Detection Limit Of Assay Research Articles

Quantification of vitamin D3 in commercial dog foods and comparison with Association of American Feed Control Officials recommendations and manufacturer-reported concentrations.

OBJECTIVE To quantify vitamin D3 (VitD3) concentrations in commercial dog foods and compare those concentrations with Association of American Feed Control Officials (AAFCO) recommendations and manufacturer-reported concentrations. DESIGN Cross-sectional study. SAMPLE 82 commercial dog foods. PROCEDURES Samples of commercially available dog foods were obtained from owners of healthy dogs in the Guelph, ON, Canada, area and owners of dogs that were patients at the Ontario Veterinary College Health Sciences Centre's Mona Campbell Centre for Animal Cancer. For each food, the VitD3 concentration was determined by high-performance liquid chromatography-tandem mass spectrometry, and adherence to AAFCO and National Research Council recommendations was assessed. Analyzed VitD3 concentrations were compared with manufacturer-reported VitD3 concentrations and between wet and dry foods, among AAFCO nutritional adequacy statements (nutrient profiles vs feeding trials and adult maintenance vs all life stages), between foods sold only by veterinarians and those sold over the counter, and between small and large manufacturers. RESULTS The analyzed VitD3 concentration was below both AAFCO and National Research Council recommendations for one sample and below the assay detection limit for another. Analyzed VitD3 concentrations did not differ significantly from manufacturer-reported VitD3 concentrations or between wet and dry foods, among foods with different AAFCO nutritional adequacy statements, between foods sold only by veterinarians and those sold over the counter, or between foods produced by small and large manufacturers. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that manufacturer-reported VitD3 concentrations were accurate and that dog owners can be confident that VitD3 intake is adequate for AAFCO-compliant commercial dog foods.

A real-time loop-mediated isothermal amplification method for rapid detection of Lawsonia intracellularis in porcine fecal samples

Porcine proliferative enteritis is a common diarrheal disease characterized by thickening of the intestinal mucosa in swine due to enterocyte proliferation, which is caused by Lawsonia intracellularis. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay was developed to detect L. intracellularis based on the conserved region of the 16S ribosomal RNA gene. The optimal reaction conditions of the real-time LAMP was 65 °C for 60 min. The LAMP products could be detected by both real-time turbidity and direct visual inspection. The assay was specific for L. intracellularis, as no cross-reaction was observed with other pathogens. The detection limit of the real-time LAMP assay was 1.4 × 10−1pg of L. intracellularis DNA, which was the same as that of real-time PCR and approximately 100 times more sensitive than that of conventional PCR. Of the 136 clinical samples, L. intracellularis DNA was identified in 60 samples by real-time LAMP, which was the same as real-time PCR and higher than conventional PCR (36.8%, 50/136). The specific, sensitive and rapid real-time LAMP assay developed in this study could be a useful alternative tool in point-of-care (POC) diagnosis of L. intracellularis infection.

Cancer risk in HIV patients with incomplete viral suppression after initiation of antiretroviral therapy.

BackgroundCancer causes significant morbidity and mortality among HIV patients in the US due to extended life expectancy with access to effective antiretroviral therapy. Low, detectable HIV RNA has been studied as a risk factor for adverse health outcomes, but its clinical impact on cancer risk remains unclear. The objective of this study was to determine whether HIV RNA <1,000 copies/mL six months after starting therapy was associated with 10-year first cancer risk.MethodsWe followed 7,515 HIV therapy initiators from a US-based multicenter clinical cohort from 1998 to 2014. We used nonparametric multiple imputation to account for viral loads that fell below assay detection limits, and categorized viral loads six months after therapy initiation into four groups: <20, 20–199, 200–999, and >999 copies/mL. We calculated estimates of the cumulative incidence of cancer diagnosis, accounting for death as a competing event. Inverse probability of exposure and censoring weights were used to control for confounding and differential loss to follow up, respectively.ResultsCrude 10-year first cancer risk in the study sample was 7.03% (95% CI: 6.08%, 7.98%), with the highest risk observed among patients with viral loads between 200 and 999 copies/mL six months after ART initiation (10.7%). After controlling for baseline confounders, 10-year first cancer risk was 6.90% (95% CI: 5.69%, 8.12%), and was similar across viral load categories.ConclusionOverall risk of first cancer was not associated with incomplete viral suppression; however, cancer remains a significant threat to HIV patients after treatment initiation. As more HIV patients gain access to treatment in the current “treat all” era, occurrences of incomplete viral suppression will be observed more frequently in clinical practice, which supports continued study of the role of low-level HIV RNA on cancer development.

2216 Characterizing physician trust and healthcare-based discrimination among long-term HIV viral trajectory groups in Washington, DC

OBJECTIVES/SPECIFIC AIMS: Discrimination within the healthcare system and physician distrust have been associated with adverse clinical outcomes for people living with HIV; however, many studies do not link these variables to biological data. We hypothesize that perceived healthcare discrimination and physician distrust associates with higher longitudinal viremia among HIV-positive women. METHODS/STUDY POPULATION: A 2006 cross-sectional survey assessed healthcare-based discrimination and physician trust in 92 HIV-positive and 46 high-risk HIV-negative women from the Washington DC Women’s Interagency HIV Study (DC-WIHS). In addition, we identified HIV viral load trajectories and demographics from the HIV-positive women who contributed≥4 semi-annual visits from 1994 to 2015. Viral suppression was defined by assay detection limits (&lt;80 to &lt;20 copies/mL). Group-based probability trajectory analyses grouped women based on longitudinal viral load patterns, and identified 3 groups: sustained viremia (n=32) with low-viral suppression over time, intermittent viremia (n=27) with varying suppression over time, and non-viremia (n=33) with high-longitudinal viral suppression. Ordinal logistic regression models assessed trajectory group and discrimination variables, controlling for demographics, using stepwise selection with significance level of α=0.05. RESULTS/ANTICIPATED RESULTS: Most women were African American (60%), insured at the time of visit (89%) and nonsmokers (56%). While physician trust did not differ by HIV viral trajectory group, trust was lower among HIV-negative women compared with HIV-positive women (p=0.03). Over 1 in 5 HIV-positive women reported discrimination in the healthcare system based on HIV status (21.3%). Report of discrimination based on drug/alcohol use was higher among HIV-negative participants (19.2% vs. 6.5%, p=0.01). Among women with longitudinal sustained viremia, report of discrimination based on race ethnicity (29%, p=0.004) and sexual orientation (15.6%, p=0.008) were higher than within the nonviremic and intermittent trajectory groups. DISCUSSION/SIGNIFICANCE OF IMPACT: Physician trust did not associate with increased longitudinal viral suppression among HIV-positive women in Washington, DC. Lack of physician trust among high-risk HIV-negative women could have implications for uptake of prevention methods. Reports of discrimination vary between HIV-positive and HIV-negative women in the Washington, DC area. The findings of healthcare system distrust among HIV-negative women has implications outside the realm of HIV, as this lack of trust may impact risk for other disease states among similar populations of women.

Paracoccidioidomycosis: Detection of Paracoccidioides brasiliensis´ genome in biological samples by quantitative chain reaction polymerase (qPCR)

Paracoccidioidomycosis (PCM) is a chronic mycosis caused by the saprobic and dimorphic species Paracoccidioides brasiliensis and P. lutzii. This disease is prevalent in Latin American countries. PCM appears as a relevant concern and challenge for the mycologists, since until now there is no a methodology suitable for an efficient and safe diagnosis and species identification. Thus, the present study aimed to validate a methodology for PCM´s diagnosis, using quantitative Polymerase Chain Reaction (qPCR) through target amplification of the gene encoding the recombinant protein Pb27, a common protein to the both species Paracoccidioides brasiliensis and P. lutzii. The experiments were performed in vitro to determine the specificity, efficiency and detection limit of qPCR assay, using specific primers and probe, which sequences were subject to a patent deposited in Brazilian CTIT, under the registration number: BR1020160078830. According to the results the technique showed sensitivity of 94% and specificity of 100%, demonstrating that it will be possible to develop a new fast and safe diagnostic PCM and can be standardized in order to present a low cost, accessible to the patient served by the public health system in Brazil and Latin America.

Validation of high-sensitivity performance for a United States Food and Drug Administration cleared cardiac troponin I assay

BackgroundHigh-Sensitivity (hs) cardiac troponin (cTn) assays are categorized by two criteria: (i) cTn values above the limit of detection (LoD) for >50% of male and female healthy cohorts of ≥300 individuals; (ii) imprecision ≤10% total %CV for sex-specific 99th-percentile clinical decision values (CDVs). No documented hs-Tn assay has yet been FDA-cleared. MethodsThe PATHFAST cTnI-II assay's LoD was 2.3 ng/L using CLSI EP-17. The AACC Universal Sample Bank of 847 healthy men (50.6%) and women (49.4%) was used to determine 99th-percentile CDVs with Nonparametric, Harrell-Davis and Robust modeling. Health/Medication questionnaires and Amino-terminal proBNP, Hemoglobin A1c and estimated Glomerular Filtration Rate surrogates excluded underlying health conditions. ResultsThe cTnI-II test's total CV was 6.1% at 29 ng/L and 7.1% at 22 ng/L; the LoD was 2.3 ng/L. Of the full 847-member healthy cohort, 113 (13.3%) were excluded by abnormal surrogate biomarkers. The final 734-member healthy population had the following (% > LoD): overall, 487 (66.3%); women, 186 (52.8%); and men, 301 (78.8%). 99th-percentile CDVs by Nonparametric modeling were: 28 ng/L (90% CI: 20–30), overall final 732-member healthy population; 20 ng/L (90% CI: 13–30), 352 women; and 30 ng/L (90% CI: 21–37), 382 men. Differences between sex-specific CDVs were not significantly different (p > .05) with Nonparametric or Harrell-Davis modeling; however, Robust Modeling did show significance (<0.05), with lower CDVs at 11 ng/L (90% CI: 9–12) and 16 ng/L (90% CI: 15–18) for the female and male cohorts, respectively. ConclusionscTnI-II is the only FDA-cleared assay that has demonstrated high-sensitivity cTn assay. Use of recommended modeling in >300 healthy subjects for determining sex-specific 99th-percentile CDVs did not show statistically significant differences except with the Robust modeling.

Performance of the Abbott RealTime MTB RIF/INH resistance assay when used to test Mycobacterium tuberculosis specimens from Bangladesh.

IntroductionThe Abbott RealTime MTB RIF/INH Resistance Assay (RT MTB RIF/INH) is an assay for the detection of rifampicin (RIF)- and/or isoniazid (INH)-resistant Mycobacterium tuberculosis (MTB). The assay can be used to test sputum, bronchial alveolar lavage, and N-Acetyl-L-Cysteine (NALC)/NaOH pellets prepared from these samples. The assay can be used in direct testing mode, or in reflex mode following a MTB positive result produced by its companion assay, Abbott RT MTB.MethodsIn this study, the direct testing mode was used to test paired sputum and NALC/NaOH pellets prepared from sputum collected from Bangladesh TB patients. One hundred and thirty two paired samples were tested.ResultsThe RT MTB RIF/INH inhibition rate was 0%. One hundred and twenty-two paired samples had results above the assay limit of detection and were analyzed by comparing with results from phenotypic drug sensitivity testing, GeneXpert MTB/RIF (Xpert), and MTBDR plus (Hain). RT MTB RIF/INH results were in good agreement with those of GeneXpert and Hain.ConclusionThe ability of this assay to detect RIF and INH resistance may contribute to the global control of multidrug resistant tuberculosis.

P-298 - Quality peroxynitrite can be produced in any laboratory Romina Medeiros

Peroxynitrite is a reactive oxygen and nitrogen specie generated in vivo when nitric oxide reacts with superoxide, being a strong oxidant and nitrating agent of biomolecules. Its preparations are used frequently in biochemical reactions, being reported many methods for its synthesis. Most of them are potentially dangerous and require special and expensive equipment. In the present work we propose a simple, fast, low cost synthesis method, with high yield and low content of contaminants; based on Hugues and Nicklin 1968. The method requires hydrogen peroxide, sodium nitrite, hydrochloric acid and sodium hydroxide as reagents, and material commonly available in any laboratory. The remaining hydrogen peroxide was removed with manganese dioxide which was also prepared in house from potassium permanganate, ethanol and water. The concentration of peroxynitrite was determined spectrophotometrically, of hydrogen peroxide with a horseradish peroxidase-based assay, and of nitrite with the Griess method. Peroxynitrite was obtained with an approximate 80% yield, which decreased to approximately 70% after treatment with manganese dioxide, a hydrogen content lower than the assay detection limit (50 μM) and a nitrite concentration of less than 5%.

Chromogenic culture media or rapid immunochromatographic test: Which is better for detecting Klebsiella pneumoniae that produce OXA-48 and can they be used in blood and urine specimens

Our goal was to compare a rapid test (OXA-48K-SeT) and four different chromogenic media (CHROMagar KPC, CHROMagar mSuperCARBA, ChromID Carba and ChromID OXA-48) for the detection of OXA-48 producing Klebsiella pneumoniae isolates and spiked urine/blood samples with these bacteria. In total 100 K.pneumoniae isolates, including 60 OXA-48 positive, 15 other carbapenemase producing, 15 Extended spectrum betalactamases (ESBL) positive and 10 carbapenem sensitive K.pneumoniae were included in the study. After all samples were inoculated into all chromogenic media, temocillin discs were placed onto the media. OXA-48K-SeT was studied according to the manufacturer's instructions and the lower detection limit was determined. Sensitivities and specificities of all chromogenic media and rapid test were detected as 100%. All of the OXA-48 producers were found resistant to temocillin on all chromogenic media. The lower detection limit of the rapid assay was determined as 106 in both direct bacterial samples and in spiked urine/blood samples. As a result, four chromogenic culture media and OXA-48 K-SeT can be used safely for detection of OXA-48 positive K.pneumoniae isolates. Although direct clinical specimens were not used, our study suggests that this media and OXA-48 K-SeT may be used in patient samples like blood and urine. Further studies are needed to assess this suggestion.

Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis

BackgroundEquine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay’s utility for regulatory testing.ResultsWe developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay’s performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5–90.7%), 90.5% (95% CI: 87.3–92.3%) and 87.4% (95% CI: 84.0–90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9–12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7–10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9–63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR.ConclusionsThe duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the reliability of the ema-1 gene-specific oligonucleotides developed in this study for confirmatory testing of non-negative serological test results for T. equi by qPCR. However, the B. caballi-specific qPCR cannot be similarly recommended as a confirmatory assay for routine regulatory testing due to the low level of agreement with serological test results demonstrated in this study. Further studies are needed to determine the transmission risk posed by PCR-negative equines with detectable antibodies to B. caballi.

Development of an immunochromatographic test based on monoclonal antibodies against surface antigen 3 (TgSAG3) for rapid detection of Toxoplasma gondii

The immunochromatographic test (ICT) is a convenient and low-cost method that can rapidly obtain results (10 min) under normal conditions. In this study, we established an ICT assay with two monoclonal antibodies: TgSAG3-3A7 and TgSAG3-4D5 based on the conserved protein of TgSAG3 that can be expressed in all the infective stages of T. gondii. 20 nm gold particles were prepared and conjugated with TgSAG3-3A7 MAb at the concentration of 12.5 μg/mL. TgSAG3-4D5 MAb were used as the capture antibody because of its higher affinity tested by ELISA. The detection limit of the ICT assay was 100 ng with visual detection under optimal conditions of analysis. Positive porcine serum of Cryptosporidium suis, Mycoplasma suis, Streptococcus suis, Salmonella choleraesuis, Cysticercus cellulosae, Isospora suis, and Trichinella spiralis were used to evaluate the specificity of this ICT and no cross-reactivity was observed. 310 porcine serum samples obtained from pig farms in Zhejiang Province, China were detected by this ICT and ELISA kit, 23 positive samples were found by the developed strip with the rate of 7.42% comparing with 22 positive samples detected by the commercially ELISA kit which the positive rate was 7.1%, the relative sensitivity and specificity of this ICT are 100% and 99.65%. Therefore, the ICT established in this study is proved effective, simple, specific and sensitive of T. gondii detection.

Collection of nasal secretions and tears and their use in allergology.

The identification of immunological markers in nasal secretions and tears is becoming essential in the study of allergic diseases. The collection procedure of nasal and ocular secretions directly influences the results, thus it is of paramount importance to validate and standardize the sampling process. Current techniques for nasal secretions sampling are mainly based on three principles: collection of spontaneous secretions, nasal washings, and absorption. Collection of spontaneous secretions is appropriate in subjects with nasal hypersecretion, whereas in healthy individuals the collected volume is frequently insufficient. Nasal washings are associated with an unpredictable, high dilution and concentrations of markers often fall below detection limits of immunological assays. Absorption seem to provide the best compromise between sufficient sample amounts and detectability of inflammatory mediators and immunoglobulin E. Tear samples can be obtained by glass capillary tubes, filter paper strips and ophthalmic sponges. Volumes are however small or highly diluted through reflex tearing. Secretions reflect the local inflammatory activity and provide valuable information about the immunological reaction to allergens at the target organ. There is increasing evidence of the potential clinical role of their analysis, for diagnosis, and monitoring of allergic rhino-conjunctivitis. Appropriate collection and processing is very important and requires special attention.

Tyrosinase-based TLC Autography for anti-melanogenic drug screening

Tyrosinase-based TLC (thin layer chromatography) was developed for screening of anti-melanogenic drugs. In particular, this technique enables researchers to identify melanogenic inhibitor(s) in tested mixtures with the naked eye. In comparison with traditional colorimetric screening assays for tyrosinase inhibitor(s), not only is tyrosinase-based TLC a more cost-effective option (nearly one-tenth the enzyme cost of colorimetric methods) but also is a more sensitive detection approach for kojic acid (KA), a standard anti-melanogenic drug. The detection limit of tyrosinase-based TLC and colorimetric tyrosinase assay for KA was 0.0125 and 1.25 μg, respectively, demonstrating that the former was 100-fold more sensitive than the latter to determine the tyrosinase inhibitory rate of KA. Furthermore, the results of this method have demonstrated excellent precision by Gage Repeatability and Reproducibility (Gage R&R), with the variation of total Gage R&R being 28.24%. To verify the applicability of tyrosinase-based TLC, this platform was employed to screen melanogenic inhibitor(s) from Ganoderma formosanum extracts and two of all fractions (GFE-EA F4, F5) obtained showed depigmenting activity. It is noteworthy that these two fractions also exerted anti-melanogenesis activity on zebrafish, therefore verifying the credibility of tyrosinase-based TLC. In sum, this technique provides new insight into the discovery of novel melanogenic inhibitor(s).

Development of a Polymerase Chain Reaction/Ligase Detection Reaction Assay for Detection of CYP2C19 Polymorphisms.

Cytochrome P450 2C19 (CYP2C19) genotypes are associated with differential drug metabolism. The aim of this study was to establish a reliable assay for CYP2C19 genotyping based on a polymerase chain reaction/ligase detection reaction (PCR-LDR). Specific primers and probes were designed to detect CYP2C19*1, *2, *3, and *17. A control for each allele was prepared and used for performance evaluation. A total of 200 clinical samples were analyzed using the PCR-LDR assay and Sanger sequencing. The detection limit of the PCR-LDR assay was 2 ng/μL of genomic DNA. Common interfering substances in the blood did not affect the results of the detection. For the clinical samples, the results of the PCR-LDR and the Sanger sequencing were identical. Among the 200 patients, 104 (52%) were wild type (*1/*1), 64 (32%) were *1/*2, 16 (8%) were *1/*3, 8 (4%) were *2/*2, 7 (3.5%) were *2/*3, and 1 (0.5%) was *1/*7. No *3/*3 genotype was detected in these patients. This PCR-LDR assay is reliable for the detection of CYP2C19 genotypes in a clinical setting. It will be a useful tool to screen for CYP2C19 loss-of-function alleles in patients before clopidogrel and proton pump inhibitor treatment.

Pharmacokinetics of amlodipine besylate at delivery and during lactation.

Amlodipine is rarely used in the treatment of pregnant hypertensive women due to limited pharmacokinetic data during pregnancy and the postpartum period. To evaluate the pharmacokinetics of amlodipine besylate in the peri-partum period including quantities of placental passage, breast milk excretion and infant exposure. This was a prospective study of pregnant women who were prescribed 5 mg of amlodipine daily for treatment of chronic hypertension and delivered at term. Cord and maternal blood samples were collected at delivery. On postpartum day 2, six paired maternal plasma and breast milk samples were obtained at 4, 6, 8, 12, 15 and 24 h following amlodipine dosing. Infant plasma samples were collected 24-48 h after delivery. All samples were analyzed for amlodipine concentration. A one compartment, first-order model was used to calculate pharmacokinetic estimates for maternal plasma. Of the 16 patients enrolled in the study, 11 had cord blood and maternal serum collected at delivery, of which only 6 produced sufficient breast milk for sampling. Amlodipine was detected in infant cord blood plasma with a mean concentration of 0.49 ± 0.29 ng/mL compared to mean maternal serum level of 1.27 ± 0.84 ng/mL. Amlodipine concentrations in both in breast milk and infant plasma were undetectable at the lower limit of assay detection (<0.1 ng/mL). In the immediate postpartum period, the amlodipine elimination half-life was 13.7 ± 4.9 h, the area under the curve was 53.4 ± 19.8 ng*h/mL and the peak concentration was 2.0 ± 1.0 ng/mL. Amlodipine does cross the placenta in measurable quantities, but is not detected in breast milk or infant plasma at 24-48 h of life indicating that it is likely safe to use during the peripartum period.

Development of a Quantitative Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Novel Goose Parvovirus.

An infectious disease characterized with short bills and protruding tongues has attacked to meat ducks in China since March 2015, which has caused ducks poor growth and enormous economic losses to duck industry of China. It was eventually proved to be caused by parvovirus after pathogen isolation and identification. As the genomic sequence analysis showed, this pathogen shared 90.8–94.6% of nucleotide identity with goose parvovirus (GPV), and it was called duck-origin novel goose parvovirus (N-GPV). In this study, a quantitative loop-mediated isothermal amplification (qLAMP) assay was developed for the rapid diagnosis of N-GPV. A set of four specific primers, two inner and two outer, were designed targeting at VP3 gene, which could be completed within 60 min at 65°C in water bath or on a real-time PCR instrument for quantitative analysis. Specificity test of LAMP assay showed that there was no cross-reactivity between N-GPV and other duck pathogens, and the detection limit of qLAMP assay was 1.0 × 102 copies/μL. The repeatability of this method was confirmed by inter-assay and intra-assay tests with variability ranging from 0.74 to 2.25%. The results have indicated that the qLAMP assay was a simple, rapid, accurate, sensitive, and specific method for detecting N-GPV, especially on field detection.

DNA detection of Trypanosoma evansi: Diagnostic validity of a new assay based on loop-mediated isothermal amplification (LAMP)

Trypanosoma evansi (T. evansi) is the most widely spread pathogenic trypanosome in the world. The control of trypanosomiasis depends on accurate diagnosis and effective treatment. Focusing on the presence of T. evansi in Asia, we developed a detection assay based on tracing phosphate ions (Pi) generated during LAMP targeting the variant surface glycoprotein (VSG) gene of Rode Trypanozoon antigenic type 1.2 (RoTat 1.2 VSG). The diagnostic potential as well as the use of the assay as a test-of-cure method after berenil treatment, was assessed in mice at different time points of infection. In addition, 67 buffalo blood collected from Tongling county, Anhui province, as well as 42 cattle sera from the Shanghai area, were used to evaluate the diagnostic validity of the test. The detection limit of the novel LAMP assay was determined to be as low as 1 fg of T. evansi DNA, while the reaction time for the test was only 30min. Hence it outperforms both microscopy and PCR. In the test-of-cure assessment, successful berenil mediated cure could be confirmed within 48h after treatment. This offers a tremendous advantage over conventional antibody-based diagnostic tools in which successful cure only can be confirmed after months. In the cattle and buffalo screening, the LAMP was able to detect a false-negative determined sample, wrongly classified in a conventional microscopy and PCR screening. Finally, no cross-reactivity was observed with other zoonotic parasites, such as T. evansi type B, T. congolense, T. brucei, Schistosoma japonicum, Plasmodium falciparum, Leishmania donovani, Toxoplasma gondii and Angiostrongylus cantonensis. We conclude that the novel LAMP assay is sensitive, specific and convenient for field use, particularly in areas where infection incidence has become extremely low. The LAMP assay could be used as a tool for trypanosomiasis control and elimination strategies in areas where T. evansi Type A infections are causing a threat to livestock farming.

Signal enhancement in ATP bioluminescence to detect bacterial pathogens via heat treatment

An ATP bioluminescence assay is frequently in the food industry as a commercially-available bacterial monitoring method because it is convenient and can monitor viable cells in food. However, such an assay suffers from low sensitivity and thus has limitations in monitoring highly pathogenic bacteria. In this study, we describe a method that improves the sensitivity by increasing the amount of ATP that is released from bacterial cells by subjecting the samples to heat treatment. When treated for 10 min at a temperature of 25 to 95°C in phosphate buffered saline (PBS) containing Escherichia coli O157:H7 or Salmonella enteritidis or Bacillus cereus at various concentrations, the relative luminescence unit (RLU) increased several times. Due to signal enhancement, the detection limit (LOD) of the ATP bioluminescence assay improved by about an order of magnitude in milk-containing microorganisms. Our results indicate that the simple heating step on the food samples before measurement is useful to improve the sensitivity of the ATP bioluminescence assay for bacterial detection.

Oligonucleotide Therapies for the Lung: Ready to Return to the Clinic?

The most promising targets for treating lung diseases are often intracellular molecules recalcitrant to conventional pharmacotherapy. In this respect, antisense oligonucleotides (ASO) would be valuable tools to treat airways disease, as the plurality of their potential mechanisms of action is well-established, their efficiency has been extensively validated in-vitro, and their potential clinical value has been demonstrated with numerous regulatory approvals. Yet in spite of high confidence in the treatment rationale and mechanism of action, it appears that oligonucleotides delivered topically to the lung either rapidly access circulation via epithelial transcytosis or are removed by alveolar macrophages, exerting minimal if any action in the cytosol of cells relevant to lung disease. Moreover, use of cell penetrating peptides, liposomes or nanoparticle delivery systems has not so far been shown to eliminate circulatory clearance, may activate immune responses, or drive macrophage phenotypic changes that, together or in isolation, may present risks to patients.

The utility of anti-Müllerian hormone in the diagnosis and prediction of loss of ovarian function following chemotherapy for early breast cancer

AimChemotherapy results in permanent loss of ovarian function in some premenopausal women. Accurate identification in women with hormone-sensitive early breast cancer (eBC) would allow optimisation of subsequent endocrine treatment. We sought to assess whether analysis of anti-Müllerian hormone (AMH) using a sensitive automated assay could identify women who would not regain ovarian function after chemotherapy. MethodsData from women in the Ovarian Protection Trial in Premenopausal Breast Cancer Patients (OPTION) trial of goserelin (a gonadotrophin-releasing hormone (GnRH) analogue) for ovarian protection were analysed. Women were assessed for premature ovarian insufficiency (POI: amenorrhoea with elevated follicle-stimulating hormone (FSH)) at 24 months after diagnosis. The accuracy of AMH for the diagnosis of POI and its prediction from measurement at the end of chemotherapy was calculated. ResultsAMH below the level of detection showed good diagnostic accuracy for POI at 24 months (n = 73) with receiver operating characteristic (ROC) area under the curve of 0.86, sensitivity 1.0 and specificity 0.73 at the assay limit of detection. In women aged >40 at diagnosis who did not receive goserelin, AMH measured at end of chemotherapy also gave good prediction of POI at 24 months (area under the curve (AUC) 0.89 95% CI 0.75–1.0, n = 32), with sensitivity 0.91, specificity 0.82, diagnostic odds ratio (DOR) 42.8. FSH gave slightly lower AUC, and specificity was low at 0.55. Age but not tamoxifen impacted on AMH levels. ConclusionUsing this sensitive AMH assay, the finding of an undetectable AMH level in women aged >40 at the end of chemotherapy for eBC gave a good prediction that ovarian function would not return. This may allow alterations in post-chemotherapy endocrine management.