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PubMed1992

[Microalbuminuria and pregnancy. Is microalbuminuria predictive of pregnancy toxemia?].

Several authors have suggested that estimating the levels of microalbuminuria will help in early screening for pre-eclampsia. The purpose of this work has been to look for the absence of microalbuminuria in normal pregnancies and to work out its predictive value for the risk of toxaemia of pregnancy when it does appear. The study was carried out on 257 women of whom 43 were controls and 214 women who were pregnant and had neither diabetes nor hypertension and had no kidney infections. The samples of urine were gathered in a 12 hour period of night and those that gave a positive reaction for albumin were rejected. RIA techniques were used to work out the levels of albuminuria and these were confirmed by immunoassay. We have compared microalbuminuria, the relationship between urine albumin and creatinine and the clearance of albumin in relationship to albuminuria (as defined by the relationship of albumin and creatinine clearance). We have calculated the sensitivity and the specificity and the prognostic value both positive and negative for these four parameters. Our results show that in a normal pregnancy there should not be any microalbuminuria, and on the other hand that if microalbuminuria does appear according to the four parameters studied, they are all equally sensitive for predicting pre-eclampsia. The relative clearance of albumin from the urine seems to be the most interesting parameter as far as we are concerned, and it could lead to early screening for toxaemia.

Adolescent↗
PubMed2003

The Cobas Amplicor MTB test for detection of Mycobacterium tuberculosis complex from respiratory and non-respiratory clinical specimens.

The Cobas Amplicor MTB test is a polymerase chain reaction (PCR) technique commonly used for direct detection of Mycobacterium tuberculosis in clinical samples. This assay is only validated for respiratory specimens, but many physicians also request PCR analyses for non-respiratory ones. 877 respiratory and 564 non-respiratory specimens were analysed by this test. Using culture results as standard, the sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of the PCR were, respectively, 97.9%, 100%, 100% and 94.4% for smear-positive respiratory specimens, 68.8%, 99.2%, 87.5% and 97.5% for smear-negative respiratory samples, 57.8%, 98.6%, 78.8% and 96.4% for all non-respiratory specimens, and 42.4%, 98.6%, 66.7% and 96.4% for smear-negative non-respiratory specimens. 154 cerebrospinal fluid samples were analysed and the sensitivity, specificity, PPV and NPV were 55.6%, 97.2%, 55.6% and 97.2%, respectively. These results indicate that the Cobas Amplicor MTB test enables detection of tuberculosis in respiratory specimens, but does not perform well enough in non-respiratory specimens. The method fails particularly in cases where a reliable and rapid test is urgently needed, e.g. in tuberculous meningitis.

Bacteriological Techniques↗
PubMed2000

Reflections on the use of controls in immunohistochemistry and proposal for application of a multitissue spring-roll control block.

The conventional positive control in immunohistochemistry consists of a separately immunostained slide containing tissue known to show positive reaction with the antibody. Although it can indicate that the reagents are sound, it cannot guarantee that the appropriate reagents have indeed been dispensed in the correct order on the test cases. Furthermore, positive controls are costly because they often account for 20% to 40% of all immunostained slides in a diagnostic surgical pathology laboratory. We describe a simple method to produce a multitissue spring-roll control block, comprising mostly normal tissues. The multitissue controls are mounted on the same slides of the test cases, and thus undergo the identical immunostaining procedures as the test tissues. Therefore they can provide the best assurance that the immunostains are working properly on the individual slides, the expected sensitivity is achieved, and the specificity is as expected. The wisdom of the conventional negative control is also questioned. When two or more immunostains are performed on a case, they should be sufficient to indicate the presence or absence of nonspecific staining.

False Negative Reactions↗
PubMed1987

Assessment of the Albuscreen microalbuminuria kit in diabetic outpatients.

Diabetic patients who have albumin excretion rates of greater than 30 micrograms/min (30 mg/L at normal urine volumes) are at increased risk of the development of diabetic nephropathy. The Albuscreen microalbuminuria kit detects albuminuria at concentrations of 30 mg/L and above by an agglutination-inhibition reaction. One hundred and ninety-five random urine samples from diabetic outpatients were assessed by Albuscreen and Albustix testing for albuminuria and the results were correlated with those of a sensitive radioimmunoassay technique. Albuscreen testing was simple, easy to use and had a sensitivity of 96%, with a specificity of 88%. Albustix testing at a detection level of 50 mg/L revealed a sensitivity of 100% and a specificity of 68% (43 samples, false-positive "trace" readings), while, at 30 mg/L, the sensitivity and specificity were 90% and 71%, respectively. Therefore, Albuscreen testing is well suited as a screening test for the presence of microalbuminuria in a diabetic outpatient setting. However, the role of Albustix in screening for microalbuminuria is less well defined, especially at the 30 mg/L level of detection, and requires further investigation.

Albuminuria↗
PubMed2005

Tuberculin skin test: estimation of positive and negative predictive values from routine data.

OBJECTIVE: To estimate, using routinely available data, the predictive values of tuberculin testing with 2TU RT23 for detection of latent tuberculosis infection (LTBI) in the Dutch population as a basis for recommendations on cut-off values at various levels of infection prevalence. DESIGN: Smoothed distributions of TST reactions among 312 tuberculosis patients and 2848 healthy non-BCG-vaccinated persons were used to estimate the sensitivity and specificity at various cut-off values. RESULTS: Sensitivity was 98.9% at 5 mm, 95.4% at 10 mm and 79.8% at 15 mm cut-off. Specificity with the corresponding cut-offs was 95.3%, 96.3% and 97.1% before and 98.0%, 98.8% and 99.6% after adjustment for presumed LTBI. At 10 mm, the positive predictive value (PPV) was > 75% if the infection prevalence in the tested population was at least 10%, but strongly declined with lower prevalences. For lower prevalences a cut-off of 15 mm was proposed, as this results in a higher PPV without greatly affecting the negative predictive value. CONCLUSION: Estimation of the predictive values of the TST from routine data can be useful for establishing cut-off values for detection of LTBI in different populations with different tuberculin preparations.

Adult↗
PubMed1987

Delayed hypersensitivity skin testing in nutritional assessment.

Skin testing is useful in nutritional assessment because decreased immunity is associated with malnutrition. Absence of positive reactions indicates anergy, reaction to one test shows some decreased immunity, and response to two or more tests is considered normal. Variation in number and type of test antigens could lead to contradictory results. Also, conventional intradermal injections vary in antigen strengths, injection techniques, and subjective evaluations, and are also uncomfortable. This study compared results from three, four, or seven antigens in relation to other nutritional indices. Fifty hospitalized men with cancer were assessed with a 23 item Protein Energy Malnutrition Scale and classified as well nourished (60%) or malnourished (40%). Number of tests did not differ in identifying the malnourished; however, the smaller battery misclassified the normal as malnourished twice as often as the seven tests. Misclassification of normal as abnormal could lead to unnecessary interventions, such as total parenteral nutrition, that increase complications and cost of care dramatically.

False Negative Reactions↗
PubMed1999

Detection of minimal residual disease in acute leukemia by flow cytometry.

Patients with acute leukemia in clinical remission may still have up to 10(10) residual malignant cells (the upper limit of detection by standard morphologic techniques). Sensitive techniques to detect minimal residual disease (MRD) may allow better estimates of the leukemia burden and help the selection of appropriate therapeutic strategies. Flow cytometry and polymerase chain reaction have emerged as the most promising methods for detecting submicrospopic levels of leukemia. Flowcytometric detection of MRD is based on the identification of immunophenotypic combinations expressed on leukemic cells but not on normal hematopoietic cells. It affords the detection of one leukemic cell among 10,000 normal bone marrow cells, and can be currently applied to at least two thirds of all patients with acute leukemia. Prospective studies in large series of patients have demonstrated a strong correlation between MRD levels during clinical remission and treatment outcome. Therefore, MRD assays can be reliably used to assess early response to treatment and predict relapse. In this review, we discuss methodologic aspects and clinical results of flowcytometric detection of MRD in patients with acute leukemia.

Adult↗
PubMed1978

Should we screen all newborns for cystic fibrosis?

To assess the value of detecting albumin in meconium as a screening procedure for cystic fibrosis [CF] 68,000 meconium samples were examined by BM Meconium Test, single radial immunodiffusion and benzidine reaction. The specificity and sensitivity of this combination of tests were 99.67% and 78.57% respectively. The prevalence of CF at birth was confirmed as 1:3600 in this country. This low prevalence resulted in a relatively high number of false positives. Therefore, a positive test result has a low predictive value [3.39%] and this is a serious drawback of the method. The experiences and opinions of 37 local paediatricians about the screening programme were evaluated by a simple questionnaire. Gold's decision rule was applied. The least relative cost of misclassification justifying a mass-screening programme was 3 times higher than the actual relative cost as suggested by the aggregate opinion of paediatricians in the region. These results support the view that with the methods used screening may have more disadvantages than not screening.

Albumins↗
PubMed2003

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.

Animals↗
PubMed1990

Evaluation of C. reactive protein values in neonatal sepsis.

C. reactive protein (CRP) estimations were performed prospectively on 30 consecutive admissions of very low birth weight infants to a Regional neonatal intensive care unit. The samples were analysed by a recently described, rapid intralipid agglutination assay and by a reference turbidimetric technique. Two hundred and ninety samples were assayed by both techniques. The intralipid agglutination was positive on two occasions when the reference method found normal levels. No false negative reactions occurred. Bacterial micro-organisms were isolated on 32 occasions but 19 of the organisms were considered to represent bacterial colonisation or contamination. The CRP remained negative in 17 cases. There were 13 episodes of clinical deterioration associated with positive bacterial cultures. In each of the six infants with severe systemic infections (septicaemia (4), meningitis (1), and osteomyelitis (1)), the levels were raised. In five of these infants the CRP was elevated before, or at the time of, the clinical deterioration. The CRP remained normal during seven (54%) of the culture positive events. We believe that the CRP estimations provide additional information in the evaluation of the infant with suspected sepsis. Serial measurements are helpful in distinguishing bacterial contamination from invasive infection but are not helpful in predicting infection during the pre-clinical phase. The intralipid agglutination technique is a rapid and reliable test and could be performed on the neonatal unit outside normal laboratory hours.

Acute-Phase Reaction↗
PubMed2001

Significance of high-risk human papillomavirus detection by polymerase chain reaction in primary cervical cancer screening.

The purposes of this study were to evaluate the incidence of high-risk human papillomavirus (HPV) infection by polymerase chain reaction (PCR) and to assess its diagnostic usefulness in primary cervical screening. PCR testing for HPV type 16, 18, 31 and 33 was performed on 1305 specimens obtained during routine cervical cancer screening. We analysed the concurrent cervical smears and biopsy, and correlated them with the HPV infection status. We also evaluated histologically-proven cases with ASCUS smears according to HPV infection. HPV DNA was identified in eight (0.7%) of 1144 cytologically normal patients; nine (10.5%) of 86 ASCUS; seven (25.0%) of 28 LSIL; 26 (78.8%) of 33 HSIL; and in all of three squamous cell carcinomas (SCC). HPV positivity was significantly associated with cytohistological diagnosis for HSIL of more. In addition, HPV-positive ASCUS cases were found to be associated with histological abnormality rather than HPV-negative. The results indicate that high-risk HPV testing by PCR could be a useful adjunct tool for Pap smear in primary cervical screening. The combination of Pap smear and high-risk HPV testing by PCR might reduce unnecessary colposcopy-guided biopsy of women with cytological diagnosis of ASCUS.

Adult↗
PubMed1991

The polymerase chain reaction: a new tool for the detection of minimal residual disease in haematological malignancies.

The polymerase chain reaction (PCR) is a novel technique for the in vitro amplification of specific short DNA fragments, which permits a selective and up to 10(7) fold enrichment of the target sequence. The method is increasingly being used for the molecular genetic analysis of hereditary, infectious and neoplastic disorders. The use of PCR for the detection of minimal residual disease in particular types of leukaemia or lymphoma, such as chronic myelogenous leukaemia expressing specific BCR/ABL-RNA and follicular non-Hodgkin lymphoma with the chromosomal translocation t(14;18) are reviewed. In acute lymphoblastic leukaemia clone-specific sequences from rearranged antigen receptor genes may be molecular markers suitable for amplification. Although PCR holds great promise for "molecular" staging and follow-up, several technical problems have to be kept in mind, and the clinical relevance of PCR-based evidence of minimal residual disease in haematological malignancies requires further investigation.

False Negative Reactions↗
PubMed2003

Molecular analysis of cerebrospinal fluid in viral diseases of the central nervous system.

The use of nucleic acid (NA) amplification techniques has transformed the diagnosis of viral infections of the central nervous system (CNS). Because of their enhanced sensitivity, these methods enable detection of even low amounts of viral genomes in cerebrospinal fluid. Following more than 10 years of experience, the polymerase chain reaction or other NA-based amplification techniques are nowadays performed in most diagnostic laboratories and have become the test of choice for the diagnosis of several viral CNS infections, such as herpes encephalitis, enterovirus meningitis and other viral infections occurring in human immunodeficiency virus-infected persons. Furthermore, they have been useful to establish a viral etiology in neurological syndromes of dubious origin and to recognise unusual or poorly characterised CNS diseases. Quantitative methods have provided a valuable additional tool for clinical management of these diseases, whereas post-amplification techniques have enabled precise genome characterisation. Current efforts are aiming at further improvement of the diagnostic efficiency of molecular techniques, their speed and standardisation, and to reduce the costs. The most relevant NA amplification strategies and clinical applications of to date will be the object of this review.

AIDS-Related Opportunistic Infections↗
PubMed2000

The role of DNA amplification technology in the diagnosis of infectious diseases.

Nucleic acid amplification and detection methods developed in the past decade are useful for the diagnosis and management of a variety of infectious diseases. The most widely used of these methods is the polymerase chain reaction (PCR). PCR assays can detect rapidly and accurately the presence of fastidious and slow-growing microorganisms, such as Chlamydia, mycoplasmas, mycobacteria, herpesviruses and enteroviruses, directly from clinical specimens. Commercial PCR assays for the diagnosis of tuberculosis and genital C. trachomatis infection are now routinely used in many diagnostic laboratories. Assays have also been developed that can detect antimicrobial resistance and are used to identify the cause of infection by organisms that cannot be cultivated. The value of viral load measurement by nucleic acid amplification in the management of patients with HIV infection or hepatitis C has also been well established. However, evaluations of this technology for rapid microbial diagnosis have generally been limited by small samples, and the cost of these assays may be as high as Can$125 per test. As nucleic acid amplification methods continue to evolve, their role in the diagnosis and management of patients with infectious diseases and their impact on clinical outcomes will become better defined.

Communicable Diseases↗
PubMed1995

Use of polymerase chain reaction for improved diagnosis of tuberculosis in children.

OBJECTIVE: To study the value of a rapid diagnostic method based on the amplification by polymerase chain reaction (PCR) of a fragment of the IS6110 insertion element for the detection of Mycobacterium tuberculosis in children. DESIGN: We tested 199 specimens obtained from 68 children referred for evaluation of suspected tuberculosis. RESULTS: In 83.3% of children with active disease and 38.9% with tuberculous infection but no evidence of disease, at least one positive PCR result was observed. No child without tuberculosis had positive PCR results (100% specificity). The sensitivity of the PCR was increased by testing of multiple samples from the same child and use of Chelex particles (Bio-Rad Laboratories, Ivry, France) rather than guanidine isothiocyanate-silica particles for DNA extraction. Bronchoalveolar lavage samples were no more useful than gastric aspirates. CONCLUSIONS: If appropriate laboratory methods are used, DNA amplification is a reliable method for the early diagnosis of tuberculosis in children and appears to be very helpful in clinical pediatric practice when the diagnosis of active tuberculosis is difficult or needs to be rapidly confirmed.

Adolescent↗
PubMedDate not supplied

AMPLICOR MTB polymerase chain reaction test for identification of Mycobacterium tuberculosis in positive Difco ESP II broth cultures.

The reliability of the Roche AMPLICOR polymerase chain reaction (PCR) Mycobacterium tuberculosis (MTB) test for identification of M. tuberculosis complex (MTBC) in Difco ESP II broth cultures was evaluated by testing aliquots from all cultures, regardless of specimen type, that were signaled positive by the ESP instrument. Polymerase chain reaction results were compared to those obtained by usual laboratory protocol, whereby MTBC was identified by DNA probe (Gen Probe, Inc., San Diego, California, USA), testing sediment from broth samples or colonies on a solid medium. Of the 242 signal-positive ESP II cultures (from 182 patients) evaluated, 98 (40.5%) contained mycobacteria, including 26 MTBC. On initial testing, 27 samples were positive by PCR; 22 of these were MTBC culture positive, and 5 were culture negative for mycobacteria. The sensitivity, specificity, and positive and negative predictive values of PCR were 84.6, 97.7, 81.5, and 98.1%, respectively. Based on review of the medical records of the patients for whom PCR and culture results were discrepant, one PCR-positive culture negative was a true PCR positive; thus the actual sensitivity, specificity, and positive and negative predictive values of PCR were 85.2, 98.1, 85.2, and 98.1%. Two of the PCR-negative, MTBC culture-positive samples were blood specimens collected in an Isolator tube; and these tubes contain sodium polyanetholsulfonate, which seems to inhibit PCR. If blood specimens are excluded from analysis, the sensitivity, specificity, and positive and negative predictive values for PCR are 92.0, 97.6, 85.2, and 98.8%, respectively. The mean times from specimen receipt to identification of MTBC were 19 (+/- 2) days (range, 6 to 39 days) for ESP II plus AMPLICOR MTB PCR and 25 (+/- 2) days (range, 8 to 42 days) for DNA probe testing of sediment from broth or colonies on solid media (p < .05). These data indicate that the AMPLICOR MTB PCR test is a rapid, reliable method for identification of MTBC in positive ESP II cultures, excluding blood specimens collected in Isolator tubes.

Bacteriological Techniques↗
PubMed1987

Immunofluorescence tests for HIV antibody and their value as confirmatory tests.

The enzyme-linked immunosorbent assay (ELISA) is currently being used as a sensitive screening test for HIV antibody. The immunoblot assay (IBA) and indirect immunofluorescence (IF) techniques are two recommended confirmation tests for EIA-positive sera. An indirect IF test has been developed by various laboratories using acetone fixed mixtures of uninfected and HIV-infected cells, which facilitated the reading, since nonspecific reactions were easily differentiated from specific staining. Similar results have been obtained with H9-, CEM-, and HUT78-HIV-infected and uninfected cells. Anti-nuclear antibodies and auto-antibodies resulting in false-positive EIA results, could easily be differentiated by the IF test. Aspecific fluorescence can be removed by absorption of the specimens with non-infected cells. However, IF is not suitable for the screening of large series of specimens. IF is especially well suited for quantitative analysis of serum antibody levels. Whereas serum antibody titers rise initially after infection, they decrease as AIDS develops. Heat inactivation of sera did not affect reactivity in IF, in contrast to a high rate of false-positive results obtained with heat inactivated sera in some ELISAs. A well characterized serum from an AIDS patient can be used to perform IF in order to monitor HIV infection of susceptible cells. It has been claimed that titers of neutralizing antibodies significantly correlate with the levels of IF anti-HIV antibodies. An overall correlation of 99% between IF and IBA was reported by different laboratories, when HIV ELISA-reactive European and North American sera were tested. The concordance with IBA was 97% when HIV ELISA-reactive African sera were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Viral↗
PubMed1997

Quality control for DNA contamination in laboratories using PCR-based class II HLA typing methods.

Quality control (QC) in laboratories performing molecular histocompatibility class II typing often includes a polymerase chain reaction (PCR) approach for monitoring DNA contamination. An oligonucleotide primer set was designed, (RBQBf/RBQBr), which is specific for nonpolymorphic regions of the DR-B, DQ-B, and DP-B consensus sequences with an expected PCR product size of 81 bp. RBQBf/RBQBr detected genomic DNA from reference cell lines LWAGS and BM21 (50 to 100 picograms) as well as DR-B, DP-B, and DQ-B amplicon (1 copy). Additionally, RBQBf/RBQBr detected SSP products from routine DR-B and DQ-B typings. Validation studies employing controlled DNA contamination of laboratory surfaces revealed that increasing amounts of wipe test samples (5% to 20% v/v) were inhibitory to the wipe test PCR, whereas lower amounts (1% to 2%), or alternatively, a diluted wipe test sample, increased the sensitivity of the test and optimized the results. Collectively, this study describes a primer set, RBQBf/RBQBr, which detects both genomic DNA and DR-B, DQ-B, or DP-B amplicon and furthermore illustrates the necessity of routine testing for potential inhibitory factors that may be introduced into the wipe test PCR.

Base Sequence↗