Type-specific Amplification Research Articles

A Novel Next-Generation Sequencing Assay for the Identification of BCR::ABL1 Transcript Type and Accurate and Sensitive Detection of TKI-Resistant Mutations.

The clinical management of chronic myeloid leukemia (CML) patients requires the identification of the type of BCR::ABL1 transcript at diagnosis and the monitoring of its expression and potential tyrosine kinase inhibitor (TKI) resistance mutations during treatment. Detection of resistant mutation requires transcript type-specific amplification of BCR::ABL1 from RNA. In this study, a custom RNA-based next-generation sequencing (NGS) assay (Dup-Seq BCR::ABL1) that enables (a) the identification of BCR::ABL1 transcript type and (b) the detection of resistance mutations from common and atypical BCR::ABL1 transcript types was developed and validated. The assay design covers BCR exon 1 to ABL1 exon 10 and employs duplicate PCR amplification for error correction. The custom data analysis pipeline enables breakpoint determination and overlapped mutation calling from duplicates, which minimizes the low-level mutation artifacts. This study demonstrates that this novel assay achieves high accuracy (positive percent agreement (PPA) for fusion: 98.5%; PPA and negative percent agreement (NPA) for mutation at 97.8% and 100.0%, respectively) and sensitivity (limit of detection (LOD) for mutation detection at 3% from 10 000 copies of BCR::ABL1 input). The Dup-Seq BCR::ABL1 assay not only allows for the identification of BCR::ABL1 typical and atypical transcript types and accurate and sensitive detection of TKI-resistant mutations but also simplifies molecular testing work flow for the clinical management of CML patients.

Serum IgA Immune Complexes Promote Proinflammatory Cytokine Production by Human Macrophages, Monocytes, and Kupffer Cells through FcαRI–TLR Cross-Talk

IgA is predominantly recognized to play an important role in host defense at mucosal sites, where it prevents invasion of pathogens by neutralization. Although it has recently become clear that IgA also mediates other immunological processes, little remains known about the potential of IgA to actively contribute to induction of inflammation, particularly in nonmucosal organs and tissues. In this article, we provide evidence that immune complex formation of serum IgA plays an important role in orchestration of inflammation in response to pathogens at various nonmucosal sites by eliciting proinflammatory cytokines by human macrophages, monocytes, and Kupffer cells. We show that opsonization of bacteria with serum IgA induced cross-talk between FcαRI and different TLRs, leading to cell type-specific amplification of proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-23. Furthermore, we demonstrate that the increased protein production of cytokines was regulated at the level of gene transcription, which was dependent on activation of kinases Syk and PI3K. Taken together, these data demonstrate that the immunological function of IgA is substantially more extensive than previously considered and suggest that serum IgA-induced inflammation plays an important role in orchestrating host defense by different cell types in nonmucosal tissues, including the liver, skin, and peripheral blood.

Molecular epidemiology of Hepatitis C Virus (HCV) in liver disease patients in Sri Lanka

Background: Globally, hepatitis C virus (HCV) is an important cause of chronic liver disease. Genotypes of HCV are associated with different profiles of pathogenicity, infectivity, and antiviral therapy. The prevalence of HCV and distribution of HCV genotypes in Sri Lanka in comparison with the rest of Asia is not well known.Objective: The objective of the study was to investigate the presence of HCV and to genotype HCV in a group of Sri Lankan patients suspected to have liver disease.Methods: A total of 1933 samples were screened for HCV antibodies using ELISA and HCV RNA with RT-PCR methods. RNA positive samples were genotyped by type specific amplification and by DNA sequencing.Results: Out Of the 1933 liver disease patients tested 219 (11.33%) were detected to be positive for anti-HCV antibodies, out of which, 54 (24.66%) were positive for HCV RNA. Furthermore out of 49 positively tested patients, 24 (48.97%) were found to be categorised as HCV genotype 1.Conclusion: This result confirms previous observations that the contribution of HCV as a causative virus in liver disease patients is low in Sri Lanka. HCV genotype 1 was found to be the most predominant genotype in studied cohort of Sri Lankan liver disease patients. DOI: http://dx.doi.org/10.3126/ajms.v6i3.10741Asian Journal of Medical Sciences Vol.6(3) 2015 1-5

Mixed Scrub Typhus Genotype, Shandong, China, 2011

Mixed Scrub Typhus Genotype, Shandong, China, 2011

Evaluation of parasite subpopulations and genetic diversity of the msp1, msp2 and glurp genes during and following artesunate monotherapy treatment of Plasmodium falciparum malaria in Western Cambodia

BackgroundDespite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic Plasmodium falciparum msp1, msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed.MethodsBlood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) and the glutamate-rich protein (glurp) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections.ResultsConsiderable variation of msp2 alleles but well-conserved msp1 and glurp were identified. At baseline, 31% of infections were polyclonal for one or more genes. Patients with recurrent malaria were significantly more likely to have polyclonal infections than patients without recurrence (seven of nine versus 36 of 127, p = 0.004). Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases.ConclusionsThe parasites responsible for artemisinin-resistant malaria in a clinical trial in Western Cambodia comprise the dominant clones of acute malaria infections rather than minority clones emerging during treatment. Additional genotyping during therapy was not beneficial. Disproportionately high rates of polyclonal infections in cases of recurrence suggest complex infections lead to poor treatment outcomes. Current research objectives should be broadened to include identification and follow-up of recurrent polyclonal infections so as to define their role as potential agents of emerging resistance.

High-risk human papillomavirus (HPV) DNA sequences in metaplastic breast carcinomas of Mexican women

BackgroundMetaplastic carcinoma, an uncommon subtype of breast cancer, is part of the spectrum of basal-like, triple receptor-negative breast carcinomas. The present study examined 20 surgical specimens of metaplastic breast carcinomas, for the presence of high-risk Human papillomavirus (HPV), which is suspected to be a potential carcinogenic agent for breast carcinoma.MethodsMastectomy specimens from patients harboring metaplastic breast carcinoma, as defined by the World Health Organization (WHO), and who attended the Instituto Nacional de Cancerologia in Mexico City, were retrieved from the files of the Department of Pathology accumulated during a 16-year period (1995–2008). Demographic and clinical information was obtained from patients’ medical records. DNA was extracted from formalin-fixed, paraffin-embedded tumors and HPV type-specific amplification was performed by means of Polymerase chain reaction (PCR). Quantitative Real-time (RT) PCR was conducted in HPV positive cases. Statistically, the association of continuous or categorical variables with HPV status was tested by the Student t, the Chi square, or Fisher’s exact tests, as appropriate.ResultsHigh-risk HPV DNA was detected in eight (40%) of 20 metaplastic breast carcinomas: seven (87.5%) HPV-16 and one (12.5%) HPV-18. Mean age of patients with HPV-positive cases was 49 years (range 24–72 years), the same as for HPV-negative cases (range, 30–73 years). There were not striking differences between HPV + and HPV– metaplastic carcinomas regarding clinical findings. Nearly all cases were negative for estrogen, progesterone and Human epidermal growth factor receptor 2 (HER2), but positive for Epidermal growth factor receptor (EGFR).ConclusionsHigh-risk HPV has been strongly associated with conventional breast carcinomas, although the subtle mechanism of neoplastic transformation is poorly understood. In Mexican patients, the prevalence of HPV infection among metaplastic breast carcinomas is higher than in non-metaplastic ones, as so the HPV viral loads; notwithstanding, HPV viral loads show wide variation and remain even lower than cervical and other non-cervical carcinomas, making it difficult to assume that HPV could play a key role in breast carcinogenesis. Further studies are warranted to elucidate the meaning of the presence of high-risk HPVDNA in breast carcinomas.

High-Risk Human Papillomavirus DNA Detected in Primary Squamous Cell Carcinoma of Urinary Bladder

We reported previously that more than one-third (37%) of primary bladder squamous cell carcinomas (SCCs) demonstrate diffuse p16 immunoreactivity independent of gender. This observation made us question whether p16 overexpression in bladder carcinoma is due to human papillomavirus (HPV)-dependent mechanisms. To determine whether the presence of high-risk HPV (HR-HPV) DNA could be detected in these tumor cells. Fourteen cases of primary bladder SCC, which were positive for p16 by immunohistochemistry, were probed for the detection of HR-HPV by in situ hybridization and the signal amplification Invader assay. Samples positive for detection of HR-HPV by Invader assay were amplified by using HR-HPV type-specific primers, and amplification products were DNA sequenced. Detection of HR-HPV by the in situ hybridization method was negative in all cases (0 of 14). However, in 3 of 14 cases (21.4%), the presence of HR-HPV DNA was detected with the Cervista HPV HR Invader assay, which was followed by identification of genotype. All positive cases were confirmed by using HR-HPV type-specific amplification followed by DNA sequencing. Identified HR-HPV genotypes included HPV 16 (2 cases) and HPV 35 (1 case). High-risk HPV DNA is detectable in a subset of primary bladder SCCs. Based on the well-documented carcinogenic potential of HR-HPV, there is a necessity for additional studies to investigate the role of HR-HPV in bladder carcinogenesis.

Identification of sex in spiny gourd (Momordica dioica) using RAPD marker analysis and its validation in four F1 progenies

SummaryMomordica dioica is an important summer vegetable crop in India and is dioecious. DNA bulks of six male and six female accessions of M. dioica were subjected to random amplified polymorphic DNA (RAPD) marker analysis using 40 primers, of which 22 primers responded for amplification. Of these, ten primers generated reproduction typespecific amplification of DNA fragments at the bulk level. This number was reduced to three RAPD primers when re-tested, individually, with six male and six female accessions. One random decamer primer, OPA-01, produced two unique amplified fragments in all female plants, while two other primers, OPB-04 and OPC-08, generated distinct amplified fragments in both male and female plants. Three amplified fragments (OPB-04430, OPC-081135, and OPC-08578) were unique to all six male accessions, while five amplified fragments (OPA-011560, OPA-01880, OPB-04880, OPB-04800, and OPC-081230) appeared uniquely and uniformly in all six female accessions. Southern hybridisation analysis was performed using five of these sex-specific amplified fragments (OPA-01880, OPB-04880, OPB-04430, OPC-08578, and OPC-081230) as probes with Eco RI and Hind III double-digested DNA of all male and female accessions. Southern hybridisation analysis with the OPC-08578 probe showed a unique hybridisation pattern for all six male accessions, as with the respective RAPD marker, whereas the other probes showed complex hybridisation patterns. Further, inheritance studies also confirmed the presence of a homologous restriction fragment targeted by this probe in F1 progenies, as expected. Hence, OPC-08578 could be used as a marker to ascertain the sex of M. dioica plants at an early stage of development.

Distribution of HPV genotypes in women with cervical cancer in Auckland, New Zealand; a review of 50 specimens between 2000-2006

In New Zealand, around two hundred women are diagnosed with cervical cancer annually, with approximately seventy deaths from cervical cancer per year. Our aim was to determine the distribution of oncogenic HPV genotypes in biopsy specimens from women with diagnosed cervical cancers in the Auckland region of New Zealand between 2000-2006. Confirmed cases of cervical carcinoma were identified from the local pathology register, and representative tissue samples were taken from these blocks. Sections were deparaffinised, and DNA was extracted according to standard protocols. Samples were subject to PCR amplification using L1 consensus primer sets MY09/11 and GP5/6. Further type-specific amplification was performed on positive samples, using an in-house primer sequence based on target sequences within the E6 gene. Remaining samples were typed by a Linear Array Assay, or by DNA sequencing. HPV DNA was detected in 100% of cases. In 49/50 samples, the HPV genotype was identified, with a total of 14 different HPV genotypes detectable. Together HPV-16 and 18 were found in 41/49 cases (83.6%) either singly or in combination. Our findings suggest that the distribution of HPV genotypes in New Zealand is similar to that of other geographic areas. Ongoing surveillance is warranted to ensure appropriate genotype selection for prophylactic HPV vaccinations.

Type-specific Amplification of 5S rRNA from Panax ginseng Cultivars Using Touchdown (TD) PCR and Direct Sequencing

Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing has often troublesome and needs more time to analyze for many samples. Since touchdown (TD) PCR can generate sufficient and highly specific amplification, it reduces unwanted amplicon generation. Accordingly, TD PCR is a good method for direct sequencing due to amplifying wanted fragment. In plants the 5S-rRNA gene is separated by simple spacers. The 5S-rRNA gene sequence is very well-conserved between plant species while the spacer is species-specific. Therefore, the sequence has been used for phylogenetic studies and species identification. But frequent occurrences of spurious bands caused by complex genomes are encountered in the product spectrum of standard PCR amplification. In conclusion, the TD PCR method can be applied easily to amplify main 5S-rRNA and direct sequencing of panax ginseng cultivars.

Detection and differentiation of human papillomavirus genotypes HPV-6 and HPV-11 by FRET-based real-time PCR

A real-time PCR (RT-PCR) assay was developed based on fluorescence resonance energy transfer (FRET) hybridization probe technology, allowing very sensitive and specific detection of HPV-6 and HPV-11, reliable differentiation of HPV-6 and HPV-11, as well as prototypic and non-prototypic HPV-6 genomic variants, in a single PCR reaction. The primers and probe were designed on the basis of multiple alignments of 74 HPV-6 E2 gene sequences and 20 HPV-11 E2 gene sequences. Testing on defined plasmid standards showed that the RT-PCR allowed simple and reliable identification of HPV-6 and HPV-11 using type specific amplification followed by probe-specific post-amplification dissociation analysis. Sensitivity, assessed by probit analysis at a 95% detection level, was 42.9, 43.4, and 25.3 DNA copies per assay for prototypic and non-prototypic HPV-6 variants and HPV-11, respectively. The results obtained by the developed assay on 51 HPV DNA-negative samples and 149 HPV DNA-positive samples, including 81 HPV-6 positive samples (19 prototypic and 62 non-prototypic HPV-6 variants), 28 HPV-11 positive samples, 10 samples of HPV-44 and HPV-74 (the closest relatives of HPV-6 and HPV-11) and 30 samples of 15 other important alpha HPV, showed complete agreement with those obtained with the INNO-LiPA human papillomavirus (HPV) Genotyping Assay and HPV-6 E2 and E6 gene sequencing.

Homogeneous Amplification of Genital Human Alpha Papillomaviruses by PCR Using Novel Broad-Spectrum GP5+ and GP6+ Primers

Human papillomavirus (HPV) DNA detection and typing are important for diagnosis and management of HPV-associated diseases. One of the most commonly used PCR methods, GP5+/6+, shows weaknesses in amplifying certain types. To circumvent this limitation, we developed and validated broad-spectrum primers targeting the GP5+/6+ region. The addition of eight upstream and two downstream BSGP5+/6+ (BS) primers improved amplification of plasmids of 14 genital HPV types 10- to 1,000-fold versus GP5+/6+ PCR without altering sensitivity for the 10 others. For these 24 types, an analytic sensitivity of < or = 1,000 plasmid copies in the presence of 100 ng cellular DNA was obtained. Additionally, we integrated an internal beta-globin PCR into both HPV PCR systems, allowing simultaneous DNA quality control without affecting the sensitivity of HPV detection. Furthermore, we describe five additional low-risk HPV probes used in multiplex HPV genotyping (MPG) for simultaneous identification of all 15 high-risk, 3 putative high-risk, and 9 low-risk HPV genotypes. The performance of BSGP5+/6+ multiplexed with beta-globin primers was compared to that of standard GP5+/6+ with DNA from 1,112 cervical scrapings. There was 79% overall agreement (kappa = 0.816). BSGP5+/6+ was significantly more sensitive than GP5+/6+ for detection of HPV 30, 39, 42, 44, 51, 52, 53, 68, 73, and 82, detecting 212 additional HPV infections and increasing the proportion of multiple infections from 17.2 to 26.9% in cancer patients. In conclusion, BSGP5+/6+ multiplexed with beta-globin PCR provides an improvement in type-specific amplification sensitivity and homogeneity compared to GP5+/6+ and offers simultaneous internal control of DNA quality. BSGP5+/6+-MPG, therefore, is suitable for epidemiologic and also diagnostic applications.

Detection and characterization of feline Bartonella henselae in the Czech Republic

The aims of the study were to characterize isolates of Bartonella henselae and to determine the prevalence of bacteremic domestic cats in urban and suburban parts of Prague, Czech Republic. Five (18%) gram-negative fastidious bacterial single-cat isolates were recovered from 27 hemocultures incubated without previous freezing. Four of these isolates originated from flea infested stray cats ( n=6) and one from a shelter cat without any ectoparasites ( n=21). None of the 34 previously frozen specimens from flea free pet cats yielded any bacteria. All five isolates were catalase and oxidase negative. Their enzymatic activity, RFLP profile of citrate synthetase gene ( gltA) and DNA–DNA hybridization results were typical of B. henselae. According to their PvuII and BglI ribotypes the isolates could be allocated to two homogeneous groups. Ribotype HindIII and RFLP of 16S–23S rRNA spacer region analysis gave unique profiles different from those of Bartonella quintana, Bartonella elizabethae and Bartonella clarridgeiae. The 16S rRNA type-specific amplification revealed an identical profile typical of B. henselae genotype II for all the cat isolates studied. Pulsed-field gel electrophoresis (PFGE) assigned a different profile to each of the isolates studied. Determination of the enzymatic activity, RFLP of gltA gene, RFLP of 16S–23S rRNA spacer region, and HindIII ribotype could be efficient tools for identification of B. henselae isolates. Ribotyping ( PvuII, BglI), 16S rRNA typing and PFGE may be useful methods to prospect ecology and epidemiology of the agent.

Human papillomavirus genotypes in rural Mozambique

We studied the genotype distribution of cervical human papillomavirus (HPV) infections in an age-stratified sample of 262 women in Mozambique using the PGMYO9-PGMY11 primer system in a reverse line-blot strip-based assay with high sensitivity in type-specific amplification. Despite the low precision of the estimates, we found that HPV-16 was not the dominant type. Instead, HPV 35 was the most commonly identified genotype among HPV-positive women (16/96 [17%]) and women with cervical neoplasia (7/23 [30%]). Certain genotypes might have been under-detected in previous studies, and type-specific HPV distributions might vary across populations. Therefore, the estimated proportion of cervical neoplasia that could be prevented by an HPV-16-based vaccine could be lower than expected.

Improved amplification of genital human papillomaviruses.

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi(2) = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40. 0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.

Prevalence of mixed infection by different hepatitis C virus genotypes in patients with hepatitis C virus—related chronic liver disease

Multiple infection by different hepatitis C virus (HCV) genotypes may be of great clinico-pathologic interest. In this study we determined the effective prevalence of coinfections by two or more HCV genotypes in 213 subjects with HCV-positive chronic hepatitis by using genotype-specific polymerase chain reaction (PCR), genotype-specific probe hybridization, and direct sequencing. The most prevalent genotype was HCV-1b (54%). HCV-2 (a/c) was also prevalent (27%), and types 1a and 3a were found in 5% and 3% of patients, respectively. A mixed infection was detected in 23 patients (10.8%): 4 out of 23 were coinfected by types 1a + 1b, while the remaining 19 patients had a 1b + 2 (a/c) mixed infection. Further analysis based on restriction fragment length polymorphism (RFLP) on type-specific PCR products was used to verify genotyping results. Only four coinfections (1a + 1b in 2 patients and 1b + 2 (a/c) in the remaining 2 patients, respectively) were confirmed by enzyme cleavage. All patients with true coinfection had long-lasting infection and liver cirrhosis. Both true and false mixed infections resulting from RFLP analysis were confirmed by direct sequencing of type-specific amplification products. We also determined a recurrent CT transversion at position 618 in all sequenced samples. In 4 cases another point mutation (GA at position 626) was found, reducing the number of mismatches between HCV-2 and HCV-1b from 4 to 3 (or 2). Interestingly, all HCV-2 isolates sequenced showed the highest degree of nucleotide homology with HCV-2 subtype c, confirming the relatively high prevalence of this subtype in Italy. In conclusion, we showed the possibility of multiple infection by different HCV types in the general population of chronically infected patients without particular risk factors, even if in a low percentage of cases. Further studies are needed to assess the clinical relevance of chronic HCV infection with multiple genotypes.

Detection of Herpes Simplex Virus in Genital Specimens by Type-Specific Polymerase Chain Reaction

Viral isolation is the standard method for the detection of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in clinical specimens. This study describes the development of a type-specific polymerase chain reaction (PCR) assay for detection and typing of HSV-1 and HSV-2, and a comparison of its sensitivity with that of isolation in a clinical setting. Specimens from patients presenting with genital ulcers were tested for the presence of HSV by both methods. Oligonucleotide primers were selected to enable type-specific amplification of HSV-1 and HSV-2 DNA. Conditions were optimized to allow detection and typing from a single reaction tube using a multi-primer PCR method. When compared with PCR, the sensitivity of isolation was 67% and the specificity 97%. This protocol allowed rapid, sensitive and accurate detection and typing of HSV with a single PCR assay.

Detection of herpes simplex virus in genital specimens by type-specific polymerase chain reaction

Viral isolation is the standard method for the detection of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in clinical specimens. This study describes the development of a type-specific polymerase chain reaction (PCR) assay for detection and typing of HSV-1 and HSV-2, and a comparison of its sensitivity with that of isolation in a clinical setting. Specimens from patients presenting with genital ulcers were tested for the presence of HSV by both methods. Oligonucleotide primers were selected to enable type-specific amplification of HSV-1 and HSV-2 DNA. Conditions were optimized to allow detection and typing from a single reaction tube using a multi-primer PCR method. When compared with PCR, the sensitivity of isolation was 67% and the specificity 97%. This protocol allowed rapid, sensitive and accurate detection and typing of HSV with a single PCR assay.

Cervical cancer in older women: A molecular analysis of human papillomavirus types, HLA types, and p53 mutations

OBJECTIVE: The purpose of this study was to evaluate cervical cancers in older women to determine whether they differed from tumors in younger women with respect to human papillomavirus types, frequencies of p53 mutations, and presence of a proposed high-risk HLA-DR2 haplotype. STUDY DESIGN: Cervical tissue was obtained from women undergoing surgical treatment of in situ or invasive carcinoma of the cervix. Viral and genomic deoxyribonucleic acid was extracted. The presence of human papillomavirus deoxyribonucleic acid was detected by polymerase chain reaction amplification. Viral subtypes were assigned by means of a combination of type-specific amplification and automated sequencing of the L1 region. The presence of p53 mutations was evaluated by direct sequencing of exons 5 through 9. The HLA-DR locus was screened for the presence of the high-risk DRB1*1501 allele by means of selective polymerase chain reaction amplification followed by agarose gel electrophoresis of HLA-DR2 types. RESULTS: Tumors from 39 women 62 to 85 years old were analyzed. Tumors from 104 younger women formed a reference group. Human papillomavirus 16 was found in 41% and 54% and human papillomavirus 18 was found in 10% and 12% of the tissue samples from older and younger women, respectively. The overall distributions of human papillomavirus types did not differ statistically between the groups. One of the 25 older patients tested had a p53 mutation. This tumor also had a positive test result for human papillomavirus 18. The DR*1501 allele was present in 33% of the older patients and 28% of the younger patients. The expected frequency of this allele in white Americans is 19.8%. The increased frequency of this allele among both older and younger women with cervical cancer was statistically significant ( P < .05). CONCLUSIONS: We hypothesized that cervical cancer in older women might differ from that in younger women with respect to human papillomavirus types, natural host immunity, or the frequency of nonviral origins of the cancer. The findings show, however, that tumors from older women are extremely similar to those from younger women with respect to the human papillomavirus types present and the infrequent occurrence of p53 mutations. In addition we found that an HLA-DR allele that is associated with a risk of cervical cancer in younger women is also associated with risk in older women. These findings are most consistent with a model similar to that in younger women but with an unusually long latency for the transforming effect of the virus in some hosts. (Am J Obstet Gynecol 1998;179:56-61.)

A three-step allele-level DRB1-DRB3-DRB4-DRB5 genotyping assay using polymerase chain reaction with immobilized sequence-specific oligoprobes.

A three-step reverse hybridization assay for allele level-resolution DRB1-DRB3-DRB4-DRB5 genotyping is described. Samples are initially amplified using a generic primer pair for all DRB1-DRB3-DRB4-DRB5 alleles and PCR products are hybridized to generic typing membranes. An intermediate-resolution level genotyping is obtained at this point. Depending on the phenotype, samples are then subjected to a DR1, DR2, DR4, DR52A, DRB3 and/or DRB5 type-specific amplification and hybridization. A third step, involving sequence-specific PCR followed by type-specific hybridization, is only performed to solve certain DR4 and DR52A heterozygous combinations. The assay allows 100% allele level-resolution DRB genotyping. Hybridization membranes contain panels of SSO probes that were optimized to all react specifically under identical stringency conditions. A computer program was written to assist in analysis of the hybridization patterns. The assay was throughly evaluated and has been used to type over 10,000 donors from the Canadian Unrelated Bone Marrow Donor Registry (UBMDR) at allele level-resolution. This method proved to be flexible, easy to update for newly described alleles, easy to perform, fast, and safe. It is also reliable and specific, as 9 novel DRB alleles so far have been detected as aberrant hybridization patterns.