A diagnostic test of real-time PCR detection in the diagnosis of clinical bloodstream infection
Introduction
Bloodstream infection is a common clinical emergency. If the diagnosis and treatment are not timely, it can seriously threaten the life of patients, and the fatality rate is extremely high (1). In recent years, the incidence of bloodstream infections has shown a significant upward trend due to the application of various antibiotics, immunosuppressants, surgical operations, and other invasive procedures and interventional treatment methods (2,3). Currently, the diagnosis of bloodstream infection mainly relies on traditional blood culture identification results. However, it takes 2–3 days or even longer from the positive blood culture of the specimen to the identification of the pathogenic bacteria in the laboratory, which seriously lags behind clinical needs (4). Furthermore, because the pathogenic bacteria cannot be identified, doctors can only use broad-spectrum antibiotics covering gram-positive cocci and gram-negative bacilli for treatment in advance, which dramatically increases the physical and economic burden of patients but also leads to bacterial resistance. In addition, an increasing number of multi-drug-resistant and pan-drug-resistant strains continue to emerge. Therefore, obtaining laboratory diagnostic results as soon as possible is particularly important for the treatment and prognosis of patients. At the same time, it can shorten the use time of clinical broad-spectrum antibiotics as soon as possible and reduce the generation of drug-resistant strains.
In recent years, real-time polymerase chain reaction (PCR) has been widely used in various molecular experiments. With the continuous development and innovation of current medical technology, real-time PCR is rapidly replacing traditional microbial detection methods (5). Real-time PCR can complete the PCR reaction and product analysis process in a reaction tube at one time. It can automatically conduct dynamic monitoring and data analysis, significantly reducing cross-infection incidence. In addition, the operation method of real-time PCR is relatively simple, the operation speed is fast, the quantification is reasonably accurate, the sensitivity is high, the operation can be repeated, and the application value is relatively high (6). Multiplex PCR is based on single real-time PCR, adding 2 or more pairs of primers to the same reaction system to amplify multiple product fragments simultaneously, fully reflecting the advantages of multiplex PCR with high efficiency and low cost (7). Multiplex PCR also has the high sensitivity and high specificity of single PCR. These advantages demonstrate that multiplex PCR is suitable for application in clinical experiments, simplifying and shortening the procedure and the time of detection processes, and providing a reference for clinical diagnosis and treatment (8,9).
In recent years, with molecular detection technology, PCR has been developed to detect DNA in the bloodstream. With the PCR detection, Selva et al. found that the sensitivity and specificity of Streptococcus pneumoniae were 94.0% and 98.4% in 128 blood culture vials (10). Arabestani et al. used multiplex PCR to identify Staphylococcus aureus, coagulase-negative Staphylococcus, Enterococcus, Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii in blood culture-positive specimens, which took significantly less time than traditional blood culture identification (11). However, Kang et al. showed that the overall sensitivity of multiplex PCR in detecting BSI pathogens could be as high as 94.6%, and the sensitivity in multiplex infection is reduced to 78.6%. Therefore, this technology is considered prone to false positive results in the mecA gene detection of Staphylococcus aureus and coagulase-negative Staphylococcus (12). Therefore, more samples are needed to study the clinical application value of PCR detection in bloodstream infection pathogens.
In this study, multiplex PCR was performed to detect pathogens in patients with bloodstream infections. This method was compared with conventional blood culture to evaluate its clinical application value. We present the following article in accordance with the STARD reporting checklist (available at https://apm.amegroups.com/article/view/10.21037/apm-22-1071/rc).
Methods
Study cohort and patients
The study included 126 cases suspected of infection detected by the microbiology laboratory of The First Hospital of Hebei Medical University laboratory department from April 2022 to June 2022 (Figure S1). There were 77 males and 49 females, with ages ranging from 18 to 98 (median age =60.5) (Table S1). The collection of all blood samples was carried out under strict relevant standards. Adults used double sets of bottles, and each set included aerobic and anaerobic bottles to ensure that the blood collection volume of each bottle was 8–10 mL. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the ethics committee of the First Hospital of Hebei Medical University (No. 20220816), and the patients and their families knew and gave informed consent.
Sample pathogen detection
The patient's sample was divided into 2 parts; one for blood culture and the other for multiplex PCR detection. The fully automatic blood culture system from Autobio company was used for blood culture (product model/item number: BC120). All samples were homogenized for nucleic acid extraction using the Zymo BIOMICS DNA/RNA Miniprep kit (Zymo R2002) according to the manufacturer’s instructions. Then, PCR was performed using the Pathogeno Elite Multiplex PCR kit (SJ0101, Shanghai Pathogeno Medical Technology Co., Ltd.) and the reaction system was configured strictly following the operating test instructions. The operation method of PCR amplification was as follows: 95 ℃ ×5 min, 1 cycle; 95 ℃ × 10 s, 35 cycles; fluorescence detection was controlled at 60 ℃, 30 s. The selected fluorescence channels were FAM, VIC, Cy5, and T0exas Red. The reaction volume was 10 µL. The final reaction system was detected using the ABI7500 instrument from Life Tech Company (Applied Biosystems). All blood culture bottles were under routine procedures.
Statistical analysis
Using blood culture as the gold standard reference method, PCR detection was used for diagnostic tests. The cases were counted as the number of cases or percentage. The number of positive results for each test in paired samples was compared in a 2-by-2 contingency table. The data were statistically analyzed using SPSS 22.0 (IBM Corp., USA) or Microsoft Excel to calculate the detection rate, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).
Results
Distribution of pathogenic bacteria in patient specimens detected by PCR
Blood samples from 126 patients (age: 18–98) were collected, including 63 samples from patients under 60 years and 63 samples from patients over 60 years. Among the pathogens detected by PCR, the overall number of pathogen-positive items was 65, and the top 5 pathogens with a higher positive rate were Epstein-Barr virus (27 cases), Human herpes virus 5 (9 cases), Klebsiella pneumoniae (5 cases), Staphylococcus (5 cases), and Stenotrophomonas maltophilia (4 cases). Among the pathogens detected by blood culture, the total number of pathogen-positive items was 7, including Klebsiella pneumoniae (4 cases), Acinetobacter baumannii (2 cases), and Escherichia coli (1 case) (Table 1). Among patients aged 60 and below, the top 3 detected items were Epstein-Barr virus (10 cases), Staphylococcus (4 cases), and Human herpes virus 5 (4 cases). Among patients over 60 years old, the top 3 detected items were Epstein-Barr virus (17 cases), Human herpes virus 5 (5 cases), and Klebsiella pneumoniae (4 cases) (Table 2).
Table 1
| Pathogen | PCR | Blood culture | |||
|---|---|---|---|---|---|
| No. | % | No. | % | ||
| Negative | 83 | 65.9% | 119 | 94.4% | |
| Epstein-Barr virus | 27 | 21.4% | 0 | 3.2% | |
| Klebsiella pneumoniae | 5 | 4.0% | 4 | 3.2% | |
| Escherichia coli | 2 | 1.6% | 1 | 0.8% | |
| Acinetobacter baumannii | 1 | 0.8% | 2 | 1.6% | |
| Stenotrophomonas maltophilia | 4 | 3.2% | 0 | 0.0% | |
| Pseudomonas aeruginosa | 2 | 1.6% | 0 | 0.0% | |
| herpesvirus 1 | 1 | 0.8% | 0 | 0.0% | |
| Klebsiella oxytoca | 1 | 0.8% | 0 | 0.0% | |
| Staphylococcus | 5 | 4.0% | 0 | 0.0% | |
| Human herpesvirus 5 | 9 | 7.1% | 0 | 0.0% | |
| VIM | 1 | 0.8% | 0 | 0.0% | |
| Rickettsia felis | 1 | 0.8% | 0 | 0.0% | |
| Enterococcus faecium | 1 | 0.8% | 0 | 0.0% | |
| ampC | 1 | 0.8% | 0 | 0.0% | |
| Mycobacterium tuberculosis complex | 2 | 1.6% | 0 | 0.0% | |
| KPC | 1 | 0.8% | 0 | 0.0% | |
| Human metapneumovirus | 1 | 0.8% | 0 | 0.0% | |
PCR, polymerase chain reaction.
Table 2
| Pathogen | No. of positive | |||
|---|---|---|---|---|
| Age 18–60 | Age >60 | Total | Percent | |
| Epstein-Barr virus | 10 | 17 | 27 | 41.54% |
| Klebsiella pneumoniae | 1 | 4 | 5 | 7.69% |
| Escherichia coli | 1 | 1 | 2 | 3.08% |
| Acinetobacter baumannii | 1 | 0 | 1 | 1.54% |
| Stenotrophomonas maltophilia | 2 | 2 | 4 | 6.15% |
| Pseudomonas aeruginosa | 0 | 2 | 2 | 3.08% |
| herpesvirus 1 | 0 | 1 | 1 | 1.54% |
| Klebsiella oxytoca | 0 | 1 | 1 | 1.54% |
| Staphylococcus | 4 | 1 | 5 | 7.69% |
| Human herpes virus 5 | 4 | 5 | 9 | 13.85% |
| VIM | 1 | 0 | 1 | 1.54% |
| Rickettsia felis | 1 | 0 | 1 | 1.54% |
| Enterococcus faecium | 1 | 0 | 1 | 1.54% |
| ampC | 0 | 1 | 1 | 1.54% |
| Mycobacterium tuberculosis complex | 1 | 1 | 2 | 3.08% |
| KPC | 0 | 1 | 1 | 1.54% |
| Human metapneumo virus | 1 | 0 | 1 | 1.54% |
| Total | 28 | 37 | 65 | |
PCR, polymerase chain reaction.
PCR detection of mixed infection of pathogens in patients
Of the 126 samples tested by PCR, 83 were positive, with a positive detection rate of 65.9% (83/126). Among them, 30 cases were infected with 1 pathogen, 8 were infected with 2 pathogens, 5 were infected with 3 pathogens, and 1 was infected with 4 pathogens. Among the patients with double and multiple infections, the proportion of co-infection with Epstein-Barr virus was higher, accounting for 75% (6/8) of patients with double infection and 60% (3/5) of patients with triple infection. One case of quadruple infection was also co-infected with Epstein-Barr virus (Table 3).
Table 3
| Pathogen | Patients number of Infection species | |||
|---|---|---|---|---|
| Single species (n=30) | Double species (n=8) | Triple species (n=5) | Quadruple species (n=1) | |
| Epstein-Barr virus | 19 | 4 | 3 | 1 |
| Klebsiella_pneumoniae | 1 | 2 | 2 | 0 |
| Escherichia coli | 1 | 0 | 1 | 0 |
| Acinetobacter baumannii | 0 | 0 | 1 | 0 |
| Stenotrophomonas maltophilia | 1 | 0 | 2 | 1 |
| Pseudomonas aeruginosa | 1 | 0 | 1 | 0 |
| herpesvirus 1 | 0 | 1 | 0 | 0 |
| Klebsiella oxytoca | 0 | 1 | 0 | 0 |
| Staphylococcus | 3 | 2 | 0 | 0 |
| Human herpes virus 5 | 1 | 4 | 3 | 1 |
| VIM | 1 | 0 | 0 | 0 |
| Rickettsia felis | 1 | 0 | 0 | 0 |
| Enterococcus faecium | 0 | 0 | 0 | 1 |
| ampC | 0 | 0 | 1 | 0 |
| Mycobacterium tuberculosis complex | 0 | 1 | 1 | 0 |
| KPC | 0 | 1 | 0 | 0 |
| Human metapneumo virus | 1 | 0 | 0 | 0 |
PCR, polymerase chain reaction.
Differences in pathogen detection performance between PCR and blood culture
Of the 126 blood samples, both methods tested negative in 81 cases and positive in 5 cases. Two specimens were positive for blood culture but negative for PCR. A total of 38 specimens were negative for blood culture and positive for PCR. The NPV of PCR and blood culture was 97.6% (81/83), the PPV was 11.6% (5/43), the sensitivity was 71.4% (5/7), the specificity was 68.1% (81/119), and the accuracy result was 68.3% (86/126). Among the 30.2% (38/126) of samples with positive PCR and negative blood culture, 66 items were specifically detected (Table 4).
Table 4
| Blood culture | Sensitivity | Specificity | Accuracy | PPV | NPV | ||||
|---|---|---|---|---|---|---|---|---|---|
| Positive | Negative | Total | |||||||
| PCR | Positive | 5 | 38 | 43 | 0.71 | 0.68 | 0.68 | ||
| Negative | 2 | 81 | 83 | 0.12 | 0.98 | ||||
| Total | 7 | 119 | 126 | ||||||
PPV, positive prediction value; NPV, negative prediction value; PCR, polymerase chain reaction.
Discussion
Blood culture is a classic method for detecting pathogenic microorganisms in blood, and it is still the gold standard for isolating and identifying pathogenic microorganisms in bloodstream infections (13). However, blood culture also has some shortcomings determined by the characteristics of the method itself, which restrict the speed and sensitivity of its detection (14). For example, it takes a certain amount of time for bacteria to divide and multiply to detectable levels. This speed is related to the bacterial species. Some bacteria grow slowly, and fastidious bacteria do not even grow, making blood cultures slow. The sensitivity for detecting neonatal bloodstream infections is low due to the small amount of blood used for detection and the low bacterial counts in the blood. In addition, blood culture has certain challenges including that the positive rate of blood cultures decreases significantly after antibiotic treatment, blood cultures can only detect viable microorganisms, and antibiotic treatment kills bacteria and reduces the detection rate (15,16). The above factors considerably reduce the speed and sensitivity of blood culture in diagnosing bloodstream infections. Therefore, clinical diagnosis and treatment urgently need a sensitive method to quickly and accurately identify pathogenic microorganisms in bloodstream infections.
PCR is a molecular biology technique used to amplify a specific DNA fragment, which can be regarded as DNA replication outside the organism. With the continuous development and innovation of current medical technology, PCR is rapidly replacing traditional microbial detection methods (17,18). This technology can complete the PCR reaction and product analysis process in one reaction tube. Conducting dynamic monitoring and analysis of corresponding data reduces the incidence of cross-infection. In addition, the operation method of fluorescent PCR technology is relatively simple, the operation speed is fast, the quantification is reasonably accurate, the sensitivity is high, the operation can be repeated, and the application value is high (19). Selva et al. used PCR to detect Streptococcus pneumoniae in 128 blood culture flasks and obtained 94.0% specificity and 98.4% specificity (10). Arabestani et al. used multiplex PCR to identify Staphylococcus aureus, coagulase-negative staphylococcus, Enterococcus, Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii in blood culture-positive specimens, and the time spent was significantly less than traditional blood culture identification (11). In another study of 285 patients with pyrexia, the positive rate of PCR detection was 17.2%, which was higher than the positive rate of ordinary blood culture. In this study, of the 126 samples, the positive detection rate of PCR detection was 65.9% (83/126) (20).
This study compared blood culture with PCR to detect pathogenic microorganisms in bloodstream infections. The PCR detection was positive, while blood culture-negative samples accounted for 30.2%. Similar detection by Lucignano et al. showed that the rate of SeptiFast detection was 36.3% (21). A study showed that the detection rate was only 19.4% (22). The results of previous study showed that the sensitivity range of PCR was 66–85%, and the specificity was 87–93% compared to blood culture (21,22). This study’s sensitivity is 71.4%, but the specificity is relatively low (68.1%). This study is a single-center observational study, which may limit the generalizability of the findings.
The PCR is positive when the corresponding pathogen is also detected in other body parts. At the same time, a negative blood culture specimen reflects the accurate result to a certain extent. Whether there is an association between bacterial DNA in the blood and the development of systemic inflammatory response syndrome and sepsis remains controversial. Some studies have found that the appearance of cell-free DNA in the blood is a risk factor for evaluating multiple organ failure in intensive care unit (ICU) patients (23,24). Therefore, PCR can not only be used to detect unknown pathogens or their DNA, but also provide valuable clinical information. This is difficult to achieve with conventional blood cultures.
Conclusions
In summary, PCR detection can rapidly and sensitively detect pathogens in patients with bloodstream infections, even if the patient has been on antibiotics. However, unlike blood cultures, PCR assays do not provide information on the resistance of infectious pathogens. Therefore, the extensive use of PCR detection combined with blood culture detection can offer an immediate treatment plan for clinical patients, reduce the generation of drug-resistant bacteria, and facilitate the recovery of patients.
Acknowledgments
Funding: This study was supported by Government-funded clinical medicine outstanding talent training project (No. LS202108).
Footnote
Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://apm.amegroups.com/article/view/10.21037/apm-22-1071/rc
Data Sharing Statement: Available at https://apm.amegroups.com/article/view/10.21037/apm-22-1071/dss
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://apm.amegroups.com/article/view/10.21037/apm-22-1071/coif). YZ and RB are from Bingyuan Medical Technology Co., LTD., Shanghai, China. They performed the data analysis and interpretation in this study, and have no financial support from their company. The other authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the ethics committee of the First Hospital of Hebei Medical University (No. 20220816), and the patients and their families knew and gave informed consent.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
References
- Karunakaran R, Raja NS, Quek KF, et al. Evaluation of the routine use of the anaerobic bottle when using the BACTEC blood culture system. J Microbiol Immunol Infect 2007;40:445-9. [PubMed]
- Baker AW, Durkin MJ, Dicks KV, et al. Methicillin-resistant Staphylococcus aureus bloodstream infection surveillance: National Healthcare Safety Network's laboratory-identified event reporting versus traditional laboratory-confirmed bloodstream infection surveillance. Infect Control Hosp Epidemiol 2014;35:1286-9. [Crossref] [PubMed]
- Kawamura H, Takahashi N, Takahashi M, et al. Differences in microorganism growth on various dressings used to cover injection sites: inspection of the risk of catheter-related bloodstream infections caused by Gram-negative bacilli. Surg Today 2014;44:2339-44. [Crossref] [PubMed]
- Book M, Lehmann LE, Zhang X, et al. Monitoring infection: from blood culture to polymerase chain reaction (PCR). Best Pract Res Clin Anaesthesiol 2013;27:279-88. [Crossref] [PubMed]
- Warhurst G, Dunn G, Chadwick P, et al. Rapid detection of health-care-associated bloodstream infection in critical care using multipathogen real-time polymerase chain reaction technology: a diagnostic accuracy study and systematic review. Health Technol Assess 2015;19:1-142. [Crossref] [PubMed]
- Cendejas-Bueno E, Romero-Gómez MP, Mingorance J. The challenge of molecular diagnosis of bloodstream infections. World J Microbiol Biotechnol 2019;35:65. [Crossref] [PubMed]
- Zhang H, Morrison S, Tang YW. Multiplex polymerase chain reaction tests for detection of pathogens associated with gastroenteritis. Clin Lab Med 2015;35:461-86. [Crossref] [PubMed]
- Caliendo AM. Multiplex PCR and emerging technologies for the detection of respiratory pathogens. Clin Infect Dis 2011;52:S326-30. [Crossref] [PubMed]
- Liu CF, Shi XP, Chen Y, et al. Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR. J Clin Lab Anal 2018; [Crossref] [PubMed]
- Selva L, Esteva C, Gené A, et al. Direct detection of Streptococcus pneumoniae in positive blood cultures by real-time polymerase chain reaction. Diagn Microbiol Infect Dis 2010;66:204-6. [Crossref] [PubMed]
- Arabestani MR, Fazzeli H, Nasr Esfahani B. Identification of the most common pathogenic bacteria in patients with suspected sepsis by multiplex PCR. J Infect Dev Ctries 2014;8:461-8. [Crossref] [PubMed]
- Kang CM, Chen XJ, Chih CC, et al. Rapid identification of bloodstream bacterial and fungal pathogens and their antibiotic resistance determinants from positively flagged blood cultures using the BioFire FilmArray blood culture identification panel. J Microbiol Immunol Infect 2020;53:882-91. [Crossref] [PubMed]
- Peters RP, van Agtmael MA, Danner SA, et al. New developments in the diagnosis of bloodstream infections. Lancet Infect Dis 2004;4:751-60. [Crossref] [PubMed]
- Briggs N, Campbell S, Gupta S. Advances in rapid diagnostics for bloodstream infections. Diagn Microbiol Infect Dis 2021;99:115219. [Crossref] [PubMed]
- Peri AM, Harris PNA, Paterson DL. Culture-independent detection systems for bloodstream infection. Clin Microbiol Infect 2022;28:195-201. [Crossref] [PubMed]
- Mwaigwisya S, Assiri RA, O'Grady J. Emerging commercial molecular tests for the diagnosis of bloodstream infection. Expert Rev Mol Diagn 2015;15:681-92. [Crossref] [PubMed]
- Bustin SA, Benes V, Nolan T, et al. Quantitative real-time RT-PCR--a perspective. J Mol Endocrinol 2005;34:597-601. [Crossref] [PubMed]
- Kubista M, Andrade JM, Bengtsson M, et al. The real-time polymerase chain reaction. Mol Aspects Med 2006;27:95-125. [Crossref] [PubMed]
- Navarro E, Serrano-Heras G, Castaño MJ, et al. Real-time PCR detection chemistry. Clin Chim Acta 2015;439:231-50. [Crossref] [PubMed]
- Leli C, Cardaccia A, D'Alò F, et al. A prediction model for real-time PCR results in blood samples from febrile patients with suspected sepsis. J Med Microbiol 2014;63:649-58. [Crossref] [PubMed]
- Lucignano B, Ranno S, Liesenfeld O, et al. Multiplex PCR allows rapid and accurate diagnosis of bloodstream infections in newborns and children with suspected sepsis. J Clin Microbiol 2011;49:2252-8. [Crossref] [PubMed]
- Reier-Nilsen T, Farstad T, Nakstad B, et al. Comparison of broad range 16S rDNA PCR and conventional blood culture for diagnosis of sepsis in the newborn: a case control study. BMC Pediatr 2009;9:5. [Crossref] [PubMed]
- Maubon D, Hamidfar-Roy R, Courby S, et al. Therapeutic impact and diagnostic performance of multiplex PCR in patients with malignancies and suspected sepsis. J Infect 2010;61:335-42. [Crossref] [PubMed]
- Cheng Z, Abrams ST, Toh J, et al. The Critical Roles and Mechanisms of Immune Cell Death in Sepsis. Front Immunol 2020;11:1918. [Crossref] [PubMed]
(English Language Editor: C. Betlazar-Maseh)