High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Blood loss and contributing factors in femoral fracture surgery

Background

Substantial blood losses frequently accompany orthopedic procedures.

Methods

We prospectively noted peri-operative hemoglobin changes in 93 patients undergoing surgery for femoral fracture with an aim of establishing blood loss and related factors.

Results

The mean total blood loss assessed 72 hours after the surgical procedure was 3.31 (SD 1.56) units of whole blood. A multiple regression analysis revealed diathermy use and a simple fracture pattern as significant factors in reducing blood loss (p<0.01).

Conclusions

Open intramedullary fixation of femur fractures leads to considerable peri-operative blood loss. This is can be reduced by use of diathermy during surgery.     

Atlanto-axial rotatory fixation: Improved demonstration using spiral CT

Atlanto-axial rotatory fixation (AARF) is an uncommon condition which is often missed at presentation because of its rarity and the relative subtlety of plain film X-ray findings, but early detection and appropriate management are vital for a cure. We describe three cases in which the use of spiral computed tomography scanning with 3D and sagittal reconstructions greatly aided diagnosis and management. The 3D images gave a more graphic picture of the overall alignment of the upper cervical spine and the skull base, while the sagittal reconstructions demonstrated the presence or absence of compensatory atlanto-occipital subluxation. The literature is briefly reviewed.     

Reproducibility and clinical value of 18F-fluorodeoxyglucose positron emission tomography in recurrent melanoma

The purpose of this study was to assess the reproducibility and clinical impact of positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) in patients with (suspected) recurrent melanoma. The clinical value of PET was prospectively measured in 58 consecutive patients referred for PET because of unresolved clinical questions after conventional work-up. Diagnostic understanding and therapy choice by referring physicians were evaluated before, directly after, and 6 months after PET. Observer agreement of PET readings was measured with respect to various parameters (interpretation, number and localization of lesions, 'clinically decisive' metastases), using intra-class correlation coefficients. FDG PET improved diagnostic understanding in 33 cases (57%). In six patients (10%), diagnostic understanding was solely based on PET information. According to the attending clinicians, PET contributed to a positive change of planned treatment in 23 patients (40%) and increased confidence in the chosen treatment in 23 (40%). Observer agreement of PET readings was very high (intra-class correlation coefficients were between 0.87 and 0.94). The diagnostic value related especially to the whole-body scan technique and the superior specificity, compared to conventional work-up. It is concluded that, in problematical cases with (suspected) recurrent melanoma, 18F-FDG PET had considerable impact on diagnostic understanding and management. Together with the excellent observer reliability, these results justify further studies to determine the optimal place of PET in routine diagnostic algorithms in recurrent melanoma.     

Real Time-PCR HBV-DNA Analysis: Significance and First Experience in Armed Forces

Background

HBV DNA quantitation is used extensively world wide for the diagnosis and monitoring of treatment of Hepatitis B virus (HBV) infection. However, it has still to be popular in India. The aim of this study was to quantitate HBV – DNA by Real time – PCR method in Hepatitis B and in immuno-compromised patients, to compare the results with HBeAg detection and to monitor the response to therapy of chronic Hepatitis B patients to antivirals.

Methods

Ninety one serum samples of Hepatitis group of patients (all HBsAg positive), 41 samples from immuno-compromised patients (all HBsAg negative) and 49 patients of Chronic Hepatitis B group (all HBsAg positive) were the subjects of this first ever study in Armed Forces. Twenty serum samples from healthy volunteers and non-hepatitis B patients served as negative controls. The amplification detection was carried out in a Rotor-Gene 2000-sequence detector

Results

Amongst Hepatitis B group, 33% (30/91) of the samples were positive for HBV-DNA and 26% (24/91) of samples were positive for HBeAg. In the immuno-compromised group of patients 14.6% (6/11) of samples were positive for HIV-DNA and 9.7% (4/41) were positive for HBeAg. Of the Chronic Hepatitis B patients on treatment, all (100%) were positive by HBV-DNA, whereas 29/49 (59.2%) were positive by HBeAg before treatment. After treatment with antivirals, 06/49 (12.2%) were positive by both tests and 11/49 (22.5%) were positive only by HBV-DNA. 32/49 (65.3%) patients became negative serologically after therapy.

Conclusion

HBeAg status did not necessarily reflect HBV-DNA level in the serum, as 10/91 (11%) in the Hepatitis B group, 2/41 (4.9%) in the immuno compromised group and 20/49 (40.8%) patients in the Chronic Hepatitis B group were positive for HBV-DNA but negative for HBeAg. HBV-DNA was not found to be positive amongst any of the negative controls. Real time – PCR is a sensitive and reproducible assay for HBV-DNA quantitation and may be started in Armed Forces referral centers in the near future.Key Words: Real time – PCR, Chronic Hepatitis B, HBV – DNA, Antivirals     

Gallstones following considerable weight loss and recommendations for their prevention

Obesity is an increasing public health problem. As a consequence, many people attempt to lose weight by dieting or participation in weight loss programmes. Weight loss is associated with an increased risk of symptomatic gallstones. Preventive measures include keeping the rate of weight loss below 1.5 kg per week, and providing a fat intake of at least 7 g a day. If it is expected that the rate of weight loss will be faster or difficult to control, for example after bariatric surgery, prophylaxis with ursodeoxycholic acid may be considered.     

Reliability,validity and psychometric properties of the Greek translation of the Major Depression Inventory

Background  

The Major Depression Inventory (MDI) is a brief self-rating scale for the assessment of depression. It is reported to be valid because it is based on the universe of symptoms of DSM-IV and ICD-10 depression. The aim of the current preliminary study was to assess the reliability, validity and psychometric properties of the Greek translation of the MDI.