Cloning and expression of portions of the 34-kilodalton-protein gene of Mycobacterium paratuberculosis: its application to serological analysis of Johne's disease

Paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle that is caused by Mycobacterium paratuberculosis, is characterized by incoercible diarrhea and fecal shedding of bacteria. The present work aimed at developing a specific serological test for this disease. We have recently shown that a 34-kDa protein belonging to the major antigen complex A36 of M. paratuberculosis is immunodominant and contains epitopes specific with respect to all mycobacteria tested, including Mycobacterium bovis and the closely related species Mycobacterium avium. From a lambda gt11 genomic library of M. paratuberculosis, three portions of the gene coding for this 34-kDa protein have been isolated. Two of them expressed cross-reacting mycobacterial epitopes. One portion (in clone a362) expressed a polypeptide which cross-reacted with all tested M. paratuberculosis strains but not with 20 other bacteria tested, including many strains of the M. avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum group. The occurrence at the M. paratuberculosis surface of epitopes corresponding to the a362 polypeptide was shown by immune electron microscopy. The recombinant a362 polypeptide was used as reagent for an enzyme-linked immunoassay for paratuberculosis. This assay correctly diagnosed all the tested blood samples from infected cattle at all stages of the disease.     

Reproducible model of a bacterial conjunctivitis

To test the efficiency of antimicrobial substances on bacterial conjunctivitis under standardized conditions a reproducible model is needed. As there are no data in the literature concerning this topic we have developed a model of bacterial conjunctivitis in the rabbit eye. Instillation of a suspension of Haemophilus influenzae, Staphylococcus epidermidis or Staphylococcus aureus into the cul-de-sac did not result in any effect. Thus, standardized techniques of conjunctival incision were used additionally. A follow-up was done for a period of 2 weeks (degree of conjunctival hyperemia, microbiology). Radial incision of the conjunctiva with injection of an S. aureus suspension (50 microliters of ATCC 29213; 1.35 x 10(8) cells) was done mimicking a branch injury. This resulted in a purulent conjunctivitis over a period of 1 week and disappeared within 14 days. The swabs were negative on day 7. This is the first model establishing a reproducible purulent conjunctivitis in the rabbit. Using this model antimicrobial substances may be tested under standardized conditions.     

Harbor seal (Phoca vitulina) C-reactive protein (C-RP): purification, characterization of specific monoclonal antibodies and development of an immuno-assay to measure serum C-RP concentrations

C-reactive protein (C-RP) was purified from harbor seal (Phoca vitulina) serum by calcium dependant phosphoryl-choline and protein A affinity chromatography. Polyacrylamide gel electrophoresis under reducing conditions revealed a single protein moiety with a molecular weight of approximately 25 kDa. An internal peptide derived from this purified protein was subjected to N-terminal amino acid sequencing. A high amino acid sequence similarity was obtained with other published mammalian C-RP molecules confirming that the purified protein was a C-RP homologue. Eight specific monoclonal antibodies (P13, P51, P87, P101, P106, P130, P157 and P219) were raised against this purified protein. All 8 monoclonal antibodies immunoblotted with the 25 kDa C-RP subunit under reducing conditions. A competitive immunoassay was developed identifying elevated C-RP concentrations in harbor seal serum samples with clinical evidence of inflammatory disease. Application of this immunoassay for the measurement C-RP may provide valuable information for the clinical assessment of harbor seal health.     

Diethyl ether. 1963

Chlamydia psittaci is a significant cause of conjunctivitis in cats, but can be difficult to diagnose owing to the small number of organisms in conjunctival swabs. In the United Kingdom laboratory diagnosis is based on three techniques: isolation of the infectious organism, amplification of chlamydial DNA by the polymerase chain reaction (PCR) or the detection of anti-chlamydial antibodies by immunofluorescence assay. To determine the most sensitive method these techniques were compared in the field. The PCR based on previously published protocols was less sensitive than isolation, but by modifying the protocol its sensitivity was increased by a factor of 25 to 1250 and it was then more sensitive than isolation. The modified PCR detected chlamydia in samples containing non-infectious organisms. Serology was of limited use in predicting which cats shed C psittaci although seronegative cats were negative by PCR and isolation. The modified PCR was the most sensitive and robust method for confirming C psittaci infection in cases of conjunctivitis in pet cats.     

Health-care technology transfer: expert and information systems for developing countries

The alkaline phosphatase immunoassay (APIA) is an antibody detection technique which permits the diagnosis of schistosomiasis using a butanolic extract preparation from adult worms. APIA has demonstrated high sensitivity and specificity in previous reports with well characterized human sera. Its potential as a diagnostic tool for epidemiological surveillance was assessed in comparison with three other diagnostic tests: stool examination, ELISA with soluble egg antigen (SEA) and the circumoval precipitin test (COPT). APIA was 100% specific in an area without Schistosoma mansoni transmission and had 89% sensitivity in an endemic area where 69% of the infected subjects excreted less than 100 eggs g of faeces. It was found to be less sensitive in children under 5 years of age who were positive by the COPT test. APIA can be applied as an initial screening test, based on its high sensitivity, specificity, absence of cross-reactivity with intestinal parasites and the fact that it is a technique suitable for use in epidemiological surveillance.     

Management of "localized" oral cancer

We have outlined the clinical manifestations of "localized" malignant lesions of the intraoral cavity, their clinical behavior after intraoral excision, and their control rate employing intraoral excision as primary therapy. We must keep in mind that these small "localized" cancers are potential "killers" and the five year mortality from disease in our series was 25 per cent. This mortality may decrease with (1) more careful selection when patients are included in the "localized" lesion group and (2) earlier use of composite procedures in questionable cases.     

Effect of three conditions of administration on "trait" and "state" measures of anxiety.

Administered a battery of test anxiety scales and 2 physiological measures to 25 undergraduates under 3 differing sets of demand characteristics in order to determine the effect of situational aspects on anxiety scale scores. Measures of academic performances were also obtained. The hypothesis that specific demand characteristics would differentially influence Ss' scoring on "trait" and "state" anxiety scales was supported, but no support for a hypothesized relationship between the operation of demand characteristics and systematic changes in the correlations between the anxiety scales and academic performance was found. Implications of findings for the construction and use of anxiety scales were discussed. (21 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Individual differences in susceptibility to the "irrelevant speech effect"

Individual differences in objective effects of noise on performance were analyzed with respect to their distribution, temporal stability, and the precision of measurement to be attained. Seventy-two subjects had to memorize sequences of visually presented digits while being exposed to one of three auditory background conditions which were randomly mixed on a trial-by-trial basis: (1) foreign speech; (2) pink noise; and (3) silence. Individual "irrelevant speech effects," operationalized by the difference in recall errors under speech and in silence, were normally distributed over a wide range extending from slight facilitation to severe disruption. When 25 subjects repeated the experiment after four weeks, the individual differences were replicated with a reliability of rtt = 0.45. Internal consistency, a measure of the precision with which individual effects can be measured in a single session, was moderate (alpha = 0.55). However, both retest, and consistency coefficients are severely attenuated by the use of (sound-minus-silence) difference scores, the reliability of which is bound to be considerably lower than that of the original error scores whenever these are correlated. Given that the original error rates in a specific auditory condition can be determined with reliabilities approaching 0.85, it may be concluded that individual performance decrements due to noise can be reliably measured in the "irrelevant speech" paradigm. Self-report measures of noise susceptibility collected to explore potential sources of the large inter-individual variation exhibited only weak relationships with the objectively measured noise effects: Subjects were quite inaccurate in assessing their individual impairment in the three auditory conditions, and a questionnaire-based measure of general noise sensitivity only accounted for a small portion of the variance in objectively measured performance decrements, although in both cases the predictive relationship was much stronger in female than in male subjects.     

"Cancer patients and health care professionals perceptions of the need for oral health education"

Reported studies indicated a need for oral health education for oncology patients. In a recent study it was concluded that septicaemia in immunosuppressed patients was attributed to an oral source in 25-50% of cases (Oral surg Jrnl 1992). "Increased susceptibility to infection accompanies both the cancer disease and treatment. The mouth provides a pool of opportunistic infection which may lead to life-threatening systemic infection in individuals whose immune system is already compromised by disease and/or treatment" (Griffiths and Boyle 1992).     

Comparison of nested polymerase chain reaction, virus isolation, and fluorescent antibody testing for identifying feline herpesvirus in cats with conjunctivitis

OBJECTIVE: To compare nested polymerase chain reaction (PCR), virus isolation (VI), and fluorescent antibody (FA) testing to detect feline herpesvirus (FHV) in cats with naturally acquired conjunctivitis or respiratory tract disease, or both. SAMPLES: Swab and microbrush specimens from the conjunctiva and throat were taken from 46 cats, allotted to 3 groups (conjunctivitis only, respiratory tract disease and conjunctivitis, and clinically normal). PROCEDURE: Cells from microbrush specimens were digested and herpesvirus DNA was amplified, using a double round of PCR. Products were detected by use of agarose gel electrophoresis. The VI and FA tests were performed in routine manner. RESULTS: Of 16 cats with conjunctivitis only, conjunctival specimens from 8 and throat specimens from 8 were FHV positive by PCR. None had positive results of VI or FA testing. Of 15 cats with respiratory tract disease and conjunctivitis, conjunctival specimens from 13 and throat specimens from 12 were FHV positive by PCR. A conjunctival specimen from 1 cat and throat specimens from 3 cats were FHV positive by VI. A conjunctival specimen from 1 cat was FHV positive by FA testing. Of 15 clinically normal cats, conjunctival and throat specimens from 2 cats were FHV positive by PCR; neither conjunctival nor throat specimens from these cats were FHV positive by VI or FA testing. CONCLUSION: For cats with respiratory tract disease and conjunctivitis, or with conjunctivitis only, nested PCR was more sensitive at detecting FHV than was VI or FA testing. CLINICAL RELEVANCE: Nested PCR is a more sensitive test than the currently available VI and FA tests for identifying FHV in cats with conjunctivitis.     

Rectal mucosal proliferation, dietary factors, and the risk of colorectal adenomas

OBJECTIVE: To evaluate a commercial microtiter enzyme immunoassay for antinuclear antibodies (ANA) by comparing the results of tests performed with this assay to an established indirect immunofluorescence method performed on human epitheliod cell substrate slides. DESIGN: Both analytical methods were used to test for the presence and levels of ANA in stored sera from 313 patients previously shown to have detectable ANA and from 102 healthy control subjects. Follow-up tests for specific autoantibodies (anti-dsDNA antibodies and antibodies to extractable nuclear antigens [ENA]) were performed on all sera from patients. The medical histories of all patients were reviewed to determine the presence of systemic rheumatic diseases (SRDs). Different cut-off levels of positivity were examined to determine the sensitivity and predictive values of positive results on the enzyme immunoassay for detecting patients with SRDs or sera with positive tests for specific autoantibodies. RESULTS: Among patients with clinically diagnosed SRDs (n = 197), the enzyme immunoassay was positive for ANA (> or =1 U) in 100% and the indirect immunofluorescence method was positive (titer > or =40) in 95.4% of cases. Among ANA-positive patients with no SRDs (n = 116), testing by enzyme immunoassay and indirect immunofluorescence yielded positive results in 97.6% and 75.6% of cases, respectively. Among healthy control subjects, each of the two methods was positive in 15% of cases. As expected, most patients with SRDs had higher levels of ANA than did ANA-positive patients with other clinical diagnoses. A cut-off level of > or =3 U on the enzyme immunoassay correctly classified 77% of patients with a SRD as "positive" and 88% of patients with other clinical diagnoses as "negative." The probability of detecting a positive result for specific autoantibodies on second-order testing increased directly with the level of ANA. A cut-off level of > or =3 U had a sensitivity of 92% for identifying sera with positive specific autoantibodies, and results > or =3 U had a predictive value of 52% for a positive second-order test result. CONCLUSION: Enzyme immunoassay is substantially equivalent to indirect immunofluorescence for detecting clinically important ANA. Cut-off levels for positive results on the enzyme immunoassay can be established that optimize the usefulness of this method in diagnostic algorithms for specific autoantibodies.     

On the meaning of "sensitivity"

The term "sensitivity" (as applied to an analytical method's performance) has again become a subject of controversy. Certain authorities (e.g., IUPAC) define a system's sensitivity as the response curve slope (or response/dose), others (e.g., IFCC) in terms of the detection limit. Many investigators have failed to perceive the contradiction between these concepts, wrongly assuming that maximizing "sensitivity" in the first sense maximizes it in the second (i.e., that they are inversely related). The existence of different meanings for this term (when used in the present context) is a source of confusion that has, among other things, led to erroneous ideas relating to immunoassay design. Such confusion should be terminated by adoption of one or the other of the definitions. However, the definitions are not of equal merit. We advance arguments against retention of the "slope" definition, which conflicts with the word's common meaning and is meaningless as an indicator of the performance of a measuring system.     

Improving patient compliance--a multicentre evaluation of the 'DDS tile test'

The feasibility and utility of the "DDS tile test" under field conditions was assessed in 112 leprosy centres in Maharashtra. About 10% of the 2952 urine samples tested negative for dapsone. Feed back information from 54 centres one year later showed that the test could be performed easily under field conditions and also that counselling of patients showing poor compliance helped to improve drug compliance in over 80% of cases.     

Purification of larval Taenia solium antigens by isoelectric focusing

By preparative isoelectric focusing in a rotating ampholine column, crude cystic membrane (M) or fluid (F) antigens of larval Taenia solium were each separated into 20 fractions. M fractions were less specific and sensitive than F fractions in detecting cysticercosis antibodies in pig serum. Among the F fractions, F15 showed the best potential to serve as a screening antigen. It contained 18 polypeptides, with pI 5.3-8.2 and a specific epitope of 25 kDa which was detected by immunoblotting. Although F15 showed slight cross-reactions with heterologous antisera in double-antibody IgG enzyme-linked immunosorbent assays (ELISA), it yielded the highest absorbance values when tested against homologous antisera. The antigen was used to screen sera samples from 4870 pigs slaughtered in Hong Kong and five other Chinese cities for cysticercosis antibodies by double-antibody ELISA, Falcon Assay Screening Test (FAST)-ELISA and enhanced chemiluminescent immunoassay. The results varied significantly between assays. However, the samples collected from Shenzhen yielded the highest positive rates. Enhanced chemiluminescent immunoassay based on camera-luminometry was found suitable for use under field conditions.     

Meningeal carcinomatosis in patient with esophagus adenocarcinoma

We assessed the practicality of using the Short Michigan Alcoholism Screening Test (SMAST) and the Alcohol Use Disorders Identification Test (AUDIT) in screening adolescents for alcoholism in a primary care setting. In addition, we sought to determine the prevalence of alcohol use among adolescents, 16-21 years of age, presenting to a private Family Medicine practice for medical care. A consecutive sample of 67 subjects presenting for medical care were asked to complete the SMAST and AUDIT questionnaires. Overall, 52 (78%) of the questionnaires were returned with complete data. Of the 52 patients, 25 (48%) admitted to drinking. Using a "positive" score on either the SMAST or AUDIT as a positive test for alcohol use yielded a sensitivity of 40% and a predictive value positive of 100%. Using a "negative" score on both the SMAST and AUDIT as a negative test for alcohol use yielded a specificity of 100% and a predictive value negative of 64%. Although alcohol use was relatively common considering the age group, using the SMAST and AUDIT to screen for alcoholism is labor intensive and is not practical in this situation. Because patients appeared to misinterpret some questions and were often accompanied to the office by their parents, their answers may not be valid. History of alcoholism taken upon typical office examination and relevant advice appears to be a better alternative to the use of questionnaires in determining the prevalence of alcohol use in this age group.     

Experience with the two-windows method for mediastinal lymph node dissection in lung cancer

A rapid dipstick test for scrub typhus was prospectively evaluated in Chiangrai, northern Thailand. Sera from 162 patients with fever of unclear etiology were tested by a dot blot immunoassay using two different antigen concentrations. Dipsticks coated with lower concentration of antigen lacked sensitivity compared with the indirect immunoperoxidase test. Dipsticks with higher antigen concentration had increased sensitivity that was equivalent to that of the immunoperoxidase test. By increasing the antigen concentration on the dipstick, sensitivity increased from 67% to 100%, positive predictive value increased from 90% to 93%, and negative predictive value rose from 92% to 100%. The specificity of both antigen concentrations was 98%. This study establishes that scrub typhus can be confirmed serologically by use of a dipstick assay and that serodiagnosis can be effectively tailored to a target population.     

Isolation of a novel human canalicular multispecific organic anion transporter, cMOAT2/MRP3, and its expression in cisplatin-resistant cancer cells with decreased ATP-dependent drug transport

Since the 1950s the U.S. military has used intramuscular injections of benzathine penicillin G (BPG) to control outbreaks of respiratory disease. In an effort to find an alternative prophylaxis, a randomized field trial was conducted among 1,016 male U.S. Marine trainee volunteers at high risk for respiratory disease. Participants were evaluated for evidence of acute respiratory infection by serological tests on pretraining and posttraining sera (63 days apart). Oral azithromycin prophylaxis (500 mg/w) outperformed BPG, preventing infection from Streptococcus pyogenes (Efficacy [E] = 84%; 95% confidence interval [CI], 63%-93%), Streptococcus pneumoniae (E = 80%; 95% CI, 50%-92%), Mycoplasma pneumoniae (E = 64%; 95% CI, 25%-83%), and Chlamydia pneumoniae (E = 58%; 95% CI, 15%-79%) in comparison with results in a no-treatment group. Azithromycin group subjects reported few side effects and less respiratory symptoms than the BPG and no-treatment groups. According to serological tests, oral azithromycin is an effective alternative prophylaxis to BPG for military populations.     

Visualization of a slow, ATP-induced structural transition in the bacterial molecular chaperone DnaK

Recent reports have shown that the binding of ATP to a 70-kDa molecular chaperone induces a rapid global conformational transition from a "high affinity" state to a "low affinity" state, where these states are defined by tight and weak binding to (poly)peptides, respectively. To complete the activity cycle, a chaperone molecule must ultimately return to the high affinity state. In this report, this return to the high affinity state was studied using a chemical cross-linking assay in conjunction with SDS-polyacrylamide gel electrophoresis. The basis for this assay is that in the absence of nucleotide or in the presence of ADP, conditions that stabilize the high affinity state, cross-linking of the Escherichia coli molecular chaperone DnaK yielded two monomeric forms, with apparent molecular masses of 70 kDa (77%) and 90 kDa (23%), whereas cross-linking yielded only the 70-kDa monomeric form in the presence of ATP. This ATP-dependent difference in cross-linking was used to follow the kinetics of the low affinity to high affinity transition under single turnover conditions. The rate of this transition (kobs = 3.4 (+/-0.6) x 10(-4) s-1 at 25 degrees C) is almost identical to the reported rate of ATP hydrolysis (khy = 2.7 (+/-0.7) x 10(-4) s-1 at 22 degrees C). These results are consistent with a two-step sequential reaction where rate-limiting ATP hydrolysis precedes the conformational change. Models for the formation of two cross-linked DnaK monomers in the absence of ATP are discussed.