Subtype-specific polymerase chain reaction for the identification of HIV-1 genetic subtypes circulating in Africa

The association between cancer and the increased tendency for bleeding and clotting is well established. Many investigators have reported on the interaction between tumor-related and plasma-related factors that may be responsible for this predisposition in cancer patients. The precise role of any of these factors in thrombosis or bleeding remains speculative. A good understanding of the course of the underlying malignancy, mechanisms of actions of the agents used for treatment, and a high degree of clinical suspicion are required to direct practicing physicians towards an objective and timely diagnosis of these complications. The goal of this article is to provide a broad overview of a few important hemostatic problems encountered in cancer patients.     

High-resolution HLA-DQB1 typing using the polymerase chain reaction and sequence-specific primers

Polymorphism at HLA-DQB1 is known to influence tissue compatibility and disease susceptibility; however, current DQB1 typing methods are unable to distinguish the 32 currently recognized DQB1 alleles. We have developed a 32-reaction PCR-SSP method capable of differentiating all DQB1 alleles that differ in amino acid sequence. This method can resolve all heterozygous combinations of DQB1 alleles, with the exception of several combinations involving alleles not thus far detected in Caucasoid populations.     

Detecting pathogens of fetal defects in paraffin embedded tissues by polymerase chain reaction

Cerebral cavernous angiomas (CCA) are rare, reportedly accounting for only 1% of all intracranial vascular lesions and 15% of all cerebral vascular malformations. Forms are sporadic or familial, and the mode of inheritance is probably autosomal dominant. We report an unusual case of an infant born at 37 weeks of gestational age following a normal pregnancy. Her birth-weight was 1560 g. The family history was negative. At 10 months of age, the child presented with the sudden onset of muscular hypotonia, motility and strength deficits, and absence of osteotendinous reflexes in the right arm. The psychomotor development of the child was normal. MRI revealed the presence of a cavernous angioma in the paramedian pontine region. The child's monoparesis quickly disappeared. This case is interesting because of the age at onset and the way in which the clinical manifestations developed.     

Comparison of human immunodeficiency virus 1 DNA polymerase chain reaction and qualitative and quantitative RNA polymerase chain reaction in human immunodeficiency virus 1-exposed infants

The endothelial lining of the blood-brain barrier tightly controls the distribution of peptide hormones between the central nervous system and the circulation. By using primary cultures of brain microvessel endothelial cells, an in vitro model of the blood-brain barrier, we report here the uptake and transport of the octapeptide angiotensin II by a specific receptor population. With the angiotensin II antagonists losartan (AT1 specific) and PD 123,319 (AT2 specific), we showed that both the uptake and transport of angiotensin II were mediated by the AT1 receptor. Western blot analysis confirmed the existence of the AT1 receptor in our cell-culture model. Rhodamine 123 studies also suggested that both angiotensin II antagonists, but not angiotensin II, were substrates for the P-glycoprotein efflux system, thus restricting the transport of these compounds. These results suggest an AT1 receptor mediates uptake and transport of angiotensin II at the blood-brain barrier and may contribute to the regulation of cerebrovascular levels of the peptide.     

Repetitive element sequence based polymerase chain reaction for typing of Brucella strains

A collection of 40 Brucella strains, including 19 clinical B.canis isolates, was analyzed using ERIC-PCR and REP-PCR. PCR amplification using primers REP 1R-I and REP 2-I provided distinct patterns for Brucella spp. All Brucella strains could be discriminated at least to the species level. ERIC-PCR, employing ERIC 1R and ERIC 2 as primers was less discriminative for Brucella, only allowing a discrimination to the genus level with occasional discrimination between individual strains. It can be concluded that rep PCR is a promising fingerprinting method for the evaluation of an outbreak.     

The polymerase chain reaction in pathology

Membrane-fixed CD14 acts as a receptor for the protein-bound endotoxin (LPS) complex and mediates the cellular effects of endotoxin. Soluble CD14 (sCD14) is suggested to neutralize circulating LPS, i.e., acting as an endotoxin antagonist. The aim of this study was to elucidate the release of both sCD14 and endotoxin in traumatized patients, starting from the earliest phase after trauma. A total of 15 patients (O ISS = 19, 9-75) suffering major trauma were enrolled in this prospective study. Blood samples were collected as early as immediately at the site of accident, on hospital admission, and thereafter hourly, then daily. For patients (O ISS = 47) died within 24 h because of their severe injuries. Immediately after the accident as well as during the first 2 h after hospital admission, the mean sCD14 levels of surviving patients did not differ from those of healthy volunteers (n = 53). Thereafter, however, sCD14 increased continuously in the trauma group. The concentrations remained elevated throughout the entire observation period. There was, however, no relation between the sCD14 release and the pattern or the severity of injury. In contrast, endotoxin levels revealed a pattern-specific release. The highest plasma concentrations of LPS were observed in patients suffering from (additional) thoracic injury. On the basis of these results we conclude that the release of sCD14 after trauma does not reflect a strict principle such as action/reaction caused by the appearance of endotoxin immediately after the injury. Soluble CD14 is more likely release by an endotoxin-independent mechanism.     

Detection of Shigella and enteroinvasive Escherichia coli using polymerase chain reaction

Two oligonucleotide primers were used in a polymerase chain reaction (PCR) procedure to amplify a region of the invasive-associated locus (ial) of Shigella and enteroinvasive Escherichia coli (EIEC). Detection of the amplified product can be done by agarose gel electrophoresis, which is specific and sensitive enough for routine diagnosis of these two pathogens. PCR is done using DNA extracted directly from faeces. The procedure can be completed in 7 h. These findings demonstrate a novel method for rapid, sensitive, specific, and simple diagnosis of diarrhoea caused by Shigella and EIEC.     

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction

Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.     

Amplification and detection of a Y-chromosome DNA sequence by fluorescence in situ polymerase chain reaction and flow cytometry using cells in suspension

In this part of Malaysia, consent of splenectomy is virtually unobtainable, so we studied the outcome of ITP without this treatment option. Thirty-two adult patients were seen, but 7 defaulted before therapy evaluation. Of the remaining 25, 17 achieved a complete remission with prednisolone, but in only 8 was this prolonged. Twelve patients, who failed to respond to prednisolone or who required > 15 mg/day as maintenance, were offered splenectomy, but all fused. Of these 12: one has died from an intracranial haemorrhage; three others have defaulted while on no treatment with platelet counts of < 16 x 10(9)/1; one has had a baby who died from intracranial bleeding. The other seven patients have platelet counts ranging from 4 - 202 x 10(9)/1 with moderate bleeding on doses of prednisolone of 0-60 mg/day: long-term corticosteroid side-effect are evident in all but one of them. This study demonstrates that ITP patients who refuse splenectomy have a high morbidity.     

Rapid isolation of cell-type-specific protein tyrosine kinases by degenerate polymerase chain reaction combined with differential hybridization technique

To identify protein tyrosine kinase (PTK) genes preferentially expressed in renal cell carcinoma cell line, we screened a PTK-cDNA-enriched library constructed from RNA of an renal cell carcinoma cell line with a PTK probe, each produced from renal cell carcinoma, gastric cancer or esophageal cancer cell lines by degenerate polymerase chain reaction. Two cDNA fragments of PTK genes, FRK and FLT-3, were isolated from the PTK-cDNA-enriched library of the renal cell carcinoma cell line by differential hybridization technique. The FRK cDNA clone represented 15.8% of the PTK-cDNA-enriched library from the renal cell carcinoma cell line, while the FLT-3 cDNA clone was 2.8% of the same library. Both of the two PTK genes were expressed preferentially in renal cell carcinoma cell lines. This method, described here, is useful for the rapid isolation of PTK cDNA fragments, including a low abundant cDNA, preferentially expressed in a specific cell line.     

Sex identification of turkey embryos using a multiplex polymerase chain reaction

A considerable portion of the W chromosome in Gallinaceous birds consists of tandem repetitive DNA. In the turkey, a 0.4-kb PstI element is repeated about 10,000 times in the female diploid genome but is undetectable as such a unit in males. In this study a multiplex polymerase chain reaction was developed to identify the sex of turkeys based upon the PstI repeat. The technique utilized two pairs of primers, the first pair was designed to amplify a region of the PstI repetitive element, resulting in the production of a 177-bp fragment in females. The other pair was designed to amplify a region of the adenosine triphosphate (ATP) synthase gene, present in both males and females. The simultaneous use of all four primers in the same reaction resulted in the coamplification of a 177-bp and a 250-bp fragment in females and a 250-bp fragment in males. This technique was used to verify the sex of 45 adults of known sex and to identify the sex of 74 embryos from Day 5 to hatch. This procedure is rapid and permits the sexing of many embryos in a short time. The ability to sex early embryos can facilitate studies on avian sex determination.     

Using polymerase chain reaction for the detection of gonorrhoea in women and children in Taiyuan

BACKGROUND: Erythroderma may result from different causes, but a proportion remains undetermined (idiopathic erythroderma). Patients with idiopathic erythroderma have often been regarded to have a pre-Sézary syndrome because some of these patients have developed a cutaneous T-cell lymphoma during follow-up. OBJECTIVE: The aim of this study was to investigate if this was true for our group and also if it is possible to identify further which patients are at high risk of developing cutaneous T-cell lymphoma. METHODS: We analyzed clinical and follow-up data and reviewed the skin histopathology of all patients who were diagnosed with idiopathic erythroderma in our clinic between 1977 and 1994. RESULTS: Twenty-eight patients, 16 males and 12 females, were diagnosed with idiopathic erythroderma. This is 27% of the patients who were diagnosed with erythroderma in our clinic, during this period. During the median follow-up of 33 months, 35% of the patients went into complete remission and 52% showed partial remission. Three patients (13%), all females, had persistent chronic erythroderma. Two of the latter group progressed to cutaneous T-cell lymphoma, i.e. 1 to Sézary syndrome and 1 to mycosis fungoides. CONCLUSION: Based on our results we conclude that only patients with persistent chronic idiopathic erythroderma, which is a minority, have an increased risk of developing cutaneous T-cell lymphoma and therefore need a close and long-term follow-up.     

Identification of selected gamma-ray induced deficiencies in zebrafish using multiplex polymerase chain reaction

The ease with which mutations can be generated in zebrafish makes this vertebrate an important resource for developmental genetics and genome studies. We have developed a PCR-based screening method that allows the efficient identification of gamma-ray induced deficiencies targeted to selected sequences. We describe three mutants characteristic of our findings and show that these mutations include deletions and translocations that can affect as much as 1% of the genome. These deficiencies provide a basis for analyzing the functions of cloned zebrafish genes using noncomplementation screens for point mutations induced by high-efficiency chemical mutagenesis.     

Species identification of intestinal microsporidiosis in HIV-positive patients using the polymerase chain reaction

BACKGROUND: Microsporidia are opportunistic parasites which, due to their morphologic characteristics, continue presenting diagnostic problems. Species-specific identification of microsporidia has become important because of varying levels of response to albendazole, which is the only effective treatment for some kinds of intestinal microsporidiosis. Although these parasites cause up to 50% of otherwise unexplained chronic diarrhea in HIV-positive patients, the number of reported cases is still very scarce in our country when compared to the existing HIV-positive population. METHODS: Intestinal microsporidiosis in HIV-positive patients with diarrhea was investigated using the modified trichrome staining technique. Microsporidia species identification was done by indirect immunofluorescence (IIF) and polymerase chain reaction (PCR) with specific primers. RESULTS: Six new cases of intestinal microsporidiosis caused by Enterocytozoon bieneusi were diagnosed in Madrid (Spain). All patients were in an advanced state of the HIV infection and they presented CD4+ values equal or inferior to 100 x 10(6)/I. CONCLUSIONS: Due to the number of cases that are accumulating, microsporidia must be included among the enteropathogens responsible for chronic diarrhea in HIV-positive individuals in Spain. The PCR technique using specific primers is a suitable determinator of the microsporidia species implicated in this intestinal pathology.     

Diagnosis of mycotic infections in oncology patients using the polymerase chain reaction]

Diagnosis of mycotic infections is despite the immense effort devoted to this problem still very inaccurate. A new and promising method is the polymerase chain reaction, PCR, which theoretically can detect a single cell. In their original study the authors decided to develop a new method for the detection of fungi by PCR and to compare this examination with post-mortem findings. Thus it was possible to determine sufficiently reliably the sensitivity and specificity of the method. For the detection of fungi the authors selected the sequence coding for a small subunit of ribosomal RNA (18S rDNA). The method is able to detect the amount of DNA from some 10-100 cells. The sensitivity was 90% and the specificity 92%. The method is so far too laborious for common practice, its simplification would be however very useful.