Usefulness of a quantitative real-time PCR assay using serum samples to discriminate between inactive, serologically positive and active human brucellosis

Abstract

Diagnosis of brucellosis can be difficult in certain scenarios where conventional microbiological techniques have important limitations. The aim of this study was to develop a LightCycler Quantitative PCR assay in serum samples to discriminate between active and past brucellosis. In total, 110 serum samples from 46 brucellosis patients and 64 controls, including persons who had recently been treated for brucellosis, asymptomatic persons exposed to brucellosis, and patients with febrile syndromes involving a differential diagnosis with brucellosis, were studied. Brucella spp.-specific sequences of the PCR primers and probe were selected from the gene encoding an immunogenic membrane protein of 31 kDa (BCSP31). The analytical sensitivity was 1 x 10(1) fg of Brucella DNA. The mean threshold cycles for brucellosis patients and controls were 31.8 +/- 1.7 and 35.4 +/- 1.1, respectively (p <0.001). The best cut-off for bacterial DNA load was 5 x 10(3) copies/mL. At this cut-off, the area under the receiver operating characteristic curves was 0.963 (95% CI 0.920-1.005), with a sensitivity of 93.5% and a specificity of 98.4%. Under the assay conditions, the LightCycler Quantitative PCR in serum samples seems to be highly reproducible, rapid, sensitive and specific. It is therefore a useful method for both the initial diagnosis and the differentiation between past and active brucellosis.

Challenges of Establishing the Correct Diagnosis of Outbreaks of Acute Febrile Illnesses in Africa: The Case of a Likely Brucella Outbreak among Nomadic Pastoralists, Northeast Kenya, March–July 2005

“…In this investigation, there was no significant difference in the IgM and IgG ELISA titers, except for specimens from three patients, two of whom had IgM titers several-folds higher than IgG suggestive of acute infection; the other positive IgG and IgM results that were the same or within one-fold titer of each other are difficult to interpret, especially in light of the fact that IgM declines faster than IgG following acute infection. 19 Although polymerase chain reaction is sensitive for Brucella , [20][21][22][23][24] it is expensive, susceptible to false positives, and still not validated for direct use on blood specimens. 20 Human leptospirosis has been documented in several parts of Kenya through clinical studies in the 1970s including isolation of leptospires from patients, which is definitive, and surveys in the 1960s.…”

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

“…Realtime PCR systems exhibit improved sensitivity, specificity, and speed; due to their combined use of fluorogenic dyes and direct detection, these methods further eliminate the need for postamplification detection procedures (11). In addition, quantitative real-time PCR is a useful tool for differentiating between inactive but serologically positive brucellosis and active brucellosis (12,13). The goals of our present study were to design a quantitative TaqMan-based real-time PCR assay and to evaluate the performance of this assay using human serum samples.…”

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

“…In our earlier findings (Mukherjee et al, 2007), compared to omp2 and 16S rRNA, the bcsp31 PCR was found to be 100% specific and was the most sensitive assay with PPv of 100% and NPv of 88%. Also numerous reports mentioned the use of bcsp31 for specific identification of genus Brucella from seropositive, active, relapsing, chronic cases in humans (Kattar et al, 2007;Mitka et al, 2007;Queipo-Ortuno et al, 2008). Recently the same gene target has been used specifically to detect Brucella in human serum, blood and cerebro-spinal fluid (Debeaumont et al, 2005;Colmenero et al, 2011;Sohrabi et al, 2011), in buffalo milk (Amoroso et al, 2011) and in clinical tissues from seals (Sidor et al, 2013).…”

“…Earlier, conventional PCR was used for the detection of Brucella from culture and clinical samples targeting the bcsp31, 16S rRNA, 16S-23S intergenic transcribed spacers (ITS), IS711, per and omp2 genes (Baily et al, 1992;Romero et al, 1995;Rijpens et al, 1996;Henault et al, 2000;Bogdanovich et al, 2004;Leal-Klevezas et al, 1995). During the past 15 years quantitation of the Brucella genome from cultures and clinical samples has been reported using qPCR targeting the bcsp-31, IS711, 16S-23S spacer, omp25, per and omp31 genes employing SYBR Green labelled probes, hydrolysis probes or systems that use fluorescence resonance energy transfer for specific hybridization with DNA template (Redkar et al, 2001;Kattar et al, 2007;Queipo-Ortuno et al, 2008;Zhang et al, 2011). Most of these reports indicated above were based on clinical studies derived from samples from human cases, and the selected gene targets were bcsp 31 or the IS711 element.…”