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1.
Human herpesvirus (HHV)--6 infections are ubiquitous, but infection or reactivation under immunocompromised conditions, such as bone marrow or solid organ transplantation, can often result in serious clinical manifestations. Two HHV-6 subtypes are known. Most primary HHV-6 infections are caused by subtype 6B, but little information is available about the prevalence, distribution, and clinical divergence of 6A and 6B. To study this, we have developed a highly sensitive and specific real-time polymerase chain reaction (PCR) assay that can detect, quantitate, and reliably differentiate HHV-6A and -6B in clinical specimens. Exploiting a single-base variation in the DNA polymerase gene of these respective subtypes, we used melting curve analysis for subtype discrimination. Moreover, this assay's ability to discriminate HHV-6 subtypes was confirmed by PCR/restriction fragment length polymorphism analysis of the HHV-6 large tegument protein gene and PCR amplicon size-discrimination analysis of the HHV-6 immediate-early gene. Using this assay, we present our findings about the prevalence and distribution of these subtypes in bone marrow transplant patients. Of 803 plasma specimens tested from 353 patients, 136 specimens (17%) from 60 patients were determined to be HHV-6 positive. We analyzed these HHV-6--positive patients for subtype identification by using our newly developed assay and determined that 58 patients (97%) were HHV-6B positive and 2 patients (3%) were HHV-6A positive. No patient was coinfected with both subtypes. This assay can be a sensitive, genotype-specific, rapid method to reliably diagnose life-threatening HHV-6 infections in immunocompromised patients and can be useful in guiding and monitoring specific therapy.  相似文献   

2.
Human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6), and human herpesvirus-7 (HHV-7) DNA in peripheral blood leukocytes (PBL) of 61 bone marrow transplant recipients was monitored weekly during the first 12 weeks post-transplantation by a nested polymerase chain reaction (PCR). Thirty-seven (61%), 17 (28%), and 32 (53%) of patients had one or more PBL specimens positive for HCMV, HHV-6 or HHV-7 DNA, respectively. HHV-7 DNA in PBL during the early post-transplant period was associated with a longer time to neutrophil engraftment (mean 28.8 days vs 19.8 days; P = 0.01). In two patients who failed to engraft, HHV-6 DNA and HHV-7 DNA was detected in plasma and PBL, respectively, early in their post-transplant period. Patients with HCMV disease were more likely to have concurrent HHV-7 DNA in PBL prior to onset of disease than were patients with asymptomatic HCMV infection, suggesting that HHV-7 may be a cofactor in the progression from HCMV infection to HCMV disease. In the 17 patients (179 specimens) in whom viral DNA in plasma was studied (in addition to PBL), a positive result was found only in 3. In each, viral DNA in plasma appeared to correlate with clinically significant disease. HHV-7 DNA in plasma was associated with encephalitis in an allograft recipient. J. Med. Virol. 53:295–305, 1997. © 1997 John Wiley & Sons, Inc.  相似文献   

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In order to evaluate the possible reactivation of human herpesviruses 6 (HHV-6) and 7 (HHV-7) after heart transplantation, buffy-coat and plasma specimens from 21 transplant patients and 56 healthy blood donors were examined for HHV-6 and HHV-7 DNA by polymerase chain reaction. Human herpesvirus 6 and HHV-7 infection or reactivation has been suggested to play a role in cytomegalovirus disease progression in renal transplant recipients. In the present study, however, no significant difference in the prevalence of HHV-6 and HHV-7 was found between the immunosuppressed and the healthy population; moreover, no viral reactivation was found in the heart transplant recipients.  相似文献   

6.
The detection of human herpesvirus 6 (HHV-6) DNA was carried out in throat swabs of adults and children by the polymerase chain reaction, and isolation of virus was also attempted from peripheral mononuclear cells. Although virus was isolated only from peripheral blood mononuclear cells of infants with exanthem subitum, HHV-6 DNA was detected in one of 30 healthy adults (3%), two of nine adults (22%) with common cold, two of 10 infants (20%) with exanthem subitum, four of 39 febrile children (10%) with antibody to HHV-6 (including three of 10 infants aged under 1 year old, and one of 29 children aged over 1 year old). However, HHV-6 DNA was not detected in samples from healthy neonates.  相似文献   

7.
Summary We explored the prevalence of human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) infections in 16 renal transplant recipients and 16 healthy controls by virus isolation, serology, polymerase chain reaction (PCR) followed by dot blot hybridization. HHV-6 variant A was isolated from one renal transplant recipient. Seven patients (44%) and six controls (38%) had HHV-6 variant B DNA in their peripheral blood mononuclear cells. The prevalence of HHV-7 DNA was found to be the same in patients and controls (19%).  相似文献   

8.
After primary infection in early life, human herpesvirus 6 (HHV-6) remains latent in the body and may reactivate in subjects with poor immune status. A 180-day longitudinal study of HHV-6 infection was carried out in 23 autologous bone marrow transplant recipients to evaluate reactivation of HHV-6; two of these patients underwent a double transplant. The patients were monitored prospectively for HHV-6 DNA in peripheral blood mononuclear cells (PBMC) by hot start nested PCR. Positive samples were typed by the enzymatic restriction protocol. Positive plasma samples were also tested for HHV-6 DNA. Antibodies against HHV-6 were measured by immunofluorescence. Five and two out of 23 patients had intermittent and persistent positivity to HHV-6 DNA in PBMCs, respectively; four patients carried variant B, and the other three patients both A and B. None of the respective plasma samples were positive. Two patients were positive for HHV-6 antibodies. Since the significance of HHV-6 DNA in PBMCs is unclear, these findings do not necessarily indicate active infection but may be due to mild immunosuppression in autologous BMT recipients.  相似文献   

9.
This report describes a PCR-based typing protocol for the HLA-A polymorphism. Locus-specific primers selectively amplified HLA-A sequences from exon 1 to exon 3 in a single PCR that avoided co-amplification of other classical and nonclassical class I genes. The allelic variation in exons 2 and 3 of the HLA-A gene was examined with a set of 44 oligonucleotide probes. According to the recognized HLA-A sequences the protocol is potentially able to distinguish all known HLA-A alleles with unique nucleotide sequences in this gene region. The related HLA-A genotypes can also be identified in both homozygous and heterozygous individuals. Thus the protocol provides the highest resolution for HLA-A typing. The PCR-SSO typing technique is accurate, reliable, and particularly suitable for a large number of samples. The DNA typing results from 42 Tenth IHWS B-cell lines are compatible with the serologic and IEF definitions. Sixty-six unrelated donors from a northern Chinese population were also tested, with 16 HLA-A alleles detected. Four subtypes of HLA-A2 were found in this population. The distribution of HLA-A subtypes in the population indicated that 40% of donor-recipient pairs thought to be matched for HLA-A by serology would be mismatched. Two novel HLA-A alleles were identified by unusual oligonucleotide hybridization patterns.  相似文献   

10.
We used the polymerase chain reaction and primers corresponding to three regions of the human cytomegalovirus (HCMV) genome to study HCMVs isolated from 16 children attending a single day-care center and the father of two children in the same center. When we analyzed isolates with primers for the pp65 and major immediate-early genes, we observed nearly uniform amplification yielding products of predicted sizes. By contrast, primers for the a sequence demonstrated variability among HCMV strains, supporting the use of these primers as an epidemiologic tool. Analysis of a-sequence products from two isolates demonstrated 50 to 70% nucleotide homology with the a sequence of HCMV Towne strain DNA. We observed 95% nucleotide homology for the two a-sequence products derived from the father-child pair. Analysis of day-care center isolates indicated that two children excreted two distinct HCMV strains during the study interval.  相似文献   

11.
Human herpesvirus-6 (HHV-6), a T-lymphotropic double-stranded DNA virus highly endemic in human populations, has been suggested to play a possible role in the development of lymphoid neoplasms, especially Hodgkin's disease. To investigate this point, we evaluated the presence and distribution of HHV-6 DNA by Southern blot, nested polymerase chain reaction, and in situ hybridization in a series of lymphoproliferative disorders including 73 Hodgkin's disease cases, 15 non-Hodgkin lymphomas, and 19 reactive lymph nodes. A high prevalence of HHV-6 infection was observed within the Hodgkin's disease category by polymerase chain reaction (38 of 52, 73%) and in situ hybridization (47 of 57, 82.4%); however, a similar prevalence was found in non-Hodgkin's lymphomas (10 of 15, 66.6%) and reactive lymph nodes (13 of 19, 68.4%). In no case did Southern blot detect viral DNA, suggesting that the neoplastic tissue contained a low number of HHV-6 copies. In situ hybridization showed that the HHV-6 positivity was restricted to lymphocytes, whereas Hodgkin and Reed-Sternberg cells were consistently negative. Immunohistochemical staining with specific monoclonal antibodies against viral structural proteins was also negative, indicating the absence of a productive infection. No relationship was observed between HHV-6 positivity and histological type, clinical parameters, and outcome of the disease. In the same series, a high proportion of cases (39 of 52, 75%) showed the presence of the Epstein-Barr virus (EBV) genome by polymerase chain reaction; In situ hybridization for Epstein-Barr-virus-encoded small RNA and immunohistochemical detection of latent membrane protein-1 gave similar results (73.6% of positive cases with both methods). In 54.9% of the cases, both sequences of HHV-6 and Epstein-Barr virus DNA were found, suggesting that a synergism of the two viruses may occur. However, the lack of detectable HHV-6 DNA in Reed-Sternberg and Hodgkin's cells seems to argue against such an interpretation. Based on these results, HHV-6 does not appear to play a specific role in the pathogenesis of Hodgkin's disease.  相似文献   

12.
Kim O  Kim SS 《Acta virologica》2003,47(2):87-90
Polymerase chain reaction (PCR) is a powerful technique of detecting Human herpesvirus 8 (HHV-8), but has a limited sensitivity and specificity. A new assay of HHV-8 based on combination of PCR with dot blot hybridization (DBH) was developed and evaluated for its sensitivity and specificity. An HHV-8-specific primer pair, ORF26out was used for amplification of target DNA. When the PCR product was detected visually the limit of detection was 0.1 ng DNA isolated from HHV-8-infected BC-3 cells. For DBH, the DNA amplified with the primer pair ORF26in specific for HHV-8 was labeled with digoxigenin (DIG). This DIG-labeled probe was capable of detecting 1.0 ng of DNA isolated from HHV-8-infected BC-3 cells. On the other hand, PCR combined with DBH (PCR/DBH) was more sensitive than PCR or DBH alone and also very specific. The sensitivity of PCR/DBH was higher than that of PCR and DBH alone. The PCR/DBH assay can be applied efficiently to confirm the presence of HHV-8 in clinical samples and to differentiate specifically HHV-8 infection from other viral infections.  相似文献   

13.
AIMS--To investigate adenovirus pulmonary infections in bone marrow transplant (BMT) recipients. METHODS--Formalin fixed, paraffin wax embedded lung tissue was examined from 13 necropsy cases after BMT using PCR and in situ hybridisation to detect adenovirus DNA. The E1A region of the adenoviral genome was targeted for PCR. In situ hybridisation was performed only in the PCR positive cases. RESULTS--Of the 13 lung specimens analysed, nine cases were negative for adenoviral nucleic acid. Four (30%) PCR and two (15%) in situ hybridisation positive cases were found. In some of the patients there were clinical and pathological indications that some diseases might be associated with adenovirus infection--haemorrhagic cystitis (three cases); necrotising pneumonia (one case). In necrotising pneumonia in which no pathogenic agents had been shown by conventional histological study, the in situ hybridisation technique showed positive staining for adenovirus. In a patient who died of renal failure caused by adenovirus nephritis, both PCR and in situ hybridisation were positive in the lung as well as in the kidney, although no histological change was found. Two PCR positive cases lacked positive sites for adenovirus by in situ hybridisation. CONCLUSIONS--The combination of PCR and in situ hybridisation could be useful for diagnosing adenovirus infection of the lung in BMT recipients. These results provide a basis for exploring further the clinical use of PCR and in situ hybridisation to diagnose adenovirus infection.  相似文献   

14.
We have used group-specific DNA amplification and sequence-specific oligonucleotide probe (SSOP) hybridization to study DRB1 sequence polymorphisms associated with DR3, DRw11(5), DRw12(5), DRw13(w6), DRw14(w6) and DRw8 alleles. Group-specific amplification of DRw52-associated DRB1 alleles was achieved using a 5' amplification primer designed to hybridize with a first hypervariable region (HVR) sequence common to all known alleles in this group, together with a 3' intron primer. Prospective SSOP typing of DR3, DRw11, DRw12, DRw13, DRw14 and DRw8 alleles was performed in 318 individuals, including 124 patients, 46 family members and 148 unrelated marrow donors. Among the 395 DRw52-associated DRB1 alleles tested in our study, a subtype corresponding to the previously defined alleles DRB1*0301-2 (DR3), DRB1*1101-4 (DR5), DRB1*1201-2 (DR5), DRB1*1301-5 (DRw6), DRB1*1401-2 and 1404 (DRw6), and DRB1*0801-4 (DRw8) could be assigned in all but 6 individuals (1.9%) tested. In addition to the 22 known alleles, we identified two new DRw6-associated alleles, DRB1*13.MW(1) and DRB1*14.GB(1). DRB1*13.MW typed serologically as DRw13 and was identical to DRB1*1301 except at codon 71 where AGG encodes arginine instead of GAG encoding glutamic acid. DRB1*14.GB represents a DRB1*1402 variant whose sequence at codon 86 encodes valine (GTG) instead of glycine (GGT). These results demonstrate that SSOP methods represent an efficient and precise approach for typing DRB1 alleles and for identifying potential novel variants previously unrecognized by conventional typing methods.  相似文献   

15.
Eight human herpesvirus 6 (HHV-6) strains were studied by Southern blot and polymerase chain reaction. DNA from infected cells was digested by a panel of restriction enzymes and hybridized with cloned BamHI fragments corresponding to about 30% of the HHV-6 strain SIE genome. In parallel, this DNA was amplified by polymerase chain reaction using pairs of primers derived from the strain SIE nucleotide sequence. Subsequently, amplification products were analyzed by hybridization, digestion with restriction endonucleases, and partial nucleotide sequencing. Overall results indicated that all strains were closely related to one another. However, concordant differences in restriction patterns allowed at least two groups to be distinguished, typified by strains SIE and HST, respectively. Differences between the two groups were found to reflect a limited number of punctual changes in nucleotide sequences. These results strengthen the idea of a unique HHV-6 species with genetic polymorphism. In addition, this study provides useful markers for the diagnosis and molecular epidemiology of HHV-6 infections.  相似文献   

16.
Preliminary studies of RAS mutational activation in human testicular germ cell neoplasms have yielded conflicting results. Whereas two studies of clinical material revealed a significant incidence of N- and KRAS mutations, two studies of a variety of germ cell lines failed to document RAS mutations. To clarify the incidence of RAS mutations in these tumors, we studied archival paraffin-embedded, formalin-fixed orchiectomy specimens from 25 nonseminomas (NSGCT), 18 seminomas (SEM), and one Leydig cell tumor. For 14 of the 44 neoplasms, DNA was also available from nonmalignant testis adjacent to the tumor. Six age-matched patients had testes removed because of nonmalignant disease and were studied as controls. Polymerase chain reaction (PCR) amplified the K-, N-, and HRAS 12, 13, and 61 codons of these specimens, and mutations were detected with mutation-specific oligonucleotide probe hybridization of Southern and slot blots. Four mutations were found in KRAS 12 (4/44;[9.1%]). One seminoma [1/18(5.6%)] contained the mutation GGT(GLY)----CGT(ARG), and three NSGCT [3/25(12%)] were found to have GGT(GLY)----GAT(ASP) mutations. One of the NSGCT mutations was detected in adjacent nonmalignant tissue, but the corresponding tumor did not contain any detectable mutation. No mutations were detected at KRAS 13 or 61, in NRAS or HRAS 12, 13, or 61, or in the control normal testes. PCR, slot blots, and hybridizations were performed twice by two separate investigators for confirmation of results. PCR-generated mutation-specific positive controls were created for all possible RAS mutations, and these along with wild-type DNA controls were integral to interpretation of the oligonucleotide mismatch hybridization assay. By using positive and negative controls, we have detected a relatively low incidence of RAS mutations in archival human testicular germ cell tumors.  相似文献   

17.
BACKGROUND: Respiratory viruses cause severe infections in lung transplant recipients, which require rapid and accurate diagnosis for appropriate management. OBJECTIVES: To evaluate the added benefit of a multiplex PCR for respiratory viruses (influenza [FLU] A and B, respiratory syncytial virus [RSV] A and B and parainfluenza virus [PIV] 1, 2, and 3) complementing rapid respiratory viral culture (RRV) and FLU-A antigen detection (EIA) in this transplant population. RESULTS: Over 6 months, 116 nasal washes and bronchoalveolar lavages, obtained from 72 lung transplant recipients with symptoms of upper or lower respiratory tract infections, were tested in real time by RRV and FLU-A EIA, and batched frozen by PCR. One or more methods recognized a respiratory virus in 31 (27%) specimens, including 15 FLU-A, nine RSV and seven PIV. PCR identified 26 of 31 positive samples demonstrating a sensitivity of 84%, higher than RRV (67%) or EIA (54%). PCR, RRV and EIA detected 60, 80 and 54%, respectively, FLU-A samples. PCR and RRV were equivalent for RSV-A, PIV-2 and 3, but PCR found a significantly higher number of RSV-B and PIV-1. CONCLUSIONS: These data indicate that routine use of PCR will enhance the number and speed with which viral respiratory tract infections are diagnosed in lung transplant recipients.  相似文献   

18.
Cardiac biopsy samples taken from transplant recipients around the time of primary toxoplasma infection were investigated by conventional histology and amplification of the P30 gene of Toxoplasma gondii by the polymerase chain reaction (PCR). Toxoplasma was detected more frequently by PCR than histology which may reflect the enhanced sensitivity of the former technique. Further studies are required to determine the optimal amount of tissue which should be examined by each technique and to develop a PCR assay capable of distinguishing between quiescent infection and active toxoplasmosis.  相似文献   

19.
Sera from 50 orthotopic liver transplant recipients were examined for antibodies to human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV), and the findings correlated with the clinical condition of the patients. Both primary and secondary HHV-6 infections were detected serologically following liver transplantation. Interpretation of serological assays is complicated by CMV and HHV-6 antibody cross reactions which were common. Sera from 5 patients became HHV-6 antibody negative following absorption with CMV infected cells. Thirty patients were initially seronegative for HHV-6 antibodies, 12 remained so following transplantation, 5 developed cross reacting antibodies, and 13 seroconverted. The seroconversions occurred at 4 to 8 weeks post-transplant in the same time period as CMV antibody rises. HHV-6 IgM was detected in only 4 of the 13. Of the 7 patients who had serological evidence of active HHV-6 infections but no evidence of CMV infection, 4 (56%) had fever, 1 (14%) hepatitis, 1 (14%) lung dysfunction, and 3 (42%) neurological disorders. In the 12 patients who remained HHV-6 antibody negative, there were fewer fevers and neurological disorders.  相似文献   

20.
Viremia in hepatitis A viral (HAV) infection is said to be limited to pre-symptomatic period. However, it is not clear how long viremia lasts in human infection due to the lack of a simple and sensitive detection system. In an attempt to find HAV genome in patients′ sera, we used the RTPCR method by setting a pair of primers in the 5′ non-coding region. While in most cases HAVRNA was detected only before alanine aminotransferase (ALT) reached peak levels with this sensitive system, the viral genome was observed in some patients′ sera even after ALT reached peak levels. Some patients also had HAV viremia after seroconversion to HAV antibody. These results show that viremia in HAV infection lasts longer than has been previously thought, and give a warning of possible secondary blood-borne infection. © 1993 Wiley-Liss, Inc.  相似文献   

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