorption ionisation time-of-flight mass spectrometry analysis on blood cultures flagging positive9 and molecular techniques10 11 may be able to predict which infections are likely to be caused by resistant organisms 12. Molecular approaches for instance can identify bacteria present in incubating blood culture bottles on average 16 and 72 h sooner than Gram stain results and conventional phenotypic identification, respectively 11. Obtaining such information out of hours is far more likely to be beneficial than Gram stain results alone.…”

Evaluation of real-time PCR and pyrosequencing for screening incubating blood culture bottles from adults with suspected bloodstream infection

“…Five hundred microliters of the drawn culture fluid was extractedusing a combination of Zirconium silica bead-based lysis and organic nucleic acid extraction as described previously [16]. Culture fluids were stored at 4°C for a maximum of 1 week before DNA extraction was completed.…”

“…DNA extracts were amplified in 25 μl reactions by PCR using SmartCycler instruments (Cepheid, Sunnyvale, CA) as previously described [16]. The customized 2.5X PCR master mix used in amplifying the 16S rRNA universal gene target (base pair location 1155–1525) [13] contained a final MgCl 2 concentration of 2 mM (catalog no.…”

“…The entire 25 μl volume of biotin-labeled PCR product was analyzed by Pyrosequencing (PyroMark ID Pyrosequencer, Qiagen, Germantown, MD) using PyroMark Gold Q96 reagents (Cat# 972804; Qiagen) as previously described [16]. The dispensation programs varied with the target and included: 11(ACTG) dNTP for the Universal, Staphylococcus, and Streptococcus rDNA targets, or AGC, 12(CTGA) dNTP for the enteric rDNA target.…”

“…On average, this approach identifies the bacterial pathogen in culture-confirmed BSI 16 hours sooner than growth is detected by the instrument and the Gram stain results is called to the unit, and a full 3 days sooner than the phenotypic identification is available [16]. In fact we found that 73% of all culture-confirmed cases of BSI are detected and accurately identified from incubating blood culture bottles after just 5 hours of enrichment using this molecular approach [16]. …”