Rapid diagnosis of human brucellosis by peripheral-blood PCR assay.

A single-step PCR assay with genus-specific primers for the amplification of a 223-bp region of the sequence encoding a 31-kDa immunogenetic Brucella abortus protein (BCSP31) was used for the rapid diagnosis of human brucellosis. We examined peripheral blood from 47 patients, with a total of 50 cases of brucellosis, and a group of 60 control subjects, composed of patients with febrile syndromes of several etiologies other than brucellosis, asymptomatic subjects seropositive for Brucella antibodies, and healthy subjects. Diagnosis of brucellosis was established in 35 cases (70%) by isolation of Brucella in blood culture and in the other 15 cases (30%) by clinical and serological means. The sensitivity of our PCR assay was 100%, since it correctly identified all 50 cases of brucellosis, regardless of the duration of the disease, the positivity of the blood culture, or the presence of focal forms. The specificity of the test was 98.3%, and the only false-positive result was for a patient who had had brucellosis 2 months before and possibly had a self-limited relapse. In those patients who relapsed, the results of our PCR assay were positive for both the initial infection and the relapse, becoming negative once the relapse treatment was completed and remaining negative in the follow-up tests at 2, 4, and 6 months. In conclusion, these results suggest that the PCR assay is rapid and easy to perform and highly sensitive and specific, and it may therefore be considered a useful tool for diagnosis of human brucellosis.

Diagnostic yield of a PCR assay in focal complications of brucellosis.

In order to evaluate the diagnostic yield of a PCR assay for patients with focal complications of brucellosis, we studied by PCR and by conventional microbiological techniques 34 nonblood samples from 32 patients with different focal forms of brucellosis. The samples from patients with brucellosis were paired to an equal number of control samples from the same locations of patients whose illnesses had different etiologies. Thirty-three of the 34 nonblood samples (97%) from the brucellosis patients were positive by PCR, whereas Brucella spp. were isolated from only 29.4% of the conventional cultures. For 11.4% of the patients, the confirmatory serological tests were either negative or showed titers below the diagnostic range. Two patients (6.2%) from the control group, both with tuberculous vertebral osteomyelitis, had a positive PCR result. The brucella PCR of blood from these two patients was also positive, and the two strains of Mycobacterium tuberculosis isolated were analyzed by the brucella PCR, with no evidence of amplification. These results show that the PCR assay is far more sensitive than conventional cultures, and this, coupled with its speed and reduction in risk to laboratory workers, makes this technique a very useful tool for the diagnosis of focal complications of brucellosis.

Comparative Study of a Real-Time PCR Assay Targeting senX3-regX3 versus Other Molecular Strategies Commonly Used in the Diagnosis of Tuberculosis.

BACKGROUND Nucleic acid amplification tests are increasingly used for the rapid diagnosis of tuberculosis. We undertook a comparative study of the efficiency and diagnostic yield of a real-time PCR senX3-regX3 based assay versus the classical IS6110 target and the new commercial methods. METHODS This single-blind prospective comparative study included 145 consecutive samples: 76 from patients with culture-confirmed tuberculosis (86.8% pulmonary and 13.2% extrapulmonary tuberculosis: 48.7% smear-positive and 51.3% smear-negative) and 69 control samples (24 from patients diagnosed with non-tuberculous mycobacteria infections and 45 from patients with suspected tuberculosis which was eventually ruled out). All samples were tested by two CE-marked assays (Xpert®MTB/RIF and AnyplexTM plus MTB/NTM) and two in-house assays targeting senX3-regX3 and the IS6110 gene. RESULTS The detection limit ranged from 1.00E+01 fg for Anyplex, senX3-regX3 and IS6110 to 1.00E+04 fg for Xpert. All three Xpert, senX3-regX3 and IS6110 assays detected all 37 smear-positive cases. Conversely, Anyplex was positive in 34 (91.9%) smear-positive cases. In patients with smear-negative tuberculosis, differences were observed between the assays; Xpert detected 22 (56.41%) of the 39 smear-negative samples, Anyplex 24 (61.53%), senX3-regX3 28 (71.79%) and IS6110 35 (89.74%). Xpert and senX3-regX3 were negative in all control samples; however, the false positive rate was 8.7% and 13% for Anyplex and IS6110, respectively. The overall sensitivity was 77.6%, 85.7%, 77.3% and 94.7% and the specificity was 100%, 100%, 90.8% and 87.0% for the Xpert, senX3-regX3, Anyplex and IS6110 assays, respectively. CONCLUSION Real-time PCR assays targeting IS6110 lack the desired specificity. The Xpert MTB/RIF and in-house senX3-regX3 assays are both sensitive and specific for the detection of MTBC in both pulmonary and extrapulmonary samples. Therefore, the real time PCR senX3-regX3 based assay could be a useful and complementary tool in the diagnosis of tuberculosis.

Exposition internationale des industries et du travail de Turin, 1911. Groupe XVI. Classe 90 (A et B). Produits de la grande culture. M. le Dr Bousquet, rapporteur. Classe 102 B. Corps gras, huiles végétales et animales, suifs, cires, etc. M. C. Artus, rapporteur. Classe 80. Météorologie, géognosie et géologie agricoles. M. le Dr Thouvenin, rapporteur

Comprend : Corps gras, huiles végétales et animales, suifs, cires

Institut Pasteur, laboratoire du professeur [Jean] Danysz, préparation du virus contre les rats, 1re préparation du bouillon de culture : [photographie de presse] / [Agence Rol]

Référence bibliographique : Rol, 57064

Vancomycin-intermediate Staphylococcus aureus selected during vancomycin therapy of experimental endocarditis are not detected by culture-based diagnostic procedures and persist after treatment arrest.

OBJECTIVES: Laboratory detection of vancomycin-intermediate Staphylococcus aureus (VISA) and their heterogeneous VISA (hVISA) precursors is difficult. Thus, it is possible that vancomycin failures against supposedly vancomycin-susceptible S. aureus are due to undiagnosed VISA or hVISA. We tested this hypothesis in experimental endocarditis.¦METHODS: Rats with aortic valve infection due to the vancomycin-susceptible (MIC 2 mg/L), methicillin-resistant S. aureus M1V2 were treated for 2 days with doses of vancomycin that mimicked the pharmacokinetics seen in humans following intravenous administration of 1 g of the drug every 12 h. Half of the treated animals were killed 8 h after treatment arrest and half 3 days thereafter. Population analyses were done directly on vegetation homogenates or after one subculture in drug-free medium to mimic standard diagnostic procedures.¦RESULTS: Vancomycin cured 14 of 26 animals (54%; P<0.05 versus controls) after 2 days of treatment. When vegetation homogenates were plated directly on vancomycin-containing plates, 6 of 13 rats killed 8 h after treatment arrest had positive cultures, 1 of which harboured hVISA. Likewise, 6 of 13 rats killed 3 days thereafter had positive valve cultures, 5 of which harboured hVISA. However, one subculture of vegetations in drug-free broth was enough to revert all the hVISA phenotypes to the susceptible pattern of the parent. Thus, vancomycin selected for hVISA during therapy of experimental endocarditis due to vancomycin-susceptible S. aureus. These hVISA were associated with vancomycin failure. The hVISA phenotype persisted in vivo, even after vancomycin arrest, but was missed in vitro after a single passage of the vegetation homogenate on drug-free medium.¦CONCLUSIONS: hVISA might escape detection in clinical samples if they are subcultured before susceptibility tests.

Distribution du bouillon de culture dans les bouteilles : [photographie de presse] / [Agence Rol]

Référence bibliographique : Rol, 57063

Ensemencement des bouteilles avec la culture virulente : [photographie de presse] / [Agence Rol]

Référence bibliographique : Rol, 57061

Point Mutations in the Monocarboxylate Transporter SLC16A12 Lead to Juvenile and Age-Related Cataract

Purpose: Previously we reported on a premature termination mutation in SLC16A12 that leads to dominant juvenile cataract and renal glucosuria. To assess the mutation rate and genotype-phenotype correlations of SLC16A12 in juvenile or age-related forms of cataract, we performed a mutation screen in cataract patients. Methods: Clinical data of approximately 660 patients were collected, genomic DNA was isolated and analyzed. Exons 3 to 8 including flanking intron sequences of SLC16A12 were PCR amplified and DNA sequence was determined. Selected mutations were tested by cell culture assays, in silico analysis and RT-PCR. Results: We found sequence alterations at a rate of approximately 1/75 patients. None of them was found in 360 control alleles. Alterations affect splice site and regulatory region but most mutations caused an amino acid substitution. The majority of the coding region mutations maps to trans-membrane domains. One mutation located to the 5'UTR. It affects translational efficiency of SLC16A12. In addition, we identified a cataract-predisposing SNP in the non-coding region that causes allele-specific splicing of the 5'UTR region. Conclusions: Altered translational efficiency of the solute carrier SLC16A12 and its allele-specific splicing strongly support a model of challenged homeostasis to cause various forms of cataract. In addition, the pathogenic property of the here reported sequence alterations is supported by the lack of known sequence variations within the coding region of SLC16A12. Due to the relatively high mutation rate, we suggest to include SLC16A12 in diagnostic cataract screening. Generally, our data recommend the assessment of regulatory sequences for diagnostic purposes.