The study was carried out at Ayub Medical Hospital, Jinnah Medical Hospital and DHQ Hospital of Abbottabad city. Abbottabad city is the capital of the district Abbottabad located in the Hazara region, province Khyber Pakhtunkhwa. It has a total area of about 1,967 km2 or 759 square miles. According to the census of 2017, the total population is 1,332,912 and the density is 680 inhabitants per km2 (25). The total number of households of the district reporting livestock such as cattle, buffaloes, sheep, goats and camels etc. is 1,263,547 and the total number of reported animals of the KPK province is 5,967,886 according to data of 2006 (27). Most of the rural population has to do livestock farming as there is little land available for agriculture in this district. Thus, a higher risk of acquiring brucellosis due to close contact to livestock can be supposed.
Study Design and Collection of Patients Detail by Questionnaire
A cross-sectional study was conducted with no follow up investigation. Blood samples (n = 500) were randomly collected from patients (both males and females) who were suspected of having brucellosis by showing any symptoms such as fever, muscle pain, fatigue etc., who visited the outdoor patient’s department (OPD) of the hospitals and agreed to take part in this study from April, 2019 to August, 2019. Ethically and professionally, neither information nor samples were collected from patients hesitant to participate in this study. Men and women included in this study were older than 20 years.
A questionnaire was filled in personally for each patient. Questions on age, gender, dwelling area, animal ownership or presence of animals in household, contact with animals, processing or handling raw animal products or meat, consumption of raw animal products, access of livestock to the household’s source of drinking water, abortion in animals or contact with aborted animals, presence or previous history of symptoms such as fever, night sweats, headache, arthralgia, generalized ache, nausea, anorexia and fatigue and presence of such symptoms or brucellosis in any other house-hold member had to be answered. Women were asked to report on previous pregnancies and abortion history.
About 4 ml blood was collected aseptically from the brachial vein with a disposable, sterile syringe. Blood was immediately injected and transferred into serum separating gel-tubes and tubes were labeled immediately. The serum was obtained by centrifugation at 25 rpm for 15 minutes. Each serum sample was divided into two parts for serum agglutination testing and DNA extraction to perform real time PCR, respectively.
The Brucella abortus antigen of the Febrile Antigen Kit (Plasmatec, Lab21 Healthcare Ltd, Bridport, Dorset, United Kingdom) was used for serum agglutination slide test as per manufacturer`s instructions. Briefly, 80 µl, 40 µl, 20 µl, 10 µl and 5 µl of undiluted serum was added onto a row of 3 cm diameter circles of a reaction slide. Then a drop of the undiluted suspension of antigen was added to each serum sample using the dropper provided with the kit. The content was mixed using a stirring stick. The slide was shaken gently for one minute and then observed for any agglutination. A test was positive when agglutination was observed at 1:80.
DNA Extraction and Quantification
DNA was extracted from seropositive serum samples (n=68) by using WizPrep gDNA Mini Kit (Wizbiosolutions Inc. Jungwon-gu, Seongnam, South Korea) according to the instructions and protocol of the kit manufacturer. After extraction of DNA from serum samples, Nanodrop-1000 UV spectrophotometer (Nano-Drop technologies, Wilmington, DE) was used for DNA quantification. DNA quantification was performed by measuring absorbance at 260 nm and DNA purity was checked with the ratio of 260/280. A value of approximately 1.8 was considered to show pure DNA. The purified DNA samples were stored at -20°C.
Real-time polymerase chain reaction
Real-time PCR was using a MJ Mini Bio-RAD Thermal cycler (Applied Biosystems, Foster City, California, USA). Genus specific primers and probes targeting the BCSP-31 gene were used according to Probert et al. (2004) (28). The BCSP-31 gene codes for a 31KDa immunogenic protein of the membrane and is conserved among all Brucella species and biovars. The sequences of PCR primers and probes used in our study are 5’-GCTCGGTTGCCAATATCAATGC-3’ (forward), 5’-GGGTAAAGCGTCGCCAGAAG -3’ (reverse) and 5’-FAM-AAATCTTCCACCTTGCCCTTG CCATCA-BHQ1-3’ (probe).
A total of 25μl of reaction mixture was prepared for the amplification of each sample. The reaction mixture was prepared by adding 5μl of 5x Amplicon qPCR master mix (Solis BioDyne, Teaduspargi, Tartu, Estonia), 0.8μl forward primer (10 pmol/μl), 0.8μl reverse primer (10 pmol/μl), 0.4μl probe (5 pmol/μl), 3μl extracted DNA sample and 15μl of nuclease free water to a final volume of 25μl.
The PCR conditions were initial denaturation for 10 minutes at 95°C, 44 cycles of 20 seconds at 95°C for denaturation, 50 seconds at 60°C for primer annealing and 50 seconds at 72°C for DNA extension. The results were considered positive when the cutoff value was ≤ 40 cycles.
For the data analysis, a patient was considered positive if she/he had positive SAT result. Data were statistically analyzed by using the online tools of Vassar Stats (Vassar College; Poughkeepsie, NY USA; http://vassarstats.net/). Collected data and results were categorized into groups. Version 2 software was used for analysis of logistic regression to determine odd ratio, risk ratio, 95% confidence interval and Chi-square test for p-value. Fisher exact test was used in case when the cross table had 5 or less counts. The data were considered to be statistically significant with a p-value ≤ 0.05.