Summary

Brucellosis is an important zoonotic disease and has public health importance. In the present study, we studied the prevalence and associated risk factors of human brucellosis in the central Indian population from tertiary care health settings. A prospective observational study was conducted from March 2015 to February 2018 in patients attending the outpatient department (OPD) of Central India Institute of Medical Sciences (CIIMS), Nagpur. A total of 7026 individuals suspected of brucellosis were recruited based on prespecified inclusion criteria, additional risk factors, and clinical symptoms. Baseline, demographic and clinical characteristics were likewise recorded. Sera samples from recruited individuals were collected and subjected to anti-brucellosis antibody (IgM) detection using a commercial kit by ELISA assay. The overall seroprevalence of brucellosis reported from tertiary care health settings was 11% (772/7026). The majority of positive cases were from the states Madhya Pradesh (58.1%), followed by (Maharashtra (38.8%) and Chhattisgarh (2.9%). Adult age (20-60) and female groups were more vulnerable. Clinical symptoms like fever, arthralgia, and myalgia risk factors like animal exposure, consumption of raw milk, vegetable, and meat were significantly associated with brucellosis in the recruited population. Among the positive cases, high seroprevalence was associated with animal handlers (66.8%) compared to other occupationally exposed groups. The present study shows a high seroprevalence of brucellosis in health care settings. We emphasize regular screening of the disease in clinical settings to develop epidemiological data and initiate appropriate control measures.

Keywords:Human Brucellosis, Seroprevalence, Risk Factors, ELISA 

Introduction

Brucellosis has been regarded as an important yet neglected zoonotic disease (Agasthya et al., 2007). Globally, more than 500,000 new cases are reported every year, with the annual incidence varying from < 2 to > 500 per 1,000,000 population among different regions (Renukaradhya et al., 2002). The disease is usually transmitted to humans through exposure to infected live stocks and consumption of raw or unpasteurized milk and milk products contaminated with Brucella species (Tembhurneet al., 2017). It is one of the causes of fever of prolonged duration in endemic areas and one of the important causes of pyrexia of unknown origin (Pathak et al., 2014).

Human brucellosis is often misdiagnosed due to overlapping clinical manifestations with other bacterial infections (Aworh et al., 2013) and therefore remains underreported in most clinical settings.  Although brucellosis is associated with febrile illness, in rare episodes, it can infect other organs such as the kidney, heart, and brain and establish chronic infection (Agasthya et al., 2012). Neurobrucellosis is among rare complications of the disease, wherein brucella bacilli can infect the Central Nervous system (CNS), leading to a high neurological sequel and morbidity rates (Tembhurneet al., 2017). 

Brucellosis in India is often regarded as a re-emerging zoonotic disease of public health importance due to the high agrarian population and their exposure to livestock. Due to this reason, the disease was included in the list of high prioritized zoonotic infections in the roadmap to combat zoonoses in India (RCZI) initiative by India  (Sekar et al., 2011). Thus screening and accurate diagnosis of brucellosis are critical for initiating appropriate public health control measures.

Various studies in different states in India have reported brucellosis as a commonly encountered disease in occupationally exposed individuals with a prevalence rate of 17-34%. (Pathak et al., 2014; Wu et al., 2013). While incidences of human brucellosis have been reported in different epidemiological studies, there is still limited data regarding estimates of brucellosis, especially from tertiary care health settings in India. Such data is critical concerning regular screening of the disease in clinical settings to develop epidemiological data and initiate appropriate control measures.

In the present study, we studied the prevalence and associated risk factors with human brucellosis in a central Indian population from a tertiary care health setting.

Materials and Methods

Study Design and Participant Recruitment

A total of 8102 participants attending the outpatient department (OPD) wards of Central India Institute of Medical Sciences (CIIMS) from March 2015 to February 2018 were prospectively enrolled. Each participant was selected using pre-specified inclusion criteria with added risk factors like unconventional food intake habits (consuming raw milk/food products) and animal exposure. Participants were screened based on clinical symptoms suggestive of brucellosis including fever, joint pain, joint swelling, chest pain, headache, back pain, and night sweating. A detailed medical history of the recruited individuals was collected using a structured questionnaire prepared beforehand by a team of expert clinicians and scientists. Baseline factors like age, gender, and other risk factors were recorded. Out of the 8102 recruited individuals, 380 were excluded due to the refusal for blood collection. Of the 7722 available participants, a total of 696 samples were excluded for the study, which included participants with  Incomplete clinical data (120), Hemolyzed samples (90), Incomplete Baseline data (150), Pregnant women and children (55), Children below 10 years (85), Blood samples not available (100), Mixed infection.  A total of 7026 participants were finally included in the seroprevalence study (Figure 1).

Fig.1. Study flow diagram for participant’s recruitment

Sample collection

2-3 ml blood was collected by a venous puncture in plain vacutainer tubes and allowed to clot at 37⁰C. Serum was collected by centrifugation of blood samples at 2000 rpm for 10 min at 4⁰C and immediately stored at -20⁰C until further use.

ELISA IgM

Detection of immunoglobulin M (IgM) antibodies using Enzyme-linked Immunosorbent assay (ELISA) was performed using a commercial kit (Novatec Immunodiagnostica GmbH, Germany) as per the manufacturer’s instructions as described elsewhere. IgM titers above 0.7 were considered positive. Sensitivity and specificity, as given by the manufacturer, were > 95% for Immunoglobulin G (IgG) and > 95% for IgM, respectively.

Data Analysis 

Descriptive and analytical statistics were used to summarize the data obtained. The frequencies (percentage) of demographics, clinical factors, and risk factors were measured on a nominal scale. The association of baseline characteristics of the study population in brucella positive and negative cases were determined by the chi-square test in MedCalc statistical software (version 10.1.2.0). Results were considered significant if the p-value was < 0.05.

Results

In the present study, we investigated the seroprevalence of brucellosis in the central India region. Out of 8102 recruited, serum samples of 7026 subjects were available for seroprevalence study. Overall and state-wise seroprevalence of brucellosis is mentioned in Table 1.Overall seroprevalence was 11% (772/7026) from hospital settings in central India. All recruited cases belonged to three states from Central India, which included Maharashtra, Madhya Pradesh, and Chhattisgarh. When overall positivity was stratified according to state-wise distribution, more than  half of positive cases from Madhya Pradesh (58.1) were found exposed to brucellosis, followed by Maharashtra (38.8%) & Chhattisgarh (2.9%) (Figure 2).

Age and sex-wise stratification of positive cases are indicated in Table 2. More than half of positive cases (53.1%) in the younger economically productive age group (20-40) were found exposed to brucellosis compared to other age groups (P > 0.001). Around a quarter of positive cases in the age group (40-60) were also found exposed to brucellosis. Based on gender-wise distribution, females were more vulnerable to their female counterparts for brucellosis, although the difference was not statistically significant. 

Table 1. Overall and state-wise seroprevalence of Brucellosis from Central India

Fig. 2. State-wise stratification of Brucella seropositivity in the study population

Among the clinical history recorded on presentation, fever was the most common symptom associated with 95% of brucellosis cases. Apart from fever, Headache (88%), Arthralgia (90%), Myalgia (86%), chronic fatigue syndrome (90%) were also major symptoms associated with brucellosis (Table 3). Among the various risk factors analyzed, direct animal exposure and consumption of raw milk and vegetables along with improperly cooked meat consumption were major factors significantly (P < 0.0001) associated with the occurrence of brucellosis in the study population (Table 4).

Table 2. Age and gender-wise distribution among study subject after brucella diagnosis

We also stratified the study population according to occupation to study vulnerable groups associated with brucellosis. Table 5 shows the occupation wise distribution of Brucella IgM titers in the study population. Among the stratified groups, animal handlers, which included zookeepers and veterinarians, showed the highest seropositivity (66.8%) rates, followed by miscellaneous groups (25.7) and farmers (7.3%) with more or less similar mean IgM levels (Figure 3).

Table 3. Clinical presentation recorded among positive brucellosis cases

Table 4. Risk factors associated with brucellosis in the study population

Table 5. Total positivity of Mean Brucella IgM levels in Occupational wise seropositivity of Brucellosis in the study population

                                       ^a veterinarians, zoo –keeper            ^b  Housewives, labours, students

Discussion

Brucellosis remains the most common zoonotic disease worldwide. Though associated with minimal mortality, its importance is realized in the substantial morbidity associated with it, in humans and animals (Patil et al., 2016). In the present study, we studied the prevalence and associated risk factors with human brucellosis in the central Indian population from tertiary care health settings using commercial IgM ELISA assay. ELISA has been used as a rapid, sensitive, and specific assay. The latest researches showed that ELISA is more reliable for diagnosing brucella infection when compared to the Rose Bengal precipitation test (RBPT)  and Serum agglutination test (SAT) (Mantur et al., 2010). ELISA is capable of readily identifying the individual IgM and IgG antibody to the surface antigens, which allows permitting a better clinical correlation, which could help for diagnosing the early stages of brucellosis; therefore, ELISA has been used for mass screening in suspected and confirmed cases. (Agasthya et al., 2007).

Fig. 3. Box whisker plots showing occupation wise distribution of IgM titers in the study population