Retrospective case–control study of haematological indices and inflammatory ratios in the diagnosis of paediatric brucellosis

Abstract

Background

Brucellosis is a common zoonotic infection in Turkey and other Mediterranean countries. In children, symptoms are usually non-specific, including fever, fatigue, arthralgia, and myalgia, which may complicate diagnosis. Haematological indices and inflammatory ratios have recently been studied as possible supportive markers in infectious diseases, but findings in brucellosis remain inconsistent. This study investigated red cell distribution width (RDW), plateletcrit (PCT), platelet indices, neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) in paediatric brucellosis.

Materials and methods

This retrospective case-control study included 42 children with brucellosis and 39 healthy controls. Patients were followed at Erzurum Regional Training and Research Hospital between September 2018 and February 2019. Diagnosis was based on compatible clinical findings together with a standard tube agglutination (Wright) titer ≥ 1:160, a positive Brucella Coombs test, or isolation of Brucella spp. from blood culture. Complete blood count parameters were measured using a Sysmex XN-series haematology analyser with a DI-60 morphology modüle. Statistical analyses included the t-test or Mann–Whitney U test for continuous variables and chi-square or Fisher’s exact test for categorical variables. Benjamini–Hochberg correction was applied for multiple comparisons. ROC curve analysis and age- and sex-adjusted binary logistic regression were also performed.

Results

Thirty patients were male and 12 were female. The median age was 14 years (range 6–17). The most common symptoms were arthralgia (85.7%), weakness (71.4%), and fever (66.7%). RDW, platelet count, and PCT differed significantly between groups in unadjusted analysis, but these differences lost significance after correction for multiple testing (all FDR-adjusted p > 0.18). ROC analysis showed low diagnostic performance overall. RDW demonstrated high specificity (92.3%) but low sensitivity (46.3%). After adjustment for age and sex, only RDW remained independently associated with brucellosis (adjusted OR 2.31, 95% CI 1.30–4.11, p = 0.005). Other haematological indices and inflammatory ratios were not significantly different.

Conclusion

Only RDW remained significantly associated with brucellosis after adjustment for age and sex. RDW may provide supportive information together with clinical and serological findings, although larger studies are needed to confirm these results.

Introduction

According to the World Health Organization, brucellosis ranks as the most prevalent zoonotic infection globally and remains endemic in the Mediterranean basin, the Middle East, and Western Asia, including Turkey, where paediatric incidence remains substantial despite eradication programs [1, 2]. Transmission to humans occurs through contact with infected animals, ingestion of raw milk or dairy foods, or inhalation of contaminated aerosols. The causative agent of brucellosis is Brucella, which are non-motile, gram-negative coccobacilli [1]. Unlike adults, almost all paediatric cases of brucellosis stem from the consumption of unpasteurized dairy products [2]. The incubation period varies from 3 to 5 days to several months, with symptoms typically manifesting 3–4 weeks after exposure to the pathogen [3]. Brucella replicates in regional lymph nodes after transmission and enters the bloodstream. It may involve multiple organ systems such as the reticuloendothelial, genitourinary, cardiovascular, respiratory, musculoskeletal, and central nervous systems [1, 4]. Patients with brucellosis present with non-specific clinical symptoms such as fever, weakness, sweating, joint pain, arthritis, myalgia, and headache [5, 6].

While the gold standard diagnostic test for brucellosis is Brucella blood culture, serological tests such as serum agglutination, Coombs test, and ELISA are more commonly used in clinical practice because of their rapid turn-around [4, 7]. However, in endemic regions a single agglutination titer may overlap with the seroprevalence of subclinical exposure, which limits the specificity of a stand-alone serological cut-off.

There is therefore a clinical need for inexpensive, readily available adjunctive markers to complement clinical and serological evaluation. Routine complete blood count parameters have attracted increasing interest as potential biomarkers in infectious, rheumatologic, and oncologic diseases [8–12]. In particular, platelet indices (MPV, PDW, PCT, P-LCR) and leukocyte-based ratios (NLR, PLR, MLR) have been investigated in adult and paediatric brucellosis with mixed and sometimes contradictory results [2, 8, 13]. To date, only a small number of paediatric studies have specifically examined RDW and PCT in brucellosis, and their independent contribution after adjustment for age and sex has not been formally evaluated.

The aim of the present study was therefore: (i) to compare a comprehensive panel of haematological indices and inflammatory ratios between children with confirmed brucellosis and healthy controls; (ii) to assess the diagnostic performance of the most promising markers through ROC analysis with AUC, sensitivity, specificity, and cut-off values; and (iii) to test whether any of these markers are independently associated with brucellosis after adjustment for age and sex. The specific novelty of this work is the focused evaluation of RDW as an adjunctive diagnostic marker in paediatric brucellosis, and the combined application of ROC analysis, multivariable logistic regression, and multiple-comparison correction in a field where previous studies have mostly relied on univariate testing.

Materials and methods

Study design and population

This was a single-center, retrospective case–control study conducted at the Paediatric Infectious Diseases Clinic of Erzurum Regional Training and Research Hospital between September 2018 and February 2019. All consecutive paediatric patients aged 1–17 years admitted during this period with suspected brucellosis were screened for eligibility, and 42 patients meeting the inclusion criteria were enrolled. A control group of 39 healthy children of comparable age, who presented to the paediatric outpatient clinic for routine check-ups during the same period and had no current complaints or abnormal physical examination findings, was included for comparison.

Inclusion criteria

Cases were included if they had clinical findings compatible with brucellosis (e.g., fever, arthralgia, fatigue) together with at least one of the following laboratory criteria: a standard tube agglutination (Wright) titer ≥ 1:160, a positive Brucella Coombs test, or isolation of Brucella spp. from blood culture. All cases underwent the same routine diagnostic work-up, including the Rose Bengal test, Wright test, Brucella Coombs test, and blood culture (BACTEC automated system). A Wright titer ≥ 1:160 was considered positive in line with national and WHO criteria.

Exclusion criteria

Both patients and controls were excluded for: (i) a previous diagnosis of brucellosis, to avoid persistent seropositivity from past exposure; (ii) a documented haematological disorder, including iron-deficiency anaemia, thalassemia, or other inherited red-cell or platelet disorders that can alter RDW, platelet indices, and PCT; (iii) any concurrent active infection at the time of sampling; (iv) a known chronic inflammatory, autoimmune, malignant, hepatic, renal, or endocrine disease; or (v) use of any medication known to affect the complete blood count (e.g., corticosteroids, anticoagulants, cytotoxic drugs) within the preceding four weeks. Alternative diagnoses such as juvenile idiopathic arthritis, acute rheumatic fever, and viral or other bacterial infections were also ruled out before enrollment.

Data collection

Demographic data (age, sex), presenting complaints, physical examination findings, and laboratory parameters were retrospectively obtained from patient records. Complete blood count parameters were measured using a Sysmex XN-series automated haematology analyser integrated with a DI-60 digital morphology module (Sysmex Corporation, Kobe, Japan), with the same calibration and quality-control protocol applied for cases and controls. For cases, laboratory data included complete blood count indices (Haemoglobin, MCV, RDW, platelet count, MPV, PDW, PCT, P-LCR), absolute neutrophil, lymphocyte, and monocyte counts, inflammatory ratios (NLR, PLR, MLR), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and liver enzymes (ALT, AST). For controls, only complete blood count parameters were available, since routine biochemistry was not requested in healthy children attending the outpatient clinic for check-ups. Group comparisons were therefore restricted to haematological parameters. Treatment regimens and outcomes of cases were also recorded.

Treatment and follow-up

Treatment regimens consisted of various antimicrobial combinations tailored to the patient’s age. Commonly used agents included oral rifampicin, oral doxycycline (in patients older than 8 years), oral trimethoprim/sulfamethoxazole, intramuscular streptomycin, intramuscular gentamicin, and intravenous ceftriaxone. Patients without joint involvement received treatment for 6 weeks, those with joint involvement for 12 weeks, and the patient with neurobrucellosis was treated for approximately one year. Relapse was defined as the recurrence of clinical symptoms after an initial symptom-free interval following completion of antimicrobial treatment, and was distinguished from treatment failure, defined as persistence of symptoms during or at the end of therapy.

Statistical analysis

Group comparisons were performed using the t-test or Mann–Whitney U test for continuous variables and the chi-square or Fisher’s exact test for categorical variables. The Benjamini–Hochberg false discovery rate (FDR) procedure was applied separately within each comparison Table (15 haematological tests in Tables 2 and 15 in Table 3) to correct for multiple comparisons. Diagnostic performance was assessed by ROC curve analysis with area under the curve (AUC), 95% confidence intervals, and Youden index–derived cut-offs. Independent associations with brucellosis were tested in age- and sex-adjusted binary logistic regression models. Because of the retrospective design, an a priori sample size calculation could not be performed; therefore, a post-hoc sensitivity power analysis was conducted for the comparison between children with brucellosis and healthy controls. With 42 patients and 39 controls, assuming a two-sided α level of 0.05 and 80% power, the study was able to reliably detect effect sizes of Cohen’s d ≥ 0.63. Analyses were performed in SPSS v21 (IBM, Chicago, IL, USA), and p < 0.05 was considered statistically significant.

Table 2.

Comparison of laboratory data between patients with brucellosis and healthy controls

Haematological parameter	Brucellosis (n = 42)	Controls (n = 39)	p	FDR-adj. p
Haemoglobin, g/dL (mean ± SD)	14.0 ± 1.4	14.2 ± 0.9	0.194	0.410
MCV, fL (median, min–max)	81.6 (70.0–87.5)	80.3 (75.5–97.3)	0.620	0.818
RDW, % (median, min–max)	12.8 (12.0–17.2)	12.8 (11.7–14.5)	0.025	0.188
Platelet count, /mm³ (mean ± SD)	289,857 ± 64,581	326,769 ± 86,109	0.021	0.188
MPV, fL (mean ± SD)	9.6 ± 0.6	9.4 ± 0.7	0.154	0.385
PDW, % (median, min–max)	10.4 (8.2–13.3)	10.4 (8.1–15.7)	0.138	0.385
PCT, % (mean ± SD)	0.27 ± 0.06	0.30 ± 0.07	0.044	0.220
P-LCR, % (mean ± SD)	21.9 ± 4.7	20.8 ± 6.1	0.566	0.818
WBC, /mm³ (mean ± SD)	6,964 ± 1,978	7,181 ± 1,972	0.654	0.818
Neutrophils, /mm³ (median, min–max)	3,420 (1,380–7,710)	3,770 (2,180–8,470)	0.138	0.385
Lymphocytes, /mm³ (mean ± SD)	2,530 ± 1,397	2,650 ± 741	0.627	0.818
Monocytes, /mm³ (median, min–max)	660 (90–1,040)	540 (240–1,060)	0.898	0.898
PLR (median, min–max)	121.4 (19.3–1,111.7)	122.8 (37.0–291.0)	0.799	0.898
NLR (median, min–max)	1.3 (0.3–11.6)	1.3 (0.76–5.76)	0.887	0.898
MLR (median, min–max)	0.27 (0.02–0.47)	0.21 (0.07–0.55)	0.558	0.818
ESR, mm/h (median, min–max)	14 (2–73)	—	—	—
CRP, mg/dL (median, min–max)	3 (0–86)	—	—	—
AST, IU/L (median, min–max)	27 (17–148)	—	—	—
ALT, IU/L (median, min–max)	25 (8–104)	—	—	—

Abbreviations: MCV, mean corpuscular volume; RDW, red cell distribution width; MPV, mean platelet volume; PDW, platelet distribution width; PCT, plateletcrit; P-LCR, platelet-large cell ratio; WBC, white blood cell count; NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio; MLR, monocyte-to-lymphocyte ratio; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; SD, standard deviation; FDR, false discovery rate

Continuous variables were compared using the independent-samples t-test (normally distributed data) or the Mann–Whitney U test (non-normally distributed data). FDR-adjusted p values were obtained by the Benjamini–Hochberg procedure across the 15 haematological comparisons. Routine biochemistry (ESR, CRP, AST, ALT) was not requested in healthy controls; group comparisons were therefore restricted to haematological parameters

Table 3.

Comparison of laboratory data between patients with Brucella arthritis and healthy controls

Haematological parameter	Brucella arthritis (n = 23)	Controls (n = 39)	p	FDR-adj. p
Haemoglobin, g/dL (mean ± SD)	14.0 ± 1.4	14.2 ± 0.9	0.455	0.698
MCV, fL (median, min–max)	81.6 (69.0–87.5)	80.3 (75.5–97.3)	0.253	0.698
RDW, % (median, min–max)	13.7 (12.0–17.9)	12.8 (11.7–14.5)	0.012	0.180
Platelet count, /mm³ (mean ± SD)	298,652 ± 49,890	326,769 ± 86,109	0.109	0.698
MPV, fL (mean ± SD)	9.6 ± 0.7	9.4 ± 0.7	0.500	0.698
PDW, % (median, min–max)	10.4 (8.4–13.3)	10.4 (8.1–15.7)	0.448	0.698
PCT, % (mean ± SD)	0.28 ± 0.04	0.30 ± 0.07	0.186	0.698
P-LCR, % (mean ± SD)	21.8 ± 6.3	20.8 ± 6.1	0.526	0.698
WBC, /mm³ (mean ± SD)	6,769 ± 1,456	7,181 ± 1,972	0.351	0.698
Neutrophils, /mm³ (median, min–max)	3,100 (1,200–6,980)	3,770 (2,180–8,470)	0.147	0.698
Lymphocytes, /mm³ (median, min–max)	2,410 (340–3,750)	2,630 (960–4,560)	0.414	0.698
Monocytes, /mm³ (median, min–max)	620 (50–970)	540 (240–1,060)	0.834	0.834
PLR (median, min–max)	116.9 (68.5–1,111.7)	122.8 (37.0–291.0)	0.710	0.819
NLR (median, min–max)	1.28 (0.32–11.65)	1.3 (0.76–5.76)	0.833	0.834
MLR (median, min–max)	0.27 (0.02–0.47)	0.21 (0.07–0.55)	0.558	0.698

Abbreviations: MCV, mean corpuscular volume; RDW, red cell distribution width; MPV, mean platelet volume; PDW, platelet distribution width; PCT, plateletcrit; P-LCR, platelet-large cell ratio; WBC, white blood cell count; NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio; MLR, monocyte-to-lymphocyte ratio; SD, standard deviation; FDR, false discovery rate

Continuous variables were compared using the independent-samples t-test (normally distributed data) or the Mann–Whitney U test (non-normally distributed data). FDR-adjusted p values were obtained by the Benjamini–Hochberg procedure across the 15 haematological comparisons

Ethics

The study was approved by the Local Institutional Ethics Committee of Erzurum Regional Training and Research Hospital (decision number 51, dated 17.02.2020) and was conducted in accordance with the principles of the Declaration of Helsinki. Given the retrospective, record-based design and the inclusion of a paediatric population, the ethics committee waived the requirement for written informed consent. All patient records were anonymised and de-identified prior to analysis, and confidentiality was maintained at every step of the study.

Results

Demographic and clinical characteristics

Among the 42 patients diagnosed with brucellosis, 30 (71.4%) were male and 12 (28.6%) were female, with a mean age of 12.4 ± 3.7 years (range 6–17). The 39 healthy controls had a mean age of 11.0 ± 3.0 years (range 6–17), of whom 18 (46.2%) were male and 21 (53.8%) were female. Patients were slightly older than controls (median 14.0 vs. 10.0 years, p = 0.048), and the proportion of males was higher among patients (71.4% vs. 46.2%, p = 0.021). These age and sex differences were therefore controlled for in the multivariable analyses.

The most common presenting complaints were arthralgia (85.7%), weakness (71.4%), and fever (66.7%). Arthritis was observed in 23 patients (54.8%), while lymphadenopathy was detected in only one patient (2.4%). One patient presented with fever, sudden vision loss, and seizures; cerebrospinal fluid analysis revealed lymphocyte predominance, and ophthalmic examination showed papillitis, leading to a diagnosis of Brucella meningitis.

On serological work-up, the Rose Bengal test was positive in all 42 patients (100%), the standard tube agglutination (Wright) test in 35 (83.3%), the Brucella Coombs test in 40 (95.2%), and blood culture in 4 patients (9.5%). The majority of patients (42.9%) were treated with rifampicin, doxycycline, and streptomycin. One case of neurobrucellosis was initially treated with ceftriaxone, rifampicin, and doxycycline but required regimen adjustments due to drug-related reactions, ultimately receiving combination therapy with ciprofloxacin for continuation. Only one patient experienced relapse during follow-up, with no sequelae observed (Table 1).

Table 1.

Demographic, clinical, diagnostic, and treatment characteristics of children with brucellosis (n = 42)

Characteristic	n (%)
Sex
Female	12 (28.6)
Male	30 (71.4)
Clinical features
Arthralgia	36 (85.7)
Weakness	30 (71.4)
Fever	28 (66.7)
Arthritis	23 (54.8)
Myalgia	22 (52.4)
Anorexia	19 (45.2)
Sweating	14 (33.3)
Abdominal pain	3 (7.1)
Lymphadenopathy	1 (2.4)
Seizure	1 (2.4)
Diagnostic tests
Rose Bengal test, positive	42 (100.0)
Wright test (titer ≥ 1:160), positive	35 (83.3)
Brucella Coombs test, positive	40 (95.2)
Blood culture, positive	4 (9.5)
Treatment regimen
Rifampicin + doxycycline	14 (33.3)
Rifampicin + doxycycline + streptomycin	18 (42.9)
Rifampicin + doxycycline + gentamicin	1 (2.4)
Rifampicin + TMP-SMZ	8 (19.0)
Ceftriaxone + doxycycline + TMP-SMZ + ciprofloxacin	1 (2.4)

Abbreviations: TMP-SMZ, trimethoprim/sulfamethoxazole. The Wright test refers to the standard tube agglutination test; a titer ≥ 1:160 was considered positive

Haematological comparison

Median RDW values were 12.8% (min–max: 12.0–17.2) in patients and 12.8% (11.7–14.5) in controls (unadjusted p = 0.025). The mean platelet count was 289,857 ± 64,581/mm³ in patients and 326,769 ± 86,109/mm³ in controls (unadjusted p = 0.021), and mean PCT was 0.27 ± 0.06% and 0.30 ± 0.07%, respectively (unadjusted p = 0.044). After Benjamini–Hochberg correction across the 15 haematological comparisons, none of these differences remained statistically significant (FDR-adjusted p = 0.188, 0.188, and 0.220, respectively). The remaining parameters — MPV, PDW, P-LCR, white blood cell count, absolute neutrophil, lymphocyte, and monocyte counts, and the inflammatory ratios NLR, PLR, and MLR — did not differ between groups in either unadjusted or adjusted analyses (Table 2). In the subgroup of patients with Brucella arthritis (n = 23), median RDW was higher than in controls (13.7% [12.0–17.9] vs. 12.8% [11.7–14.5]; unadjusted p = 0.012, FDR-adjusted p = 0.180), while no other parameter differed significantly (Table 3).

ROC analysis

ROC analysis was performed for the three markers that showed differences in unadjusted univariate analysis. Discriminative performance was modest in all cases: RDW yielded an AUC of 0.646 (95% CI: 0.531–0.749), with an optimal cut-off of > 13.6% providing 46.3% sensitivity and 92.3% specificity (Youden index 0.39); platelet count yielded an AUC of 0.617 (95% CI: 0.503–0.723), with a cut-off of ≤ 394,000/mm³ providing 100.0% sensitivity but only 28.2% specificity; and PCT yielded an AUC of 0.615 (95% CI: 0.501–0.721), with a cut-off of ≤ 0.32% providing 83.3% sensitivity and 43.6% specificity. Pairwise comparison of the three ROC curves did not show statistically significant differences in AUC between any pair (all p > 0.66) (Table 4; Fig. 1).

Table 4.

Receiver operating characteristic (ROC) analysis of haematological markers for the diagnosis of brucellosis

Marker	AUC	95% CI	Optimal cut-off (Youden)	Sensitivity (%)	Specificity (%)
RDW (%)	0.646	0.531–0.749	> 13.6	46.3	92.3
Platelet count (/mm³)	0.617	0.503–0.723	≤ 394,000	100.0	28.2
PCT (%)	0.615	0.501–0.721	≤ 0.32	83.3	43.6

Abbreviations: AUC, area under the curve; CI, confidence interval; RDW, red cell distribution width; PCT, plateletcrit. Optimal cut-off values were determined using the Youden index (J = sensitivity + specificity − 1)

Fig. 1.

Receiver operating characteristic (ROC) curves of red cell distribution width (RDW), platelet count, and plateletcrit (PCT) for discriminating paediatric brucellosis from healthy controls. Among the evaluated haematological markers, RDW showed the highest, although still modest, discriminative ability (AUC = 0.646, 95% CI: 0.531–0.749), followed by platelet count (AUC = 0.617, 95% CI: 0.503–0.723) and PCT (AUC = 0.615, 95% CI: 0.501–0.721). The diagonal dashed line represents the reference line for a non-discriminatory test (AUC = 0.50)

Multivariable logistic regression

In age- and sex-adjusted binary logistic regression models, only RDW remained independently associated with brucellosis. Each 1-unit increase in RDW was associated with 2.3-fold higher odds of disease (adjusted OR 2.31, 95% CI: 1.30–4.11, p = 0.005); male sex was also independently associated with brucellosis (adjusted OR 2.87, 95% CI: 1.04–7.94, p = 0.042). Neither platelet count (adjusted OR per 100,000/mm³: 0.57, 95% CI: 0.30–1.08, p = 0.085) nor PCT (adjusted OR 0.004, 95% CI: 0.000–4.64, p = 0.125) was independently associated with brucellosis (Table 5).

Table 5.

Age- and sex-adjusted binary logistic regression models for the diagnosis of brucellosis

Model	Variable	Adjusted OR	95% CI	p value
Model 1 (RDW)	Age	1.148	0.990–1.331	0.068
	Male sex	2.872	1.039–7.936	0.042
	RDW	2.306	1.295–4.107	0.005
Model 2 (PCT)	Age	1.114	0.970–1.280	0.127
	Male sex	2.472	0.950–6.434	0.064
	PCT (per 0.1%)	0.576	0.284–1.166	0.125
Model 3 (Platelets)	Age	1.107	0.963–1.273	0.152
	Male sex	2.399	0.916–6.285	0.075
	Platelet count (per 100,000/mm³)	0.567	0.297–1.082	0.085

Abbreviations: OR, odds ratio; CI, confidence interval; RDW, red cell distribution width; PCT, plateletcrit. Three separate binary logistic regression models were constructed, each including age, sex, and one of the candidate haematological markers (RDW, PCT, or platelet count) as predictors. The dependent variable was brucellosis status (case vs. control)

Discussion

Brucellosis is one of the most commonly encountered zoonotic diseases. Despite strict control measures in many developed countries, it still remains a significant disease in the Mediterranean region, the Middle East, and Western Asia [14].

The most commonly reported symptoms in childhood brucellosis include fever, arthralgia, fatigue, anorexia, and abdominal pain, which can mimic many other diseases [5]. In our study, the most frequent presenting complaints were arthralgia (85.7%), weakness (71.4%), and fever (66.7%), in keeping with the literature. Consistent with previous reports, the mean age of children in our cohort was 12 years and males predominated, which is most often attributed to greater participation of boys in animal husbandry from age 10 onward [14, 15].

Arthritis has been reported in 10% to 71.4% of brucellosis patients [16]. In our study, arthritis was detected in 23 patients (54.8%), all presenting as monoarthritis. The proportion of neurobrucellosis among brucellosis patients is usually less than 5%, with symptoms typically manifesting as meningitis or meningoencephalitis [6]. One of our patients had neurobrucellosis, presenting with fever, sudden vision loss, and seizures.

Definitive diagnosis of brucellosis is made by isolation of the organism from blood or tissue cultures, or by molecular methods such as polymerase chain reaction. Blood culture positivity in childhood brucellosis has been reported to range between 7.1% and 27.4% [15], and a similar rate (9.5%, 4/42) was observed in our study. Among the serological tests, the Brucella Coombs test showed the highest positivity rate (95.2%), followed by the standard tube agglutination (Wright) test (83.3%) and the Rose Bengal test (100%, used as a screening test), supporting the high sensitivity of Coombs-based serology in paediatric brucellosis.

Diagnostic performance of haematological indices was evaluated by Yorulmaz et al., who reported significant differences between neonatal sepsis and control groups in neutrophil count, lymphocyte count, NLR, MCV, RDW, platelet count, MPV, PDW, and PCT [17]. Similarly, Dursun et al. found higher NLR and MPV values in paediatric patients with sepsis [18]. Aslan and Çiçek also showed that MPV and NLR were useful in differentiating cholesteatoma from chronic active otitis media [19]. These findings show that haematological indices behave differently across various infectious and inflammatory diseases, which may partly explain the inconsistent results in paediatric brucellosis.

In our study, RDW, platelet count, and PCT differed between cases and controls in unadjusted analysis. However, after FDR correction for multiple comparisons these differences did not retain statistical significance, and the ROC analysis showed only modest discriminative performance (AUCs 0.61–0.65). RDW showed high specificity (92.3%) but limited sensitivity (46.3%), suggesting that it may be more useful as a confirmatory adjunct than as a screening tool. Although RDW did not remain significant after FDR correction in the univariate comparison, it retained an independent association with brucellosis after adjustment for age and sex, indicating that multivariable modelling provided additional information beyond univariate testing. Accordingly, RDW was the only haematological marker independently associated with brucellosis (adjusted OR 2.31, p = 0.005).

Our findings differ from those of Bozdemir et al., who studied a comparable population of 60 Turkish children with brucellosis and 55 healthy controls and reported significantly lower MPV but higher NLR values in patients than in healthy controls [2]. In our cohort, neither MPV (9.6 ± 0.6 vs. 9.4 ± 0.7, p = 0.154) nor NLR (1.3 vs. 1.3, p = 0.887) differed between groups. Several factors may account for this discrepancy, including differences in disease stage at sampling, prior antibiotic exposure, analyser platform and pre-analytical conditions, and our stricter exclusion of children with iron-deficiency anaemia or concurrent infections.

Beyond the points raised above in relation to Bozdemir et al., several biological considerations apply. NLR primarily reflects a stress-induced neutrophilia coupled with lymphopenia and is therefore most informative in acute bacterial infections with marked systemic inflammation, such as sepsis or pyogenic infections [17, 18]. Brucellosis, in contrast, is an intracellular infection that often runs a subacute or chronic course with relatively preserved leukocyte and lymphocyte counts; in our cohort the mean WBC (6,964 ± 1,978/mm³) and absolute lymphocyte count (2,530 ± 1,397/mm³) were within reference ranges and not different from controls. This biology is consistent with our finding that NLR did not distinguish cases from controls, and aligns with reports in adult brucellosis where NLR has shown variable performance [8]. The negative finding should also be interpreted in light of the limited statistical power for small effect sizes in our sample.

Aktar et al. reported an increase in NLR and PLR in paediatric Brucella arthritis cases compared with healthy controls [9]. In our subgroup of 23 patients with Brucella arthritis, RDW differed from controls (median 13.7 vs. 12.8, p = 0.012), whereas NLR (p = 0.833) and PLR (p = 0.710) did not.

The biological rationale for RDW as the most informative marker in our cohort deserves further consideration. RDW reflects anisocytosis and is increased in conditions characterized by ineffective erythropoiesis, oxidative stress, and chronic low-grade inflammation [20]. Brucella species are facultative intracellular pathogens with a particular tropism for the reticuloendothelial system, including the bone marrow, and haematological involvement — including anaemia, leukopenia, and thrombocytopenia — has been documented in a substantial proportion of paediatric brucellosis cases [21, 22]. Pro-inflammatory cytokines such as IL-6, which are elevated in active brucellosis, suppress erythropoiesis through hepcidin-mediated impairment of iron utilization and direct inhibition of erythroid precursors in the bone marrow, both of which contribute to a wider distribution of erythrocyte size and consequently to higher RDW values [23]. The fact that RDW remained independently associated with brucellosis after adjustment for age and sex — and was also elevated in the Brucella arthritis subgroup. The persistence of the RDW association after adjustment for age and sex supports a disease-related rather than demographic explanation.

Limitations

This study has several limitations that should be considered when interpreting the results. First, the single-center, retrospective design limits generalizability and precludes prospective control of confounding. Second, the sample size (42 patients and 39 controls) was modest; the post-hoc sensitivity power analysis showed that the study could reliably detect only effect sizes of Cohen’s d ≥ 0.63, so the negative findings for MPV, NLR, PLR, MLR, P-LCR, and PDW may reflect insufficient statistical power (Type II error) rather than true absence of effect. Third, multiple statistical comparisons were performed; after FDR correction, the previously significant differences in RDW, platelet count, and PCT no longer reached the 0.05 threshold, and the results should therefore be regarded as exploratory and hypothesis-generating. Fourth, ESR, CRP, AST, and ALT were not available for healthy controls because these tests are not part of routine paediatric outpatient check-ups, and obtaining them solely for research purposes in asymptomatic children was not appropriate; this precluded between-group comparisons for these parameters. Fifth, iron status parameters (serum iron, ferritin, transferrin saturation) were not measured, and although patients with overt iron-deficiency anaemia were excluded, latent iron deficiency — a known modifier of RDW — could not be ruled out. Sixth, residual confounding from other undocumented subclinical conditions cannot be excluded despite the application of broad exclusion criteria. Seventh, blood cultures had a positivity rate of 9.5%, which is within the range reported in paediatric brucellosis (7.1–27.4%) [15], but most diagnoses relied on serology combined with clinical compatibility, introducing a small risk of misclassification in this endemic region. Finally, the study was conducted during a single seasonal period (autumn–winter), which may limit the generalizability of the findings to other times of the year.

Conclusion

In this paediatric cohort, only RDW remained independently associated with brucellosis after adjustment for age and sex (adjusted OR 2.31, p = 0.005), whereas none of the inflammatory ratios (NLR, PLR, MLR) or platelet indices (MPV, PDW, PCT, P-LCR) discriminated between groups in adjusted analyses. These exploratory findings suggest that RDW may serve as a supportive adjunctive marker alongside clinical and serological evaluation in paediatric brucellosis, particularly given its high specificity (92.3%) at a cut-off of > 13.6%. However, its limited sensitivity (46.3%) and the loss of significance after multiple-testing correction in univariate analysis indicate that RDW cannot replace standard diagnostic testing. Confirmation in adequately powered, multicentre prospective studies with standardised analyser platforms is warranted.

Abbreviations

ALT

Alanine Aminotransferase

aOR

Adjusted Odds Ratio

AST

Aspartate Aminotransferase

AUC

Area Under the Curve

CBC

Complete Blood Count

CI

Confidence Interval

CRP

C-Reactive Protein

ELISA

Enzyme—Linked Immunosorbent Assay

ESR

Erythrocyte Sedimentation Rate

FDR

False Discovery Rate

Hb

Haemoglobin

IL6

Interleukin—6

MCV

Mean Corpuscular Volume

MLR

Monocyte—to—Lymphocyte Ratio

MPV

Mean Platelet Volume

NLR

Neutrophil—to—Lymphocyte Ratio

OR

Odds Ratio

PCR

Polymerase Chain Reaction

PCT

Plateletcrit

PDW

Platelet Distribution Width

P-LCR

Platelet-Large Cell Ratio

PLR

Platelet-to-Lymphocyte Ratio

RDW

Red Cell Distribution Width

ROC

Receiver Operating Characteristic

SD

Standard Deviation

TMP-SMZ

Trimethoprim/Sulfamethoxazole

WBC

White Blood Cell

WHO

World Health Organization

Author contributions

Emine Erdeniz: Conceptualization, study design, data collection, data analysis, interpretation of results, manuscript drafting, and critical revision of the manuscript. Yeter Düzenli Kar: Contributed to study design, patient data collection, and interpretation of results. Participated in the review and approval of the final version of the manuscript, excluding the writing process. Both authors read and approved the final manuscript.

Funding

This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by the Local Institutional Ethics Committee of Erzurum Regional Training and Research Hospital (decision no. 51, dated 17 February 2020) and was conducted in accordance with the Declaration of Helsinki. Because of the retrospective design, the requirement for informed consent was waived by the ethics committee.

Consent for publication

Not applicable. No identifying information or images of any participant are included in this article.

Note

The corresponding author (E.H.E.) was affiliated with Erzurum Regional Training and Research Hospital during the study period (September 2018 – February 2019) and is currently at Ondokuz Mayıs University, Samsun. Similarly, the second author (Y.D.K.) was affiliated with Erzurum Regional Training and Research Hospital during the study period and is currently at Bursa Uludağ University.

Competing interests

The authors declare no competing interests.