Abstract
A simple polymerase chain reaction-enzyme immunoassay (PCR-EIA) was employed for the rapid laboratory diagnosis of human brucellosis directly from peripheral blood. Whole blood and serum specimens were collected from 243 patients with acute brucellosis as determined by blood culture, serological tests, and the patients’ clinical characteristics and from a control group of 50 healthy individuals. Diagnosis of brucellosis was established in 179 cases by isolation of Brucella spp. in blood culture and in 64 cases by clinical signs and serological investigation. Following the amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenic membrane protein specific for the Brucella genus, the amplified product was detected in a microtiter plate by hybridization. Two hundred forty-one of the 243 patients tested had detectable Brucella DNA in either whole blood or serum specimens: 149 (61.3%) patients were positive in both whole blood and serum specimens, 43 (17.7%) were positive in serum specimens only, and 49 (20.2%) were positive in whole blood specimens only. The diagnostic specificity of the PCR-EIA assay for both specimen categories was 100%, while the sensitivity was 81.5% for whole blood specimens, 79% for serum specimens, and 99.2% for whole blood and serum specimens combined. The results suggest that the detection of Brucella DNA in whole blood and serum specimens by PCR-EIA assay is a sensitive and specific method that could assist the rapid and accurate diagnosis of acute human brucellosis.
Similar content being viewed by others
References
Corbel MJ (1997) Brucellosis: an overview. Emerg Infect Dis 3:213–221
World Health Organization (1997) Fact sheet N173. WHO, Geneva
Centers for Disease Control and Prevention (2003) Summary of notifiable diseases. Morb Mortal Wkly Rep 50:1–108
Young EJ (1995) An overview of human brucellosis. Clin Infect Dis 21:283–290
Ikonomopoulou E, Skordopoulou K, Petropoulos Ch, Regli-Pantazopoulou A (2000) Brucellosis: a disease in elevation during the period 1997–’A’ half of 1999. Acta Microbiol Hellenica 45:548–552
Kostoula A, Bobojianni C, Levidiotou-Stefanou S, Paraschou P, Dea A, Vrioni G, Gitona E, Papadopoulou C (2001) Ten-year follow-up of human brucellosis in the rural area of NW Greece. In: Program and abstracts, 2nd Balkan Conference of Microbiology, Thessaloniki, Greece, p 185
World Health Organization (2000) WHO surveillance programme for control of foodborne infections and intoxications in Europe. Seventh report, 1993–1998. WHO, Geneva, pp 45–147
Araj GF (1999) Human brucellosis: a classical infectious disease with persistent diagnostic challenges. Clin Lab Sci 12:207–212
Yagupsky P (1999) Detection of brucellae in blood cultures. J Clin Microbiol 37:3437–3442
Dabdoob A, Abdulla ZA (2000) A panel of eight tests in the serodiagnosis and immunological evaluation of acute brucellosis. East Mediterr Health J 6:304–312
Ariza J, Pellicer T, Pallares R, Foz A, Gudiol F (1992) Specific antibody profile in human brucellosis. Clin Infect Dis 14:131–140
Young EJ. (1991) Serologic diagnosis of human brucellosis: analysis of 214 cases by agglutination tests and review of the literature. Rev Infect Dis 13:359–372
Rossetti OL, Aresse AI, Boschiroli ML, Cravero SL (1996) Cloning of Brucella abortus gene and characterization of expressed 26-kilodalton periplasmic protein: potential use for diagnosis. J Clin Microbiol 34:165–169
Matar GM, Khneisser IA, Abdelnoor AM (1996) Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31-kilodalton Brucella antigen DNA. J Clin Microbiol 34:477–478
Queipo-Ortuno MI, Morata P, Ocon P, Manchado P, Colmenero JD (1997) Rapid diagnosis of human brucellosis by peripheral-blood PCR assay. J Clin Microbiol 35:2927–2930
Morata P, Queipo-Ortuno MI, Reguera JM, Garcia-Ordonez MA, Pichardo C, Colmenero JD (1999) Posttreatment follow-up of brucellosis by PCR assay. J Clin Microbiol 37:4163–4166
Morata P, Queipo-Ortuno MI, Reguera JM, Miralles F, Lopez-Gonzalez JJ, Colmenero JD (2001) Diagnostic yield of a PCR assay in focal complications of brucellosis. J Clin Microbiol 39:3743–3746
Morata P, Queipo-Ortuno MI, Reguera JM, Garcia-Ordonez MA, Cardenas A, Colmenero JD (2003) Development and evaluation of a PCR-enzyme-linked immunosorbent assay for diagnosis of human brucellosis. J Clin Microbiol 41:144–148
Alton GG, Jones LM, Pietz DE (1975) Laboratory techniques in brucellosis, 2nd edn. World Health Organization, Geneva
Solomon HM, Jackson D (1992) Rapid diagnosis of Brucella melitensis in blood; some operational characteristics of the BACT/ALERT. J Clin Microbiol 30:222–224
Shapiro DS, Wong JD (1999) Brucella. In: Murray P, Baron E, Pfaller M, Tenover F, Yolken R (eds) Manual of clinical microbiology. American Society for Microbiology, Washington DC, pp 625–631
Baily GG, Kranhn JB, Drasar BS, Stoker NG (1992) Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 95:271–275
Mayfield JE, Bricker BJ, Godfrey H, Crosby RM, Knight DJ, Halling SM, Ballinsky D, Tabatabai LB (1988) The cloning and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein. Gene 63:1–9
Ministry of Health (2000) Report of communicable diseases, October 1998–October 2000. National Epidemiological Surveillance Center, Athens, Greece
Zerva L, Bourantas K, Mitka S, Kansouzidou A, Legakis NJ (2001) Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR. J Clin Microbiol 39:1661–1664
Navarro E, Fernabdez JA, Escribano J, Solera J (1997) PCR assay for diagnosis of human brucellosis. J Clin Microbiol 37:1654–1655
Morata P, Queipo-Ortuno MI, Colmenero JD (1998) Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis. J Clin Microbiol 36:2443–2446
Romero C, Gamazo C, Pardo M, Lopez-Goni I (1995) Specific detection of Brucella DNA by PCR. J Clin Microbiol 33:615–617
Jantos CA, Roggendorf R, Wuppermann FN, Hegemann JH (1998) Rapid detection of Chlamydia pneumoniae by PCR-enzyme immunoassay. J Clin Microbiol 36:1890–1894
Luk JMC, Kongmuang U, Tsang RW, Lindberg AA (1997) Enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype. J Clin Microbiol 35:714–718
Zambardi G, Druetta A, Roure C, Fouque B, Girardo P, Chypre C, Marchand J, Frency J, Fleurette J (1995) Rapid diagnosis of Mycobacterium tuberculosis infections by ELISA-like detection polymerase chain reaction products. Mol Cell Probes 9:91–99
Matsiota-Bernard P, Wasser S, Vrioni G (2000) Detection of Legionella pneumophila DNA in urine and serum samples from patients with pneumonia. Clin Microbiol Infect 6:223–225
Andreoletti L, Hober D, Belaich S, Lobert PE, Dewilde A, Wattre P (1996) Rapid detection of Enterovirus in clinical specimens using PCR and microwell capture hybridization assay. J Virol Methods 62:1–10
Bougnoux ME, Dupont C, Mateo J, Saulnier P, Faivre V, Payen D, Nicolas-Chanoine MH (1999) Serum is more suitable than whole blood for diagnosis of systemic candidiasis by nested PCR. J Clin Microbiol 37:925–930
Brown PD, Gravenkamp C, Carrington DG, Kemp H van de, Harrtskeeri RA, Edwards CN, Everard CO, Terpstra WJ, Levett PN (1995) Evaluation of the polymerase chain reaction for early diagnosis of leptospirosis. J Med Microbiol 43:110–114
Kawamura S, Maesaki S, Noda T, Hirakata Y, Tomono K, Tashiro T, Kohno S (1999) Comparison between PCR and the detection of antigen in sera for diagnosis of pulmonary aspergillosis. J Clin Microbiol 37:218–220
Murdoch DR, Walford EJ, Jennings LC, Light GJ, Schousboe MI, Chereshsky AY, Chambers ST, Town GI (1996) Use of the polymerase chain reaction to detect Legionella DNA in urine and serum samples from patients with pneumonia. Clin Infect Dis 23:475–480
Casanas MC, Queipo-Ortuno MI, Rodriguez-Torres A, Orduna A, Colmero JD, Morata P (2001) Specificity of a polymerase chain reaction assay of a target sequence on the 31-kilodalton Brucella antigen DNA used to diagnose human brucellosis. Eur J Clin Microbiol Infect Dis 20:127–131
Romero C, Pardo M, Grillo MJ, Diaz R, Blasco JM, Lopez-Goni I (1995) Evaluation of PCR and direct enzyme-linked immunosorbent assay on milk samples for diagnosis of brucellosis in dairy cattle. J Clin Microbiol 33:3198–3200
Ezzedine H, Mourad M, van Osel C, Logge C, Squifflet JP, Renaul F, Wauters G, Giggi J, Wilmotte L, Haxhe JJ (1994) An out break of Ochrobactrum anthropi bacteraemia in five organ transplant patients. J Hosp Infect 27:35–42
Gransden WR, Eykyn SJ (1992) Seven cases of bacteremia due to Ochrobactrum anthropi. Clin Infect Dis 15:1068–1069
Yagupsky P, Peled N, Riesenberg K, Banai M (2000) Exposure of hospital personnel to Brucella melitensis and occurrence of laboratory-acquired disease in an endemic area. Scand J Infect Dis 32:31–35
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Vrioni, G., Gartzonika, C., Kostoula, A. et al. Application of a Polymerase Chain Reaction Enzyme Immunoassay in Peripheral Whole Blood and Serum Specimens for Diagnosis of Acute Human Brucellosis. Eur J Clin Microbiol Infect Dis 23, 194–199 (2004). https://doi.org/10.1007/s10096-003-1082-4
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10096-003-1082-4