This study was designed as a feasibility study, not as an outcome-based study, to compare the utility of a 16S rRNA PCR assay to that of the BACTEC 9240 system for detecting bacteria in blood obtained from neonates suspected of having bacterial sepsis. The comparison of 548 paired neonatal blood samples revealed a high level of agreement between the two methodologies, with sensitivity, specificity, and positive and negative predictive values of 96.0, 99.4, 88.9, and 99.8%, respectively, for PCR. The high negative predictive value that was calculated for the PCR assay compared to that of culture is indicative of the assay's usefulness in accurately ruling out the diagnosis of bacterial sepsis in the uninfected term neonate admitted to the NICU for such an evaluation.
This study reiterates the current problem; the vast majority of term infants admitted to the NICU for suspected sepsis are not infected but have symptoms consistent with those of other medical conditions that mimic sepsis, such as hypoglycemia, delayed transition, or transient tachypnea. Despite this fact, these term infants are treated with antibiotics for at least 48 h while awaiting the results of the preliminary blood culture report. Automated blood culturing systems are good given time, but if a laboratory test could be developed that would rule out bacterial septicemia in less time than blood culturing, those infants whose symptoms had resolved could be taken off of antibiotics and discharged from the NICU sooner.
NICU admissions and intravenous antibiotic therapies result in expensive hospital stays for infants that separate newborns from their mothers and create potential difficulties in successful bonding and breast-feeding, while exposing infants to antibiotics which are increasingly expensive and overused. At best these practices increase the financial burden on our health care system, and at worst they contribute to the increasingly serious problem of antibiotic resistance. Although blood culturing will not be completely replaced by a nucleic acid amplification technology anytime soon, as pure isolates remain essential for antimicrobial drug susceptibility testing, PCR does appear to be an excellent diagnostic test choice for a rapid means of ruling out bacterial sepsis in certain select patient populations.
There are numerous examples of PCR-based assays for detecting bacteria in blood, including Streptococcus pneumoniae
DNA from whole blood (37
) or inoculated Peds Plus bottles (11
) and coagulase-negative Staphylococcus
sp. from blood culture bottles (3
). A different PCR-based assay that detected Candida
sp. DNA directly from 26 of 27 blood samples obtained from neonates with culture-proven candidemia was developed (19
). Another study illustrated the close agreement between PCR and bacterial culture in 15 of 16 culture-positive amniotic fluid samples obtained from women in premature labor (18
). Recently, a multiplex approach was developed to detect neonatal sepsis, coamplifying portions of the 16S rRNA gene along with the housekeeping gene for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (25
). In that study, among the 33 newborn infants classified as being at risk for early-onset sepsis, Laforgia et al. was able to detect the 16S rRNA gene by PCR in all four of the culture-proven sepsis cases, as well as in two samples with negative culture results. Finally, a PCR assay using primers which recognize an 861-bp fragment of the 16S rRNA gene was suggested for use in triaging bacterial sepsis (27
). That study revealed the successful amplification of the rRNA gene from 12 different species of bacteria, including gram-negative and gram-positive organisms, without amplifying human genomic DNA.
Over the course of this study, a number of technical challenges were identified and successfully overcome. First of all, we came to appreciate the benefits of prefiltering the PCR master mix using the Centricon YM-100 centrifugal filter device (Millipore Corporation). Implementing this prefiltration step allowed the use of 28 cycles, compared to the originally indicated 25 cycles (14
), without our experiencing the occasional false-positive result in the no-DNA control that had occurred with 28 and 30 cycles when prefiltering was not included. This change resulted in an increased assay sensitivity, permitting detection of 13 CFU/ml with 28 cycles, compared to 25 CFU/ml with 25 cycles (data not shown).
Second, a dramatic loss in sensitivity was observed when we attempted to detect the bacterial 16S rRNA gene from whole-blood specimens with volumes of appreciably less than 200 μl. More specifically, 19 culture-positive blood specimens whose paired samples for PCR testing had volumes ranging from 25 to 75 μl lacked detectable levels of the 16S rRNA gene (data not shown). This observation led us to establish a minimum blood volume requirement of 200 μl for 16S rRNA PCR testing.
Implementing a PCR-based assay for ruling out bacterial sepsis in the uninfected term neonate could potentially save significant health care dollars and reduce the emotional impact for families. In fact, Escobar et al. developed an algorithm for predicting which newborns admitted to the NICU to rule out sepsis are eligible for discontinuation of antibiotic treatment at 24 h based on 15 parameters, including clinical assessments, maternal risk factors, laboratory test results, and demographic data (9
). In a retrospective analysis, their decision rule correctly identified the vast majority of babies with positive blood cultures, the persistently symptomatic babies with negative culture results, and a group of low-risk infants eligible for only 24 h of antibiotic treatment. More recently, Harrell et al. developed a clinical model for predicting outcomes for young infants which includes laboratory and diagnostic markers (15
A similarly based algorithm containing a PCR-based assay for detecting the 16S rRNA gene in blood specimens might be used as an effective diagnostic tool in rapidly identifying uninfected term infants. The approximate time required to test neonatal blood for bacterial 16S rRNA gene is roughly 9 h. This includes 5 h of TSB incubation, followed by 1.5 h for sample preparation, 2 h for 28 cycles of DNA amplification, and 0.5 h for gel electrophoresis of the amplified PCR master mix. Attempts are under way to reduce still further the entire assay time, without sacrificing assay sensitivity.
The issue of organizing and managing sample processing is also important for minimizing the test turnaround time. Neonatal blood samples arrive in the laboratory continuously throughout the day. Samples should be set up immediately upon arrival into the laboratory, with two runs being completed each day. Blood samples inoculated into TSB by 8 a.m. could produce results by 5 p.m. Samples received after 8 a.m. would be incubated overnight in TSB, rather than the minimum 5 h and processed immediately the following morning, with final results being available before 12 noon. This approach would allow the NICU to receive finalized results on each neonatal blood sample from 9 to 28 h after receipt of the sample. On average, this work flow scheme could provide test results 1 full day earlier than the 48-h preliminary blood culture report would be received in the NICU.
For the term infant with a PCR-negative test result, discharge from the NICU might become possible if symptoms have resolved and vital signs have stabilized. In general, most term infants admitted to the NICU are there because of symptoms due to a poor transition immediately following birth and not because of bacterial sepsis. For these infants, symptoms usually resolve within the first 24 h of admission to the NICU.
In the three cases where the infants' blood specimens were found to be PCR positive but culture negative, the neonatalogists would have most likely treated them with a 7-day course of antibiotics for presumed sepsis, identical to that of an infant with a positive blood culture result.
In summary, the only way to prove the value of this PCR-based assay is to develop an algorithm with the NICU clinicians and to perform an outcome-based study. If found to be successful, this type of patient management could lead to shorter antibiotic courses and NICU stays for the otherwise healthy newborn infants. This approach is currently under evaluation at MWH for term infants suspected of having sepsis who are admitted to the NICU.