High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites-Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis-were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites.     

Problems of the effective energy used as a quality expression of diagnostic X-ray

The effective energy has been generally used as a method of handily expressing an X-ray quality by one numerical value. The effective energy is a concept derived from "Half Value Layer (HVL)" that is the expressing parameter of beam quality based on the attenuation of the primary X-ray by a material. When beam quality is expressed by using HVL and / or the effective energy, it is necessary to describe the tube potential, the rectification method, and the homogeneity coefficient, etc. in parallel. However, recently feelings are that the effective energy should be handled like an absolute numerical value to physical characteristics of X-rays. In this paper, it was theoretically clarified that the effective energy had a different value depending on the absorber material used for the HVL measurement. In addition, the errors when physical characteristics of the X-rays were evaluated using the effective energy was also examined. Physical characteristics, such as interactions to the material of mono-energetic X-ray, are not equal to that of X-rays with a wide energy spectrum. It is not an easy comparison to express the quality of the diagnostic X-rays, and to calculate physical characteristics of the X-rays by using the effective energy. It is necessary to design a new method of expressing the quality of X-rays that takes the place of the "effective energy."     

Therapeutic factors causing hallucination in Parkinson's disease patients, especially those given selegiline

Selegiline protects nigral dopaminergic neurons and is recommended for the treatment of patients in the early stage of Parkinson's disease (PD). We treated 112 PD patients and noted that those given selegiline had a high incidence of hallucination. Our objective was to determine which clinical therapeutic factors cause such hallucinations. The Kruskal-Wallis and chi-square test showed that in 94 patients, the severity of the hallucinations was significantly related to the duration of illness, Hoehn and Yahr stage, doses of levodopa and cabergoline, whether or not selegiline was used, and whether or not medication for constipation was required. In addition, patients who were treated with a low dose of levodopa (< or =300 mg/day), who had a low Hoehn and Yahr stage, and a short duration of illness (< or =8 years) together with a high dose of selegiline or cabergoline also tended to have hallucinations. MRI findings were not related to the incidence of hallucination. When selegiline is given to patients who have PD of long duration and a high Hoehn and Yahr stage, and who already are receiving levodopa and a dopamine agonist, the doses of levodopa and the dopamine agonists given, as well as the presence of constipation, may be related to the incidence of hallucination.     

Significant improvement in MRI-proven bone edema is associated with protection from structural damage in very early RA patients managed using the tight control approach

Objective

To identify the value of magnetic resonance imaging (MRI)-proven bone edema in patients with very early rheumatoid arthritis (RA).

Methods

All of the 13 patients included in the study were positive at entry for MRI-proven bone edema of the wrist and finger joints and anti-cyclic citrullinated peptide antibodies or IgM-rheumatoid factor. A tight control approach was applied for 12 months. Plain MRI and radiographs of both wrist and finger joints were examined every 6 months. MRI was scored by the RA MRI scoring (RAMRIS) technique and plain radiographs were scored using the Genant-modified Sharp score. Variables that were correlated with plain radiographic changes at 12 months were examined.

Results

Simplified disease activity index (SDAI) remission was achieved in 7 patients, and a significant reduction in the RAMRIS bone edema score, which declined to <33 % as compared with the baseline, was achieved in 8 out of 13 patients. Four patients showed plain radiographic progression while 9 patients did not. Significant reductions in the RAMRIS bone edema score (p = 0.007) and the time-integrated SDAI (p = 0.031) were the variables involved in plain radiographic progression.

Conclusions

Improvement in bone edema may be associated with protection against structural damage in very early RA patients managed using the tight control approach.     

Evaluation of PCR-RFLP in the Pre-S Region as Molecular Method for Hepatitis B Virus Genotyping

Background

Hepatitis B virus (HBV) infection is a public health problem in developing countries. HBV genotypes play major role in the evolution of infection since they were involved in different clinical presentations and response to treatment.

Objectives

This study was conducted to evaluate the efficiency of restriction fragment length polymorphism (RFLP) analysis for HBV genotyping.

Patients and Methods

We investigated 98 samples collected from patients chronically infected with HBV. HBV genotypes were determined by analysis of patterns obtained after amplification in Pre-S region and digestion of the amplicon by two endonucleases AvaII and DpnII. Obtained results were confirmed by partial sequencing in the same region.

Results

Two different HBV genotypes were detected in this study, Genotype D (in 95. 9%) and Genotype A (in 4.1%). Seventy-four samples (75.5%) were successfully genotyped with RFLP analysis and all classified as genotype D. The remaining 24 samples (24.5%) which were un-genotyped by RFLP analysis, were classified by partial sequencing of the pre-S region as HBV genotype D (20 samples, 20.4%) and genotype A (4 samples, 4.1%). Atypical profiles were significantly associated with advanced liver disease (P = 0.001) as well as older age (P < 0.05).

Conclusions

Several previous studies used PCR-RFLP to genotype HBV; however, we showed the high risk to obtain atypical profiles, especially in advanced stages of chronic infection, with as results difficulties to genotype the virus. These profiles resulted from the accumulation of mutations during natural course of infection resulting in a modification in restriction sites for enzymes. So, we recommended completing the investigation by partial sequencing to confirm obtained results.