Polymerase chain reaction for the detection of Cryptosporidium spp. in cat feces

The objective of this study was to compare a polymerase chain reaction (PCR) assay and a monoclonal antibody-based immunofluorescence assay (IFA) for detection of Cryptosporidium parvum in cat feces. Eight C. parvum-naive DSH cats were orally inoculated with 1 x 10(6) oocysts of a C. parvum human isolate. Fecal samples were collected before inoculation, daily for the next 30 days, and twice weekly until day 85. Methylprednisolone acetate was administered at 20 mg/kg i.m. on days 85, 92, and 99. From days 86 to 115, feces were collected daily and then up to twice weekly until day 126. Immunofluorescence assay was performed after collection of the samples, and then the samples were frozen at -70 C until assayed by PCR. Cryptosporidium parvum was detected by PCR in 101 of 353 samples and by IFA in 52 of 353 samples: 27 samples were PCR positive, IFA positive; 74 samples were PCR positive, IFA negative; 25 samples were PCR negative, IFA positive; and 227 samples were PCR negative, IFA negative. The percentage of concordance between IFA and PCR was 72%. Results of this study suggest that this PCR assay is more sensitive than IFA for detection of C. parvum in cat feces.     

Reliability of non-lethal surveillance methods for detecting ranavirus infection

Ranaviruses have been identified as the etiologic agent in many amphibian die-offs across the globe. Polymerase chain reaction (PCR) is commonly used to detect ranavirus infection in amphibian hosts, but the test results may vary between tissue samples obtained by lethal and non-lethal procedures. Testing liver samples for infection is a common lethal sampling technique to estimate ranavirus prevalence because the pathogen often targets this organ and the liver is easy to identify and collect. However, tail clips or swabs may be more practicable for ranavirus surveillance programs compared with collecting and euthanizing animals, especially for uncommon species. Using PCR results from liver samples for comparison, we defined false-positive test results as occurrences when a non-lethal technique indicated positive but the liver sample was negative. Similarly, we defined false-negative test results as occurrences when a non-lethal technique was negative but the liver sample was positive. Using these decision rules, we estimated false-negative and false-positive rates for tail clips and swabs. Our study was conducted in a controlled facility using American bullfrog Lithobates catesbeianus tadpoles; false-positive and false-negative rates were estimated after different periods of time following exposure to ranavirus. False-negative and false-positive rates were 20 and 6%, respectively, for tail samples, and 22 and 12%, respectively, for swabs. False-negative rates were constant over time, but false-positive rates decreased with post-exposure duration. Our results suggest that non-lethal sampling techniques can be useful for ranavirus surveillance, although the prevalence of infection may be underestimated when compared to results obtained with liver samples.     

Cat scratch disease. Survey on the presence of Bartonella henselae among cats of Tuscany

To verify the presence of Bartonella henselae-infection in cats living in Tuscany (central Italy) serological and bacteriological surveys were carried out. The blood serum samples of 427 cats, 254 living in private houses and gardens and 173 in public or private catteries, were tested for anti-B. henselae antibodies by indirect immunofluorescence assay (IFA). Among these samples, 35 were examined by IFA to detect antibodies against Bartonella quintana. Bacteriological examinations were performed on the blood samples, collected in EDTA (ethylene diaminetetraacetic acid), of 18 cats (10 seropositive to B. henselae and 8 negative). From each of the same 18 specimens DNA was extracted and used as template in polymerase chain reaction (PCR). The primers p24E and p12B were employed in the PCR assay to amplify a 296 bp fragment of the Bartonella 16S rRNA gene. IFA detected 98 (22.95%) B. henselae-positive serum samples (40-40.82% from cats living in houses and gardens and 58-59.18% from cats of catteries) at different antibody titers (70 at 1:64 titer, 4 at 1:128, 22 at 1:256, 2 at 1:512). Among the 35 sera tested to detect antibodies against B. quintana, 9 (25.71%) resulted positive at 1:64 titer; all these samples showed higher antibody titers to B. henselae. Out of the 26 negative sera, 20 were negative to B. henselae too and 6 had antibodies against B. henselae at 1:64. Hemocultures gave negative results. PCR scored positive with DNA of 4 B. henselae-seropositive cats, two of which belonged to two children with cat scratch disease (CSD).     

Evaluation of indirect fluorescent antibody assays compared to rapid influenza diagnostic tests for the detection of pandemic influenza A (H1N1) pdm09

Performance of indirect fluorescent antibody (IFA) assays and rapid influenza diagnostic tests (RIDT) during the 2009 H1N1 pandemic was evaluated, along with the relative effects of age and illness severity on test accuracy. Clinicians and laboratories submitted specimens on patients with respiratory illness to public health from April to mid October 2009 for polymerase chain reaction (PCR) testing as part of pandemic H1N1 surveillance efforts in Orange County, CA; IFA and RIDT were performed in clinical settings. Sensitivity and specificity for detection of the 2009 pandemic H1N1 strain, now officially named influenza A(H1N1)pdm09, were calculated for 638 specimens. Overall, approximately 30% of IFA tests and RIDTs tested by PCR were falsely negative (sensitivity 71% and 69%, respectively). Sensitivity of RIDT ranged from 45% to 84% depending on severity and age of patients. In hospitalized children, sensitivity of IFA (75%) was similar to RIDT (84%). Specificity of tests performed on hospitalized children was 94% for IFA and 80% for RIDT. Overall sensitivity of RIDT in this study was comparable to previously published studies on pandemic H1N1 influenza and sensitivity of IFA was similar to what has been reported in children for seasonal influenza. Both diagnostic tests produced a high number of false negatives and should not be used to rule out influenza infection.     

Detection of antibodies to cytomegalovirus, herpes simplex virus and rubellavirus with the micro-enzyme immunoassay.

Serum samples of pregnant women (200), non-pregnant women (100) and children (36) were tested for the presence of antibodies to cytomegalovirus (CMV), herpes simplex virus (HSV) and rubellavirus with the micro-enzyme immunoassay (micro-EAI), the indirect immunofluorescence antibody (IFA) technique and the haemagglutination-inhibition (HI) test. Micro-EIA gave the highest positivity rate: 78% for CMV, 86% for HSV and 85% for rubellavirus compared to 67% (CMV) and 84% (HSV) of the IFA technique and 79% (rubellavirus) of the HI test, respectively. Among IFA and HI positive sera the occurrence of micro-EIA negative ones was 6% for CMV, 4% for HSV and 9% for rubellavirus. It is concluded that the introduction of micro-EIA will improve the diagnostic and screening work of virus laboratories.     

Detection of Pneumocystis carinii DNA in clinical samples from children with respiratory diseases

Method of Pneumocystis carinii DNA detection in clinical samples (sputum) is presented. Primers to one fragment of 16S rRNA gene were used for detection of DNA. Isolation and amplification of DNA were performed in presence of internal DNA control. Analytical sensitivity of method was 200 copies of DNA per 1 ml. Analytical and diagnostic specificities were 100% and 95% accordingly. Sputum from 176 children with frequent respiratory infections were sampled before start of antibacterial therapy and studied simultaneously by the polymerase-chain reaction (PCR) and immunofluorescent assay (IFA). Results of PCR and IFA coincided in 167 (94.89%) children. From them, P.carinii was detected in sputum in 5 (2.85%) children. All children with positive results were treated with antibiotics. Repeated tests of sputum 8 days after start of treatment were all negative. PCR could be recommended as part of complex of clinical diagnostics and control of treatment.     

Comparative evaluation of antibody positive titer by ELISA and IFA in Theileria annulata vaccinated cattle in Iran

An enzyme linked immunosorbent assay (ELISA) was used to evaluate antibody positive titer in vaccinated and non-vaccinated cattle using schizont infected myeloid cells as an antigen. The result was compared with indirect fluorescent antibody level in the same animals. For this study 116 milking cows, 95 vaccinated and 21 non-vaccinated, were bleeded in order to prepare sera. They were tested with both ELISA and IFA tests. 94 sera had positive antibody titer and 22 sera were negative through ELISA test but, with IFA test, only 89 sera showed positive antibody titer and 27 were negative. Thereby, it was concluded that the sensitivity and specificity of ELISA test in comparison with IFA test was 95.5% and 66.6% respectively. This study generally indicated that ELISA could be an effective test for sero-epidemiological investigations of bovine tropical theileriosis, and it is considered to be valid as an additional test to distinguish the vaccinated from the non vaccinated cattle in order to schedule vaccination programs.     

Evaluation of a Semiautomated System for Direct Fluorescent Antibody Detection of Salmonellae

A semi-automatic system under development by Aerojet Medical and Biological Systems for the direct fluorescent antibody detection of salmonellae was evaluated with various food, feed, and environmental samples. All samples were simultaneously examined by Automated Bioassay System (ABS), manual direct fluorescent antibody procedures and cultural procedures. The ABS gave satisfactory results with the processed samples. It detected all of the culturally positive powdered egg and candy samples with no false negative results and gave only 6.6 and 5.3% false positive rates, respectively. With meatmeal samples the ABS failed to detect one culturally positive specimen that was also positive by manual fluorescent antibody and gave one (1.1%) false-positive result. A high rate of false-negative results was obtained by ABS on unprocessed samples of creek water, poultry, and sausage. Adding another enrichment step to the protocol reduced the false-negative rate considerably but severely increased the false-positive rate. The instruments worked reasonably well, but research is needed to improve enrichment procedures for samples to be processed by the system.     

Statistical analysis of fine needle aspiration cytology of the breast. A review of 678 cases plus 4,265 cases from the literature

Between 1979 and 1984, 678 breast fine needle aspiration (FNA) cytologic specimens were received in our laboratory; tissue follow-up was available for 159 cases (23%). The diagnoses rendered in cases with subsequent tissue study were benign (41%), insufficient (10.5%), atypical and/or suspicious for carcinoma (10.5%) and malignant (38%). Using the tissue diagnosis as the standard, there were four false-negative cytologic results and one false-positive result. Considering only cases with a definitive diagnosis of benign or malignant, the sensitivity of cytologic interpretation for diagnosing malignancy on adequate material was 94% while the specificity was 98%; the overall efficiency of the test was thus 96%. The predictive values were 98% for a positive test and 94% for a negative test. Of the total number of submitted cases, a correct and definitive diagnosis was rendered 76% of the time. Calculation of similar statistics from six other series in which the FNA cytologic diagnoses were compared with the histologic diagnoses yielded data almost identical to our findings. The high degree of uniformity in the data indicates that these statistical parameters can be used as standards for evaluating the efficiency of breast FNA cytology.     

Clinicopathologic management of tumors of the thyroid gland in an endemic goiter area. Combined use of preoperative fine needle aspiration biopsy and intraoperative frozen section

In total, 15,325 fine needle aspiration (FNA) biopsies of the thyroid were examined in the Department of Pathology of the University of Innsbruck, Austria, between 1976 and 1985, with the cytologic results histologically verified in 3,112 cases. Since (1) it is frequently impossible to distinguish benign from malignant encapsulated follicular thyroid tumors by cytologic criteria and (2) there is a high level of follicular thyroid carcinoma in our endemic goiter area, we have adopted a diagnostic strategy that accepts a high percentage of false-positive cytologic results in order not to miss highly differentiated follicular carcinoma. To avoid unnecessarily extensive surgical treatment, 1,079 intraoperative frozen section examinations of the thyroid were performed in the same time period in (1) patients with preoperative suspicious or positive FNA cytologic findings, (2) cases with suspicious clinical and anamnestic data and (3) tumors with a suspicious macroscopic appearance without preoperative FNA or with negative or unsatisfactory cytologic findings. In 48 cases (4.5%), the frozen section diagnosis had to be revised after examination of paraffin-embedded tissue. An intraoperative false-positive diagnosis was obtained in 3 cases (0.3%) while a false-negative diagnosis was made in 45 cases (4.2%). The main effort in examining frozen sections should be concentrated on avoiding false-positive errors, which can lead to unnecessary thyroidectomies.     

Limitations of the direct immunofluorescence test for antibody-coated bacteria in determining the site of urinary tract infections in children.

The results of the direct immunofluorescence test for antibody-coated bacteria to determine the site of a urinary tract infection do not always correlate with the clinical data. When this test was performed on urine specimens from 282 children with significant bacteriuria, false-negative and false-positive results were observed in 20% (19/94) and 52% (19/188) of the specimens. Contamination of the specimen during collection and the presence of Fc receptors (receptors for the crystallizable fragment of the immunoglobulin molecule) on the surface of some strains of Staphylococcus aureus yielded false-positive results, and stools and vaginal secretions were shown to be potential sources of antibody-coated bacteria. It is suggested that for children this test be run on urine collected by bladder puncture. The use of conjugated anti-IgG antiserum containing only F(ab'')2 (the antigen-binding fragments of the IgG molecule) is also recommended to eliminate false-positive results due to the presence of Fc receptors on the bacterial surface.     

Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells II. Test validation and interpretation

The L5178Y Mouse Lymphoma TK assay was studied extensively to determine if this mammalian cell assay for gene mutations at the thymidine kinase (TK) locus could provide valid, interpretable determinations of mutagenic potential, and whether this information is of value in the safety evaluation of chemicals. We first determined that test-derived TFT^R mutants were phenotypically stable, possessing little or no thymidine kinase activity as measured by labeled thymidine uptake, but demonstrating 100% cross resistance to bromodeoxyuridine. Common solvent vehicles such as acetone, dimethylsulfoxide and ethanol were shown to produce little cytotoxicity and no mutagenic activity when present at 1% levels. Out of a total of 10 noncarcinogens tested, all were negative when results were analyzed by a 2-sample log_et test on control and treated mutant count means. Of the 13 putative animal carcinogens tested, 10 were positive, 2 were negative (auramine O and sodium phenobarbital), and 1 showed sporadic activity (hydrazine sulfate) in the TK assay on the basis of test-derived t statistics. 2 compounds, 1,2-epoxybutane and ICR 191, which have been described as Ames positive non-carcinogens, were also positive in the TK assay. Although this sampling of a total of 29 compounds is insufficient for precise estimations of expected false-positive or false-negative frequencies, these data indicate the TK assay can be expected to detect a majority of carcinogens as mutagens including some missed by more established point-mutation assays.     

Detecting cervical cancer by quantitative promoter hypermethylation assay on cervical scrapings: a feasibility study

Current morphology-based cervical cancer screening is associated with significant false-positive and false-negative results. Tumor suppressor gene hypermethylation is frequently present in cervical cancer. It is unknown whether a cervical scraping reflects the methylation status of the underlying epithelium, and it is therefore unclear whether quantitative hypermethylation specific PCR (QMSP) on cervical scrapings could be used as a future screening method augmenting the current approach. Cervical scrapings and paired fresh frozen cervical tissue samples were obtained from 53 cervical cancer patients and 45 controls. All scrapings were morphologically scored and analyzed with QMSP for the genes APC, DAPK, MGMT, and GSTP1. To adjust for DNA input, hypermethylation ratios were calculated against DNA levels of a reference gene. Hypermethylation ratios of paired fresh frozen tissue samples and scrapings of cervical cancer patients and controls were strongly related (Spearman correlation coefficient, 0.80 for APC, 0.98 for DAPK, and 0.83 for MGMT; P < 0.001). More cervical cancer patients than controls were DAPK positive (P < 0.001). When cutoff levels for ratios were defined to be above the highest ratio observed in controls, QMSP in cervical scrapings identified 32 (67%) of 48 cervical cancer patients. This feasibility study demonstrates that QMSP on cervical scrapings holds promise as a new diagnostic tool for cervical cancer. The addition of more genes specifically methylated in cervical cancer will further improve the assay.     

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax and its use in a seroepidemiological survey of the Eastern Caribbean Basin

An enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against a South American (New World) strain of Trypanosoma vivax was developed and used for mass screening of cattle from 20 islands in the Eastern Caribbean Basin. The sensitivity and specificity of antigens prepared from a bovine-derived field strain and a murine-adapted laboratory strain of T. vivax, both of New World origin, were compared using an indirect fluorescent antibody (IFA) test, and an antigen prepared from the murine-adapted strain was subsequently used to develop an ELISA test. The results of the ELISA test were then compared with the results of a concurrently run IFA test. There was no cross-reactivity with either test using serum from a Trypanosoma theileri-infected cow. Both tests were weakly cross-reactive with sera from a T. brucei-infected steer, and the IFA test was moderately cross-reactive with several serum samples from a T. evansi-infected steer. For bovine sera collected from herds on islands in the Eastern Caribbean region, only five of 640 tested positive with the ELISA test. Thirty five of 653 sera tested were positive by IFA although the fluorescence elicited was weak as compared to that elicited by sera from known infected animals. Sera collected from 27 cattle in a region known to be free of T. vivax (OH, U.S.A) were negative with the ELISA test, whereas seven of 30 sera from a herd in French Guiana known to be infected with T. vivax were positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anaplasma phagocytophilum infection in a fallow deer (Dama dama) population in a preserve of central Italy

From autumn 2004 to spring 2005, 70 fallow deer (Dama dama), 27 female and 43 male, living in a natural preserve of central Italy were examined by indirect immunofluorescence assay (IFA) to detect specific antibodies to Anaplasma phagocytophilum. Thirty-one (44.28%) sera scored positive: in particular 10 fallow deer (8 male and 2 female) scored positive at 40 antibody titer, 21 deer (8 male and 13 female) at > or = 80 titer. EDTA anticoagulated blood samples collected from 29 of the 70 deer examined were tested by a nested-PCR assay to disclose a 546 bp fragment, specific of A. phagocytophilum 16S rRNA gene. Twenty (72.41%) blood samples (8 male and 13 female deer) resulted positive. Fifteen PCR-positive deer also resulted positive to IFA, whereas the remaining six did not show specific antibodies. Three serologically positive animals gave negative results at the nested PCR. Five deer scored negative both to serological tests and PCR.     

Detection of human herpesvirus 7 infection in young children presenting with exanthema subitum

In this study, we assessed the prevalence of human herpesvirus-7 (HHV-7) in 141 serum samples from children less than four years of age with exanthematic disease. All samples were negative for measles, rubella, dengue fever and parvovirus B19 infection. Testing for the presence of human herpesvirus-6 (HHV-6)-specific high avidity IgG antibodies by indirect immunofluorescence assay (IFA) revealed two main groups: one composed of 57 patients with recent primary HHV-6 infection and another group of 68 patients showing signs of past HHV-6 infection. Another 16 samples had indeterminate primary HHV-6 infection, by both IgG IFA and IgM IFA. Serum samples were subjected to a nested polymerase chain reaction to detect the presence of HHV-7 DNA. Among patients with a recent primary HHV-6 infection, HHV-7 DNA was present in 1.7% of individuals; however, 5.8% of individuals tested positive for HHV-7 DNA in the group with past primary HHV-6 infection. Among the 16 samples with indeterminate diagnosis, 25% (4/16) had HHV-7 DNA (p < 0.002). We hypothesise that HHV-7 might be the agent that causes exanthema. However, a relationship between clinical manifestations and the detection of virus DNA does not always exist. Therefore, a careful interpretation is necessary to diagnose a primary infection or a virus-associated disease. In conclusion, we detected HHV-7 DNA in young children from the state of Rio de Janeiro, Brazil.     

Detection of methicillin-resistant <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers

In this study, we reported on the design of a multiplex real-time PCR assay based on SYBR Green I, incorporating dual priming adenine-thymine (AT)-rich primers for direct detection of MRSA from nasal samples. The multiplex real-time polymerase chain reaction (RT-PCR) assay reported in this study is based on SYBR Green I with incorporation of six dual priming AT-rich primers designed from the SCCmec/orf junction. A string (4–6 bp) of low-melting bases, such as adenine and thymine, was incorporated into the primers, which virtually divided a single primer in two functional regions, thus decreasing non-specific PCR products. The analytical sensitivity and specificity of the RT-PCR assay was determined with genomic DNA of reference strains (MRSA, MSSA, and MRCoNS). RT-PCR assay was performed for analysis of 72 nasal swab specimens, and the results were confirmed by use of a culture method. Furthermore, the results of RT-PCR were compared with LightCycler MRSA advance test. The multiplex RT-PCR assay reproducibly detected a minimum of 1 pg genomic DNA (31.5 copy of genome) of MRSA reference strains and clinical isolates, with a specific melting peak at 83.5 ± 1.5°C, and neither fluorescence nor a melting peak was detected in non-target isolates. The concordance rate between RT-PCR assay and culture method was 87.5% with Cohen's kappa value (κ) 0.75, which showed good agreement between the two assays. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 93.5%, 82.9%, 80.5%, and 94.4%, respectively. In a comparative study for the detection of 72 nasal samples, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex RT-PCR assay with respect to LightCycler MRSA advance test was 84.2%, 88.2%, 89%, and, 83.3%, respectively. The results of RT-PCR assay demonstrated high specificity (88.2%) and positive predictive value (89%) for the direct detection of MRSA from nasal samples.     

Comparison of four enzyme immunoassays for the detection of cytomegalovirus IgG antibodies

Four commercial enzyme immunoassays (EIA), namely the Behring Enzygnost EIA (BE-EIA), Abbott IMx, Whittaker CMV STAT Test Kit and Diamedix assay, were evaluated for the detection of CMV IgG. The methods were compared as to sensitivity, specificity, positive and negative predictive values, global agreement, ease of performance and, for a small number of specimens, reproducibility. Discordant results were resolved by using the Gull CMV indirect fluorescent antibody (IFA) method. Our data suggest that all four assays were valuable screening tools for the detection of CMV IgG based on their high sensitivity and high negative predictive value. However, differences were noted in the reproducibility level and in the incidence of false-positive, equivocal and nonspecific results regarding certain tests in particular. In our hands, the Abbott IMx and the BE-EIA ranked high in the performance characteristics for a good screening test, yet the Abbott IMx offers the added advantages of being the easiest to perform and having the most rapid turnaround time.     

DNA breaks as measured by the alkaline comet assay in exfoliated cells as compared to voided urine cytology in the diagnosis of bladder cancer: a study of 105 subjects

In this study we evaluated the clinical usefulness of identifying urothelial cells with increased DNA damage with the alkaline comet assay and compare it with voided urine cytology for the assessment of markers indicative of bladder cancer. The analysis was carried out on 105 subjects having clinical suspicion of bladder cancer, and who had undergone cytology for the first time. Urine cytology and alkaline comet assay were performed on the same fresh urine samples obtained from each patient. The subjects were divided according to negative or positive cytology. The Mann-Whitney U-test showed that the comet parameters (tail moment, tail length, and % of DNA in the tail) and the numbers of comets (cells with an arbitrary cut-off value of head intensity <90% of DNA content) in subjects positive in both tests were significantly higher than in the negative group. Sensitivity, specificity, and positive and negative predictive value of the comet assay were compared with those of cytology, which is regarded as the gold standard. Sensitivity was 71.4%, specificity was 91.8%, positive and negative predictive values were 38.5 and 97.8, respectively. Two subjects negative in the comet assay were positive in cytology. Eight patients were positive in the comet assay and negative for cytology. Interestingly, one of these eight patients was later found positive for cytology. Logistic regression analysis indicates that the tail moment is significantly associated with an increased risk for positive cytology.