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KUFA JOURNAL FOR NURSING SCIENCES Vol. No. 2 May through August 201
Diagnostic and Epidemiologic Study of Human Brucellosis at
Al-Najaf province,
Dr.Thikra Abdullah Mahmood(1), Dr.Salam J. Mohammed (2), Dr.Wisam
S. Abbood(3), Dr. Sabah N. Mohammed(4), Dr. Suaad M. Hashim (5), Dr.
Kareem G. Mohammed (6)
RT- PCR
;
CDC
Abstract
Background: Maltese fever is one of the dangerous occupational diseases for dairy producers, processor and
staff cattle slaughterhouse, veterinarians and laboratory personnel. Infection accompanies a wide range of
clinical symptoms that require the use of effective diagnostic tests. The choice of the epidemiological
diagnosis in the region and the decision of the infection depends on the size of the infestation., Clinical
information provided by the patient must include the geographical area, work type Was traveling to rural
areas or affected countries this disease Is a family member with the disease, treatment he takes the patient
what is positive and negative impact on the patient. Germ Malta fever tend to tissue rich in blood vessels
Aims of study: The study aims to evaluate the epidemiological aspects of the population according to gender
and age. And the study of the property and the sensitivity of the two tests, the first is the use of a conventional
test (Rose Bengal), and modern molecular technique (RT- PCR) and compare the two results.
Methodology: Type of study is a diagnostic study includes 41 blood samples from patients with suspected
Malta fever, from both gender diagnosed questionable, the ages were between 14-45 years. Patients were
attending to the province of Al-Najaf Al-Ashraf from rural area surrounding the governorate and the center
of the province, and attend private clinic from January to December 2015 in order to evaluate serum infection
of Malta carried out using serological testing and technology of molecular test method
Results: A total of 41 patients with suspected brucellosis had been included in this study. The most frequent
age group was more than 35(29.3%) years. The percentage to an urban cases were (58.54%) and the percent
of rural area. Brucellosis, variations during one year shows positive predictive value=50%, Negative
KUFA JOURNAL FOR NURSING SCIENCES Vol. No. 2 May through August 201
predictive value=95.2% and Accuracy =73.1 % from diagnosis of serological compared to Real time PCR
proportion of 41 patients. High sensitivity (90.9 %) has detection of Brucellosis by Real Time PCR, and the
Specificity=66.7%.
Conclusion Estimated incidence in most regions injury (rural areas) may be less compared to the younger
population. Medical safety is very important in the laboratory. Organization of the CDC classifies Iraq from
high incidence areas with Malta fever.
Recommendations: Avoid unpasteurized hand milk products and fresh milk which is not boiled. Clinical
laboratories must be under the terms of health safety when isolate the pathogen. And early treatment of the
disease at the onset of clinical signs of the disease eliminates protect against chronic doubled.
_____________________________________________________________
___
1. PhD. , Department of Community Medicine, College of Medicine/University of Kufa.
2. M.B.Ch.B.,FIBM, Department of Community Medicine , College of Medicine , University of Kufa.
3. PhD. , Department of Medical Microbiology, College of Medicine /University of Qadisiyia.
4. M.B.Ch.B. , FIBM-Immunology /Al-Sadder Medical City/ Kidney Transplant Center.
5. PhD., Department of Community Medicine, College of Medicine/University of Kufa.
6. PhD., Department of Community Medicine , College of Medicine/ University of Kufa.
Htt://www.med@uokufa.edu.iq/dr/thikra; thakraas @gmail .com
INTRODUCTION
Brucellosis is the commonest human zoonotic disease. It is caused by Brucella species:
Gram- negative, aerobic, facultative intracellular coccobacilli bacteria. It is transmitted to human
through consumption of unpasteurized dairy products or undercooked meat from infected animals
and direct contact with infected animals (1), it can enter the body via skin wounds, mucous
membranes, or inhalation the bacteria that causes brucellosis may also lead to infection. This risk
is generally greater for people in laboratories that work with the bacteria. Person-to-person
transmission is rare, infected mothers who are breast-feeding may transmit the infection to their
infants. Sexual transmission, tissue transplantation or blood transfusions have been rarely
reported(2). Initial symptoms can include: Fever, sweats, malaise, anorexia, headache, pain in
muscles, joint, back fatigue and spontaneous abortion in pregnant woman. Some signs and
symptoms may persist for longer periods of tissue, that may continue to trouble patients for as long
as 25 years (3.
These High-risk regions include the South and Central America, Eastern Europe, Asia,
Africa, and the Middle East. In these areas(4) . brucellosis is primarily enzootic in cattle, sheep, and
goat populations, who is exposed to the bacteria that cause brucellosis is at risk for infection
(5).Interest in brucellosis has been increasing because of the growing phenomena of international
tourism and migration, in addition to the potential use of Brucella as a biologic weapon(6).
Diagnosis of brucellosis is complicated because the disease may have an incubation period
varying from five days to five months and can progress in various forms: acute, chronic or
asymptomatic (7). Symptoms of the acute phase of brucellosis fever and weakness in humans is
common to a wide range of different diseases. Therefore, the final diagnosis of brucellosis has to
be obligatorily confirmed by laboratory testing. Brucellosis diagnostics is based on bacteriological
and molecular methods (direct tests), and serological in vitro and in vivo methods (indirect tests)
(8). The choice of the diagnostic method depends on the overall epidemiological situation in the
region and the objectives of the study: validation of the diagnosis, screening (monitoring), cross-
sectional studies or confirmation of brucellosis-free status of the region (9). Rose Bengal is a broadly
used simple method of brucellosis diagnostics is the test). It is a simple spot agglutination test where
drops of stained antigen and serum are mixed on a plate and any resulting agglutination signifies a
positive reaction. The results are received in several minutes(10).
KUFA JOURNAL FOR NURSING SCIENCES Vol. No. 2 May through August 201
Molecular biological techniques, often based on the polymerase chain reaction (PCR)
amplification, are successfully used for Brucella identification and typing. The first crucial step of
PCR based methods is DNA isolation from biological samples (11). The most simple and reliable
method of Brucella identification is PCR with a single pair of primers, specific to the bacterial
DNA sequences, such as 16S - 23S rRNA operon, IS711 or BCSP31 genes (12) .
Bosphore, Real Time- PCR Brucella detection kit detects Brucella spp. DNA in human
biological sample .The analytic sensitivity is 7.5*102 copies /ml. Amplification and fluorescence
detection as a novel approach to assess the molecular response to various infectious diseases (13).
OBJECTIVE
The study aims to evaluate the epidemiological aspects of the population according to gender
and age. And the study of the property and the sensitivity of the two tests, the first is the use of a
conventional test (Rose Bengal), and modern molecular technique (RT- PCR) and compare the two
results.
MATERIALS AND METHODS
Ethics statementHuman Brucellosis case data were extracted from same patients by
present and past case history. The study was approved by Research and Development department
of faculty of medicine.
Study design and Patients: Sample size of diagnostic study was from 41 patients blood
samples, from both genders with suspected Brucellosis according to professional clinical
assessment, their ages were between 14-45 years. Patients were attending in Al- Najaf province
from rural area and urban area, and patients attended private clinics between January and December
from 2015.
Collection of samples: Five milliliters (ml) of venous blood were drawn from each patient
by venipuncture (5ml disposable syringe). Blood was divided into 2 groups.
1. 3 ml of blood were collected in sterile gel serum tube for using Rose Bengal test and left for
serum collection, which was stored at-20 C˚.
2. 1 ml of blood were collected in EDTA tube and extraction of DNA from blood was done in each
sample in the Eppendorf tubes (1-2) ml and were done immediately for RT- PCR test.
Serological DiagnosisA-Agglutination for slide and tube tests Qualitative and Quantities Determination of Serum
Antibody. Complete Agglutination Kits were used. This kit was supplied by (Biorex Diagnostic
Company / United Kingdom) which included:
1. Agglutination reaction for Qualitative and quantities detection of Brucellaabortus in
human was measured by agglutination technique using Kit.
2. Agglutination reaction for Qualitative and quantities detection of Brucellamelitensis in
human was measured by agglutination technique using Kit.
Molecular Diagnosis A- Extraction and Estimation.
1. DNA Extraction: DNA extraction kit was supplied by Bosphore Anatoligene works Company
(Istanbul -Turkey), DNA extraction was performed according to the manufacturer҆ s
instructions.
B- Real Time PCR
KUFA JOURNAL FOR NURSING SCIENCES Vol. No. 2 May through August 201
1-Real – Time PCR test for qualitative detection of Brucella in human was measured by RT-PCR
Kit (Bosphore Anatoli Biotechnologies –company –Turkey) .Programming the Real-Time PCR
Thermo cycler conditions (Amplification):Real-Time PCR Thermo cycler conditions were set
according to kit instructions .
RESULTS :
1. Distribution of age
Table (1): Distribution of age with positive Brucellain the study sample (n=41) .
Age group
Frequency
Percent
<=20
7
17.1
21-25
9
22.0
26-30
8
19.5
31-35
5
12.2
>35
12
29.3
Total
41
100.0
A total of 41 patients with suspected brucellosis had been included in this study. The age of patients
shows in table 1. The most frequent age group was more than 35(29.3%) years; the second most
frequent age group was 21-25 years (22.0%).
2-Residence distribution
.
Figure (2): Residence of studied patients.
This figure showed the human Brucellosis cases at the rural and urban from 2015, and
compared between them (Figure: 2). the percentage to an urban cases were 58.54% and the percent
of rural were 41.44% determine statistically significant differences between each from the rural
area and urban.
KUFA JOURNAL FOR NURSING SCIENCES Vol. No. 2 May through August 201
3-Gender distribution
Figure 2: Gender distribution of study sample
In this figure (figure: 2) showed the difference of gender was the most frequent at female was about
(58.54%).
MOLECULARRESULT Table (2) Diagnostic accuracy of serology compared to Real time PCR proportion of
patients (n=41).
Real time PCR
Total
positive
Negative
Serology
Positive
10
10
20
90.9%
33.3%
48.8%
Negative
1
20
21
9.1%
66.7%
51.2%
Total
11
30
41
100.0%
100.0%
100.0%
In this table(Table 2) showed , Predictive variables have been responsible for 41 patients
Brucellosis, variations during one year showed positive predictive value=50%, Negative predictive
value=95.2% and Accuracy =73.1 % from diagnosis of serological compared to Real time PCR
proportion of 41 patients. High sensitivity (90.9 %) has detection of Brucellosis by Real Time PCR,
and the Specificity=66.7% .
DISCUSSION High sensitivity (90.9 %) has detection of Brucellosis by Real Time PCR, and the
Specificity=66.7% .Serology test of human Brucellosis must take into checking individuals develop
antibodies but do not become infected, that agreement with Chen, et al., (2013) (14).Serological test
KUFA JOURNAL FOR NURSING SCIENCES Vol. No. 2 May through August 201
result and presence of clinical signs with brucellosis are essential to screening, specificity was
reduced to 90.9 %that nearby Gómez MC,et al(2008)( 15), positive Real Time PCR results were
specificity to 66.7% ( 16).
Residence of patients was conducted of human Brucellosis, the rural area (41.44%) was considered
an appropriate for the study of human Brucellosis due to its provision of a suitable environment for
animals .In this study found an urban area (58.54%) was more than rural due to milk production
unsterile ,due to colonized of bacteria leading to frequent milk shedding (17). In urban, consumption
of home-made milk products is a risk factor for brucellosis infections. Sero-positivity for
brucellosis and age, sex, and the consumption of fresh cheese and cream made from unpasteurized
milk (18).Important role in the types of vegetation that can grow in certain areas, temperature ranges,
and other environmental conditions important to the survival and transmission of Brucella(19) .
The most frequent at female was about (58.54%), that disagreement with larger numbers of reported
human cases during 2008 occurred predominantly in a specific gender (male, 70.2%), andthe most
frequent age group was more than 35(29.3%) years, that agreement with age range (30–59 years
old, 64.7%)(20).
CONCLUSION
1. Estimated incident in higher areas ( rural ) may be compared by the younger population.
2. Medical safety is very important in the laboratory.
3. Organization The CDC classifies Iraq from high incidence areas.
RECOMMENDATIONS
1. Avoid unpasteurized milk and fresh milk products is boiled.
2. Clinical laboratory, when isolated pathogen must be under the terms of health safety.
3. Early treatment of the disease when the onset of clinical signs that eliminates the disease.
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