Pleural and peritoneal infections cause substantial morbidity and mortality. Traditional diagnostic methods rely on the cultivation of clinical samples, which usually takes days to obtain report and holds a low detection sensitivity. In this study, we evaluated a 5-fluorescent-channel droplet digital PCR (ddPCR) system and 5 assay panels for culture-independent rapid pathogen detections directly from pleural and peritoneal fluid samples. Traditional culture of the same sample was used as reference. A total of 40 pleural fluid samples and 19 peritoneal fluid samples were tested in this study. Twenty-five positives including 4 polymicrobial infections by culture and 26 positives including 11 polymicrobial infections by ddPCR were detected for pleural fluid samples; 14 positives including 2 polymicrobial infections by culture and 15 positives including 3 polymicrobial infections by ddPCR were detected for peritoneal fluid samples. Klebsiella pneumoniae was the most common bacterium detected both in pleural and in peritoneal fluid samples. The sensitivity of the ddPCR assay for pleural and peritoneal fluid samples was 96% (95% confidence interval (CI) = 79.65 to 99.90%) and 92.86% (95% CI = 66.13 to 99.82%), respectively. The turnaround time of the ddPCR assay was approximately 3 h comparing with 38.30 ± 22.44 h for culture-based identifications. Our results demonstrated that the ddPCR assay is a rapid and sensitive method for identifying pathogens responsible for pleural and peritoneal infections and would be a promising approach for early diagnosis and optimizing treatment of infections.

College of Medical Technology Zhejiang Chinese Medical University Hangzhou 310053 Zhejiang People's Republic of China

Department of Clinical Laboratory The 1st Affiliated Hospital of Zhejiang Chinese Medical University 54 Youdian Road Hangzhou 310006 Zhejiang People's Republic of China

The 1st Clinical Medical College of Zhejiang Chinese Medical University Hangzhou 310053 Zhejiang People's Republic of China

Zobrazit více v PubMed

Amin M, Yousef Pour S, Navidifar T. Detection of the major bacterial pathogens among children suffering from empyema in Ahvaz city, Iran. J Clin Lab Anal. 2019;33:e22855. doi: 10.1002/jcla.22855. PubMed DOI PMC

Bianchi DW, Parker RL, Wentworth J, Madankumar R, Saffer C, Das AF, Craig JA, Chudova DI, Devers PL, Jones KW, Oliver K, Rava RP, Sehnert AJ. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med. 2014;370:799–808. doi: 10.1056/NEJMoa1311037. PubMed DOI

Brogden KA, Guthmiller JM, editors. Polymicrobial diseases. Washington (DC): ASM Press; 2002. PubMed

Click ES, Murithi W, Ouma GS, McCarthy K, Willby M, Musau S, Alexander H, Pevzner E, Posey J, Cain KP. Detection of apparent cell-free M. tuberculosis DNA from plasma. Sci Rep. 2018;8:645. doi: 10.1038/s41598-017-17683-6. PubMed DOI PMC

Corcoran JP, Wrightson JM, Belcher E, DeCamp MM, Feller-Kopman D, Rahman NM. Pleural infection: past, present, and future directions. Lancet Respir Med. 2015;3:563–577. doi: 10.1016/S2213-2600(15)00185-X. PubMed DOI

Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A. Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol. 2013;10:472–484. doi: 10.1038/nrclinonc.2013.110. PubMed DOI

Elshimali YI, Khaddour H, Sarkissyan M, Wu Y, Vadgama JV. The clinical utilization of circulating cell free DNA (CCFDNA) in blood of cancer patients. Int J Mol Sci. 2013;14:18925–18958. doi: 10.3390/ijms140918925. PubMed DOI PMC

Franchetti L, Schumann D, Tamm M, Jahn K, Stolz D. Efficacy of a multiplex bacterial PCR in identifying pathogens in pleural empyema. Eur Respir J. 2018;52(suppl 62):PA1966. doi: 10.1183/13993003.congress-2018.PA1966. DOI

Grumaz S, Stevens P, Grumaz C, Decker SO, Weigand MA, Hofer S, Brenner T, von Haeseler A, Sohn K. Next-generation sequencing diagnostics of bacteremia in septic patients. Genome Med. 2016;8:73. doi: 10.1186/s13073-016-0326-8. PubMed DOI PMC

Hardick J, Won H, Jeng K, Hsieh Y-H, Gaydos CA, Rothman RE, Yang S. Identification of bacterial pathogens in ascitic fluids from patients with suspected spontaneous bacterial peritonitis by use of broad-range PCR (16S PCR) coupled with high-resolution melt analysis. J Clin Microbiol. 2012;50:2428–2432. doi: 10.1128/jcm.00345-12. PubMed DOI PMC

Hughes JA, Cabilan CJ, Williams J, Ray M, Coyer F. The effectiveness of interventions to reduce peripheral blood culture contamination in acute care: a systematic review protocol. Syst Rev. 2018;7:216. doi: 10.1186/s13643-018-0877-4. PubMed DOI PMC

Jiménez D, Díaz G, García-Rull S, Vidal R, Sueiro A, Light RW. Routine use of pleural fluid cultures. Are they indicated? Limited yield, minimal impact on treatment decisions. Respir Med. 2006;100:2048–2052. doi: 10.1016/j.rmed.2006.02.008. PubMed DOI

Li X, Du W, Wang Y, Liu Z, Li K, Chen H, Liu R, Ma L, Zhang L, Dong Y, Che N, Gao M. Rapid diagnosis of tuberculosis meningitis by detecting Mycobacterium tuberculosis cell-free DNA in cerebrospinal fluid. Am J Clin Pathol. 2019;153:126–130. doi: 10.1093/ajcp/aqz135. PubMed DOI

Maskell NA, Batt S, Hedley EL, Davies CWH, Gillespie SH, Davies RJO. The bacteriology of pleural infection by genetic and standard methods and its mortality significance. Am J Respir Crit Care Med. 2006;174:817–823. doi: 10.1164/rccm.200601-074OC. PubMed DOI

Menzies SM, Rahman NM, Wrightson JM, Davies HE, Shorten R, Gillespie SH, Davies CWH, Maskell NA, Jeffrey AA, Lee YCG, Davies RJO. Blood culture bottle culture of pleural fluid in pleural infection. Thorax. 2011;66:658–662. doi: 10.1136/thx.2010.157842. PubMed DOI

Picazo JJ, Contreras JR, Ríos E, Culebras E, Rodríguez-Avial I, Méndez C, Betriu C. Rapid diagnosis of invasive pneumococcal disease in pediatric population. J Microbiol Methods. 2013;93:116–120. doi: 10.1016/j.mimet.2013.03.001. PubMed DOI

She RC, Romney MG, Jang W, Walker T, Karichu JK, Richter SS. Performance of the BacT/Alert Virtuo Microbial Detection System for the culture of sterile body fluids: prospective multicentre study. Clin Microbiol Infect. 2018;24:992–996. doi: 10.1016/j.cmi.2017.12.011. PubMed DOI

Suo T, Liu X, Feng J, Guo M, Hu W, Guo D, Ullah H, Yang Y, Zhang Q, Wang X, Sajid M, Huang Z, Deng L, Chen T, Liu F, Xu K, Liu Y, Zhang Q, Liu Y, Xiong Y, Chen G, Lan K, Chen Y. ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens. Emerg Microbes Infect. 2020;9:1259–1268. doi: 10.1080/22221751.2020.1772678. PubMed DOI PMC

Ushio R, Yamamoto M, Nakashima K, Watanabe H, Nagai K, Shibata Y, Tashiro K, Tsukahara T, Nagakura H, Horita N, Sato T, Shinkai M, Kudo M, Ueda A, Kaneko T. Digital PCR assay detection of circulating Mycobacterium tuberculosis DNA in pulmonary tuberculosis patient plasma. Tuberculosis (Edinb) 2016;99:47–53. doi: 10.1016/j.tube.2016.04.004. PubMed DOI

Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A. 1999;96:9236–9241. doi: 10.1073/pnas.96.16.9236. PubMed DOI PMC

Wan JCM, Massie C, Garcia-Corbacho J, Mouliere F, Brenton JD, Caldas C, Pacey S, Baird R, Rosenfeld N. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017;17:223–238. doi: 10.1038/nrc.2017.7. PubMed DOI

Wang D, Hu Y, Li T, Rong H-M, Tong Z-H. Diagnosis of Pneumocystis jirovecii pneumonia with serum cell-free DNA in non-HIV-infected immunocompromised patients. Oncotarget. 2017;8:71946–71953. doi: 10.18632/oncotarget.18037. PubMed DOI PMC

Weerakoon KG, McManus DP. Cell-free DNA as a diagnostic tool for human parasitic infections. Trends Parasitol. 2016;32:378–391. doi: 10.1016/j.pt.2016.01.006. PubMed DOI

White PL, Barnes RA, Springer J, Klingspor L, Cuenca-Estrella M, Morton CO, Lagrou K, Bretagne S, Melchers WJG, Mengoli C, Donnelly JP, Heinz WJ, Loeffler J. Clinical performance of Aspergillus PCR for testing serum and plasma: a study by the European Aspergillus PCR Initiative. J Clin Microbiol. 2015;53:2832–2837. doi: 10.1128/jcm.00905-15. PubMed DOI PMC

Wouters Y, Dalloyaux D, Christenhusz A, Roelofs HMJ, Wertheim HF, Bleeker-Rovers CP, Te Morsche RH, Wanten GJA. Droplet digital polymerase chain reaction for rapid broad-spectrum detection of bloodstream infections. Microb Biotechnol. 2019;13:657–668. doi: 10.1111/1751-7915.13491. PubMed DOI PMC

Wu YD, Li W, Wei Y, Gao HH, Shang SQ, Du LZ. Rapid and sensitive identification of bacterial infection and bacteria gram types in pleural fluid of children. Glob Pediatr Health. 2015;2:2333794X15569302. doi: 10.1177/2333794x15569302. PubMed DOI PMC