Detection Limit Of Assay Research Articles

A visual method to detect meat adulteration by recombinase polymerase amplification combined with lateral flow dipstick

Determining the animal source in meat and meat products is crucial to prevent meat adulteration and fraud. Conventional methods require considerable operator skills, expensive instruments and are unable to provide fast mobile on-site detection systems to detect contamination of meat products. We developed a visual method based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) to identify beef (Bos taurus), sheep (Ovis aries), pork (Sus scrofa), duck (Anas platyrhynchos) and chicken (Gallus gallus). The reaction was completed within 20 min. The results were determined by the naked eye. The detection limits of the RPA-LFD assays for duck, beef, sheep, chicken and pork were 101/µL, 102/µL, 102/µL, 101/µL and 101/µL, respectively. Furthermore, the RPA-LFD assays could differentiate species in boiled, microwaved, pressure-cooked or fried samples. These RPA-LFD assays represent a rapid, mobile detection system for determining meat product contamination.

Charge-Transfer Complex of Linifanib with 2,3-dichloro-3,5-dicyano-1,4-benzoquinone: Synthesis, Spectroscopic Characterization, Computational Molecular Modelling and Application in the Development of Novel 96-microwell Spectrophotometric Assay.

BackgroundLinifanib (LFB) is a multi‐targeted receptor tyrosine kinase inhibitor used in the treatment of hepatocellular carcinoma and other types of cancer. The charge-transfer (CT) interaction of LFB is important in studying its receptor binding mechanisms and useful in the development of a reliable CT-based spectrophotometric assay for LFB in its pharmaceutical formulation to assure its therapeutic benefits.PurposeThe aim of this study was to investigate the CT reaction of LFB with 2,3-dichloro-3,5-dicyano-1,4-benzoquinone (DDQ) and its application in the development of a novel 96-microwell spectrophotometric assay for LFB.MethodsThe reaction was investigated, its conditions were optimized, the physicochemical and constants of the CT complex and stoichiometric ratio of the complex were determined. The solid-state LFB-DDQ complex was synthesized and its structure was analyzed by UV-visible, FT-IR, and 1H-NMR spectroscopic techniques, and also by the computational molecular modeling. The reaction was employed in the development of a novel 96-microwell spectrophotometric assay for LFB.ResultsThe reaction resulted in the formation of a red-colored product, and the spectrophotometric investigations confirmed that the reaction had a CT nature. The molar absorptivity of the complex was linearly correlated with the dielectric constant and polarity index of the solvent; the correlation coefficients were 0.9526 and 0.9459, respectively. The stoichiometric ratio of LFB:DDQ was 1:2. The spectroscopic and computational data confirmed the sites of interaction on the LFB molecule, and accordingly, the reaction mechanism was postulated. The reaction was utilized in the development of the first 96-microwell spectrophotometric assay for LFB. The assay limits of detection and quantitation were 1.31 and 3.96 μg/well, respectively. The assay was successfully applied to the analysis of LFB in its bulk and tablets with high accuracy and precision.ConclusionThe assay is simple, rapid, accurate, eco-friendly as it consumes low volumes of organic solvent, and has high analysis throughput.

Rapid highly sensitive general protein quantification through on-chip chemiluminescence.

Protein detection and quantification is a routinely performed procedure in research laboratories, predominantly executed either by spectroscopy-based measurements, such as NanoDrop, or by colorimetric assays. The detection limits of such assays, however, are limited to M concentrations. To establish an approach that achieves general protein detection at an enhanced sensitivity and without necessitating the requirement for signal amplification steps or a multicomponent detection system, here, we established a chemiluminescence-based protein detection assay. Our assay specifically targeted primary amines in proteins, which permitted characterization of any protein sample and, moreover, its latent nature eliminated the requirement for washing steps providing a simple route to implementation. Additionally, the use of a chemiluminescence-based readout ensured that the assay could be operated in an excitation source-free manner, which did not only permit an enhanced sensitivity due to a reduced background signal but also allowed for the use of a very simple optical setup comprising only an objective and a detection element. Using this assay, we demonstrated quantitative protein detection over a concentration range of five orders of magnitude and down to a high sensitivity of , corresponding to pM concentrations. The capability of the platform presented here to achieve a high detection sensitivity without the requirement for a multistep operation or a multicomponent optical system sets the basis for a simple yet universal and sensitive protein detection strategy.

A powerful qPCR-high resolution melting assay with taqman probe in Plasmodium species differentiation

BackgroundThe use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, including the two Plasmodium ovale species and zoonotic variants of the disease, will be important for the understanding of changing epidemiological patterns of the disease.MethodsA qPCR-high resolution melting (HRM) method was to detect and differentiate all human Plasmodium species with one forward and one reverse primer set. The HRM detection method was further refined using a hydrolysis probe to specifically discriminate Plasmodium falciparum.ResultsOut of the 113 samples tested with the developed HRM-qPCR- P. falciparum probe assay, 96 (85.0 %) single infections, 12 (10.6 %) mixed infections, and 5 (4.4 %) were Plasmodium negative. The results were concordant with those of the nested PCR at 98.2 %. The assay limit of detection was varied from 21.47 to 46.43 copies /µl, equivalent to 1–2.11 parasites/µl. All P. falciparum infections were confirmed with the associated Taqman probe.ConclusionsAlthough the dependence on qPCR currently limits its deployment in resource-limited environments, this assay is highly sensitive and specific, easy to perform and convenient for Plasmodium mono-infection and may provide a novel tool for rapid and accurate malaria diagnosis also in epidemiological studies.

Development of a Rapid and Fully Automated Multiplex Real-Time PCR Assay for Identification and Differentiation of Vibrio cholerae and Vibrio parahaemolyticus on the BD MAX Platform.

Vibrio cholerae and Vibrio parahaemolyticus are common diarrheal pathogens of great public health concern. A multiplex TaqMan-based real-time PCR assay was developed on the BD MAX platform; this assay can simultaneously detect and differentiate V. cholerae and V. parahaemolyticus directly from human fecal specimens. The assay includes two reactions. One reaction, BDM-VC, targets the gene ompW, the cholera toxin (CT) coding gene ctxA, the O1 serogroup specific gene rfbN, and the O139 serogroup specific gene wbfR of V. cholerae. The other, BDM-VP, targets the gene toxR and the toxin coding genes tdh and trh of V. parahaemolyticus. In addition, each reaction contains a sample process control. When evaluated with spiked stool samples, the detection limit of the BD MAX assay was 195–780 CFU/ml for V. cholerae and 46–184 CFU/ml for V. parahaemolyticus, and the amplification efficiency of all genes was between 95 and 115%. The assay showed 100% analytical specificity, using 63 isolates. The BD MAX assay was evaluated for its performance compared with conventional real-time PCR after automated DNA extraction steps, using 164 retrospective stool samples. The overall percent agreement between the BD MAX assay and real-time PCR was ≥ 98.8%; the positive percent agreement was 85.7% for ompW, 100% for toxR/tdh, and lower (66.7%) for trh because of a false negative. This is the first report to evaluate the usage of the BD MAX open system in detection and differentiation of V. cholerae and V. parahaemolyticus directly from human samples.

23-valent polysaccharide vaccine (PPSV23)-targeted serotype-specific identification of Streptococcus pneumoniae using the loop-mediated isothermal amplification (LAMP) method.

Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 103 to 106 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (103 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (102 copies per reaction), compared with 103 to >106 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.

Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections.

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

The Limit of Detection Matters: The Case for Benchmarking Severe Acute Respiratory Syndrome Coronavirus 2 Testing.

BackgroundResolving the coronavirus disease 2019 (COVID-19) pandemic requires diagnostic testing to determine which individuals are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard is to perform reverse-transcription polymerase chain reaction (PCR) on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of approximately 100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss infected patients. However, the relative clinical sensitivity of these assays remains unknown.MethodsHere we model the clinical sensitivities of assays based on their LoD. Cycle threshold (Ct) values were obtained from 4700 first-time positive patients using the Abbott RealTime SARS-CoV-2 Emergency Use Authorization test. We derived viral loads from Ct based on PCR principles and empiric analysis. A sliding scale relationship for predicting clinical sensitivity was developed from analysis of viral load distribution relative to assay LoD.ResultsCt values were reliably repeatable over short time testing windows, providing support for use as a tool to estimate viral load. Viral load was found to be relatively evenly distributed across log10 bins of incremental viral load. Based on these data, each 10-fold increase in LoD is expected to lower assay sensitivity by approximately 13%.ConclusionsThe assay LoD meaningfully impacts clinical performance of SARS-CoV-2 tests. The highest LoDs on the market will miss a majority of infected patients. Assays should therefore be benchmarked against a universal standard to allow cross-comparison of SARS-CoV-2 detection methods.

Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR.

In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method—nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18–23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020.

Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

Establishment of recombinase polymerase amplification technology for detecting Viral Hemorrhagic Septicemia Virus (VHSV) in cultured aquatic animals

We established a recombinase polymerase amplification (RPA) assay for the rapid detection of viral hemorrhagic septicemia virus (VHSV) using primers designed to the N gene in this species. Optimization experiments showed that the optimal amplification temperature of the RPA assay was 37 °C and that the reaction could be completed within only 15 min. Specificity tests showed that the RPA assay did not exhibit any cross-reactivity with other infective fish viruses. Sensitivity tests further showed that the detection limit of the new RPA assay was 8.3 copies/μL, indicating that this assay was more sensitive than traditional RT-PCR method. The method was verified by detecting VHSV in 1924 batches of samples collected from domestic and imported fishes. The detection results were consistent with that of traditional RT-PCR, and the specificity and sensitivity of the method met the detection requirements for aquatic animal diseases. Collectively, our findings indicate that the novel RPA assay is fast, simple, specific, sensitive, and has significant potential for the clinic, on-site testing, and a wide range of other applications.

Mining and evaluating novel serovar-specific Salmonella C1 serogroup genes by polymerase chain reaction analysis

Salmonella is a food-borne pathogen that causes worldwide infections in humans and animals. Over 2600 Salmonella serovars have been identified, and serogroup C1 (O:6, 7 epitopes) contains highly diverse serovars associated with high pathogenicity. Here, we aimed to develop a reliable polymerase chain reaction (PCR) method for detecting serovar-specific markers of Salmonella serogroup C1, with greater reliability and lower complexity than current methods. We identified three, two, four, and one novel genes unique to the Infantis, Rissen, Braenderup, and Virchow serovars. The PCR primers targeting these genes were specific for the target serovars. The detection limits of the PCR assays using 9 pairs of primers were 102–103 colony-forming units (CFUs)/mL and 104–105 CFU/g in pure bacterial cultures and artificially contaminated raw pork samples, respectively. No false-positive or false-negative results were obtained with 45 food samples, as determined by comparison with biochemical tests and traditional serotyping methods. The potential functions of all serovar-specific genes identified are unknown, except for one Rissen-specific gene that encodes a secreted protein. Our results imply that all 10 novel, specific genes can be considered as useful targets for detecting the Salmonella Infantis, Rissen, Braenderup, and Virchow serovars in food.

Rapid detection of the New Delhi metallo-β-lactamase (NDM) gene by recombinase polymerase amplification.

New Delhi metallo-β-lactamase (NDM) is a series of enzyme conferring resistance to β-lactam antibiotics including the carbapenems. The blaNDM gene has been reported in a variety of Gram-negative bacilli, especially in the Enterobacteriaceae and Acinetobacter spp., which is deeply disconcerting for public health worldwide. In this study, recombinase polymerase amplification assays using a basic detection (Basic-RPA) and a real-time fluorescent detection (Exo-RPA) were established for detecting blaNDM gene. The RPA reactions were performed at 39°C and finished within 20min. Using different copy numbers of pMD18T-NDM plasmid DNA as templates, we identified the detection limit of Basic-RPA assay (1.85×103 copies/μL), conventional PCR assay (1.85×104 copies/μL), Exo-RPA assay (1.85×102 copies/μL) and real-time PCR assay (1.85×102 copies/μL). Both Basic-RPA and Exo-RPA assays were highly specific for detecting blaNDM, as there were no cross-reactions with the strains without blaNDM gene. Examination of 62 clinical samples by RPA assays and PCR assays showed the same results, suggesting that RPA assays are reliable in clinical diagnosis. The amplification time of RPA is much shorter than that of other molecular techniques, it is easyto implement and has the potential for clinical application.

Colorimetric/fluorescent/Raman trimodal sensing of zinc ions with complexation-mediated Au nanorod

Accurate and selective in-field detection of metal ions in complex media has gained wide interests due to the complexed matrices and weak affinity towards sensing surface. Herein, we develop a first trimodal method for sensing of Zn2+ in complex matrices by stimuli-responsive N-[6-piperazinyl-2-pyridinyl]-N-(2-pyridinylmethyl)-2-Pyridinemethanamine dithiocarbamates (DPY) modified gold nanorods (GNRs-DPY). The presence of Zn2+ triggers the aggregation of GNRs-DPY, leading to increment of color and fluorescence intensity of the sensing system, which could be visually discerned with bare eye. Moreover, the intensive electromagnetic enhancement among “hot spots” of GNRs, generated during self-aggregation of the GNRs-DPY caused by Zn2+, lowers the detection limit of SERS assay to 6 × 10−3 pM. It is noteworthy that GNRs-DPY based sensing platform not only enables distinguishing Zn2+ from Cd2+, with simplicity and rapidity, but also demonstrates as trimodal nanoprobe for sensitive and selective quantitative determination of Zn2+ in different matrices. Therefore, the GNRs-DPY provides a new strategy for accurate and credible on-spot determination of Zn2+ in complicated specimens, as well as offering multiple applications in point-of-care monitoring.

Effects of differing withdrawal times from ractopamine hydrochloride on residue concentrations of beef muscle, adipose tissue, rendered tallow, and large intestine.

Ractopamine hydrochloride (RAC) is a beta-agonist approved by the U.S. Food and Drug Administration (FDA) as a medicated feed ingredient for cattle during the final days of finishing to improve feed efficiency and growth. Maximum residue limits and U.S. FDA residue tolerances for target tissues have defined management practices around RAC usage in the U.S. However, many countries have adopted zero tolerance policies and testing of off-target tissues, presenting a major challenge for international export. Therefore, the objective this study was to determine the necessary withdrawal time among cattle group-fed RAC to achieve residue concentrations below tolerance levels in muscle and off-target tissues. Specifically, both total and parent RAC residues were quantified in muscle, adipose tissue, rendered tallow, and large intestines from animals group-fed RAC and subjected to withdrawal 2, 4, or 7 days before harvest. Ractopamine (parent and total) residues were below the assay limit of detection (< 0.12 ng/g) in all muscle and adipose tissue samples from animals in control groups (no RAC). However, RAC residues were detectable, but below the limit of quantitation, in 40% of tallow and 17% of large intestine samples from control animals. As expected, mean RAC residue concentrations in muscle, adipose tissue, and large intestine samples decreased (P < 0.05) as the RAC withdrawal duration (days) was extended. Irrespective of RAC withdrawal duration, mean parent RAC residue concentrations in muscle, adipose tissue, and large intestine ranged from 0.33 to 0.76 ng/g, 0.16 to 0.26 ng/g, 3.97 to 7.44 ng/g, respectively and all tallow samples were > 0.14 ng/g (detectable but below the limit of quantitation). Results of this study provide a baseline for the development of management protocol recommendations associated with withdrawal following group-feeding of RAC to beef cattle in countries that allow RAC use and intend to export to global markets which may be subject to zero tolerance policies and off-target tissue testing.

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

BackgroundEarly detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.MethodsIn the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.ResultsThe detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6–98.2) and 100% (95% CI = 78.5–100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P < 0.001).ConclusionThe RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

Clinical phenotype and risk factors for severe efavirenz-associated neurotoxicity amongst inpatients in Cape Town, South Africa

Background: Efavirenz, widely used as first line antiretroviral therapy (ART), is frequently associated with mild transient neuropsychiatric symptoms, usually occurring early in therapy. More severe neurotoxicity has been reported, but the clinical phenotype and risk factors are poorly defined. Methods and materials: We identified adult patients with an efavirenz concentration >4 mg/L (target 1–4 mg/L) at four hospitals in Cape Town, South Africa. Data were extracted from medical records, including clinical symptoms and signs and laboratory abnormalities at the time of efavirenz quantification; severe neurotoxicity was defined as Division of AIDS score > 3. We performed logistic regression to identify associations with neuropsychiatric symptoms and severe neurotoxicity. Results: Data were included from 82 patients: 62 had neuropsychiatric symptoms, 19 had hepatitis, and 1 had no apparent indication for efavirenz quantification. Median age was 38 years (interquartile range (IQR) 29–45), median weight 53 kg (IQR 44–63), 29 (34%) were male, and 49 (63%) patients had concomitant isoniazid exposure. Median efavirenz concentration was 12.1 mg/L (IQR 6.6–20.0). Neuropsychiatric manifestations included ataxia (n = 20) and other cerebellar signs (n = 13), psychomotor slowing (n = 24), psychosis (n = 17), mood disturbance (n = 11), and insomnia/sleep disturbance (n = 4). Compared with hepatitis, neurotoxicity was associated with male sex (adjusted odds ratio (aOR) 20.0, 95% CI 2.0–199.9), higher efavirenz concentrations (aOR 1.2 per 1 mg/L increase, 95% CI 1.0–1.4) and isoniazid exposure (aOR 5.1, 95% CI 1.1–23.5). Severe neuropsychiatric symptoms occurred in 47 (75%) patients with neurotoxicity, after a median of 25 weeks (IQR 9–175) on efavirenz-based ART. Median efavirenz concentration was 20.0 mg/L (the upper limit of assay detection, IQR 13.0–20.0) in those with severe versus 7.0 mg/L (4.5–11.8) in those with mild neurotoxicity (p < 0.01). Amongst patients with severe neurotoxicity, there was complete resolution of symptoms within 1 month in 29/31 (94%) who discontinued efavirenz and in 2/5 (40%) who underwent dose-reduction. 3-month mortality was 11% (n = 45) for patients with severe neurotoxicity. Conclusion: Cerebellar signs were the most frequent clinical manifestation of efavirenz-associated neurotoxicity. There was a concentration-effect relationship for severe neurotoxicity, which occurred late and resolved in most patients after efavirenz discontinuation.

A pilot study demonstrating the identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using a multiplexed high-resolution melt qPCR.

Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104 tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces results in less than 2 hours, avoiding gel electrophoresis and subjective analysis. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.

Optimizing the Use of High-Sensitivity Troponin Assays for the Early Rule-out of Myocardial Infarction in Patients Presenting with Chest Pain: A Systematic Review.

We assessed the accuracy and clinical effectiveness of high-sensitivity cardiac troponin (hs-cTn) assays for early rule-out of non-ST-segment elevation myocardial infarction (NSTEMI) in adults presenting with acute chest pain. Sixteen databases were searched to September 2019. Review methods followed published guidelines. The bivariate model was used to estimate summary sensitivity and specificity with 95% confidence intervals for meta-analyses involving 4 or more studies, otherwise random-effects logistic regression was used. Thirty-seven studies (124 publications) were included in the review. The hs-cTn test strategies evaluated in the included studies were defined by the combination of 4 factors (assay, number of tests, timing of tests, and threshold concentration or change in concentration between tests). Clinical opinion indicated a minimum acceptable sensitivity of 97%. A single test at presentation using a threshold at or near the assay limit of detection could reliably rule-out NSTEMI for a range of hs-cTn assays. Serial testing strategies, which include an immediate rule-out step, increased the proportion ruled out without loss of sensitivity. Finally, serial testing strategies without an immediate rule-out step had excellent sensitivity and specificity, but at the expense of the option for immediate patient discharge. Test strategies that comprise an initial rule-out step, based on low hs-cTn concentrations at presentation and a minimum symptom duration, and a second step for those not ruled-out that incorporates a small absolute change in hs-cTn at 1, 2, or 3 hours, produce the highest rule-out rates with a very low risk of missed NSTEMI. CRD42019154716.

Development and optimization of a loop-mediated isothermal amplification (LAMP) assay for the species-specific detection of Penicillium expansum

Penicillium expansum is the main cause of Blue Mold Decay, which is the economically most significant postharvest disease on fruits. It occurs especially on pomaceous fruits such as apples and pears but also on a wide range of other fruits such as grapes or strawberries. Besides its negative economic effects on the industry, the fungus is also of health concern as it produces patulin, a mycotoxin known to provoke harmful effects in humans. A specific and rapid detection of this fungus therefore is required. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized for the species-specific detection of P. expansum. The assay showed high specificity during tests with genomic DNA of 187 fungal strains. The detection limit of the developed assay was 25 pg genomic DNA of P. expansum per reaction. The assay was successfully applied for the detection of the fungus on artificially contaminated apples, grapes, apple juice, apple puree, and grape juice. The developed assay is a promising tool for rapid, sensitive, specific, and cost-efficient detection of P. expansum in quality control applications in the food and beverage industry.