Evaluation Of Conventional Castaneda Biphasic And Lysis Centrifugation Blood Culture Techniques And Polymerase Chain Reaction In The Diagnosis Of Human Brucellosis

Abstract

BACKGROUND: Brucellosis is an endemic zoonotic disease with a wide spectrum of non-specific symptoms, Serological methods being the mainstay of diagnosis, are characterized by low specificity especially in areas with high base line titres. Though blood culture is gold standard of diagnosis, low rate of culture yield and long doubling time of the organism are the main limitations .Molecular diagnostic modalities like PCR for detection of DNA are being increasingly used. Hence this study was undertaken to evaluate the blood culture techniques – Conventional Castaneda Biphasic Medium and Lysis Centrifugation Technique and PCR for diagnosis of human Brucellosis. Objective: To evaluate Conventional blood culture methods (Castaneda Biphasic Medium and Lysis Centrifugation) and PCR in diagnosis of human brucellosis Material and Methods: The study was a cross sectional study carried out in in the serology section of department of Microbiology, Jawaharlal Nehru Medical College, Belgaum. A total of 1545 serum samples were screened. 50 serum samples were positive for Brucellosis by slide agglutination test using Rose Bengal Card Test and were included in the study. 10-12 ml of blood was collected for both the blood culture methods while serum was used as the sample for PCR. The culture isolates of Brucella were identified by colony morphology, staining and biochemical reactions. DNA extraction was done from serum samples and PCR was done targeting the IS711 genome of Brucella. Results: Out of the 50 samples positive by RBPT, blood culture was positive in 10 cases (20%) by the Conventional Castaneda Biphasic Medium and in 21 cases (42%) by the Lysis Centrifugation Technique. The mean duration of culture positivity by Conventional method was 6 days while the mean duration of culture positivity by Lysis Centrifugation was 3 days. Sensitivity of the Castaneda Biphasic Medium compared to the Lysis Centrifugation Technique was 47.6% while the specificity was 100%. PCR was positive in 47 cases (94%). The sensitivity of PCR compared to Lysis Centrifugation Technique was 100%. Conclusion: Isolation of the organism by blood culture though being the gold standard, has a low sensitivity and also the duration of incubation required is long. Blood culture by the Lysis Centrifugation Technique not only increases the rate of isolation but also decreases the duration of incubation. PCR by amplifying even the intracellular DNA can detect the presence of the organism in the body within 24 hours thus giving conclusive evidence of infection especially in chronic cases and focal complications. However PCR can detect even non-viable bacilli. Also the cost, technical expertise and infrastructure required may preclude its use at all centres