Efficient Diagnosis and Treatment Follow-Up of Human Brucellosis by a Novel Quantitative TaqMan Real-Time PCR Assay: a Human Clinical Survey

Abstract

cRapid and effective diagnosis of brucellosis is a challenge for clinicians. Even when diagnosis is on time and therapy is initiated, meticulous follow-up appointments are crucial for ensuring the efficacy of the treatment. Due to shortcomings of serological methods, molecular diagnosis, especially real-time PCR, is becoming a main approach in laboratory diagnostics. Thus, the development of efficient procedures and standardization of the PCR tests will have a great impact on the precise detection and quantification of bacterial DNA loads, which is valuable for the medical management of brucellosis patients. We developed a new TaqMan real-time PCR directed to bcsp31, a shared gene of the brucellae. The bcsp31 gene fragment was cloned into pJET1.2. Recombinant pJET1.2-bcsp31 was linearized by HindIII digestion, and the product was used for the preparation of a standard curve. A panel of Brucella spp. and non-Brucella pathogens was tested. No bacterial genomes other than those of the brucellae were detected. According to the results, specificity of the method was 100%. In a clinical assessment, the positive-control group comprised 37 patients with microbiologically confirmed brucellosis, and 25 healthy individuals served as the negative-control group. By the end of the treatment period, there was a significant decrease in the DNA load of the 37 brucellosis patients, which persisted for the 4 weeks of monitoring after treatment, suggesting that our proposed method is an efficient monitoring tool. Serum samples prior to any treatment were collected from the 25 serologically suspicious patients and assessed by our method; 72% of these patients tested positive for brucellosis.T he brucellae are facultative, intracellular, Gram-negative bacteria that infect animals and humans (1). Although it has been eradicated in some developed countries, brucellosis still remains a major problem worldwide. Diagnosis of the disease presents a major challenge to clinical and veterinary services (2, 3). Brucella spp. are able to survive within host cells, leading to relapses and focal complications (1). Early diagnosis of brucellosis results in early initiation of therapy, which plays a key role in the success of the control and eradication programs; nonetheless, accurate diagnosis requires reliable and sensitive diagnostic tools (4). The intracellular localization of the bacteria and the slow evolution of the disease hamper the usefulness of blood cultures (5). Despite being the gold standard, isolation of brucellae from blood culture or other sterile body fluids has a sensitivity of about 70% (4, 6). Serodiagnostic assays are principally based on antibodies against the Brucella lipopolysaccharide (LPS) and have high sensitivity but low specificity. The low specificity may be due to the crossreaction of antibodies with LPS from other bacterial species and subsensitive or high-immunity reactions, depending on the subclinical or endemic prevalence of the disease (4, 6, 7). The presence of IgG and IgM for months after therapy complicates...

Optimization and Validation of a Diagnostic Real-Time PCR for Bovine Brucellosis

“…Thus some proportion of samples that were positive by culture was negative by qPCR (Debeaumont et al, 2005), similarly samples positive by IS711 PCR assay have been shown negative by culture (Sanjuan-Jimenez et al, 2013). Choice of media selected for isolation by culture may also affect sensitivity (Her et al, 2009;Sohrabi et al, 2014;Dean et al, 2014). The range of C q values of clinical samples positive for brucellosis reported in the current study were similar (C q -33.3±4.6) to those reported earlier (Colmenero et al, 2011).…”

“…Their studies had indicated that the DSe could vary from 64.7% to 88% and DSp 98.3% to 100%. Previously the estimates of diagnostic qPCR for brucellosis had been calculated based on case-wise (Surucuoglu et al, 2009) and also on sample-wise status in humans and in animals (Debeaumont et al, 2005;Sanjuan-Jimenez et al, 2013;Sohrabi et al, 2014), and on comparison with status by culture (Amoroso et al, 2011;El Behiry et al, 2014), serology (Gwida et al, 2011;Menshawy et al, 2014;Sohrabi et al, 2014) and In the present study, we have optimized and validated the diagnostic estimates of qPCR by animal-wise and sample-wise approach comparing the culture status in the first instance and the combined culture and serology status in the second instance. The multiple approach adopted for derivation of diagnostic estimates led to interesting observations.…”

Guidelines for the management of human brucellosis in the State of Paraná, Brazil

“…The Rose Bengal test has high sensitivity and specificity, but positive results can occur in asymptomatic patients after exposure to Brucella or vaccination 20 . Real-time PCR was considered as the gold-standard method for diagnosis because Brucella can only be cultured in laboratories with at least a biosafety level for three 21,22 . Symptomatic patients (symptoms suggesting brucellosis) with positive a PCR or a reagent EIA IgM should receive treatment, after excluding other potential causes of the symptoms.…”

<b>Desenvolvimento, padronização e validação de reação em cadeia pela polimerase (PCR) em tempo real para diagnóstico da brucelose canina</b>

“…A resolução do sistema de detecção dos produtos amplificados na PCR em tempo real é considerada maior do que na PCR convencional, já que a primeira é baseada na emissão de fluorescência pelos produtos amplificados que são marcados com corantes intercalantes fluorescentes ou pelo uso de sondas marcadas com fluoróforos, a qual é detectada por um leitor de fluorescência. Já na PCR convencional, a verificação dos produtos amplificados é feita visualmente, pela observação dos produtos amplificados em gel de agarose após a coloração com brometo de etídio, sob luz ultravioleta (DEBEAUMONT; FALCONNET; MAURIN, 2005;KATTAR et a., 2007;BOUNAADJA et al, 2009;SOHRABI et al, 2014;HÄNSEL et al, 2015;KHAMESIPOUR, 2015;KADEN et al, 2017;LINDAHL-RAJALA et al, 2017).…”

“…Tabela 4-Resultados dos testes de hemocultura, PCR convencional (PCRc) e PCR em tempo real utilizando o protocolo B (PCRtr-B) realizado à temperatura de annealing de 59ºC e utilizando-se 200 nM de primers em oito amostras de DNA obtidas de sangue canino, para a detecção de Brucella spp. BOUNAADJA et al, 2009;SOHRABI et al, 2014;HÄNSEL et al, 2015;KHAMESIPOUR, 2015;KADEN et al, 2017;LINDAHL-RAJALA et al, 2017), no entanto, ao longo deste estudo foi observado que o método apresentou falhas, uma vez que houve amplificação de alguns controles negativos da extração e também amplificação de diversas amostras que tinham sido negativas nos outros testes usados para diagnóstico de infecção por B. canis (hemocultura e PCR convencional).…”