A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortusinfection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a “gold standard,” was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus(samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortusshedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and ofB. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.
The role of genotypic analysis in disease diagnostics and drug response assessment is continually expanding. New genomic discoveries combined with new, novel technologies may provide a greater range of testing capabilities in the near future. We describe the application of nanotechnology, in which DNA microarrays have been placed in a microchannel environment that can be read and analyzed in an optical (CD/DVD) disc drive system. The potential exists to combine molecular and immunological applications together into a rapid, low-cost, high-capacity screening platform. The relevance of this technology is discussed in respect to infectious agent detection, pharmacogenomics, neurogenomics and genetic variations associated with neurologic diseases.
This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5′ untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634–639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.
The ability to detect estrogen and progesterone receptors by immunocytochemical analysis in formalin-fixed, paraffin-embedded sections has clear advantages over other techniques, including the ability to assay small biopsy specimens, fine needle aspirate samples, and archival material. Twenty-two cases of breast carcinoma were evaluated for estrogen and progesterone receptors by immunocytochemical analysis and enzyme immunoassay. Using a true color-based image analysis system, histograms of area versus the optical density of the positive staining nuclei were generated. A binary decision algorithm was derived from these histogram parameters by the Classification and Regression Trees (CART) computer program. Estimates generated by the algorithm for image analysis/immunocytochemical analysis had a 90% concordance with the enzyme immunoassay values. We conclude that quantitative immunocytochemical results for estrogen and progesterone receptor content in formalinfixed, paraffin-embedded tissue can be generated using image analysis.Abstract The number of tests available for the prognostication of patients with breast cancer, (e.g., estrogen and progesterone receptor, DNA ploidy, % S-phase analysis, HER-2/neu, EGFR, p53, cathepsin D, pS2, PCNA, etc.) is staggering. Many published studies statistically prove the prognostic significance for each independent test, but the situation becomes confusing and empirical for the clinician making a decision for a particular patient, particularly when test utilization and cost considerations must be weighed into the equation. Other factors such as the pathological stage, histological grade, vascular and lymphatic invasion, and the age and wishes of the patient should all be taken into consideration in arriving at the optimal treatment protocol. We have applied a Bayesian probability approach to published data in order to derive a branched tree algorithm to predict the survival rates for both lymph node-positive and lymph node-negative women with breast cancer. Specimen quality and test results suggested which subsequent tests were most clinically useful. The size of the algorithm was reduced to minimize the number of tests requested and thus reduce costs. This type of analysis is necessary to ensure that the most information is obtained at the lowest cost, and serves as a model for other diagnostic situations. 0 1993 Wiley-Liss, Inc.
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