Polymerase chain reaction for the detection of Bordetella pertussis in clinical nasopharyngeal aspirates

A PCR procedure for the detection of Bordetella pertussis in nasopharyngeal aspirates (NPAs) was developed with primers derived from the pertussis toxin promoter region. The amplification resulted in a 191-bp PCR product specific for B. pertussis. A total of 681 NPAs collected from children with cough lasting >7 days was evaluated by PCR and culture; 104 aspirates were positive by PCR and 93 by culture. Sixteen cases were positive only by PCR and five culture positive aspirates were negative by PCR. An internal control was included in the assay to monitor the performance of the PCR and to identify possible inhibitory components in clinical samples. The PCR method was more efficient than culture in detecting B. pertussis in samples collected late in the disease, in antibiotic-treated children and in patients with mild disease.     

Polymerase chain reaction: a link to the future

Polymerase chain reaction (PCR) is a multi-faceted technology that is advancing disease detection. For pathology and clinical laboratories, PCR will be the tool of choice into the next century as scientists continue to develop and refine new uses. An amazingly simple process that amplifies nucleic acid sequences, PCR will change the practice of medicine because it will play a role in all aspects of the patient care continuum from diagnosis to treatment monitoring. Some striking applications range from detecting infectious and inherited diseases to assessing pharmacologic interventions. Additionally, the remarkable sensitivity of PCR will allow determination of a patient's genetic predisposition to diseases, thereby improving prevention modalities. Thus, medical practitioners should gain a basic understanding of this phenomenal cornerstone of molecular diagnostics. This article briefly reviews the history, theory, and uses of PCR and discusses relevant applications for military medicine.     

Polymerase chain reaction determination of RhD blood type: an evaluation of accuracy

OBJECTIVE: To evaluate the accuracy of polymerase chain reaction (PCR) amplification of a portion of the RhD gene by testing a large number of DNA samples derived from individuals whose RhD status was established by the standard serologic method. METHODS: Seven hundred sixty-five samples were obtained from two sources: subjects taking part in studies at the University of Iowa Hospitals and Clinics (n = 107), and Centre d'Etude du Polymorphisme Humain (CEPH) families used for studies of genetic variation (n = 658). Deoxyribonucleic acid was extracted from blood samples of University of Iowa volunteers and from CEPH families by standard techniques. With few modifications, published primers, reaction and electrophoresis conditions, which yield a 1200-bp fragment in all individuals and a 600-bp fragment in RhD-positive individuals, were used. RESULTS: By standard serologic techniques, we identified samples from 632 RhD-positive and 133 RhD-negative individuals. Two (both from CEPH) of the 632 RhD-positive individuals were characterized as RhD-negative by PCR. Seven of the 133 RhD-negative samples were judged to be RhD-positive by PCR because of the presence of a light 600-bp band. Despite repeated attempts, no bands or DNA were identified in one RhD-negative sample. Thus, the sensitivity of RhD typing by PCR was 99.7%, the specificity 94.0%. CONCLUSION: Based on our data, it would appear that the use of PCR to establish RhD type can be introduced cautiously into current management schemes in the evaluation of RhD sensitization.     

Polymerase chain reaction approaches for the detection of Neospora caninum and Toxoplasma gondii

This review summarizes existing knowledge on the development and use of the polymerase chain reaction for the detection of DNA from Neospora and Toxoplasma. Several strategies which utilise the polymerase chain reaction for the diagnosis of toxoplasmosis in humans and livestock have been described and they principally target the B1 repetitive sequence, the P30 gene or ribosomal DNA. Experience has shown that the polymerase chain reaction has proven insufficiently robust to serve as a diagnostic test alone although when used in conjunction with other diagnostic techniques it does prove to be a useful aid. The marketing of a commercial polymerase chain reaction kit may well solve some of the inadequacies seen using "home made" polymerase chain reaction technology which are commonly used in diagnostic laboratories around the world. Recent progress on the development of polymerase chain reaction diagnostics for Neospora has been rapid and is discussed in detail.     

Polymerase chain reaction amplification analyses of clonality in T-cell malignancy including peripheral T-cell lymphoma

Clonality in T-cell malignancy was investigated using T-cell receptor (TcR) V beta 1-20 family primers and polymerase chain reaction amplification (PCR) of cDNA prepared from tissue biopsies and blood involved with tumour. Secondary PCR amplification of the VDJ joints of primary PCR products was performed to distinguish clonal from polyclonal products, and clonal V beta gene products were confirmed by direct PCR sequencing in the majority of cases. In eight T-cell malignancies including T-cell acute lymphoblastic leukaemia (T-ALL) and T-cell chronic lymphocytic leukaemia (T-CLL) shown to be clonal by Southern blot analysis, one or two primary PCR products were identified and shown to be clonal. In five cases of peripheral T-cell lymphoma (PTCL) all V beta 1-20 families were identified after primary PCR amplification, and clonal products were identified in two cases after secondary amplification; TcR V beta clonal families could not be demonstrated in the remaining three cases. These data were in agreement with previous Southern blot analysis of these cases, and confirmed the presence of reactive T cells in PTCL as well as providing further evidence for the genotypic heterogeneity of this entity. In the remaining case, a blood lymphocytosis, primary PCR amplification produced predominant TcR V beta 6 and V beta 12 family products, of which the V beta 6 family proved clonal after secondary PCR amplification. There was no evidence for overrepresentation of TCR V beta families by the tumour populations in this study, furthermore the data confirm the involvement of reactive cells in T-cell malignancy and the genetic heterogeneity of PTCL.     

Polymerase chain reaction to detect Mycobacterium tuberculosis in histologic specimens

BACKGROUND: Whether acute MS lesions are primarily inflammatory or demyelinative is unresolved. Our study examined acute MS lesions longitudinally by quantitative magnetization transfer (MT), an MRI technique that identifies tissue integrity and destruction. METHODS: Four MS patients were studied by serial MRI including MT, conventional T2-weighted images, and postgadolinium T1-weighted images for 9 to 12 months. In 15 new lesions, the MT ratio (MTR) was calculated retrospectively. RESULTS: In 13 lesions, a marked decrease in the MTR was present early during the first 2 months after the onset of the lesion and was followed by a variable increase. In two other lesions, the MTR progressively declined. CONCLUSIONS: These results suggest that major early structural changes compatible with demyelination and followed by remyelination and gliosis, or by continuous demyelination, occur in new MS lesions. The various MTR profiles provide in vivo confirmation of the current knowledge of the progression in MS lesions. Furthermore, MTR may be used to monitor in vivo drug efficacy in new MS lesions.     

Polymerase chain reaction analysis of distal vaginal specimens: a less invasive strategy for detection of Trichomonas vaginalis

We compared polymerase chain reaction (PCR) analysis of specimens obtained from the distal vagina with wet mount microscopy and culture of specimens from the posterior vaginal fornix. One or all three techniques revealed that 61 (20.3%) of 300 women tested were positive for Trichomonas vaginalis. PCR analysis of distal vaginal specimens detected 56 (91.8%) of 61 infections, while wet mount microscopy and culture detected 49 (80.3%) of 61 infections. Results of this study may impact the approach to testing for T. vaginalis by eliminating the requirement of a vaginal speculum examination. The distal vagina is an appropriate testing site for T. vaginalis by PCR analysis.     

Nested polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples

The nested polymerase chain reaction technique was compared with the conventional smear and culture methods for detection of Mycobacterium tuberculosis. The nested polymerase chain reaction used in this study showed excellent specificity, sensitivity, and agreement with the conventional methods for 417 clinical samples, indicating a contribution to the rapid diagnosis of mycobacterial infectious diseases.     

Use of polymerase chain reaction to assess efficacy of leprosy chemotherapy

The assessment of chemotherapy efficacy in leprosy is difficult, since the only reliable method for determining whether the causative organism, Mycobacterium leprae, is viable depends on its growth in mouse foot pads. In an attempt to replace this expensive, time-consuming test, methods based on the polymerase chain reaction (PCR) have been developed. These methods depend on detection of DNA, which is more susceptible to degradation on cell death than are other cell components, so should be a more accurate indicator of viability. We have used a specific PCR assay to detect M leprae DNA in skin biopsy samples from leprosy patients. By use of limiting dilution PCR (LD-PCR), the concentration of M leprae DNA in the original sample could be measured. The DNA concentration was more closely correlated with the morphological index (derived from a staining technique that distinguishes morphologically intact and damaged bacteria) than with the number of bacteria visible (bacterial index, BI, which counts both alive and dead bacteria). In a longitudinal study of multibacillary patients on multi-drug therapy, skin biopsy samples were collected before treatment and 3, 6, 12, and 24 months after the start of therapy. While the BI showed little or no change during treatment, the number of genomes detected by PCR fell sharply, in parallel with the MI. We propose that PCR can be used as a rapid measure of M leprae viability and that this approach can be used for monitoring individual leprosy patients and for assessment of existing and new regimens. The method may be applicable to other infectious diseases in which culture of the causative organism is slow or impossible.     

Detection of colorectal cancer cells in peripheral blood by reverse-transcriptase polymerase chain reaction for cytokeratin 20

In this study we have determined systemic and local antibody responses against different Helicobacter pylori antigens in H. pylori-infected and noninfected subjects. In addition, we studied whether differences in antibody responses between patients with duodenal ulcers and asymptomatic H. pylori carriers might explain the different outcomes of infection. Sera and in most instances gastric aspirates were collected from 19 duodenal ulcer patients, 15 asymptomatic H. pylori carriers, and 20 noninfected subjects and assayed for specific antibodies against different H. pylori antigens, i.e., whole membrane proteins (MP), lipopolysaccharides, flagellin, urease, the neuraminyllactose binding hemagglutinin HpaA, and a 26-kDa protein, by enzyme-linked immunosorbent assay. The H. pylori-infected subjects had significantly higher antibody titers against MP, flagellin, and urease in both sera and gastric aspirates compared with the noninfected subjects. Furthermore, the antibody titers against HpaA were significantly elevated in sera but not in gastric aspirates from the infected subjects. However, no differences in antibody titers against any of the tested antigens could be detected between the duodenal ulcer patients and the asymptomatic H. pylori carriers, either in sera or in gastric aspirates.     

Polymerase chain reaction to detect infectious laryngotracheitis virus in conjunctival swabs from experimentally infected chickens

The polymerase chain reaction (PCR) was standardized and assessed as a potential diagnostic test for infectious laryngotracheitis using conjunctival swabs collected from experimentally infected chickens. Polymerase chain reaction primers based on the sequence of a 1.1-kb BamHI restriction enzyme fragment of the Ontario 1598 (Ont 1598) strain of infectious laryngotracheitis virus were selected and 300 fg of purified viral DNA were detected by ethidium bromide staining of agarose gels or 30 fg were detected by DNA hybridization. At least five different strains (Ont 1598, ATCC N-71851, LT-IVAX, Laryngo-Vac, and a local strain 322) were amplified whereas other avian pathogens and uninfected cell cultures tested negative. Swabs collected from experimentally infected chickens were analyzed by both PCR and virus isolation on various days postinfection. A comparison of virus isolation to PCR indicated that, in the mid-postinfection phase, PCR and virus isolation appeared to be comparable with a kappa value of greater than 0.8. The polymerase chain reaction was shown to be the better test later in infection, when clinical recovery had occurred.     

Polymerase chain reaction in the diagnosis of tuberculosis. Comparison of two target sequences for amplification

Amplification of a 340 bp sequence of the 38 kDa protein gene of Mycobacterium tuberculosis by the polymerase chain reaction has been developed. The sensitivity of this PCR was shown to be 10 fg both by agarose gel electrophoresis and Southern blot hybridisation being equivalent to 2-3 organisms and highly specific to M. tuberculosis and excluding even M. tuberculosis H37Ra and Mycobacterium bovis BCG. Sputum samples from patients with pulmonary tuberculosis gave a positivity rate of 45%. PCR was also performed using pt8 and pt9 primers which amplified a 541 bp sequence of IS6110. 41% of the above samples gave positive amplification. Three samples that were positive for 38 kDa sequence were negative for IS6110.