Serial Invasive Signal Amplification Reaction Research Articles

Knockdown resistance allele frequencies in North American head louse (Anoplura: Pediculidae) populations.

The study examines the extent and frequency of a knockdown-type resistance allele (kdr type) in North American populations of human head lice. Lice were collected from 32 locations in Canada and the United States. DNA was extracted from individual lice and used to determine their zygosity using the serial invasive signal amplification technique to detect the kdr-type T917I (TI) mutation, which is most responsible for nerve insensitivity that results in the kdr phenotype and permethrin resistance. Previously sampled sites were resampled to determine if the frequency of the TI mutation was changing. The TI frequency was also reevaluated using a quantitative sequencing method on pooled DNA samples from selected sites to validate this population genotyping method. Genotyping substantiated that TI occurs at high levels in North American lice (88.4%). Overall, the TI frequency in U.S. lice was 84.4% from 1999 to 2009, increased to 99.6% from 2007 to 2009, and was 97.1% in Canadian lice in 2008. Genotyping results using the serial invasive signal amplification reaction (99.54%) and quantitative sequencing (99.45%) techniques were highly correlated. Thus, the frequencies of TI in North American head louse populations were found to be uniformly high, which may be due to the high selection pressure from the intensive and widespread use of the pyrethrins- or pyrethroid-based pediculicides over many years, and is likely a main cause of increased pediculosis and failure of pyrethrins- or permethrin-based products in Canada and the United States. Alternative approaches to treatment of head lice infestations are critically needed.

Determination of knockdown resistance allele frequencies in global human head louse populations using the serial invasive signal amplification reaction

Pediculosis is the most prevalent parasitic infestation of humans. Resistance to pyrethrin- and pyrethroid-based pediculicides is due to knockdown (kdr)-type point mutations in the voltage-sensitive sodium channel alpha-subunit gene. Early detection of resistance is crucial for the selection of effective management strategies. Kdr allele frequencies of lice from 14 countries were determined using the serial invasive signal amplification reaction. Lice collected from Uruguay, the United Kingdom and Australia had kdr allele frequencies of 100%, while lice from Ecuador, Papua New Guinea, South Korea and Thailand had kdr allele frequencies of 0%. The remaining seven countries investigated, including seven US populations, two Argentinian populations and populations from Brazil, Denmark, Czech Republic, Egypt and Israel, displayed variable kdr allele frequencies, ranging from 11 to 97%. The newly developed and validated SISAR method is suitable for accurate monitoring of kdr allele frequencies in head lice. Proactive management is needed where kdr-type resistance is not yet saturated. Based on sodium channel insensitivity and its occurrence in louse populations resistant to pyrethrin- and pyrethroid-based pediculicides, the T917I mutation appears to be a key marker for resistance. Results from the Egyptian population, however, indicate that phenotypic resistance of lice with single or double mutations (M815I and/or L920F) should also be determined.

Pyrethroid Pediculicide Resistance of Head Lice in Canada Evaluated by Serial Invasive Signal Amplification Reaction

Most people in the United States and Canada with pediculosis will be treated with neurotoxic pediculicides containing pyrethrins or pyrethroids. Their widespread use led to significant resistance reported from various countries. Although treatment failures are frequently observed in Canada, the resistance frequency to pyrethroid pediculicide of human head lice (Pediculus humanus capitis) has not been determined. To determine the knockdown resistance (kdr) allele frequency in human head louse populations in Canada. Patients infested with Pediculus humanus capitis, aged 4 to 65 years, residents of Ontario, Quebec, and British Columbia, were participants. Head lice were collected by combing and picking the enrolled subjects' hair. Lice were analyzed by serial invasive signal amplification reaction (SISAR) for genotyping the T917I mutation of lice indicating permethrin resistance. The permethrin-resistant kdr allele (R allele) frequency could then be evaluated in the head lice collected in Canada. Of the head louse populations analyzed, 133 of 137 (97.1%) had a resistant (R) allele frequency, whereas only 4 of 137 (2.9%) had a susceptible (S) allele frequency. The 97.1% resistant (R) allele frequency in head lice from Canada could explain the treatment failures encountered with pyrethrin and pyrethroid pediculicide treatments in Canadian populations infested with Pediculus humanus capitis as the latter will not be eliminated by those pediculicides.

Permethrin Resistance Due to Knockdown Gene Mutations is Prevalent in Human Head Louse Populations

Permethrin resistance in head lice is mainly conferred by the knockdown resistance (kdr) trait, a voltage- sensitive sodium channel (VSSC) insensitivity factor. Three VSSC mutations have been identified and confirmed to reduce the sensitivity of VSSC to permethrin. A step-wise resistance monitoring system has been established based on molecular resistance detection techniques. Quantitative sequencing (QS) predicts the kdr allele frequency in head lice on a population basis. The speed, simplicity and accuracy of QS made it an ideal candidate for a routine primary resistance monitoring tool to screen a large number of wild louse populations as an alternative to conventional bioassay. As a secondary monitoring method, real-time PASA (rtPASA) provides a more precise determination of low resistance allele frequencies. To obtain more detailed information on resistance allele zygosity, as well as allele frequency, serial invasive signal amplification reaction (SISAR) is utilized as an individual genotyping method. Using this approach, kdr alleles were detected in head lice from 10 of the 14 countries examined and an overall kdr allele frequency of 73.8% determined.

Assessment of 14 functional gene polymorphisms in Japanese patients with oral lichen planus: a pilot case-control study

Oral lichen planus (OLP) is a refractory mucosal disease. Its pathogenesis is thought to involve immunologic and genetic alterations. To gain a better understanding of the genetic risk factors, the authors evaluated associations between 14 functional gene polymorphisms and OLP. 32 Japanese patients with OLP and 99 unrelated healthy Japanese controls were genotyped for 14 single nucleotide polymorphisms (SNPs) of genes that regulate host immune responses. Genotyping was performed with a modified version of the serial invasive signal amplification reaction. A trend towards over-representation of tumor necrosis factor receptor 2 (TNFR2) +587 G allele was found in the patients compared with the controls (allele frequency: P=0.049). The other 13 SNPs were unassociated with OLP. These results suggest that TNFR2 +587 gene polymorphism may be associated with susceptibility to OLP.

Molecular mechanisms and monitoring of permethrin resistance in human head lice

Head lice resistance to permethrin is mainly conferred by the knockdown resistance ( kdr) trait, a voltage-sensitive sodium channel (VSSC) insensitivity factor. Three VSSC mutations (M815I, T917I and L920F) have been identified. Functional analysis of the mutations using the house fly VSSC expressed in Xenopus oocytes revealed that the permethrin sensitivity is reduced by the M827I (M815I) and L932F (L920F) mutations when expressed alone but virtually abolished by the T929I (T917I) mutation, either alone or in combination. Thus, the T917I mutation is primarily responsible for permethrin resistance in head lice. Comparison of the expression rates of channel variants indicates that the M815I mutation may play a role in rescuing the decreased expression of channels containing T917I. A step-wise resistance monitoring system has been established based on molecular resistance detection techniques. Quantitative sequencing (QS) has been developed to predict the VSSC mutation frequency in head lice at a population basis. The speed, simplicity and accuracy of QS made it an ideal candidate for a routine primary resistance monitoring tool to screen a large number of wild louse populations as an alternative to conventional bioassay. As a secondary monitoring method, real-time PASA (rtPASA) has been devised for more precise determination of low resistance allele frequencies. To obtain more detailed information on resistance allele zygosity, as well as allele frequency, serial invasive signal amplification reaction (SISAR) has been developed as an individual genotyping method. Our approach of using three tiers of molecular resistance detection should facilitate large-scale routine resistance monitoring of permethrin resistance in head lice using field-collected samples.

Development of High Throughput PCR-Invader Assay to Detect the JAK2 V617F Mutation in Japanese Myeloproliferative Disorders

The classic myeloproliferative disorders (MPD) of polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) are hematopoietic ones characterized by the overproduction of one or more mature myeloid lineages. Although their clonal nature and cytokine independence were recognized a couple of decades ago, their molecular abnormality was not clarified until the identification of the activating JAK2 V617F mutation. Around 95% of patients with PV have the JAK2 V617F mutation. The JAK2 V617F mutation is found in approximately half of ET and IMF patients. ET patients with JAK2 V617F resemble PV patients such as in their increased erythropoiesis and granulopoiesis, and have increased rates of venous thrombosis compared to JAK2 V617F-negative patients. Accurate genotyping of JAK2 is critical for a diagnosis of MPD and can be useful for treatment decisions. However, compared to Western populations, there is limited information available about JAK2 mutation in a Japanese population. This prompted us to analyze JAK2 genotyping utilizing a new method. Although various methods such as direct sequencing, PCR-RFLP, SSCP and DHPLC have been developed for genotyping, none of these provide a rapid, accurate and reproducible system. In this study, a simple assay based on a serial invasive signal amplification reaction assay (PCR-Invader assay) was developed for the detection of JAK2 V617F. The PCR-Invader assay was originally developed for typing SNPs in the human genome. It can detect genotypes in simple 96-well plates without sequence analysis, enzyme digestion or electrophoresis. We analyzed 86 Japanese MPD patients (PV 21, ET 52, IMF 10, others 3). Genomic DNA was extracted from whole blood and the JAK2 V617F mutation then analyzed by the PCR-Invader method. To confirm the accuracy of genotyping by PCR-Invader, the direct sequencing method was also applied to all samples. The percentage of JAK2 V617F mutation-positive patients was PV 81.0%, ET 48.1% and IMF 80.0%. Both white blood cell and platelet counts were higher in V617F-positive PV compared with V617F-negative PV samples (p<0.05). V617F-positive ET samples were accompanied by higher counts of white blood cells and platelets compared with V617F-negative ET ones. However, we did not observe the any relationship between blood cell counts and V617F mutation in IMF; consistent with previous reports. We confirmed that this PCR-Invader method was approximately 20 times more sensitive than direct sequencing. There were no false positive results by PCR-Invader in all 86 samples tested; indicating that PCR-Invader has cost-effectiveness advantages. It has been reported that the activating MPL mutation is observed in some populations of IMF and ET; therefore, we analyzed exon 10 of the MPL genome by direct sequencing. We found that only two V617F-negative ET patients had MPL mutations; W515K and W515-P518delinsKT. There were no MPL mutations in V617F-positive ET patients or all IMF patients. The two ET patients with MPL mutations displayed lower hemoglobin and higher platelets at diagnosis, which is in line with the recently report of the PT-1 study group. In this study we analyzed the JAK2 V617F mutation utilizing the new PCR-Invader assay and undertook MPL mutation by direct sequencing in a Japanese population. Given that some JAK2 inhibitors for MPD patients are now in clinical trials, we expect that this simple PCR-Invader assay and the clinical information about JAK2 and MPL mutations in Japanese MPD patients will be useful to develop clinical trials for JAK2 inhibitors in Japan.

Association of interleukin‐1 receptor antagonist +2018 gene polymorphism with Japanese chronic periodontitis patients using a novel genotyping method

Genetic variants at multiple loci have been shown to be associated with periodontitis risk. In this study, we have focused on nine functional gene polymorphisms encoding immunoregulation-related molecules such as cytokines (interleukin-1 (IL-1), transforming growth factor-beta1 (TGF-beta1)) and cell surface receptors (immunoglobulin G and A Fc receptors (Fc gamma R and Fc alpha R)). In total, 113 Japanese patients with chronic periodontitis (CP) and 108 race-matched healthy controls were genotyped with the modified serial invasive signal amplification reaction. There was a significant difference in the distribution of IL-1 receptor antagonist (RN) +2018 T/C allele between the patient and control groups, with enrichment of the +2018 C in controls (P = 0.021, odds ratio = 0.38). An increased frequency of the IL-1 haplotype comprising IL-1A +4845 G, IL-1B -31 C, and IL-1RN +2018 C was observed in controls (P = 0.004). Moreover, a multivariate logistic regression analysis revealed that subjects with IL-1RN +2018 C allele were less likely to have CP (P = 0.016, odds ratio = 0.29). These findings document the association of IL-1RN +2018 C with reduced susceptibility to CP in the Japanese.

Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes

Hepatitis B virus is a worldwide public health problem. A simple and effective test to identify viral genotypes would greatly aid efforts to understand and control the spread of this disease. A serial invasive signal amplification reaction assay (PCR-Invader assay) was developed for distinguishing the known eight genotypes (A–H) and four subgenotypes (Aa, Ae, Ba, Bj) of hepatitis B virus (HBV). The preS/S and core regions were amplified by multiplex PCR and delivered to 12 wells containing genotype-specific Invader probes. By observing the fluorescence patterns in the wells, HBV sub/genotypes can be assigned. A total of 505 serum samples containing HBV/HBsAg in Japan was examined by PCR-Invader and compared the results with those from ELISA assays with monoclonal antibodies against epitopes on gene products of the preS2 region and with a genotype-specific probe assay (GSPA) based on the preS1 region. Genotypes determined by the PCR-Invader agreed with those of the ELISA method in 98.2% of cases and with the GSPA method in 97.1% of cases. Co-infection with two distinct genotypes was correctly identified by the PCR-Invader in four serum samples, as determined by GSPA. Thus, the PCR-Invader assay is a useful tool for detecting the 10 known HBV sub/genotypes.

Influence of CYP2C19 Polymorphism and Helicobacter pylori Genotype Determined From Gastric Tissue Samples on Response to Triple Therapy for H pylori Infection

The relationship between single nucleotide polymorphisms (SNPs) and clinical outcomes has been intensively studied. We intended to determine SNPs of CYP2C19 and 23S rRNA of Helicobacter pylori by using rapid urease test (RUT)-positive gastric mucosal samples. One hundred thirty-nine patients with H pylori -positive results based on RUT completed 1-week treatment with lansoprazole 30 mg twice a day, clarithromycin 200 mg 3 times daily, and amoxicillin 500 mg 3 times daily. SNPs from adenine to guanine at positions 2142 and 2143 of 23S rRNA of H pylori (A2142G and A2143G) and SNPs from guanine to adenine at positions 681 in exon 5 (* 2 ) and 636 in exon 4 (* 3 ) of CYP2C19 were determined by the serial invasive signal amplification reaction assay by using DNAs extracted from gastric tissue samples already used for RUT. Minimum inhibitory concentrations of clarithromycin for H pylori were determined by culture test. CYP2C19 genotypes were classified into the rapid metabolizer (* 1 /* 1 ), intermediate metabolizer (* 1 /* 2 or * 1 /* 3 ), and poor metabolizer (* 2 /* 2 , * 2 /* 3 , or * 3 /* 3 ) groups. H pylori strains with A2142G or A2143G mutation had higher minimum inhibitory concentrations for clarithromycin. Cure rates in rapid, intermediate, and poor metabolizer groups were 57.8% (95% confidence interval, 42.1%-72.4%), 88.2% (78.1%-94.8%), and 92.3% (74.9%-99.1%), respectively ( P < .001). Cure rates in strains with and without A2142G or A2143G mutation were 48.3% (29.4%-67.5%) and 87.3% (79.5%-92.7%), respectively ( P < .001). SNPs of CYP2C19 and 23S rRNA of H pylori using RUT-positive gastric mucosal samples could be predictable determinants for H pylori eradication by triple therapy.

Serial invasive signal amplification reaction for genotyping permethrin-resistant ( kdr-like) human head lice, Pediculus capitis

Permethrin resistance in the human head louse, Pediculus capitis De Geer (Anopulura: Pediculidae), has been reported worldwide, is associated with the knockdown phenotype, and elicits cross-resistance to DDT and the pyrethrins. Two point mutations, T929I and L932F, in the voltage-sensitive sodium channel α-subunit gene are responsible for permethrin resistance as a resistant haplotype ( kdr-like). We have optimized a serial invasive signal amplification reaction (SISAR) protocol for the detection of these mutations using PCR amplified DNA fragments. SISAR distinguished all genotypes with high accuracy in a head louse population from Texas that was heterogeneous in terms of permethrin sensitivity. Using SISAR, resistance-conferring mutations are detected in a high throughput format, facilitating the efficient monitoring of permethrin resistance allele frequency in field populations.

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction.

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10(7) reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.