Assessment of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Pathogenic Bacteria from Respiratory Samples in Patients with Hospital-Acquired Pneumonia
Abstract
:1. Introduction
2. Material and Methods
2.1. Study Design
2.2. Collection of Samples
2.3. Routine Microbiological Methods
2.4. Extraction of DNA
2.5. LAMP Protocol
2.6. Determination of the Limits of Detection
2.7. Statistical Analysis
2.8. Ethical Aspects
3. Results
3.1. BAL Samples
3.2. BAS/EA Samples
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
- Kohbodi, G.N.A.; Noor, A. Pneumonia, Ventilator-Associated. [Updated 2018 November 18]. In StatPearls [Internet]; StatPearls Publishing: Treasure Island, FL, USA, January 2019. Available online: https://www.ncbi.nlm.nih.gov/books/NBK507711/ (accessed on 14 January 2019).
- Li Bassi, G.; Ferrer, M.; Marti, J.D.; Comaru, T.; Torres, A. Ventilator-Associated Pneumonia. Semin. Respir. Crit. Care Med. 2014, 35, 469–481. [Google Scholar]
- Torres, A.; Niederman, M.S.; Chastre, J.; Ewig, S.; Fernandez-Vandellos, P.; Hanberger, H.; Marin, K.; Li Bassi, G.; Luna, C.M.; Martin-Loeches, I.; et al. International ERS/ESICM/ESCMID/ALAT guidelines for the management of hospital-acquired pneumonia and ventilator-associated pneumonia: Guidelines for the management of hospital-acquired pneumonia (HAP)/ventilator-associated pneumonia (VAP) of the European Respiratory Society (ERS), European Society of Intensive Care Medicine (ESICM), European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and Asociación Latinoamericana del Tórax (ALAT). Eur. Respir. J. 2017, 50, 1700582. [Google Scholar] [PubMed]
- Aykac, K.; Ozsurekci, Y.; Basaranoglu, S.T. Future Directions and Molecular Basis of Ventilator Associated Pneumonia. Can. Respir. J. 2017, 2017, 2614602. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chi, S.Y.; Kim, T.O.; Park, C.W.; Yu, J.Y.; Lee, B.; Lee, H.S.; Kim, Y.I.I.; Lim, S.C.; Kwon, Y.S. Bacterial pathogens of ventilator associated pneumonia in a tertiary referral hospital. Tuberc. Respir. Dis. 2012, 73, 32–37. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Injac, V.; Batranovic, U.; Matijasevic, J.; Vukoja, M.; Hadnadjev, M.; Bukumiric, Z.; Trajkovic, G.; Jankovic, S. Etiology and resistance patterns of bacteria causing ventilator-associated pneumonia in a respiratory intensive care unit. Vojn. Pregl. 2017, 74, 954–962. [Google Scholar] [CrossRef]
- Ferrer, M.; Difrancesco, L.F.; Liapikou, A.; Rinaudo, M.; Carbonara, M.; Li Bassi, G.; Gabarrus, A.; Torres, A. Polymicrobial intensive care unit-acquired pneumonia: Prevalence, microbiology and outcome. Crit. Care 2015, 19, 450. [Google Scholar] [CrossRef] [Green Version]
- Bouza, E.; Giannella, M.; Torres, M.V.; Catalán, P.; Sánchez-Carrillo, C.; Hernandez, R.I.; Muñoz, P.; Marañón, G. Task Force for Pneumonia. Herpes simplex virus: A marker of severity in bacterial ventilator-associated pneumonia. J. Crit. Care 2011, 26, 432.e1–432.e6. [Google Scholar] [CrossRef]
- Osman, N.M.; Sayed, N.M.; Abdel-Rahman, S.M.; Hamza, S.A.; Abd al Aziz, A.A. The impact of cytomegalovirus infection on mechanically ventilated patients in the respiratory and geriatric intensive care units. Egypt J. Chest Dis. Tuberc. 2014, 63, 239–245. [Google Scholar] [CrossRef] [Green Version]
- Klompas, M. Does this patient have ventilator-associated pneumonia? JAMA 2007, 297, 1583–1593. [Google Scholar] [CrossRef]
- Kalanuria, A.A.; Zai, W.; Mirski, M. Ventilator-associated pneumonia in the ICU. Crit. Care 2014, 18, 208. [Google Scholar] [CrossRef] [Green Version]
- Herkel, T.; Uvizl, R.; Doubravska, L.; Adamus, M.; Gabrhelik, T.; Htoutou Sedlakova, M.; Kolarc, M.; Hanulikc, V.; Pudovac, V.; Langovad, K.; et al. Epidemiology of hospital-acquired pneumonia: Results of a Central European multicenter, prospective, observational study compared with data from the European region. Biomed. Pap. 2016, 160, 448–455. [Google Scholar] [CrossRef] [PubMed]
- Roberts, K.L.; Micek, S.T.; Juang, P.; Kollef, M.H. Controversies and advances in the management of ventilator associated pneumonia. Expert Rev. Respir. Med. 2017, 11, 875–884. [Google Scholar] [CrossRef] [PubMed]
- Millot, G.; Voisin, B.; Loiez, C.; Wallet, F.; Nseir, S. The next generation of rapid point-of-care testing identification tools for ventilator-associated pneumonia. Ann. Transl. Med. 2017, 5, 451. [Google Scholar] [CrossRef] [Green Version]
- Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe, K.; Amino, N.; Hase, T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000, 28, E63. [Google Scholar] [CrossRef] [Green Version]
- Dhama, K.; Karthik, K.; Chakrabort, S.; Tiwari, R.; Kapoor, S.; Kumar, A.; Thomas, P. Loop-mediated Isothermal Amplification of DNA (LAMP): A New Diagnostic Tool Lights the World of Diagnosis of Animal and Human Pathogens: A Review. Pak. J. Biol. Sci. 2014, 17, 151–166. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kang, Y.; Deng, R.; Wang, C.; Deng, T.; Peng, P.; Cheng, X.; Wang, G.; Qian, M.; Gao, H.; Han, B.; et al. Etiologic Diagnosis of Lower Respiratory Tract Bacterial Infections Using Sputum Samples and Quantitative Loop-Mediated Isothermal Amplification. PLoS ONE 2012, 7, e38743. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Petrone, B.L.; Wolff, B.J.; DeLaney, A.A.; Diaz, M.H.; Winchell, J.M. Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens. J. Clin. Microbiol. 2015, 53, 2970–2976. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Altman, D.G.; Bland, J.M. Diagnostic tests. 1: Sensitivity and specificity. BMJ 1994, 308, 1552. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Altman, D.G.; Bland, J.M. Diagnostic tests 2: Predictive values. BMJ 1994, 309, 102. [Google Scholar] [CrossRef] [Green Version]
- Li Bassi, G.; Senussi, T.; Aguilera, E. Prevention of ventilator-associated pneumonia. Curr. Opin. Infect. Dis. 2017, 30, 214–220. [Google Scholar] [CrossRef]
- Torres, A.; Lee, N.; Cilloniz, C.; Vila, J.; Van der Eerden, M. Laboratory diagnosis of pneumonia in the molecular age. Eur. Respir. J. 2016, 48, 1764–1778. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Jamal, W.; Al Roomi, E.; AbdulAziz, L.R.; Rotimi, V.O. Evaluation of Curetis Unyvero, a Multiplex PCR-Based Testing System, for Rapid Detection of Bacteria and Antibiotic Resistance and Impact of the Assay on Management of Severe Nosocomial Pneumonia. J. Clin. Microbiol. 2014, 52, 2487–2492. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kunze, N.; Moerer, O.; Steinmetz, N.; Schulze, M.H.; Quintel, M.; Perl, T. Point-of-care multiplex PCR promises short turnaround times for microbial testing in hospital-acquired pneumonia—An observational pilot study in critical ill patients. Ann. Clin. Microbiol. Antimicrob. 2015, 14, 33. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ozongwu, C.; Personne, Y.; Platt, G.; Jeanes, C.; Aydin, S.; Kozato, N.; Gant, V.; O’Grady, J.; Enne, V.I. The Unyvero P55 ‘sample-in, answer-out’ pneumonia assay: A performance evaluation. Biomol. Detect. Quantif. 2017, 13, 1–6. [Google Scholar] [CrossRef]
- Kaneko, H.; Kawana, T.; Fukushima, E.; Suzutani, T. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J. Biochem. Biophys. Methods 2007, 70, 499–501. [Google Scholar] [CrossRef]
- Vergara, A.; Zboromyrska, Y.; Mosqueda, N.; Morosini, M.I.; García-Fernández, S.; Roca, I.; Cantón, R.; Marco, F.; Vila, J. Evaluation of a Loop-Mediated Isothermal Amplification-Based Methodology to Detect Carbapenemase Carriage in Acinetobacter Clinical Isolates. Antimicrob. Agents Chemother. 2014, 58, 7538–7540. [Google Scholar] [CrossRef] [Green Version]
- Garcia-Fernandez, S.; Morosini, M.I.; Marco, F.; Gijón, D.; Vergara, A.; Vila, J.; Ruiz-Carbajosa, P.; Cantón, R. Evaluation of the eazyplex(R) SuperBug CRE system for rapid detection of carbapenemases and ESBLs in clinical Enterobacteriaceae isolates recovered at two Spanish hospitals. J. Antimicrob. Chemother. 2015, 70, 1047–1050. [Google Scholar] [CrossRef] [Green Version]
- Zboromyrska, Y.; Vergara, A.; Cosgaya, C.; Verger, G.; Mosqueda, N.; Almela, M.; Pitart, C.; Roca, I.; Marco, F.; Vila, J. Rapid detection of β-lactamases directly from positive blood cultures using a loop-mediated isothermal amplification (LAMP)-based assay. Int. J. Antimicrob. Agents 2015, 46, 355–356. [Google Scholar] [CrossRef]
Microorganisms Identified by Culture | N | Concordance | Minor Errors | Major Errors | Comments |
---|---|---|---|---|---|
S. aureus | 19 | 15 | 2 A,B | 2 C,D | A LAMP: SAUR and SMAL detected B LAMP: SAUR and PAER detected (GNB in gram stain) C,D Culture: Few CFU of SAUR |
P. aeruginosa | 11 | 9 | - | 2 E,F | E Culture: < 1000 CFU of PAER, LAMP: SAUR detected F Culture: <1000 CFU of PAER, LAMP negative |
S. maltophilia | 6 | 6 | - | - | - |
K. pneumoniae | 4 | 2 | - | 2G | G Culture: <1000 CFU of KPNE (two cases) |
E. coli | 3 | 3 | - | - | |
A. baumannii | 2 | 1 | - | 1 H | H Culture: 100,000 CFU of ABAU |
Negative | 7 | 6 | 1 I | - | I LAMP: KPNE detected (GNB in gram stain) |
Mixed flora | 6 | 3 | 3 J,K,L | - | J LAMP:PAER detected K LAMP:SAUR detected L LAMP:KPNE detected |
TOTAL | 58 | 45 | 6 | 7 |
Minor Error as Wrong | Minor Error as Right | |||
---|---|---|---|---|
Value (%) | 95%CI | Value (%) | 95%CI | |
BAL | ||||
Sensitivity | 76.5 | 62.5–87.2 | 86.3 | 73.7–94.3 |
Specificity | 85.7 | 42.1–99.6 | 100 | 59–100 |
PPV | 97.5 | 86.3–99.6 | 100 | 89–100 |
NPV | 33.3 | 21.9–47.2 | 50 | 33.5–66.6 |
Accuracy | 77.6 | 64.7–87.5 | 87.9 | 76.7–95 |
Kappa coefficient | 43.3 | 17.9–68.8 | 70.9 | 51.5–90.2 |
BAS/EA | ||||
Sensitivity | 68.9 | 57.1–79.2 | 94.6 | 86.7–98.5 |
Specificity | 100 | 66.3–100 | 100 | 66.4–100 |
PPV | 100 | 91.3–100 | 100 | 93.5–100 |
NPV | 28.1 | 21.8–35.5 | 69.2 | 46.5–85.4 |
Accuracy | 72.3 | 61.4–81.6 | 95.2 | 88.1–98.7 |
Kappa coefficient | 46.3 | 29.2–63.5 | 89.7 | 80–99.5 |
Microorganisms Identified by Culture | N | Concordance | Minor Errors | Major Errors | Comments |
---|---|---|---|---|---|
K. pneumoniae | 13 | 10 | 3 A,B | - | A LAMP: KPNE and ECOL detected (two cases) B LAMP: KPNE and PAER detected |
S. aureus | 10 | 7 | 2 C,D | 1 E | C LAMP: SAUR and KPNE detected D LAMP: SAUR and ECOL detected ECulture: <1000 CFU of SAUR |
P. aeruginosa | 11 | 9 | 2 F | - | F LAMP: PAER and ECOL detected (two cases) |
E. coli | 8 | 3 | 5 G,H,I,J | - | G LAMP: ECOL and PAER detected H LAMP: ECOL, PAER, SAUR and SMAL detected I LAMP: ECOL and KPNE detected J LAMP: ECOL, PAER and SMAL detected (two cases) |
S. maltophilia | 2 | 2 | - | - | - |
Polymicrobial | 10 | 3 | 4 | 3 | See Table S1 in Supplementary Material. |
Other | 10 | 7 | 3 K,L,M | - | K LAMP: ECOL and KPNE detected L LAMP: PAER detected M LAMP: PAER, SAUR and SMAL detected |
Mixed flora | 10 | 10 | - | - | - |
Negative | 9 | 9 | - | - | - |
TOTAL | 83 | 60 | 19 | 4 |
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Vergara, A.; Boutal, H.; Ceccato, A.; López, M.; Cruells, A.; Bueno-Freire, L.; Moreno-Morales, J.; Puig de la Bellacasa, J.; Castro, P.; Torres, A.; et al. Assessment of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Pathogenic Bacteria from Respiratory Samples in Patients with Hospital-Acquired Pneumonia. Microorganisms 2020, 8, 103. https://doi.org/10.3390/microorganisms8010103
Vergara A, Boutal H, Ceccato A, López M, Cruells A, Bueno-Freire L, Moreno-Morales J, Puig de la Bellacasa J, Castro P, Torres A, et al. Assessment of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Pathogenic Bacteria from Respiratory Samples in Patients with Hospital-Acquired Pneumonia. Microorganisms. 2020; 8(1):103. https://doi.org/10.3390/microorganisms8010103
Chicago/Turabian StyleVergara, Andrea, Hervé Boutal, Adrián Ceccato, Míriam López, Adrià Cruells, Leticia Bueno-Freire, Javier Moreno-Morales, Jorge Puig de la Bellacasa, Pedro Castro, Antoni Torres, and et al. 2020. "Assessment of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Pathogenic Bacteria from Respiratory Samples in Patients with Hospital-Acquired Pneumonia" Microorganisms 8, no. 1: 103. https://doi.org/10.3390/microorganisms8010103
APA StyleVergara, A., Boutal, H., Ceccato, A., López, M., Cruells, A., Bueno-Freire, L., Moreno-Morales, J., Puig de la Bellacasa, J., Castro, P., Torres, A., Marco, F., Casals-Pascual, C., & Vila, J. (2020). Assessment of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Pathogenic Bacteria from Respiratory Samples in Patients with Hospital-Acquired Pneumonia. Microorganisms, 8(1), 103. https://doi.org/10.3390/microorganisms8010103