Comparative Clinical Study of Different Multiplex Real Time PCR Strategies for the Simultaneous Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis

<div><p>Background</p><p>Both brucellosis and tuberculosis are chronic-debilitating systemic granulomatous diseases with a high incidence in many countries in Africa, Central and South America, the Middle East and the Indian subcontinent. Certain focal complications of brucellosis and extrapulmonary tuberculosis are very difficult to differentiate clinically, biologically and radiologically. As the conventional microbiological methods for the diagnosis of the two diseases have many limitations, as well as being time-consuming, multiplex real time PCR (M RT-PCR) could be a promising and practical approach to hasten the differential diagnosis and improve prognosis.</p><p>Methodology/Principal Findings</p><p>We designed a SYBR Green single-tube multiplex real-time PCR protocol targeting <i>bcsp31</i> and the <i>IS711</i> sequence detecting all pathogenic species and biovars of <i>Brucella</i> genus, the <i>IS6110</i> sequence detecting <i>Mycobacterium</i> genus, and the intergenic region senX3-regX3 specifically detecting <i>Mycobacterium tuberculosis complex</i>. The diagnostic yield of the M RT-PCR with the three pairs of resultant amplicons was then analyzed in 91 clinical samples corresponding to 30 patients with focal complications of brucellosis, 24 patients with extrapulmonary tuberculosis, and 36 patients (Control Group) with different infectious, autoimmune or neoplastic diseases. Thirty-five patients had vertebral osteomyelitis, 21 subacute or chronic meningitis or meningoencephalitis, 13 liver or splenic abscess, eight orchiepididymitis, seven subacute or chronic arthritis, and the remaining seven samples were from different locations. Of the three pairs of amplicons (senX3-regX3+ bcsp3, senX3-regX3+ IS711 and IS6110+ IS711) only senX3-regX3+ IS711 was 100% specific for both the <i>Brucella</i> genus and <i>M. tuberculosis complex</i>. For all the clinical samples studied, the overall sensitivity, specificity, and positive and negative predictive values of the M RT-PCR assay were 89.1%, 100%, 85.7% and 100%, respectively, with an accuracy of 93.4%, (95% CI, 88.3—96.5%).</p><p>Conclusions/Significance</p><p>In this study, a M RT-PCR strategy with species-specific primers based on senX3-regX3+IS711 sequences proved to be a sensitive and specific test, useful for the highly efficient detection of <i>M. tuberculosis</i> and <i>Brucella</i> spp in very different clinical samples. It thus represents an advance in the differential diagnosis between some forms of extrapulmonary tuberculosis and focal complications of brucellosis.</p></div

Tm (°C) and Ct (cycle) values with the three strategies of M RT-PCR studied by type of clinical sample.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002593#s3" target="_blank">Results</a> are given as mean ± SD with the number of samples assayed in parentheses.</p

Results of M RT-PCR according to clinical sample, microorganism, and culture result.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002593#s3" target="_blank">Results</a> of M RT-PCR according to clinical sample, microorganism, and culture result.</p

Specificity of PCR products based on the Tm.

<p><b>Panel A</b>, M RT-PCR assay IS6110+IS711. Black triangles lines, positive control of <i>B. abortus</i>; Black stars, positive controls of <i>M. tuberculosis</i>; blue lines and orange lines, false positive results due to <i>M. intracellulare</i> and <i>M. simiae</i>, respectively. <b>Panel B</b>, M RT-PCR assay senX3-regX3+bcsp31. Black triangles lines, positive control of <i>B. abortus</i>; Black stars, positive controls of <i>M. tuberculosis</i>; green lines, false positive results related with <i>Ochrobactrum anthropi</i>.</p

M RT-PCR results with DNA from different microorganism included in this study.

<p>M RT-PCR results with DNA from different microorganism included in this study.</p

Sample type and diagnosis of the study patients.

a<p><i>S. aureus</i>, 4 cases, <i>E. coli</i> and <i>S. epidermidis</i> 2 cases, <i>S. agalactiae, Peptoestreptococcus</i>, <i>S. intermedius</i>, and <i>M. xenopi</i> one case respectively,</p>b<p>Meningoencephalitis 4 cases (<i>T. whippelii</i>, V-Z, JC virus and toxoplasma), <i>criptococcu</i>s meningitis 2 cases and neurosyphilis, giant cell arteritis, neurosarcoidosis, meningeal carcinomatosis one case respectively,</p>c<p><i>E. coli</i> 4 cases, <i>P. aeruginosa</i>, <i>S. intermedius</i> and <i>B. fragilis</i> one case each respectively.</p>d<p><i>P. mirabilis</i> and seminoma one case respectively,</p>e<p><i>S. aureus</i> 3 cases and <i>N. meningitides</i> one case,</p>f<p>Sternal osteomyelitis due to <i>Mycobacterium avium</i>.</p

Amplicons Tm of the three M RT-PCR assayed in different clinical samples and collection strains.

<p>Amplicons Tm of the three M RT-PCR assayed in different clinical samples and collection strains.</p

DNA concentrations and purity from the different clinical samples studied.

<p>Sample type: VT, vertebral or paravertebral tissue; CSF, cerebrospinal fluid; HET, hepatic or splenic tissue; SF, synovial fluid; OS, other samples.</p

Diagnostic yield of M RT-PCR in clinical specimens from patients with focal complications of brucellosis or extrapulmonary tuberculosis.

<p>PPV, positive predictive value; NPV, negative predictive value; Positive LR, positive likelihood ratio; Negative LR, negative likelihood ratio; 95% CI = 95% confidence interval, ND*, not done for mathematical reasons (division by zero).</p