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Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology.

A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures. The assay uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5' terminus and biotin at the 3' terminus. The CPT reaction occurs at a constant temperature, which allows the probe to anneal to the target DNA. RNase H cuts the RNA portion of the probe, allowing the cleaved fragments to dissociate from the target DNA, making the target available for further cycling. The strip detection step uses a nitrocellulose membrane with streptavidin and immunoglobulin G antibody impregnated on the surface. In the absence of the mecA gene, the uncut probe is bound to an antifluorescein-gold conjugate and is then captured by the streptavidin to form a test line. In the presence of the mecA gene, the probe is cut and no test line is formed on the strip. A screen of 324 S. aureus clinical isolates by the CPT-strip assay showed a 99.4% sensitivity and a 100% specificity compared to the results of PCR for the detection of the mecA gene. Specificity testing showed that the CPT-strip assay did not exhibit any cross-reactivity with a panel of mecA-negative non-S. aureus isolates. The CPT-strip assay is simple and does not require sophisticated equipment. Furthermore, the assay takes 1.5 h starting from a primary culture to the time to detection of the mecA gene in S. aureus isolates.

Bacterial Proteins↗

Breakpoints for predicting Pseudomonas aeruginosa susceptibility to inhaled tobramycin in cystic fibrosis patients: use of high-range Etest strips.

Inhaled administration of tobramycin assures high concentrations in cystic fibrotic lungs, improving the therapeutic ratio over that of parenteral tobramycin levels, particularly against Pseudomonas aeruginosa. Conventional Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) breakpoints only consider parenteral levels and do not take into account these high antimicrobial concentrations. The Spanish Antibiogram Committee (The MENSURA Group) has tentatively defined specific breakpoint values for inhaled tobramycin when testing P. aeruginosa isolates from cystic fibrosis (CF) patients (susceptible, < or =64 microg/ml; resistant, > or =128 microg/ml). The antimicrobial susceptibilities of 206 prospectively collected CF P. aeruginosa isolates were determined by the reference agar dilution method. For tobramycin, the performance of high range tobramycin Etest strips (AB Biodisk, Solna, Sweden) and conventional tobramycin disks were assessed with the same collection. Applying MENSURA proposed breakpoints, 95.1% of the strains were categorized as susceptible to tobramycin, either using agar dilution or Etest high-range strips (99% categorical agreement between both methods). With CLSI breakpoints, susceptibility rates decreased to 79.1 and 81.1% for agar dilution and Etest strips, respectively (83.5% categorical agreement). Minor, major, and very major errors for Etest strips (CLSI criteria) were 13.6, 1.2, and 14.8%, respectively. Upon applying the new proposed criteria for inhaled tobramycin, only one major and one very major error were observed with Etest strips. Whenever inhaled tobramycin is considered for therapy, we suggest that P. aeruginosa strains from CF patients categorized as intermediate or resistant to tobramycin according to the CLSI criteria should be retested with high-range Etest strips and recategorized using MENSURA interpretive criteria. CLSI breakpoints should still be followed when intravenous tobramycin is used in CF patients, particularly during the course of exacerbations.

Anti-Bacterial Agents↗

Evaluation of rK39 strip test for the diagnosis of visceral leishmaniasis in infants.

This study estimated the sensitivity and specificity of the rK39 strip test compared with the immunofluorescent antibody test and microscopy of bone marrow aspirate smears (the gold standard) in 47 children with suspected visceral leishmaniasis. A control group of children with other diagnoses (tuberculosis, toxoplasmosis, systemic lupus erythematosus, malaria or cutaneous leishmaniasis) were also tested to check false positive results. The sensitivity and specificity of the strip test were 82.4% and 100% and that of immunofluorescent antibody were 100% and 92.7%. The rK39 strip test is reliable where there is no access to laboratory facilities.

Animals↗

Personal blood glucose testing in children.

The value of blood glucose testing over urine sugar testing is emphasized, and techniques of self-monitoring of blood glucose in children are presented. Test strips and reflectance meters currently on the market are described and evaluated. Useful suggestions are made for introducing blood glucose testing to children, stressing flexibility in the frequency of testing, at least initially, and proper education in the interpretation of testing results.

Adolescent↗

Strip ELISA for the detection of IgG antibodies to Toxoplasma gondii.

An enzyme linked immunosorbent assay (ELISA) has been developed using a polycarbonate coated strip and penicillinase for the detection of IgG antibodies to T. gondii. Of 40 serum samples from patients clinically suspected to have toxoplasma infection, 24 were positive for IgG antibodies to T. gondii. Strip ELISA showed 95.83 per cent sensitivity and 89.4 per cent specificity. This method is simple (end result assessed visually), sensitive (detection of antitoxoplasma IgG antibodies to a level of 15 IU/ml) and reproducible (5.9 and 11.6 intra and inter percentage of coefficient of variation respectively).

Adult↗

[Clinical applicability of an enzyme micromethod for the rapid determination of blood sugar].

The clinical applicability of an enzymatic micromethod for the fast determination of glycaemia is discussed. The method is based on the use of an optical reflectometer for the quantitative reading of the variation in colour intensity of specific reactive strips for the semi-quantitative evaluation of blood glucose. Thanks to the method's rapidity and simplicity, it can be used for mass screening and is also very useful for the routine investigations of Diabetologic Centres. It is also invaluable for the identification of emergency clinical situations.

Blood Glucose↗

Rapid accurate field diagnosis of Indian visceral leishmaniasis.

BACKGROUND: A firm diagnosis of visceral leishmaniasis (kala-azar) requires demonstration of the parasite in organ aspirates or tissue biopsy samples. The aim of this prospective study was to assess the diagnostic usefulness of non-invasive testing for antibody to the leishmanial antigen K39 by means of antigen-impregnated nitrocellulose paper strips adapted for use under field conditions. METHODS: One drop of peripheral blood is applied to the hitrocellulose strip. Three drops of test buffer (phosphate-buffered saline plus bovine serum albumin) are added to the dried blood. The development of two visible bands indicates presence of IgG anti-K39. 323 consecutive patients with suspected kala-azar referred to two specialist units in India, and 25 healthy controls, provided fingerstick blood samples for the test. Spleen aspirates were taken from 250 patients. FINDINGS: Kala-azar was confirmed by microscopy of spleen-aspirate smears in 127 patients. The K39 strip test was positive in all 127; the estimated sensitivity was therefore 100% (95% CI 98-100). Four patients had positive strip tests but negative aspirate smears; all four responded to treatment for leishmaniasis. 217 individuals, including the 25 healthy controls, 73 patients with malaria or tuberculosis, and 119 spleen-aspirate-negative patients who had presumed malaria or cirrhosis (79) or no final diagnosis (40), had negative strip-test results. None of the 119 aspirate-negative patients developed evidence of kala-azar during 3-6 months of follow-up. The estimated specificity of the strip test was 98% (95-100; 217/221). INTERPRETATION: Detection of anti-K39 by immunochromatographic strip testing is a rapid and non-invasive method of diagnosing kala-azar, which has good sensitivity and specificity and is well suited for use in field conditions.

Adolescent↗

Development of paper-strip test for 3-hydroxybutyrate and its clinical application.

A rapid paper-strip test for the semiquantitating 3-hydroxybutyrate (3-OHBA) has been developed. The color develops within 5 min after applying the serum to the paper strip, and the purple color was read either visually or by reflectance meter. This can detect 3-OHBA levels as low as 0.1 mmol/L up to 2.0 mmol/L. The more concentrated sample can be measured on serial dilution. Clinical usefulness has been tested in a summer camp for insulin-dependent diabetic children as well as in a routine diabetes clinic. Serum 3-OHBA levels ranged from greater than 100 mumol/L to 4 mmol/L in all the subjects before breakfast in a summer camp. In four subjects, 3-OHBA was elevated to the level of 2-4 mmol/L, and only one of these four subjects exhibited ketonuria by nitroprusside test. In a diabetes clinic, a new paper-strip test for 3-OHBA has revealed ketonemia in 34 (74%) of 46 diabetic subjects, while nitro-prusside test revealed ketonemia in only 4 (13%). The present paper-strip test for 3-OHBA is sensitive enough to detect levels as low as 0.1 mmol/L and is clinically useful for rapid detection of ketosis proneness as well as for monitoring of diabetes control.

3-Hydroxybutyric Acid↗

Unusual behaviour of Klebsiella rhinoscleromatis strains on API 20E strips.

Seven Klebsiella rhinoscleromatis standard cultures and two wild isolates were examined for their responses to Api 20E (API System, S.A.) strips. Several strains yield incostant results for arabinose fermentation test in Api strips and, when positive, they were not identified. The arabinose positive test indeed, led to the numerical profiles 0004773 (not mentioned in the analytical catalogue), or 0004553 (two strains) corresponding to a Klebsiella ozaenae identification. The mathematical analysis of the biochemical results confirmed the identity of the strains as K. rhinoscleromatis.

Arabinose↗

Proteome resolution by two-dimensional gel electrophoresis varies with the commercial source of IPG strips.

By facilitating reproducible first dimension separations, commercial immobilized pH gradient (IPG) strips enable high throughput and high-resolution proteomic analyses using two-dimensional gel electrophoresis (2DE). Amersham, Biorad, Invitrogen, and Sigma all market linear pH 3-10 IPG strips. We have applied optimized 2DE protocols with both membrane and soluble brain protein extracts to critically evaluate all four products. Resolved protein spots were quantitatively evaluated after carrying out these protocols using IPG strips from the four companies. Biorad and Amersham IPG strips resolved a high number of membrane and soluble proteins, respectively. Furthermore, Amersham IPG strips eluted the largest amount of protein into the second dimension gels and had the most protein remaining in the strip after 2DE. Biorad and Amersham IPG strips maintained a consistent linear pH 3-10 gradient, whereas those from Invitrogen appeared nonlinear or "compressed" within the central pH region. The gradient range within Sigma IPG strips appeared to be slightly less than pH 3-10, due to one extended pH unit within the gradient. Overall, all four commercially available IPG strips have the ability to resolve both membrane and soluble brain proteomes. The difference is that Amersham and Biorad do so more consistently and with better spot resolution. It appears that the physical/chemical nature of commercially available IPG strips can vary considerably, leading to marked differences in subsequent protein resolution in 2DE. These differences likely reflect variations in the uptake of proteins into the strips, and differences in the focusing and elution of proteins from the first to the second dimension. These differences would appear, in part, to underlie some inter-lab variations in the effective resolution of proteomes.

Animals↗

Evaluation of a new urinary amylase test strip in the diagnosis of acute pancreatitis.

We have developed a novel rapid test strip for detecting pancreatic amylase in urine and prospectively evaluated its accuracy in screening for acute pancreatitis (AP). The test strip is based on the immunochromatography principle and uses two monoclonal antibodies specific for pancreatic amylase. Urine samples were collected from 500 consecutive patients with acute abdominal disease (52 with AP) and prospectively tested with the strip. The accuracy of the test strip was compared with that of two quantitative urine amylase determinations and a urinary dipstick test for amylase (Rapignost). Sensitivity of the test was 69% and specificity was 97% in differentiating patients with AP from those with acute abdominal extrapancreatic disease at admission. The negative predictive value was 0.986. The test showed moderate agreement both with an assay measuring total amylase activity and with another measuring pancreatic amylase immunoreactivity. At similar high specificity (97%), quantitative determination of total amylase activity (cut-off 3960 U/L) and pancreatic amylase (cut-off 2180 micrograms/L) showed lower sensitivity (54% and 41%) than the test strip (69%). The test is specific and rapid to perform, and it rules out AP with high probability. It could therefore be useful in an emergency setting without laboratory facilities in the differential diagnosis of acute abdominal pain.

Abdominal Pain↗

Rapid biotyping of Haemophilus influenzae and Haemophilus parainfluenzae with PathoTec strips and spot biochemical tests.

PathoTec strips and spot biochemical tests were evaluated for the ability to biotype Haemophilus influenzae and Haemophilus parainfluenzae. Indole, urease, and ornithine decarboxylase reactions were tested. The results of PathoTec strips compared favorably with those conventional methods; the percent agreements were as follows: indole, 100; urease, 99.5; and ornithine, 95.5. Spot tests were simple and rapid, and the results also compared favorably with those of conventional tests; the percent agreements were as follows: indole, 99; urease, 100; and ornithine, 96.

Bacteriological Techniques↗

Evaluation of a rapid test for HIV antibodies in saliva and blood.

OBJECTIVE: To test whole blood and saliva for HIV antibodies (anti-HIV) using a rapid test strip capillary flow immunoassay, and to correlate the test strip results with blood specimen results obtained from routine diagnostic anti-HIV assays. DESIGN: A prospective pilot study of selected HIV-positive and HIV-negative individuals, children and medico-legal cases from Gauteng, South Africa. METHODS: Whole blood specimens taken from every individual and medico-legal case (total study population 153) and saliva specimens taken from 76 selected cases were tested for anti-HIV using the respective Hema-Strip HIV-1/2, Sero-Strip HIV-1/2 and Saliva-Strip HIV-1/2 (Saliva Diagnostic Systems Inc.) rapid test strip methodology. All results were correlated with the currently recommended anti-HIV assays. RESULTS: The whole blood test strip results correlated 100% with the traditional diagnostic results. Only two saliva test strip results tested false-negative, both from marasmic and severely dehydrated babies, while the other results were in concordance. All test strip results on postmortem blood and saliva were fully concordant with the diagnostic assay results. CONCLUSION: The anti-HIV test strip methodology for whole blood and saliva specimens is rapid, reliable and easy to perform and interpret. Saliva specimens can be readily collected from any individual, and there is a reduction in hazard risk. Anti-HIV saliva testing using the test strip methodology is recommended for South Africa, particularly in high-risk situations such as the paediatric and forensic medicine settings. A large field study obtaining specimens from different regions in South Africa is advised.

Adult↗

Evaluation of a new molecular system for simultaneous identification of four Enterococcus species and their glycopeptide resistance genotypes.

We compared the performances of a new DNA-based strip assay and the VITEK 2 system for identification of 105 enterococcal strains and differentiation of their van gene-associated resistance levels. Both methods provided excellent results. The molecular assay showed advantages in time to result for identification of van-associated genes.

Anti-Bacterial Agents↗

A simple method for monitoring mutans streptococci in margins of restorations.

Plaque material collected from margins of restorations using a sharp toothpick was streaked across one and the same plastic strip from a commercially available test kit and inoculated in a liquid medium containing bacitracin. The reproducibility of the method was good. The method is simple, inexpensive and suitable for the chairside demonstration of the colonization of mutans streptococci in localized areas of the mouth, such as margins of restorations.

Adult↗

Identification of human semenogelin in membrane strip test as an alternative method for the detection of semen.

Semenogelin (Sg), a protein originating in the seminal vesicles and a substrate for prostate specific antigen (PSA or p30), is a useful marker for the identification of semen. And detection of Sg has been available commercially in a membrane test recently. PSA is commonly used to detect semen in forensic significant samples taken from sexual assault cases. The strip PSA test has been available commercially from various manufacturers for many years. In this study, we evaluated two immunochromatographic membrane tests, one for Sg and the other for PSA by analyzing human semen, other human bodily fluids/materials including urine, blood, saliva, sweat, breast milk, vaginal secretion and fecal materials, semen from various animals and forensic casework samples. The data demonstrate that both Sg and PSA strip tests provide rapid and sensitive method for identification of seminal plasma. These results show that the immunochromatographic method for Sg detection is useful for the identification of seminal plasma in forensic samples, an alternative to the method for PSA detection.

Animals↗

[Self-control of blood sugar with a new blood sugar test strip (author's transl)].

Blood sugar self-control was assessed in 20 in-patients using a new blood sugar test strip, Haemo-Glukotest 20--800. Comparative estimations of 445 strip tests with values obtained by the hexokinase method showed good correlations particularly in the low range below the renal threshold. Nearly 70% of measurements were assessed correctly in this range. Mean deviations of test strip results from the corresponding laboratory value were 0.61 to 0.89 mmol/l (11--16 mg/dl). Haemo-Glukotest 20--800 is thus well suited for blood glucose self-control particularly in the range below the renal threshold. In a survey most patients estimated the importance of blood sugar to be more than that of urinary sugar.

Activities of Daily Living↗