Comparison between Polymerase Chain Reaction and Blood Culture for Diagnosis of Neonatal Sepsis

1. Introduction

Neonatal sepsis can be defined as any systemic bacterial infection confirmed by a positive blood culture in the first month of life ( 1 ). Neonatal septicemia remains one of the main causes of mortality and morbidity despite the progress in hygiene, the introduction of new and potent antimicrobial agents for treatment and advanced measures for diagnosis. Up to 10% of infants have infections in the first month of life, resulting in 30‐50% of neonatal deaths in developing countries ( 2 ). Early onset neonatal sepsis (EOS) occurring in the first 72 hours of life remains a significant cause of illness and death among very low birth weight (VLBW) preterm infants ( 3 ). Late-onset sepsis (LOS) is present during 7-28 days of age. The source of infection in LOS is either nosocomial (hospital-acquired) or community-acquired, and neonates usually present with septicemia, pneumonia or meningitis. The spectrum of infecting organisms has evolved over the last two decades from a predominance of Gram-negative organisms to Gram-positive organisms. Most community-acquired infections are due to Streptococcus spp. and Escherichia coli, other Enterobacteriaceaeand non-ferment Gram-negative bacteria like Pseudomonas and Acinetobacter spp. ( 4 ).

Group B streptococcus (GBS, Strep. agalactiae) remains the leading cause of neonatal sepsis and meningitis in the United States. Although less common, Listeria monocytogenes is associated with invasive disease in newborns. Enterobacter spp, Klebsiellapneumoniae, P. aeruginosa and Staphylococcus aureus is also microorganisms commonly isolated from infants with EOS ( 5 ). The bacteria responsible for late-onset septicemia include those acquired from the maternal genital canal and organisms acquired after birth from human contacts or contaminated equipment or materials ( 6 ).

The clinical diagnosis of sepsis in neonates is difficult because many signs of sepsis are nonspecific and are observed with other noninfectious conditions. Although a routine physical examination is evidence that sepsis is not present, bacteremia can occur without clinical signs ( 7 ). Microbial cultures of blood or other sterile body fluids are the gold standard in diagnosing neonatal sepsis. Blood cultures are generally assumed to have low sensitivity in neonates.

Molecular assays have the advantage of direct pathogen detection in a more rapid turnover time than blood cultures, with results available within a few hours. Molecular assays might eventually replace blood cultures but will continue as a supplement to blood cultures until they are adequately evaluated ( 8 ).

2. Materials and Methods

2.1. Patients

This study was conducted on 85 neonates (1-28 days) with clinical or laboratory findings suggestive of sepsis admitted at NICUs and premature infant unit of the Babylon Hospital for Pediatric and Gynecology from November 2014 to March 2015. Complete history taking, thorough clinical examination and routine laboratory investigations were made on all neonates with suspected sepsis.

2.2. Specimens

From each neonate, a minimum of 1–3 ml of blood was collected by standard sterile procedures, 2 ml for blood culture, while 1 ml was used for DNA extraction.

2.3. Blood Culture

A minimum of 2 ml of blood is taken through venipuncture and injected into two or more "blood bottles" with specific media for aerobic and anaerobic organisms. The blood is collected using an aseptic technique. This requires that both the tops of the culture bottles and the venipuncture site of the patient are cleaned prior to collection by swabbing with 70% isopropyl alcohol (povidone and left to dry before venipuncture).

2.4. Polymerase Chain Reaction

DNA (extracted from bacterial cells) was used as a template in specific PCR to detect 16s RNA, rpoB and ITS genes. A pair of specific primers were used to amplify a fragment gene. While the conditions of PCR methods are listed in table 1. A single reaction mixture contained 2.5 µl of upstream primers, 2.5 µl of downstream primer, 5 µl of DNA extraction, 12.5 µl of master mix and 2.5µl of nuclease-free water to obtain a total volume of 25 µl. The resulting PCR products were run in 2% agarose gel.

3. Results and Discussion

3.1. Etiologic Agents of Septicemia

Out of the 85 samples were collected from the neonatal unit. Only 6 (7.06 %) samples yielded bacterial growth, of which Staphylococcus aureus recorded 1 isolate (16.67%), Klebsiella sp. 3 (50.0%), Enterobacter species1 (16.67%), E. coli 1 (16.67%)and 79 showed no bacterial growth (Table 2).

The low frequency of blood culture is attributed to the differences in blood volume withdrawn ( 9 ), blood culture techniques and exposure to antimicrobials of the mother or the possibility of infection with viruses, fungi or anaerobes ( 10 ). Another factor that may highly influence the sensitivity of the blood culture is starting antibiotic treatments at birth in all neonates with risk factors for early sepsis prior to diagnosis (11).

The isolation rate of bacteria in this study is comparable to rates reported in previous studies done by Fanaroff, Stoll ( 1 ) (8.7%), and Ahmed, Chowdhury ( 12 ) (4.2%). However, in contrast with many studies done by Nathoo, Mason ( 13 ) (31.75%), Aftab and Iqbal ( 14 ) (54.0%), and Kellogg, Ferrentino ( 15 ) (52.6%), where isolation rates are high, because of large samples that are used in their studies. The frequency of isolation of Gram-positive and harmful bacteria from blood culture in this study was 16.67% and 83.34%, respectively.

This finding is similar to other studies, which showed that Gram-negative bacteria were responsible for most cases of neonatal sepsis ( 16 ). This was in contrast to other studies where Gram-positive bacteria were the most typical cause of neonatal sepsis ( 17 , 18 ), while another study showed that the frequency of isolation of both Gram-positive and negative was equal ( 19 ).

Klebsiella spp were the most common isolates (50.0%) causing neonatal sepsis in this study. Previous studies have reported similar findings ( 11 , 20 , 21 ). Klebsiella species have often been isolated in a hospital setting and are often implicated in nursery outbreaks ( 7 ).

3.2. Molecular Detection of Neonatal Sepsis

3.2.1. Detection Using 16s RNA Gene

The results of gel electrophoresis for DNA bands based on genes specific for bacterial 16S RNA revealed that 17:85 patients (20%) were positive for target specific for a bacterial gene (16S rRNA) in their blood samples with length (861) base pairs (Figure 1).

Figure 1. Gel electrophoresis of PCR products of 16s rRNA gene in neonatal sepsis

The result in this study shows that six neonates gave positive bacterial blood cultures with the isolation of clinically significant microorganisms, while 17 neonates gave positive PCR results. However, 79 neonates gave a negative bacterial blood culture result, and 68 neonates gave negative PCR results. Six neonates had positive bacterial blood culture / PCR results, while 68 had negative bacterial blood culture / PCR results. Eleven neonates with a positive PCR had a negative bacterial blood culture result, as shown in table 3.

This difference includes intermittent seeding of low numbers of bacteria within the bloodstream, the extremely small blood volumes obtained from infants for culture, and the increasingly common practice of providing intrapartum antibiotics to mothers of high-risk deliveries ( 13 ). It is well known that the smaller volume of blood obtained for culturing lower the chances of recovering the organisms ( 5 , 9 ). Although some infants may have a somewhat greater magnitude of bacteremia than adults (10 to 300 and 1 to 30 CFU/ml, respectively), the amount of blood sampled from newborn infants is significantly less than that taken from adults (0.5 to 1.0 and 10 to 30 ml, respectively) ( 8 ).

Haque, Jahan ( 11 ) found that bacterial DNA consensus sequences, e.g., the 16S rRNA gene, have been identified to define an organism as a bacterium. With such sequence information available, numerous DNA primers and probes have been described for PCR-based assays to diagnose bacterial sepsis. These assays may prove helpful for detecting or ruling out septicemia in term infants born to mothers who received intrapartum antibiotics. However, data describing the use of molecularly-based testing for diagnosing viral, bacterial, or candidal infections in the adult and pediatric populations exist.

The result of this study agreed with Shang, Chen ( 22 ), who found that PCR amplification of 861 bp was positive in (13%) and 4% with negative cultures.

Schuchat, Whitney ( 23 ) found that the number of positive specimens identified by PCR by using 16 sRNA (9.88%) was significantly higher than the number of positives identified by blood culture (4.65%).

3.2.2. Detection by Using the rpob Gene

Primers of the rpoB gene were used to detect Enterobacteriaceae in neonate blood samples. It has been found that 16:85 patients (18.8%) were positive for target-specific bacterial genes (rpoB) with a length of 512 base pairs. The amplicon was detected in gel electrophoresis and compared with an allelic ladder, as shown in figure 2.

Figure 2. Gel electrophoresis product of rpoB gene

The rpoB gene coding for the beta subunit of the RNA polymerase has emerged as a core gene candidate for phylogenetic analyses and identification of bacteria, especially when studying closely related isolates. rpoB has been proposed as an alternative biomarker for microbial community studies. This gene is described as possessing the same key attributes as 16s rRNA in that it is common in all bacteria and is a mosaic of conserved as well as variable sequence domains ( 24 ).

Bindayna, Jamsheer ( 25 ) demonstrate that rpoB displays other important characteristics as an ecological marker, including (i) its universal presence in all prokaryotes, (ii) the presence of slowly and quickly evolving regions for the design of probes and primers of differing specificities, (iii) having a housekeeping function, making it less susceptible to some forms of lateral gene transfer, and (iv) a large enough size to contain phylogenetic information, even after removal of regions that are difficult to align.

Elamreen ( 18 ) showed that the rpoB gene is the most promising target for detecting Enterobacteriaceae by PCR amplification for the detection of neonatal sepsis.

3.2.3. Detection of Fungi Using its Gene

After specific DNA extraction from blood samples, DNA was amplified with primer ITS F and ITSR in 25µl reaction mixtures. Then, the PCR products were purified by agarose gel electrophoresis, and it was found that no samples were producing fungal DNA with primer in pairs (400) bp fragments.

Sequence comparison of the ITS region is widely used in taxonomy and molecular phylogeny because it is easy to amplify even from small quantities of DNA (due to the high copy number of rRNA genes) and has a high degree of variation even between closely related species. This can be explained by the relatively low evolutionary pressure acting on such non-functional sequences ( 26 ).

Detection of fungemia by means of fungal blood culture is notoriously difficult, and invasive fungal infections are diagnosed better by molecular assay as PCR can detect naked DNA due to the presence of dead and degrading fungi within circulating phagocytes ( 27 ).

Unfavourable results can be from (i) the need for an appropriate gene sequence or (ii) faulty reagents and/or thermal cycler ( 28 ).

Authors' Contribution

Study concept and design: M. E. H.

Acquisition of data: M. H. H.

Analysis and interpretation of data: M. E. H.

Drafting of the manuscript: M. H. H.

Critical revision of the manuscript for important intellectual content: I. K. A.

Statistical analysis: M. H. H.

Administrative, technical, and material support: M. H. H.

Ethics

The study was conducted in accordance with the ethics committee of the Al Mustaqbal University College, Babylon, Iraq.

Conflict of Interest

The authors declare that they have no conflict of interest.

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