brucellosis is endemic in low socio - economic countries like india and the prevalent species causing human infections are brucella melitensis and brucella abortus . contact with the infected animals , consumption of raw milk and ignorance regarding the disease , are the major risk factors . since clinical manifestations of this disease are protean in nature , laboratory help is a must in the diagnosis . in the laboratory , brucellosis although an array of serological tests is available , rose bengal plate test ( rbpt ) and serum agglutination test ( sat ) are the most widely used . the sensitivity of rbpt is considerably high , but its specificity is much lower when used to test individuals residing in an endemic area . in the same way , sat , used to confirm rbpt results , also has limitations of lack of sensitivity as well as specificity . apart from this , serological testing does not provide direct evidence for the presence of the pathogen , hence , isolation of brucella spp . from the clinical specimen many of the methods have been developed for culturing brucella spp . from blood specimen , including the conventional ruiz - castaneda ( castaneda ) method , automated systems , lysis concentration ( lc ) and clot culture . in general , blood culture for brucella is performed by conventional castaneda method where the blood specimen is directly inoculated in the liquid phase of the castaneda medium . although the results by this method are satisfactory in acute untreated cases , the incubation time required is very long . the isolation rate is markedly reduced in treated cases or sub - acute / chronic brucellosis patients . the earlier reports have shown an increase in isolation rate and decrease in recovery time by adapting lc technique . however , both conventional and lc methods require one more blood sample for culture once the serological test is found positive . as it is difficult to obtain one more blood specimen in ignorant patients , clot culture could be a better option . , an attempt has been made to compare lc , clot culture and conventional culture techniques for the isolation of brucella from blood specimen in an endemic area . blood culture by all the three techniques was performed in 169 out of 191 patients showing sat titer of 160 international units ( iu ) after obtaining written consent . depending on the duration of the disease , patients were divided into three groups as : acute ( < 8 weeks ) , sub - acute ( 8 - 52 weeks ) and chronic ( > 52 weeks ) brucellosis . patients were considered to have relapse / re - infection if symptoms reappeared within a year after the completion of antibrucellar treatment . five ml of blood was collected aseptically from patients with the clinical symptoms resembling brucellosis ( fever , malaise , joint pain , low backache , headache , and weight loss ) in a sterile , plastic screw capped bottle with 6 - 7 glass beads of 2 mm diameter . the specimen was centrifuged at 2000 rpm for 15 min ; the serum was separated aseptically and used for serology . blood sample for conventional and lc culture technique was collected from patients who had significant sat titers ( 160 iu ) . about 10 ml of blood was collected in adults and 5 ml in case of children . the specimen was equally distributed and processed for conventional blood culture and lc technique . the blood specimen was inoculated aseptically into the broth phase of castaneda 's biphasic medium consisting of brain heart infusion agar and broth with brucella selective supplement ( hi - media ) . , was employed with a minor modification . 5 ml of blood was added to 40 ml of sterile distilled water with 1.5 ml of 4% sodium citrate in adults and 2.5 ml was added to 20 ml of sterile distilled water with 0.75 ml of 4% sodium citrate in children , in a sterile screw caped centrifuge tube . the contents were gently mixed and the tubes were centrifuged at 7,000 rpm for 30 min . the supernatant was discarded and the sediment was inoculated in the castaneda 's medium , instead of culture plate the blood clot preserved in the sterile screw capped plastic tube with glass beads after removal of serum was used for this method . the media were incubated at 37c with 10% co2 for a maximum of 30 days . sub - culturing was done by allowing the blood broth mixture to flow over the solid phase . the day of appearance of the first colony was recorded for comparison of growth rates . the isolates were identified based on colony morphology , gram stain , modified  zn  staining , co2 requirement , biochemical tests like oxidase and urease , h2s production for 4 days and growth in the presence of basic fuchsin ( 1:50,000 and 1:100,000 ) and thionin ( 1:25,000 , 1:50,000 and 1:100,000 ) . provisional confirmation and bio - typing of the isolate was done by performing slide agglutination test using b. abortus and b. melitensis monospecific antisera ( murex biotech ) . statistical analysis of the data was performed by graphpad instat software designed by graphpad software , inc . the blood specimen was inoculated aseptically into the broth phase of castaneda 's biphasic medium consisting of brain heart infusion agar and broth with brucella selective supplement ( hi - media ) . . 5 ml of blood was added to 40 ml of sterile distilled water with 1.5 ml of 4% sodium citrate in adults and 2.5 ml was added to 20 ml of sterile distilled water with 0.75 ml of 4% sodium citrate in children , in a sterile screw caped centrifuge tube . the contents were gently mixed and the tubes were centrifuged at 7,000 rpm for 30 min . the supernatant was discarded and the sediment was inoculated in the castaneda 's medium , instead of culture plate . the blood clot preserved in the sterile screw capped plastic tube with glass beads after removal of serum was used for this method . the media were incubated at 37c with 10% co2 for a maximum of 30 days . sub - culturing was done by allowing the blood broth mixture to flow over the solid phase . the day of appearance of the first colony was recorded for comparison of growth rates . the isolates were identified based on colony morphology , gram stain , modified  zn  staining , co2 requirement , biochemical tests like oxidase and urease , h2s production for 4 days and growth in the presence of basic fuchsin ( 1:50,000 and 1:100,000 ) and thionin ( 1:25,000 , 1:50,000 and 1:100,000 ) . provisional confirmation and bio - typing of the isolate was done by performing slide agglutination test using b. abortus and b. melitensis monospecific antisera ( murex biotech ) . statistical analysis of the data was performed by graphpad instat software designed by graphpad software , inc . blood culture by all the three techniques was performed in 169 out of 191 patients . overall blood culture positivity was found to be 24.8% , 43.1% and 34.9% by conventional , lc and clot culture techniques respectively . the isolation rates in patients with acute , sub - acute , chronic brucellosis and relapse / reinfection by various culture techniques are given in table 1 . culture positivity by various culture techniques in different stages of brucellosis when compared with the conventional technique lc and clot culture techniques yielded growth in significantly more number of cases ( p < 0.0001 ) . the mean recovery time for conventional technique was 9.61 days ( standard deviations [ sd ] 1.68 ) , whereas with lc and clot culture techniques it was 4.08 ( sd  0.87 ; p < 0.0001 ) and 5.83 ( sd  1.37 ; p < 0.0001 ) respectively . in the acute stage , earliest growth by the conventional technique was seen on the 6 day where as it was on the 3 day with both lc and clot culture techniques . lc technique showed earlier growth in 96% blood cultures , before the first positive culture by conventional method was noted , whereas clot culture yielded earlier growth in 42.55% of cultures . maximum incubation time required for culture to be positive by lc , clot culture and conventional technique was 6 , 9 and 16 days respectively . after 16 day , no culture was found positive , though the cultures were incubated for 1 month . despite maximum aseptic precautions taken during collection and processing of the sample , contamination rate was high for clot culture ( 5.32% ) followed by lc ( 2.95% ) , may be due to more number of manipulations required for the procedure . in the present study , blood culture positivity was found to be 24.8% , 43.1% and 34.9% and the recovery time was 10 , 4 and 6 days by conventional , lc and clot culture techniques respectively . our findings suggest that lc and clot culture methods are more sensitive and require less time than conventional culture thereby helping in early detection of infection . among the lc and clot culture techniques , lc was better due to increased isolation rates and decreased recovery time . , the mean recovery time by lc in this study was 4.08 days , which is in agreement with espinosa et al . , but slightly more as compared to the results of 2 and 2.7 days by etemadi et al . , and mantur and mangalgi . in a study done by escamilla et al . , clot culture technique was found to be less sensitive and more labor - intensive when compared with the conventional method . conversely in our study , clot culture technique was more sensitive and also the recovery time was less when compared with the conventional method . , another advantage of this technique is that need for one more blood sample is alleviated . in a study by espinosa et al . , isolation rate was higher in sub - acute cases than in patients with acute brucellosis by lc technique . whereas far higher isolation rates in both acute and chronic stages have been reported by mantur and mangalgi . in our study , culture positivity rate was high in patients with acute followed by sub - acute brucellosis and none of the chronic cases grew brucella . as the patients in our study were not relieved of their symptoms over a long time , they switched on to many doctors before being diagnosed as brucellosis cases at our hospital , which could be an attribute for our lower isolation rates . history of minimum 3 - 4 courses of antibiotics was elicited in 116 ( 68.63% ) patients . in general , blood cultures are attempted to confirm the presumptive serological diagnosis . in india , especially in rural areas , due to the misperception that blood once lost is lost forever and blood loss enhances the disease ; it is difficult to obtain one more blood specimen for culture in seropositive individuals . in such situations and also in field conditions ( camps ) , wherein obtaining a second sample for culture is difficult , clot culture could be the optimal alternative . both lysis and clot culture require manipulations , which enhance the chances of laboratory infection and contamination , thereby necessitating strict biosafety and aseptic measures . we propose that for the isolation of brucella from blood specimen , lc method is better than conventional castaneda 's method as the isolation rate is high and the recovery time is less . clot culture is a better option when a second blood sample can not be obtained for culture . as lysis and clot culture techniques are sensitive , simple and inexpensive and yield earlier results , they can be adapted in the technically and economically backward areas where automated systems are far from reach . however , to prevent laboratory acquired infection , these methods are to be carried out in class ii biosafety cabinet with bsl-3 precautions .
background : isolation of brucella is the gold standard in the laboratory diagnosis of brucellosis . as brucella is intracellular and the number of circulating bacteria is usually low , removal / dilution of antibacterial substances , concentration of bacteria and optimal culture conditions may enhance the rate of isolation.aims and objectives : the objective of the following study was to compare the lysis concentration ( lc ) , clot culture and conventional castaneda blood culture techniques for the isolation rate and recovery time in the diagnosis of human brucellosis.materials and methods : blood cultures by lc , clot culture and conventional method were performed in 169 patients who had antibody titers 160 international units by the serum agglutination test.results:overall blood culture positivity was found to be 24.8% , 43.1% and 34.9% by conventional , lc and clot culture techniques in that order . 
 the mean recovery time by lc and clot culture techniques was significantly less than conventional method , resulting in an overall difference of nearly 6 and 4 days respectively.conclusions:for the isolation of brucella from blood , lc and clot culture techniques are better than the conventional technique .