Abstract
Introduction:
Brucellosis, which is among the endemic regions of Turkey, is a common zoonotic disease. The gold standard in diagnosing brucellosis is culture. We aimed to compare demographic characteristics, risk factors, and clinical and laboratory variables between cases with culture positivity and undetected in culture.
Materials and Methods:
This single-center study was conducted between January 2007 and April 2022. Clinical and laboratory data of patients with brucella growth in blood culture and patients without growth were compared.
Results:
A total of 150 patients were included in the study. The median age was 10 (1–18 years). Of the patients, 66 (44%) were female and 84 (56%) were male. Forty (26.7%) of the patients were bacteremic and 110 (73.3%) were nonbacteremic. In the bacteremic group, white blood cell count, platelet, and hemoglobin counts were lower, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) values were higher. In clinical evaluation, fever, hepatomegaly, splenomegaly, and abdominal pain were more common in the bacteremic group.
Conclusion:
The distinction between bacteremic and nonbacteremic brucellosis can be predicted using laboratory values such as white blood cells, hemoglobin counts, platelet, ALT, and AST, and clinical findings such as fever, abdominal pain, hepatomegaly, and splenomegaly.
Introduction
The most prevalent zoonotic illness, brucellosis, is caused by the bacterium Brucella, a tiny, nonmotile, Gram-negative coccobacillus, and has 500,000 cases per year worldwide (Kimberlin, 2021; Pappas et al., 2006). Children make up between 10% and 30% of all brucellosis cases (Roushan and Amiri, 2013). There are 12 known species of Brucella bacteria, and the four most significant in terms of their ability to infect both humans and animals with the disease are Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis (Yagupsky et al., 2019). Humans who have brucellosis can experience a wide range of symptoms, from nonspecific ones like mild flu-like symptoms to significant ones, including heart, musculoskeletal, and nerve system involvement. Most commonly, nonspecific symptoms of systemic brucellosis include fever, malaise, anorexia, headache, muscle pains, arthralgia, lethargy, and sweating (Galińska and Zagórski, 2013; Shakir, 2021; Tao et al., 2021).
Since the isolation of Brucella organisms in cultures remains the gold standard of diagnosis, the clinician faces a difficult situation in the event of a negative culture due to inherent challenges like the slow growth characteristics of Brucella species, safety concerns in the laboratory, and reduced sensitivity in prolonged illness and focal infections. Although new diagnostic techniques have been developed, such as nucleic acid amplification tests (NAAT), the diagnosis of nonbacteremic (or low bacteria to detect) conditions in children remains a key concern (Yagupsky et al., 2019).
In this study, we compared patients with brucellosis who were bacteremic with those who were not, as well as the risk factors, symptoms, clinical variations, and laboratory findings.
Materials and Methods
This retrospective single-center study was conducted in a Pediatrics and Pediatric Surgery Training and Research Hospital, which is a referral center for pediatric patients. The study included patients diagnosed with Brucella, who were hospitalized in the pediatric infectious diseases service or admitted to the outpatient clinic between January 2007 and April 2022. All patients included in the study were found to have a positive serum agglutination test, in addition to epidemiological linkage such as drinking raw unpasteurized milk and dairy product consumption, cattle contact, medical history, and physical examination findings supporting the diagnosis of brucellosis. Data such as age, gender, symptoms, risk factors such as unpasteurized milk and dairy product consumption or cattle contact, family history for Brucella, laboratory values, standard agglutination test (SAT), and culture result, through electronic medical record system and patient files, were collected and evaluated.
Laboratory tests include complete blood count (white blood cells, hemoglobin, and platelet counts), C-reactive protein (CRP), erythrocyte sedimentation rate, serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT) concentrations, and blood culture. The standard tube agglutination test for Brucella was evaluated. Serum Brucella agglutination test ≥1/160 titer and above were considered positive. All patients diagnosed with Brucella between 1 and 18 years of age were included in the study. Patients whose data could not be accessed and who did not have blood cultures were excluded from the study.
The patients were regrouped into two groups, including Group I, which constitutes the bacteremic cases, and Group II, which constitutes nonbacteremic cases. The blood samples collected by venipuncture were inoculated into pediatric blood culture bottles and they were incubated in an automated blood culture system (BD BACTEC Peds Plus/F; Becton Dickinson Company, USA). The incubation period of blood samples taken from brucellosis suspected cases was extended up to 7 days. When the growth signal was detected, blood samples were inoculated onto sheep blood agar, chocolate agar, and eosin methylene blue agar plates. In addition, smears prepared from positive blood culture bottles were stained with Gram stain. Identification of isolated bacteria was carried out using an automated system (VITEK 2; bio Merieux, France).
Serological analysis for brucellosis was performed by SAT. Serum samples were incubated with Brucella abortus antigen (Brucella abortus Tube Antigen supplied from Public Health Institution of Turkey, Ankara, Turkey). Brucella antigen for 24 h at 37°C was evaluated for the presence of agglutinin particles and samples with titers greater than 1/80 were considered positive. Clinical compliance was evaluated and SAT was repeated in patients with scores between 1/80 and 1/160, and those with titer increases were considered significant.
Statistical analyses
Statistical analysis was performed using SPSS statistical software (version 26; SPSS, Chicago, IL, USA). The categorical variables were analyzed using relative frequencies, and numerical variables using median or mean (depending on whether they show normal distribution) values. Categorical variables were compared using Pearson chi-squared and Fisher's exact tests. Numerical variables between the bacteremic and nonbacteremic groups were compared using the t-test or the nonparametric Mann–Whitney U test. A p value of <0.05 was considered to be statistically significant.
Results
Patient overview
A total of 167 patients with Brucella diagnoses were included in the preliminary evaluation. Among them, 17 patients, including 3 patients with Brucella isolation and 14 patients without Brucella isolations, were excluded, due to the unavailability of data. At last, a total of 150 patients who followed up with the diagnosis of brucellosis were included in this study. There were 66 (44%) females and 84 (56%) males among the 150 patients. The mean age was 9.5 ± 4.2 years (ranging from 1 month to 17 years).
Regarding the age distribution, 34 (22.7%) were between 1 and 5 years of age, 67 (44.6%) were between 5 and 12 years, and 49 (32.7%) were 12 years of age and older. When the patients were evaluated according to risk factors, 53 (35.3%) had unpasteurized milk consumption and 20 (13.3%) had a history of animal contact. Of the patients, 29 (19.3%) had a family history.
Due to the growth of Brucella in the blood culture, 40 (26.7%) patients were considered bacteremic, while 110 (73.3%) were deemed nonbacteremic since Brucella was not detected in the culture.
Demographic comparison
While 14 (35%) of the patients were girls and 26 (65%) of them were boys in group 1, 52 (47.3%) patients were girls and 58 (52.7%) were boys in group 2 (p > 0.05). The mean age in group 1 was 9.2 ± 4.5 years, while group 2′s mean age was 9.6 ± 4.2 years. There was no significant difference between both groups (p > 0.05).
Consumption of unpasteurized milk was present in 12 (30%) of the bacteremic patients and 41 (37.3%) of the nonbacteremic patients. When the patient was evaluated in terms of animal contact, 2 (5%) of the bacteremic patients and 18 (16.4%) of the nonbacteremic patients had a contact history. When compared in terms of family history, 23 (20.9%) of the bacteremic patients and 6 (15%) of the nonbacteremic patients had a family history, with no significance (p > 0.05).
When compared in terms of prior antibiotic use, 18 (45%) patients in the bacteremic group and 51 (46.1%) patients in the nonbacteremic group had a history of prior antibiotic use, and there was no statistical difference (p > 0.05) (Fig. 1). While fever and abdominal pain were statistically significantly higher in the bacteremic group, there was no difference between the two groups in terms of other symptoms. In the physical examination findings, hepatomegaly and splenomegaly were highly significant in the bacteremic group (p < 0.01). The symptoms and physical examination findings are reviewed in Table 1.
Comparison of prior antibiotic use between bacteremic and nonbacteremic patients.
Comparison of Symptoms and Physical Examination of Bacteremic and Nonbacteremic Patients
When both groups were compared, it was seen that white blood cell, platelet, and hemoglobin count were lower (p = 0.015, p = 0.02, and p = 0.01); AST and ALT values were higher in the bacteremic group compared to the nonbacteremic group (p = 0.047 and p = 0.012). Table 2 reviewed the laboratory findings.
Comparison of Laboratory Findings Between Bacteremic and Nonbacteremic Patients
ALT, alanine aminotransferase; AST, aspartate aminotransferase.
Discussion
In our study, bacterial isolation was detected in culture in 26.7% of the patients with Brucella. Our study showed that laboratory values such as white blood cell count, hemoglobin level, platelet count, AST, and ALT can be useful in distinguishing between bacteremic and nonbacteremic patients.
The diagnosis of brucellosis requires the isolation of organisms from blood or body tissues or a combination of clinical presentation and positive serology (Memish et al., 2000). In recent years, it has been one of the diagnostic tests used in the diagnosis of NAAT (Pappas and Papadimitriou, 2007; Yagupsky et al., 2019). However, the gold standard for the diagnosis of brucellosis is still the identification of Brucella species from blood or bone marrow cultures. Since bone marrow aspiration is a more invasive method, blood culture is preferred in the first place (Qie et al., 2020).
Studies have shown that positive Brucella infection rates from blood cultures can vary between 16% and 90%, and most studies reported the rate as accumulated between 26.7% and 27.9% (Kara and Cayir, 2019; Özdem et al., 2022; Shi et al., 2021). Culture growth of Brucella can be detected at a low rate in the early period due to low concentration and in the late period due to the organism being removed from the bloodstream and isolated in macrophages (Yagupsky et al., 2019). Therefore, the higher rate of nonbacterial Brucella may be related to the hospital admission process, such as early admission due to the knowledge of the disease in the immediate environment, or delayed admission due to recurrent applications.
In our study, 40 (26.7%) of the 150 patients we followed up with the diagnosis of brucellosis had Brucella growth in the blood culture, while 110 (73.3%) were not bacteremic and were consistent with these studies. In a previous study from our center, the culture positivity rate was reported to be around 34.4%, while this study found a lower rate. The first study, including the first 10-year period in our center, revealed that a total of 44.8% of the Brucella patients had a story of previous antimicrobial usage, while in the latter one, 46.8% of the patients had antimicrobial usage, supporting the lower rate might not be associated with previous antimicrobial usage (Apa et al., 2013).
SATs continue to be the most guiding tests in the diagnosis because they do not always provide culture growth; a titer of ≥1/160 is considered positive and its sensitivity ranges from 65% to 95% (Roushan and Amiri, 2013). Although a study indicated that as the titer of the SAT increased, the rate of culture positivity elevated, many studies found that there was no correlation between them, as in our study (Apa et al., 2013; Kara and Cayir, 2019; Pappas and Papadimitriou, 2007).
Unlike some studies in which bacteremic patients were younger than nonbacteremic patients, there was no significant age difference between bacteremic and nonbacteremic patients in our patient group (Kadanali et al., 2009; Özdem et al., 2022). In a study conducted in adult patients with a diagnosis of brucellosis, it was observed that there was no significant age difference between the bacteremic and nonbacteremic groups (Qie et al., 2020).
The primary mode of transmission of brucellosis is the consumption of unpasteurized dairy products in endemic countries. In some epidemiological studies from Turkey, a history of raw milk product consumption was reported for 62.6% to 94.6% of cases (Buzgan et al., 2010). The most common risk factor in our study was the consumption of unpasteurized dairy products with 35.3%, which was the primary risk factor.
The most common symptoms of patients diagnosed with Brucella were fever, arthralgia, myalgia, abdominal pain, and sweating, similar to other studies (Memish et al., 2000; Roushan and Amiri, 2013). When the clinical and examination findings were evaluated, fever, hepatomegaly, and splenomegaly were found to be high in our patient group, similar to studies in which fever, arthralgia, anorexia, hepatomegaly, and splenomegaly were found to be significantly higher in the culture-positive group. Unlike other studies, abdominal pain was also significantly higher in the bacteremic group (Özdem et al., 2022; Shi et al., 2021).
Different values were found to be significant in different studies on laboratory values. According to the studies, when significant values were evaluated in the bacteremic group compared to the non-bacteremic group, leukopenia and high AST values were detected in one study, high CRP, AST and ALT values were detected in one study, low hemoglobin, high CRP and AST values were detected in one study, and low hemoglobin and high AST, ALT and CRP values were detected in one study (Apa et al., 2013; Kara and Cayir, 2019; Özdem et al., 2022; Shi et al., 2021). In our study, white blood cell, platelet, and hemoglobin counts were lower, and AST and ALT values were higher in the bacteremic patients, supporting possible initial steps of the systemic inflammatory response of bacteria.
The limitations of the study are that all data cannot be evaluated because it is retrospective and it has a relatively low sample size. Although the sample size is relatively low, the results are consistent with other studies. In addition, we could only reach data on the presence of previous antimicrobial usage from the patients' history, but could not have the generic names of antimicrobial drugs, and thus could not evaluate if they are effective against Brucella.
In conclusion, in the diagnosis and follow-up of brucellosis, markers such as fever, abdominal pain, hepatomegaly, splenomegaly, white blood cell count, hemoglobin, platelet count, AST, and ALT may be significant in distinguishing between bacteremic and nonbacteremic Brucella.
Footnotes
Authors' Contributions
I.D. conceptualized and designed the study, carried out the initial analyses, and reviewed and revised the article. M.Y.Ç. collected data, carried out the initial analyses, and drafted the initial article. S.N.B. conceptualized and designed the study, coordinated and supervised data collection, and reviewed and revised the article. E.B., E.K., S.Ş., E.C., M.G., P.K., E.Ö., A.A.K., G.G.Ö., F.Y.A.,Y.S., and H.A. designed the data collection instruments, collected data, and contributed essential intellectual content.
All authors approved the final article as submitted and agree to be accountable for all aspects of the work.
Ethical Approval and/or Institutional Review Board (IRB)
Ethical approval was received for this research from Dr. Behçet Uz Child Disease and Pediatric Surgery Training and Research Hospital Ethics Committee.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
No funding was received for this article.