Detection of a novel HLA-A allele by polymerase chain reaction-sequence-specific oligonucleotide typing: HLA-A*0252

Anew human leukocyte antigen (HLA) class I allele, HLA-A*0252, has been found during routine typing of samples for the Australian Bone Marrow Donor Registry. A*0252 differs from A*020101 at four codon positions, with all the new polymorphisms resulting in an amino acid change. The amino acids involved are located in the antigen-binding region of the HLA protein.     

Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.

We have noticed that suboptimal specimen processing and storage conditions may cause false-negative results in the detection of hepatitis C virus (HCV) RNA in plasma or serum. To establish the influence of specimen handling in a serological laboratory on the rate of detection of HCV RNA by the cDNA polymerase chain reaction (cDNA-PCR), we tested routine serum samples and fresh-frozen plasma samples from the same bleeding from confirmed anti-HCV-positive blood donors. When primers from the NS3/NS4 region were used, HCV RNA was detected in fresh-frozen plasma from 67% of the donors, whereas positive results were obtained with only 50% of the serum samples that had been subjected to routine serological procedures. Analysis of the same samples with primers from the highly conserved 5'-terminal region (5'-TR) revealed an HCV RNA detection rate of 92% for both the routine and the fresh-frozen samples. However, the yield of the amplification product in routine samples was strongly reduced compared with that in fresh-frozen plasma. Comparison of both primer sets for cDNA-PCR showed that the 5'-TR primer set was 10- to 100-fold more effective in detecting HCV RNA. We also analyzed the effect of storage of whole EDTA-blood and serum at room temperature and at 4 degrees C on the yield of the amplification product. A rapid decline in detectable HCV RNA of 3 to 4 log units was observed within 14 days when whole blood and serum were stored at room temperature. By contrast, no perceptible reduction in the cDNA-PCR signal was found in freshly prepared serum stored at 4 degrees C.     

Combined use of blood and oropharyngeal samples for noninvasive diagnosis ofPneumocystis carinii pneumonia using the polymerase chain reaction

To evaluate the clinical use of a polymerase chain reaction (PCR) assay for diagnosis ofPneumocystis carinii pneumonia (PCP) using samples collected non-invasively, the Internal Transcribed Spacers (ITSs) nested PCR was performed on 148 samples from 40 subjects. Bronchoalveolar lavage (BAL) fluid sera, gargled oropharyngeal washes, and peripheral blood mononuclear cells (PBMC) from 14 AIDS patients (mean age, 35.6 years; mean CD4+ cell count, 49.2 cells/mm³) with proven PCP and from 13 HIV-seropositive controls (mean age, 34.6 years; mean CD4+ cell count, 107.3 cells/mm³) with other AIDS-related opportunistic infections were evaluated. Sera and oropharyngeal samples were also collected from 13 HIV-seronegative health care personnel working in an infectious disease ward for use as negative controls. The ITSs nested PCR confirmed the morphological diagnosis of PCP in all patients when BAL fluid was tested (100% sensitivity). This technique also detectedPneumocystis carinii DNA in oropharyngeal samples from 78.6% of patients, in sera from 71.4% of patients, in PBMC from 35.7% of patients. When all results obtained after ITSs nested PCR were considered together for the same patient, the sensitivity for PCP diagnosis was 100% for blood and oropharyngeal samples (gargled saline), as confirmed by subsequent BAL. All samples collected noninvasively from 26 of 26 controls were negative using ITSs nested PCR (100% specificity).     

Comparison of a conventional polymerase chain reaction with real-time polymerase chain reaction for the detection of neurotropic viruses in cerebrospinal fluid samples

Purpose: To compare a conventional polymerase chain reaction (PCR) and real-time PCR for the detection of neurotropic DNA viruses. Materials and Methods: A total of 147 cerebrospinal fluid (CSF) samples was collected from patients attending a tertiary care hospital in South India for a period from 2005 to 2008. All these samples were tested using a conventional multiplex/uniplex PCR and a real-time multiplex/uniplex PCR. This technique was used to detect a large number of herpes viruses responsible for central nervous system infections, including HSV-1, HSV-2, VZV, CMV and EBV and the polyoma virus JCV. Results: Overall, in the entire set of samples, the real-time PCR yielded 88 (59.9%) positives and conventional PCR had six (4.1%) positives. Conclusion: Our results suggest that the real-time PCR assay was more sensitive compared with the conventional PCR. The advantage of real-time PCR is that it can be performed much faster than conventional PCR. Real-time PCR is less time-consuming, less labour-intensive and also reduces the chance of contamination as there is no post-amplification procedure. In the entire study population, the major viruses detected using real-time PCR were EBV (34%), HSV-2 (10.8%) and VZV (6.8%).     

Further studies on the use of a polymerase chain reaction test for the diagnosis of infectious coryza

Further information is reported on the use of a polymerase chain reaction (PCR) test for the diagnosis of infectious coryza in China. The majority of sinus swabs taken from artificially infected chickens and stored in glycerol-enriched phosphate-buffered saline were still positive by PCR after storage for 180 days at either 4 degrees C or - 20 degrees C. Storage of swabs in either saline or nutrient broth was not as effective. Traditional culture failed to detect H. paragallinarum after storage for 3 days, regardless of storage medium or storage temperature. With dry swabs, the PCR could detect H. paragallinarum after storage for 7 days or longer at either 4 degrees C or - 20 degrees C, while traditional culture could not. In PCR tests on 64 artificially-challenged chickens, all were positive by PCR at the six sampling dates up to 18 days post-challenge. Traditional culture gave a similar result. Both PCR and culture detected 50% or less of chickens as positive at 21 and 24 days post challenge. Antibiotic treatment reduced the ability of both culture and the PCR test to detect H. paragallinarum. The value of the PCR test and its superiority over traditional culture for the diagnosis of infectious coryza has been confirmed in these experiments.     

Storage and preservation of whole blood samples for use in detection of human immunodeficiency virus type-1 by the polymerase chain reaction.

Methods used in the diagnosis of human immunodeficiency virus type-1 (HIV-1) infection by the polymerase chain reaction (PCR) usually require the separation of lymphocytes from a whole-blood sample within 24 hours of patient sampling. A method is described in which blood samples are mixed with a cryopreservative ('Glycigel'), stored frozen, and DNA suitable for use in an HIV PCR recovered. Samples can be stored at -20 degrees C for up to 3 months and still give positive results with all samples from infected patients; storage at -80 degrees C for at least 3 months shows no loss of titre. The method shows no loss of sensitivity compared to previously described sample preparation methods. Deglycerolised Glycigel supernatants were found to be suitable for conventional anti-HIV-1 serological studies and loss of sensitivity only represented the dilution effect due to sample preparation. Application of the method as a means of storing samples frozen at the point of sampling and transporting them to a central laboratory for processing is demonstrated using samples taken from HIV-1-infected mothers and their babies.     

The gel test: use in the identification of unexpected antibodies to blood group antigens

The IgG GEL test was compared with the LISS tube test (L?w and Messeter's low-ionic-strength saline) for antibody identification. The suitability of red blood cells (RBCs) pretreated with ficin, dithiothreitol (DTT), or chloroquine diphosphate (CDP) also was assessed for use in the GEL test. In addition, time-in-motion studies were performed comparing GEL (12 panels per batch) with polyethylene glycol (PEG) tube tests (3 panels per batch). In 57 antibody identification studies, there were 63 GEL+ LISS+, 2 GEL+ LISS-, and 6 GEL-LISS+ antibodies. Among the GEL+ LISS+ antibodies were 19 that yielded stronger reactions in GEL than in LISS; by virtue of their specificity, 14 of these are considered potentially significant: D, 5 E, 2 e, 2 Jka, 2 S, K, and Fya. There were 38 antibodies that yielded equivalent results by both methods, including 31 that are considered potentially significant. Of six antibodies with significantly greater reactivity in LISS, there were three anti-Rh and three that are considered harmless with respect to transfusion management. The two GEL+ LISS- antibodies (anti-Jkb) were potentially significant. GEL- LISS+ reactions involved only harmless antibodies. Of the 50 antibodies of potential significance, GEL yielded equivalent or superior results in 47 (94%) instances. Additionally, GEL failed to detect 6 of 21 harmless antibodies. Expected results were obtained with normal serum or plasma and antibodies of known specificity in tests with RBCs treated with ficin, DTT, or CDP. Hands-on-time required for each GEL panel was 2 to 21/2 minutes compared with 12 minutes for PEG. These data document the suitability of GEL for use in antibody identification studies.     

Evaluation of a multiple peptide assay for typing of antibodies to the hepatitis C virus: Relation to genomic typing by the polymerase chain reaction

A panel of 16 type-specific synthetic peptides corresponding to variable antigenic regions within the hepatitis C virus (HCV) core, nonstruc-tural 4 (NS4), and NS5 proteins was synthesised. The peptide panel was used to develop an enzyme immunoassay (EIA) for the detection of antibodies directed to HCV type 1 (genotypes I/1a and II/1 b), type 2 (genotypes III/2a and IV/2b), and type 3 (genotype V/3). The peptides corresponded to residues 68–81 of the HCV core (types 1,2, and 3), residues 1692–1705 and 1710–1728 of HCV NS4 (types 1 a, 1 b, 2a, 2b, and 3), and residues 2303–2319 of HCV NS5 (types 1a, 1b, 2a, and 2b). The 16-peptide panel was evaluated using human sera from 46 carriers of HCV, which were genotyped in parallel by the polymerase chain reaction (PCR) using primers specific for types I, II, III, IV, and V of HCV core. Of the 46 carriers, 14 (30%) were infected by HCV genotype I, 7 (15%) by genotype II, 16 (35%) by HCV genotype IV, and 6 (13%) by HCV of genotype V. Two carriers had double infections of types I and II, and the HCV strain of one carrier could not be genotyped. Using the serotyping system, 40 (89%) out of the 45 genotyped carriers were found to contain type-specific antibodies corresponding to the genotypes identified by PCR. In 5 of the 23 carriers infected by genotypes I and/or II, antibodies specific for HCV type 1 could not be detected, whereas all 16 carriers infected by genotype IV were serologically typed as type 2. Out of the six carriers infected by HCV of genotype V, all were found to have antibodies of serotype 3, but in most cases together with antibodies to NS5 type 1, indicating sequence homologies between types 1 and 3 of this NS5 region. In the one patient serum where the HCV strain could not be genotyped, a mixture of types 1, 2, and 3 antibodies were found. In conclusion, a serotyping system with a sensitivity and specificity of 89% was developed. It is confirmed that at least three distinct serotypes of antibodies to HCV exist. The major advantage of using four different antigenic regions is that we often obtain high absor-bance values which are easily interpreted, or multiple reactions which confirm each other. © 1995 Wiley-Liss, Inc.     

The use of nested polymerase chain reaction and restriction fragment length polymorphism for the detection and typing of mucosal human papillomaviruses in samples containing low copy numbers of viral DNA

Mucosal human papillomaviruses (HPVs) that infect the genital area have also been shown to infect the oral cavity. In this study a restriction fragment length polymorphism (RFLP) method was developed on a nested polymerase chain reaction (PCR) product to identify ten high risk HPV types 16, 18, 31, 33, 35, 45, 51, 52, 58 and 59 as well as the low risk HPV 11. HPV DNA was detected in 23/31 (74%) of buccal specimens using a sensitive nested PCR employing degenerate consensus primers (Williamson and Rybicki, 1991). Consensus PCR using the PGMy09/11 primers. was able to detect HPV in only 29% of the specimens that had tested positive using the nested HPV PCR primers. HPV 11 type specific primers detected HPV 11 DNA in only 66% of the specimens showing HPV 11 DNA by means of nested PCR and RFLP. A Genbank search revealed that the PCR primers could detect a wide range of mucosal HPV types including types HPV 70, 72 and 73 which have all been isolated from immunocompromised patients. Of the 23 buccal specimens that were positive for HPV DNA, 13 were single infections, five were dual infections and three were triple infections. The HPV types identified by RFLP were: HPV 11 (18/23), HPV 18 (8/23), HPV 16 (3/23), and HPV 33 (1/23). HPV 13 (2/23) was identified by direct sequencing of the inner amplicon of the PCR product.     

HLA-DR typing for kidney transplants: advantage of polymerase chain reaction with sequence specific primers in a routine hospital laboratory.

AIMS--To determine whether the polymerase chain reaction with sequence specific primers (PCR-SSP) can assign HLA-DR type more accurately than serology in a routine hospital laboratory. METHODS--The 93 patients currently awaiting kidney transplants have been DR typed by serology over the past 14 years, 82% within the past five years. They have now been retyped using the PCR-SSP method described by Bein et al. Where the two results differed, PCR-SSP was repeated, once by the same method and once using the primer set of Olerup and Zetterquist. RESULTS--There were 13 (14%) discrepancies between the results. Of these, two were PCR-SSP failures, later overcome: three were failure to detect DRB1*0103 by serology; five assignment of other alleles by PCR-SSP to serological "blanks"; and three alleles were differently assigned by serology and PCR. The serological typing of the final patient when repeated for this study was at variance with the original findings (14 years ago), but in agreement with PCR. In the remaining patients, serology had not determined the split of 36 DR3 alleles (all DR17 by PCR-SSP) or 13 DR6 alleles (12 DR13 and one DR14 by PCR-SSP). One patient in each case had their antigen splits of DR2 and DR5 assigned by PCR-SSP (DR15 and DR11, respectively) but not by serology. CONCLUSIONS--PCR-SSP provides more reliable and detailed information on HLA-DR polymorphism than serology, and does so within a routine tissue typing laboratory.     

Development of a polymerase chain reaction (PCR) test for the detection of virulent forms of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a marine bacterium and some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Use of randomly amplified polymorphic DNA (RAPD)-PCR produced a unique 600 bp amplicon (band Y) in the majority of clinical isolates and rarely in environmental isolates tested. The DNA from band Y was cloned and sequenced and found to code for an outer membrane protein (OMP). Two polymerase chain reaction (PCR) primers were designed to specifically amplify a 200 bp unique sequence from presumptive virulent strains (PCR-OMP). The virulence of 23 clinical and 32 environmental isolates was assessed in cytotoxicity tests by treatment of Caco-2 cells with extracellular products (ECPs). All but two of the clinical isolates (91%) were positive for the 200 bp PCR-OMP and their ECPs produced a significantly higher (p < 0.05) lactate dehydrogenase (LDH) release (mean 72.88%) than the ECPs of environmental isolates (mean 15.3%) with the exception of one environmental isolate that produced the 200 bp amplicon. A positive 200 bp PCR-OMP is strongly correlated with virulence, as determined by the cytotoxicity assay, and identified virulent forms better than current PCR tests for tdh, trh or T3SS2.     

Analysis of blood CD4+ and CD8+ T-lymphocyte subsets with monoclonal antibodies. Comparison of the Genetic Systems CD4/CD antigen typing kit with conventional indirect immunofluorescence microscopy and the automated Technicon-H1 Enzyme Immunoassay (EIA)

The Genetic Systems Technique (GS: direct immunofluorescence microscopy with ethidium bromide counterstaining of nuclei) was tested for quantitative analysis of T-lymphocyte subsets in human peripheral blood. The monoclonal antibodies anti-CD4 and anti-CD8 were used for detection of T-helper and T-suppressor cells respectively and the results compared to those obtained by conventional indirect immunofluorescence microscopy and the Technicon Enzyme Immunoassay (EIA) with automated reading. The GS technique provided results correlating well with both indirect immunofluorescence and EIA techniques. Moreover, this method has two advantages: it is less time-consuming than the indirect immunofluorescence microscopy and necessitates less expensive and more commonly available equipment than the automated EIA technique.     

Development and clinical significance of a diagnostic assay based on the polymerase chain reaction for detection of human cytomegalovirus DNA in blood samples from immunocompromised patients.

The presence of human cytomegalovirus (HCMV) DNA in blood was investigated by the polymerase chain reaction (PCR) in 293 blood samples from 86 immunocompromised patients. Of the 86 patients, 23 underwent clinical and virologic follow-up for HCMV infection. In parallel, blood samples were examined for viremia and antigenemia. Concordant results between PCR and assays for viremia and antigenemia were obtained on 124 positive and 110 negative samples, with an overall concordance of 79.8%, while 59 samples (most from patients with HCMV infection) were positive by PCR alone. PCR is a new powerful tool for detection of HCMV infections in blood samples from immunocompromised patients. However, its clinical significance appears to be restricted to the indication of a risk of reactivation of HCMV infection.     

Quantitation of cytomegalovirus DNA by real-time polymerase chain reaction in peripheral blood specimens of patients with solid organ transplants: comparison with end-point PCR and pp65 antigen test

The polymerase chain reaction (PCR) for cytomegalovirus (CMV) DNA quantitation provides sensitive and specific data for detecting CMV as well as monitoring the infection and determining the appropriate antiviral strategy. A recently introduced real-time PCR assay for CMV DNA quantitation was applied on 158 peripheral blood leukocytes (PBLs) from 32 liver-transplanted patients with CMV asymptomatic infection and correlated with a commercial quantitative end-point PCR (COBAS AMPLICOR CMV Monitor) and CMV pp65 antigenemia. A good correlation was found between real-time PCR and pp65 antigen test (r2 = 0.691) and the two PCR assays (r2 = 0.761). Real-time PCR data were higher in pre-emptive treated patients (>20 pp65 + positive cells, median CMV DNA value: 3.8 log(10) copies/500,000 PBLs) than in not-treated ones (2.9 logs). According to pp65 levels of 0, 1-10, 11-20, 21-50, 51-100, and >100 positive cells/200,000 PBLs, median CMV DNA by real-time PCR was 2.6, 3.0, 3.6, 4.0, 4.2, and 4.8 logs, respectively, (CMV DNA levels by COBAS AMPLICOR: 2.8, 2.9, 3.8, 3.7, 3.9, and 4.0 logs). For samples with >20 pp65 + cells, real-time PCR gave significantly higher values than in groups with <20 pp65 + cells, whereas the COBAS AMPLICOR results showed a slower progression rate. Dilutions of CMV AD169 strain were used to probe real-time PCR reproducibility (between and intra-assay variability <2%) and sensitivity (100% detection rate at 10 copies/reaction, 28.5% with end-point PCR). In conclusion, real-time PCR significantly improves the study of CMV DNA dynamics due to a more reliable quantitation of CMV DNA for moderate and high DNA level compared to end-point PCR with better sensitivity and specificity. Real-time PCR provides more precise information for evaluating infection progress and assessing antiviral response, simplifying and accelerating the process of producing a reliable quantitation of CMV DNA for clinical purposes.     

Sequence-based typing of the HLA-A10/A19 group and confirmation of a pseudogene coamplified with A*3401

The strategy for sequencing human leukocyte antigen (HLA)-A was based on separate amplification of exons 2 and 3, followed by forward and reverse heterozygous sequencing of the alleles. Validation of the method was obtained by sequencing 11 individuals carrying alleles from all different HLA-A allele groups, except *43. All alleles could be correctly identified except A*3401. Unexpected polymorphic positions were identified in exon 3, even in individuals homozygous for A*3401. In addition, the pseudogene HLA-COQ or HLA-DEL linked to A*3401 was coamplified and sequenced. The problem was solved by using different amplification primers for exon 3 with mismatches for the two pseudogenes. A total of 252 unrelated individuals with at least one allele belonging to the A10 or A19 group were typed for HLA-A by this strategy. Ten different alleles were identified in the A10 group and 14 in the A19 group. As second allele a further 30 different subtypes from all different groups were sequenced. In 21 individuals, sequencing exon 1 was necessary to distinguish A*7401 from A*7402. The sequencing strategy, with separate amplification of the exons, has proven to be a robust method, resulting in reliable and efficient high-resolution HLA-A typing.     

Detection and typing of human papillomavirus using the Vira Type "in situ" kit: comparison with a conventional dot blot technique

A new commercial kit (Vira Type "in situ", Life Technologies, Inc., Molecular Diagnostics Division, Guithersburg, Maryland, USA) for the detection of human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33 and 35 in routinely processed human anogenital tissue was compared with a conventional dot blot assay for HPV 6, 11, 16 and 18. Both systems use double-stranded genomic DNA probes for the detection of type specific HPV DNA. The probes used on the dot blots were labelled with 32P and visualised autoradiographically. The Vira Type probes were labelled with biotin and visualised using a streptavidin-alkaline phosphatase conjugate with NBT-BCIP substrate. Biopsy specimens from the cervix, vagina, and vulva of 46 women were processed by both methods and compared. The histological diagnoses ranged from benign changes, to dysplasia, and invasive carcinoma. Overall, 50% of biopsy specimens were positive for HPV DNA by dot blot hybridisation; only 39% were positive by Vira Type in situ hybridisation. Three of the specimens positive by the Vira Type "in situ" kit showed no cross hybridisation and were the same HPV type as the dot blot. A further 13 showed hybridisation, but the showed cross hybridisation, but the to the dot blot results. One biopsy specimen was positive for different HPV types by the two tests and one was positive by Vira Type and negative by dot blot. Six biopsy specimens were negative by Vira Type but positive by dot blot. It is concluded that the Vira Type "in situ" kit has a similar specificity but lower sensitivity than the dot blot hybridisation method for the detection of HPV DNA.     

Clinical trials: specific problems associated with the use of a placebo-control group

In order to test hypotheses prospectively with hard data and against placebo, the scientific method of clinical trials has been developed. The present paper focuses on specific problems associated with the use of a placebo control group. (a) The placebo highlights the ethical dilemma that a controlled clinical trial can place us in. (b) Also, in a trial an atmosphere is created of enhanced risks of placebo effects. (c) Significantly different from placebo does not necessarily mean clinically relevant. (d) A biased placebo period due to carry-over effect is a common problem of controlled trials with a cross-over or self-controlled design. (e) Likewise an asymmetric placebo group is also a common problem in parallel-group designs. (f) The response to a placebo is generally small in comparison with the response to active treatment and is therefore sometimes more susceptible to bias. It is emphasized that routinely accounting for such problems may further improve the powerful method of controlled clinical trials.