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1.
Testing of pooled samples has been proposed as a low-cost alternative for diagnostic screening and surveillance for infectious agents in situations where the prevalence of infection is low and most samples can be expected to test negative. The present study extends our previous work in pooled-sample testing (PST) to evaluate effects of the following factors on the overall PST sensitivity (SEk) and specificity (SPk): dilution (pool size), cross-contamination, and cross-reaction. A probabilistic model, in conjunction with Monte Carlo simulations, was used to calculate SEk and SPk, as applied to detection of bovine viral diarrhea virus (BVDV) persistently infected (PI) animals using RT-PCR. For an average prevalence of BVDV PI of 0.01 and viremia in each animal between 102 and 107 virus particles/mL, the pool size associated with the lowest number of tests, and lowest cost, corresponded to eight samples/pool. However, the least-cost pool size (lowest number of tests) was associated with a SEk of 0.90 (0.75–1), which corresponded to a decrease of 0.04, relative to the assay sensitivity for a single sample. The SPk for the same pool size, considering the effect of detection of BVDV acutely infected animals and cross-contamination as source of false positive results, was 0.90 (0.85–0.95). The effect of a hypothetical cross-reacting agent was to markedly decrease SPk, especially as the prevalence of the cross-reacting agent increased. For a pool size of eight samples and a prevalence of the cross-reacting agent of 0.3, SPk ranged from 0.67 to 0.86, depending on the probability that the assay would detect the cross-reacting agent. The methods presented offer a means of evaluating and understanding the various factors that can influence overall accuracy of PST procedures.  相似文献   

2.
The purpose of this report is to offer concepts for consideration in developing infectious disease surveillance systems, defined here as active, formal, and systematic processes intentionally directed to rapidly seek out and identify infectious disease agents or disease. Performance of surveillance systems can be judged by their accuracy (sensitivity and specificity), precision (repeatability), timeliness, multiple utility, and value. Surveillance system operation and function necessary to achieve high performance are defined in part by characteristics of the specific infectious disease, including disease transition state dynamics, that define probabilities of being in the latent, infectious, or clinical phase of disease. Two key components of surveillance are the sampling scheme, which is intended to maximize the probability of capturing an infected animal or specimen as soon as possible after the herd has been exposed, and the diagnostic assays, which should maximize the probability of detecting the agent, or evidence of the agent, if it is present in the specimen, while minimizing the likelihood of a false-positive result. Proportional risk sampling, targeted sampling, and repeated sampling are strategies that can improve overall surveillance system accuracy and particularly the temporal sensitivity related to early detection. Hierarchical sampling schemes and multiplexed assays can maximize efficiency and improve utility by serving multiple surveillance systems and purposes. Development of the surveillance systems needed to address emerging and foreign animal diseases will necessarily require design and architecture that are highly probability-driven to maximize surveillance sensitivity and specificity and to minimize cost.  相似文献   

3.
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4.
In a cross-sectional study on milk samples from 1155 cows from 22 Danish dairy herds, selected risk factors for paratuberculosis were identified. The diagnostic procedure used was an indirect enzyme-linked immuno-sorbent assay (ELISA) to detect antibodies against Mycobacterium avium subsp. paratuberculosis. A sample was considered test-positive if it had a corrected optical density >/=0. 025 (test sensitivity 71.4% and test specificity 89.7%). Of the 1155 samples, 8.8% (102/1155) were test-positive, and 19 out of the 22 dairy herds had >/=1 test-positive cows. The significant risk factors in a multiple logistic regression analysis were: Jersey versus large breeds, high parity versus low parity, the first month after calving versus other months of lactation, and a large herd size compared to a small herd size. The highest probability (37-38%) of a positive test was observed among older cows (parity >4) and tested within the first month after calving (irrespective of breed). The lowest probability (2%) of a positive test-result was observed among first parity, large-breed cows tested before calving or later than one month after.  相似文献   

5.
Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.  相似文献   

6.
The study investigated the effect of gamma-irradiation on bovine serum samples on the ability of enzyme-linked immunosorbent assay (ELISA) methods to detect trypanosomal antibodies. The serum samples were analysed using two standardised indirect ELISA systems. Higher measurement values were observed for most gamma-irradiated antibody positive and negative test samples. Using cut-off points, determined from the analysis of a non-irradiated trypanosomal antibody-negative population, the gamma-irradiated sera data showed that there was an increased risk of misclassifying samples as false positive or cross-reactive due to increased analytical sensitivity and decreased analytical specificity. The intraplate precision and agreement between tested and expected values of measurements were not altered throughout. The impact on the assays' diagnostic performance was estimated by analysing diagnostic sensitivity, diagnostic specificity and related parameters. The data demonstrated that although there was a bias of higher measurement values after gamma-irradiation, this could be compensated after readjustment of the cut-off points to obtain best separation of antibody-positive and -negative samples. Thus, for each assay, no significant difference of the diagnostic proficiency was found before and after gamma-irradiation. The practical implications are discussed of a serum sterilisation procedure using (60)Co gamma-rays for routine sample testing, assay validation and trypanosomosis monitoring and tsetse-fly control and eradication programmes.  相似文献   

7.
Part of the requirements for demonstrating disease freedom usually will be that sufficient testing be done to give a specified confidence of detecting the disease if it were present at a specified level. Often, this requirement is translated into a fixed testing regime that must be followed (an inflexible approach that might not be the most economic or practical solution).

A more flexible approach is to specify the capabilities of the various tests that can be used to detect the disease, and let the party hoping to demonstrate disease freedom decide upon the testing regime. The question then arises as to how to combine information that can come from a variety of sources over a period of time to give an overall level of confidence.

Two methods are given. The first, an exact method based on multiplying probabilities, would be more appropriate for a survey of an area in which no disease is thought to be present. The second method (more appropriate for a herd-assurance program within an infected area) is a point-based system that takes into account the different sensitivities of the methods used to detect disease and the change in prevalence over time. It allocates points for each test done proportional to the sensitivity of the test and the prevalence at the time of testing.  相似文献   


8.
Evaluation of heartworm immunodiagnostic tests   总被引：1，自引：0，他引：1  
In this report, the use of appropriate statistical methods for the evaluation of heartworm immunodiagnostic tests is discussed. The evaluation of these tests is complicated by factors causing variation in sensitivity, specificity, accuracy, and predictive values of positive and negative test results. The primary sources of inconsistency are variation in the prevalence of heartworm infection among populations of dogs and the sensitivity of immunodiagnostic tests to various categories of heartworm infections (ie, patent, immune-mediated occult, unisex occult, and immature occult). Sample size (ie, number of dogs tested) affects the confidence limit values of sensitivity and specificity. At least 100 dogs should be used in each testing group (infected and uninfected) to generate values of sensitivity or specificity within reasonably narrow confidence limits. Use of more than 200 dogs in each testing group contributes little to further narrowing of confidence limits. The selection of appropriate statistical tests for comparison of tests or comparison of the sensitivity or specificity of a single diagnostic test to various categories of heartworm infections is critical. The McNemar paired chi 2 test is appropriate for comparison of diagnostic tests, but it must be done by use of duplicate sera from each animal. A chi 2 test of independence, or, in the case of a small sample size, the Fisher exact test, is appropriate for comparing the sensitivity or specificity of a single diagnostic test to various categories of heartworm infection.  相似文献   

9.
Switzerland has been officially free of bovine tuberculosis (OTF) since 1960. Since 1980 the control of bovine tuberculosis (bTB) has been reduced to passive abattoir surveillance. Isolated cases of bTB, partly due to reactivation of human Mycobacterium bovis infections with subsequent transmission to cattle, have been noticed in the last years. In Europe, the overall prevalence of bTB is slightly increasing. Both OTF and non-OTF countries report increases in the proportion of bTB positive cattle herds. Current bTB eradication and control programs in Europe are facing a range of challenges. Whole herd depopulation is becoming a less attractive option for economic reasons and due to animal welfare concerns. Live animal trade is increasing both at national and international levels. Regarding these tendencies and taking into account the chronicity of bTB infection, pre-movement testing is becoming increasingly important as a central tool for eradication and for protection against re-introduction of bTB. Pre-movement testing, however specifically focuses on the infection status in individuals, requiring a high level of diagnostic accuracy to correctly diagnose infected animals. Current screening tests for bTB, however, have been designed to meet demands as herd tests. This illustrates that the modification of existing and/or the development of new diagnostics for bTB might be needed. The tuberculin skin test (TST), the primary screening test for bTB may in certain situations have low sensitivity. The interferon gamma (IFN-γ) assay is accepted to be more sensitive compared to TST. Reduced specificity, however, especially in areas of low bTB prevalence raises concerns. New antigen combinations including Rv3615c, OmpATb and others have been shown to complement ESAT-6 and CFP-10 in the whole blood IFN-γ assay and resulted in improved sensitivity (compared to ESAT-6 and CFP-10) and specificity (compared to tuberculins). Lesion detection after slaughter represents a cost-effective procedure for passive surveillance of bTB, especially in areas of low prevalence or in regions free of bTB; however, its sensitivity is very low. This illustrates that trade is linked with a certain risk to re-introduce bTB in OTF regions or countries and that there may be delays in detecting a re-introduction of bTB. In conclusion, regarding the fact that some parameters linked with bTB programs are changing, the development of improved diagnostic tests with a high reliability for use as individual animal tests will be important for future eradication of bTB, in line with international commitment to high standard animal health programs.  相似文献   

10.
An indirect enzyme-linked immunosorbent assay (ELISA) was developed for differentiation of animals infected with foot-and-mouth disease virus (FMDV) from vaccinated animals. The test was based on a highly pure and concentrated preparation of recombinant 3AB1 protein obtained by expression in a prokaryotic system, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electro elution. Experimental- and field-serum samples from naive, vaccinated, and infected cattle were tested for anti3AB1 antibody using the ELISA. A cutoff level was set at 35% of the maximum absorbance obtained with a positive control serum (FMDV-infected animal, 21 days postinfection [dpi]). This assay could detect antibodies from sera of animals experimentally infected by contact (n = 118) with a sensitivity of 97.5%. The specificity was 100%, based on negative test results obtained on 109 sera from naive animals. Remarkably, all sera from animals vaccinated either once (n = 102) or twice (n = 30) were negative. In addition, this 3AB1-ELISA could detect seroconversion at 7 dpi in animals inoculated intradermolingually. This assay constitutes an important tool for the rapid detection of FMDV outbreaks in a vaccinated population. In addition, it presents a reliable, economical, and simple method for testing large numbers of serum samples.  相似文献   

11.
AIM: To determine the diagnostic capability of a newly developed Western blot (WB) assay for the detection of serum antibodies against Mycoplasma agalactiae compared with conventional serological tests, and to identify the best test for routine diagnostic use. METHODS: The serological test methods used were: two commercial indirect enzyme-linked immunosorbent assays (ELISA), viz ELISA-1, using a bacterial antigen preparation, and ELISA-2, using a recombinant protein (lipoprotein p48) antigen; the complement fixation test (CFT); and a newly developed WB assay, the latter both using a bacterial antigen preparation. Thirty sera from goats infected with M. agalactiae and 97 sera from non-infected sheep were tested using all four methods. RESULTS: Staining patterns in the WB were quite variable. An immuno-dominant band of 41 kDa was detected in 63% of sera from infected animals. The same band also appeared, although mostly very weakly, in 10% of sera from non-infected animals. When suspicious or very weak reactors were omitted, the diagnostic sensitivity (DSE) and diagnostic specificity (DSP), respectively, for the four assays were: WB=56.7%, 97.9%; ELISA-1=76.7%, 99.0%; ELISA-2=56.7%, 100%; and CFT=40.0%, 94.8%. CONCLUSIONS: ELISA-1 performed best in this comparison. While the WB can be used, it did not have a technical advantage over the ELISA. The CFT should be discouraged as the primary screening method for contagious agalactia and should be replaced by ELISA-1. Results from this study confirm that serological test methods for contagious agalactia are useful for the detection of infected flocks but will not detect every individual infected animal.  相似文献   

12.
OBJECTIVE: To develop a method of probability diagnostic assignment (PDA) that uses continuous serologic measures and infection prevalence to estimate the probability of an animal being infected, using Neospora caninum as an example. ANIMALS: 196 N caninum-infected beef and dairy cattle and 553 cattle not infected with N caninum; 50 dairy cows that aborted and 50 herdmates that did not abort. PROCEDURE: Probability density functions corresponding to distributions of N caninum kinetic ELISA results from infected and uninfected cattle were estimated by maximum likelihood methods. Maximum likelihood methods also were used to estimate N caninum infection prevalence in a herd that had an excessive number of abortions. Density functions and the prevalence estimate were incorporated into Bayes formula to calculate the conditional probability that a cow with a particular ELISA value was infected with N caninum. RESULTS: Probability functions identified for infected and uninfected cattle were Weibull and inverse gamma functions, respectively. Herd prevalence was estimated, and probabilities of N caninum infection were determined for cows with various ELISA values. CONCLUSIONS AND CLINICAL RELEVANCE: Use of PDA offers an advantage to clinicians and diagnosticians over traditional seronegative or seropositive classifications used as a proxy for infection status by providing an assessment of the actual probability of infection. The PDA permits use of all diagnostic information inherent in an assay, thereby eliminating a need for estimates of sensitivity and specificity. The PDA also would have general utility in interpreting results of any diagnostic assay measured on a continuous or discrete scale.  相似文献   

13.
In order to test if disease is present in a large herd, an investigator will often subject only a small sample of animals to a fallible diagnostic test. The herd is declared positive for disease if the number of test-positive animals is greater than or equal to a previously chosen cut-off value. Such a test, called an aggregate test, has a sensitivity and specificity that depends on the sample size, the cut-off point and the sensitivity and specificity of the individual test. It also depends on the distribution of the disease among the herds being tested and on the fact that factors such as herd-level seropositivity may cause some herds to be more prone to testing errors than others. In this paper, we use the beta-binomial distribution to model all these factors and thereby calculate and tabulate aggregate test sensitivities and specificities under a variety of conditions. Receiver operating characteristic (ROC) curve methodology permits the choice of optimum sample sizes and cut-off values. We also investigate the situation in which an investigator may be willing to miss detecting the disease if the prevalence in the herd is low. A compiled FORTRAN program for the calculation of aggregate test cut-off point properties, including positive and negative predictive values, is available from the authors.  相似文献   

14.
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15.
Blood collection from small exotic pocket pets can be difficult to achieve. The individual collecting the blood must know both the anatomy and behavior of the species to obtain suitable amounts of blood for diagnostic testing. Given the animals' small size, it is often difficult to collect large volumes of blood. A clinician serious in developing an exotic small mammal practice should understand the limitations of blood sample collection and the risks involved with the procedure. Unlike domestic animals, these pets are often not comfortable with being handled and are often prone to induced complications when presented to a veterinary clinic and restrained for examination. For some cases, the clinician will have to determine if the risk of getting the sample is better achieved by anesthetizing the patient, and if doing so will have a detrimental effect on the animal. One will also need to consider the effect of the anesthetic versus the stress the restraint may have on the blood results.  相似文献   

16.
The present study was conducted to evaluate the sensitivity and specificity of an immunochromatography-based diagnostic kit for Salmonella. The analytical sensitivity of the test when using pure colonies of different Salmonella species was in the range of 1 X 10(4) to 1 x 10(5) colony-forming units per milliliter. The strip detected 19 of 22 strains of Salmonella spp. but failed to detect S. worthington, S. choleraesuis var. kunzendorf and S. johannesburg. The strip did not detect 27 different enteric bacteria, including Escherichia coli O157:H7, Campylobacter jejuni, Shigella sonnei, and Vibrio parahaemolyticus. In direct testing of feces (n = 66) from chickens infected with Salmonella typhimurium, the strip had a sensitivity of 12.3% and a specificity of 100%. Evaluation of the strip assay (n = 510) after sample pre-enrichment in 2% buffered peptone water (BPW) yielded a sensitivity of 93.8% and specificity of 89% when compared to isolation and identification with xylose-lysine-tergitol 4 (XLT4) selective plating media. Subsequent enrichment in Hajna tetrathionate (TT) broth yielded a higher sensitivity (94.7%) and specificity (96.8%). The agreement (kappa) between the strip test and isolation was 0.004 in direct fecal testing, 0.82 in BPW, and 0.89 in TT broth. The assay could detect Salmonella sp. as early as 18-48 hours during pre-enrichment and enrichment compared to isolation on XLT4, which required an overnight incubation step for the presumptive isolation and identification of Salmonella.  相似文献   

17.
The feasibility of using a SYBR Green-based real-time RT-PCR assay (SYBR Green ReTi RT-PCR) followed by melting curve analysis (MCA) for detecting and genotyping porcine reproductive and respiratory syndrome virus (PRRSV) was assessed. The SYBR Green ReTi RT-PCR and a previously reported two-step, non-nested RT-PCR assays were simultaneously tested on selected European (EU) and North American (US) PRRSV strains and isolates collected from diverse clinical, temporal, and geographical origins. The validation experiments showed that the optimised SYBR Green ReTi RT-PCR can sensitively and specifically detect PRRSV, consistently detecting as little as 0.03TCID(50)/sample of each virus genotype, with no type-bias and no amplification signal for other swine pathogens. After MCA, two well-differentiated melting temperature (T(m)) profiles for each virus genotype were obtained, as sequencing confirmed it. High repeatability was obtained for the T(m) values, with intra-run coefficients of variation (CoVs) of 0.25 and 0.32 and inter-run CoVs of 0.42 and 0.52 for EU and US genotypes, respectively. The sensitivity of the SYBR Green ReTi RT-PCR (100%) was higher than that of the RT-PCR (95.7%) when testing field isolates. This greater sensitivity of the SYBR Green ReTi RT-PCR was further confirmed by the detection of a higher proportion of PRRSV-positive diagnostic specimens (29.7%) than by the RT-PCR (28.5%). The SYBR Green ReTi RT-PCR test detected infection as early as 2 dpi in the sera of experimentally infected pigs regardless of virus genotype, and discriminated negative (non-inoculated), EU- and US-infected pigs. In conclusion, the reported SYBR Green ReTi RT-PCR assay coupled with MCA can detect and type PRRSV and may be useful as an alternative diagnostic assay in diverse PRRSV epidemiological circumstances.  相似文献   

18.
AIM: To determine the performance characteristics of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against liver fluke (Fasciola hepatica) in bovine milk. METHODS: Serum and milk from liver fluke infected and non-infected cattle was assayed in a commercially available enzyme-linked immunosorbent assay. Serum test results were used to determine the "gold standard" infection status of cattle and milk ELISA results assessed by ROC analysis. RESULTS: ROC analysis suggested changes to the ELISA protocol, arriving at milk dilutions assayed considerably higher than those suggested by the manufacturer. With those changes, the ELISA performed with high sensitivity and specificity, 95 and 98.2%, respectively, for individual bovine milks (relative to sera). For bovine tank milks, sensitivity was lower, with bulk milks only testing positive if 60% or more of cattle milking in the herd were infected. CONCLUSIONS: The analysis of the ELISA's performance when used on individual bovine milks demonstrated high sensitivity and specificity. ROC analyses optimised the assay conditions and cut-off point suggested by the manufacturer for this commercial diagnostic assay. This would help with the identification and control of fasciolosis, enabling simpler sample collection.  相似文献   

19.
Although the interferon-gamma (IFN-γ) assay for measurements of cell-mediated immune (CMI) responses to paratuberculosis PPD (johnin) has been available for close to 20 years, the assay has not yet emerged as the long desired test to identify infected animals at an early time point. Among other issues, this relates to problematic interpretation of the test results and maybe an over-expectation of what can be deducted from this kind of test given the chronic nature and slow development of infection of paratuberculosis. Over a number of years a modified IFN-γ assay with addition of recombinant bovine IL-12 to the PPDj stimulation of blood samples from the heifer group in more than 20 Danish dairy herds which also perform surveillance of MAP antibodies in milk have been performed. The results indicate that IFN-γ assay results are specific for paratuberculosis, but the IFN-γ assay result of an individual animal cannot establish whether the animal is infected or predict the future progression of disease in this animal. The IFN-γ assay should thus be used on a group of animals to test the level of exposure to paratuberculosis bacteria the animals have experienced, and thereby assist in maintaining rational in-herd management procedures and in the establishment of paratuberculosis status of a given herd. Indeed, for any diagnostic test applied in paratuberculosis, both the diagnostic target condition and the purpose of the diagnostic testing must be considered before any meaningful estimates of sensitivity or specificity can be given.  相似文献   

20.
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