Shalini Thakur1, Jasbir S Bedi2, Randhir Singh3, Jatinder P S Gill4, Anil K Arora5, Neeraj Kashyap6. 1. School of Public Health and Zoonoses, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India. Electronic address: 1723shalini@gmail.com. 2. School of Public Health and Zoonoses, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India. Electronic address: jasbirsbedi@gadvasu.in. 3. School of Public Health and Zoonoses, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India. Electronic address: randhirsingh@gadvasu.in. 4. Director Research, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, Punjab, India. Electronic address: jpsgill@gadvasu.in. 5. Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India. Electronic address: akarora@gadvasu.in. 6. Department of Veterinary Animal Genetics and Breeding, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.
Abstract
BACKGROUND: Brucellosis is one of the neglected zoonotic diseases in humans. The serological methods based on antibody detections are unable to detect the effectiveness of treatment in humans as antibodies persist for long time in humans even after therapy. Therefore, we developed qPCR technique to overcome such discrepancy and device a rapid and efficient test for both diagnosis and follow up of the brucellosis affected individuals. METHODS: High risk suspected individuals with positive serology (RBPT, STAT and iELISA) and PCR were mainly analyzed for DNA quantification by qPCR assay. The bcsp-31 gene, a shared gene of Brucella species was amplified by genus specific primers and cloned to pGEMT™ easy vector and the cloned plasmid were used to construct a standard curve (R2=0.99, efficiency=1.98) over 7 orders of magnitude with sensitivity of ≈10 copy number. The assay was found 100% specific. RESULTS: Overall 85 individuals were found positive out of 188. Out of them, 23 serological, PCR and qPCR positive individuals were recommended for 45days therapy according to WHO regimen (Doxycycline and Rifampin) and each case was further followed by qPCR. The mean threshold cycle (Cq) before treatment was 26.05±0.347 (3940.5copies/μl), which increased significantly to 32.7±0.66 (259.13copies/μl) on 4th week during treatment, 35.12±3.12 (38.52copies/μl) at 6th week on day of treatment completion, 35.6±0.66 (34.21copies/μl) on 21st day after treatment depicting a significant reduction in DNA load over the course of treatment. Serological follow up showed that only 3 individuals had decreased STAT titre but no change in RBPT results. Out of 17 symptomatic individuals under therapy, 10 improved clinically, 5 improved clinically with persistent weakness and 2 had no effect of therapy. CONCLUSION: The study suggests that qPCR is more useful and rapid test to follow treated individuals than serology.
BACKGROUND:Brucellosis is one of the neglected zoonotic diseases in humans. The serological methods based on antibody detections are unable to detect the effectiveness of treatment in humans as antibodies persist for long time in humans even after therapy. Therefore, we developed qPCR technique to overcome such discrepancy and device a rapid and efficient test for both diagnosis and follow up of the brucellosis affected individuals. METHODS: High risk suspected individuals with positive serology (RBPT, STAT and iELISA) and PCR were mainly analyzed for DNA quantification by qPCR assay. The bcsp-31 gene, a shared gene of Brucella species was amplified by genus specific primers and cloned to pGEMT™ easy vector and the cloned plasmid were used to construct a standard curve (R2=0.99, efficiency=1.98) over 7 orders of magnitude with sensitivity of ≈10 copy number. The assay was found 100% specific. RESULTS: Overall 85 individuals were found positive out of 188. Out of them, 23 serological, PCR and qPCR positive individuals were recommended for 45days therapy according to WHO regimen (Doxycycline and Rifampin) and each case was further followed by qPCR. The mean threshold cycle (Cq) before treatment was 26.05±0.347 (3940.5copies/μl), which increased significantly to 32.7±0.66 (259.13copies/μl) on 4th week during treatment, 35.12±3.12 (38.52copies/μl) at 6th week on day of treatment completion, 35.6±0.66 (34.21copies/μl) on 21st day after treatment depicting a significant reduction in DNA load over the course of treatment. Serological follow up showed that only 3 individuals had decreased STAT titre but no change in RBPT results. Out of 17 symptomatic individuals under therapy, 10 improved clinically, 5 improved clinically with persistent weakness and 2 had no effect of therapy. CONCLUSION: The study suggests that qPCR is more useful and rapid test to follow treated individuals than serology.