Further, if proteinuria is identified, uAPR check details may provide useful insights into whether the problem lies with the cART regimen, requiring regimen change, or elsewhere, requiring further enquiry into comorbidity. In our cohort, those with biopsy-proven cART-associated damage were also identified by a high uPCR but a low uAPR, proteinuria resolved after switching cART regimen. In summary, it is important to consider the screening protocol used for urinary protein estimation in HIV-infected individuals. The use of uACR or dipstick urinalysis alone as a screening test for proteinuria may not detect significant tubular dysfunction or alert the clinician

to potential cART-related problems. Our results suggest that measuring both uPCR and uACR on a single sample (and hence obtaining a uAPR) may be both practical and helpful in evaluating proteinuria in selected HIV-infected patients, and may help to identify those in whom a more careful evaluation of tubular dysfunction is warranted. Conflicts of interest: AS has received travel bursaries and scholarships from Boehringer Ingelheim, Bristol Myers Squib, Gilead, Merck Sharp

and Dohme, Tibotec and Viiv Healthcare. KN has received funding for travel, consultancies and teaching purposes from Bristol Myers Squibb, Gilead Sciences and Viiv Healthcare. CS has received funding Daporinad mouse for travel, consultancies and teaching purposes from Gilead Sciences, Bristol Myers Squibb and Janssen-Cilag. MF has received honoraria and/or travelling scholarships from Abbott, Bristol Myers Squibb, Gilead, Janssen, Merck and Viiv Healthcare. YG has received travel bursaries and educational grants from Abbott, Gilead, Tibotec and Viiv Healthcare. SH has received honoraria

from Gilead in the past. “
“The Malawi antiretroviral therapy (ART) programme uses the public health approach to identify ART failure. Advanced disease progression may occur before switching to second-line ART. We report outcomes for patients evaluated and initiated on second-line treatment in Malawi. Patients meeting Malawi immunological or clinical criteria for ART failure in two large urban ART clinics Acesulfame Potassium were evaluated for virological failure (viral load >400 HIV-1 RNA copies/mL) and, if failure was confirmed, initiated on second-line ART (zidovudine/lamivudine/tenofovir/lopinavir/ritonavir). Patients were seen monthly and laboratory evaluations were performed quarterly and as needed. We performed logistic regression modelling to identify factors associated with mortality, mortality or new HIV illnesses, and virological suppression at 12 months. Of the 109 patients with confirmed virological failure, five patients died prior to initiation, three declined switching and 101 patients initiated second-line treatment. Over 12 months, 10 additional patients died, 34 patients experienced 45 HIV-related events, and 19 patients experienced grade 3 or 4 toxicities. Among survivors, 85.2% had HIV-1 RNA<400 copies/mL at 12 months.

However, more years of education may reflect better understanding of find more the improved prognosis for HIV infection

treatment and hence greater treatment optimism. More years of education might also be associated with greater resources and better access to medical treatment. The other socio-demographic predictors in our model – younger age, substance use and engagement with care – have been previously demonstrated in the literature [4–9,11]. Two individual questions from the ACASI interview, along with Treatment Optimism scale scores, were statistically significant predictors of TRBs in our model. Thus we suggest that these questions, combined with socio-demographic variables that would already be known to a medical provider (younger age, greater educational attainment, greater engagement with care and substance use history), could be put together as an effective brief screener for patients who have recently engaged in TRBs and who may be at risk of continuing to do so. The most robust single item was a question regarding concern about having infected Linsitinib datasheet someone else in the past 6 months. Used in conjunction

with two other questions about risk of re-infection and treatment optimism, patients could effectively be identified as needing more intensive and focused prevention resources. The proposed TRB screener asks for level of agreement with the following statements: ‘I am concerned about the risk of being re-infected with HIV’, ‘The availability of combination HIV drug treatments makes me less worried about having unprotected sex’, and ‘I am worried that I could have infected someone else with HIV in the past 6 months.’ [We chose item 2 (see Table 2) from the Treatment Optimism scale because it had the highest corrected item-total correlation.] This brief screener could be easily Enzalutamide chemical structure implemented during the course of a medical clinic visit, helping the busy medical provider identify those HIV-infected patients in

need of more intensive prevention resources. In addition to its potential temporal advantage, the screener does not require an exhaustive set of questions about sexual TRBs that could generate denial, social-desirability biases, or defensiveness. Given that these initial development and validation data came from a convenience sample, the screener will benefit from additional validation in other samples of HIV-infected patients and in samples tracked across time. Seattle has a lower proportion of African-American and Hispanic persons compared with other large urban areas which may also limit the generalizability of the findings. That said, the proportion of African-American and Hispanic patients at the Madison Clinic is significantly higher than in Seattle generally, which reflects the demographic trends in HIV infection in the United States.

[1, 2] Subcutaneous lumbar see more or abdominal localizations are exceptional and are almost exclusively secondary to local extension of tuberculosis (Pott’s disease, psoas abscess, and lymphadenitis) or to hematogenous dissemination.[3] Our patient had neither concurrent active tuberculosis (local or distant) nor a history of tuberculosis. Treatment is poorly defined. Although most thoracic wall abscesses (the most common) were treated surgically,[1] some authors proposed exclusive medical therapy.[2] Our patient received a multidrug regimen and underwent three needle aspirations and remains relapse free 2 years after

stopping treatment. “
“A 54-year-old Japanese man without underlying disease developed pneumococcal bacteremia and meningitis after traveling to the Philippines. The isolate demonstrated high affinity to the lung and invasiveness in vivo. The international travelers can

import indigenous high virulent strains even if the bacterium is commonly isolated in the home country. Streptococcus pneumoniae is an important bacterium which causes not only pneumonia but also invasive pneumococcal diseases such as bacteremia and meningitis. Invasive pneumococcal disease often occurs in immunocompromised patients and can be life-threatening in some cases. We report here a case with lethal pneumococcal disease that occurred in a seemingly healthy individual after international travel. Moreover, to confirm the virulence of the isolated strain, we experienced its invasiveness and lethality using the pneumococcal airway infection mouse model. A 54-year-old Japanese man visited the Philippines from December 29, 2007 to January 5, 2008, but his itinerary and foods during his selleck inhibitor stay were unknown. After coming back to Japan, he had sore throat, headache, and temperature. On January

7, he was referred to Kurume University Hospital by a local hospital for further examination as his laboratory findings represented bicytopenia. After his arrival at 15:30, suddenly, a clonic convulsion attacked him when he was waiting for results of his blood examination, and then his respiration and heartbeat were Reverse transcriptase arrested at 16:30 and he died at 21:30 despite of resuscitation. In his laboratory data, the white blood cell count was 1,100 cells per mL and platelet count was 5,000 per mL. C-reactive protein and procalcitonin were dramatically elevated at 31.89 mg/mL and 177.47 ng/mL, respectively. Biochemical data represented features of multiple organ failure and disseminated intravascular coagulation. Immunoglobulin G (IgG) slightly decreased at 700 mg/dL, but there were no findings of diabetes, syphilis, hepatitis B or C virus infection, adult T cell leukemia, and human immunodeficiency virus-1 (HIV-1) infection. The influenza virus antigen and the urine antigen of Legionella were negative. In radiological examination, no abnormal opacity was shown in head and chest. To determine the reason for the convulsion, the cerebrospinal fluid and the blood were sampled.

Between 2005 and 2010 between 1100 and 1300 children were born each year in the UK to diagnosed HIV-positive women. Since

virtually all diagnosed women in the last decade have taken ART to reduce the risk of MTCT, almost all of these children are uninfected. However, this means there are, in 2011, over 11 000 HIV-exposed uninfected children in the UK whose mothers conceived on combination ART (cART), or started ART during pregnancy [5]. The number of children Selleckchem DZNeP diagnosed with vertically acquired HIV infection in the UK increased from about 70 a year in the early 1990s to a peak of 152 in 2004, and declined to 82 in 2009 [6]. During the last decade, about two-thirds of newly diagnosed children were born abroad. Owing to the

increasing prevalence of maternal infection, combined with increasing maternal diagnosis rates and decreasing MTCT rates, the estimated number of infected children born in the UK has remained stable over the last decade, at about 30–40 a year. More than 300 children have also been reported, mostly in the early years of the epidemic, with non-vertically acquired infection, the majority from blood or blood products. Among HIV-positive children with follow-up care in the UK and Ireland, the rate of AIDS and mortality combined declined from 13.3 cases per 100 person years before 1997 to 2.5 per 100 person years in 2003–2006 [7]. With improving survival, the median age Selleckchem PD-1/PD-L1 inhibitor of children in follow-up increased from 5 years in 1996 to 12 years in 2010, by which time over 300 young people had transferred to

adult care [8]. Pregnancies in vertically infected young women are now occurring [9]. Before the widespread implementation of the routine offer and recommendation of antenatal HIV screening in the UK, detection rates before delivery were poor. In the mid-1990s only about one-third of infected pregnant women were diagnosed, and most of those were aware of their infection status before they became pregnant [10]. In England, the routine offer and recommendation policy was implemented in 2000, and similar policies were subsequently adopted elsewhere in the UK. By the end of 2003, virtually all maternity units had implemented the antenatal screening policy, and over two-thirds had achieved >80% uptake, with about one-third reaching oxyclozanide the 90% target [11]. Standards for monitoring antenatal screening were revised and updated in 2010 [12]. National uptake of antenatal HIV screening was reported to be 95% in 2008, up from 89% in 2005, and all regions reported at least 90% [13]. Between 2000 and 2004 the majority of HIV-positive women diagnosed before delivery were identified through antenatal screening. However, since 2005 the situation has reversed and in 2010 about three-quarters of women diagnosed before delivery were already aware of their infection before they conceived, many of them diagnosed in a previous pregnancy [5].

Hypertension was defined as see more resting systolic blood pressure >130 mmHg, resting diastolic blood pressure >85 mmHg (on three occasions) or current use of an anti-hypertensive agent. Exclusion criteria included: chronic hepatitis B or active hepatitis C virus infection, diabetes, male hypogonadism (<7.0 nmol/L), hypo- or hyperthyroidism (<0.2 or >12 μIU/mL), pregnancy or plans to become pregnant, prior myocardial infarction (MI), unstable angina, heart failure, coronary artery disease, resting ST-segment (segment between the S-wave and T-wave on the electrocardiogram) depression >1mm, coronary

artery bypass graft, stroke and active substance abuse. Both groups received monthly nutrition counselling (American Heart Association (AHA) guidelines [34]) from a research dietician. Standard of care included regular routine visits to the participant’s infectious disease physician, no added physical activity, no changes in cART and no added medications for hyperglycaemia, hyperlipidaemia or hypertension. All participants ZD1839 price signed an informed consent document and the study was approved by the Human Research Protection Office at Washington University School of Medicine. At baseline and 20 weeks, participants were examined by a physician-investigator. Waist circumference was measured at the midpoint between

the costal margin and the anterior superior iliac crest. After an overnight fast (8–10 h), resting electrocardiogram (EKG) and blood pressures (average of three resting measures), serum lipid/lipoprotein levels (total and HDL cholesterol, triglyceride, and calculated LDL and non-HDL cholesterol levels), a comprehensive metabolic panel (e.g. liver and kidney function tests), CD4 T-cell count (flow cytometry), plasma HIV RNA level (Roche Amplicor™ HIV-1 Monitor Test;

Roche, Branchburg, NJ, USA) and a 75-g, 2-h oral glucose tolerance test (oGTT) with plasma glucose and insulin monitoring at 0, 30, 60, 90 and 120 min were obtained. Whole-body and regional body composition were quantified using enhanced-array whole-body dual energy X-ray absorptiometry (software v12.4; Hologic Discovery, Waltham, MA, USA). Participants completed the Medical Outcomes Study (MOS) Short Form (SF)-36 health-related QOL (HIV-QOL) inventory and SPTLC1 a 3-day diet record to evaluate energy, macro- and selected micronutrient intakes. Fasting serum lipid/lipoproteins were quantified as described previously [35]. The accuracy of these analytical methods has been verified and standardized by participation in the Centers for Disease Control and Prevention (CDC) Lipid Standardization Program, the CDC Cholesterol Reference Method Laboratory Network, and the College of American Pathologists external proficiency programme. Blood glucose levels were quantified using the glucose oxidase reaction (Yellow Springs Instruments, Yellow Springs, OH, USA).

MR technician and assistant roles were modified to ensure advanced dispensing for discharge and increased time for MR, ward-based labelling and pre-pack selection. Changes made to the electronic discharge form allowed pharmacists to indicate prescription urgency and dispensary processes were redesigned for optimal workflow and prioritisation. MR rates, dispensing times and the proportion Bleomycin cost of ward-based prescription processing were measured monthly, using a combination of manual and electronic data collection methods. Ethics committee approval was not required. Over the period Apr 2013 to Feb 2014, the proportion

of TTOs completed at ward level on the focus wards increased from 5% to

22%. The average time for completion of these prescriptions was 12 minutes. Over the same time period, for all wards in Selleck BIBF1120 the hospital, the average turnaround time for dispensary completion of urgent TTOs reduced from 125 minutes to 32 minutes. The table below shows dispensing times in Feb 2014 compared to a baseline in Apr 2013: Pharmacist prioritisation No. of prescriptions Average dispensary turnaround time (hours) Proportion completed within target   Feb 14 Apr 13 Feb 14 Apr 13 Feb 14 Apr 13 Urgent (1–2 hours) 848 (48%) Total 1646 prescriptions with no priority assigned 0.53 2.08 100% 412 (25%) within 90 minute target MR within 24 hours of admission improved from 56% to 82% on the focus wards. We have reduced medicines related delays at discharge by optimising pharmacy activity along the entire discharge prescription pathway, moving activity from the dispensary to wards, reviewing the roles and skill mix of ward-based staff and fully integrating ward-based and dispensary processes. Improving MR rates was considered important to assist with accurate writing of discharge prescriptions and assessment of patients’; own medicines in Pazopanib ic50 preparation for discharge. Emphasis was placed on

increasing ward-based clinical screening and medicines supply to reduce bottlenecks in the dispensary and eliminate delivery delays. The hospital is now focussing on other causes of discharge delay, but whilst all wards have benefitted from this project, only six currently receive the new comprehensive ward-based service. Further work and investment is required to extend this to other wards and clinical specialities and particularly to improve availability of pharmacy services to support weekend discharges. 1. NHS England. High quality care for all now, and for future generations: Transforming urgent and emergency care services in England – Urgent and Emergency Care Review end of Phase 1 Report. Nov 2013. 2. Care Quality Commission Inspection Report. Southampton General Hospital. Dec 2012. L. Lewisa, K.

Studying individual parts of the system does not provide a complete perspective and may further weaken the evidence and undermine interventions. The aim of this review is to estimate the scale of medication errors as a problem across the medicines management system in primary care. Objectives were: To review studies addressing the rates of medication errors, and To identify studies on interventions to prevent medication errors in primary care. A systematic search of the literature was performed Selumetinib nmr in PubMed (MEDLINE), International Pharmaceutical Abstracts (IPA), Embase, PsycINFO, PASCAL, Science Direct, Scopus, Web of Knowledge, and CINAHL PLUS from 1999

to November, 2012. Bibliographies selleck chemical of relevant publications were searched for additional studies. Thirty-three studies estimating the incidence of medication errors and thirty-six studies evaluating the impact of error-prevention interventions in primary care were reviewed. This review demonstrated that medication errors are common, with error rates between <1% and >90%, depending on the part of the system studied, and the definitions and methods used. The prescribing stage is the most susceptible, and that the elderly (over 65 years), and children

(under 18 years) are more likely to experience significant errors. Individual interventions demonstrated marginal improvements in medication safety when implemented on their own. Targeting the more susceptible population groups and the most dangerous aspects of the system may be a more effective approach to error management and prevention. Co-implementation of existing interventions at points within the system

may offer time- and cost-effective options to improving medication safety in primary care. Medical error and patient Nitroxoline safety have been the subjects of discussions for government bodies, healthcare organizations, the media, researchers and patients in the past decade. The American Institute of Medicine report, ‘To err is human,’ describes the harmful, common, expensive and, importantly, the preventable nature of medical errors.[1] A UK Department of Health report, ‘An organization with a memory: learning from adverse events in the NHS (National Health Service),’[2] emphasises the importance of learning from errors based on their potential for reoccurrence. These government reports underscore the need for a paradigm shift in safety culture within healthcare teams and organisations, the role of teamwork and active reporting. The USA, UK, World Health Organization, and many developed countries including Australia and Denmark have identified that priority needs to be given to improving patient safety and outcome.[2–6] Medication errors are one of the most common types of medical errors resulting in patient morbidity and mortality.

C.M., who is also a recipient of grant R01 DA012609 from the National Institutes of Health. Abbreviations ACEA arachidonyl-2-chloroethylamide aCSF artificial cerebrospinal fluid AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) AM281 (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) CGP-55845 ((2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)

Akt inhibitor phosphinic acid) CGRP calcitonin gene-related peptide CI confidence interval CTAP D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 DMSO dimethyl sulfoxide DRG dorsal root ganglion GPCR G protein-coupled receptor NK1R neurokinin 1 receptor TRPV1 transient receptor potential cation channel, subfamily V, member 1 “
“Glutamate is the major excitatory neurotransmitter in the central nervous system. Considerable evidence suggests that both ionotropic and metabotropic glutamate receptors are involved in pain hypersensitivity. However, glutamate receptor-based therapies are limited by side-effects because the activities of glutamate receptors are essential for many important physiological functions. Here, we review recent key findings in molecular and cellular mechanisms of glutamate receptor regulation and their roles in triggering

and sustaining pain hypersensitivity. Targeting these molecular mechanisms could form the basis for new therapeutic strategies for the treatment of chronic pain. “
“Stress exposure resulted in brain induction

of immediate-early selleck compound genes (IEGs), considered as markers of neuronal activation. Upon repeated exposure to the same stressor, reduction of IEG response (adaptation) has been often observed, but there are important discrepancies in literature that may be in part related to the particular IEG and methodology used. We studied the differential pattern of adaptation of the IEGs c-fos and arc (activity-regulated cytoskeleton-associated protein) after repeated exposure to a severe stressor: immobilization on wooden boards (IMO). Rats repeatedly exposed to IMO showed reduced c-fos mRNA levels in response to acute BCKDHB IMO in most brain areas studied: the medial prefrontal cortex (mPFC), lateral septum (LS), medial amygdala (MeA), paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus. In contrast, the number of neurons showing Fos-like immunoreactivity was only reduced in the MeA and the various subregions of the PVN. IMO-induced increases in arc gene expression were restricted to telencephalic regions and reduced by repeated IMO only in the mPFC. Double-labelling in the LS of IMO-exposed rats revealed that arc was expressed in only one-third of Fos+ neurons, suggesting two populations of Fos+ neurons. These data suggest that c-fos mRNA levels are more affected by repeated IMO than corresponding protein, and that arc gene expression does not reflect adaptation in most brain regions, which may be related to its constitutive expression.

5% w/v. This is in contrast to glucose, where concentrations above 0.2% w/v resulted in the saturation of growth (Fig. 1a). Casamino concentrations higher than 0.5% w/v were not tested because the resulting OD of more than 0.7 is already rather high for turbidity measurements and higher values Pirfenidone clinical trial would be imprecise. When high cell masses are needed, for example for biochemical experiments, casamino acid concentrations higher than 0.5% w/v should be used. As a next application of growth in microtiter plates, the usage of seven different carbon sources was investigated (Fig. 1c). Haloferax volcanii did not grow at all on mannose, but to a variable extent on the other six carbon

sources. The best growth was obtained on glucose and fructose, followed by glycerin (and pyruvate, data not shown), xylose and arabinose, and the slowest growth was obtained with acetate as the sole carbon and energy source. These results, together with the very fast growth on casamino acids (Fig. S2), underscore the versatile metabolism of H. volcanii that can grow on a variety of different sugars, sugar alcohols, acids, amino acids and peptides. It will be interesting to test further and more unusual carbon sources like various polymers

or man-made chemicals. The next aim was to SCH772984 clinical trial unravel the vitamin dependence of H. volcanii. About 20 years ago, it was reported that H. volcanii stops growing after two or three serial dilutions in a synthetic medium, suggesting that vitamins are missing, and that the addition of biotin and thiamine is enough to allow prolonged growth in a synthetic medium (Kauri et al., 1990). At that time, we were working with H. volcanii strain WR340 and found that the addition of biotin and thiamine did not yield reproducible and satisfactory results; therefore, we started to add 0.01% w/v yeast extract Quisqualic acid as a vitamin source. However, several groups regularly reported the growth of H. volcanii in a synthetic medium with biotin and thiamine as the sole vitamin sources (e.g. Allers et al., 2004; Blaby et al., 2010); therefore, we used microtiter-based

growth to reinvestigate the vitamin dependence of H. volcanii. Much to our surprise, repeated serial dilutions of precultures in the absence of added vitamins did not lead to growth arrest and H. volcanii and it grew rather well in the absence of vitamins (Fig. 2), in contrast to earlier observations (Kauri et al., 1990). This clearly showed that H. volcanii is able to synthesize all coenzymes and prosthetic groups and does not depend on vitamin addition. However, the addition of both biotin and thiamine enhanced the growth rate, indicating that the biosynthesis rates of both substances limited the maximal growth rate. However, the effect was not additive; the addition of both biotin and thiamine led to a growth rate lower than that obtained with the addition of thiamine alone, but the difference was rather small (Fig. 2).

After vortexing, the samples were boiled in a water bath for 10 min and subsequently refrigerated at 4 °C for 10 min. Finally, the samples were centrifuged at 16 000 g for 2 min and 100 μL of the

supernatant was used as template DNA. All samples were immediately used for multiplex http://www.selleckchem.com/products/fg-4592.html and real-time PCR assays after preparation. The PCR assay was optimized using an MJ PTC 100 thermocycler (Bio-Rad, Hercules, CA). The primer sets for the PCR assay are listed in Table 2. All primers were synthesized by Integrated DNA Technologies (IDT, Coralville, IA). The reactions resulted in a 90-bp fragment for C. jejuni, a 150-bp fragment for E. coli O157:H7 (Sharma et al., 1999), and a 262-bp fragment for S. Typhimurium. (Cheng et al., 2008). The Campylobacter spp. primers were designed

by targeting a conserved region of the hsp60 gene. Reactions specific for each pathogen were first carried out independently and each reaction consisted of a 25-μL total volume mixture with 12.5 μL of SYBR Green Premix Ex Taq™ (Takara, Fisher Scientific, Pittsburg, PA), 800 nM of each primer, 1.6 μL of bovine serum albumin (BSA, 20 mg mL−1), 1 μL of DNA template, and water to volume. After BGB324 each PCR reaction was optimized independently, an m-PCR reaction was optimized to detect all three pathogens simultaneously and three independent experiments were performed to verify the reproducibility. The m-PCR reaction consisted of 25 μL of total volume mixture with 12.5 μL of SYBR Green Premix Ex Taq™ (Takara, Fisher Scientific), 400 nM of Campylobacter spp.-specific primers, 400 nM of E. coli O157:H7-specific primers, 960 nM of Salmonella spp.-specific primers, 1.6 μL of BSA (20 mg mL−1), 3 μL of three DNA templates, and water to volume. The PCR reaction was optimized to conditions of 94 °C for 2 min. and then 35 cycles

of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension cycle at 72 °C for 5 min. The PCR products were separated in a 2% agarose gel at 100 V for 20 min. Gels were stained with ethidium bromide (10 mg mL−1) and viewed using a UV transilluminator. The SYBR green real-time PCR assay was optimized using an Eppendorf Masterplex thermocycler ep (Eppendorf, Westbury, NY). Gradient Technology in the Eppendorf unit was used to optimize annealing and extension temperatures and times. Real-time PCR assays were conducted oxyclozanide as three independent experiments and triplicate samples per experiment. The same primer sets utilized for conventional PCR, listed in Table 1, were also used for the SYBR green real-time PCR reaction. A 25-μL total volume reaction mixture consisted of 12.5 μL of SYBR Green Premix Ex Taq™ (Takara, Fisher Scientific), 800 nM of each primer, 1.6 μL of BSA (20 mg mL−1), 1 μL of DNA template, and water to volume. The PCR reaction was optimized to the conditions of 95 °C for 2 min., followed by 40 cycles of 95 °C for 15 s, 55 °C for 15 s, and 68 °C for 20 s, with fluorescence being measured during the extension phase.