Polymerase Chain Reaction-high Resolution Melting Research Articles

Vitamin D Deficiency and Its Association With Vitamin D Receptor Gene Variants Among Malaysian Women With Hypertensive Disorders in Pregnancy: Protocol for a Nutrigenomics Study.

Vitamin D deficiency has been associated with hypertensive disorders in pregnancy (HDP). The risk of developing HDP was reported to be further augmented among individuals with a vitamin D receptor (VDR) genetic variant. However, the reported roles of VDR variants in hypertensive disorders are inconsistent among different populations. Given the relatively higher incidence of vitamin D deficiency among Malaysian pregnant women and the high incidence of HDP in this population, we hypothesize that there may be associations between the risk of vitamin D deficiency and HDP with VDR genetic variants. This paper outlines the protocol for a study to determine the association of vitamin D status and VDR sequence variants among Malaysian pregnant women with HDP. This prospective study consists of two phases. The first phase is a cross-sectional study that will entail gathering medical records, a questionnaire survey, and laboratory testing for vitamin D status, with a planned recruitment of 414 pregnant women. The questionnaire will be utilized to assess the risk factors for vitamin D deficiency. The vitamin D status will be obtained from measurement of the vitamin D (25-hydroxyvitamin D3) level in the blood. The second phase is a case-control study involving a Malay ethnic cohort with vitamin D deficiency. Participants will be divided into two groups with and without HDP (n=150 per group). Genomic DNA will be extracted from the peripheral blood monocytes of participants using the Qiagen DNA blood kit, and VDR sequence variants will be determined using polymerase chain reaction-high-resolution melting (PCR-HRM) analysis. Sanger sequencing will then be used to sequence randomly selected samples corresponding to each identified variant to validate our PCR-HRM results. The VDR genotype and mutation frequencies of BsmI, ApaI, TaqI, and FokI will be statistically analyzed to evaluate their relationships with developing HDP. As of December 2023, 340 subjects have been recruited for the phase 1 study, 63% of whom were determined to have vitamin D deficiency. In the phase 2 study, 50 and 22 subjects have been recruited from the control and case groups, respectively. Recruitment is expected to be completed by March 2024 and all analyses should be completed by August 2024. The outcome of the study will identify the nonmodifiable genetic components contributing to developing vitamin D deficiency leading to HDP. This will in turn enable gaining a better understanding of the contribution of genetic variability to the development of HDP, thus providing more evidence for a need of customized vitamin D supplementation during pregnancy according to the individual variability in the response to vitamin D intake. ClinicalTrials.gov NCT05659173; https://clinicaltrials.gov/study/NCT05659173. DERR1-10.2196/53722.

Associations between Selected ADRB1 and CYP2D6 Gene Polymorphisms in Children with Ventricular and Supraventricular Arrhythmias.

Background and Objectives: Tachycardia is a common cardiovascular disease. Drugs blocking β1-adrenergic receptors (ADRB1) are used in the therapy of arrhythmogenic heart diseases. Disease-related polymorphisms can be observed within the ADRB1 gene. The two most important are Ser49Gly and Arg389Gly, and they influence the treatment efficacy. The family of the cytochrome P450 system consists of the isoenzyme CYP2D6 (Debrisoquine 4-hydroxylase), which is involved in phase I metabolism of almost 25% of clinically important drugs, including antiarrhythmic drugs. A study was conducted to detect the ADRB1 and CYP2D6 gene polymorphisms. Materials and Methods: The material for the test was whole blood from 30 patients with ventricular and supraventricular tachycardia and 20 controls. The samples were obtained from the Department of Pediatric Cardiology. The first to be made was the extraction of DNA using a GeneMATRIX Quick Blood DNA Purification Kit from EURx. The selected ADRB1 and CYP2D6 gene polymorphisms were detected by high-resolution melting polymerase chain reaction (HRM-PCR) analysis. Results: Based on the analysis of melt profile data for each PCR product, the identification of polymorphisms was carried out. Heterozygotes and homozygotes were found in the examined alleles. Conclusions: The frequency of the Arg389Gly polymorphism differs statistically significantly between the control group and patients with supraventricular and ventricular arrhythmias, as well as between these two groups of patients. Moreover, the Arg389Gly polymorphism was statistically more prevalent in the group of girls with SVT arrhythmia compared to girls with VT. A few carriers of homozygous and heterozygous systems of the S49G polymorphism were detected among patients with arrhythmias, as well as control group. The percentage of individuals carrying the CYP2D6 4 allele as either homozygous or heterozygous was observed in the study and control groups. The high prevalence of the CYP2D6*4 allele carriers in both groups prompts the optimization of beta-1 blocker therapy.

Solute Carrier Family 45 Member 2 Germline Variants in the Colombian Population with Melanoma

Background: The solute carrier family 45-member 2 (SLC45A2) gene, located on chromosome 5p13.2, is involved in melanin biosynthesis. Single-nucleotide variants (SNVs) in this gene are associated with skin, eye, and hair color variations in the population. SNVs p.L374F (C/T) and p.E272K (C/G) are additionally associated with protection against melanoma. Objectives: This study aimed to evaluate the association of SNVs p.L374F and p.E272K with the development of melanoma in a sample of individuals from Bogotá, Colombia. Methods: In this case-control study, DNA samples were obtained from individuals after signing an informed consent form. Genotyping was performed by real-time polymerase chain reaction high-resolution melting (PCR-HRM). Results: Phototype II was found in 19% of the subjects, phototype III in 70%, and phototype IV in 11%. Eighty percent of the subjects had brown eyes and dark brown hair color. Both SNVs were in Hardy-Weinberg equilibrium. The p.Glu272Lys variant was found to be a protective factor for melanoma, while the p.Phe374Leu variant was found to be a risk factor. The study also found that a CG haplotype was a risk factor for melanoma [odds ratio (OR), 2.75; 95% CI, 1.22 - 6.22; P = 0.021]. The study also found a strong correlation between variants and phototypes, as well as the sex of the patients. The study also analyzed the impact of these variants on the protein structure and found that the p.Phe374Leu variant has a probable pathogenic effect on the function of the protein. Conclusions: The frequencies found for the SLC45A2 gene variants are consistent with other studies conducted in the Latin American population, where the predominant phototypes are II and III. The CG haplotype is associated with a higher risk of melanoma.

TBX 5 gene mutation analysis among Tanzanian children with congenital heart diseases using high-resolution melting assays

Early cardiac development is governed by transcription factor genes. TBX5, a T-box transcription factor gene, plays an important role in the development of the second heart field during cardiac septation by promoting cell cycle progression through the enhancement of Cdk6 and hedgehog signaling pathways. TBX5 binds to the promoter region of genes, enhancing the expression of alpha cardiac myosin heavy chain 6 (MYH6), which is a predominant isoform found in human cardiac tissue. TBX5 gene mutations are postulated to cause congenital heart diseases. A casecontrol TBX5 mutational analysis was performed to provide insight into the etiology of sporadic congenital heart diseases in our setting. We used a magnetic induction cycler (mic-PCR), which is a next-generation tool for polymerase chain reaction-high resolution melting assays, to detect mutations in children with sporadic isolated congenital heart diseases. A retrospective casecontrol study was conducted at the Jakaya Kikwete Cardiac Institute. The peripheral blood samples were collected, and DNA was extracted using the Quick-DNA Miniprep Kit. The primers were designed using Primer 3 software, validated using the program BLAST, and checked for hairpin and homo-hetero-dimerization using the IDT oligo analyzer. Real-time polymerase chain reaction (PCR)-high-resolution melting assays for screening TBX5 gene mutations were done using a magnetic induction cycler. We found two (2) TBX5 mutations in exon 5, among patients with Atrial-Ventral Septal Defects (ASVD) and Atrial-Septal Defects (ASD) and none among controls. TBX5 exon 5 is a molecular hotspot for isolated congenital heart diseases.

Association of APOBEC3A/B deletion variant with endometrial cancer risk in Norwegian population: A case-control study (242)

<b>Objectives:</b> APOBEC3 family of proteins play important roles in intrinsic responses to viral infection by introducing mutations into the viral nucleic acids. APOBEC3 activity targeting the host cell's DNA may lead to mutations contributing to tumorigenesis. Uterine cancers are found to be linked with three APOBEC associated signatures; single-base substitution signatures 2 and -13 and double-base substitution signature 11. A germline deletion of 30Kb in chromosome 22 affecting the APOBEC3A and -B genes leads to the formation of a hybrid transcript, APOBEC3A/B, found to be more stable than the wild-type APOBEC3A and causes more DNA damage through higher protein levels. To the best of our knowledge, the potential association between the APOBEC3A/B deletion variant and the risk of endometrial cancer has not yet been assessed. This study aimed to evaluate the APOBEC3A/B deletion as a potential risk modulating factor for endometrial cancer. <b>Methods:</b> DNA extracted from blood samples of Norwegian (Caucasian) women diagnosed with primary uterine adenocarcinoma (<i>n</i>=1,470), and healthy controls (<i>n</i>=1,918) were screened for germline APOBEC3A/B deletion variant using quantitative polymerase chain reaction high-resolution melting (qPCR-HMR) curves. For technical validation, 35.4% of cases were genotyped for SNP rs12628403 (Chr 22:38962032), an SNP in proximity to the APOBEC3A/B-deletion start point that is found in strong linkage disequilibrium with the deletion allele. The potential difference in results from APOBEC3A/B deletion and rs12628403-analyses were considered negligible due to a very low recombination rate of 5.8x10-3. <b>Results:</b> Cases and controls were in Hardy-Weinberg equilibrium for genotype distribution (p=0.78 and p>0.4, with a MAF of 0.072 and 0.094, respectively). Our study cohort showed a reduced risk of endometrial cancer in dominant and allele models (OR: 0.75, 95 % CI: 0.62-0.91, p=0.003 and OR: 0.75, 95 % CI: 0.63-0.90, p=0.0002, respectively). In a subgroup analysis by histology, endometrioid endometrial cancer showed similar patterns (dominant model: OR: 0.64, 95% CI: 0.51-0.79, p=0.1x10-3 and allele model: OR: 0.64, 95% CI: 0.52-0.79, p=0.1x10-3). No clear risk association was observed in non-endometrioid endometrial cancer subtype. Among age subgroups generated with a 10-year cut-off, dominant and allele models yielded a significantly reduced cancer risk in individuals in the age range 50-59 (OR: 0.51, 95% CI: 0.33-0.78, p=0.002 and OR: 0.54, 95% CI: 0.35-0.81, p=0.002, respectively) and 60-69 (OR = 0.62, 95% CI=0.43-0.88, p=0.8x10-3 and OR=0.62, CI=0.44-0.88, p=0.005, respectively). <b>Conclusions:</b> APOBEC3A/B deletion variant is associated with reduced endometrial cancer risk in Norwegian women.

Molecular characterization of Burkholderia mallei strains isolated from horses in Brazil (2014-2017).

Glanders is an infectious zoonosis caused by Burkholderia (B.) mallei that mainly affects equids. The objective of this work was to provide additional knowledge on the diversity of the strains circulating in Brazil. Six Burkholderia mallei isolates obtained during necropsies of glanderous horses between 2014 and 2017 in two different states (Pernambuco and Alagoas) were analyzed by polymerase chain reaction-high-resolution melting (PCR-HRM). While four strains (9902 RSC, BM_campo 1, BM_campo 3 and UFAL2) clustered in the L3B2 branch, which already includes the Brazilian 16-2438_BM#8 strain, two strains (BM_campo 2.1 and BM_campo 2.2) clustered within the L3B3sB3 branch, which mostly includes older isolates, from Europe and the Middle East. Whole genome sequencing of two of these strains (UFAL2 and BM_campo 2.1), belonging to different branches, confirmed the HRM typing results and refined the links between the strains, including the description of the L3B3Sb3Gp1SbGp1 genotype, never reported so far for contemporary strains. These results suggest different glanders introduction events in Brazil, including a potential link with strains of European origin, related to colonization or trade.

High-resolution melting PCR analysis for genotyping the gene polymorphism of TNF-α, TGF-β1, IL-10, and IFN-γ in lung transplant recipients

High-resolution melting (HRM) analysis is a genotyping method which has the advantages of simple, rapid, low-cost and closed-tube operation.This study evaluated HRM analysis as an option for detecting the single nucleotide polymorphism (SNP) of cytokine, and profiled the distribution of cytokine gene polymorphism in the lung transplant recipients (LTRs).High-resolution melting-polymerase chain reaction (HRM-PCR) assays for genotyping tumor necrosis factor alpha (TNF-α) (-308 A/G), tumor growth factor beta 1 (TGF-β1) (+869 T/C), interleukin 10 (IL-10) (-592 C/A, -819 T/C, -1082 G/A), and interferon gamma (IFN-γ) (+874 T/A) SNPs were developed on the LightCycler® 480. The SNPs of the aforementioned cytokine genes in 322 LTRs and 266 normal controls were detected using HRM-PCR approach. To confirm the accuracy of the HRM-PCR assay, we randomly selected 100 samples from the LTRs and detected the aforementioned SNPs with sequence-specific primer-polymerase chain reaction (SSP-PCR) method, using a commercial kit.The data show that the HRM-PCR assay can distinguish all the cytokine SNPs, and the results of HRM-PCR analysis are in complete concordance to the genotyping results obtained using a commercial kit (κ = 1.0). Our data also show that the allele and genotype frequencies of the abovementioned cytokine are not significantly different between the LTRs and the control groups (p > 0.05). In addition, we found the genotypes of TGF-β1 +869 associated with high expression phenotype were prevalent in the LTRs. On the contrary, for TNF-α -308, IL-10 and IFN-γ, the genotypes associated with low expression phenotype were most common in the LTRs.In this study, we described a rapid, low-cost and high-throughput HRM-PCR technology for genotyping cytokine SNPs. Our data may be utilized for future studies examining the associations of cytokine gene polymorphisms with the prognosis of the LTRs.

Comparison of Direct Sequencing with Real-time PCR High Resolution Melt and PCR Restriction Fragment Length Polymorphism Analysis to Identify Clinically Important Candida Species

Background: Candida albicans is the predominant yeast reported from human infection. Non-albicans Candida species have been recently developed as medically vital fungi. Therefore, it is essential to detect and identify the pathogens at the species level to prescribe appropriate treatment. Methods: This study assessed two complementary methods, including real-time polymerase chain reaction-high resolution melt (PCR-HRM) and polymerase chain reaction-restriction fragment length morphism (PCR-RFLP) with standard PCR and Sanger sequencing as the benchmark. Results: In total, 66 samples were tested, and two newly-advanced assays were more effective and displayed comprehensive concordance (66/66, 100%) with Sanger sequencing outcomes. Moreover, accurate and economical tests were positively advanced by real-time PCR-HRM for C. albicans and C. parapsilosis complexes. Conclusions: Given the number of studies performed on the comparison of sensitivity and specificity of phenotypic and genotypic methods to diagnose and identify invasive fungal pathogens and the findings of this study, it could be stated that the correlative PCR-HRM and PCR-RFLP methods were effectively advanced as substitutes for conventional Sanger sequencing for the reasonable identification. However, supplementary evaluations and confirming studies should be carried out with a broad range of samples to standardize this method for routine application in medical laboratories.

Real-time PCR High-resolution Melting Analysis for the Species Identification of Meat Products: Focusing on Food Safety and Detection of Meat Adulterations

Background: Real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis are currently considered as reliable techniques for the species identification of meat-based products and widely used to detect meat adulteration. Objectives: To examine the validity of real-time PCR and HRM analysis to identify meat species in meat-based products. Methods: Meat samples from five species (i.e., cattle, sheep, chicken, turkey, and wild pig) were purchased. Minced meat from the animal species of interest was prepared at the purities of 10%, and 20% and also were prepared as single and mixtures of two species. For molecular assessments, DNA samples were extracted from all the meat samples and subjected to real-time PCR by amplifying a mitochondrial cytochrome b specific for each species. Results: All the meat species studied in this research were successfully detected in the mixed meat samples when separately examined by real-time PCR. High-resolution melting analysis showed that all the meat species of interest were efficiently distinguished when examined simultaneously. Conclusions: The data presented here shows that the real-time PCR and HRM analysis are reliable methods for the identification of meat species used in meat products.

PCR-HRM for Detecting JAK2V617F Gene Mutation: Is It a Sensitive Assay?

Background: A substitution of G to T at nucleotide 1849 in exon 14 of the Janus kinase2 (JAK2) gene is well recognized in myeloproliferative neoplastic disorders (MPNs). Based on WHO guidelines, detection of the mutation is very important to confirm the disease in suspected patients.&#x0D; Methods: Eighty-seven patients with different background diseases were tested for JAK2 V617F mutation by four different methods, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), polymerase chain reaction-high resolution melting (PCR-HRM), and two different commercial kits.&#x0D; Results: The mean age of patients was 53.38±17.43 years, 72.4% were males, and 37.6% were females. JAK2 mutation was detected in 16 patients (18.3%). Of those, 7 (43.75%) suffered from PV, 5 (31.25%) from ET, 3 (18.75%) from PMF, and 1 (6.15%) from unclassified neoplastic disorders. The frequency of JAK2 mutation was 71.4% (5/7) in PV, 80% (4/5) in ET, and 66.7% (2/3) in PMF patients. The sensi- tivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and GE of PCR-HRM for detection of the JAK2 mutation was 86.7%, 100%, 100%, 97.3%, and 97.7%, respectively. While the sensitivity, specificity, PPV, NPV, and GE of PCR-RFLP were 93.3%, 80.5%, 50%, 98.3%, and 82.7%, respectively. On the other hand, the sensitivity, specificity, PPV, NPV, and GE of ARMS assays were evaluated by about 80%, 96%, 100%, 96%, and 96.5%, respectively.&#x0D; Conclusion: This study showed that PCR-HRM was a more sensitive assay to detect the JAK2 V617F mutation than the other assays. So, it can be used as a quick, easy, and effective method for screening the JAK2 V617F mutation in patients with MPNs disorders. PCR-RFLP must accompany it as a gold standard method for confirmation of the mutation of JAK2 V617F.

Flow cytometry in detection of Nucleophosmin 1 mutation in acute myeloid leukemia patients: A reproducible tertiary hospital experience.

Nucleophosmin 1 (NPM1) mutation is one of the most frequent gene mutations in adult acute myeloid leukemia (AML), being detected in 35% of all cases and in up to 60% of patients with normal karyotype AML. AML with mutated NPM1 has distinct pathology, immunophenotyping, and confirmed favorable prognostic significance. Hence, AML with mutated NPM1 is a separate entity in the revised 2016 World Health Organization classification. This study aimed to evaluate the use of a reproducible flow cytometry approach in the assay of mutant NPM1 protein in AML patients and to correlate flow cytometric results with the NPM1 gene mutation. Eighty-nine newly diagnosed AML patients were evaluated for the expression of mutant NPM1 using flow cytometry and for the presence of NPM1 exon 12 mutations using high-resolution melting polymerase chain reaction (HRM PCR). The NPM1 mutation was found in 35 (39.3%) patients by HRM PCR. These patients showed a significantly higher level of percentage of positive-stained cells (% positive cells) and normalized median fluorescence intensity (MFI) for mutant NPM1 by flow cytometry than the negative mutation group. Flow cytometric detection of mutant NPM1 offers a possible tool to indicate NPM1 mutational status.

Genetic identification of flax, chia and sesame seeds in processed foods

Chia, sesame and flax seeds are becoming increasingly more frequent components of functional foods because of their proven health benefits. The identification of plant components in complex matrices is generally performed using DNA typing. This method is a fast and economic tool widely applied to assess the genetic origin and authenticity of products from the food supply chain. The aim of this study was to compare two DNA-based methods, quantitative polymerase chain reaction high-resolution melting (qPCR-HRM) and endpoint PCR, used to identify chia, flax and sesame seeds in laboratory-made and commercial products. For this purpose, an endpoint PCR and qPCR-HRM methods were developed. DNA was extracted from chia, sesame and flax seeds, laboratory-made and commercial products. The analysis of flax, chia and sesame melting profiles using HRM post-PCR analysis allowed the identification of chia, sesame and flax in processed foods. The results obtained showed that qPCR-HRM is a cost-effective and efficient method for the identification, authentication and/or detection of seeds in processed food and complex matrices containing chia, sesame and flax.

High Resolution Melt Assays to Detect and Identify Vibrio parahaemolyticus, Bacillus cereus, Escherichia coli, and Clostridioides difficile Bacteria.

Over one hundred bacterial species have been determined to comprise the human microbiota in a healthy individual. Bacteria including Escherichia coli, Bacillus cereus, Clostridioides difficile, and Vibrio parahaemolyticus are found inside of the human body and B. cereus and E. coli are also found on the skin. These bacteria can act as human pathogens upon ingestion of contaminated food or water, if they enter an open wound, or antibiotics, and environment or stress can alter the microbiome. In this study, we present new polymerase chain reaction (PCR) high-resolution melt (HRM) assays to detect and identify the above microorganisms. Amplified DNA from C. difficile, E. coli, B. cereus, and V. parahaemolyticus melted at 80.37 ± 0.45 °C, 82.15 ± 0.37 °C, 84.43 ± 0.50 °C, and 86.74 ± 0.65 °C, respectively. A triplex PCR assay was developed to simultaneously detect and identify E. coli, B. cereus, and V. parahaemolyticus, and cultured microorganisms were successfully amplified, detected, and identified. The assays demonstrated sensitivity, specificity, reproducibility, and robustness in testing.

Genetic association of E26 transformation specific sequence 1 polymorphisms with the susceptibility of primary biliary cholangitis in China

Primary biliary cholangitis (PBC) is a chronic and cholestatic liver disease characterized by an autoimmune-mediated destruction of intrahepatic bile ducts. E26 transformation specific sequence 1 (ETS-1) is a transcription factor regulating the expression of various immune-related genes. The aim of our study was to identify the associations between the gene polymorphisms of ETS-1 with the susceptibility and clinical characteristics of PBC in Chinese Han population. Three single nucleotide polymorphisms (rs4937333, rs11221332 and rs73013527) of ETS-1 were selected based on relevant studies. Genotyping was executed with polymerase chain reaction-high resolution melting (PCR-HRM) assay. SNP rs4937333 of ETS-1 was prominent correlation with the susceptibility of PBC (P = 0.007, OR = 1.44, 95%CI = 1.10–1.88). For rs4937333, PBC patients carrying the allele T assumed high-level TP (P = 0.020), and homozygous genotype TT assumed low-level RDW (P = 0.033). For rs11221332, PBC patients carrying the allele T assumed high-level TP and HDLC (P = 0.004, P = 0.015, respectively). For rs73013527, PBC patients carrying the allele T assumed low-level PLT (P = 0.002), and homozygous genotype TT assumed high-level RDW (P = 0.021). In conclusion, Gene polymorphisms of ETS-1 present relevant with the susceptibility of PBC, and affect the expression of TP, HDLC, PLT and RDW concentrations in patients with PBC.

Resistance profile and molecular characterization of pyrethroid resistance in a Rhipicephalus microplus strain from Colombia.

Intensive use of chemical acaricides for the control of cattle ticks (Rhipicephalus microplus) has led to the development of multiple acaricide resistance in Colombia. The present study aimed to characterize, using toxicological bioassays and molecular biology techniques, the resistance profile of a tick strain isolated from the Arauca state, Northeast Colombia. Commercial acaricides were used in adult immersion tests to determine its in vitro efficacies. Deltamethrin showed very low activity (4-7.3%), a mixture of cypermethrin and chlorpyrifos had intermediate efficacy (64-75.2%), and ethion presented the highest activity (88.5-100%). A colony (Arauquita strain) was established and larval immersion tests confirmed high resistance level to deltamethrin (241-fold) and susceptibility to ivermectin. A quantitative polymerase chain reaction-high resolution melt technique was used to identify single nucleotide polymorphisms (SNPs) in the para-sodium channel gene. All of the genotyped individuals were mutant, presenting one (n= 7), two (n= 7) or three (n= 9) SNPs previously associated with pyrethroid resistance. Sequencing revealed a novel mutation (F712L), that was found for the first time in R. microplus ticks from South America. This is the first description of mutations associated with pyrethroid resistance in R. microplus from Colombia. The acaricide resistance pattern found in the Arauquita strain is similar to other parts of Colombia.

First Molecular Report of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) guyanensis in Paraguayan Inhabitants Using High-Resolution Melt-PCR.

American tegumentary leishmaniasis is an endemic anthropozoonosis undergoing expansion on the American continent. The disease is caused by several Leishmania species and it is manifested as cutaneous and mucocutaneous leishmaniasis. In this study, we evaluate the viability of high-resolution melt polymerase chain reaction (HRM-PCR) analysis to differentiate four closely related Leishmania species as a routine tool for the diagnosis of leishmaniasis. For this purpose, biopsy specimens from cutaneous and mucocutaneous lesions were taken from 132 individuals from endemic and non-endemic areas for leishmaniasis. Each sample was processed for parasitological, histopathological, and molecular analysis. Positive biopsy samples were analyzed by HRM-PCR of a 144-bp heat-shock protein (hsp70) gene fragment, and new cases were confirmed by sequencing. Of the 132 samples analyzed, 36 (27%) were positive for Leishmania spp., of which 86% were from cutaneous lesions and 14% from mucocutaneous lesions. We identified Leishmania (Viannia) braziliensis (84%), Leishmania (Leishmania) infantum (13%), and Leishmania (Leishmania) amazonensis (3%) in cutaneous lesions, and L. (V.) braziliensis (40%), L. (L.) infantum (20%), L. (L.) amazonensis (20%), and Leishmania (Viannia) guyanensis (20%) in mucocutaneous lesions. The main purpose of this research was to report for the first time in Paraguay the presence of L. (L.) amazonensis and L. (V.) guyanensis in patients with cutaneous and mucocutaneous lesions, using the HRM-PCR technique. In addition, we report the presence of additional new cases of L. (L.) infantum in cutaneous lesions.

Application of High-Resolution Melting PCR to Detect the Genomic Fungal ITS 2 Region.

Invasive fungal infections (IFIs) are a main cause of morbidity and mortality. High-resolution melting polymerase chain reaction (HRM PCR) is promising for the identification of fungal species via the detection of internal transcribed spacer 2 (ITS2). To assess the sensitivity and specificity of HRM PCR in diagnosing IFIs, compared with blood culture. Our study included 100 patients who were suspected of having IFIs; we analyzed their specimens via blood culture and HRM PCR. Blood culture results were positive in 57 cases and negative in 43 cases. HRM PCR results were positive in 14 cases and negative in 86 cases. The 14 cases with positive results included 4 with Candida tropicalis, 4 with Candida glabrata, and 6 with Candida krusei. HRM PCR sensitivity was 24.6%, specificity was 100%, positive predictive value (PPV) was 100%, and negative predictive value (NPV) was 50%. HRM PCR is specific but not sensitive. Blood culture is more sensitive and cannot be replaced by HRM PCR.

Development of a PCR High-Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional "Unregulated Legal High" Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose).

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an "unregulated legal high" in higher concentrations. The European Union limits thujone from Artemisia species to 35mg/kg while the U.S. Food and Drug Administration requires less than 10ppm to be "thujone-free." However, individuals can smoke or ingest A.absinthium in different forms. This study developed a polymerase chain reaction (PCR) high-resolution melt (HRM) assay to detect and identify A.absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three "unregulated legal high" species: Datura stramonium, Merremia tuberosa, and A.absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42±0.20°C, 83.88±0.22°C, and 87.77±0.15°C, respectively. The primer set developed distinguished A.absinthium from a variety of plant species and was successfully triplexed.

Cellular Aspects of Muscle Specialization Demonstrate Genotype - Phenotype Interaction Effects in Athletes.

IntroductionGene polymorphisms are associated with athletic phenotypes relying on maximal or continued power production and affect the specialization of skeletal muscle composition with endurance or strength training of untrained subjects. We tested whether prominent polymorphisms in genes for angiotensin converting enzyme (ACE), tenascin-C (TNC), and actinin-3 (ACTN3) are associated with the differentiation of cellular hallmarks of muscle metabolism and contraction in high level athletes.MethodsMuscle biopsies were collected from m. vastus lateralis of three distinct phenotypes; endurance athletes (n = 29), power athletes (n = 17), and untrained non-athletes (n = 63). Metabolism-, and contraction-related cellular parameters (such as capillary-to-fiber ratio, capillary length density, volume densities of mitochondria and intramyocellular lipid, fiber mean cross sectional area (MCSA) and volume densities of myofibrils) and the volume densities of sarcoplasma were analyzed by quantitative electron microscopy of the biopsies. Gene polymorphisms of ACE (I/D (insertion/deletion), rs1799752), TNC (A/T, rs2104772), and ACTN3 (C/T, rs1815739) were determined using high-resolution melting polymerase chain reaction (HRM-PCR). Genotype distribution was assessed using Chi2 tests. Genotype and phenotype effects were analyzed by univariate or multivariate analysis of variance and post hoc test of Fisher. P-values below 0.05 were considered statistically significant.ResultsThe athletes demonstrated the specialization of metabolism- and contraction-related cellular parameters. Differences in cellular parameters could be identified for genotypes rs1799752 and rs2104772, and localized post hoc when taking the interaction with the phenotype into account. Between endurance and power athletes these concerned effects on capillary length density for rs1799752 and rs2104772, fiber type distribution and volume densities of myofibrils (rs1799752), and MSCA (rs2104772). Endurance athletes carrying the I-allele of rs1799752 demonstrated 50%-higher volume densities of mitochondria and sarcoplasma, when power athletes that carried only the D-allele showed the highest fiber MCSAs and a lower percentage of slow type muscle fibers.DiscussionACE and tenascin-C gene polymorphisms are associated with differences in cellular aspects of muscle metabolism and contraction in specifically-trained high level athletes. Quantitative differences in muscle fiber type distribution and composition, and capillarization in knee extensor muscle explain, in part, identified associations of the insertion/deletion genotypes of ACE (rs1799752) with endurance- and power-type Sports.

Expansion of urban cutaneous leishmaniasis into rural areas of southeastern Iran: Clinical, epidemiological and phylogenetic profiles explored using 7SL high resolution melting-PCR analysis.

Cutaneous leishmaniasis (CL) has increased remarkably in Iran and has expanded into new areas. The present study aimed to assess the emerging CL outbreak in southeastern Iran using high resolution melting-polymerase chain reaction (HRM-PCR) and phylogenetic analysis using the 7SL RNA gene marker. A cross-sectional and analytical survey was conducted during a house-to-house census of 11,021 inhabitants in Narmashir County in southeastern Iran in 2016. The cases were detected by direct smear microscopic examination and sequencing and were characterized using the 7SL RNA gene. All age groups and sexes were equally affected. Most were single lesions (70.7%). The hands (55.2%) and face (37.9%) were the main sites of involvement. The disease was more common among illiterate persons. Sequencing and HRM-PCR revealed that Leishmania tropica (accession no. MH632168 Qale-Shahid) was the principal causative agent of anthroponotic CL (ACL) in new areas of expansion. This is the first emergence of ACL in rural areas of Narmashir County. Based on the molecular data, the causative parasite species confirmed to be L. tropica. Sequencing and phylogenetic analysis indicated that a single clone of the organism derived from a single source has spread into the affected villages. Construction of a main road, population movement and recent urbanization in the area are likely the major factors associated with the establishment of this new outbreak. This study was essential to enable the planning of effective therapeutic and prophylactic measures to control the disease.