Can Hematological Inflammatory Indices Be Used to Differentiate Modic Type 1 Changes from Brucella Spondylodiscitis?

Background and Objectives: Differentiation between brucella spondylodiscitis and Modic type I changes (MC1) includes difficulties. Hematological inflammatory indices (HII) such as neutrophil to lymphocyte ratio (NLR) and aggregate index of systemic inflammation (AISI) are suggested as indicators of inflammation and infection and have diagnostic, prognostic, and predictive roles in various diseases. This study aimed to evaluate differences between brucella spondylodiscitis and MC1 in terms of HII. Materials and Methods: Thirty-five patients with brucella spondylodiscitis and thirty-seven with MC1 were enrolled in the study. Brucella spondylodiscitis and MC1 were diagnosed by microbiological, serological, and radiological diagnostic tools. HII (NLR, MLR, PLR, NLPR, SII, SIRI, AISI) were derived from baseline complete blood count. Results: The two groups were similar for age (p = 0.579) and gender (p = 0.092), leukocyte (p = 0.127), neutrophil (p = 0.366), lymphocyte (p = 0.090), and monocyte (p = 0.756) scores. The Brucella spondylodiscitis group had significantly lower pain duration (p < 0.001), higher CRP and ESR levels (p < 0.001), and lower platelet count (p = 0.047) than the MC1 group. The two groups had similarity in terms of HII: NLR (p = 0.553), MLR (p = 0.294), PLR (p = 0.772), NLPR (p = 0.115), SII (p = 0.798), SIRI (p = 0.447), and AISI (p = 0.248). Conclusions: Increased HII can be used to differentiate infectious and non-infectious conditions, but this may be invalid in brucellosis. However, pain duration, CRP and ESR levels, and platelet count may be useful to distinguish brucella spondylodiscitis from MC1.

Brucellosis screening and follow-up of seropositive asymptomatic subjects among household members of shepherds in China.

To measure the seroprevalence of high-exposure populations in brucellosis endemic areas and report the outcome and duration of seropositive asymptomatic subjects, we screened 595 family members of shepherds in Jilin Province, China and then followed up 15 seropositive asymptomatic subjects for 18 months. We found that the seropositive rate of 15.5%. Nearly half of seropositive asymptomatic subjects (7/15) developed into brucellosis in the short term; others were still seropositive asymptomatic or had decreased SAT titer in a longer time.

Prevalence, distribution and risk factors for brucellosis infection in goat farms in Ningxiang, China.

BACKGROUND: In south China, goats are the major source of Brucellosis for human infection. However, there are few studies on the prevalence of and risk factors for goat brucellosis in south China. In this study, we conducted a cross-sectional study to investigate the herd prevalence, spatial distribution and relevant risk factors for goat brucellosis in Ningxiang county, south China. Commercial goat farms (n = 457) were randomly selected, and their disease status was ascertained by testing serum samples of chosen individuals using the Rose Bengal Test (screening test) and the Serum Agglutination Test (confirmatory test) in series. A farm with at least two positive individuals was defined as a case farm. Standardized questionnaires were used to collect information on management and hygiene practices in farms. A logistic model with a binomial outcome was built to identify risk factors for being seropositive. RESULTS: The true herd prevalence in commercial goat farms was 4.5% (95%CI: 0.2%-12.2%) and the townships in the centre of the county had higher herd prevalence. The risk factors associated with seropositive on local goat farms include "Introduction in the past 12 months" (OR= 61, 95%CI: 16-333), "Improperly disposal of the sick or dead goats" (OR= 33, 95%CI: 5-341) and "Poor hygiene in lambing pen" (OR= 25, 95%CI: 5-192). CONCLUSIONS: These findings will aid in the development of control strategies of Brucellosis in south China and risk factors identified in this study should be taken into consideration when designing a control strategy.

Sero-epidemiology and associated risk factors of brucellosis among sheep and goat population in the south western Nepal: a comparative study.

BACKGROUND: Brucellosis is a zoonotic disease caused by Brucella spp. In Nepal, the presence of brucellosis in small ruminants, namely sheep and goats, has impacted farmers' livelihood and the food safety of consumers. A cross-sectional study was conducted in Rupandehi district of Nepal during January to March 2020 to investigate the seroepidemiology and associated risk factors of brucellosis in the sheep and goat population. Altogether, 19 sheep and 60 goat farms in the district were visited. Owners were interviewed to get information on animals, including their management and movement patterns. Three hundred fifty-seven samples (80 sheep and 277 goat samples) were collected proportionately based on farm sizes. Each serum sample was tested with Rose Bengal Test and ELISA to estimate the seropositivity of brucellosis. Logistic regression was carried out to calculate corresponding odds ratios of each variable associated with detection of brucellosis. RESULTS: At the farm level, 31.6% (6/19; 95% CI: 12, 54%) of sheep farms and 3.3% (2/60, 95% CI: 0.9, 11.4%) of goat farms were seropositive to brucellosis. Out of 80 sheep serum samples, 12 (15%; 95% CI: 8.79-24.41%) and out of 277 goat serum samples, three (1.1%; 95% CI: 0.37-3.14%) were seropositive to brucellosis. Age greater than 1.5 years (OR = 5.56, 95% CI: 1.39, 29.38; p = 0.02) and herd size of greater than 100 (OR = 4.74, 95% CI: 1.23, 20.32, p = 0.03) were identified as significant risk factors for seropositivity of brucellosis in the sheep population. While in the goat population, none of the variables was identified as a significant risk factor. CONCLUSION: The study provides evidence that the older sheep and the sheep from the large herds were at higher risk of brucellosis. A control program should be put in place immediately in the sheep population because they may transmit infections to other livestock as they were regularly moved for grazing and selling purposes. Also, strict biosecurity measures should be implemented among pastoralists to prevent brucellosis transmission in them. We suggest further one health-based study to reveal the transmission dynamics of brucellosis between animals and humans.

Roles of novel IL-1 family (IL-36, IL-37, and IL-38) members in chronic brucellosis.

The secretion of interleukin (IL)-1 family cytokines is one of the most potent and earliest pro-inflammatory responses triggered by brucellosis. However, the roles of the most recently discovered IL-1 family members, IL-36, IL-37, and IL-38, in the transition into the chronic form of brucellos is remain largely unknown. Therefore, in this study, the roles of IL-36, IL-37, and IL-38 in brucella infections and their effects on the transition from the acute to chronic form of the disease were investigated. Using peripheral blood samples from 40 patients with acute brucellosis, 40 patients with chronic brucellosis, and 40 healthy control subjects, we analysed the serum concentrations of secreted IL-36, IL-37, and IL-38 using ELISA. The findings were confirmed by using RT-qPCR to analyse the mRNA levels of the genes encoding IL-36, IL-37, and IL-38 in peripheral blood mononuclear cells (PBMCs) from 10 randomly selected patients from each of the three groups. Our results showed that serum IL-37 (p < 0.001) and IL-38 (p < 0.001) concentrations were lower in patients with brucellosis than in the healthy controls. In addition, serum IL-37 and IL-38 concentrations were higher in the chronic patient group than in the acute patient group. The mRNA expression levels of IL-37 and IL1F10, genes that encode IL-38, did not affect serum cytokine secretion levels. This result suggests that the high secretion levels of IL-37 and IL-38 may be related to the progression into the chronic form of brucellosis. Our findings will aid in clarifying the mechanism of the transition of brucellosis from the acute to the chronic form of the disease.

ELISA is superior to bacterial culture and agglutination test in the diagnosis of brucellosis in an endemic area in China.

BACKGROUND: Brucellosis is endemic in many areas in China. The current diagnosis of Brucellosis predominantly relies on the traditional bacterial culture and serum agglutination test. In this study, we aimed to explore the value of ELISA in the diagnosis of Brucellosis in Chinese population. METHODS: We recruited 235 patients with a diagnosis of Brucellosis at different clinical stages: 117 in acute, 78 in subacute, and 40 in chronic. We also recruited 248 control patients who presented with similar clinical symptoms but with a different diagnosis other than Brucellosis. In addition, 90 healthy volunteers were also recruited. Bacterial culture, agglutination test and ELISA assay were performed to detect Brucella spp. RESULTS: Among 235 patients with Brucellosis, 51 (21.7%) was positive for bacterial culture, 150 (63.8%) were positive by agglutination test, and 232 (98.7%) were positive by ELISA (IgG and/or IgM). When we stratified the patients based on the disease stages (acute, subacute and chronic), ELISA was the most sensitive method and showed a highest positive rate in all stages. By Receiver Operating Characteristic Curve analysis of ELISA results, we found that measurement of IgG level was superior to measurement of IgM level (AUC, 0.993 versus 0.877). Since the measurement of IgG itself missed rare cases in acute phase, we recommended measuring IgG and IgM simultaneously by ELISA for the diagnosis of Brucellosis. In term of the specificity of ELISA in the diagnosis of Brucellosis, our study showed that only 1.6% (4/248) non-Brucellosis patients were positive by ELISA; all positive cases were IgM only and none showed positive IgG. Similar results were found in healthy volunteers. In summary, our study concluded that ELISA is the most sensitive and specific method to detect Brucellosis in Chinese population. CONCLUSIONS: ELISA assay is sensitive, fast, and convenient to detect Brucellosis. It shows the high sensitivity and specifity and should be used as a routine lab test when Brucellosis is suspected in clinical practice.

A novel, rapid and simple method for detecting brucellosis based on rapid vertical flow technology.

AIMS: To prevent the spread of brucellosis, a simple and rapid vertical flow technology (RVFT) for the detection of antibodies targeting brucellosis was developed. METHODS AND RESULTS: In this study, Brucella sp. lipopolysaccharide was purified and used to detect brucellosis antibodies. Sheep IgG was used as a negative control. Colloidal gold-labeled recombinant staphylococcus aureus protein A was sprayed on a fibreglass membrane to prepare immunogold pads. Rapid vertical flow technology was used to detect Brucella in 1668 Sheep, 2743 bovine, 674 red deer and 420 human samples. The results indicated that the accuracy of this assay can reach 98%. CONCLUSIONS: The established RVFT uses a single multifunctional buffer that can be used to detect antibodies in serum, plasma, whole blood and other biological samples while preserving the advantages of lateral-flow immunoassays. SIGNIFICANCE AND IMPACT OF THE STUDY: This technology would be of great use in primary medical units and veterinary stations, and it is of great significance for the control of epidemic diseases.

Serological and molecular evidence of Brucella species in the rapidly growing pig sector in Kenya.

BACKGROUND: Brucellosis is an emerging yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi region, Kenya were screened in parallel, using both Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 gene and real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes. RESULTS: A prevalence of 0.57% (n = 4/700) was estimated using RBT; none of these samples was positive on cELISA. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a real-time multiplex PCR. CONCLUSION: The detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs confirm the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

Comparison of the serum level of interleukin-4 in patients with brucellosis and healthy controls.

INTRODUCTION: Evaluation of cytokines such as interleukin-4 (IL-4) can be an important tool in examining immune responses to brucellosis. Also, determining the response rate to treatment is necessary for controlling and eradicating of disease. The review of previous studies reveals contradictory results that require further research in this regard. The aim of this study was to compare the serum level of IL-4 in patients with brucellosis and healthy controls. MATERIAL AND METHODS: In this descriptive-analytical study for comparison of two groups, a total of 165 participants, including 83 patients with brucellosis and 82 non-infected people, were evaluated after matching of sex and age in Hamadan (northwest of Iran) in 2017 and the serum level of IL-4 was compared by ELISA method. The collected data were analyzed by SPSS software version 21 at 95% significant level. RESULTS: Mean of age in the case and control groups were 50.25 ± 16.01 and 43.26 ± 15.6 years, respectively. The serum levels of IL-4 in the case and control groups were 1.42 ± 0.51 pg/mL and 1.31 ± 1.02 pg/mL, respectively. Based on the non-parametric Mann-Whitney test, the IL-4 level was significantly higher in the case group, compared with the control (P < .001), but no statistically significant relationship was found between serum levels of IL-4 with age, sex, and serologic titers of Wright and 2ME. CONCLUSION: In patients with brucellosis, the level of IL-4 increases independently of the duration and severity of the disease, which indicates the role of this cytokine of immune system in this infectious disease.

Cholinesterase's activities of infected mice by Brucella ovis.

The role of cholinesterase in inflammatory reactions has been described in several infectious diseases. However, in Brucella spp. this has not yet been studied. Therefore, the objective of this study was to evaluate whether experimental infection by Brucella ovis alters the cholinergic activity in pro- or anti-inflammatory responses to the disease. For the study 48 mice were used, 24 infected by B. ovis and 24 non-infected. We collected samples of whole blood on days 7, 15, 30 and 60 post-infection (PI) by B. ovis. Acetylcholinesterase (AChE) activity in the blood increased on days 15 and 60 PI (P < 0.05). Butyrylcholinesterase (BChE) activity in serum increased on days 7 and 60 PI (P < 0.05). An increase in serum free radical levels occurred on days 7, 15 and 60 PI (P < 0.05), and consequently superoxide dismutase activity increased on day 15 PI (P < 0.05). A reduction in catalase activity occurred when the infection became chronic (60 PI). The increase in AChE and BChE characterized a pro-inflammatory response, since these enzymes regulate levels of acetylcholine (ACh) and butyrylcholine (BuSCh), molecules with anti-inflammatory properties. Therefore, with the increase of cholinesterase activity, there was an extracellular reduction of ACh, an inhibitor of several inflammatory mediators. This proinflammatory response of B. ovis infection leads to oxidative stress, and consequently to cellular damage.

A potential marker in brucellosis, long non coding RNA IFNG-AS1.

Brucellosis is the most common bacterial zoonotic infection. This pathogen may survive and sustain in host. The aim of this study is to define relationship between long noncoding (lnc) RNA-IFNG-AS1 and interferon gamma (IFN-γ) in different groups of patients with brucellosis compared to control group. In this study, associations of lncRNA IFNG-AS1 expression with secretion of IFN-γ level in Sixty patients with brucellosis, which were divided into 3 groups (acute, chronic and relapse groups), as a case group were compared with 20 subjects with negative serological tests and brucellosis clinical manifestation as a control group. In this regard, RNA were extracted from isolated peripheral blood mononuclear cells (PBMCs). LncRNA IFNG-AS1, T-box transcription factor (T-bet) and IFN-γ expressions were detected using quantitative polymerase chain reaction (qPCR). Serum level IFN-γ was assessed using enzyme linked immunosorbent assay (ELISA). The results showed that expression level of LncRNA IFNG-AS1, T-bet and IFN-γ increased significantly in all patient groups in compared to healthy subjects (P < 0.0001, P < 0.01, P < 0.001). However, there was no significant difference in T-bet expression between chronic and healthy groups (P = 0.98). Additionally, further analysis revealed that the serum level of IFN-γ in acute and relapsed groups were higher than control group (P < 0.0001, P < 0.001). The effective role of IFNG-AS1 in many protective actions, including enhancing the expression of INF-γ in the immune response of brucellosis patients, revealed new potential marker, LncRNA IFNG-AS1 in screening, diagnosis or treatment of brucellosis.

Protein-G-based lateral flow assay for rapid serodiagnosis of brucellosis in domesticated animals.

This study was conducted to develop and evaluate protein-G-based lateral flow assay (LFA) for rapid serodiagnosis of brucellosis in various domesticated animal species. The assay diagnostic performance was tested with 144 reference and 356 field sera samples and then compared with other serological assays. Results revealed that LFA showed 89% and 99% sensitivity and specificity, respectively, when compared with competitive ELISA as the gold standard. This study demonstrated LFA alone as a potential serodiagnostic assay for rapid serodiagnosis of brucellosis in various domesticated animal species.

Serum prolidase level in patients with brucellosis and its possible relationship with pathogenesis of the disease: a prospective observational study

Background/aim: Changes in collagen metabolism and fibroblastic activity may play a role in the pathogenesis of brucellosis. The prolidase enzyme plays an important role in collagen synthesis. We aimed to investigate the association of prolidase levels with brucellosis. Materials and methods: Serum prolidase levels in 20 patients newly diagnosed with brucellosis were compared with levels in 30 healthy control subjects. Patients with brucellosis were reassessed 3 months later for prolidase, other laboratory measurements, and response to treatment. Results: The levels of serum prolidase were significantly higher in brucellosis patients compared with those of healthy controls. Prolidase, sedimentation, and C-reactive protein levels were significantly lower after antibrucellosistreatment than before treatment. Conclusion: The current study is the first to demonstrate significantly increased serum prolidase levels in patients with brucellosis compared with healthy controls. Prolidase levels also significantly decreased with antibrucellosis treatment. This finding provides a new experimental basis to understand the pathogenesis of brucellosis in relation to collagen metabolism. The increase in serum prolidase levels might be related to several factors such as tissue destruction, increased fibroblastic activity, and granuloma formation, all of which are involved in the natural history of brucellosis.

[PECULIARITIES OF THE PREVALENCE OF POLYMORPHISM (C-589T) OF THE IL-4 GENE AND ITS RELATIONSHIP WITH SERUM IL-4 LEVEL IN PATIENTS WITH ACUTE BRUCELLOSIS IN THE REPUBLIC OF AZERBAIJAN].

The aim of the study was to determine the frequency of polymorphism of IL-4 (C-589T) in patients with acute brucellosis in the Republic of Azerbaijan and to establish its correlation with serum IL-4 levels. One hundred seventy eight patients with clinical symptoms of brucellosis were examined. According to the criteria for inclusion in a study of 178 patients, only 120 persons- (the main group) - fully met all criteria. The control group consisted of 30 practically healthy persons. Also, all patients in both groups were tested for IL-4 (C-589T) polymorphism and level of interleukin-4 (IL-4). It has been established that carriers of the allele T of the polymorphic (C-589T) IL-4 gene are at increased risk for brucellosis (OR=4.26, 95% CI [2.01-9.05]], whereas in the case of carrier allele C, on the contrary, is a reduced risk of developing brucellosis. The combination of genotypes C/T + T/T among patients with brucellosis was determined 3.1 times more than in practically healthy individuals (OR=10.31, 95% CI [1.55-19.18], χ2=29.21, p<0.0001). It was found that among the carriers of the C/C genotype, IL-4 level was 1.44 higher in brucellosis than in T/T genotype carriers and 1.2 times higher in comparison with the C/T genotype. Among carriers of the C/T genotype, there is a significantly increased risk of brucellosis (χ2=29.73; p=4.0E-7; OR=9.63; 95% CI [3.43-27.03] while cariousness of the homozygous genotype C/С, on the contrary, has a protective effect on the development of brucellosis (OR=0.10, 95% CI 0.04-0.25).

Brucellosis in Dogs and Public Health Risk.

Brucella canis infects dogs and humans. In dogs, it can cause reproductive failure; in humans, it can cause fever, chills, malaise, peripheral lymphadenomegaly, and splenomegaly. B. canis infection in dogs is underrecognized. After evaluating serologic data, transmission patterns, and regulations in the context of brucellosis in dogs as an underrecognized zoonosis, we concluded that brucellosis in dogs remains endemic to many parts of the world and will probably remain a threat to human health and animal welfare unless stronger intervention measures are implemented. A first step for limiting disease spread would be implementation of mandatory testing of dogs before interstate or international movement.

An Autoimmune Disease-Associated Risk Variant in the TNFAIP3 Gene Plays a Protective Role in Brucellosis That Is Mediated by the NF-κB Signaling Pathway.

Naturally occurring functional variants (rs148314165 and rs200820567, collectively referred to as TT>A) reduce the expression of the tumor necrosis factor alpha-induced protein 3 (TNFAIP3) gene, a negative regulator of NF-κB signaling, and predispose individuals to autoimmune disease. In this analysis, we conducted a genetic association study of the TT>A variants in 1,209 controls and 150 patients with brucellosis, an infectious disease, and further assessed the role of the variants in brucellosis. Our data demonstrated that the TT>A variants were correlated with cases of brucellosis (P = 0.002; odds ratio [OR] = 0.34) and with individuals who had a positive serum agglutination test (SAT) result (titer of >1/160) (P = 4.2 × 10-6; OR = 0.23). A functional study demonstrated that brucellosis patients carrying the protective allele (A) showed significantly lower expression levels of the TNFAIP3 gene in their peripheral blood mononuclear cells and showed increased NF-κB signaling. Monocytes from individuals carrying the A allele that were stimulated with Brucella abortus had lower mRNA levels of TNFAIP3 and produced more interleukin-10 (IL-10), IL-6, and IL-1ß than those from TT allele carriers. These data showed that autoimmune disease-associated risk variants, TT>A, of the TNFAIP3 locus play a protective role in the pathogenesis of brucellosis. Our findings suggest that a disruption of the normal function of the TNFAIP3 gene might serve as a therapeutic target for the treatment of brucellosis.

Seroprevalence and risk factors of brucellosis among slaughtered indigenous cattle, abattoir personnel and pregnant women in Ngaoundéré, Cameroon.

BACKGROUND: Brucellosis is a neglected debilitating zoonosis with a high prevalence in many developing countries. Bovine brucellosis is widespread in Cameroon but the epidemiological situation of human brucellosis is not known. A cross sectional study was carried to determine the seroprevalence and factors associated with bovine and human Brucellosis among abattoir personnel and pregnant women in Ngaoundéré, Cameroon. METHODS: Serum sample from 590 abattoir cattle and 816 plausible occupational risk and vulnerable humans to brucellosis (107 abattoir personnel and 709 pregnant women) were collected and screened for anti-brucella antibodies using Rose Bengal Plate Test (RBPT) and ELISA tests. Structured questionnaires were used to collect data on socio-demographics and risk-factors. The differences in proportions between seropositive and seronegative reactors were tested using odds-ratio and χ2tests. RESULTS: Bovine brucellosis seroprevalence was at 3.40% (n = 590; 3.4% for RBPT, 5.93% for i-ELISA). Human Brucella seroprevalence was at 5.6% among abattoir personnel (n = 107; 5.6% for RBPT, 12.15% for Brucella IgG ELISA) and 0.28% in pregnant women (n = 709; both tests). Breed (P < 0.00001) was associated with increased risk of brucellosis in cattle and the seroprevalence was highest among the Djafoun (OR = 16.67, 95%CI: 4.49-28.85) and Akou (OR = 16.96, 95% CI: 0.10-23.91) cattle compared to the other breeds. There was a moderate positive correlation (R2 = 0.5025) of Brucella IgG concentrations (> 200 U/ml) and clinical data for Brucella IgG ELISA seropositive humans. Several potential factors were associated (P > 0.05) with increased risk of human brucellosis seroprevalence among the abattoir personnel. The abattoir personnel were essentially males; the seropositive respondents were male and did not use protective equipment at work. Handling of foetus and uterine contents (OR = 13.00, 95%CI: 1.51-111.88) was associated with increased risk of human brucellosis. CONCLUSIONS: Antibrucella antibodies are prevalent in cattle (3.40%), among abattoir personnel (5.60%) and in pregnant women (0.28%) in Ngaoundéré, Cameroon. The study reports the first evidence of human brucellosis in Cameroon and therefore, an indication of a real public health problem. Public awareness campaigns and health education especially among livestock professional and in agropastoral communities should be highlighted to disseminate knowledge, associated risk factors and control measures of brucellosis.

Comparison of inflammatory markers between brucella and non-brucella epididymo-orchitis.

OBJECTIVES: Brucellosis is a multi-system infectious disease that is associated with inflammation, which causes an increase in acute phase reactants. Hematological inflammatory markers of brucellosis include mean platelet volume (MPV), red cell distribution width (RDW), neutrophil/lymphocyte ratio (NLR), and platelet/lymphocyte ratio (PLR). In this study, we aimed to evaluate the diagnostic value of hematological inflammatory markers in Brucella epididymo-orchitis (BEO), and to investigate the utility of these markers for differential diagnosis from non-Brucella epididymo-orchitis (non-BEO). MATERIALS AND METHODS: We retrospectively reviewed the records of 22 BEO and 50 non-BEO patients. Hematological parameters were recorded and compared between the two groups. The main diagnostic criteria for BEO were positive clinical findings (i.e., testicular pain, tenderness and scrotal swelling), a positive Rose Bengal test result, standard tube agglutination (STA) titer ≥ 1/160, and/or a positive blood culture. RESULTS: The most decisive factors in discriminating between BEO and non-BEO were NLR, RDW, and MPV, in decreasing order of their strength. Regardless of other factors, NLR values < 2.3 significantly increased the odds of BEO (OR=8.080, 95% CI: 1.929- 33.843, p=0.004). After adjusting for other factors, RDW values >14.45% significantly increased the odds of BEO (OR=7.020, 95% CI: 1.749-28.176, p=0.006). Independent of the other factors, patients with MPV < 7.65 fL had a 6.336 times higher risk for BEO (95% CI: 1.393 - 28.822, p=0.017). CONCLUSION: Hematological inflammatory markers such as NLR, RDW, and MPV can aid in the differential diagnosis of BEO and non-BEO.

[Comparison of two commercial DNA extraction kits and PCR master mixes for the detection of Brucella from blood samples and blood culture bottles]. / Kandan ve kan kültür siselerinden Brucella saptanmasinda iki farkli ticari DNA ekstraksiyon kiti ve PCR master kiksinin karsilastirilmasi.

The diagnosis of human brucellosis requires culture or serological tests for the conformation of the clinical findings. Isolation of the bacteria is used as a gold standard, however it is time consuming and processing of positive cultures has a potential risk for laboratory acquired human brucellosis. Polymerase chain reaction (PCR) based methods have offered new approaches for early diagnosis of brucellosis and reduce the risk of laboratory acquired human brucellosis. A major limitation of the PCR method is the difficulty to remove the inhibitors in specimens. The aim of this study was to determine the performance of two DNA extraction kits by using two separate PCR master mixes and to determine appropriate "extraction kit - PCR master mix" combination for the diagnosis of Brucella from whole blood samples and blood culture bottles. Two commercial DNA extraction kits, NORGEN Blood DNA isolation kit (Norgen) and Thermo Scientific GeneJet Whole blood genomic DNA purification kit (Thermo Fisher Scientific, USA) and two PCR master mixes, QuantiTect multiplex PCR (QuentiTect, Qiager, Almanya) and Ampliqon Multiplex TEMPase (Amliqon, Denmark) were assessed on 30 simulated blood samples with known concentrations (102-104 cfu/ml) of Brucella melitensis ATCC 23456 strain and 10 blood culture bottles that gave positive signal. By using different combinations of extraction kits and PCR master mixes, a total of 160 different multiplex real-time PCR (Rt-PCR) trials were performed with probes and primers specific to Brucella spp., B.melitensis, and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All the 120 Rt-PCR trials performed on the DNA samples extracted from blood samples gave positive results with GAPDH probe/primers. The rate of positive PCR results for Brucella spp. was 96.7% for the combination of Norgen-QuantiTect, 93.3% for Thermo-Ampliqon, 93.3% for Thermo-QuantiTect, and 86.7% for Norgen-Ampliqon. The frequency of positive B.melitensis results for these combinations were 96.7%, 93.3%, 56.7% and 90%, respectively. In the samples with the bacterial density of 102 cfu/ml, Brucella spp. detection rates were 80% for Thermo-Ampliqon and Norgen-Ampliqon, and 90% for Thermo-QuantiTect and Norgen-QuantiTect; and for B.melitensis positivite rates were 90%, 70%, 20%, and 90%, respectively. Rt-PCR assays with the DNA samples extracted from blood culture bottles using Norgen isolation kit yielded 80% positivite result. However, the frequency of PCR positivite results was only 20% in the DNA samples extracted by Thermo DNA extraction kit. PCR result for GAPDH gene was also negative in 80% of the samples extracted by Thermo kit. Our results revealed that for the removal of inhibitors and detection of even low number of Brucella spp./B.melitensis in blood samples and blood culture bottles, NORGEN Blood DNA isolation kit can be used with a combination of QuantiTect multiplex PCR or Ampliqon Multiplex TEMPase.

Molecular prevalence of putative virulence-associated genes in Brucella melitensis and Brucella abortus isolates from human and livestock specimens in Iran.

Molecular prevalence of nine putative virulence factors in two more prevalent Brucella species in Iranian patients and livestock was investigated. During five years (2010-2015), 120 human and animal specimens were collected from three geographical areas of Iran. All samples were cultured in blood culture media and subcultured into Brucella agar medium. Nine primer pairs were designed for detection of VirB2, VirB5, VceC, BtpA, BtpB, PrpA, BetB, BPE275 and BSPB virulence factors using PCR and sequence analysis. Totally, 68 Brucella isolates including 60 B. melitensis and 8 B. abortus were isolated from the human and animal specimens examined. Approximately, all B. melitensis and B. abortus strains were positive (100%) regarding btpA, btpB, virB5, vceC, bpe275, bspB, and virB2 genes except for prpA and betB that were detected in 86% and 97% of the strains, respectively. Significant relationships were found between the presence of prpA and human B. melitensis isolates (P = 0.04), and also between the presence of betB and human isolates of B. abortus (P = 0.03). In conclusion, our results revealed that Iranian Brucella strains, regardless of human or animal sources, are extremely virulent due to high prevalence of virulence attributes in almost all strains studied.