Recessive multiple epiphyseal dysplasia (rMED) with homozygosity for C653S mutation in the DTDST gene - phenotype, molecular diagnosis and surgical treatment of habitual dislocation of multilayered patella: case report.

BACKGROUND: Multiple epiphyseal dysplasia (MED) is one of the more common generalised skeletal dysplasias. Due to its clinical heterogeneity diagnosis may be difficult. Mutations of at least six separate genes can cause MED. Joint deformities, joint pain and gait disorders are common symptoms. CASE PRESENTATION: We report on a 27-year-old male patient suffering from clinical symptoms of autosomal recessive MED with habitual dislocation of a multilayered patella on both sides, on the surgical treatment and on short-term clinical outcome. Clinical findings were: bilateral hip and knee pain, instability of femorotibial and patellofemoral joints with habitual patella dislocation on both sides, contractures of hip, elbow and second metacarpophalangeal joints. Main radiographic findings were: bilateral dislocated multilayered patella, dysplastic medial tibial plateaus, deformity of both femoral heads and osteoarthritis of the hip joints, and deformity of both radial heads. In the molecular genetic analysis, the DTDST mutation g.1984T > A (p.C653S) was found at the homozygote state. Carrier status was confirmed in the DNA of the patient's parents. The mutation could be considered to be the reason for the patient's disease. Surgical treatment of habitual patella dislocation with medialisation of the tibial tuberosity led to an excellent clinical outcome. CONCLUSIONS: The knowledge of different phenotypes of skeletal dysplasias helps to select genes for genetic analysis. Compared to other DTDST mutations, this is a rather mild phenotype. Molecular diagnosis is important for genetic counselling and for an accurate prognosis. Even in case of a multilayered patella in MED, habitual patella dislocation could be managed successfully by medialisation of the tibial tuberosity.

Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood.

When a bloodstream infection (BSI) is suspected, most of the laboratory results-biochemical and haematologic-are available within the first hours after hospital admission of the patient. This is not the case for diagnostic microbiology, which generally takes a longer time because blood culture, which is to date the reference standard for the documentation of the BSI microbial agents, relies on bacterial or fungal growth. The microbial diagnosis of BSI directly from blood has been proposed to speed the determination of the etiological agent but was limited by the very low number of circulating microbes during these paucibacterial infections. Thanks to recent advances in molecular biology, including the improvement of nucleic acid extraction and amplification, several PCR-based methods for the diagnosis of BSI directly from whole blood have emerged. In the present review, we discuss the advantages and limitations of these new molecular approaches, which at best complement the culture-based diagnosis of BSI.

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR.

Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ≥ 10 DNA copies per reaction (1000 copies ml- 1). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in large-scale screening programs and also during outbreaks.

Ten years of R&D and full automation in molecular diagnosis.

A 10-year experience of our automated molecular diagnostic platform that carries out 91 different real-time PCR is described. Progresses and future perspectives in molecular diagnostic microbiology are reviewed: why automation is important; how our platform was implemented; how homemade PCRs were developed; the advantages/disadvantages of homemade PCRs, including the critical aspects of troubleshooting and the need to further reduce the turnaround time for specific samples, at least for defined clinical settings such as emergencies. The future of molecular diagnosis depends on automation, and in a novel perspective, it is time now to fully acknowledge the true contribution of molecular diagnostic and to reconsider the indication for PCR, by also using these tests as first-line assays.

Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

Molecular diagnosis of Anaplasmataceae organisms in dogs with clinical and microscopical signs of ehrlichiosis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Coagglutination for viral DNA preparation of canine parvovirus for molecular diagnosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of histological and molecular diagnosis of Helicobacter pylori in benign lesions and gastric adenocarcinoma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MOLECULAR DIAGNOSIS of LEISHMANIA AMAZONENSIS IN A CAPTIVE SPIDER MONKEY IN BAURU, São Paulo, BRAZIL

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Rapid molecular diagnosis of tuberculosis and other mycobacteriosis in smear-negative clinical specimens

Delay in diagnosis of pulmonary and other forms of tuberculosis (TB) can be fatal, particularly in HIV-infected patients. Hence, techniques based on nucleic acid amplification, which are both rapid and of high specificity and sensitivity, are now widely used and recommended for laboratories that diagnose TB. In the present study, diagnostic methods based on mycobacterial DNA amplification were evaluated in comparative trials alongside tradicional bacterial methods, using negative smear samples from patients with clinically-suspected TB (sputum samples from 25 patients with suspected pulmonary TB, urine samples from two patients with suspected renal TB and cerebrospinal fluid samples from one patient with suspected meningeal TB). A specificity of 100% was achieved with DNA amplification methods and tradicional culture/identification methods, in relation to clinical findings and treatment results. For the smear-negative sputa, conventional PCR for M. tuberculosis was positive in 62% of suspected lung TB case, showing the same sensitivity as bacterial identification. Both techniques failed in the detection of extra-pulmonary samples. Nested PCR showed, after species-specific amplification, a sensitivity of 100% for M. avium and 85% for M. tuberculosis. For extra-pulmonary smear-negative samples, only Nested PCR detected M. tuberculosis and all cases were confirmed clinically. Nested PCR, in which two-step amplification reactions are performed, can identify the two most important mycobacteria in human pathology quickly and directly from clinical spicimens.

Large-Scale Population Analysis Challenges the Current Criteria for the Molecular Diagnosis of Fascioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (<= 8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4-8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1-8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.

Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis

Bovine mastitis, an inflammatory disease of the mammary gland, is one of the most costly diseases affecting the dairy industry. The treatment and prevention of this disease is linked heavily to the use of antibiotics in agriculture and early detection of the primary pathogen is essential to control the disease. Milk samples (n=67) from cows suffering from mastitis were analyzed for the presence of pathogens using PCR electrospray-ionization mass spectrometry (PCR/ESI-MS) and were compared with standard culture diagnostic methods. Concurrent identification of the primary mastitis pathogens was obtained for 64% of the tested milk samples, whereas divergent results were obtained for 27% of the samples. The PCR/ESI-MS failed to identify some of the primary pathogens in 18% of the samples, but identified other pathogens as well as microorganisms in samples that were negative by culture. The PCR/ESI-MS identified bacteria to the species level as well as yeasts and molds in samples that contained a mixed bacterial culture (9%). The sensitivity of the PCR/ESI-MS for the most common pathogens ranged from 57.1 to 100% and the specificity ranged from 69.8 to 100% using culture as gold standard. The PCR/ESI-MS also revealed the presence of the methicillin-resistant gene mecA in 16.2% of the milk samples, which correlated with the simultaneous detection of staphylococci including Staphylococcus aureus. We demonstrated that PCR/ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease.

Molecular diagnosis of diphyllobothriasis in Spain, most presumably acquired via imported fish, or sojourn abroad

Human diphyllobothriasis is sporadically detected in Spain. Diphyllobothrium latum and Diplogonoporus balaenopterae have been identified. In the study, four cases of presumably imported diphyllobothriasis in Spanish patients were appraised. Molecular diagnosis allowed us to identify 'exotic' fish tapeworms such as Diplogonoporus balaenopterae in one patient and Diphyllobothrium pacificum in the others.

Use of multiple biomarkers for a molecular diagnosis of prostate cancer

The identification of biomarkers capable of providing a reliable molecular diagnostic test for prostate cancer (PCa) is highly desirabie clinically. We describe here 4 biomarkers, UDP-N-Acetyl-alpha-D-galactosamine transferase (GalNAc-T3; not previously associated with PCa), PSMA, Hepsin and DD3/PCA3, which, in combination, distinguish prostate cancer from benign prostate hyperplasia (BPH). GalNAc-T3 was identified as overexpressed in PCa tissues by microarray analysis, confirmed by quantitative real-time PCR and shown immunohistochemically to be localised to prostate epithelial cells with higher expression in malignant cells. Real-time quantitative PCR analysis across 21 PCa and 34 BPH tissues showed 4.6-fold overexpression of GalNAc-T3 (p = 0.005). The noncoding mRNA (DD3/PCA3) was overexpressed 140-fold (p = 0.007) in the cancer samples compared to BPH tissues. Hepsin was overexpressed 21-fold (p = 0.049, whereas the overexpression for PSMA was 66-fold (p = 0.047). When the gene expression data for these 4 biomarkers was combined in a logistic regression model, a predictive index was obtained that distinguished 100% of the PCa samples from all of the BPH samples. Therefore, combining these genes in a real-time PCR assay represents a powerful new approach to diagnosing PCa by molecular profiling. (c) 2005 Wiley-Liss, Inc.