Diagnostic usefulness of bone marrow aspiration material for the amplification of IS6110 insertion element in extrapulmonary tuberculosis: comparison of two PCR techniques.

SETTING: In many cases of extra-pulmonary tuberculosis (EPTB), with the exception of paucibacillary analysed specimens, the suspected site of mycobacterial infection is relatively inaccessible or unknown, making laboratory confirmation of TB laborious and problematic. OBJECTIVE: Two different polymerase chain reaction (PCR) based methods were compared to investigate the validity of bone marrow aspiration material as an easily accessible alternative sample for molecular analysis in EPTB. DESIGN: We amplified the same sequence of IS6110 of Mycobacterium tuberculosis complex in 19 confirmed cases of EPTB using two different nested PCR techniques: one in-house 'classic' PCR and another based on LightCycler technology. RESULTS: Both methods demonstrated the same reliability when performed in samples of infected tissue. However, the LightCycler protocol was superior to the in-house system when applied in bone marrow aspiration material, revealing positivity in 18/19 compared to 13/19 samples of 'classic' PCR. CONCLUSION: The application of an optimised LightCycler nested amplification protocol in bone marrow aspirates may promote diagnostic accuracy in difficult and/or urgent cases of EPTB.

Autoimmune manifestations in human myelodysplasia: a positive correlation with interferon regulatory factor-1 (IRF-1) expression.

BACKGROUND: Patients with myelodysplasia may have autoimmune manifestations (AIM). Interferon regulatory factor-1 (IRF-1) is a transcription factor involved in interferon signalling, leukaemogenesis, and the development of the immune system. OBJECTIVES: To determine whether IRF-1 is implicated in the pathophysiology of AIM in myelodysplasia. METHODS: 14 patients with myelodysplasia were studied, seven with AIM and seven without. Five patients with vasculitis and seven normal subjects served as controls. The expression of IRF-1 was studied in bone marrow mononuclear cells taken from patients and controls, using a relative quantitative reverse transcriptase polymerase chain reaction. RESULTS: A 10-fold reduction in full length IRF-1 mRNA was detected in the myelodysplasia patients without AIM compared with the normal controls. In contrast, the group with AIM had increased IRF-1 transcripts, to a level almost equal to that observed in patients with vasculitis and normal controls. CONCLUSIONS: Myelodysplasia patients without IRF-1 expression had a decreased incidence of AIM. Thus the absence of IRF-1 transcription factor appears to protect against the development of autoimmunity in myelodysplasia.

Non-isotopic RNase cleavage assay for mutation detection in MEFV, the gene responsible for familial Mediterranean fever, in a cohort of Greek patients.

BACKGROUND: The MEFV gene is responsible for familial Mediterranean fever (FMF). Several disease associated mutations have been identified. The range of genetic variation in MEFV in Greek patients has not been determined. OBJECTIVE: To describe a method that facilitates the routine screening of the entire coding sequence of MEFV (excluding exon 1). METHODS: The non-isotopic RNase cleavage assay (NIRCA) was optimised and used as a first step screening method to screen exons 2 to 10 of MEFV. Exons 2 and 10 were analysed separately at DNA level, while exons 3 to 9 were analysed together at cDNA level. The sample group consisted of 26 FMF patients diagnosed using established clinical criteria, six asymptomatic relatives, 12 patients with atypical clinical manifestations, nine patients suffering from various inflammatory diseases, and three normal individuals. All were analysed by NIRCA for mutations in the MEFV gene and direct sequencing was applied subsequently to confirm the results. RESULTS: MEFV mutations were identified in 25 of 26 typical FMF patients and in two of 12 patients with atypical manifestations. NIRCA results were in concordance with sequencing findings in all sequences analysed, suggesting that the method is highly reliable in this disease. Sixteen alterations of MEFV were identified (eight missense mutations and eight single nucleotide polymorphisms). CONCLUSIONS: NIRCA can be used for rapid screening of the coding sequence of the MEFV gene in patients suspected of suffering from FMF.

Detection of Mycobacterium tuberculosis complex DNA in pericardial fluid, bone marrow and peripheral blood in a patient with pericardial tuberculosis. A case report.

Definitive diagnosis of tuberculous pericarditis requires identification of bacilli in pericardial fluid or tissue. Conventional diagnostic methods are time-consuming and have a low sensitivity making bacteriological confirmation of the disease very difficult. Hereby, we report the case of molecular detection of Mycobacterium tuberculosis in pericardial fluid, bone marrow and peripheral blood from a 63-year-old woman with pericardial tuberculosis, using a nested PCR assay specific for IS6110 insertion element of M. tuberculosis complex. The patient had an excellent response to a three-drug combination anti-tuberculous regimen and 1 year later was asymptomatic, without evidence of constrictive pericarditis.

Rapid mutational analysis of N-ras proto-oncogene in hematologic malignancies: study of 77 Greek patient.

BACKGROUND AND OBJECTIVES: N-ras mutations are the most commonly detected molecular abnormalities in hematologic malignancies, especially in those of myeloid origin. Different techniques have been used to detect N-ras mutations; however, most of them are either labor intensive or provide sequence data for only a limited number of codons. Consequently, study of the N-ras oncogene has not been convenient in every day clinical practice being restricted, as a rule, to retrospective analysis of patients. DESIGN AND METHODS: In this study we used a recently developed method that enables rapid and reliable detection of mutations at the cDNA level, namely, the non-isotopic RNase cleavage assay (NIRCA). Using this method we were able to screen the N-ras oncogene rapidly and determine the incidence and prognostic significance of N-ras mutations in 77 Greek patients with acute leukemia, myelodysplastic syndromes and chronic myeloproliferative disorders, both at the presentation and during relapse or progression of the disease. RESULTS: Activating N-ras mutations were detected in 7 patients and our results were confirmed by direct sequencing. Interestingly, two novel alterations were identified, a mutation at codon 8 (characterized by a substitution of valine by leucine) in a patient with chronic myeloid leukemia during hematologic relapse of the disease and a polymorphism at codon 92 (1002T-->C, without amino acid substitution) in a patient with chronic myelomonocytic leukemia. INTERPRETATION AND CONCLUSIONS: A rapid and easy protocol that allows the analyses of N-ras sequences has been developed. This reverse transcription-polymerase chain reaction (RT-PCR)/NIRCA protocol can allow the study of this proto-oncogene in every day clinical practice, rapidly facilitating the validation of the diagnostic and prognostic value of N-ras mutational analyses in patients with hematologic malignancies.

Amplification of IS6110 sequence for detection of Mycobacterium tuberculosis complex in HIV-negative patients with fever of unknown origin (FUO) and evidence of extrapulmonary disease.

OBJECTIVES: Extrapulmonary tuberculosis (TB) constitutes the main cause of classic fever of unknown origin (FUO) in many populations. The aim of this study was to improve the diagnostic field of the disease using a nested polymerase chain reaction (PCR) assay, specific for the IS6110 insertion element of Mycobacterium tuberculosis complex, in order to achieve a more timely diagnosis and treatment. SETTING: Twenty-four, HIV-negative classic FUO patients who were admitted to the Regional Hospital of Alexandroupolis between April 1997 and July 1999. SUBJECTS AND DESIGN: The above patients were considered as putative extrapulmonary TB after 3 weeks of in-patient investigation and underwent exhaustive examination for diagnosis of the disease. For this purpose, specimens were obtained from peripheral blood and bone marrow from these patients, as well as from damaged tissues, and analysed by both PCR and conventional methods. Anti-tuberculous treatment was initiated in 16 out of 24 patients and the response to this regimen was considered as the final criterion for diagnosis of tuberculosis. RESULTS: Extrapulmonary TB was established in 11 patients. The PCR-based methodology, when applied to samples derived from bone marrow aspirations and suspected damaged tissues, was able to diagnose 10 of them, whereas the conventional methods were able to detect only two. CONCLUSIONS: Our results confirm the diagnostic value of molecular detection of M. tuberculosis in cases of FUO, thus supporting the application of PCR in tissue samples suspected of bacillus infection. Furthermore, our studies demonstrate that bone marrow aspiration specimens constitute an alternative, easy, safe and reliable source for such PCR analysis.