seroprevalence of human Brucellosis in and Around Puducherry: Application of an Immunocapture Agglutination Technique (Brucellacapt) selvaraj stephen*, Jothimani Pradeep, Balakrishnan sangeetha and shanmugam Jayaraman

The objective of the present study was the serological diagnosis of human brucellosis in and around Puducherry by application of Immunocapture-agglutination technique (Brucella Capt), which has been validated and found to be quite satisfactory. The study has included febrile patients, patients with chronic back pain/arthritis, healthy pregnant women, personnel exposed to domestic livestock and voluntary blood donors. Prevalence of brucellosis in these different categories was studied. Overall seroprevalence of brucellosis among different categories of individuals from Puducherry is very low, viz., 3.27%. Among febrile patients, the prevalence is almost the same with 3.33% positivity. Among 100 healthy pregnant women, only one had antibodies to Brucella abortus (1.00%). The category of animal handlers as expected had higher seroprevalence rate of 8.00%. All the 45 voluntary blood donors were seronegative for brucellosis.


MAteRiAls AND MethODs
An investigative laboratory based study was conducted with archived and anonymised samples in the Department of Microbiology,

Mahatma Gandhi Medical College & Research Institute (MGMC & RI), Sri Balaji Vidyapeeth
(Deemed-to-be-University), Pondicherry. After getting approval from the Institutional Human Ethical Committee (IHEC), MGMC & RI. The study period was between December 2013 to August 2018. Sample size calculation was based on the national average as well as average in the southern states. The present seroprevalence of brucellosis in India and different parts of South India is different and much lower when compared to early seventies and eighties. Sample size (n) was calculated using the formula: "n = [t 2 x p (1-p)]/ m 2 ", Where t = confidence level at 95%, p = estimated prevalence of the disease, m = margin of error at 5%.
With the prevalence rate of 11.26%, based on the seroprevalence of human brucellosis in the last 15 years (2000-2015), the calculated sample size was 154. We have taken a larger size of 300 patients and 220 as controls. Thus, 520 archived serum samples were included in the study after anonymising.

Categories of participants
Three hundred blood samples left over after completion of the laboratory investigations from patients with PUO/Hepatomegaly/ Splenomegaly/Patients with neurological disease/ chronic back pain/arthritis Seventy five serum samples from animal handlers (veterinarians, butchers, slaughter-house workers, household contacts with domestic livestock) Forty five healthy voluntary blood donors One hundred healthy ante-natal women Brucella Capt (Vircell Microbiologists, Spain) One-step Immunocapture assay was used for the identification of total brucella antibodies (IgM/IgG/IgA). The procedure was as outlined in the technical brochure of the kit. Briefly, all the reagents, test plates and serum samples were brought to room temperature before performing the test. The test plate contains U-bottom wells which are coated with anti-human immunoglobulins. Based on the kit recommendation for screening procedure, two doubling dilutions of serum samples were prepared viz., 1:160 and 1:320. Fifty microlitre of diluted serum samples, positive, negative controls were added to the plate. This was followed by the addition of 50µl of the bacterial suspension (stained Brucella abortus suspension treated with formaldehyde) into all the wells and mixed well by shaking the plates. The plates were sealed with adhesive tape and incubated for 24 hours at 37°C, in a dark humidity chamber protected from light exposure.
The test results were positive if the agglutination covers the whole well surface.
A button at the center of the well indicates the negative result (Fig. 1). Titre ≥1:160 was taken as positive.

ResUlts
Overall seroprevalence of total Brucella antibodies was 17 (3.27%) out of 520 subjects. Table 1 shows the details of antibody titres to Brucella abortus by Brucella Capt among different participants. Among the 300 febrile patients 10 (3.33%) were positive. Eight patients had titres of 1:160, one patient each had 1:320 and 1:2560 respectively. Out of 75 animal handlers, six were seropositive (8.00%) which comprises of five butchers who had titres of 1:160 and one farmer with a titre of 1:320. Only one healthy pregnant woman was seropositive for brucellosis (1.00%) with a titre of 1:160. None of the voluntary blood donors were positive in Brucella Capt agglutination test. Remaining 503 participants were negative for brucella antibodies.

Validation of Brucella Capt by different Researchers
Brucella Capt, an Immunocapture agglutination test was validated by both overseas as well as Indian researchers with highly satisfactory  In Indian subcontinent, brucellosis is considered to be an endemic zoonosis, which has been reported from every state of India.  Thus a seroprevalence of 11.5% was found in Andhra Pradesh and 6.5% in Orissa. [21][22] In South and North Karnataka, 2.14%, 0.92% seropositivity was observed respectively among patients with or without fever and those presenting other symptoms for brucellosis 17 . Mangalgi et al. 27 conducted a seroepidemiological survey in three districts of Karnataka viz., Bijapur, Bagalkot, and Gulbarga. They divided into two groups namely, directly exposed and indirectly exposed to animal contacts. Of the 1,733 subjects screened, 998 were grouped in group I and 735 in group II. Significant titers by SAT (≥160) and 2-ME (≥80) test were noted in 133 (13.3%) and 99 (9.9%) individuals in group I and three (0.4%) individuals in group II. In the present study a very low seroprevalence of 3.3% was observed in people of Puducherry and neighbouring Tamil Nadu with symptoms of PUO.

Seroprevalence of brucellosis among ante-natal women
Mantur et al. 3 from Belgaum, reported three cases of ante-natal women who suffered from Brucellosis. In our study, only one out of 100 healthy pregnant women was seropositive for brucellosis with a titre of 1:160. It is to be stressed here that even the next higher titre of 1:320 does not necessarily confirm active brucellosis and needs clinical correlation.

seroprevalence of brucellosis among animal handlers
In Central Kerala, the overall seroprevalence is about 1.6% which comprises of general population and veterinary students with 2.45% and 1.14% seropositivity respectively 7 . Recently, Shome et al. 17 from Karnataka reported overall prevalence of 7.04% among veterinary health care professionals. In our study 8.00% animal handlers were seropositive for brucellosis. Mangalgi et al. 15 reported that the incidence rate was high when ingestion of raw milk regularly than the direct contact with domestic livestock.

seroprevalence of brucellosis among Blood donors
Vaishnavi et al. 20 from Chandigarh reported that only 0.36% of voluntary blood donors were seropositive for brucellosis with a titre of 1:160 by STAT. A study from Maharashtra found 52.63% RBPT positive donors with background low titre at 1:80 in STAT and 5.91% positivity was confirmed by RBPT 15 . Sero-prevalence study from Bangalore documented a very low seropositivity of brucellosis (1.1%) by using STAT 10 . An Iranian study found that 0.33% had low level of titres but 0.11% were confirmed as true positive for Brucella antibodies 28 . In the present study none of the voluntary blood donors were seropositive for Brucellosis.

CONClUsiON
The seroprevalence of human brucellosis in Puducherry is very low. This could be because of consumption of pasteurized milk and other dairy products by the people. Drinking unpasteurized raw milk is not the common custom in this part of South India. Brucella Capt kit has performed well in our experience like similar reports from Indian and Overseas researchers.