Brucellosis 2003 proceedings - PHIDIAS

Short Oral Communications
Human brucellosis
the four remaining sera whose reactivity was unaffected by absorption with anti-RF
Ab corresponded to acute cases and had high levels of IgM antibodies.
Although interference by RF potentially poses a serious problem to diagnostic
tests based on IgM-detection, our results show that this phenomenon does not hinder
achieving a correct serological diagnosis of human brucellosis as long as
simultaneous performance of IgG tests are carried out. However, considering a
potential interference by RF in patients with relapse and/or patients with long-lasting
brucellosis is important because, as we show, specific test detecting IgM antibodies
can yield false positive results due to the presence of RF.
HO4- RAPID DIAGNOSIS OF HUMAN BRUCELLOSIS BY SERUM
QUANTITATIVE REAL-TIME PCR.
J. D. Colmenero 1 , M. I. Queipo-Ortuño 2 , María E. Pachón 3 , M. Gonzalez 4 , J. Mª Reguera 1 , G. Baeza 2 ,
M. A. García-Ordoñez 1 and P. Morata 2 . (1) U. E. Infecciosas Hospital Universitario Carlos Haya,
Málaga, Spain. (2) Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Málaga,
Spain. (3) Servicio E. Infecciosas, Hospital Universitario Virgen del Rocío, Sevilla, Spain. (4) U. E.
Infecciosas, Hospital Universitario Virgen de la Victoria, Málaga, Spain.
In order to simplify molecular diagnosis of human brucellosis and make it more
accessible to any clinical laboratory, we have now developed a Real-Time PCR
assay and have evaluated its diagnostic yield in serum samples.
From April 2002 to May 2003, serum samples from 60 patients with active
brucellosis were analyzed by quantitative Real-Time PCR. Two of the patients
provided two samples each; one corresponding to the initial episode and the other
corresponding to a relapse. Control samples were obtained from 55 subjects with
febrile syndromes of other defined etiologies which had initially involved a differential
diagnosis with brucellosis, asymptomatic patients with a history of brucellosis treated
correctly during the previous 12 months, asymptomatic subjects professionally
exposed to Brucella infection, with persistent high titers of antibrucella antibodies and
healthy subjects.
A 223-base pair PCR target sequence present in the gene encoding a 31 kDa
Brucella abortus antigen was selected for amplification. The LightCycler detection
system and SYBR Green I Dye were used for amplification and online quantification.
of PCR products.
Of the 60 patients included in the study, 36 (60%) had positive blood cultures
and the other 24 (40%) were diagnosed based on clinical and serological criteria.
Forty (66.6%) had fever with no apparent focus and 20 (33.3%) had one or more
focal complications. Of the 62 samples from the patients with brucellosis, 57 (91.9%)
were positive in the Real-Time PCR. The amplification threshold was 29.9+3.3 cycles
(range 20.1-37.7 cycles). Two control samples (3.6%) had a false positive Real-Time
PCR test. Thus, Real-Time PCR assay sensitivity, specificity and positive and
negative predictive values were 91.9%, 96.4, 96.6, 91.4%, respectively. Positive and
negative likelihood ratios were 25.3 and 0.08 respectively.
In conclusion, the high sensitivity and specificity of this Real-Time PCR assay,
together with its speed, versatility, and risk reduction for laboratory personnel, makes
this technique a very useful tool for the diagnosis of human brucellosis.
Brucellosis 2003 International Research Conference
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