JMD etoc alert
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jordan, J. A.
Right arrow Articles by Brozanski, B. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jordan, J. A.
Right arrow Articles by Brozanski, B. S.
JMD 2006, Vol. 8, No. 3
Copyright © 2006 American Society for Investigative Pathology & Association for Molecular Pathology

Evaluating the Near-Term Infant for Early Onset Sepsis

Progress and Challenges to Consider with 16S rDNA Polymerase Chain Reaction Testing

Jeanne A. Jordan*,{dagger}{ddagger}, Mary Beth Durso{ddagger}, Allyson R. Butchko{ddagger}, Judith G. Jones{dagger}§ and Beverly S. Brozanski{dagger}§

From the Departments of Pathology * and Pediatrics, § Magee-Women’s Hospital, {dagger} and Magee-Women’s Research Institute, {ddagger} University of Pittsburgh, Pittsburgh, Pennsylvania


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the rate of early onset sepsis in the near-term neonate is low (one to eight of 1000 cases), the rate of mortality and morbidity is high. As a result, infants receive multiple, broad-spectrum antibiotic therapy, many for up to 7 days despite blood cultures showing no growth. Maternal intrapartum antibiotic prophylaxis and small blood volume collections from infants are cited as reasons for the lack of confidence in negative culture results. Incorporating an additional, more rapid test could facilitate a more timely diagnosis in these infants. To this end, a 16S rDNA polymerase chain reaction (PCR) assay was compared to blood culturing for use as a tool in evaluating early onset sepsis. Of 1751 neonatal intensive care unit admissions that were screened, 1233 near-term infants met inclusion criteria. Compared to culture, PCR demonstrated excellent analytical specificity (1186 of 1216, 97.5%) and negative predictive value (1186 of 1196, 99.2%); however, PCR failed to detect a significant number of culture-proven cases. These findings underscore the cautionary stance that should be taken at this time when considering the use of a molecular amplification test for diagnosing neonatal sepsis. The experience gained from this study illustrates the need for changes in sample collection and preparation techniques so as to improve analytical sensitivity of the assay.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Early onset sepsis (EOS) in the near-term infant (>34 weeks gestation) is difficult to diagnose because the infant’s signs and symptoms may be subtle or may mimic other medical conditions such as hypoglycemia, delayed transition, or transient tachypnea.1, 2, 3, 4 Even though the incidence of culture-positive sepsis is low (1 to 8 cases of 1000 live births), the risk of mortality is high, ranging from 15 to 50%.3, 5 Thus, many infants are evaluated for EOS and receive multiple broad-spectrum antibiotics, although few have culture-proven sepsis. In fact, it is estimated that between 5% and 10% (189,000 to 489,000) of all newborns in the United States receive systemic antibiotics annually.6

Early-onset disease occurs before the first 5 to 7 days of life and presents as a fulminate, multisystemic illness.7 Typically, the infant has acquired the organism during the intrapartum period from the maternal genital tract. The most common etiologies of EOS include group B Streptococcus, Escherichia coli, Enterococcus sp., and Listeria monocytogenes.3

At Magee-Women’s Hospital, a tertiary care center with a 65-bed neonatal intensive care unit (NICU), 70% of near-term infants undergo a sepsis evaluation on admission to the NICU although only ~1% of them have culture-proven sepsis. These infants are managed clinically with bacterial cultures, complete blood count (CBC), and combination antimicrobial therapy for presumed sepsis; ampicillin and gentamicin, or ampicillin and cefotaxime, are usually given to the infant.8, 9, 10, 11 Although this regimen is effective in decreasing bacterial sepsis, there are significant risks associated with using multiple, broad-spectrum antibiotics. There risks include adverse drug reaction or toxicity, bacterial superinfection with drug resistant organisms, and/or overgrowth of yeast and/or fungi.

Among the diagnostic procedures used currently, growth in culture is considered the gold standard for detecting blood-borne bacteria. However, culturing requires significant time and has a low sensitivity.2, 12 In addition, maternal intrapartum antimicrobial prophylaxis and/or small blood volume collections from infants are cited as possible reasons for the lack of confidence in negative culture results.13, 14, 15, 16 At Magee-Women’s Hospital, preliminary results are generated after 48 hours for blood cultures lacking detectable growth. However, final results for negative blood cultures are not finalized until after 5 days. Using a more rapid assay for ruling out sepsis in the near-term infant who is stable and asymptomatic would provide valuable information sooner for medical decision making.

Nucleic acid amplification tests such as the polymerase chain reaction (PCR) assay are rapid to perform and have been used successfully to diagnose a wide range of infectious diseases, including bacterial, yeast, viral, and protozoal infections.17 In fact, the nucleic acid amplification test is now considered the gold standard test for diagnosing neonatal meningitis due to herpes simplex virus.18 PCR detection of blood-borne bacteria using highly conserved targets within the 16S ribosomal RNA gene (16S rDNA) has been described.19, 20, 21, 22, 23, 24, 25 Our own laboratory published the first large-scale study for detecting sepsis in neonates. The study tested 548 blood samples collected from NICU-based infants being evaluated for either EOS or hospital-acquired sepsis, regardless of their gestational age.26 The results revealed a high level of overall agreement between PCR and culture (99.3%), with less time needed for reporting results.

The objective of the current study was to determine the robustness of the same PCR assay in a population with a very low rate of culture-proven sepsis: near-term infants (>34 weeks gestation) admitted to the NICU for EOS evaluation. The purpose of this study was to compare the performance of a 16S rDNA PCR assay to that of an automated blood culturing system for ruling out sepsis in the near-term infant (ie, negative predictive value). If the PCR assay could be shown to have a high level of agreement with culture, then using a more rapid test in conjunction with culture could provide valuable information sooner for medical decision making in the case of the uninfected, stable, and asymptomatic infant.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patient Selection and Sample Rejection Criteria
We received hospital institutional review board approval before initiation of this study. To be eligible, an infant had to be >34 weeks gestation at the time of birth, be admitted to the NICU within a few hours of birth for EOS evaluation, and have both a blood culture and CBC ordered by the attending physician. The 16S rDNA PCR analyses were performed from the unused portions of each CBC. Sample rejection for PCR included blood volumes of <100 µl or grossly hemolyzed and/or clotted specimens. Sample rejection occurred in only 10 specimens (0.8%). To be considered clinically stable, the infant needed to fulfill the following criteria for a minimum of 24 hours: adequate urine output (>1 cc/kg/hour), full volume oral feedings (>100 cc/kg/day), and breathing in room air (<60 breaths/minute).

Blood Culturing and Phenotypic Identification of Culture-Proven Cases
Blood for culture was collected either by a venous or arterial draw, with 0.5 to 1.0 ml being inoculated directly into a single pediatric sample-sized resin-containing blood culture bottle (Peds Plus; Becton Dickinson, Sparks, MD). Each blood culture bottle was sent immediately to the clinical microbiology laboratory where it was loaded into a Bactec 9240 blood culture instrument (Becton Dickinson) in 1 hour or less from the time of receipt in accordance with the manufacturer’s recommendation. The Bactec 9240 instrument is an automated blood culture system that uses a fluorescent sensor for detecting growth of the microorganisms.

When the Bactec 9240 instrument detected bacterial growth, fluid from the bottle was withdrawn, gram-stained, and subcultured on the appropriate agar-based culture plates. After overnight incubation, the purified colonies were identified either using an automated identification system (MicroScan Dade, West Sacramento, CA) or manually using the appropriate reagents.

CBC Whole Blood Collection
Whole blood for CBC analysis was collected either by venous or arterial draw or by a heel stick method. A pediatric-sample-sized ethylenediaminetetraacetic acid-containing Vacutainer tube (Becton Dickinson) was filled with 300 to 900 µl of whole blood. The specimens were sent immediately to the hospital’s clinical hematology laboratory for analysis on a Coulter Counter instrument (Coulter Inc., South Beach, FL). The unused portion of blood was then released for 16S rDNA PCR analysis once the CBC result was finalized.

Whole Blood Enrichment and Sample Processing for 16S rDNA PCR
The protocol for sample enrichment and processing was described previously.26 Briefly, the entire volume (100 to 600 µl) of the remaining CBC was added to 4 ml of tryptic soy broth (Difco Laboratories, Detroit, MI) and incubated at 37°C for up to 5 hours. The pre-enriched specimen was treated with 4°C red cell lysis buffer (0.32 mol/L sucrose, 10 mmol/L Tris-HCl, pH 7.5, 5 mmol/L MgCl2, and 1% Triton X-100). Nucleated cells were pelleted at 13,000 x g for 5 minutes at 4°C and treated with 100 µl of phosphate-buffered saline (PBS) containing 50 U Mutanolysin (Sigma, St. Louis, MO) and 10 mg/ml of proteinase K for 30 minutes at 70°C. After enzymatic digestion, the crude cell lysate was heated to boiling for 10 minutes to inactivate the proteinase K.

Universal 16S rDNA PCR Assay
The PCR assay used the well-characterized and highly conserved RW01 and DG74 primers.19, 26 The amplified 380-bp product was visualized using an ethidium-stained, gel-based detection method, followed by Southern blot analysis using a digoxigenin-labeled RDR245 probe as described previously.26 Two controls were included with each run, a ß-globin control26 and a spiking control. The former control tested for presence of DNA in the specimen whereas the latter control assessed the specimen for evidence of inhibitors. For spiking, 100 colony-forming units of group B Streptococcus were added to the 16S rDNA PCR master mix containing the specimen.

Initial 16S rDNA Sequencing Reaction
Each 380-bp amplicon generated by the 16S rDNA PCR was partially sequenced using RDR245 as the sequence primer (Pyrosequencing; Biotage, Uppsala, Sweden).26 The sequence information obtained by pyrosequencing was used to provide partial identity for the bacteria contained within each PCR-positive blood sample as previously described.27

Culture-Based Identification of the Staphylococcus sp.
Bacteria were grown on sheep blood agar plates (BBL Division, Becton Dickinson) to obtained purified colonies. The bacteria were gram-stained; those appearing gram-positive and resembling cocci in clusters under microscopic examination were tested for their catalase reaction using a 3% solution of H2O2. Catalase-positive colonies were further analyzed for their coagulase reaction using Staphaurex (Remel, Lenexa, KS). Coagulase-positive, catalase-positive, gram-positive cocci in clusters were identified as Staphylococcus aureus (SA), whereas the coagulase-negative, catalase-positive, gram-positive cocci in clusters were identified as coagulase-negative Staphylococcus species (CoNS).

Preparation of Crude Cell Lysates from Purified Isolates of Staphylococcus sp.
Individual colonies of SA and CoNS were used to prepare individual crude cell lysates for Staphylococcus sp.-specific 16S rDNA PCR analysis. An isolated colony was touched with a sterile loop and the resulting bacteria resuspended in a solution containing 89 µl of PBS (pH 7.6) and 5 µl (50 U) of Mutanolysin (Sigma) and then incubated for 30 minutes at 37°C. To this mixture, 5 µl of proteinase K (10 mg/ml) (Sigma) and 1 µl of 10% sodium dodecyl sulfate (Sigma) were added, and the solution was incubated for 30 minutes at 37°C and then heated to 95°C for 10 minutes. A 1-µl volume of the resulting crude cell lysate was added to 99 µl of the PCR master mix for amplification of the 16S rDNA Staphylococcus sp.-specific target.

Staphylococcus sp. 16S rDNA PCR Assay
The 16S rDNA PCR-positive specimens identified initially by pyrosequencing as Staphylococcus sp. were further analyzed to distinguish SA from CoNS using a different set of primers targeting a separate region of the 16S rDNA gene. To accomplish this, GenBank database was queried for available Staphylococcus sp. 16S rRNA gene sequences. Staphylococcus epidermidis (AF397060 and AY167864), Staphylococcus hominis (X66101 and AY030318), Staphylococcus haemolyticus (X66100), Staphylococcus caprae (Y12593), Staphylococcus capitis (AY030321), and S. aureus (X68417) were aligned using the software program Sequencer (Gene Codes Inc., Ann Arbor, MI). The goal was to identify both a region within the 16S rRNA gene containing complete homology for primer recognition as well as a region with bp differences to differentiate SA from CoNS. The search revealed a 243-bp region within the 16S rRNA gene (bp no. 46 to 288, relative to GenBank reference number X70648). The PCR and sequencing primers were designed to recognize both SA and CoNS with an internal sequence containing differences in 3 of 23 bases to distinguish SA from CoNS.

The PCR primers included a biotinylated forward primer: 5'-TGC CTA ATA CAT GCA AGT CGA GCG-3' (bp no. 46 to 69) and an unlabeled reverse primer: 5'-GTT GCC TTG GTA AGC CGT TAC CTT-3' (bp no. 288 to 265). The PCR master mix consisted of 10 mmol/L Tris-HCl, pH 8.3; 2 mmol/L MgCl2; 200 mmol/L each dATP, dCTP, dTTP, and dGTP; 0.2 mmol/L of each primer; and 2.5 U Taq polymerase (Promega, Madison, WI). A volume of 1 µl of crude cell lysate was added to 99 µl of master mix. The PCR cycling parameters consisted of an initial denaturing step (10 minutes at 95°C), followed by 40 cycles of the following: 1 minute at 95°C, 2 minutes at 60°C, and 1 minute at 72°C. After amplification, 10 µl of material were analyzed by agarose gel electrophoresis. The ethidium-stained gel was evaluated for the presence of the 243-bp DNA fragment compared to a 100-bp ladder molecular weight standard (BioWhitaker, Rockland, ME).

Differentiation Between SA and CoNS Using Pyrosequencing
A 40-µl aliquot of the Staphylococcus sp.-specific 16S rDNA PCR generated amplicon was purified using streptavidin-coated Sepharose beads (3 µl) (Amersham, Piscataway, NJ) in an annealing buffer provided by Biotage. The unlabeled DNA strand was dissociated and discarded after alkaline denaturation according to the manufacturer’s instructions. The pyrosequencing vacuum prep tool was used to perform the washing (10 mmol/L Tris acetate, pH 7.6) and bead transferring steps. The bead-bound, biotinylated DNA strand was added to a well in a 96-well microtiter plate along with annealing buffer and 0.5 µmol/L of the complementary sequencing primer, 5'-GTG TTA CTC ACC CGT CCG CCG CTA-3' (bp no. 122 to 99). The microtiter plate containing the samples was heated to 95°C for 2 minutes and then allowed to cool to room temperature on the bench top, at which time the primer annealed to its complementary target.

The plate containing the annealed products was placed into the PSQ 96MA system (Biotage) where the direct sequencing reaction occurred using sequence analysis reagents (Biotage) and a cyclic dispensation program of nucleotides according to the manufacturer. The pyrosequencing reaction utilizes a four-enzyme cascade system that produces visible light read by a charge-coupled device camera. The light detected by the camera is displayed as peaks, with the height of the peak being proportional to the number of bases of a specific dNTP incorporated during the reaction. All four bases are added individually, one dNTP base at a time with the unincorporated nucleotide being destroyed enzymatically before the next NTP is added (dATP, dCTP, dTTP, and finally dGTP). The software program provides both a pyrogram figure and a text-based sequence for analysis.

Full-length sequencing of several SA and CoNS-based 243-bp amplicons was also performed using the ABI 3100 instrument for comparison (data not shown). The resulting sequences from both the ABI and pyrosequencing reactions were compared to one another and to the Staphylococcus sp. sequences found in GenBank, EMBL Nucleotide Sequence Database, and the DNA Data Bank of Japan28, 29 using the BLAST tool30 from the National Center for Biotechnology Information. Sequences were aligned using Sequencer software (Gene Codes Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enrollment Numbers for the Near-Term Infants Being Evaluated for EOS
In this study, a total of 1751 NICU admissions were screened between September 1, 2000 and April 1, 2004. Of the infants screened, 1233 met the inclusion criteria for enrollment and were included in this study. Compared to culture, the 16S rDNA PCR demonstrated a high negative predictive value for ruling out EOS in the asymptomatic and stable near-term infant.

In all, 1233 infants who had paired blood culture and 16S rDNA PCR results were enrolled (Table 1)Go . The comparison revealed an overall level of agreement of 96.8% (1193 of 1233) between culture and PCR results. The percentage of near-term infants with culture-negative results was 98.6% (1216 of 1233), whereas those with PCR-negative results was 97% (1196 of 1233). Compared to blood culture, PCR demonstrated a high negative predictive value (99.2%) and analytical specificity (97.5%). The 1186 infants with culture-negative/PCR-negative results had stabilized and were asymptomatic, having met the criteria listed for a minimum of 24 hours.


View this table:
[in this window]
[in a new window]
  		Table 1. Comparison of Blood Culture and 16S rDNA PCR Results from Near-Term Infants Admitted to the NICU for EOS Evaluation

 
The 16S rDNA PCR Assay Failed to Detect a Significant Number of Culture-Proven Cases of EOS in Near-Term Infants
There were a total of 17 culture-proven cases of EOS detected in near-term infants enrolled in this study (Table 1)Go ; 5 cases have been previously described.27 The culture-based identification of all 17 bacterial isolates included group B Streptococcus (n = 6), CoNS (n = 6), E. coli (n = 2), Streptococcus viridans (n = 2), and Micrococcus sp. (n = 1) (Tables 2Go and 3)Go and are representative of bacteria responsible for EOS.7


View this table:
[in this window]
[in a new window]
  		Table 2. Details of Culture-Positive/PCR-Positive Results

 

View this table:
[in this window]
[in a new window]
  		Table 3. Details of the Culture-Positive/PCR-Negative Results

 
The 16S rDNA PCR assay failed to detect 10 of the 17 cases of culture-proven sepsis. Dividing the culture-proven cases of EOS into concordant (Table 2)Go and discordant (Table 3)Go results revealed differences in the average times required to detect bacterial growth in culture: 12 hours 6 minutes for concordant results, and 23 hours 15 minutes for discordant results. In addition to the longer detection time needed for the discordant results, other issues including sample collection and preparation techniques are also responsible for the failure of PCR to detect bacterial DNA in these cases.

16S rDNA PCR Assay Detected Bacterial DNA from Blood Collected on a Significant Number of Culture-Negative Cases in Near-Term Infants
There were 30 culture-negative/PCR-positive results from near-term infants enrolled in this study. There was sufficient DNA in 27 of the 30 amplified samples to perform pyrosequencing using the RDR245 oligonucleotide as the sequencing primer as previously described.27 Table 4Go illustrates the pyrosequencing results for the 27 amplicons; they were identified as Staphylococcus sp.(n = 20), Streptococcus sp.(n = 5) and enteric gram-negative rods (n = 2).


View this table:
[in this window]
[in a new window]
  		Table 4. Pyrosequencing Results of Blood Culture-Negative/PCR-Positive Discordant Results

 
Differentiation Between SA and CoNS by Direct Sequence Analysis
The Staphylococcus sp.-specific 16S rDNA PCR amplification and subsequent pyrosequencing reaction was useful in differentiating SA from CoNS. A total of 258 purified isolates of Staphylococcus sp. were analyzed. These isolates consisted of 106 S. aureus, including 26 methicillin-resistant S. aureus, and 152 coagulase-negative Staphylococcus sp. that had been identified using culture-based methods. Figure 1Go illustrates representative pyrograms for the SA (Figure 1A)Go and CoNS (Figure 1B)Go . The SA and CoNS sequences revealed three base differences (underlined) that could be used to differentiate SA from CoNS. All of the 106 SA isolates tested had the following 23-bp sequence: 5'-ACA TCA GAG AAG CAA GCT TCT CG-3', whereas all of the 152 CoNS isolates tested had the following 23-bp sequence: 5'-ACG TCA G/A AG GAG CAA GCT CCT CG-3'. The seventh base varied in the group of CoNS, being either a guanine or an adenine.


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		Figure 1. Representative pyrograms of SA and CoNS. The SA (A) and CoNS (B) sequences revealed three base differences (underlined) that could be used to differentiate SA from CoNS. A: 5'-ACA TCA GAG AAG CAA GCT TCT CG-3'; and B: 5'-ACG TCA AAG GAG CAA GCT CCT CG-3'.

 
The Staphylococcus sp.-specific 16S rDNA PCR assay using crude cell lysates from non-Staphylococcal bacteria failed to generate an amplicon (data not shown). The analysis included several isolates each of Bacillus sp., Corynebacterium sp., Listeria monocytogenes, E. coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus sp., Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae.

The Staphylococcus sp. Detected by PCR and Identified by Pyrosequencing Were Mainly CoNS
Table 4Go illustrates the success of using pyrosequencing directly from amplified clinical materials for differentiation between SA and CoNS. Nineteen of the twenty (95%) Staphylococcus sp. amplicons generated from PCR-positive/culture-negative blood samples had a pyrogram consistent with CoNS, whereas 1 of 20 (5%) was consistent with SA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge, this is the largest study to date comparing the results of PCR to culture in a population of near-term infants being evaluated for EOS. Our data confirms the fact that EOS in the near-term infant occurs at a very low incidence, regardless of the method used to detect bacteria. More importantly however, the results from this study emphasize the need for improvements to study design and methodologies that must be incorporated into any future studies comparing PCR and culture before a molecular-based test can be considered for use as a screening tool for neonatal sepsis.

In retrospect, our study had several design flaws that compromised our efforts to adequately compare PCR to culture. The primary design flaw was using the remaining CBC sample for PCR analysis. This choice, however, was not made lightly; the time frame between an infant’s delivery and his/her admission to the NICU for suspicion of EOS can be extremely short. This fact made obtaining parental consent onerous; in fact, the mother may still have been in recovery, having just left the labor suite. Therefore, to achieve adequate study participation of the near-term infant being evaluated for EOS, we felt justified in using the remaining CBC sample for PCR analysis.

A second weakness to our study design was variation in sample collection; a significant percentage of the CBC specimens were not collected concurrently with blood drawn for culture, and/or were collected using a heel-stick method. The former variation could have produced dissimilar levels of potential bacteria present in the two samples, whereas the latter variation could have introduced bacterial contamination into the sample used for PCR analysis. Using heel-stick collected blood for PCR analysis likely contributed to many of the 30 culture-negative/PCR-positive discrepancies that occurred in this study. Heel stick collection of blood for CBC analysis is a common practice in our NICU; up to 30% of CBC samples are collected using a heel-stick method, a practice that carries the risk of acquiring normal skin flora in the sample, which does not pose a problem for CBC analysis but could lead to false-positive PCR results. In fact, 20 of 30 (66.7%) of the culture-negative/PCR-positive discrepancies were identified using pyrosequencing to be Staphylococcus sp., with 19 of 20 (95%) being CoNS, organisms that can be considered skin contaminants.

For future studies, we strongly recommend using a dedicated blood sample for PCR analysis, one that is collected aseptically from an arterial or venous draw at the same time as the blood being drawn for culture. This approach will help to eliminate sample-to-sample variation and provide a more accurate comparison between PCR and culture methods. This approach, however, will require that parental consent be obtained. Therefore, to achieve adequate enrollment numbers, those infants being evaluated for acquired sepsis will be targeted for enrollment rather than the near-term infants being evaluated for EOS. Here the time frame for obtaining consent will be more manageable, and the rate of sepsis significantly higher.

Improving analytical sensitivity of the PCR assay is critical. All 10 cases of culture-proven sepsis that PCR failed to detect were due to a gram-positive cocci; this observation points to a need to change sample preparation protocols. Effective lysis of bacterial cells to liberate their DNA is crucial in achieving an assay with adequate sensitivity. Because of their cell wall composition, gram-positive organisms are more difficult to lyse than gram-negative ones. Perhaps including an additional enzymatic treatment targeting gram-positive bacteria, using labiase or achromopeptidase31 or using glass beads to disrupt the cell wall more effectively would improve cell lysis.

Although the use of crude cell lysates did not appear to contain significant levels of PCR inhibitors, sample preparation protocols including a DNA extraction step would be an improvement; DNA extracted from whole blood using either a manual method (eg, QIAamp column; Qiagen Inc., Valencia, CA) or an automated one should be considered. A DNA purification method that can extract DNA efficiently from small sample volumes and elute DNA in small fraction volumes to facilitate sample concentration is recommended.

Nucleic acids released from whole blood contain not only possible bacterial DNA but also genomic DNA from the patient’s white blood cells (WBCs) as well. Human genomic DNA from WBCs is normally present at much higher concentration than bacterial DNA, and this difference creates a competitive advantage for binding of human genomic DNA rather than bacterial DNA onto the column or bead matrix. Recently, we demonstrated that DNA contained in whole blood samples collected from infants with elevated white blood cell counts (≥39 x 103/µl) out-competed the exogenously added bacterial DNA for binding to a Qiagen column, resulting in a false-negative PCR result.32

The results from this study have allowed us to identify several key factors that likely contributed to the low analytical sensitivity of our PCR. The hurdles outlined above can be overcome. In fact, recent attempts to improve assay sensitivity have met with success when using purified DNA and a real-time PCR platform for testing.32 However, many challenges remain before PCR can be recommended for the diagnosis of sepsis, and the success of this approach must be proven on a much larger scale using multiple sites. Then, and only then, will we have confidence in the test. Implementing the modifications outlined above should improve the assay’s analytical sensitivity, something that is essential before PCR can be considered a reliable and useful diagnostic tool in detecting neonatal sepsis.


   Footnotes
 
Address reprint requests to Jeanne A. Jordan, Magee-Women’s Research Institute, 204 Craft Ave., Pittsburgh, PA 15213. E-mail: rsijaj{at}mwri.magee.edu

Supported by the National Institute of Child Health and Human Development (grant R01 HD038559).

Accepted for publication January 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cerase PA: Neonatal sepsis. J Perinatal Neonatal Nurs 1989, 3:48-57[Medline]
  2. Gerdes JS: Clinicopathologic approach to the diagnosis of neonatal sepsis. Clin Perinatol 1991, 18:361-381[Medline]
  3. Klein JO, Marcy SM: Bacterial sepsis and meningitis. Infectious Diseases of the Fetus and Newborn. 1990 W. B. Saunders Philadelphia
  4. Witek-Janusek L, Cusack C: Neonatal sepsis: confronting the challenge. Crit Care Nurs Clin North Am 1994, 6:405-419[Medline]
  5. Freedman RM, Ingram DL, Gross I, Ehrenkranz RA, Warshaw JB, Baltimore RS: A half century of neonatal sepsis at Yale: 1928 to 1978. Am J Dis Child 1981, 135:140-144[Abstract]
  6. Wegman ME: Annual summary of vital statistics—1992. Pediatrics 1993, 92:743-754[Abstract/Free Full Text]
  7. Stoll BJ, Gordon T, Korones SB, Shankaran S, Tyson JE, Bauer CR, Fanaroff AA, Lemons JA, Donovan EF, Oh W, Stevenson DK, Ehrenkranz RA, Papile LA, Verter J, Wright LL: Early-onset sepsis in very low birth weight neonates: a report from the National Institute of Child Health and Human Development Neonatal Research Network. J Pediatr 1996, 129:72-80[CrossRef][Medline]
  8. Chadwick EG, Yogev R, Shulman ST: Combination antibiotic therapy in pediatrics. Am J Med 1986, 80:166-171[CrossRef][Medline]
  9. de Louvois J, Harvey D: Strategies for the treatment of bacterial infections in the newborn. Ann Acad Med Singapore 1985, 14:631-641[Medline]
  10. Fanos V, Dall’Agnola A: Antibiotics in neonatal infections: a review. Drugs 1999, 58:405-427[CrossRef][Medline]
  11. Odio CM: Cefotaxime for treatment of neonatal sepsis and meningitis. Diagn Microbiol Infect Dis 1995, 22:111-117[CrossRef][Medline]
  12. Brown DR, Kutler D, Rai B, Chan T, Cohen M: Perinatal/neonatal clinical presentation: bacterial concentration and blood volume required for a positive blood culture. J Perinatol 1995, 15:157-159[Medline]
  13. Cavaliere TA: Pharmacologic treatment of neonatal sepsis: antimicrobial agents and immunotherapy. J Obstet Gynecol Neonatal Nurs 1995, 24:647-658[CrossRef][Medline]
  14. Dietzman DE, Fischer GW, Schoenknecht FD: Neonatal Escherichia coli septicemia—bacterial counts in blood. J Pediatr 1974, 85:128-130[Medline]
  15. Heimler R, Nelin LD, Billman DO, Sasidharan P: Identification of sepsis in neonates following maternal antibiotic therapy. Clin Pediatr 1995, 34:133-137[Abstract/Free Full Text]
  16. Schuchat A, Whitney C, Zangwill K: Prevention of perinatal group B streptococcal disease: a public health perspective. MMWR Recomm Rep 1996, 45:1-24[Medline]
  17. Ehrlich GD, Greenberg SJ: PCR-Based Diagnostics in Infectious Disease. 1994 Blackwell Scientific Publications Boston
  18. Kimberlin D: Herpes simplex virus, meningitis and encephalitis in neonates. Herpes 2004, 11 Suppl 2:S65A-S76A
  19. Greisen K, Loeffelholz M, Purohit A, Leong D: PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol 1994, 32:335-351[Abstract/Free Full Text]
  20. Hitti J, Riley DE, Krohn MA, Hillier SL, Agnew KJ, Krieger JN, Eschenbach DA: Broad-spectrum bacterial rDNA polymerase chain reaction assay for detecting amniotic fluid infection among women in premature labor. Clin Infect Dis 1997, 24:1228-1232[Medline]
  21. Laforgia N, Coppola B, Carbone R, Grassi A, Mautone A, Iolascon A: Rapid detection of neonatal sepsis using polymerase chain reaction. Acta Paediatr 1997, 86:1097-1099[Medline]
  22. Ley BE, Linton CJ, Bennett DM, Jalal H, Foot AB, Millar MR: Detection of bacteraemia in patients with fever and neutropenia using 16S rRNA gene amplification by polymerase chain reaction. Eur J Clin Microbiol Infect Dis 1998, 17:247-253[Medline]
  23. McCabe KM, Khan G, Zhang YH, Mason EO, McCabe ER: Amplification of bacterial DNA using highly conserved sequences: automated analysis and potential for molecular triage of sepsis. Pediatrics 1995, 95:165-169[Abstract/Free Full Text]
  24. Qian Q, Tang YW, Kolbert CP, Torgerson CA, Hughes JG, Vetter EA, Harmsen WS, Montgomery SO, Cockerill FR, III, Persing DH: Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rRNA gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results. J Clin Microbiol 2001, 39:3578-3582[Abstract/Free Full Text]
  25. Rothman RE, Majmudar MD, Kelen GD, Madico G, Gaydos CA, Walker T, Quinn TC: Detection of bacteremia in emergency department patients at risk for infective endocarditis using universal 16S rRNA primers in a decontaminated polymerase chain reaction assay. J Infect Dis 2002, 186:1677-1681[CrossRef][Medline]
  26. Jordan JA, Durso MB: Comparison of 16S rRNA gene PCR and BACTEC 9240 for detection of neonatal bacteremia. J Clin Microbiol 2000, 38:2574-2578[Abstract/Free Full Text]
  27. Jordan JA, Butchko AR: Use of pyrosequencing 16S rRNA fragments to detect and classify bacteria responsible for neonatal sepsis. J Mol Diagn 2005, 7:105-110[Abstract/Free Full Text]
  28. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Rapp BA, Wheeler DL: GenBank. Nucleic Acids Res 2000, 28:15-18[Abstract/Free Full Text]
  29. Galperin MY: The molecular biology database collection: 2004 update. Nucleic Acids Res 2004, 32 Database issue:D3-D22[Abstract/Free Full Text]
  30. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215:403-410[CrossRef][Medline]
  31. Niwa T, Kawamura Y, Katagiri Y, Ezaki T: Lytic enzyme, labiase for a broad range of gram-positive bacteria and its application to analyze functional DNA/RNA. J Microbiol Methods 2005, 61:251-260[CrossRef][Medline]
  32. Jordan JA, Durso MB: Real-time polymerase chain reaction for detecting bacterial DNA directly from blood of neonates being evaluated for sepsis. J Mol Diagn 2005, 7:575-581[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jordan, J. A.
Right arrow Articles by Brozanski, B. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jordan, J. A.
Right arrow Articles by Brozanski, B. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS