|Year : 2016 | Volume
| Issue : 3 | Page : 146-150
Nucleic acid-based methods for the early detection of sepsis in heart transplant recipients
Sarvesh Pal Singh
Department of CTVS, AIIMS, New Delhi, India
|Date of Web Publication||2-Mar-2017|
Sarvesh Pal Singh
Department of CTVS, AIIMS, New Delhi
Source of Support: None, Conflict of Interest: None
Nucleic acid-based tests (NABTs) were developed to decrease the time required to identify microorganism in the pathological specimens. The commercially available NABTs are of two types – one those can be applied to positive cultures grown in blood culture bottles and other those can be applied directly to blood samples. The latter tests are polymerase chain reaction (PCR)-based assays which can amplify the existing load of microorganism nucleic acids many a times like a culture in a blood culture bottle. Both tests then identify the pathogenic organisms with the use of specific nucleic acid probes. These tests have proven useful in management of heart transplant sepsis management.
Keywords: Molecular diagnostics, pathogens, polymerase chain reaction
|How to cite this article:|
Singh SP. Nucleic acid-based methods for the early detection of sepsis in heart transplant recipients. J Pract Cardiovasc Sci 2016;2:146-50
|How to cite this URL:|
Singh SP. Nucleic acid-based methods for the early detection of sepsis in heart transplant recipients. J Pract Cardiovasc Sci [serial online] 2016 [cited 2017 Oct 18];2:146-50. Available from: http://www.j-pcs.org/text.asp?2016/2/3/146/201387
| Introduction|| |
The new definition of sepsis defines sepsis as “life-threatening organ dysfunction due to a dysregulated host response to infection.” The primary determinants of survival in patients with sepsis are time to diagnose sepsis, prompt initiation of antibiotics within 1 h of diagnosis, and resuscitation in the first 6 h of presentation. In heart transplant recipients, this time duration is even more important, because once a recipient contracts an infection, the clinical deterioration is very fast. Therefore, heart transplant recipients need a high index of suspicion and prophylaxis against common pathogens. Furthermore, any unexplained organ dysfunction in a transplant recipient with evidence of infection should be considered as sepsis.
The laboratory diagnosis of sepsis relies on documenting the increase in markers of inflammation such as procalcitonin and C-reactive protein, leukocytosis or leukopenia, thrombocytopenia, antigen assays for galactomannan or 1,3 beta-D-glucan, and other parameters which imply end-organ dysfunction. However, the culture of an organism still remains the gold standard for confirming the presence of an infection. It takes 48–72 h to grow and test the antibiotic susceptibility of organisms by conventional methods (phenotyping and biochemical testing).
Nucleic acid-based tests (NABTs) were developed to decrease the time required to identify microorganism in the pathological specimens. The commercially available NABTs are of two types – one those can be applied to positive cultures grown in blood culture bottles and other those can be applied directly to blood samples. The latter tests are polymerase chain reaction (PCR)-based assays which can amplify the existing load of microorganism nucleic acids many a times like a culture in a blood culture bottle. Both tests then identify the pathogenic organisms with the use of specific nucleic acid probes. The use of PCR-based techniques for the detection of nonbacterial infections has also been established in heart transplant recipients.,,
Nucleic acid-based technologies in sepsis may be categorized as follows:
- Hybridization assays (probe hybridization, fluorescent in situ hybridization (FISH)-based arrays)
- Amplification assays (broad range PCR, real-time PCR, multiplex PCR)
- Postamplification detection assays (PCR-sequencing, PCR-hybridization, and PCR MALDI-TOF).
| Commercially Available Nucleic Acid-Based Tests|| |
Those can be applied on positive cultures in blood culture bottles.
Only those tests which have been used in humans will be discussed.
QuickFISH (AdvanDx, Woburn, MA, USA)
QuickFISH  is a Food and Drug Administration-approved test that identifies pathogen from positive blood culture using peptide nucleic acid (PNA)-FISH technology. In comparison to the original PNA-FISH test which took 90 min, QuickFISH takes only 20 min. PNA probes are DNA or RNA molecules in which sugar backbone (negatively charged) is replaced by peptide backbone (neutral) so that there is no electrostatic repulsion between negatively charged backbones. PNA probes are complementary base pair sequences to the target pathogen molecule. They are used in combination with quenchers which decrease their fluorescent signal intensity. When this PNA-quencher complex comes in contact with the rRNA of bacteria, the PNA probe dissociates from quencher and attaches to the rRNA with complementary base sequences. Once the quencher molecule is left, the PNA probe fluorescence increases and can be readily identified on a fluorescence microscopy.
Pathogens detected by PNA-FISH assay are Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Coagulase-negative Staphylococcus, Enterococcus faecalis, other enterococci, Candida albicans, Candida parapsilosis, Candida glabrata, Candida krusei, and Candida tropicalis [Figure 1]. QuickFISH detects S. aureus, coagulase-negative Staphylococcus, E. faecalis, other enterococci, E. coli, K. pneumoniae, and P. aeruginosa.
Hyplex BloodScreen (BAG, Lich, Germany)
Hyplex BloodScreen  is a multiplex PCR-based method hybridized with enzyme-linked immunosorbent assay for subsequent identification of bacteria. It can be used to identify 10 bacteria – six Gram-positive (Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium, methicillin-sensitive and methicillin-resistant S. aureus, Staphylococcus epidermidis) and four Gram-negative (Pseudomonas aeruginosa, E. coli, Klebsiella spp., and Enterobacter aerogenes) organisms. It can also detect mecA gene in positive blood cultures. In a study of 82 samples of blood, Hyplex BloodScreen showed an overall sensitivity of 100% for Gram-negative bacilli and 96.6%–100% for Gram-positive cocci. The specificity for Gram-negative bacilli was between 92.5% and 100% and for Gram-positive cocci was between 97.7% and 100%.
Prove-it Sepsis (Mobidiag, Helsinki, Finland)
Prove-it Sepsis , assay is a multiplex PCR-based method which is available on a strip array platform. First, the pathogen nucleic acid is amplified by PCR and then detected in microarray wells. The strip array consists of eight reaction wells, each containing a microarray capable of analyzing one sample. Each microarray can identify more than 60 bacteria, vancomycin and methicillin resistance markers, and 13 fungi in a single assay. It has automated software for result detection and analysis. The time taken is 3 h. A large study of 2107 samples found Prove-it to have a specificity of 99% and sensitivity of 95%. Following pathogens may be detected using Prove-it [Table 1].
Those applied on blood directly can be divided into two groups:
- Those which target selected group of pathogens: SeptiFast, VYOO, Magicplex
- Those which detect broad range of pathogens using universal targets: SepsiTest, IRIDICA.
Those Which Target Selected Group of Pathogens: Septifast, Vyoo, Magicplex
LightCycler SeptiFast test (Roche Molecular Systems, Branchburg, NJ)
LightCycler SeptiFast test uses multiplex real-time PCR technology to identify internal transcribed spacer region and distinguish 25 different species of bacteria and fungi, including Staphylococcus, Pseudomonas, Acinetobacter, Klebsiella, E. coli, Streptococcus, Enterococcus, Enterobacter, Proteus, Serratia, Candida spp., and Aspergillus fumigatus. It takes up to 6 h for a final report and is approved for the use in Europe. The 25 microorganisms targeted by SeptiFast account for >90% of all bloodstream infections. It is one of the most extensively used sepsis screen tests., Reinhart et al. reported the inability of SeptiFast to identify organisms in 20%–30% of culture-positive results by multiplex PCR. The main advantage of this test is its real-time format which decreases the risk of contamination.
VYOO (SIRS-Lab, Jena, Germany)
VYOO uses multiplex PCR technology with subsequent gel electrophoresis. VYOO can detect 34 bacteria, 7 fungi (including Aspergillus fumigatus), and 5 antibiotic resistance genes (blaSHV, blaCTX-M, mecA, vanA, and vanB) in about 7 h. In addition to the organisms detected by SeptiFast, it also detects Clostridium, Haemophilus, Neisseria, and Prevotella spp., The company claims to cover 99% of sepsis-relevant species plus major resistance genes. This assay has a sensitivity of 38%–60%.
Magicplex Sepsis test (Seegene, Korea)
Magicplex also uses multiplex PCR technology to detect 90 sepsis-causing pathogens in 3 h after DNA extraction. More than seventy Gram-positive (30 staphylococci, 40 streptococci, 3 enterococci), 12 Gram-negative, and 6 types of fungi may be identified using this test. It also detects three genes for antibiotic resistance (mecA, vanA, vanB). Magicplex increased the diagnostic accuracy by 143% in children with sepsis.,
Those Which Detect Broad Range of Pathogens Using Universal Targets: Sepsitest, Iridica
SepsiTest (Molzym, Bremen, Germany)
SepsiTest uses broad-range PCR to amplify the species-specific internal regions in the genome of a pathogen. If they found positive, these amplicons are subjected to sequence analysis to identify the microorganism. The broad-range binding of primers to 16S and 18S rRNA allows detection of more than 345 pathogens by SepsiTest. A specific feature of SepsiTest is the degradation of the contaminant human DNA before analysis, therefore decreasing the incidence of false positive results. Sample analysis is performed in duplicate to increase the sensitivity and reliability of pathogen detection. The turnaround time is about 8–12 h. In India, it is being used in the name of Sepsiscreen (SRL Lab).
IRIDICA system (Abbott Laboratories, USA)
IRIDICA system uses a combination of two technologies – PCR and electrospray ionization time-of-flight mass spectrometry (PCR/ESI-MS). In addition to the 16S rRNA gene, the PCR/ESI-MS targets the 23S RNA polymerase and other genes for bacterial detection and quantification. IRIDICA currently has five panels – sepsis, bacterial, fungal, and viral infections and ventilator-associated pneumonia. It can identify more than 1000 pathogens in 6 h including more than 750 bacteria and 200 fungi. In a study by Metzgar et al., the IRIDICA BAC BSI assay yielded approximately twice as many positive detections as culture in a set of 285 clinical blood specimens from patients with symptoms of sepsis. In the majority of cases, the IRIDICA BAC BSI assay-positive, culture-negative detections were either of species commonly associated with BSI or of clinically relevant human pathogens that would be difficult to grow in blood culture bottles (e.g., Ehrlichia chaffeensis).
False positive results from NABTs are due to contamination of samples, especially in laboratories with increased workload. A low organism load in the sample can lead to false negative results; therefore, newer versions of some test recommend increasing the sample volume from 1 ml to 5 ml. The molecular methods use less volume of blood 1–5 ml of blood because human DNA may inhibit the PCR reaction or lessen the probability of pathogen detection. Hemoglobin may also inhibit PCR amplification. The above tests have become available due to advancement in technology, but they are not a replacement for clinical acumen or culture-based methods.
| Our Experience|| |
In our experience, since Sepsiscreen was introduced in 2015, we have used it in 4 out of 20 heart transplants. The use of Sepsiscreen is limited by cost in Indian setup which is approximately Rs. 13000/test. The patient characteristics, type of specimen, and result are depicted in [Table 2], [Table 3]. The concurrence of the results of Sepsiscreen with cultures was good. Although in one patient (patient 4) there was no growth on culture with both samples of bronchoalveolar lavage and blood, Sepsiscreen identified Aspergillus spp. in the blood. The serum galactomannan levels in bronchoalveolar lavage were high, and the computed tomography of the chest showed a pulmonary nodule in the left upper lobe with perinodular halo [Figure 2]. The patient was treated with liposomal amphotericin B for 6 weeks and his lesion resolved.
|Table 2: Different features of heart transplant recipients who developed sepsis and were investigated using Sepsiscreen|
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|Table 3: Comparison of Sepsiscreen results with conventional culture method|
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|Figure 2: Noncontrast computed tomography of the chest of patient 4 showing single nodular opacity in upper lobe of the left lung with perinodular halo suggestive of fungal lesion. An additional cavitary lesion is seen which is a remnant of treated fungal pneumonia. On SepsiScreen test, the patient was found to have invasive aspergillosis and was treated with intravenous liposomal amphotericin B for 6 weeks.|
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| Conclusion|| |
The positive results on molecular diagnostic tests should be interpreted with respect to patients' clinical condition and not in isolation. Molecular diagnostic tests are complementary and not a replacement for culture methods.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 2], [Figure 1]
[Table 1], [Table 2], [Table 3]