Standard Polymerase Chain Reaction Methods Research Articles

Spherical Nucleic Acid-Mediated Spatial Matching-Guided Nonenzymatic DNA Circuits for the Prediction and Prevention of Malignant Tumor Invasion.

Detection of intracellular miRNAs, especially sensitive imaging of in vivo miRNAs, is vital to the precise prediction and timely prevention of tumorgenesis but remains a technical challenge in terms of nuclease resistance and signal amplification. Here, we demonstrate a gold nanoparticle-based spherical nucleic acid-mediated spatial matching-guided nonenzymatic DNA circuit (SSDC) for efficient screening of intracellular miRNAs and, in turn, finding cancerous tissues in living organisms before the appearance of clinical symptoms. Due to the substantially enhanced nuclease resistance, the false positive signal is avoided even in a complex biological medium. Target miRNA can straighten out the hairpin DNA probe to be linear, allowing the probe to penetrate into the internal region of a core/shell DNA-functionalized signal nanoampfilier and initiate a strand displacement reaction, generating an amplified fluorescence signal. The detection limit is as low as 17 pM, and miRNA imaging is in good accordance with the gold standard polymerase chain reaction method. The ability to image intracellular miRNAs is substantially superior to that of conventional fluorescence in situ hybridization techniques, making in vivo SSDC-based imaging competent for the precise prediction of tumorigenesis. By intratumoral chemotherapy guided by SSDC-based imaging, tumorigenesis and progression are efficiently controlled before the onset of clinical symptoms.

Seems fishy: environmental DNA impacts on sketa22 quality control in salmonidae dominated waterbodies using qPCR and ddPCR

Globally, water resources used for recreation and drinking water are threatened by fecal pollution. These pollutants can cause gastrointestinal illness and environmental degradation. Additionally, most sources of fecal pollution are non-point sources stemming from multiple species. Identifying these sources is vital to categorizing the exposure risk from contact and improving remediation efforts. A common technique to provide species-specific information for fecal source identification is microbial source tracking (MST). MST quantifies DNA of host or host-associated microorganisms through polymerase chain reaction (PCR) technologies such as quantitative PCR (qPCR) or droplet digital PCR (ddPCR). MST techniques have been implemented globally and are used for routine monitoring. In the United States (US), the US Environmental Protection Agency has provided several approved standard PCR methods for MST and other recreational water quality applications. These methods have specified quality controls including sample processing controls (SPC) and assessments for sample inhibition. A standard SPC used in EPA methods involves spiking samples with salmon testes DNA (nominally originating from Chum Salmon, Oncorhynchus keta and quantifying them using Sketa22, a genus specific TaqManTM assay). This quality control (QC) behaves similarly to the microbial species being monitored. MST testing in Fall 2022 indicated elevated Sketa22 recoveries and re-analysis of samples indicated the detection of external Salmonidae DNA on both qPCR and ddPCR platforms. Our research was designed to identify the cause of this interference. Results indicate that the primer probe set may react with wild Salmonidae DNA. Analyzing the Sketa22 sequence using BLAST indicated matches with many species of Salmonidae present in the sampled stream system. Consequently, further research is required to identify the effectiveness of Sketa22 as a QC when native and migratory Salmonidae are present. General recommendations are provided to account for excess ambient Salmonidae DNA.

The ratio of microbial-to-host nucleic acid impacts detection of microbial nucleic acid in placental tissue

The placental microbiome is characterized metagenomically as a low-biomass, sparse, reliably detected collection of genetic material that is vastly outnumbered by source host DNA. This complicates its molecular detection, leading some research teams to achieve detection while others attribute detection to contamination. Contributing to this misclassification is the failure to appreciate microbial:host nucleic acid stoichiometry in amplicon generation via polymerase chain reaction (PCR). We hypothesized that germ-free pregnant mice inoculated with specific microbes during gestation will have placentas with detectable microbial signatures via targeted quantitative PCR (qPCR) but not untargeted standard PCR methods.

Exanthema on background of COVID-19 in children: is additional therapy needed?

Goal . To assess the frequency of skin manifestations in children, the need and effectiveness of pathogenetic therapy for skin rash associated with COVID-19 in children. Materials and methods . The data of the case histories of 55 children aged 8 months to 18 years with confirmed infection caused by the SARS-CoV-2 virus were analyzed. Of these, 36 were hospitalized with a diagnosis of community-acquired pneumonia of coronavirus etiology (SARS-CoV-2 virus identified) in the children’s infectious diseases department of the Mytishchi City Clinical Hospital. Another 19 children received treatment on an outpatient basis for rhinopharyngitis and/or bronchitis or tracheobronchitis against the background of laboratory-confirmed COVID-19. In most cases, there was a moderate course (64 % or 35 children) and a mild form (36 % or 20 patients). The SARS-CoV-2 virus was identified in pharyngeal and nasal swabs using the standard polymerase chain reaction method. All patients received therapy in accordance with the current temporary guidelines, and if indicated, other drugs. Results . In 7 (13 %) children in the acute period of coronavirus infection, urticarial rashes were found, in 3 (5 %) – maculopapular. Itching was mild in most cases. According to children and (or) their parents, elements of exanthema appeared in the period from 1st to 8th days from the time the first symptoms of COVID-19 developed. The rash persisted for 1 to 7 days. In 2 children who had a pronounced widespread exanthema against the background of rhinopharyngitis in the absence of pulmonary lesions, a prolonged period of significant astheno-vegetative disorders was noted. In one of the observed children on day 1, exanthema was the only manifestation of coronavirus infection. When prescribing additional antihistamines, there was no significant result. When using enterosorbents (Zosterin Ultra 30 or 60), the appearance of new elements stopped, the rash regressed within 2–5 days. Conclusions . 18 % of children treated for mild to moderate COVID-19 developed skin rashes. Exanthema with coronavirus infection in children should be considered one of the symptoms of the disease. Despite the more frequent detection of urticaria-type rash, no direct relationship with allergic history has been identified. The use of enterosorbents contributes to the relief of exanthema.

Evaluation of a wet reagent based loop isothermal amplification test for the detection of Mycobacterium ulcerans in clinical sample

Background: Buruli ulcer is a disease of poverty globally reported in >37 countries across the world. The disease affects people of all age group with >65% of cases usually examined in the advanced stage, a situation which has been shown to increase the cost of treatment. Enumerated among six research priority areas of the world health organization (WHO) is the development of simple and sensitive diagnostic tools which holds relevant keys to unlocking the other 5 research areas. In meeting the diagnostic gap, various assays have been developed including five Loop isothermal amplification test (LAMP) for detecting M. ulcerans. Exceptional among these LAMP test is a wet reagent base-LAMP test designed by Ablordey and co-workers which generates real-time results; a deviation from the earlier colourimetric based LAMP designs. The study aimed at evaluating the newly improved LAMP assay for the detection of Mycobacterium ulcerans in clinical samples. Methods and materials: Protocol for the wet reagent based LAMP (WRB-LAMP) test was optimized. Using deoxyribonucleic acid (DNA) samples of M. ulcerans prepared from culture and 75 archived clinical swab samples, technical and clinical characteristics of the test was determined. Results: WRB-LAMP detected as low as 31.4 ag/uL and was shown to be highly specific with 97% (95% CI: 84%–100%) clinical sensitivity and 100% (95% CI: 92%–100%) clinical specificity yielding an overall accuracy of 99% (95% CI: 95%–99%). Amplification properties such as constant incubation temperature and short test duration are an added advantage for field use. The assay gave an almost perfect cohen kappa agreement (p < 0.05) with the gold standard polymerase chain reaction method. The assay is rapid and meets the recommended 90% sensitivity requirement expected by WHO for new developed tools for the diagnosis of Buruli ulcer and also have similar performance as the PCR test. Conclusion: WRB-LAMP has the potential for consideration as a test deployable in district health facilities for diagnosis of Buruli ulcer disease.

ВИКОРИСТАННЯ ТЕСТІВ БАЗОВАНИХ НА АМПЛІФІКАЦІЇ НУКЛЕЇНОВИХ КИСЛОТ ПРИ КОНТРОЛІ КОРОНАВІРУСНОЇ (COVID-19) ПАНДЕМІЇ

Background. COVID-19 pandemic highlighted an importance of sensitive and specific tests that would be cost-efficient, fast and scalable. There are more than 200 COVID-19 detection tests available worldwide, with every country developing own assays. Sample collection, preparation for a test, tests itself and interpretation of results have a strong impact on the clinical value of the testing. Diversity of tests and workflows requires an analysis of their performance in clinics. Methods. Literature review, analysis of clinical reports, online resources, public and commercial reports were used to collect information about the tests. Collected information was processed for extraction of relevant to this review information. Results. Here we review COVID-19 detection tests that are based on amplification of nucleic acids. The tests employ polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP). Clinical value of the tests depends on used technologies that differ for LAMP, real-time and standard PCR methods. Diversity of sample preparation protocols, different designs of the tests, used chemistries and protocols have significant impact on performance of the tests. Tailoring of a testing workflow to available infrastructure and selection of the most efficient combination of tests and protocols for each step in a testing workflow is crucial for the success. Conclusion. Reviewed here strong and weak sides of different tests and protocols can be of help in selection of a testing workflow for achieving maximal clinical utility.

Perspectives on meeting the COVID‐19 testing challenge: A dental school collaborative

Perspectives on meeting the COVID‐19 testing challenge: A dental school collaborative

Electrochemical Sensors to Monitor Multi-Drug Resistant Organisms in the Gut Microbiome

Infections caused by multi-drug resistant organisms (MDROs) are responsible for over 2 million of hospitalization and 23,000 deaths annually in the United States, with direct financial impact of $20 billion a year. Microbiome dysbiosis (imbalance) is considered to be the main cause for MDRO colonization and the resulting infections. MDRO outbreaks need to be quickly detected and monitored for effective intervention to provide relief to the stressed patient community and healthcare facility. Unfortunately, current diagnostic methods for MDRO detection are slow and costly. Polymerase chain reaction (PCR) and enzyme immunoassay (EIA) techniques or the next generation sequencing (NGS) methods do not provide the low cost and rapid platform necessary for such MDRO pathology for a patient care facility. These methods often require several days to complete, use many reagents, and need skilled technicians in a resource intensive laboratory. In this work, we report a rapid, accurate and cost-effective electrochemical sensor capable of MDRO detection down to 104 colony forming units (CFU)/g in mice and human stool samples. To achieve this, we utilize disposable screen printed electrodes (SPEs) modified with highly specific oligonucleotide probes capable of hybridizing (duplex formation) select target gene sequences associated with MDROs. In the presence of a redox mediator, the hybridized probe/target complex generates an electrochemical current signal that differs from a non-hybridized probe molecule. The signal is highly sensitive and selective of target gene sequences and can detect as little as 3.5 picomols of target DNA. Furthermore, the SPEs can be pre-functionalized, stored, and used when needed, reducing individual sample analysis time to less than an hour. Several target sequences from two exemplary genes (AmpC and AcrB found in MDRO E. coli) have been identified and successfully detected in clinical stool samples with results comparable to the standard quantitative PCR method. Additional target genes associated with drug resistance (TEM-1, VanA, and SHV) have also been successfully detected in vitro and are ready to be tested in clinical samples. Our gut microbiome sensor is currently being developed for multiplexed operation to simultaneously detect up to eight MDROs genes, including the ones that are responsible for breaking down b-lactam containing antibiotics. This novel sensor platform will be an economical point-of-care device with little requirement of reagent handling or technical training. Figure 1

Protein mass spectrometry extends temporal blood meal detection over polymerase chain reaction in mouse-fed Chagas disease vectors.

BACKGROUND Chagas disease is highly prevalent in Latin America, and vector control is the most effective control strategy to date. We have previously shown that liquid chromatography tandem mass spectrometry (LC-MS/MS) is a valuable tool for identifying triatomine vector blood meals.OBJECTIVES The purpose of this study was to determine blood meal detection ability as a function of method [polymerase chain reaction (PCR) vs. LC-MS/MS], time since feeding, and the effect of molting in mouse-fed triatomine insect vectors targeting hemoglobin and albumin proteins with LC-MS/MS and short interspersed nuclear elements (SINE)-based PCR.METHODS We experimentally fed Triatoma protracta on mice and used LC-MS/MS to detect hemoglobin and albumin peptides over time post-feeding and post-molting (≤ 12 weeks). We compared LC-MS/MS results with those of a standard PCR method based on SINEs.FINDINGS Hemoglobin-based LC-MS/MS detected blood meals most robustly at all time points post-feeding. Post-molting, no blood meals were detected with PCR, whereas LC-MS/MS detected mouse hemoglobin and albumin up to 12 weeks.MAIN CONCLUSIONS In our study, the hemoglobin signature in the insect abdomen lasted longer than that of albumin and DNA. LC-MS/MS using hemoglobin shows promise for identifying triatomine blood meals over long temporal scales and even post-molting. Clarifying the frequency of blood-feeding on different hosts can foster our understanding of vector behavior and may help devise sounder disease-control strategies, including Ecohealth (community based ecosystem management) approaches.

Prevalence and risk factors for asymptomatic malaria and genotyping of glucose 6-phosphate (G6PD) deficiencies in a vivax-predominant setting, Lao PDR: implications for sub-national elimination goals

BackgroundLao People Democratic Republic (PDR; Laos), a landlocked country in Southeast Asia, has made important progress in reducing malaria morbidity and mortality in the past 5–6 years, and the northern provinces have very low reported incidence. To support national progress towards elimination, it is critical to verify and understand these changes in disease burden.MethodsA two-stage cluster cross-sectional survey was conducted in four districts within four northern provinces (Khua, Phongsaly Province; Paktha, Bokeo Province; Nambak, Luang Prabang, and Muang Et, Huaphanh Province). During September and October 2016, demographics and malaria risk factors were collected from a total of 1492 households. A total of 5085 persons consented to collection of blood samples for testing, by rapid diagnostic test (RDT) and polymerase chain reaction (PCR)-based testing. Risk factors for infection were examined using logistic regression; and a randomized subset of males was tested for glucose-6-phosphate dehydrogenase (G6PD) deficiencies using a combined PCR and sequencing approach.ResultsThere were zero positives by RDT, and PCR detected Plasmodium infections in 39 (0.77%; 95% CI 0.40–1.47%) of 5082 analysable samples. The species distribution was Plasmodium vivax (28 total); Plasmodium falciparum/P. vivax (5); P. falciparum (3), Plasmodium malariae (2), and P. vivax/P. malariae (1). In multivariable analysis, the main risk factors included having any other cases within the household [aOR 12.83 (95% CI 4.40 to 37.38), p < 0.001]; and lack of bed net ownership within the household [aOR 10.91 (95% 5.42–21.94), p < 0.001]; age, sex and forest-travel were not associated with parasitaemia. A total of 910 males were tested for the six most common G6PDd in SE Asia; and 30 (3.3%; 95% CI 2.1–5.1%) had a G6PD variant allele associated with G6PD deficiency, with the majority being the Union (14) and Viangchan (11) polymorphisms, with smaller numbers of Canton and Mahidol.ConclusionThis is the first rigorous PCR-based population survey for malaria infection in Northern Lao PDR, and found a very low prevalence of asymptomatic Plasmodium infections by standard PCR methods, with P. vivax predominating in the surveyed districts. Clustering of cases within households, and lack of a bed nets suggest reactive case detection, and scale-up of coverage should be prioritized. The predominance of infections with P. vivax, combined with moderate levels of serious G6PD deficiencies highlight the need for careful rollout of primaquine towards elimination goals.

Specific Magnetic Isolation of E6 HPV16 Modified Magnetizable Particles Coupled with PCR and Electrochemical Detection.

The majority of carcinomas that were developed due to the infection with human papillomavirus (HPV) are caused by high-risk HPV types, HPV16 and HPV18. These HPV types contain the E6 and E7 oncogenes, so the fast detection of these oncogenes is an important point to avoid the development of cancer. Many different HPV tests are available to detect the presence of HPV in biological samples. The aim of this study was to design a fast and low cost method for HPV identification employing magnetic isolation, polymerase chain reaction (PCR) and electrochemical detection. These assays were developed to detect the interactions between E6-HPV16 oncogene and magnetizable particles (MPs) using commercial Dynabeads M-280 Streptavidin particles and laboratory-synthesized “homemade” particles called MANs (MAN-37, MAN-127 and MAN-164). The yields of PCR amplification of E6-HPV16 oncogene bound on the particles and after the elution from the particles were compared. A highest yield of E6-HPV16 DNA isolation was obtained with both MPs particles commercial M-280 Streptavidin and MAN-37 due to reducing of the interferents compared with the standard PCR method. A biosensor employing the isolation of E6-HPV16 oncogene with MPs particles followed by its electrochemical detection can be a very effective technique for HPV identification, providing simple, sensitive and cost-effective analysis.

Correlation of mutation profile and response in patients with myelofibrosis treated with ruxolitinib

AbstractAlthough most patients with myelofibrosis (MF) derive benefit from ruxolitinib, some are refractory, have a suboptimal response, or quickly lose their response. To identify genes that may predict response to ruxolitinib, we performed targeted next-generation sequencing (NGS) of a panel of 28 genes recurrently mutated in hematologic malignancies in a cohort of patients with MF who were treated with ruxolitinib in a phase 1/2 study. We also tested for CALR deletions by standard polymerase chain reaction methods. Ninety-eight percent of patients had a mutation in ≥1 gene. Seventy-nine (82.1%) patients had the JAK2V617F mutation, 9 (9.5%) had CALR mutations (7 type 1, 2 type 2), 3 (3.1%) had MPL mutations, and 4 (4.2%) were negative for all 3. ASXL1/JAK2 and TET2/JAK2 were the most frequently comutated genes. Mutations in NRAS, KRAS, PTPN11, GATA2, TP53, and RUNX1 were found in <5% of patients. Spleen response (≥50% reduction in palpable spleen size) was inversely correlated with the number of mutations; patients with ≤2 mutations had ninefold higher odds of a spleen response than those with ≥3 mutations (odds ratio = 9.37; 95% confidence interval, 1.86-47.2). Patients with ≥3 mutations also had a shorter time to treatment discontinuation and shorter overall survival than those with fewer mutations. In multivariable analysis, only number of mutations and spleen response remained associated with time to treatment discontinuation. Patients with ≥3 mutations had the worst outcomes, suggesting that multigene profiling may be useful for therapeutic planning for MF.

Analysis of interspecific relationships between Funneliformis mosseae and Fusarium oxysporum in the continuous cropping of soybean rhizosphere soil during the branching period

This study analysed the interspecific relationships between the dominant arbuscular mycorrhizal (AM) fungus, Funneliformis mosseae, and the major soybean root rot pathogen, Fusarium oxysporum, in the rhizosphere soil of continuous cropped soybean. Our aim was to provide theoretical evidence on the AM fungi to overcome the obstacles of soybean continuous cropping. We selected soybean cultivars, including Kenfeng 16 (an intermediate cultivar), Heinong 44 (a high-fat cultivar) and Heinong 48 (a high-protein cultivar), and sowed in the soybean continuous cropping soil under different treatments. The infection status of the soybean roots during the branching period by Fu. mosseae and F. oxysporum was estimated using the standard polymerase chain reaction method, as well as their colonisation status in rhizosphere soil. The AM fungal colonisation rates and F. oxysporum disease incidence of soybean roots were determined, respectively. Quantitative polymerase chain reaction was applied to analyse the DNA content of Fu. mosseae and F. oxysporum to investigate the relationship between Fu. mosseae and F. oxysporum. The results show that both Fu. mosseae and F. oxysporum can infect the soybean roots during the branching period and colonise the rhizosphere. However, the DNA content of F. oxysporum clearly decreased in soybean root and rhizosphere samples after the inoculation with Fu. mosseae. In addition, the disease incidence of F. oxysporum significantly decreased after inoculation with Fu. mosseae, which might indicate inhibitive effects of Fu. mosseae over F. oxysporum.

Real-time PCR assay to detect Verticillium albo-atrum and V. dahliae in hops: development and comparison with a standard PCR method

Verticillium wilt, caused by Verticillium albo-atrum and V. dahliae, is a devastating disease that causes considerable economic crop losses in hop fields. The fungus can survive in soil for several years by producing resting structures, and due to the lack of effective fungicides, the disease is spreading. Thus, a fast and sensitive detection system is urgently needed. In this study we report a novel routine detection method for the identification of V. albo-atrum and V. dahliae on a molecular basis. The standard polymerase chain reaction (PCR) assay includes isolation of the fungus from affected tissue by following DNA extraction from fungal cultures and PCR identification by specific primers. In order to improve this detection assay, DNA was isolated directly from the hop bine using a commercially available DNA isolation kit. A multiplex real-time PCR assay for the simultaneous detection of V. albo-atrum and V. dahliae was established using a specific primer pairs/TaqMan probes combination. Ninety-six plants were collected from different culti-vars and locations in Germany and tested by standard and real-time PCR assay. The new detection system is more accurate, sensitive and time-saving than the standard PCR method and is suitable for routine use. The test provides a valuable tool for rapid and sensitive detection of V. albo-atrum and V. dahliae in plants and gives farmers and plant protection offices the chance to react accordingly and to evaluate measures for plant disease management against Verticillium.

Marzipan: Polymerase Chain Reaction-Driven Methods for Authenticity Control

According to German food guidelines, almonds are the only oilseed ingredient allowed for the production of marzipan. Persipan is a marzipan surrogate in which the almonds are replaced by apricot or peach kernels. Cross-contamination of marzipan products with persipan may occur if both products are produced using the same production line. Adulterations or dilutions, respectively, of marzipan with other plant-derived products, for example, lupine or pea, have also been found. Almond and apricot plants are closely related. Consequently, classical analytical methods for the identification/differentiation often fail or are not sensitive enough to quantify apricot concentrations below 1%. Polymerase chain reaction (PCR)-based methods have been shown to enable the differentiation of closely related plant species in the past. These methods are characterized by high specificity and low detection limits. Isolation methods were developed and evaluated especially with respect to the matrix marzipan in terms of yield, purity, integrity, and amplificability of the isolated DNA. For the reliable detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea, qualitative standard and duplex PCR methods were developed and established. The applicability of these methods was tested by cross-reaction studies and analysis of spiked raw pastes. Contaminations at the level of 0.1% could be detected.

HPV typing and its relation with apoptosis in cervical carcinoma from Indian population

Definite progress in understanding the etiology of cervical cancer has been achieved, and some types of human papillomavirus have been established as the central cause of cervical cancer worldwide. This study investigates the human papillomavirus infection and its correlation with apoptosis and clinicopathologic characteristics in squamous cell carcinoma of uterine cervix. Human papillomavirus typing was done by type-specific primers for high-risk human papillomavirus using standard polymerase chain reaction method. Programmed cell death (apoptosis) was determined by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. Human papillomavirus infection in tissue biopsy of cervical carcinoma was detected in 131 of 135 (97%) cases. Among the positive cases of human papillomavirus, 123 (94%) cases were human papillomavirus type 16, and five (4%) cases were human papillomavirus type 18. Out of 135 cervical carcinoma cases, 81 (60%) cases showed presence of apoptosis. The phenomenon of apoptosis was seen slightly higher in squamous cell carcinoma than in adenocarcinoma (40% in squamous cell carcinoma and 33% in adenocarcinoma). The human papillomavirus infection in cervical cancer might not play any role in the occurrence of apoptosis.

Polymerase chain reaction detection of Lactobacillus acidophilus in human oral cavity and fecal samples after 2-week consumption of yoghurt

Objective. To investigate whether short-term daily consumption of yoghurt leads to colonization by Lactobacillus acidophilus in a group of human subjects who were initially totally devoid of L. acidophilus in their oral cavities. Material and methods. Twenty-three volunteers consumed yogurt containing L. acidophilus during a 14-day trial stage. Oral and fecal samples were collected at the clearance stage and at the post-yoghurt intake stage until L. acidophilus was found. Standard polymerase chain reaction methods using specific primers were adopted for the detection and identification of L. acidophilus. Results. The isolation frequency decreased rapidly 72 h after stopping intake of yoghurt. After 1 week, L. acidophilus was absent in all oral samples. Non-significant differences were found between the survival rates of L. acidophilus in samples of saliva, plaque, tongue surface, and buccal mucosa. L. acidophilus was also found to remain in the gastrointestinal tract for longer than in the oral cavity. Conclusion. Allochthonous L. acidophilus is not likely to permanently colonize the oral cavity and intestine.

Association Analysis of Endothelial Nitric Oxide Synthase G894T Gene Polymorphism and Erectile Dysfunction Complaints in a Population-Based Survey

Erectile dysfunction (ED) is a common disorder leading to a serious, negative impact on the quality of the patient's life. The gene encoding endothelial nitric oxide synthase (eNOS) is an interesting candidate gene for understanding the physiopathology of ED, as it is involved in the catalytic production of nitric oxide (NO), the neurotransmitter that plays a critical role in penile tumescence and erection. To evaluate a potential association between the G894T polymorphism in the eNOS gene and ED complaints in a population-based sample in São Paulo, Brazil. The prevalence of ED complaints was estimated according to the answer to the question "How would you describe your ability to get and keep an erection that is adequate for satisfactory intercourse?" ED was considered to be present if the response was "sometimes" or "never." A total of 449 men were enrolled in the study and answered an eight-item questionnaire to ascertain sexual performance/ED and satisfaction. The eNOS G894T polymorphism was genotyped using a standard polymerase chain reaction method. Univariate analysis demonstrated that ED was associated with diabetes, hypertension, sleep apnea severity, increasing age and body mass index, as well as testosterone levels (P < 0.05). Forward multiple regression models indicated that age was the only independent factor associated with ED in this population (odds ratio = 1.09; 95% CI 1.06-1.11; P < 0.0001). Genotypic and allelic analyses provided no evidence for an association between this polymorphism and the risk for ED complaints in this sample. Population stratification did not affect the association test results. This is the first study to examine the effect of polymorphisms in the eNOS gene and the risk for ED utilizing a case-control approach in the Brazilian population. Our results do not support a major role for eNOS gene polymorphisms in ED in this population.

Benthic ecology of Vibrio spp. and pathogenic Vibrio species in a coastal Mediterranean environment (La Spezia Gulf, Italy)

We carried out a 16-month in situ study to investigate the ecology of Vibrio spp. and pathogenic Vibrio species in coastal sediments of the Mediterranean Sea, employing multiple-regression analysis to reveal the major environmental factors controlling their occurrence in the benthic environment. In addition, association between vibrios and sediment-inhabiting meiofauna, which is a major component of benthic ecosystems, was investigated. Culturable and total Vibrio spp. estimates by most-probable-number technique coupled with standard polymerase chain reaction (PCR) and real-time PCR methods, respectively, were at least one order of magnitude higher in sediment than in seawater. In addition, potential human pathogenic species Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus occurred in the sediment with V. parahaemolyticus being the most frequently found. In the pelagic environment, 60% of total variance in culturable Vibrio data was explained by sea surface temperature (40%), salinity (13%) and organic matter concentration (7%). In the benthic environment, sea surface temperature was the only factor that significantly affected culturable Vibrio occurrence although it explained only 25% of total variance, suggesting that additional unexplored factors may play a role as well. No correlation was found between culturable Vibrio spp. concentrations and the abundance of harpacticoid copepods in the sediment whilst a negative correlation was found between Vibrio spp. and nematode abundance which accounted for almost 90% of the total meiofaunal density. Taxonomic analysis revealed that selective bacterial feeders accounted for nearly 50% of the total nematode community and included genera such as Terschellingia, Molgolaimus and Halalaimus, suggesting that top-down control by nematode grazing may be an important factor affecting Vibrio occurrence in these sediments. It is concluded that the benthic marine environment may function as a reservoir of Vibrio spp. and potential pathogenic vibrios whose ecological features appeared substantially different from the ones recognised in the pelagic environment.

Detection of the Dihydrofolate Reductase–164L Mutation in Plasmodium falciparum Infections from Malawi by Heteroduplex Tracking Assay

Standard polymerase chain reaction methods often cannot detect drug-resistance mutations in Plasmodium falciparum infections if the mutation is present in < or = 20% of the parasites. A heteroduplex tracking assay was developed that can detect dihydrofolate reductase 164-L mutations in variants representing 1% of the parasites in an individual host. Using this assay, we confirmed the presence of the mutation in P. falciparum infections in Malawi.