Human brucellosis is a common zoonotic infection and is still prevalent in many countries of Africa, Middle East, the Mediterranean area, Indian subcontinent, Central America and Central Asia (Papas et al. 2006). In the Middle East, the incidence of human Brucellosis was the highest during the 1990s, although a gradual decline in incidence has been witnessed afterward; still, Saudi Arabia is considered an endemic zone for Brucellosis. The clinical manifestation of the disease constitutes a broad range of signs and symptoms. Patients commonly present with fever, chills, fatigue, joint, muscle and back pain. The fact that symptoms are non-specific and can be shared by other infectious diseases makes it even more difficult for clinicians to diagnose it clinically.
Although the diagnosis is confirmed by isolation of the
Serologically, ELISA is the most popular and widely used diagnostic assay.
A retrospective search was performed on the serum samples that were analyzed by ELISA for the presence of IgG and IgM anti-
A total number of 1051 patients were evaluated for the presence of
IgG and IgM ELISA were performed using Abcam kits (Cambridge, UK; Cat #ab100547 and Cat #ab214568, respectively). All the steps were performed following instructions from the manufacturer. Sera and controls (50 µl) were dispensed to the antigen-coated wells of micro-test plates followed by first incubation for 60 min at 37°C. The wells were then washed followed by addition of anti-human IgG or IgM antibodies conjugated with an alkaline phosphatase enzyme. Later ELISA plates were incubated for another 30 min at 37°C and well were washed again. Enzyme substrate (50 µl) was then added to all wells followed by another incubation for 30 min and finally the stopping solution was added to all wells to inhibit the reaction. The color intensity was measured by ELISA reader.
IgG and IgM values above 11 standard units were considered positive. Values between 9 and 11 were considered uncertain while antibody levels below 9 standard units were considered negative for both IgM and IgG
Blood culture was performed using a minimum of two culture set (one aerobic and one anaerobic) from two different venipuncture sites using a volume of 7–10 ml blood per vial for adult and 1–5 ml blood per vial for pediatric. All the vials were loaded to the Baltic system, which detected any CO2 level change in blood culture vial as an indicator of the growth of the organisms. On identifying specific vial with CO2 level change, subculture was performed for the sample using four plates (5% blood agar plate, Chocolate blood agar, MacConkey agar plate, and
RSAT (Rapid Slide Agglutination Assay Test) was performed using Atlas Medical Kits (Cambridge, UK; Cat #8.01.15.0.0010). All the steps were performed following instructions from the manufacturer. One drop (50 µl) each of serum and control was dispensed into separate circles on the slide test followed by one drop (50 µl) of the antigen into each well. Slides were placed on a mechanical rotator for one minute and agglutination was noted in bright indirect light.
A total of 1102 samples were processed in the Immunology Laboratory of KFHU over a period of two years from January 2015 to December 2017 and ELISA serology was performed on them for IgM and IgG anti-
Out of these samples, 991 were negative for both IgG and IgM antibodies, 68 samples were positive for both IgG and IgM, 28 samples were positive for IgG and negative for IgM while 15 samples were positive for IgM and negative for IgG antibodies against
68 double-positive (IgG- and IgM-positive) samples belonged to 38 patients. The number of times serology was repeated for these patients and the number of times the result was the same (IgG and IgM positive) ranged between 2 and 5. The
The serology patterns for
Serology Pattern | Number of Samples | Number of Patients | Number of times serology was repeated with the same results | |
---|---|---|---|---|
1 | IgG−IgM− | 991 | 976 | 1 |
2 | IgG+IgM+ | 68 | 38 | 2–5 |
3 | IgG+IgM− | 28 | 22 | 1–5 |
4 | IgG−IgM+ | 15 | 15 | 0 |
We have further divided single-positive patients into two groups. The first group included patients with serology positive for IgG and negative for IgM and the second group included patients who were positive for IgM and negative for IgG against
Of the 22 patients, two had additional risk factors of possible exposure to
Most common presenting complaint was fever followed by musculoskeletal pain. 12 patients presented with fever while five patients had joint-related symptoms that led the physician to request serology. Other symptoms leading to a serological assessment of
RSAT test was requested for only two patients and the serology was negative for both. Blood culture for
The definite clinical diagnosis of these patients is presented in Table II. Out of 12 patients that presented with fever, the infectious cause was identified in seven with only one patient diagnosed with active brucellosis. Out of five patients with musculoskeletal pains, three were diagnosed as having connective tissue disease, while polyarthralgia and mechanical neck pain was a diagnosis in remaining two. Out of three patients with the symptoms of abdominal pain, one patient with accompanying fever was diagnosed as having syphilis, while the other two were diagnosed with self-limited abdominal pain and prostate malignancy. The diagnosis of lymphoproliferative disorder was established in one patient with pancytopenia. Stroke was the diagnosis in one patient with the weakness of limbs. No final diagnosis could be established in the remaining two patients who presented with dizziness and were treated symptomatically by the local physician.
Patients with positive IgG and negative IgM anti-
Age | Sex | Risk factors | Symptoms | Other diagnostic tests | Final diagnosis | |
---|---|---|---|---|---|---|
1 | 48 yr | M | No | Arthralgia | Culture negative, ANA positive | Connective tissue disease |
2 | 45 yr | F | No | Fever, knee joint pain | Culture negative, Rubella IgG positive, ANA positive | Connective tissue disease |
3 | 59 yr | M | No | Paralysis (TIA) | Culture negative | Stroke |
4 | 36 yr | M | No | Fever, cough, | Culture negative | Tuberculosis |
5 | 31 yr | M | No | Fever and fatigue | Culture negative, Monospot positive, VCA-IgM positive | EBV – infectious mononucleosis |
6 | 35 yr | F | No | Joint pain | Culture negative, ANA positive | Polyarthralgia |
7 | 7 yr | M | No | Fever | Culture negative, RSAT negative | Meningitis |
8 | 34 yr | M | Worker at slaughter-house | Fever | Culture positive, VCA-IgM negative | Brucellosis |
9 | 35 yr | M | No | Fever, fatigue, malaise | Culture negative | PUO |
10 | 45 yr | M | No | High-grade fever | Culture negative | Self-limited febrile syndrome |
11 | 64 yr | M | No | Fever and weight loss | Culture negative, HIV-Ab positive | HIV |
12 | 27 yr | M | No | Fever and low BP | Culture and RSAT negative | Septic shock of unknown origin |
13 | 17 yr | M | No | Fatigue and dizziness | Culture negative | – |
14 | 26 yr | M | No | dizziness | – | – |
15 | 46 yr | M | No | Pancytopenia | Culture negative | Lymphoproliferative disease |
16 | 17 yr | F | No | Joint pain | Culture negative, ANA positive | Connective Tissue Disease |
17 | 31 yr | F | No | Fever | Culture negative | PUO |
18 | 17 yr | M | No | Fever and abdominal pain | Culture negative, Syphilis-Ig positive | Syphilis |
19 | 28 yr | M | No | Abdominal pain | Culture negative | Self-limited unspecified abdominal pain |
20 | 44 yr | M | Medical laboratory Technologist | Generalized abdominal pain | Culture negative | Prostate malignancy |
21 | 22 yr | M | No | Neck and right shoulder pain | Culture negative, ANA negative | Mechanical neck pain |
22 | 32 yr | M | No | Low-grade fever | Culture negative, HBsAg positive | Hepatitis-B |
Other than the risk of living in a high prevalence country, one patient had an additional risk of working as a nurse in the infectious diseases clinic for two years.
Fever was the most common presenting symptom and the reason behind requesting Brucellosis workup in eight out of 15 patients. Joint pains became the second most common cause leading to request ELISA for anti-
RSAT was performed for 13 patients and turned out positive for four patients and culture was performed for 13 patients and turned out negative for all of them. The final diagnosis for each patient with IgM positive and IgG negative anti-
Patients with positive IgM and negative IgG anti-
Age | Sex | Risk factors | Symptoms | Other diagnostic tests | Final diagnosis | |
---|---|---|---|---|---|---|
1 | 14 yr | F | No | Chest pain, cough and fever | Negative blood culture and RSAT | URTI |
2 | 30 yr | M | No | Shoulder pain | Negative blood culture and RSAT | Cervicalgia |
3 | 40 yr | F | No | Fever and arthralgia | Negative blood culture and positive RSAT | Acute cystitis |
4 | 32 yr | F | No | Abdominal pain and splenomegaly | Negative blood culture and RSAT, ANA positive | SLE |
5 | 22 yr | F | No | Arthralgia | – | Multiple sclerosis |
6 | 37 yr | F | No | Fever and back ache | – | Spondylarthrosis |
7 | 25 yr | M | No | Fever and body aches | Positive Syphilis-Ig, Negative blood culture and RSAT | Syphilis, HTN |
8 | 17 yr | M | No | Myalgia and arthralgia | Positive ANA, Negative blood culture and RSAT | Connective tissue disease/SLE |
9 | 31 yr | F | No | Arthralgia | Positive ANA and dsDNA, Negative blood culture and positive RSAT | SLE |
10 | 24 yr | M | Nurse in a Medical Unit | Fever and fatigue | Negative blood culture and positive RSAT | PUO |
11 | 30 yr | M | No | Fever and Myalgia | Influenza PCR positive, Negative blood culture and positive RSAT | Influenza |
12 | 30 yr | F | No | Backache | Negative blood culture and negative RSAT, ANA positive | CTD/SLE |
13 | 39 yr | M | No | Fever cough chest pain hemoptysis | Negative RSAT | Community acquired pneumonia |
14 | 22 yr | M | No | Hematuria | Negative blood culture and negative RSAT | Renal stones |
15 | 24 yr | M | No | Fever | Negative blood culture and negative RSAT | PUO |
Not a single patient was diagnosed as having brucellosis. Of all the patients that presented with fever, one had influenza while another had syphilis as definite diagnosis after demonstration of positive the Influenza PCR and the Syphilis-Ig, respectively. Pyrexia of unknown origin (PUO) was diagnosed in two patients with fever. In another two patients, the diagnosis of acute cystitis and spondylarthrosis was established. Tuberculosis and community-acquired pneumonia remained the final diagnosis in two patients that presented with a cough and chest pain. In four patients, the demonstration of antinuclear antibodies (ANA) led to the diagnosis of systemic lupus erythematosus (SLE). Cervicalgia was diagnosed in one patient who presented with shoulder pain while another patient with arthralgia was diagnosed with multiple sclerosis. One patient with hematuria as the chief presenting complaint was diagnosed as having renal stones.
Saudi Arabia is considered a high prevalence zone for brucellosis and the prevalence is higher in a rural community as compared to urban areas. Non-specific presentation and a high index of suspicion on part of local physicians enabled us to describe a large series of patients who presented to the KFUH ID clinic with the clinical picture suggestive of brucellosis and variable patterns of serology results. Most common symptoms were fever and musculoskeletal pains.
Serology was performed by ELISA for all patients. Different patterns of positivity were observed for IgG and IgM anti-
ELISA has a diagnostic advantage over other serological assays in an endemic setting where is a need to process a huge number of samples. However, sensitivity and specificity of IgG and IgM anti-
Out of 53 patients who had IgM-antibodies in their sera, 38 were also positive for IgG and all of these double-positive patients were diagnosed for brucellosis based on suggestive clinical picture and isolation of the organism from the blood culture. None of the 15 patients who had only IgM antibodies against
Furthermore, the other possible reason for false posi tive IgM antibodies could be the presence of rheumatoid factor (ISCIII 2018). Therefore, it is recommended to remove rheumatoid factor by pre-absorption before the determination of IgM anti-
Mantecón et al. (2006) have described IgG anti-
Only one patient with IgG only antibodies detected in his serum was diagnosed with brucellosis on confirmation by isolation of
The RSAT is considered a suitable screening test for the diagnosis of brucellosis; however, considering a great proportion of false-positive and false-negative results reported by RSAT, it is recommended to use a supplementary laboratory technique like ELISA or MAT to further confirm the results of RSAT (Geresu et al. 2016). Four out of 13 patients with IgM only antibodies against
Different studies carried out on the sensitivity and specificity of IgG and IgM anti-
Being retrospective research, our study is subjected to some limitations. It was not possible to compare ELISA results with the MAT, the gold standard for serological diagnosis, to rule out false-positive and false-negative results for the determination of IgG and IgM anti-
To conclude, the combined sensitivity of IgG and IgM against