Reverse Hybridization Assay Research Articles

KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay

KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay

Rapid detection of ethambutol-resistant genes in Mycobacterium tuberculosis clinical isolates

An oligonucleotide probe has been designed and compounded for detecting the ethambutol (EMB) resistance gene of Mycobacterium tuberculosis. It was placed on a nitrocellulose membrane for use in the reverse dot-blot hybridization assay, with the PCR product labeled with biotin. Analysis was carried out on 82 M. tuberculosis clinical isolates. The results indicate that among the 31 EMB-sensitive strains, the single-strand conformational polymorphism (SSCP) spectrum and reverse dot-blot (RDB) assay results on the embB gene in 26 strains were completely identical to those of the standard strain (H37Rv), testing positive for E1 hybridization; the SSCP spectrum of the remaining five strains indicated that migration had occurred. Among the 51 EMB-resistant strains, 24 strains tested positive for E1 hybridization; of the remaining 27 strains, 18 strains tested positive for E1b hybridization, two tested positive for E1c hybridization, five tested positive for E1d hybridization, one tested positive for E1e hybridization, and one tested positive for E1f hybridization. The low-concentration (64.70%) drug-resistant strains did not have the mutation at the codon 306 of embB. Among the 18 high-concentration drug-resistant strains, 15 strains tested positive for E1b hybridization, two tested positive for E1c hybridization, and one tested positive for E1f hybridization. The mutation detection rate was 52.94%. The RDB technology may be an easy and rapid method for detecting the codon 306 mutation of embB gene in some M. tuberculosis clinical isolates. We conclude that the ATG → GTG and ATG → CTG mutations of codon 306 of embB are one of the main causes of stron g EMB drug resistance in M. tuberculosis.

A case of transplacental transmission of Theileria equi in a foal in Trinidad

Equine piroplasmosis due to Theileria equi and Babesia caballi is endemic in Trinidad. A case of equine piroplasmosis due to T. equi was diagnosed in a thoroughbred foal at 10h post-partum. A high parasitaemia (63%) of piroplasms was observed in a Wright-Giemsa® stained thin blood smear from the foal. In addition, the 18S rRNA gene for Babesia/Theileria was amplified from DNA extracted from the blood of the foal and the mare. Amplified products were subjected to a reverse line blot hybridization assay (RLB), which confirmed the presence of T. equi DNA in the foal. The mare was negative by RLB but was positive for T. equi using a nested PCR and sequence analysis.In areas where equine piroplasmosis is endemic, severe jaundice in a post-partum foal may be easily misdiagnosed as neonatal isoerythrolysis. Foals with post-partum jaundice should be screened for equine piroplasmosis, which may be confirmed using molecular methods if available.

The Diagnosis of α-Thalassaemia: A Case of Hemoglobin H-α Deletion.

We report a case of hemolytic anemia that was subsequently identified to be a case of α-thalassaemia harboring the common rightward 3.7kb deletion/HbH. The diagnosis was based on sequential analyses using BioRad D10 HPLC, Alkaline gel electrophoresis, GPO α THAL-IC strips and the identification of the specific genetic lesion using an α Globin reverse dot blot hybridization assay. Supravital stain of RBCs helped in identifying classical HbH inclusions. In a background of a variable clinical presentation, lack of definitive hematological markers, and general under-diagnosis of α-thalassaemias we have used this case to highlight the features and sequence of techniques involved in identifying and characterizing an α-globin chain mutation, starting from a diffuse clinical history and presentation up to the identification of a specific genetic lesion involved.

Re-analysis of 178 previously unidentifiable Mycobacterium isolates in the Netherlands in 1999–2007

Nontuberculous mycobacteria (NTM) that cannot be identified to the species level by reverse line blot hybridization assays and sequencing of the 16S rRNA gene comprise a challenge for reference laboratories. However, the number of 16S rRNA gene sequences added to online public databases is growing rapidly, as is the number of Mycobacterium species. Therefore, we re-analysed 178 Mycobacterium isolates with 53 previously unmatched 16S rRNA gene sequences, submitted to our national reference laboratory in 1999–2007. All sequences were again compared with the GenBank database sequences and the isolates were re-identified using two commercially available identification kits, targeting separate genetic loci. Ninety-three out of 178 isolates (52%) with 20 different 16S rRNA gene sequences could be assigned to validly published species. The two reverse line blot assays provided false identifications for three recently described species and 22 discrepancies were recorded in the identification results between the two reverse line blot assays. Identification by reverse line blot assays underestimates the genetic heterogeneity among NTM. This heterogeneity can be clinically relevant because particular sub-groupings of species can cause specific disease types. Therefore, sequence-based identification is preferable, at least at the reference laboratory level, although the exact targets needed for clinically useful results remain to be established. The number of NTM species in the environment is probably so high that unidentifiable clinical isolates should be given a separate species status only if this is clinically meaningful.

Could genetic analysis be useful in reducing cerebrovascular risk in hypertensive subjects with hyperhomocysteinemia and patent foramen ovale? A 2-year follow-up study

Objectives Potential causes of cryptogenic cerebrovascular (CV) events are patent foramen ovale (PFO) and hyper homocysteinemia (H-Hcys), this latter a well-established risk factor for thrombosis particularly in the presence of mutation for the methylenetetrahydrofolate reductase (MTHFR) gene. This study investigated if in uncomplicated hypertensive subjects (HTs) with isolated PFO and H-Hcys, a different MTHFR polymorphism pattern for C667 → T gene mutation could influence PFO management and to reduce the CV risk. Methods In thirty-two HTs aged 55.6 ± 14.4 years, PFO was diagnosed by echocardiography. MTHFR genotype was evaluated by a multiplex polymerase chain reaction with reverse line blot hybridization assay. In relation to the T allele distribution, HTs were divided in normal (CC), heterozygote (CT) and homozygote (TT) for the MTHFR genotype. All subjects received a supplementation of oral folate (5 mg daily) and were evaluated yearly for 2 years. Analysis of variance for repeated measures (ANOVA) was used to compare changes of Hcys at baseline and at the end of follow-up and differences between continuous variables were evaluated in the three MTHFR groups with the Tukey's post hoc test after adjustment for confounders. Results At the follow-up, Hcys levels significantly normalized from baseline both in TT (38.1 ± 6.7 vs. 15 ± 3.6, p < 0.01) and CT (26.6 ± 2.3 vs. 9.2 ± 1.6, p < 0.01) but not in CC subjects (18.2 ± 1.8 vs. 16.0 ± 1.6, NS). Independently of age, BMI, vitamin treatment both systolic and diastolic blood pressure (BP) significantly decrease at the follow-up in all the MTHFR genotypes. No CV events were observed during the follow-up. Conclusions In HTs with isolated PFO and H-Hcys, oral folate supplementation reduces Hcys levels both in TT and CT subjects with C667→T mutation of MTHFR. In addition the BP normalization probably contributed to reduce CV risk in these genotypes.

A REPORT OF 8 CASES WITH HEMOGLOBIN H DISEASE IN AN IRANIAN FAMILY

α-Thalassemia is a common genetic disorder in Iran. However, no comprehensive data on epidemiology of severe forms of α-thalassemia, including hemoglobin H (HbH) or hydrops fetalis, is available in this population. This is a first case report of an Iranian family with large number of HbH individuals. The proband is a 48-year-old woman, referred to our center with anemia and no history of previous blood transfusions. Similar clinical phenotype has been observed in all of her 5 siblings, 2 of her 4 children, and her granddaughter, whose parents are first cousins. A reverse hybridization assay covering 21 α globin mutations was performed to determine the genotype in 11 members of this family and a fetus. HbH genotype was identified in 9 individuals, representing 3 generations, including a fetus. The high prevalence of α-thalassemia carriers together with the high rate of consanguineous marriages could lead to a large number of individuals with HbH or even hydrops fetalis in Iranian families. Therefore, to avoid the risk of having affected offspring, carrier detection, genetic counseling, and prenatal diagnosis would be of vital importance for individuals with low red blood cell (RBC) indices, normal iron status, and normal HbA2 level, who are suspected to be α-thalassemia carriers.

Genetic diversity of equine piroplasms in Greece with a note on speciation within Theileria genotypes ( T. equi and T. equi-like)

Equine piroplasms in Greece were studied using the reverse line blot hybridization (RLB) assay. Three genotypes consisting of two Theileria ( T. equi and T. equi-like) and one Babesia ( B. caballi-like) were identified. Of 787 samples tested, 371 (47.14%) hybridised to catchall probe (probe specifically designed to capture any piroplasm species present in a sample), 346 (43.96%) to T. equi probe, 364 (46.25%) to T. equi-like probe, 0 (0%) to B. caballi probe and 3 (0.38%) to B. caballi-like probe. Seven samples gave faint signals with the catchall probe only, indicating the presence of known or unknown piroplasm species, or a novel genotype or a known genotype occurring at a very low level of parasitemia. A partial sequence (509 bp) of the V4 region of the 18S rRNA gene of a T. equi-like isolate showed only 99% similarity with the reference T. equi-like isolates from Northern Spain from which the detecting probe used in the present study was designed but showed 100% similarity with the T. equi-like variants from Southern Spain. This indicated a noticeable degree of polymorphism within the population of T. equi-like. No unusual parasites previously reported in horses, such as B. canis canis and B. bovis were detected in this study. The values of the bioclimatic variables were very similar between the geographic locations for T. equi and T. equi-like genotypes, suggesting the two are not yet different species as hypothesized by some authors but are possibly undergoing a speciation process within Theileria genotypes. Both T. equi and T. equi-like were found in predominantly forest type land cover.

Multiplex polymerase chain reaction-based reverse line blot hybridization assay to detect common genital pathogens

The objective of the present paper is to develop and apply a multiplex polymerase chain reaction (mPCR) based reverse line blot (RLB) hybridization assay to facilitate the diagnosis of genital infections by detection of seven recognized or putative genital pathogens (Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma genitalium, Mycoplasma hominis and Trichomonas vaginalis). Species-specific biotin-labelled primer pairs were used in a single mPCR to amplify target regions in each of seven pathogens. The amplified biotin-labelled PCR products were hybridized with membrane-bound-specific oligonucleotide probes and were detected by chemiluminescence. Two hundred and eleven specimens (104 male urethral and 107 female vaginal swabs), collected from patients with suspected genital infections attending the Wuhan First Hospital Sexually Transmitted Diseases (STD) clinic, were tested by mPCR/RLB and results were confirmed by single PCR using different species-specific targets. The sensitivity of the assay was assessed using dilutions of positive DNA controls with known copy numbers, for each target. The assay correctly identified all reference strains and detected potential pathogens in a high proportion of clinical specimens. There was no cross-reaction between the seven pathogens. The mPCR/RLB can detect <or= 10(2) copies of the target gene fragments. Comparison of mPCR/RLB and single PCR assays showed discrepant results in six of 211 (2.8%) clinical specimens, which were positive by mPCR/RLB assay, but negative by the corresponding single PCR. Nested PCR on the six discrepant specimens gave results consistent with those of mPCR/RLB. In conclusion, the mPCR/RLB hybridization assay is sensitive and specific, and able to rapidly detect genital pathogens in clinical specimens.

Molecular Variants of HPV Type 16 E6 Among Honduran Women

In this study, we investigated the prevalence of human papillomavirus 16 (HPV-16) variants in Honduran women with normal cytology and with dysplasia and cervical cancer. Samples identified as positive for HPV-16 by SPF10-LiPA were tested for intratypic subtypes and variants by analysis of the E6/E7 region using a novel reverse hybridization assay (line probe assay). We found that most infections in all clinical groups belong to the E6 European variants, suggesting that HPV-16 non-European variants do not represent an additional factor associated with increased occurrence of high-grade cervical lesions in the studied population. Among the 106 HPV-16-positive women analyzed, E-350G was the most prevalent variant in all different disease stages, being present in 18% of cervical cancer, 13% of cervical intraepithelial neoplasia grade III (CIN III), 5% of CIN II, 5% of CIN I, and 20% of control samples. Mixed variants of HPV-16 infections were detected in 7.7% of the samples, mostly in women with normal cytology. This study shows for the first time the diversity of HPV-16 variants in cervical samples of Honduran women.

HPV genotyping and HPV16 variant analysis in glandular and squamous neoplastic lesions of the uterine cervix

Objective The objective of the study was to compare the distribution of HPV genotypes and HPV16 variants in glandular and squamous cervical neoplasia. Methods Cases of endocervical adenocarcinoma in-situ (AIS, n = 33) invasive adenocarcinoma (ADCA, n = 55), cervical intraepithelial neoplasia-3 (CIN3, n = 130) and squamous cell carcinoma (SCC, n = 60) were collected at the New York Hospital and tested for HPV using SPF 10PCR-LIPA 25 (version 1) assays and for HPV16 variants using a multiplex PCR and reverse hybridization assay. Results There was a difference between the spectrum of HPV genotypes detected in glandular and squamous neoplasia: 13 different HPV genotypes were detected in CIN3 as single infections and 11 in SCC, while only 4 single genotypes were detected in AIS and 3 in ADCA. The most common single HPV types in CIN3 were HPV16, 31, and 52 (56.9%, 10%, 8.4%, respectively). In SCC the most common were HPV16, 18 and 31 (70%, 6.5%, 4.9%). In AIS, HPV16, 18, 45 and 35 accounted for 69.7%, 27.2%, 3%, 3% of cases. The three single types in ADCA were HPV16 (43.6%), HPV18 (41.8%) and HPV45 (10.9%). European variants of HPV16 were the most common in CIN3 (83.8%), SCC (71.4%) and AIS (73.9%). In ADCA the Asian American (AA) variant was the most common (41.7%) followed by European variants (33.3%). AA variant was also detected in 17.4%, 4.1%, and 2.4% of HPV16 positive AIS, CIN3 and SCC, respectively. Conclusion Asian American variant of HPV16, HPV18 and HPV45 are preferentially associated with cervical adenocarcinoma as compared to squamous cell carcinoma .

Replacing Reverse Line Blot Hybridization Spoligotyping of the Mycobacterium tuberculosis Complex

Spoligotyping is a tool for the molecular characterization/typing of Mycobacterium tuberculosis complex (MTBC) strains based on target sequences (spacers) in the direct repeat (DR) region (14). The standard spoligotyping assay involves the hybridization of amplified sample DNA to nylon membrane-immobilized oligonucleotides whose sequences are representative of 43 spacer regions. Variations in the number of spacers as a result of deletions of adjacent blocks of repetitive units allow the differentiation of clinical isolates. In the present study, we developed a new multiplexed primer extension-based spoligotyping assay using automated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that improves the classical reverse line blot hybridization assay with respect to reproducibility, throughput, process flow, ease of use, and data analysis. Validation of the MALDI-TOF MS-based spoligotyping assay with two sample sets with a total of 326 samples resulted in 96.6% concordance (315/326) when the full spoligotype patterns were compared with the results of standard spoligotyping and 99.9% concordance when the results were compared with those of individual primer extension assays. Ten strains (including two Mycobacterium canettii strains) showed discordant results with one or two spacer differences from the membrane-based spoligotyping result. Most discordant samples were identified to be the result of ambiguities in the interpretation of weak hybridization signals in the reverse line blot assay and sequence variations in the spacer regions. We established a new automated primer extension assay and successfully validated it for use for the routine typing of MTBC strains in the research and public health laboratory environments. The present multiplex levels of up to 30 are extendable and allow the additional incorporation of controls and antibiotic resistance markers.

Detection and identification of mycobacteria in sputum from suspected tuberculosis patients

BackgroundDetection of Tuberculosis agent like nontuberculous mycobacteria (NTM) species by culture and microscopic methods remains difficult and time consuming. A fast and reliable diagnosis of tuberculosis would greatly improve the control of the disease. The purpose of this study is to compare the conventional multiplex PCR and multiplex PCR reverse cross blot hybridization assay to culture method in terms of mycobacteria species detection.FindingsAmong the 117 positively cultured samples, nontuberculous mycobacteria (NTM) species were found in 9 samples of multiplex PCR reverse cross blot hybridization assay; compared to only 3 NTM species found in our conventional multiplex PCR, and 13 NTM species were successfully identified among 162 negatively cultured samples compared to only 5 NTM species identification in conventional multiplex PCR results.ConclusionsThe sensitivity of the multiplex PCR reverse cross blot hybridization assay comparing to culture method was 86.03%, the specificity is 35.46%, the positive predictive value was 41.94% and the negative predictive value was 82.41%. For conventional multiplex PCR these values are 81.62%, 38.65%, 41.89%, 79.51%, respectively. Furthermore, in terms of mycobacteria species detection, the conventional multiplex PCR was relatively equal compared to the multiplex PCR reverse cross blot hybridization assay, and to be particularly having no significant discrepant results on the identification of Mycobacteria tuberculosis in both methods.

Site‐Specific Gingival Bleeding on Probing in a Steady‐State Plaque Environment: Influence of Polymorphisms in the Interleukin‐1 Gene Cluster

We previously reported a high prevalence of a combination of alleles 2 at positions interleukin (IL)-1A(-889) and IL-1B(+3954) in an Arabic population with gingivitis. In a steady state, the proportion of sites with bleeding on probing (BOP) was consistently reduced. The aim of the present study is to expand this observation by considering the site level. Fifty healthy non-smoking volunteers, 19 to 28 years of age, participated in this study. Clinical examinations included probing depth, BOP, plaque index (PI), and calculus. Examinations were repeated after 2 and 4 weeks, and subjects were advised not to change oral hygiene habits. Polymorphisms in the IL-1 gene cluster were assessed using a reverse hybridization assay. Twenty-six subjects (52%) carried alleles 2 at positions IL-1A(-889) and IL-1B(+3954) and were designated genotype positive. In multivariate multilevel models of BOP, bleeding tendency in subjects with positive IL-1 genotype was only reduced at sites with PI 0 (odds ratio of 0.98, 0.65, and 0.56 at baseline and 2 and 4 weeks, respectively; chi(2)((3)) = 11.946; P = 0.008) or 1 (chi(2)((3)) = 6.027; P = 0.110). A decreased bleeding tendency at certain tooth types in subjects who were IL-1 positive was largely related to the relative cleanliness of these areas. Random parts of the models revealed very low biserial correlations of 0.13 to 0.15 at the site level, whereas correlations were considerably higher (0.76 to 0.83) at the subject level. Multilevel modeling of site-specific data yielded new information about the influence of the IL-1 genotype in plaque-induced gingivitis.

The Nonserotypeable Pneumococcus: Phenotypic Dynamics in the Era of Anticapsular Vaccines

Nonserotypeable pneumococci (NSP) are commonly carried by Australian Indigenous children in remote communities. The purpose of this study was to characterize carriage isolates of NSP from Indigenous children vaccinated with the seven-valent pneumococcal conjugate vaccine (PCV7) and to use these data to guide decisions on reporting of NSP. A total of 182 NSP were characterized by BOX typing, antibiogram analysis, and multilocus sequence typing (MLST) of common BOX types. NSP positive for the wzg capsule gene were analyzed by a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB-H) targeting capsule genes to determine the serotype. Among 182 NSP, 49 BOX types were identified. MLST of 10 representative isolates found 7 STs, including ST448 (which accounted for 11% of NSP). Non-penicillin susceptibility was evident in 51% of the isolates. Pneumococcal wzg sequences were detected in only 23 (13%) NSP, including 10 that contained an approximately 1.2-kb insert in the region. mPCR/RLB-H identified serotype 14 wzy sequences in all 10 NSP, and 1 also contained a serotype 3-specific wze sequence. Among the remaining 13 wzg-positive NSP, few belonged to the serotypes represented in PCV7. It appears that most NSP identified in Australian Indigenous children are from a true nonencapsulated lineage. Few NSP represented serotypes in PCV7 that suppress capsular expression. High rates of carriage and penicillin resistance and the occasional presence of capsule genes suggest a role for NSP in the maintenance and survival of capsulated pneumococci. To avoid the inflation of pneumococcal carriage and antibiotic resistance rates, in clinical trials, we recommend separate reporting of rates of capsular strains and NSP and the exclusion of data for NSP from primary analyses.

Efficacy of an in-feed formulation containing ivermectin for the control of intestinal strongyles in captive zebras ( Equus burchelli (Gray, 1824))

The efficacy of medicated food pellets (containing 10 mg of ivermectin per kg, UNIFEED, Veronesi, Verona, Italy) was evaluated for the control of intestinal strongyles in a group of captive zebras ( Equus burchelli) at the Safari Park, Fasano (Apulia region, Brindisi Province, southern Italy). The egg reappearance period and the faecal egg counts in terms of eggs per gram of faeces were investigated. From day 0 until day +9, 30 zebras were fed with medicated pellets and pooled faecal samples were collected from the floor of paddocks. The drug showed an efficacy of 100% for up to 78 days post-treatment, with one exception. Strongyle eggs collected prior to the treatment were used to perform coprocultures and larvae harvested were molecularly identified as Cylicostephanus minutus and Cylicocyclus leptostomum with a reverse line blot hybridization assay. The administration of ivermectin with medicated food pellets was effective in controlling intestinal strongylosis in captive zebras. The opportunity to treat captive ungulates with an in-feed anthelmintic is discussed in relation to the fractious nature of these animals, which often impairs helminth control programs in zoo-parks.

Hepatitis C Virus (HCV) Genotype 1 Subtype Identification in New HCV Drug Development and Future Clinical Practice

BackgroundWith the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination.Methodology/Principal FindingsA large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5′ non-coding region (5′NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%–29.5% of cases, and HCV subtype 1b in 9.5%–8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5′NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5′NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases.Conclusions/SignificanceIn the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5′NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.

Abstract C59: Human papillomavirus genotype distribution in northern India: Implications for vaccination

Abstract Background: Cervical cancer caused by human papilloma virus (HPV), is the commonest cancer in Indian women, with an estimated 132000 new cases and 76000 deaths annually. More than 100 HPV genotypes have been identified in humans of which at least 40 HPV types are found in the genital tract. Several high-risk HPV types are known to cause the disease. There are only limited data available on the genotype distributions in this region. Methods: During the period three different cross-sectional studies were carried out: (1) a hospital-based cross-sectional study in which 106 women presenting with invasive cervical cancer; (2) a hospital-based cross-sectional study in which 524 women presenting to have an unhealthy cervix were invited to participate; (3) a community-based cross-sectional study in which 465 women also underwent HPV testing. HPV typing in these studies was done by using L1 consensus PCR followed by reverse line blot hybridization assay (Roche). Results: In the hospital-based population, among 106 invasive cervical cancers the six commonest HPV types were HPV-16, -18, -45, -59, -33 and 73. HPV-16/18, singly or in combination, were seen in 83% of cases. (2) In the hospital-based population, among the 524 symptomatic hospital subjects, the overall HPV prevalence was 15.5 % and the six commonest HPV types were as follows: HPV -16, -89, -39, -52, -33 and 18. Among the HPV positive women accounted for HPV-16/18 were associated with 34.3%, 45.4% and 65.7% of normal, low-grade and high-grade disease respectively. Among (3) the community-based women, the overall HPV prevalence was 7 % and the six commonest HPV types were as follows: HPV- 16, - 45, -52, -31, -66, and -18. Conclusions: A wide spectrum of HPV types is seen in north Indian women, with HPV-16/18 are the commonest types in all populations. However, thereafter there is considerable variation. It is estimated that the HPV -16/18 vaccine would reduce over 75% of the total cancer burden in this region. The community population is from a confined geographical area whereas the hospital population includes not only residents of Delhi and neighbouring areas but also further states of North India as well as neighboring countries of Nepal and Bangladesh. Thus more studies are required to establish the true picture of HPV type-distribution in this large and diverse region. Citation Information: Cancer Res 2009;69(23 Suppl):C59.

Sensitive Detection of KRAS Mutations in Archived Formalin-Fixed Paraffin-Embedded Tissue Using Mutant-Enriched PCR and Reverse-Hybridization

Recently, evidence has emerged indicating that assessment of KRAS mutations before anti-epidermal growth factor receptor therapy improves outcome in patients with metastatic colorectal cancer (CRC). We report here a novel reverse-hybridization (RH) assay to screen for KRAS mutations in formalin-fixed paraffin-embedded colorectal tissue samples. We combined mutant-enriched PCR based on peptide nucleic acid clamping and RH of amplification products to nitrocellulose test strips that contained a parallel array of oligonucleotide probes targeting 10 frequent mutations in codons 12 and 13 of the KRAS gene. DNA mixing experiments, which included eight different tumor cell lines with known KRAS mutations, were performed to examine the sensitivity of mutation detection. All KRAS mutations present in tumor cell lines were unambiguously identified by the RH assay with 1% of each cell line DNA diluted in normal DNA. RH was then used to screen for KRAS mutations in 74 colorectal tumor and 4 normal control samples. Twenty-six (35%) of the 74 tumor samples showed KRAS mutations. No mutation was found in the four samples of normal colorectal tissue. DNA sequencing without previous mutant enrichment, however, failed to detect four (15%) out of 26 KRAS-positive formalin-fixed paraffin-embedded samples (FFPE). This finding suggests that even after microdissection, mutant sequences in a given DNA isolate can be rare and more sensitive methods are needed for mutation analysis.

High prevalence ofblaCTX-Mextended-spectrum β-lactamase genes inEscherichia coliisolates from pets and emergence of CTX-M-64 in China

Nontuberculous mycobacteria (NTM) that cannot be identified to the species level by reverse line blot hybridization assays and sequencing of the 16S rRNA gene comprise a challenge for reference laboratories. However, the number of 16S rRNA gene sequences added to online public databases is growing rapidly, as is the number of Mycobacterium species. Therefore, we re-analysed 178 Mycobacterium isolates with 53 previously unmatched 16S rRNA gene sequences, submitted to our national reference laboratory in 1999–2007. All sequences were again compared with the GenBank database sequences and the isolates were re-identified using two commercially available identification kits, targeting separate genetic loci. Ninety-three out of 178 isolates (52%) with 20 different 16S rRNA gene sequences could be assigned to validly published species. The two reverse line blot assays provided false identifications for three recently described species and 22 discrepancies were recorded in the identification results between the two reverse line blot assays. Identification by reverse line blot assays underestimates the genetic heterogeneity among NTM. This heterogeneity can be clinically relevant because particular sub-groupings of species can cause specific disease types. Therefore, sequence-based identification is preferable, at least at the reference laboratory level, although the exact targets needed for clinically useful results remain to be established. The number of NTM species in the environment is probably so high that unidentifiable clinical isolates should be given a separate species status only if this is clinically meaningful.