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PCR in laboratory diagnosis of human Borrelia burgdorferi infections.

The laboratory diagnosis of Lyme borreliosis, the most prevalent vector-borne disease in the United States and endemic in parts of Europe and Asia, is currently based on serology with known limitations. Direct demonstration of Borrelia burgdorferi by culture may require weeks, while enzyme-linked immunosorbent assays for antigen detection often lack sensitivity. The development of the PCR has offered a new dimension in the diagnosis. Capable of amplifying minute amounts of DNA into billions of copies in just a few hours, PCR facilitates the sensitive and specific detection of DNA or RNA of pathogenic organisms. This review is restricted to applications of PCR methods in the diagnosis of human B. burgdorferi infections. In the first section, methodological aspects, e.g., sample preparation, target selection, primers and PCR methods, and detection and control of inhibition and contamination, are highlighted. In the second part, emphasis is placed on diagnostic aspects, where PCR results in patients with dermatological, neurological, joint, and ocular manifestations of the disease are discussed. Here, special attention is given to monitoring treatment efficacy by PCR tests. Last, specific guidelines on how to interpret PCR results, together with the advantages and limitations of these new techniques, are presented.

Borrelia burgdorferi Group↗

[Laboratory diagnosis of infection caused by Borrelia burgdorferi].

The laboratory diagnosis of Lyme disease in Denmark is reviewed with recommendations for serological testing. In Denmark the laboratory testing is performed with an ELISA technique. Most laboratories use an assay based on purified flagella antigen. The two-tier approach with Western Blot as confirmatory testing is not recommended since the contribution to the diagnostic specificity is only marginal. Predictive values of Lyme serology are presented, based on the estimated prevalence of the different stages of Lyme disease in Denmark.

Antibodies, Bacterial↗

[Current methods for the laboratory diagnosis of campylobacteriosis].

An analytical review of recent publications of home and foreign authors on the problem of laboratory diagnosis of campylobacteriosis is presented. The commercial nutrient media, methods of creation of the microaerophilic conditions for cultivation of campylobacter are presented. The filtration method is preferable for isolation of these agents from the studied material highly contaminated by accompanying microflora. A special attention is paid to immunodiagnosis of campylobacteriosis: agglutination reaction, coagglutination reaction, passive hemagglutination reaction, immunoenzyme and radioimmune analyses. Seroepidemiological examination of the staff at one of meat-packing factories in the Republic carried out by the method of indirect immunoenzyme analysis has revealed high levels of anticampylobacteriosis antibodies in 17.9% of examinees. The promising trends in perfection of the methods for laboratory diagnosis of campylobacteriosis are outlined.

Antibodies, Bacterial↗

Laboratory diagnosis of hepatitis B virus infection by nucleic acid hybridization analyses and immunohistologic detection of gene products.

The laboratory diagnosis of hepatitis B virus (HBV) infection is based on two types of analyses: (i) Immunochemical detection in serum or liver of viral antigens or their respective antibodies; (ii) Demonstration of HBV DNA in serum or liver by nucleic acid hybridization techniques (dot and transfer blots, in situ hybridization). These hybridization analyses have proven to be more sensitive than immunochemical assays for the detection of viral markers and have revealed HBV DNA in serum and liver of patients without serologic evidence of HBV infection. With appropriate controls, probes and hybridization conditions the hybridization assays are exquisitely specific. They are particularly useful in studying the molecular biology of the virus, its extrachromosomal replication or integration into the cellular genome and transformation of the infected hepatocytes. Further, recent studies have identified new target cells for HBV in liver (bile duct epithelial cells, endothelial cells, smooth muscle cells in blood vessel walls), spleen, bone marrow and white blood cells. While serological and immunohistochemical assays are still the basis of routine laboratory diagnosis of HBV infection, nucleic acid hybridization provides novel information useful in establishing the etiology of certain cases of viral hepatitis without serologic evidence for known viral agents (so-called non-A, non-B hepatitis), for demonstrating active viral replication in infected individuals, and for selecting and monitoring patients undergoing antiviral therapy.

DNA, Viral↗

[Rational laboratory diagnosis of cholestasis syndrome].

Laboratory diagnostics efficiently applied is of decisive importance for a great deal of questions in spite of the technical and endoscopic methods which are available today in the field of examinations. By means of a screening programme consisting of ALAT, gamma-GT, ChE at a sensitivity of > 90% alterations in both the hepatobiliary system and cholestasis can be recognized with sufficient reliability. Clinical data and a defined laboratory routine programme as a second step in diagnostics results in reliable indication for distinction between obstructive and non-obstructive cholestasis which can be promoted by computer-aided techniques. On the basis of such pre-selections special laboratory methods (differential diagnosis of consecutive non-obstructive cholestasis in liver diseases) or invasive methods to clarify their reason and localization diagnostics of biliary obstruction can then be applied in a well-directed manner to obtain a definite nosologic diagnosis. Effective diagnostic procedures in this three-step programme are described.

Alanine Transaminase↗

Evaluation of a commercial dengue NS1 antigen-capture ELISA for laboratory diagnosis of acute dengue virus infection.

A commercial dengue NS1 antigen-capture ELISA was evaluated to demonstrate its potential application for early laboratory diagnosis of acute dengue virus infection. Dengue virus NS1 antigen was detected in 199 of 213 acute serum samples from patients with laboratory confirmation of acute dengue virus infection but none of the 354 healthy blood donors' serum specimens. The dengue NS1 antigen-capture ELISA gave an overall sensitivity of 93.4% (199/213) and a specificity of 100% (354/354). The sensitivity was significantly higher in acute primary dengue (97.3%) than in acute secondary dengue (70.0%). The positive predictive value of the dengue NS1 antigen-capture ELISA was 100% and negative predictive value was 97.3%. Comparatively, virus isolation gave an overall positive isolation rate of 68.1% with a positive isolation rate of 73.9 and 31.0% for acute primary dengue and acute secondary dengue, respectively. Molecular detection of dengue RNA by RT-PCR gave an overall positive detection rate of 66.7% with a detection rate of 65.2 and 75.9% for acute primary dengue and acute secondary dengue, respectively. The results indicate that the commercial dengue NS1 antigen-capture ELISA may be superior to virus isolation and RT-PCR for the laboratory diagnosis of acute dengue infection based on a single serum sample.

Acute Disease↗

Laboratory diagnosis of central nervous system infections with herpes simplex virus by PCR performed with cerebrospinal fluid specimens.

Until recently, the laboratory diagnosis of central nervous system (CNS) infections with herpes simplex virus (HSV) has been limited by poor sensitivity and/or specificity. We assessed the diagnostic utility of PCR for detection of HSV in over 2,100 specimens referred to the Mayo Clinic from August 1993 to May 1996. DNA extracted from cerebrospinal fluid (CSF) samples with IsoQuick was amplified by PCR with oligonucleotide primers directed to the DNA polymerase gene of HSV, yielding a 290-bp amplicon. HSV DNA was detected in 150 (135 by gel electrophoresis, 15 by Southern blotting only) of 2,106 (7.1%) specimens. PCR-positive CNS disease occurred in patients ranging in age from 13 days to 89 years; 59% of the cases occurred in patients between the ages of 30 and 69, and 21 (14%) of the patients were infants. Genotype analysis was not routinely performed; however, amplification of a 335-bp product within the thymidine kinase gene of HSV revealed 13 positions within a span of 80 nucleotides that accurately identified the two serotypes of the virus according to 14 reference strains. We conclude that PCR detection of HSV DNA in CSF specimens should be considered an emerging "gold standard" for the laboratory diagnosis of CNS infections with this virus.

Adolescent↗

A stepwise approach to the laboratory diagnosis of Buruli ulcer disease.

OBJECTIVE: In view of technical and financial limitations in areas of endemicity, the current practice and recommendations for the laboratory diagnosis of Buruli ulcer disease (BUD) may have to be reconsidered. We reviewed diagnostic results in order to explore options for a modified, more practicable, cost-effective and timely approach to the laboratory diagnosis of BUD. METHODS: Diagnostic specimens from 161 clinically diagnosed BUD patients from four different treatment centres in Ghana were subjected to laboratory analysis. The positivity rates of the laboratory assays were compared. RESULTS: The number of laboratory-confirmed clinically diagnosed BUD cases with one positive confirmative test was 20% higher than that with two positive confirmative tests. The specificity of microscopy (MIC) and PCR was 96.6% and 100%, respectively. Subsequent analysis of specimens from surgically excised pre-ulcerative tissue-by-tissue MIC and tissue PCR rendered 65% laboratory-confirmed BUD cases. Subsequent analysis of diagnostic swabs from ulcerative lesions by swab smear MIC and swab PCR rendered 70% of laboratory-confirmed BUD cases. CONCLUSIONS: The specificity of the diagnostic tests used in this study suggests that one positive diagnostic test may be considered sufficient for the laboratory confirmation of BUD. Subsequent application of different diagnostic tests rendered a laboratory confirmation of 65% pre-ulcerative and of 70% ulcerative lesions. Implementation of a stepwise, subsequent analysis of diagnostic specimens will result in considerable cost saving compared with simultaneous testing of specimens by several diagnostic assays.

Cost-Benefit Analysis↗

[The laboratory diagnosis of spring-summer infections in Yekaterinburg in the epidemic season for tick-borne encephalitis].

The authors propose a comprehensive approach to laboratory diagnosis of seasonal transmissible infections, based on modern methods permitting etiological deciphering of disease. A universal diagnostic algorithm notably accelerated the laboratory diagnosis due to cutting the period between collection of material from a patient and consecutive screening for antibodies to agents of tick-borne encephalitis, Lyme disease, and California encephalitis.

Algorithms↗

[Cyclospora cayetanensis infection. Laboratory diagnosis].

Cyclospora cayetanensis is an Apicomplexa protozoa which was found to cause gastroenteritis in humans in 1979. This paper reviews the laboratory diagnosis of the disease. The usefulness of direct examination of fresh fecal matter with special acid-fast stains is emphasized as well as the morphometric differentiation between this organism and other similar coccidia. The paper reviews the sporulation technique of Cyclospora cayetanensis for the recognition and differentiation of artifacts and green-blue algae. Another aspect discussed is the morphology of the parasite in histological sections and with electromicroscopic examination whereby tissue morphology changes caused by the microorganism can be identified. The experience of the Service and Laboratory of Parasitology of the Instituto Nacional de Pediatría of Mexico in the laboratory diagnosis of this protozoosis is described. The paper is oriented towards the inclussion of Cyclospora cayetanensis in the diagnostic protocols for the study of diarrheas in our health Institutions.

Animals↗

A new test for the laboratory diagnosis of spherocytosis.

A new test for the laboratory diagnosis of spherocytosis, conventionally called 'Pink test', is presented. This test, semi-quantitatively or quantitatively, determines the hemolysis of small blood samples in a solution containing glycerol (135 mmol/l), NaCl (25 mmol/l), NaN3 (1.5 mmol/l), buffered to pH 6.66 with Bis-Tris (70 mmol/l) and HCl. 'Pink test', as well as 'acidified' glycerol lysis test, were positive in 100% of 42 patients suffering from hereditary spherocytosis, and optimally discriminated them from healthy subjects, showing a diagnostic sensitivity greater than 'standard' glycerol lysis test and osmotic fragility in hypotonic saline solutions of fresh or incubated blood. 'Pink test' was also positive in some cases of renal failure, immunohemolytic anemia, chronic hemoproliferative disorders, normal pregnant women, and negative in other microcytic anemias (beta-thalassemia, iron deficiency anemia). The results do not critically depend on pH of the solution (differently from those obtained with 'acidified' glycerol lysis test), and for this reason they show a good reproducibility.

Buffers↗

[Laboratory diagnosis of human herpes virus infection].

Reviews modern data on laboratory diagnosis (virological, immunobiochemical, molecular biological methods) and differential diagnosis of herpesvirus infection induced by various Herpesviridae.

Clinical Laboratory Techniques↗

[Laboratory diagnosis and practical treatment of patients].

The practical treatment of patients requires a reliable diagnosis and prognosis followed by therapy. Laboratory medicine is an essential scientific basis to rational treatment in each of these three phases. The significance, usefulness and extent of laboratory tests are largely dependent upon the disease spectrum. The possibilities and aims of laboratory medicine are: 1. Knowledge of and research into both the pathobiochemical and statistical connections between biochemical data and diseases leading to the selection of diagnostically reliable and relevant parameters. 2. Knowledge of the sensitivity, specificity and predictive values of relevant biochemical parameters and their application to the different clinical problems. 3. Evaluation of reliable analytical methods with details of their accuracy and precision. 4. Critical evaluation of pre-analytical factors. The translation of accurate laboratory data into clinically relevant information must always be coupled with a critical assessment of its diagnostic value, validity and range of error. An uncritical "consumption" of laboratory data is dangerous for both the patient and the clinician as is the suppression of these data when it does not fit the supposed diagnosis. Laboratory medicine is indispensible in proper patient care since it guarantees adequate medical action. The information that it delivers is binding and therefore offers help as well as protection.

Clinical Laboratory Techniques↗

Laboratory diagnosis of laryngeal disease.

The most important method used in the laboratory diagnosis of laryngeal disease is histological examination of material removed at biopsy. Surgical and technical aspects of this procedure are discussed. A review of diagnoses made of laryngeal biopsies in a single year by the writer's laboratory is given together with a brief description of the pathology of the commonest non-malignant conditions. The handling of laryngectomy and block dissection specimens in the laboratory is described. The parts played by the cytology, bacteriology and virology laboratories in laryngeal diagnosis are briefly reviewed.

Biopsy↗

Enhanced laboratory diagnosis of foot and mouth disease by real-time polymerase chain reaction.

The performance of an automated real-time reverse transcription polymerase chain reaction (RT-PCR) was compared to virus isolation (VI) in cell culture and antigen detection enzyme-linked immunosorbent assay (ELISA) for the laboratory diagnosis of foot and mouth disease (FMD). The World Reference Laboratory for FMD in Woking, the United Kingdom, examined a collection of 334 epithelia received from eighteen countries between August 2002 and January 2004. The results showed that all VI positive (n = 195) and VI and ELISA positive samples combined (n = 204) were also positive by RT-PCR. Depending on the cut-off used, FMD virus genome was detected in a minimum of an additional 60 samples (18% of all samples tested). Furthermore, the RT-PCR generated results in less than one day from test commencement in contrast to up to 4 days to define some positive and all negative samples by VI. The study demonstrates that real-time RT-PCR provides an extremely sensitive and rapid procedure for improved laboratory diagnosis of FMD.

Animals↗

[The comparative characteristics of direct microscopic methods in the laboratory diagnosis of chlamydial infection in newborn infants].

Methods of laboratory diagnosis of chlamydial infection in newborns are compared. Two methods are highly effective: cytological and immunofluorescent. If the material for investigation is properly collected and the laboratory assistant is highly qualified, a cheaper and available cytological method of staining after Romanovsky-Giemsa can be used in practical public health. The method should be used in modification according to May-Grünwald with addition of DMSO into staining solution.

Chlamydia Infections↗

Comparative evaluation of enzyme immunoassay and culture for the laboratory diagnosis of gonorrhea.

A commercially available, solid-phase enzyme immunoassay (EIA), called Gonozyme, was compared to Martin-Lewis medium in Jembec plates for the laboratory diagnosis of gonorrhea. A total of 577 clinical specimens (419 urethral and 158 endocervical) were collected from a high-risk, walk-in patient population attending a sexually transmitted disease clinic. The results showed that EIA was comparable to a conventional cultural procedure for identifying infected and noninfected males. In addition, the system may be used reliably for performing test of cure on urethral samples obtained from this male population. Gonozyme was also comparable to culture in identifying females who had gonococcal infection. However, because of the high incidence of false positive test results, most likely attributable to antigen persistence in endocervical secretions, EIA is not recommended for performing test of cure in the female. Overall, the Gonozyme system is an easily performed, rapid, and reliable system that provides for a noncultural alternative for the laboratory diagnosis of gonorrhea.

Cervix Uteri↗