Line Blot Assay Research Articles

Evaluation of a Modified Reverse Line Blot Assay for Detection and Typing of Human Papillomavirus

Detection and typing of human papillomaviruses (HPVs) are requested by clinicians with growing frequency as part of the overall management of patients. A new method using consensus L1 amplification and a reverse line blot hybridization detection method has been described for broad spectrum HPV genotype identification. This method involves hybridization at 53 degrees C in a shaking water bath, which many laboratories might find cumbersome. We describe a modified hybridization step, using a dry-air incubator, that simplifies the detection protocol. Overall, comparable results were obtained by the modified, more easily performed protocol, indicating that laboratories might validate and use this modified method.

Evaluation of a Modified Reverse Line Blot Assay for Detection and Typing of Human Papillomavirus

Detection and typing of human papillomaviruses (HPVs) are requested by clinicians with growing frequency as part of the overall management of patients. A new method using consensus L1 amplification and a reverse line blot hybridization detection method has been described for broad spectrum HPV genotype identification. This method involves hybridization at 53 degrees C in a shaking water bath, which many laboratories might find cumbersome. We describe a modified hybridization step, using a dry-air incubator, that simplifies the detection protocol. Overall, comparable results were obtained by the modified, more easily performed protocol, indicating that laboratories might validate and use this modified method.

Modifiable Risk Factors Associated with Clearance of Type-Specific Cervical Human Papillomavirus Infections in a Cohort of University Students

Previous findings regarding risk factors for human papillomavirus (HPV) persistence, other than viral determinants, identified from prospective cohort studies have been inconsistent in part because study designs have differed with respect to differing HPV detection methods and varying lengths of follow-up time. Therefore, the objectives of this study were to continue the search for epidemiologic risk factors of persistent cervical HPV infections and determine what behaviors differed between those women with transient HPV infections and those women who cannot clear their type-specific HPV infections. Female university students (n = 621) in Montreal were followed for 24 months at 6-month intervals. At each visit, a cervical cell specimen was collected. HPV DNA was detected using the MY09/MY11 PCR protocol and 27 HPV genotypes were identified by the line blot assay (Roche Molecular Systems, Inc., Alameda, CA). Proportional hazards regression was used to estimate the crude and adjusted hazard ratios of clearing a type-specific high-risk (n = 222) or low-risk (n = 105) HPV infection over time according to specific baseline and time-dependent covariates. Daily consumption of vegetables seemed to increase the rate of HPV clearance independent of type. The use of tampons was associated with a reduced rate of high-risk HPV clearance, whereas regular condom use was associated with an increased rate of low-risk HPV clearance only. Some proactive measures can be taken to increase the rate of HPV clearance, and there may be some differences between the sets of predictors of low-risk and high-risk HPV clearance.

The Natural Course ofChlamydia trachomatisInfection in Asymptomatic Colombian Women: A 5‐Year Follow‐Up Study

The natural course of Chlamydia trachomatis infection and its risk factors were studied in Colombian women with normal cytological results, during a 5-year period. Eighty-two women who were found to be positive for C. trachomatis at the start of the study were studied at 6-month intervals. At each visit, a cervical scrape sample was obtained for detection of C. trachomatis by use of C. trachomatis endogenous-plasmid polymerase chain reaction (PCR)-enzyme immunoassay and VD2-PCR-reverse line blot assay. Of the women studied, 67% had a single-serovar infection, 10% had a mixed-serovar infection, and 23% had an infection with an unidentified type. An inversed rate of clearance of C. trachomatis infection was observed with oral contraceptive use (hazard ratio [HR], 1.7 [95% confidence interval {CI}, 1.1-2.7]) and first sexual intercourse at >/=20 years of age (HR, 4.3 [95% CI, 2.3-8.0]). Serovars of group B (B, D, and E) and C (H, I, J, and K) had a decreased rate of clearance (rate ratio, 0.4 [95% CI, 0.1-0.9]), compared with that for serovars of the intermediate group (F and G). At 4 years of follow-up, 94% of the women had cleared their infections.

Is the perceived association between Chlamydia pneumoniae and vascular diseases biased by methodology?

Inter- and intralaboratory inconsistencies in detection rates of Chlamydia pneumoniae in vascular specimens have been demonstrated. In this study, 66 vascular tissue specimens from 66 patients with vascular disease were tested by three PCR assays: a 16S PCR-based reverse line blot (RLB) assay, a single-step PCR, and a nested PCR. Also, we explored the impacts of different DNA polymerase enzymes on the results based on gel electrophoresis and hybridization. The PCR results by gel electrophoresis in the single-step PCR depended on which DNA polymerase was used. All samples were negative with AmpliTaq Gold DNA polymerase, and 54.5% (36 of 66) were positive with the conventional Taq DNA polymerase. All samples were negative after hybridization with a C. pneumoniae-specific probe. In the nested PCR, all specimens were negative by gel electrophoresis and after hybridization. The RLB assay failed to detect C. pneumoniae in any specimen; however, 20 specimens were Chlamydia sp. positive. The sequence analysis of six of these samples demonstrated Chlamydia-like organisms. RLB detected Chlamydia sp. DNA in water and in the elution buffer after passage of the Qiagen columns (11 of 40). This study identified factors that may influence the detection of C. pneumoniae DNA in vascular tissues and consequently bias the perception of a link between C. pneumoniae and vascular diseases. The following are strongly recommended: to use DNA polymerases that have to be activated, to decontaminate with dUTP-uracil-DNA glycosylase, to hybridize with specific probes, to include sufficient controls, and to use molecular grade water.

Detection and identification of equine Theileria and Babesia species by reverse line blotting: epidemiological survey and phylogenetic analysis

Specific oligonucleotide probes were designed to develop a new and highly sensitive reverse line blot assay to detect and identify simultaneously different Theileria and Babesia species in horses. The amplified hypervariable V4 region of the 18S rRNA gene was hybridised against different generic and species-specific probes. The survey was conducted over 243 samples of equine blood divided into three different groups: group 1, 24 horses presented as possible clinical piroplasmosis; group 2, 181 clinically healthy free-ranging horses exposed to ticks; group 3, 38 riding horses with unrelated pathologies and low or no contact with ticks. The study demonstrated a high piroplasm prevalence in the first two groups of animals. Two Theileria genotypes sharing 96.8% similarity between their 18S rRNA gene sequences and two Babesia genotypes sharing 97.4% similarity, were identified. The biologic meaning of such genotypes is discussed in terms of their phylogenetic relationships and potential pathogenicity.

Combination of PCR targeting the VD2 of omp1 and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrape specimens.

In this study we developed and evaluated a new PCR-based typing assay, directed to the VD2 region of the omp1 gene, for the detection and typing of urogenital Chlamydia trachomatis infections. A nested VD2 PCR-reverse line blot (RLB) assay was developed for the typing of nine different urogenital serovars of C. trachomatis. The assay developed was tested with reference strains of C. trachomatis serovars and cervical scrapes of 86 Colombian women previously found to be positive for C. trachomatis by using plasmid PCR. Two sets of primers directed to the VD2 region of the omp1 gene of C. trachomatis were designed, and fragments of 220 and 166 bp were generated in the primary and nested PCRs, respectively. In addition, an RLB assay was developed to identify nine different urogenital serovars of C. trachomatis (Ba, D, E, F, G, H, I, J, and K) and group controls, including group B (Ba, D, and E), group C (I, J, K, and H), and an intermediate group (F and G). Using this assay, we were able to type 81 of the 86 samples (94.2%). Of these samples, 91.3% were single C. trachomatis infections, and 8.7% were multiple infections. The most common serovars identified were serovars D (22.2%), F (18.5%), G (13.6%), and E (12.3%). Of the women with multiple C. trachomatis infections, >50% contained both serovars D and E. The nested VD2 PCR-RLB developed is a simple, fast, and specific method for the identification of individual urogenital C. trachomatis serovars previously detected by using plasmid PCR. Moreover, it is an appropriate method for studying multiple C. trachomatis infections and for use in large epidemiological studies.

The persistence of component Theileria parva stocks in cattle immunized with the 'Muguga cocktail' live vaccine against East Coast fever in Uganda.

The 'Muguga cocktail' live vaccine comprises three Theileria parva stocks (Muguga, Kiambu 5 and the buffalo-derived Serengeti-transformed) and has been used extensively in Eastern, Central and Southern Africa with an infection and treatment protocol to protect cattle against East Coast fever. We report the characterization of the three component vaccine stocks using a panel of polymorphic micro-satellite and mini-satellite markers and the development of a stock-derived PCR method that distinguishes two of the vaccine stocks. These markers, with the use of a recently developed Reverse Line Blot assay, have enabled us to address four important questions in relation to vaccination. First, how closely related are the vaccine stocks, secondly do all three stocks persist post-vaccination and induce a carrier state, thirdly is there evidence for the transmission of the vaccine stocks and fourthly does vaccination prevent infection with local genotypes? The results show that Muguga and Serengeti-transformed stocks are highly related but very distinct from Kiambu 5 that persists in vaccinated cattle establishing a carrier state. No evidence was obtained for the transmission of vaccine stocks to co-grazed animals, although these animals were infected with up to 8 different T. parva genotypes showing there was a significant level of tick challenge. Some of the vaccinated animals become infected with a subset of local genotypes providing evidence for limited vaccine 'breakthrough'.

Lyme borreliosis spirochetes in questing ticks from mainland Portugal.

In Portugal, Ixodes ricinus ticks have been shown to contain DNA of several spirochetes belonging to the Borrelia burgdorferi sensu lato complex, with major differences in the genetic diversity between ecozones. Some isolates have been obtained since 1999, confirming the circulation of pathogenic strains in these ticks. Ixodes ricinus is considered to be a widespread species, however, in Portugal it is found only in a few habitats. Here we present preliminary results from a nationwide survey of questing I. ricinus (n = 4,001) and other Ixodidae (n = 1,534) in Portugal, initiated in 2001. The sampling points (so far 41) were selected using a Geographic Information System, according to the type of vegetation, accessibility and prevalence of human cases. The spatial and temporal of tick abundance and the infection of B. burgdorferi sensu lato in ticks were determined in selected areas. Ticks were examined for the presence of B. burgdorferi sensu lato by culturing (719 out of 4,001 I. ricinus), and direct PCR amplification of the 5S-23S intergenic spacer region (1,870 out of 5,535) followed by RFLP analysis, the reverse line blot assay and nucleotide sequencing of PCR amplicons. The most abundant tick genus was Rhipicephalus (53%), followed by Dermacentor (34%), I. ricinus and Hyalomma (7%, each). The Mafra and Grândola sites, where a more intensive study was carried out, were excellent habitats for I. ricinus. However, a clear difference of the prevalence of Borrelia infection and the genetic diversity of circulating spirochetes was observed in these two sites. Genotyping of all I. ricinus isolates revealed 5 B. garinii, 8 B. lusitaniae and 1 B. valaisiana strains, which were obtained for the first time in these regions along with a considerable percentage of tick-derived PCR amplicons. Two hard-tick species other than I. ricinus in Grândola were also B. lusitaniae positive, thus seeming to take part in the transmission cycle of Borrelia. The seasonal dynamics of I. ricinus in Mafra was bimodal, more pronounced in nymphs than in adults. The present findings indicate that B. burgdorferi sensu lato agents are differentially maintained in nature in local tick populations in different geographic areas across Portugal and that the risk of acquiring Lyme borreliosis in certain areas of Portugal is higher than previously assumed.

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.

Application of a reverse line blot assay to the study of haemoparasites in cattle in Uganda

Recent advances in genomic technology have focused many veterinary researchers on the possibility of producing one multivalent recombinant vaccine against all the haemoparasites that infect cattle in the tropics. Before such a vaccine is developed it is essential to define target cattle populations as well as the range of anti-pathogen vaccines required in order to control disease. To further this objective, we have evaluated a reverse line blot (RLB) assay, which simultaneously detects the principal tick transmitted protozoan and rickettsial cattle pathogens, in different epidemiological scenarios in Uganda. A critical question is the sensitivity, particularly in relation to detecting carrier animals. As Theileria parva is considered to be the most important pathogen in the region, we assessed the sensitivity of the RLB assay for T. parva and showed that 1–2×10 3 parasites per ml of blood could be detected—a level comparable with previously developed PCR methods and well below conventional microscopic detection. We applied the RLB assay to evaluate the differences in pathogen profiles between crossbred and indigenous cattle and show that there were different profiles, with a low prevalence of T. parva and Theileria taurotragi in the indigenous cattle compared to a high prevalence in the crossbred cattle. In contrast, we show higher prevalences of Theileria mutans and Theileria velifera in the indigenous compared to the crossbred cattle. Interestingly Anaplasma marginale, Babesia bovis and Babesia bigemina were of low prevalence but a high prevalence of Ehrlichia bovis was seen, raising the question of whether this rickettsial species could be pathogenic in cattle. Analysis of animals with clinical symptoms of East Coast Fever showed that, while T. parva is a major cause of these symptoms, T. mutans and possibly T. taurotragi and T. velifera, may also cause clinical disease. Overall, the results presented here highlight the complexity of tick-borne pathogen infections in cattle in Uganda.

HLA is a candidate region for psoriatic arthritis: evidence for excessive HLA sharing in sibling pairs

Psoriatic arthritis (PsA) is an inflammatory arthritis that may affect as many as 30% of patients with psoriasis (Ps). Genetic factors play an important role in the susceptibility to and the expression of PsA. The objective of this study was to identify whether haplotype sharing among affected sibling pairs of individuals with PsA is increased compared with unaffected sibling pairs. We collected 182 sibling pairs of probands affected with PsA. Extracted genomic DNA was amplified in polymerase chain reactions using locus specific primers homologous to nucleotide sequences for each of the HLA-A, -B, -C, -DR, and -DQ loci. Polymerase chain reaction amplicons were identified by reverse line blot assay using sequence-specific oligonucleotide probes. Evidence for excessive haplotype sharing was examined through Green and Woodrow's test. Results indicate that of the 182 sibling pairs, 46 were affected by PsA, 48 by Ps, and 88 were unaffected. The sharing of 2, 1, and 0 haplotypes for the PsA affected sibling pairs was 14, 27, and 5, respectively ( p = 0.04); whereas the haplotype sharing for the Ps affected sibling pairs was 12, 26, and 10, respectively ( p = 0.38). In conclusion, the human leukocyte antigen region on chromosome 6p is implicated as one of the candidate regions in PsA.

Différences génétiques entre les populations métisses de Bolivie et du Pérou

Data on the genetic background of Peruvian and Bolivian populations is still scarce. Studies have shown that mestizos (i.e. mixed, but of predominantly Native American ancestry) present several HLA alleles originally described in genetically isolated Amerindian tribes, and that Peruvian and Bolivian mestizos are closely genetically related. Using Reverse Line Blot Assay, HLA typing for Class I (HLA-A, HLA-B, and HLA-Cw) as well as for Class II (HLA-DRB1 and HLA-DQB1) alleles was performed in 57 Bolivian mestizos. Subjects came mainly from La Paz (the main city in the Bolivian Andes) and three other important cities located in the Bolivian Central Valley (Sucre, Tarija and Tupiza). Comparison of the allele frequencies of Bolivian mestizos with available data from the literature from Peruvian mestizos, revealed that Class I HLA-Cw*01, and B*40 alleles were higher in the Peruvian mestizo group (Chi-sq = 4,87 p<0,05; and chi-sq = 7,56 p<0,01 respectively). Regarding Class II alleles, HLA-DRB1*1101 and DRB1*1302 alleles were higher in Peruvian mestizos (Chi-sq = 5,12 p<.05; chi-sq = 3,8 p = 0.05 respectively), while HLA-DQB1*0402 was significantly higher in Bolivian mestizos (Chi-sq = 47,2 p<.0001). Differences in allele frequencies, and the absence or presence of certain alleles in either group could be due to a higher grade of admixture with Andean ethnic groups in the mestizos from Bolivia and/or less rural traits and more white (Caucasian) admixture in the Peruvian mestizos reported in the literature. These findings may have an impact regarding efficacies of modern forms of immunization (DNA vaccines) in Andean population, as well as in the identification of groups at risk for certain medical conditions.

Peculiar HLA polymorphisms in Italian patients with primary biliary cirrhosis

Background/Aims: Primary biliary cirrhosis (PBC) is an autoimmune cholestatic liver disease of unknown etiology with a highly variable progression rate and prevalence among different geographical areas. Data concerning human leukocyte antigen (HLA) polymorphisms in PBC come from a limited number of geographical areas, from which the association with the HLA-DRB1*08 allele has been consistently reported. Methods: To investigate whether HLA polymorphisms contribute toward disease susceptibility, we compared 186 well-defined Italian PBC patients with 558 healthy subjects matched by age, gender and geographical area (Northern, Central and Southern Italy). Patients and controls were HLA typed at low resolution by PCR-sequence specific oligonucleotides for the loci A and B; HLA-DRB1 alleles were typed by reverse line blot assay of PCR-amplified DNA. Results: HLA-DRB1*11 was associated with a markedly reduced risk of developing PBC (OR: 0.3; 95% CI: 0.2–0.5). No association was found with HLA-DRB1*08. The B*15 (2.5; 1.3–4.6), B*41 (12.0; 2.7–72.1), B*55 (2.9; 1.1–7.5) and B*58 alleles (6.8; 1.1–46.3) were more frequent in PBC. The frequency of HLA polymorphisms was similar in PBC patients with progressive or non-progressive disease, and in those with or without anti-mitochondrial antibodies. Conclusions: Our data on a large series of Italian patients suggest that PBC may have a peculiar genetic background in the Mediterranean area.

Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichiaruminantium in Amblyomma variegatum ticks by reverse line blot hybridization

The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.

Use of PGMY primers in L1 consensus PCR improves detection of human papillomavirus DNA in genital samples.

The novel PGMY L1 consensus primer pair is more sensitive than the MY09 and MY11 primer mix for detection and typing with PCR of human papillomavirus (HPV) DNA in genital specimens. We assessed the diagnostic yield of PGMY primers for the detection and typing of HPV by comparing the results obtained with PGMY09/PGMY11 and MY09/MY11/HMB01 on 299 genital samples. Amplicons generated with PGMY primers were typed with the line blot assay (PGMY-line blot), while HPV amplicons obtained with the degenerate primer pool MY09/MY11/HMB01 were detected with type-specific radiolabeled probes in a dot blot assay (standard consensus PCR test). Cervicovaginal lavage samples (N = 272) and cervical scrape samples (N = 27) were tested in parallel with both PCR tests. The PGMY-line blot test detected the presence of HPV DNA more frequently than the standard consensus PCR assay. The concordance for HPV typing between the two assays was 84.3% (214 of 255 samples), for a good kappa value of 0.69. Of the 177 samples containing HPV DNA by at least one method, 40 samples contained at least one HPV type detected only with PGMY-line blot, whereas positivity exclusively with the standard consensus PCR test was found for only 7 samples (P < 0.001). HPV types 45 and 52 were especially more frequently detected with PGMY than MY primers. However, most HPV types were better amplified with PGMY primers, including HPV-16. Samples with discordant results between the two PCR assays more frequently contained multiple HPV types. Studies using PGMY instead of MY primers have the potential to report higher detection rates of HPV infection not only for newer HPV types but also for well-known genital types.

Genotyping of human papillomavirus in liquid cytology cervical specimens by the PGMY line blot assay and the SPF(10) line probe assay.

A comparison of two PCR-based human papillomavirus (HPV) DNA detection and genotyping systems (PGMY LBA and SPF(10) LiPA) was conducted in two laboratories. Both systems are based on broad-spectrum PCR for the detection of HPV DNA, followed by reverse hybridization with type-specific probes. A total of 400 selected cervical scrape specimens in PreservCyt solution (55% normal cytology, 18% atypical squamous cells of unknown significance, 14.8% low-grade squamous intraepithelial lesions [SIL], and 12.5% high-grade SIL) were tested for the presence of HPV DNA. In this selected group of specimens, the overall agreement between the two methods for the detection of any HPV DNA was high (kappa = 0.859). When the 20 common HPV genotypes identified by both methods were considered (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 56, 58, 59, 66, and 68), compatible genotype-specific results were observed in 96.5% of the samples, even when multiple HPV genotypes were present. However, for some specific HPV genotypes, there were significant differences in HPV detection by the two methods. PGMY LBA detected more HPV type 42 (P = 0.002), HPV type 56 (P = 0.039), and HPV type 59 (P < 0.001), whereas SPF(10) LiPA detected more HPV type 31 (P < 0.001) and HPV type 52 (P = 0.031). For the remaining genotypes, including HPV types 16 and 18, the results obtained by the two methods were not significantly different. In general, both genotyping methods are highly suitable for clinical and epidemiological studies.

Detection and identification of eight Trichinella genotypes by reverse line blot hybridization.

A reverse line blot (RLB) assay was developed to identify different Trichinella genotypes. The RLB assay accomplishes detection and specific identification of the different Trichinella genotypes and relies on hybridization of the amplified 5S ribosomal DNA intergenic spacer regions to specific, membrane-bound oligonucleotide probes. After one single amplification, we were able to detect and genetically identify six sibling species, i.e., T. spiralis, T. britovi, T. nativa, T. murrelli, T. nelsoni, and T. pseudospiralis, and two additional Trichinella genotypes, T6 and T8. Twenty-four Trichinella strains of different genotypes were unequivocally identified evaluated using one simple PCR-based assay based on single larvae. This assay allows the specific identification of Trichinella species without the need to passage larvae in laboratory animals.

The Streptococcus anginosus species comprises five 16S rRNA ribogroups with different phenotypic characteristics and clinical relevance.

A collection of 267 consecutively isolated Streptococcus anginosus strains was screened for the prevalence of previously described 'motile' strains by hybridization with oligonucleotide probes in a reverse line blot assay. The motile strains represented 101 (37.8%) of the S. anginosus strains. The vast majority of these strains fermented mannitol and raffinose, whereas most other S. anginosus strains did not (P<0.001). Most (83/101) of the motile strains were recovered from the urogenital tract (including five strains from neonatal surveillance cultures) and only a minority (36) of them were associated with infection-related samples (P<0.001). Strains that hybridized with the same oligonucleotide probes as the type strain S. anginosus ATCC 33397T (= NCTC 10713T) were designated ATCC-like strains. They accounted for 74 (27.7%) of the strains examined, were commonly distributed among the different body parts and were significantly more associated with infection-related samples. Three other hybridization patterns were recognized in the reverse line blot assay, ribogroup I (n = 51), ribogroup II (n = 21) and ribogroup III (n = 19). Ribogroup II strains were significantly more frequently recovered from the abdominal cavity and were associated with infection-related samples. Ribogroup I included the majority of the S. anginosus strains that carried Lancefield group C. Comparison of the nearly complete 16S rRNA sequence of two representative strains of each ribogroup revealed that all five ribogroups were closely related (>97% sequence similarities), and that most sequence divergences between the ribogroups occurred in the 1024-1064 bp region of the 16S rRNA gene. The present data confirm the heterogeneity within the S. anginosus species.

Nonisotopic detection and typing of human papillomavirus DNA in genital samples by the line blot assay. The Canadian Women's HIV study group.

The line blot assay, a gene amplification method that combines PCR with nonisotopic detection of amplified DNA, was evaluated for its ability to detect human papillomavirus (HPV) DNA in genital specimens. Processed samples were amplified with biotin-labeled primers for HPV detection (primers MY09, MY11, and HMB01) and for beta-globin detection (primers PC03 and PC04). Amplified DNA products were hybridized by a reverse blot method with oligonucleotide probe mixtures fixed on a strip that allowed the identification of 27 HPV genotypes. The line blot assay was compared to a standard consensus PCR test in which HPV amplicons were detected with radiolabeled probes in a dot blot assay. Two hundred fifty-five cervicovaginal lavage specimens and cervical scrapings were tested in parallel by both PCR tests. The line blot assay consistently detected 25 copies of HPV type 18 per run. The overall positivity for the DNA of HPV types detectable by both methods was 37.7% (96 of 255 samples) by the line blot assay, whereas it was 43. 5% (111 of 255 samples) by the standard consensus PCR assay. The sensitivity and specificity of the line blot assay reached 84.7% (94 of 111 samples) and 98.6% (142 of 144 samples), respectively. The agreement for HPV typing between the two PCR assays reached 83.9% (214 of 255 samples). Of the 37 samples with discrepant results, 33 (89%) were resolved by avoiding coamplification of beta-globin and modifying the amplification parameters. With these modifications, the line blot assay compared favorably to an assay that used radiolabeled probes. Its convenience allows the faster analysis of samples for large-scale epidemiological studies. Also, the increased probe spectrum in this single hybridization assay permits more complete type discrimination.