Human Brucellosis: An Observational Study From a Tertiary Care Centre in North India

Aim: The main aim/objective of this study was to detect and characterize the Brucella species from patients having complaints of joint pain and also to know the potential causes of human brucellosis. In our study, we focused on joint pain symptoms that may be due to arthralgia or arthritis. Introduction: Brucellosis is a neglected zoonotic disease that affects both humans and animals. In humans, brucellosis begins with chronic illness leading to great financial losses from not being able to work well and continued treatment costs, but few such studies have come from northern India. Joint pain is the common presentation of brucellosis and there are several risk factors associated with brucellosis. Methods: A total of 200 blood samples were collected from the participants having joints pain from September 2019 to September 2021 at Gandhi Memorial & Associated Hospitals of King George’s Medical University, Lucknow, India, and tested by serology for anti-Brucella IgM and IgG enzyme-linked immunosorbent assay (ELISA), molecular tests byreverse transcriptase-polymerase chain reaction (RT-PCR), conventional polymerase chain reaction (PCR), and automated blood culture system. The anti-Brucella IgM and IgG ELISA were performed using the kit from NovaTec Immundiagnostica GmbH (Dietzenbach, Germany). Isolation of DNA was carried out using the QIAamp DNA Mini kit (QIAGEN, Hilden, Germany), and the primers and probes specific for targeted regions (BCSP31 and IS711 gene) in the Brucella genome were procured from Eurofins Scientific SE (Luxembourg, France), and for internal control from CDC. Result: The study showed 19 (9.5%) and 23 (11.5%) positive results by anti-Brucella IgM ELISA and anti-Brucella IgG, respectively, and of these, one (0.5%) was positive for both anti-Brucella IgM and anti-Brucella IgG ELISA. Out of 19 anti-Brucella IgM ELISA positive, eight (4%) samples were positive for PCR/RT-PCR and that was negative for anti-Brucella IgG ELISA. All blood culture reports of all patients were negative. Conclusion: Anti-Brucella IgM ELISA was more accurate than anti-Brucella IgG ELISA in detecting human brucellosis. Consumption of animal products (i.e. milk, a dairy product of cow, buffalo, goat, and meat of goat) and contact with animals were the main risk factors that were identified for Brucella disease.


Introduction
Brucellosis is the most prevalent and neglected bacterial zoonotic disease, affecting both domestic and wild animal species, besides humans for decades worldwide [1].In humans, brucellosis begins with chronic illness leading to great financial losses from not being able to work well and continued treatment costs; chronic illness is defined broadly as conditions that last one year or more.With regard to brucellosis, joint pain for more than one year is usually considered as chronic disease [2].
Brucellosis is caused by the Gram-negative, facultative intracellular bacteria belonging to the genus Brucella [3].Of 12 known Brucella species, only four species are pathogenic in humans: Brucella melitensis, B. abortus, B. suis, and B. canis [4].These Brucella species are resistant to antibiotics because of their surviving capability within phagocytic cells [3,5,6].
Transmission of brucellosis in humans occurs either directly or indirectly through inhalation of an affected animal's infectious material or consumption of unprocessed animal products [7].Typically, two to four weeks are required for human brucellosis to develop (starts five days after infection and lasts for six months).Initial brucellosis symptoms in humans include recurrent fever, malaise, bone pain or back pain, tiredness, headache, and night sweats.Signs of human brucellosis include lymphadenopathy, hepatomegaly, and splenomegaly, and it is underdiagnosed worldwide.Lymphadenopathy is misdiagnosed with autoimmune disease, malignancies, drug reactions, etc., hepatomegaly is misdiagnosed with tumors, anemias, heart failure, metabolic disorders, etc., and splenomegaly is misdiagnosed with tuberculosis and endocarditis, etc. [5,7,8].
To identify human brucellosis, several laboratory tests are available including serological and molecular tests like polymerase chain reaction (PCR).Culture is very difficult and takes a long time.PCR is the most accurate test for brucellosis and also it is used as an estimation marker for the course of the disease [9,10].
The goal of this investigation was to detect and characterize the Brucella species from symptomatic patients having complaints of joint pain and also to know the risk factors.
This article was previously presented as an abstract at the 45th Annual Conference of the Indian Association of Medical Microbiologists conducted at Kalinga Institute of Medical Sciences (KIMS), Kalinga Institute of Industrial Technology (KIIT) University, Bhubaneshwar, Odisha, India, on November 25, 2022.
The serological and blood culture data used in this article was also published in a previous article by the authors (Kumari et al., 2023) [11].
This article was published as a Preprint on the Access Microbiology server on October 28, 2022 [12].

Materials And Methods
This study included 200 patients aged 7-86 years attending the outpatient and inpatient departments of the Gandhi Memorial & Associated Hospitals of King George's Medical University, Lucknow, India, with a complaint of joint pain over the period of two years from September 2019 to August 2021.The study was approved by the Institutional Ethics Committee of King George's Medical University, Lucknow, Uttar Pradesh, India (approval number: 1168/Ethics/2019).
We collected 200 blood samples in which the serological and molecular test was performed for all samples.A total of 5 ml of venous blood was collected from each patient in two different vials (a plain vial for serology and an ethylenediamine tetraacetic acid (EDTA) vial for molecular).Data collections of all patients including clinical symptoms, sociodemographic characteristics, and occupations were recorded.Centrifugation, performed for 10 minutes at roughly 1500 rpm, was used to separate the serum from the blood.The serum was aliquoted into two labelled clean cryo vials.One tube was used immediately for serology and another serum vial and EDTA whole blood was immediately frozen at -80 degree freezer till further testing (ELISA and molecular).

Sampling method and sample size
Probability sampling methods and the sample size calculation were made by using the below formula.n = Z2P (1-P)/d2 n=22 x0.06x94/ (0.5)2 =90.24 for one year The sample size for one year study is 91 approximately so the sample size for two years will be 200.Where, n= sample size, Z= Z score for a level of confidence (considered 1.96 or 2 at 95% level), P= expected prevalence, and d=precision (considered to be 5%).The seroprevalence of brucellosis is considered to be 6% as per the study of Kaur et al. [13].

Serological assay
The anti-Brucella IgM and IgG ELISA were performed as per the manufacturer's instructions using kits from NovaTec Immundiagnostica GmbH (Dietzenbach, Germany).The calculation was done by following the formula as specified by the manufacturer; for this, absorbance values measured were first converted into NovaTec Units (NTU).
Interpretation of the result was done by following cut-off values, > 11 NTU was considered positive, <9 NTU was considered negative, and 9-11 NTU was considered equivocal as specified by the manufacturer [14,15].

DNA Extraction
DNA extraction was carried out using the QIAamp DNA Mini kit (QIAGEN, Hilden, Germany).A total of 200 µl of whole blood, 20 µl of proteinase K, and 200 µl of lysis buffer were added, and the mixture was incubated at 56 o C for 10 minutes in a water bath.After 10 minutes, 200 µl of absolute ethanol was put into the lysate.After that, the spin column was cleaned and centrifuged by the manufacturer's instructions.Nucleic acid was eluted with 60 µl of elution buffer in the fresh recovery tube provided in the kit, after five minutes of incubation.For positive control, culture isolates of B. abortus and B. melitensis were obtained from Indian Veterinary Research Institute, Izatnagar, Bareilly, India.Primers and probes specific for targeted regions (BCSP31 and IS711 gene) in the Brucella genome were procured from Eurofins Scientific SE (Luxembourg, France) and for internal control from CDC.

Quantitative Real-Time (qRT)-PCR Assay
The primers and probe employed for the identification of genus Brucella were targeted to Brucella cell surface protein-31 kDa (BCSP31-genus-specific) genes; those are conserved within the genus Brucella and as well as all of its biovars.Target gene Rnase P (RNP) was performed as an internal control for the extraction of Brucella (housekeeping gene).Primers as well as fluorescently labeled probes employed in this research are displayed in Table 1.Uniplex RT-PCR tests were executed in a total volume of 10 µl with QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, Massachusetts, United States).PCR reaction 96 well plates contained 8.5 µl Takara uniplex PCR master mixes (Takara Bio Inc., Kusatsu, Shiga, Japan), l.5 µl of primer probes mix of 10 pmol for Brucella spp, and 5 µl templates DNA.In the case of RNP, a 10-µl reaction containing 6.25 µl of buffer (Thermo Fisher Scientific Inc.), 1 µl of the primer-probe mix at a concentration of 10 pmol, 0.5 µl of Taq polymerase enzyme, 2.25 µl of H 2 O, and 5 µl of extracted DNA were used.The procedure for the reaction was carried out in Applied Biosystems QuantStudio 5 (0.2ml).The following conditions existed for RT-PCR: an initial denaturation at 95 o C for 10 minutes, followed by 44 cycles at 95 o C for 20 seconds, and then at 60 o C for 90 seconds.Positive and negative controls were used for each experimental study.

Blood culture
For culture, only 130 patients' blood samples were collected as the remaining patients did not agree to give their samples for culture tests due to them being outdoor patients and the fact that they were not so sick.For culture, 20 ml venous blood from adults and 5-10 ml from children was collected into BacT/ALERT blood culture bottles (bioMérieux SA, Marcy-l'Étoile, France) and subjected to an automated blood culture system (BacT/ALERT) and the cultures were incubated for 21 days at 37°C.Positive BacT/ALERT culture bottles were processed.The methodology is shown in Figure 1.

Statistical analysis
Statistical analysis was done using IBM SPSS Statistics for Windows, Version 21.0 (Released 2012; IBM Corp., Armonk, New York, United States).The prevalence of brucellosis was detected differently depending on the age groups, gender, risk factors, and clinical features.Chi-square test was employed to assess the tests used.Statistical significance was defined as a p-value of <0.05.
Out of the 200 samples, 19 (9.5%) were found positive for anti-Brucella IgM and 23 (11.5%) for anti-Brucella IgG; of these, 1(0.5%) were positive for both anti-Brucella IgM and anti-Brucella IgG ELISA.Out of 19 anti-Brucella IgM ELISA positive, eight (4%) samples were positive for PCR/RT-PCR and negative for anti-Brucella IgG ELISA (Table 3).The sensitivity of anti-Brucella IgM ELISA and anti-Brucella IgG ELISA was 100%, and 62.5%, respectively, while the specificity was 92.7% for anti-Brucella IgM ELISA and 53.1% for anti-Brucella IgG ELISA (Table 4).ELISA is the best tool to identify anti-Brucella antibodies during an earlier phase of the infection.Among 200 samples, eight (4%) were positive for brucellosis by using genus-specific BCSP31 RT-PCR (Figure 2) and by using species-specific gene IS711 conventional PCR.Five samples (2.5%) were found positive for B. abortus (Figure 3) and three (1.5%) for B. melitensis by PCR (Figure 4).Analysis showed that brucellosis mostly affects the age group of 7-69 years.The major group affected was housewives (four patients (5.5%) by PCR and anti-Brucella IgG, and 10 patients (13.7%) by anti brucella IgM) (Table 3).History of contact with animals (buffaloes, cows, dogs, etc.) was in seven (9.2%) for anti-Brucella IgM, eight (10.5%) for anti-Brucella IgG, and four (5.3%) for PCR positivity.Consumption of animal products (i.e.milk, a dairy product of cow, buffalo, goat, and meat of goat) was reported in eight (13.8%) for anti-Brucella IgM, eight (13.8%) for anti-Brucella IgG, and three (5.2%) for PCR (Table 5).The symptoms were fever, headache, chills, and myalgia.All the patients presented with joint pain (n=200; 100%), followed by fever, headache, chills, and myalgia.There was a significant correlation between clinical signs such as night sweats (p = 0.026)) with the positive serology anti-Brucella IgG (Table 6).Age (p= 0.032), and sex (p= 0.043) with the positive patients for anti-Brucella IgG (Table 3) were both significantly associated with each other.No blood culture was found positive for Brucella.

Discussion
Brucellosis is a disease that affects both wild and domestic animals that could be spread among humans by either direct or indirect exposure to infected animals and via unpasteurized milk [17,18].Signs and symptoms of human brucellosis are not specific.RT-PCR might be the most efficient test for identifying brucellosis brought on by B. abortus and B. melitensis because of the increased determination limit [19][20][21].
As a result of the specificity of the chosen genes for detection of Brucella in the genus and species levels, the PCR method was appraised as a gold standard technique in the fast and secure diagnosis of brucellosis except the serological testing to determine a more authentic diagnosis of brucellosis notably in cases which are not decisive.Some other studies like that of Kuila et al. correlate with our findings for anti-IgM ELISA and PCR [9].Similarly, brucellosis prevalence has been widely reported in other parts of India, such as in the Goa region (6.02%), and 4.96% of specimens tested positive by indirect ELISA for anti-Brucella IgM and anti-Brucella IgG, respectively, in the study by Pathal et al. [21].Kaur et al. reported in their study that the

FIGURE 2 :
FIGURE 2: Real-time uniplex Brucella species (Genus specific) PCR amplification plot PCR: polymerase chain reaction; PC: positive control; Δ Rn value: Rn value of experimental reaction − the Rn value of the baseline signal generated by the instrument

TABLE 1 : RT-PCR oligonucleotides primers and probes of BCSP31 for Brucella species
Thermo Fisher Scientific Inc.), 1 µl of each primer at a concentration of 10 pmol, enzyme 0.5 µl (SuperScript III taq mix; Thermo Fisher Scientific Inc.), DNase free water 5.0 µl, and 5 µl of extracted DNA.Conventional PCR was carried out in a thermal cycler 2720.The cyclic conditions for B. abortus and B. melitensis were slightly different.For B. abortus, it was primary denaturation at 95°C for five minutes followed by 35 cycles of denaturation for the template at 94°C for one minute, 60 seconds of annealing at 61°C, and 60 seconds of extension at 72 °C with a final extension cycle at 72°C for seven minutes.For B. melitensis, it was primary denaturation at 95°C for five minutes followed by 36 cycles of denaturation for the template at 94°C for one minute, 60 seconds of annealing at 64°C, and 60 seconds of extension at 72°C with a final extension cycle at 72°C for seven minutes.
RT-PCR: real time-polymerase chain reactionPCR AmplificationTable2shows primer sequences used for the characterization of B. abortus and B. melitensis for conventional PCR.Primers were used in a 25 µl reaction that contained 12.5 µl of PCR Master Mix (SuperScript™ III;

TABLE 2 : PCR oligonucleotides primers of IS711 for Brucella species
[16]ysis of the PCR ProductsPCR products were examined on 2% agarose gel (Thermo Fisher Scientific Inc., Waltham, Massachusetts, United States) in a dilution of 10X TBE Buffer (Tris-borate-EDTA) (1X TBE buffer solution) at room temperature.For gel electrophoresis, 5 µl of the PCR products plus 1 µl 6x loading dye were placed into each gel slot.A ladder of 100 bp (BR Biochem Lifesciences Pvt Ltd, New Delhi, India) was often used to estimate the size of the fragments.The amplified PCR products in agarose gel were viewed and pictured from a gel document system (Alpha-Imager Private Limited, Bengaluru, India).DNase-free water was employed as a negative control, and DNA extracted from B. abortus and B. melitensis was used as a positive control[16].
Four separate PCR estimations were carried out.The first was for the Brucella genus-specific identification, the second PCR for the internal gene or housekeeping gene, and the third and fourth for the species-specific PCR reactions detection of B. abortus and B. melitensis, respectively.

TABLE 3 : Association between demographic factors and anti-Brucella IgM, anti-Brucella IgG, and RT-PCR/PCR results
RT-PCR: real time-polymerase chain reaction

Table 4
shows the receiver operating characteristic (ROC) analysis for anti-Brucella IgM and anti-Brucella IgG ELISA, with RT-PCR/PCR as a gold standard.

TABLE 6 : Association between clinical features and anti-Brucella IgM, anti-Brucella IgG, and RT- PCR/PCR results
RT-PCR: real time-polymerase chain reaction