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Biomedical subjects

D J Marshall

Publications and source records attributed to D J Marshall.

At least 19 recordsLinked to original sources
PubMed2005

The benefits of polyandry in the free-spawning polychaete Galeolaria caespitosa.

In many species, females are thought to benefit from polyandry due to the reduced risks of fertilization by genetically incompatible sperm. However, few studies that have reported such benefits have directly attributed variation in female reproductive success to the interacting effects of males and females at fertilization. In this paper, we determine whether male x female interactions influence fertilization in vitro in the free-spawning, sessile polychaete Galeolaria caespitosa. Furthermore, we determined whether polyandry results in direct fertilization benefits for females by experimentally manipulating the number of males contributing towards staged spawning events. To test for male x female interaction effects we performed an initial experiment that crossed seven males with six females (in all 42 combinations), enabling us to assess fertilization rates for each specific male-female pairing and attribute variation in fertilization success to males, females and their interaction. This initial experiment revealed a strong interaction between males and females at fertilization, confirming that certain male-female combinations were more compatible than others. A second experiment tested the hypothesis that polyandry enhances female reproductive success by exposing each female's eggs to either a single male's sperm (monandry) or the sperm from three males simultaneously (polyandry). We performed this second experiment at two ecologically relevant sperm concentrations. This latter experiment revealed a strong fertilization benefit of polyandry, independent of the effects of sperm concentration (which were also significant). We suggest that these direct fertilization gains arising from polyandry will constitute an important source of selection on females to mate multiply in nature.

Analysis of Variance↗
PubMed2005

Does egg competition occur in marine broadcast-spawners?

When the availability of sperm limits female reproductive success, competition for sperm, may be an important broker of sexual selection. This is because sperm limitation can increase the variance in female reproductive success, resulting in strong selection on females to compete for limited fertilization opportunities. Sperm limitation is probably common in broadcast-spawning marine invertebrates, making these excellent candidates for investigating scramble competition between broods of eggs and its consequences for female reproductive success. Here, we report our findings from a series of experiments that investigate egg competition in the sessile, broadcast-spawning polychaete Galeolaria caespitosa. We initially tested whether the order in which eggs encounter sperm affects their fertilization success at two ecologically relevant current regimes. We used a split-clutch-split--ejaculate technique to compare the fertilization success of eggs from individual females that had either first access (competition-free treatment) or second access (egg competition treatment) to a batch of sperm. We found that fertilization success depended on the order in which eggs accessed sperm; eggs that were assigned to the competition-free treatment exhibited significantly higher fertilization rates than those assigned to the egg competition treatment at both current speeds. In subsequent experiments we found that prior exposure of sperm to eggs significantly reduced both the quantity and quality of sperm available to fertilize a second clutch of eggs, resulting in reductions in fertilization success at high and low sperm concentrations. These findings suggest that female traits that increase the likelihood of sperm-egg interactions (e.g. egg size) will respond to selection imposed by egg competition.

Animals↗
PubMed2003

Evaluation of an absorbed ELISA and an agar-gel immuno-diffusion test for ovine paratuberculosis in sheep in Australia.

The sensitivities and specificities of an absorbed enzyme-linked immunosorbent assay (ELISA) and an agar-gel immuno-diffusion (AGID) test for the detection of Johne's disease in sheep were estimated using data from six known infected and 12 assumed uninfected sheep flocks. Sensitivities were estimated for all histologically positive sheep, as well as by histological lesion score, lesion type (paucibacillary or multibacillary) and sheep body-condition score, with ELISA sensitivities estimated at 95 and 99% specificity. Logistic-regression analysis was used to test for significant effects of lesion score and condition score, with flock included in the model as a random effect. Estimated specificities were 95% (95% CI: 93.4, 95.6%) and 99% (98.4, 99.4%) for ELISA cut-point ratios of 2.4 and 3.6, respectively, and 100% (99.7, 100.0%) for the AGID. Estimated sensitivities for all infected sheep were 41.5% (35.0, 48.3%), 21.9% (16.6, 27.9%) and 24.6% (19.1, 30.7%) for ELISA cut-point ratios of 2.4 and 3.6 and for AGID, respectively. Sensitivities of all tests and cut-points varied significantly between flocks and between categories of lesion score and condition score. Sensitivity ranged from 25 to 73, 10 to 47 and 9.2 to 63% between flocks, for the ELISA with cut-points of 2.4 and 3.6, and for the AGID, respectively. Sensitivity was highest in thin sheep and in sheep with multibacillary lesions. The effects of lesion type and condition score on test sensitivity were significant in the logistic regressions for the AGID and ELISA at both cut-points and the flock effect was significant for the AGID but not for the ELISA at either cut-point.

Animals↗
PubMed2003

Isolation of Mycobacterium avium subsp paratuberculosis from environmental samples collected from farms before and after destocking sheep with paratuberculosis.

OBJECTIVE: To determine whether Mycobacterium avium subsp paratuberculosis could be isolated from soil-pasture, faecal, water and sediment samples on farms before and after removal of sheep with paratuberculosis. A feasibility study and subsequent field survey. PROCEDURE: First the analytical sensitivity of radiometric culture of the organism from two types of soil was determined relative to faeces. Then soil-pasture, faecal, water and sediment samples were collected for culture from a range of sites from 6 farms with paratuberculosis affected sheep and goats. Similar samples were collected from 20 farms at least 9 months after removal of infected stock. RESULTS: The analytical sensitivity of culture of M a paratuberculosis from soil samples was 2 orders of magnitude less than that from faeces, and environmental samples required longer incubation periods to yield significant growth in radiometric culture (BACTEC) medium. However, the organism was recovered from approximately 20% of 163 soil-pasture, water and sediment samples from 6 properties with clinically-affected animals with paratuberculosis. The positive samples were from a range of topographic sites, including open exposed and dry areas, however, low lying areas tended to have larger numbers of organisms. When the same sites were sampled again about 5 months later, only 1 was culture positive, and none were culture positive > 12 months later. Of 17 water and dam sediment samples collected from farm 6, which had long-standing high prevalence OJD infection, only one water sample and one sediment from the same dam were culture positive. None of the 5 water samples from the other farms were culture positive. Of 96 water samples, 90 sediment samples and 93 soil samples from farms that had been destocked of infected sheep/goats for 9 to 24 months, one sediment sample from a farm in Victoria (destocked for 12 months) and two sediment samples from a farm in New South Wales (10, 19 months) were culture positive. Recontamination from cattle or water could not be excluded as a cause of the positive cultures from the second farm. CONCLUSION: M a paratuberculosis can be detected by radiometric culture in environmental samples from farms grazed by sheep or goats with paratuberculosis. There is a relatively low likelihood of recovery of the organism from water samples from such farms, and at 5 or more months after removing stock with paratuberculosis the likelihood of positive cultures from environmental samples is very low. Although the analytical sensitivity of culture from environmental samples is less than that from faeces, surveys of environmental sites are nevertheless feasible. However, improved culture methods are needed for critical surveys and to study the movement and fate of the organism in the environment.

Animals↗
PubMed2002

Correlations between metal uptake in the soft tissue of Perna perna and gill filament pathology after exposure to mercury.

The accumulation of metal in soft tissues, filtration rate and gill filament morphology are correlated in the southern African rock mussel, Perna perna, during exposure to mercury (24 days) and recovery (24 days). The amount of Hg in soft tissues increased from 0.13 to 87.5 microg/g dry weight after 24 days exposure, and declined to 13 microg/g during recovery. Mean filtration rate fell from 3,979 to 1,818 ml/h/g dry weight by day 2, but recovered slightly through days 4 and 8 (3,037 ml/h/g), with a higher average rate (5,030 ml/h/g) being maintained over the 24-48 days recovery period. The initial decline in filtration coincided with epithelial cell deterioration presented as interstitial oedema, neural and epithelial cell degeneration and reduced ciliation. Between days 8 and 24, cilia regenerated and there was a general improvement in cell morphology. Gill filament morphology returned to near normal during the metal-free recovery period. The usefulness of P. perna as an indicator of pollution is discussed.

Animals↗
PubMed2002

Metal accumulation, filtration and O(2) uptake rates in the mussel Perna perna (Mollusca: Bivalvia) exposed to Hg(2+), Cu(2+) and Zn(2+).

Tissue metal concentrations, filtration and oxygen uptake rates were investigated for Perna perna (Bivalvia: Mollusca) during exposure to Hg(2+), Cu(2+) and Zn(2+) (50 microg/l for 24 days, and 24 days recovery with no metal). Hg and Cu tissue levels increased with exposure time, reaching maximum levels after 24 days (87.5 microg Hg/g dry mass and 45 microg Cu/g dry mass, respectively). Zn levels peaked after 4 days exposure (to 233 microg Zn/g dry mass) and stabilized thereafter. Accumulated metal was rapidly lost from tissues when mussels were returned to uncontaminated seawater, suggesting that tissue concentration data may be of limited use in biomonitoring situations where environmental metals fluctuate to low levels. Filtration rates fell below control rates during Hg(2+) exposure, and became elevated again during the recovery period. Cu(2+) and Zn(2+) exposure had little effect on filtration, but suppressed oxygen uptake. During recovery, oxygen uptake of Cu(2+) and Zn(2+) exposed mussels was elevated above the controls. Filtration and oxygen uptake rates were not correlated, but rather responded in different ways to metal pollution. While these physiological responses of P. perna may be of limited use in biomonitoring, they could indicate how populations may respond to marine pollution.

Animals↗
PubMed2000

A continuous assay for DNA cleavage: the application of "break lights" to enediynes, iron-dependent agents, and nucleases.

Although extensive effort has been applied toward understanding the mechanism by which enediynes cleave DNA, a continuous assay for this phenomenon is still lacking. In fact, with the exception of assays for DNase, continuous assays for most DNA cleavage events are unavailable. This article describes the application of "molecular break lights" (a single-stranded oligonucleotide that adopts a stem-and-loop structure and carries a 5'-fluorescent moiety, a 3'-nonfluorescent quenching moiety, and an appropriate cleavage site within the stem) to develop the first continuous assay for cleavage of DNA by enediynes. Furthermore, the generality of this approach is demonstrated by using the described assay to directly compare the DNA cleavage by naturally occurring enediynes [calicheamicin and esperamicin), non-enediyne small molecule agents (bleomycin, methidiumpropyl-EDTA-Fe(II), and EDTA-Fe(II]), as well as the restriction endonuclease BamHI. Given the simplicity, speed, and sensitivity of this approach, the described methodology could easily be extended to a high throughput format and become a new method of choice in modern drug discovery to screen for novel protein-based or small molecule-derived DNA cleavage agents.

Aminoglycosides↗
PubMed2000

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction.

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10(7) reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.

Base Sequence↗
PubMed2000

Biocompatibility of cardiovascular gene delivery catheters with adenovirus vectors: an important determinant of the efficiency of cardiovascular gene transfer.

Gene therapy approaches hold promise for the treatment of a wide variety of cardiovascular diseases. Many strategies for cardiovascular gene therapy involve catheter-mediated vector delivery via intramyocardial injection, intracoronary infusion, or direct gene transfer into the vessel wall. Several different gene delivery catheters have been developed and utilized in preclinical and clinical studies of cardiovascular gene therapy. However, rigorous studies of the biocompatibility of these catheters with gene therapy vectors have not yet been reported. In this report, we have examined the compatibility of cardiovascular gene therapy catheters and catheter constituents with first-generation E1/E3-deleted adenovirus vectors. We show that (i) currently available catheters rapidly and efficiently inactivate adenovirus vector infectivity; (ii) this inactivation is mediated by a variety of commonly used catheter constituents including stainless steel, nitinol, and polycarbonate; (iii) catheter-mediated inactivation of adenovirus vectors can be prevented by preflushing catheters with solutions of serum albumin; and (iv) it is possible to identify a set of catheter materials that are compatible with current adenovirus vectors. These results underscore the importance of catheter/vector compatibility and suggest methods for increasing the efficiency of catheter-mediated cardiovascular gene therapy.

Adenoviridae↗
PubMed2000

Use of pooled fecal culture for sensitive and economic detection of mycobacterium avium subsp. paratuberculosis infection in flocks of sheep.

Ovine Johne's disease, or paratuberculosis, occurs in many countries. In Australia, surveillance using serology is used as part of a control program, but the testing regime is costly relative to its sensitivity. For this reason, culturing of Mycobacterium avium subsp. paratuberculosis in fecal samples pooled from a number of sheep was evaluated. Initially, the effect of pooling on the sensitivity of fecal culture was evaluated using samples from 20 sheep with multibacillary paratuberculosis and 20 sheep with paucibacillary paratuberculosis, each confirmed histologically. All multibacillary cases and 50% of paucibacillary cases were detected by culturing of feces at a pooling rate of 1 infected plus 49 uninfected sheep. In a pilot-scale study in 1997, M. avium subsp. paratuberculosis was detected by pooled fecal culture on 93% of 27 infected farms which were identified originally based on history, clinical signs, and one or more rounds of testing using serologic and histopathologic examinations. Pooled fecal culture was compared with serologic examination for submissions from 335 farms where both tests had been conducted on the same sheep and was significantly more sensitive (P<0.001). Computer simulation of random sampling indicated that the testing of 6 pools of 50 sheep would provide 95% confidence in detecting > or =2% prevalence of infection. The estimated laboratory cost of pooled fecal culture when applied as a flock test is approximately 30% that of serologic examination, and sample collection costs are lower. It is recommended that pooled fecal culture replace serologic examination for detection of M. avium subsp. paratuberculosis infection at the flock level.

Animals↗
PubMed2000

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia.

The distribution and prevalence of strains of Mycobacterium avium subsp. paratuberculosis were determined among sheep, cattle, and other species with Johne's disease in Australia. A total of 328 isolates were evaluated from numerous farms in New South Wales, Victoria, Tasmania, and South Australia, Australia. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA using BstEII and an IS900 probe and IS1311 polymorphism analysis using PCR and restriction endonuclease analysis (PCR-REA) was used to classify isolates as cattle (C) or sheep (S) strains. IS1311 PCR-REA provided similar information to IS900 RFLP analysis but was more useful than RFLP analysis where DNA was degraded or scarce. Twelve IS900 RFLP types were found. Johne's disease in sheep was always due to S strains, while cattle were infected only with C strains. RFLP type S1 was the dominant strain in sheep in New South Wales (97% of isolates) and was the only strain found in sheep from Victoria. Seven RFLP types were present in cattle. RFLP types C3 and C1 were most common (collectively, 85% of isolates), but C1 was not found in New South Wales and C3 was present in dairy cattle but not in beef cattle in Victoria. These differences may be explained by restricted livestock trading patterns between different segments of the cattle industry. Up to five RFLP types were present in some geographic regions in Victoria, while up to three RFLP types were found among cattle on some farms. Individual cattle usually were infected with only one RFLP type, but one animal was infected with both C5 and CU4. Two isolates from goats were C type as were three from alpacas, one from a rhinoceros, and two from a human with Crohn's disease. The prevalences of specific RFLP types in Australia differ from those reported in Europe and elsewhere. Given the existence of geographical and farm enterprise differences in IS900 RFLP type, this technique may be applied selectively to trace the spread of Johne's disease, at least in the cattle industries. As these observations reflect past exposure of livestock to M. avium subsp. paratuberculosis, the monitoring of strains present in animals in Australia is continuing.

Animals↗
PubMed1999

Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinant adeno-associated virus vectors.

BACKGROUND: The delivery of recombinant genes to cardiomyocytes holds promise for the treatment of a variety of cardiovascular diseases. Previous gene transfer approaches that used direct injection of plasmid DNA or replication-defective adenovirus vectors have been limited by low transduction frequencies and transient transgene expression due to immune responses, respectively. In this report, we have tested the feasibility of using intramyocardial injection or intracoronary infusions of recombinant adeno-associated virus (rAAV) vectors to program transgene expression in murine cardiomyocytes in vivo. METHODS AND RESULTS: We constructed an rAAV containing the LacZ gene under the transcriptional control of the cytomegalovirus (CMV) promoter (AAVCMV-LacZ). We then injected 1x10(8) infectious units (IU) of this virus into the left ventricular myocardium of adult CD-1 mice. Control hearts were injected with the AdCMV-LacZ adenovirus vector. Hearts harvested 2, 4, and 8 weeks after AAVCMV-LacZ injection demonstrated stable beta-galactosidase (beta-gal) expression in large numbers of cardiomyocytes without evidence of myocardial inflammation or myocyte necrosis. In contrast, the AdCMV-LacZ-injected hearts displayed transient beta-gal expression, which was undetectable by 4 weeks after injection. Explanted C57BL/6 mouse hearts were also perfused via the coronary arteries with 1.5x10(9) IU of AAVCMV-LacZ and assayed 2, 4, and 8 weeks later for beta-gal expression. beta-Gal expression was detected in <1% of cardiomyocytes at 2 weeks after perfusion but was detected in up to 50% of cardiomyocytes 4 to 8 weeks after perfusion. CONCLUSIONS: Direct intramyocardial injection or coronary artery perfusion with rAAV vectors can be used to program stable transgene expression in cardiomyocytes in vivo. rAAV appears to represent a useful vector for the delivery of therapeutic genes to the myocardium.

Animals↗
PubMed1999

Evaluation of modified BACTEC 12B radiometric medium and solid media for culture of Mycobacterium avium subsp. paratuberculosis from sheep.

Definitive diagnosis of Johne's disease in ruminants depends on confirming the presence of the causative bacterium, Mycobacterium avium subsp. paratuberculosis, in tissues of the host. This is readily achieved in most ruminant species by culture. However, culture of clinical specimens from sheep in many countries has been unrewarding. Such a culture from sheep was achieved recently in Australia by using a radiometric culture medium. The aims of the present study were to evaluate the culture of M. avium subsp. paratuberculosis from sheep by using modified BACTEC 12B radiometric medium, to determine the sensitivity of culture in relation to histopathology, and to evaluate a range of solid media. Culture of M. avium subsp. paratuberculosis from sheep with Johne's disease is a sensitive method of diagnosis: intestinal tissues from all 43 animals with multibacillary disease and all 22 animals with paucibacillary disease were culture positive, while 98% of feces from 53 animals with multibacillary disease and 48% of feces from 31 animals with paucibacillary disease were culture positive. Of sheep without histological evidence of Johne's disease from infected flocks, intestinal tissue from 32% of 41 were culture positive, while feces from 17% of 41 were culture positive. Consequently, culture is recommended as the "gold standard" test for detection of ovine Johne's disease. Of the wide range of solid media that were evaluated, only modified Middlebrook 7H10 and 7H11 agars, which were very similar in composition to modified BACTEC 12B medium, yielded growth of ovine strains of M. avium subsp. paratuberculosis. The sensitivity of detection of M. avium subsp. paratuberculosis on solid media was slightly lower than that in modified BACTEC 12B radiometric medium. Both egg yolk and mycobactin J were essential additives for growth of ovine strains of M. avium subsp. paratuberculosis in both liquid and solid media.

Animals↗
PubMed1999

Experimental infection of calves with bovine viral diarrhea virus genotype II (NY-93).

To ascertain the virulence of bovine viral diarrhea virus (BVDV) genotype II, isolate NY-93 was inoculated intranasally into 3 calves, 2 of which were treated with a synthetic glucocorticoid prior to and after virus inoculation. Anorexia, fever (up to 42 C), dyspnea, and hemorrhagic diarrhea developed 6 days after intranasal inoculation with BVDV NY-93. The condition of all calves deteriorated further until the end of the study on day 14 postinoculation. The most significant postmortem macroscopic changes in all calves were limited to the gastrointestinal tract and consisted of moderate to severe congestion of the mucosa with multifocal hemorrhages. Microscopic lesions found in the gastrointestinal tract were similar to those observed in mucosal disease, including degeneration and necrosis of crypt epithelium and necrosis of lymphoid tissue throughout the ileum, colon, and rectum. The basal stratum of the epithelium of tongue, esophagus, and rumen had scattered individual necrotic cells. Spleen and lymph nodes had lymphocytolysis and severe lymphoid depletion. Severe acute fibrinous bronchopneumonia was present in dexamethasone-treated calves. Abundant viral antigen was detected by immunohistochemistry in the squamous epithelium of tongue, esophagus, and forestomachs. BVDV antigen was prominent in cells of the media of small arteries and endothelial cells. The presence of infectious virus in tissues correlated with an absence of circulating neutralizing antibodies. These findings highlight the potential of BVDV genotype II to cause severe disease in normal and stressed cattle.

Animals↗
PubMed1998

Recent advances in skeletal-muscle-based gene therapy.

Gene transfer into skeletal muscle holds promise for the treatment of a variety of serum protein deficiencies, muscular dystrophies, and chronic ischemic limb syndromes. The past two years have seen the development of new and improved vectors for programming recombinant gene expression in skeletal muscle. Important advances include first, novel plasmid DNA, adenovirus, and adeno-associated virus vectors that can be used to stably express therapeutic levels of recombinant proteins in the skeletal muscle of immunocompetent hosts and second, the development of vector systems that enable regulated and tissue-specific transgene expression in skeletal muscle in vivo.

Animals↗
PubMed1998

Rapid detection of Mycobacterium paratuberculosis in clinical samples from ruminants and in spiked environmental samples by modified BACTEC 12B radiometric culture and direct confirmation by IS900 PCR.

The suitability of a radiometric culture medium consisting of BACTEC 12B with PANTA PLUS, mycobactin J, and egg yolk was evaluated for detection of Mycobacterium paratuberculosis in feces, mesenteric lymph nodes, and intestinal walls from cattle, sheep, and goats. In addition, a simple method that would enable the rapid identification of Mycobacterium paratuberculosis by IS900 PCR in the primary cultures was sought so that subculture to secondary egg-free radiometric medium could be avoided. An ethanol extraction followed by differential centrifugation was used to separate M. paratuberculosis from PCR inhibitors in the primary culture. PCR was then undertaken with the pellet, after boiling to lyse the mycobacteria; if this test was negative, the DNA in the lysate was purified with guanidine thiocyanate and silica. Cultures of feces, ilea, and mesenteric lymph nodes from cattle, sheep, and goats known to have or suspected of having Johne's disease yielded positive PCR results 1 to 7 weeks after inoculation. Similar results were obtained with soil and pasture samples that had been spiked with M. paratuberculosis. The results suggested that radiometric culture was more sensitive than histopathology in detecting M. paratuberculosis infection in sheep and goats and more sensitive than culture on Herrold's egg yolk medium for the detection of the infection in cattle. Of 259 individual PCR tests with samples from cultures with growth indices of > or = 10,237 (91.5%) were positive, with only 28 (11.8%) requiring both ethanol and silica preparation to yield a positive result. Of the 22 negative PCR results for samples from cultures with growth indices of > or = 10, 18 were for samples from cultures that had only just developed evidence of growth. PCR-positive cultures tended to remain PCR positive over successive weeks. Flexibility in the timing of the sampling for PCR is thus possible, facilitating batch processing of samples in large-scale disease control programs for ruminants.

Animals↗
PubMed1998

Algorithmic approach to high-throughput molecular screening for alpha interferon-resistant genotypes in hepatitis C patients.

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.

Algorithms↗