Brucella PCR Research Articles

Seronegative brucella meningitis diagnosed by CSF PCR: report on seven cases

IntroductionNeurobrucellosis (NB) can be associated with meningitis and present as a headache with or without meningeal signs. Pseudotumor presentation of NB has been reported to be accompanied by lymphocytic predominant cerebrospinal fluid(CSF) pleocytosis. NB is diagnosed by means of isolation of Brucella from blood or CSF and/or the presence of anti-Brucella antibodies in the CSF. Molecular techniques have been used in chronic or challenging cases of NB.Clinical findingsWe report on seven cases of NB presenting with different types of headache and signs of meningeal involvement. In five cases, signs of intracranial hypertension were evident in the form of papilledema, sixth nerve palsy and blurred vision.DiagnosisMRIs of the brain revealed signs of intracranial hypertension in three patients, basal meningeal enhancement in one patient and white matter lesions in one patient. Brucella serology in the blood and CSF was negative in all patients. It was interesting that four patients had normocellular CSF analysis with normal glucose and protein results. The diagnosis was made by Brucella PCR in all patients.ConclusionNB should be considered in the differential diagnoses of pseudotumor cerebri syndrome in endemic areas. It is important to employ molecular techniques using sterile CSF samples in the investigation of Brucella.

Molecular Characterization and Analysis of Risk Factors Associated with Brucellosis in Central Indian and Meghalaya Population.

Objective: In this study we evaluated the utility of Abortus Melitensis Ovis Suis Brucella PCR (AMOS PCR) for the molecular characterization of Brucella species and analyzed the associated risk factors for brucellosis in Central Indian and Meghalayan population. Methods: AMOS PCR was carried out in a total of 160 BSCP-31 PCR-positive DNA samples isolated previously from the blood of Central Indian (n = 90) and Meghalayan cohorts (n = 70). Clinical and associated risk factors recorded earlier were used to establish strain-specific disease outcomes in study cohorts. Results: Brucella melitensis was found to be the dominant strain in both Central Indian and Meghalayan cohorts (57.7% and 54.28%, respectively) followed by Brucella abortus (42.22% and 38.57%). Although rare, brucellosis cases in the Meghalayan population also showed the presence of Brucella suis (7.14%) and Brucella ovis (2.85%). Febrile illness was a major clinical risk factor in both study cohorts, while occupational risk factors like exposure to animals and raw milk consumption were major mediating factors for brucellosis in Central Indian cohorts. On the contrary, meat consumption was found to be significant predisposing factor for brucellosis in Meghalaya. Conclusion: Molecular characterization of Brucella species provides important public health data for mitigation, advocacy, and antimicrobial stewardship.

Detection of Brucella in Dermacentor Ticks of Wild Boar with Brucellosis

Brucellosis is a sanitary and economically relevant disease affecting humans, livestock, and wildlife. Ticks have been suggested as vectors, long-term carriers, and amplifiers of Brucella. In this study, ticks from wildlife ungulate hosts living in hunting reserves of a central region of Spain were collected during a 6-year period, pooled, and screened for Brucella spp. by PCR. Aiming to correlate Brucella spp. DNA presence in ticks with Brucella spp. infections in wildlife ungulate hosts, liver samples from deceased wildlife ungulates coming from the hunting reserves showing a positive result for Brucella in ticks were tested using a commercial ELISA. In total, 229 tick pools from wild boar (Sus scrofa, n = 176; 76.8%, 95% CI 70.9%–81.8%), red deer (Cervus elaphus, n = 40; 17.4%, 95% CI 13.1%–22.9%), mouflon (Ovis orientalis musimon, n = 7; 3.06%, 95% CI 1.49%–6.17%), and fallow deer (Dama dama, n = 6; 2.62%, 95% CI 1.21%–5.60%) were analyzed. PCR results showed that 3.93% (95% CI 2.08%–7.30%) tick pools (9/229) from 16.6% hunting reserves (7/41) screened yielded a positive PCR result for Brucella. All positive ticks were Dermacentor (Dermacentor marginatus or Dermacentor reticulatus) collected from wild boar. Ticks collected from wild boars were positive to Brucella in a relative percentage of 5.10% (95% CI = 1.61–11.4) in 2018 and of 7.59% (95% CI = 2.79–15.6) in 2021 (6-year prevalence of 5.17%, 9/176). ELISA showed positive results in three wild boars coming from two out of seven hunting reserves (28.5%) with a positive PCR for Brucella in ticks. To conclude, Brucella spp. DNA can be detected in Dermacentor ticks parasitizing wild boars living in hunting reserves harboring Brucella spp.-seropositive wild boars. This study provides evidence that the contribution of arthropod vectors should be considered in the epidemiology of brucellosis in wildlife.

A novel, highly sensitive, one-tube nested quantitative real-time PCR for Brucella in human blood samples.

Brucellosis is a zoonotic disease caused by Brucella spp. and is a major threat to human and livestock health. The accurate and rapid detection of Brucella DNA is essential for the diagnosis and treatment of this disease. However, traditional diagnostic methods, such as culture and serological tests, have limitations in terms of sensitivity, specificity, and time consumption. To address these challenges, we developed a highly sensitive one-tube nested quantitative real-time PCR (qPCR) method to detect Brucella DNA using a closed-tube approach with two primers and two probes that sequentially react with encoding a Brucella outer membrane protein. The analytical sensitivity of this one-tube nested qPCR approach was 100 fg/μL, which is 100-fold higher than that of a conventional qPCR. Intra-batch and inter-batch replicates showed low coefficients of variation, both less than 5%. The study included 250 clinical samples, showing that this one-tube nested qPCR method has a specificity of 100% and a sensitivity of 98.6%, exceeding the sensitivity of conventional qPCR (84.1%) at a cycle threshold (CT) cutoff of 38. More importantly, the one-tube nested qPCR reduced the CT values by an average of 6.4 compared to conventional qPCR, significantly improving the detection rate of low-load samples (CT > 35). In conclusion, the bcsp31 one-tube nested qPCR method is a promising tool for the detection of brucellosis, owing to its high sensitivity and operational simplicity. This study highlights the potential of qPCR-based methods to improve the accuracy and speed of brucellosis diagnoses. IMPORTANCE This study developed a highly sensitive and efficient method for the detection of brucellosis by introducing a one-tube nested quantitative real-time PCR (qPCR) approach, representing a remarkable advance in the field. The method demonstrated an impressive analytical sensitivity of 100 fg/μL, surpassing conventional qPCR and enabling the detection of even low levels of Brucella DNA. In addition, the study's comprehensive evaluation of 250 clinical samples revealed a specificity of 100% and a sensitivity of 98.6%, underscoring its reliability and accuracy. Most importantly, the new method significantly improved the detection rate of low-burden samples, reducing cycle threshold values by an average of 6.4. These results underscore the immense potential of this approach to facilitate rapid and accurate brucellosis diagnosis, which is critical for effective disease management and control.

Molecular epidemiology of Brucella species in mixed livestock-human ecosystems in Kenya

Brucellosis, caused by several species of the genus Brucella, is a zoonotic disease that affects humans and animal species worldwide. Information on the Brucella species circulating in different hosts in Kenya is largely unknown, thus limiting the adoption of targeted control strategies. This study was conducted in multi-host livestock populations in Kenya to detect the circulating Brucella species and assess evidence of host–pathogen associations. Serum samples were collected from 228 cattle, 162 goats, 158 sheep, 49 camels, and 257 humans from Narok and Marsabit counties in Kenya. Information on age, location and history of abortion or retained placenta were obtained for sampled livestock. Data on age, gender and location of residence were also collected for human participants. All samples were tested using genus level real-time PCR assays with primers specific for IS711 and bcsp31 targets for the detection of Brucella. All genus positive samples (positive for both targets) were further tested with a speciation assay for AlkB and BMEI1162 targets, specific for B. abortus and B. melitensis, respectively. Samples with adequate quantities aggregating to 577 were also tested with the Rose Bengal Test (RBT). A total of 199 (33.3%) livestock and 99 (38.5%) human samples tested positive for genus Brucella. Animal Brucella PCR positive status was positively predicted by RBT positive results (OR = 8.3, 95% CI 4.0–17.1). Humans aged 21–40 years had higher odds (OR = 2.8, 95% CI 1.2–6.6) of being Brucella PCR positive compared to the other age categories. The data on detection of different Brucella species indicates that B. abortus was detected more often in cattle (OR = 2.3, 95% CI 1.1–4.6) and camels (OR = 2.9, 95% CI 1.3–6.3), while B. melitensis was detected more in sheep (OR = 3.6, 95% CI 2.0–6.7) and goats (OR = 1.7, 95% CI 1.0–3.1). Both B. abortus and B. melitensis DNA were detected in humans and in multiple livestock host species, suggesting cross-transmission of these species among the different hosts. The detection of these two zoonotic Brucella species in humans further underpins the importance of One Health prevention strategies that target multiple host species, especially in the multi-host livestock populations.

352. Seronegative Relapse of Brucellosis in the Central Nervous System

BackgroundBrucellosis is the most common zoonotic infection in the world. High risk areas include the Mediterranean Basin, Eastern Europe and the Middle East. Clinical presentation is quite heterogenous.CNS involvement (neurobrucellosis) varies widely, from 0,5 to 25%. This reflects a high prevalence in endemic areas and the lack of established criteria for a diagnosis. We present a patient with a seronegative relapse of brucellosis, confined in the CNS, identified by Brucella IgG ELISA and 16S rRNA sequencing.MethodsCase ReportResultsA 60-year-old man, farmer, with a history of systematic brucellosis two years before admission, presented with high fever, headache and agitation. A serum agglutination test and anti-Brucella IgG ELISA were negative at baseline. Spinal tap revealed lymphocytosis and low glucose. CSF culture was negative. The patient received ceftriaxone, ampicillin and acyclovir with an initial remission. A week later the patient’s symptoms relapsed. Administration of ceftriaxone and acyclovir was reinitiated with clinical improvement, however, lymphocytic meningitis persisted even after 15 days of treatment.Brain MRI demonstrated nonspecific white matter hyperintensities and severe meningitis as identified by contrast-enhanced 3D Flair MRI. CSF oligoclonal bands showed intrathecal immunoglobin synthesis. CSF agglutination tests and CSF IgG ELISA were positive for Brucella. Though CSF PCR for Brucella was negative, 16S rRNA sequencing revealed the presence of Brucella spp. Patient was treated with ceftriaxone and dexamethasone and despite an initial worsening of neurological symptoms of tremor, loss of balance, hearing loss and diplopia, a clinical remission was achieved after a month and a laboratory remission after eight months of treatment.Brain MRI: 3D Flair Sequence with Contrast Enhancement Table. CSF Parameters ConclusionNeurobrucellosis presents with a variety of clinical symptoms and it should always be considered in neurological patients in highly endemic areas. Establishing a diagnosis is challenging. In our patient, CSF oligoclonal bands and the agglutination test in the CSF helped in achieving a diagnosis, suggesting their possible role in the diagnostic criteria. Although still under debate, the use of corticosteroids in our patient as well as the prolonged use of ceftriaxone in the therapeutic regime were crucial.Disclosures All Authors: No reported disclosures

Human brucellosis outbreaks in Klang district, Malaysia

Background: Human brucellosis is a rare zoonotic disease in Malaysia. Case description: We report two outbreaks of eight human brucella cases which occurred from October 2018 till February 2019 in the Klang district. A total of five confirmed and three probable brucella cases were reported. Of this, four patients had brucellosis after consuming unpasteurized goat's milk from a single goats farm while the other four are farm workers from a cattle farm. Among eight cases, two were children aged 6 and 10 years old. All eight cases presented with fatigue, anorexia and prolonged fever. Four of them presented with headache, myalgia and weight loss while three had arthralgia and one experienced night sweats. Mean duration from onset of symptoms to diagnosis was 45 days, longest duration being 87 days. Six of them developed transaminitis and four of them had haematological abnormalities. Two of those with hematological abnormalities had pancytopenia. Two had hepatomegaly and two had splenomegaly. Two patients were treated as neurobrucellosis as they presented with severe headache and hyperreflexia respectively. One had positive Brucella PCR from his cerebrospinal fluid analysis. Five patients were confirmed brucellosis by PCR detection from blood culture. Three probable Brucella cases were diagnosed based on clinical presentation, positive Brucella IgG titre and an epidemiological link. All the patients required an average of 14 days of hospitalizations and received at least 6 weeks of combination antibiotic. All eight recovered without long term sequalae. Discussion: From these two outbreaks, we find that all patients had prolonged fever and non-specific symptoms with routine laboratory investigations being inconclusive. In both outbreaks, a positive epidemiological link to consuming unpasteurized goat milk and handling of cattle was the important clue to diagnosis. In addition, timely recognition of neurobrucellosis is crucial as the choices and duration of antibiotic might differ from our usual regime. Conclusion: In conclusion, assessment of exposure risk to brucella is prudent while taking history for all patients presenting with fever of unknown origin as it will lead to early diagnosis, early treatment and minimised risk of laboratory staff exposure.

Post-mortem findings in southern right whales Eubalaena australis at Península Valdés, Argentina, 2003-2012.

Between 2003 and 2012, 605 southern right whales (SRW; Eubalaena australis) were found dead along the shores of Península Valdés (PV), Argentina. These deaths included alarmingly high annual losses between 2007 and 2012, a peak number of deaths (116) in 2012, and a significant number of deaths across years in calves-of-the-year (544 of 605 [89.9%]; average = 60.4 yr(-1)). Post-mortem examination and pathogen testing were performed on 212 whales; 208 (98.1%) were calves-of-the-year and 48.0% of these were newborns or neonates. A known or probable cause of death was established in only a small number (6.6%) of cases. These included ship strike in a juvenile and blunt trauma or lacerations (n = 5), pneumonia (n = 4), myocarditis (n = 2), meningitis (n = 1), or myocarditis and meningitis (n = 1) in calves. Ante-mortem gull parasitism was the most common gross finding. It was associated with systemic disease in a single 1-2 mo old calf. Immunohistochemical labeling for canine distemper virus, Toxoplasma gondii and Brucella spp., and PCR for cetacean morbillivirus (CeMV), influenza A, and apicomplexan protozoa were negative on formalin-fixed, paraffin-embedded lung and brain samples from a subset of whales; PCR for Brucella spp. was positive in a newborn/neonate with pneumonia. Skin samples from whales with gull parasitism were PCR negative for CeMV, poxvirus, and papillomavirus. This is the first long-term study to investigate and summarize notable post-mortem findings in the PV SRW population. Consistent, significant findings within or between years to explain the majority of deaths and those in high-mortality years remain to be identified.

Application of Fluorescence Based Molecular Assays for Improved Detection and Typing of Brucella Strains in Clinical Samples

Abstract Bacteria from the genus Brucella are causative agents of brucellosis - a zoonotic disease which affects many wild and domestic animal species and humans. Taking into account the significant socio-economic and public health impact of brucellosis, its control is of great importance for endemic areas. The chosen control strategy could be successful only if adapted to the current epidemiological situation. This implies that a choice of appropriate diagnostic procedures for detection and typing of Brucella spp. strains are of essential importance. Significant advancement of molecular techniques and their advantages compared to classical methods, give strong arguments in promotion of these techniques as a powerful tool for comprehensive diagnostics of brucellosis. Considering this, the major tasks of the study were to select and implement molecular tests for detection and genotyping Brucella spp. and evaluate their performances using DNA from cultivated brucellae (islolates) and limited number of tissue samples from seropositive animals. The obtained results confirmed that implemented real time PCR for Brucella spp. detection, as well as MLVA-16 used for genotyping, have excellent analytical sensitivity (4.2 fg of Brucella DNA were successfully detected and genotyped). Furthermore, compared to bacteriological cultivation of Brucella spp., real time PCR and MLVA-16 protocols showed superior diagnostic sensitivity and detected Brucella DNA in tissues from which Brucella could not be cultivated. Based on the summarized study results, we propose a diagnostic algorithm for detection and genotyping of Brucella spp. bacteria. Routine use of proposed diagnostic algorithm will improve the effectiveness of infection confirmation and help for accurate evaluation of epidemiological situation.

Two-stage PCR assay for detection of human brucellosis in endemic areas

BackgroundBrucellosis is a common zoonosis that can cause a severe febrile illness in humans. It constitutes a persistent health problem in many developing countries around the world. It is one of the most frequently reported diseases in Saudi Arabia and incidence is particularly high in the Central region, and around the city of Riyadh. The aim of this study was to evaluate a two-stage PCR assay for detection of human brucellosis particularly in endemic areas.MethodsA total of 101 serum samples were collected from patients with acute febrile illness (AFI) of unknown cause from two different locations in the Western region of Saudi Arabia. The first location (Northern) is characterized by a nomadic rural population while the second (Central) is a modern urban city. All samples were subjected to DNA extraction and Brucella genus-specific PCR amplification using B4/B5 primers of the bcsp31 gene. Positive B4/B5 samples were subjected to multiplex species-specific Brucella PCR amplification.ResultsIn the Northern location, 81.9% of the AFI samples were confirmed Brucella positive, while all the samples collected from the Central region proved to be Brucella negative. Samples positive for Brucella were subjected to multiplex species-specific Brucella amplification. B. abortus was detected in 10% and B. melitensis in 8% of the samples, while the majority (82%) of samples showed both B. abortus and B. melitensis. As expected, B. suis was not detected in any of the samples.ConclusionsThis study concluded that a two-stage PCR assay could be useful as a rapid diagnostic tool to allow the consideration of brucellosis as a possible cause of AFI, particularly in non-urban locations. It also recommends the collection of epidemiological data for such patients to obtain further information that may help in rapid diagnosis.

Serological and molecular diagnosis of human brucellosis in Najran, Southwestern Saudi Arabia

This study aimed to investigate the prevalence of human brucellosis in Najran, southwestern Saudi Arabia, and to assess the performances of ELISA and PCR as diagnostic tools for brucellosis with respect to conventional methods. The study included 340 patients with clinical characteristics of brucellosis. Blood samples from cases and controls were subjected to culture, standard tube agglutination test (SAT), ELISA for IgM and IgG, and brucella PCR. The diagnosis of brucellosis was confirmed in 54 (15.9%) of the 340 provisionally diagnosed brucellosis patients. Blood culture identified only 14 (25.9%) cases. The SAT was positive for 50 (92.6%) cases, whereas ELISA IgM, IgG and PCR were found positive in 46, 52 and 38 cases respectively. The sensitivities of ELISA IgM and IgG were 85.2% and 96.3% respectively and the specificity was 100% for each. For PCR, the sensitivity and specificity were 70.4% and 100% respectively. In conclusion, ELISA offers a significant advantage over conventional serological methods in the diagnosis of brucellosis in endemic areas. The PCR test results can be particularly important in patients with clinical signs and symptoms, and negative serological results, allowing the early and rapid confirmation of the brucellosis.

Pautas diagnósticas y actitud terapéutica ante un bruceloma hepático

Liver involvement due to brucellosis can occur during the acute or the chronic phase of the disease. The marked tendency toward circumscription can give rise to hepatic pseudotumoral lesions with a calcified granulomatous appearance, called brucellomas. Surgery is the only correct treatment, including Brucella PCR in the central calcified lesion, because serological studies are often inconclusive.

Large-scale evaluation of a single-tube nested PCR for the laboratory diagnosis of human brucellosis in Kuwait

A single-tube nested PCR assay identifying a 52 bp fragment from the genus-specific Brucella IS711 gene was used prospectively in clinical practice for the diagnosis of human brucellosis in Kuwait. Patients with suspected brucellosis and with other infections were investigated as clinically indicated but in all of them culture, serology and PCR for Brucella were carried out. Out of 263 suspected cases of brucellosis, diagnostic tests were positive in 199, serology was positive in 199 and culture in 89, while the Brucella PCR was positive in 193 (sensitivity 96.98%, 95% confidence interval 94.5-99.5%). Chronic brucellosis, involving symptoms for more than 1 year, was diagnosed in 49 out of these 193 patients. Diagnoses in four out of the six patients with positive serology but negative PCR and culture were non-Brucella bacterial meningitis and viral meningitis. False-negative PCR results were possible in the remaining two; both had been on long-term antibiotics for previously diagnosed brucellosis but their adherence may have been questionable, allowing a relapse. The PCR was negative in 244 patients with other infections (specificity 100%) and in 180 control subjects with negative Brucella culture and serology. PCR results were available within 24 h of sample receipt, providing diagnostic information rapidly. The PCR is expensive and technically demanding and may not be appropriate for all cases. Future studies will help define how it may best be used. For example Brucella and tuberculous meningitides can be very similar, Brucella PCR may allow prompt distinction between the two, avoiding the need for prolonged empirical treatment for both.