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Prognostics and Health Management (PHM) models aim to estimate remaining useful life (RUL) of complex systems, enabling lower maintenance costs and increased availability. A substantial body of work considers the development and testing of new models using the NASA C-MAPSS dataset as a benchmark. In recent work, the use of ensemble methods has been prevalent. This paper proposes two adaptations to one of the best-performing ensemble methods, namely the Convolutional Neural Network – Long Short-Term Memory (CNN-LSTM) network developed by Li et al. (IEEE Access , 2019, 7 , pp 75464–75475)). The first adaptation (adaptable time window, or ATW) increases accuracy of RUL estimates, with performance surpassing that of the state of the art, whereas the second (sub-network learning) does not improve performance. The results give greater insight into further development of innovative methods for prognostics, with future work focusing on translating the ATW approach to real-life industrial datasets and leveraging findings towards practical uptake for industrial applications. [ABSTRACT FROM AUTHOR]

In five experiments, we investigated college students' use of base rate and case cue information in estimating likelihood. The participants reported that case cues were more important than base rates, except when the case cues were totally uninformative, and made more use of base rate information when the base rates were varied within subjects, rather than between subjects. Estimates were more Bayesian when base rate and case cue information was congruent, rather than contradictory. The nature of the 'witness' in case cue information (animate or inanimate) did not affect the use of base rate and case cue information. Multiple trials with feedback led to more accurate estimates; however, this effect was not lasting. The results suggest that when base rate information is made salient by experience (multiple trials and within-subjects variation) or by other manipulations, base rate neglect is minimized. [ABSTRACT FROM AUTHOR]

Background: To meet the WHO target of eradicating yaws by 2030, highly sensitive and specific diagnostic tools are needed. A multiplex Treponema pallidum-Haemophilus ducreyi loop-mediated isothermal amplification (TPHD-LAMP) test holds promise as a near-patient diagnostic tool for yaws and H ducreyi. We conducted a prospective evaluation in Cameroon, Côte d'Ivoire, Ghana, and the Republic of the Congo to determine the diagnostic accuracy of the TPHD-LAMP test, as well as to assess its acceptability, feasibility, and cost., Methods: Active case searching within schools and communities was used to locate participants with clinically suspicious laws-like lesions. Individuals with serologically confirmed active yaws provided paired lesion swabs between March, 2021, and April, 2023. For each participant, one swab was tested with the TPHD-LAMP at a local district laboratory and the other with reference quantitative PCR (qPCR) tests conducted at national reference laboratories. The primary outcome was TPHD-LAMP test sensitivity and specificity compared with qPCR. Laboratory technicians were interviewed using a multiple-choice survey to gauge acceptability and feasibility of the TPHD-LAMP test. Costs of each test were calculated., Findings: Of 3085 individuals with at least one suspected yaws lesion, 531 (17%) were serologically confirmed. We enrolled 493 participants with seropositive yaws and a further 32 with negative serology. The sensitivity of the TPHD-LAMP test for detecting T pallidum was 63% (95% CI 56-70) and the specificity was 66% (95% CI 61-71). Sensitivity and specificity for T pallidum improved to 73% (63-82; p=0·0065) and 75% (68-80; p=0·0003), respectively, in H ducreyi-negative samples. Interviews highlighted challenges in user-friendliness and practicality of the TPHD-LAMP test. The cost of the test per sample was one third of that of qPCR, although the TPHD-LAMP test entailed higher costs to establish the assay., Interpretation: This was the first multi-country diagnostic evaluation of a molecular test for yaws. The TPHD-LAMP testing, in its current form, falls short of the WHO target product profile criteria for yaws diagnostics. These findings highlight the importance of assessing new diagnostics in real-world conditions to ensure their suitability for programmatic use., Funding: The EDCTP2 programme supported by the EU., Competing Interests: Declaration of interests SF, MB, and EL are employees of Mast Diagnostica, which produces and sells LAMP products. A patent covering the mediator displacement probe technique, a component of the TPHD-LAMP assay described in this study, has been applied for by the University of Freiburg and Hahn-Schickard. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Integrated approaches to mapping skin Neglected Tropical Diseases (NTDs) may be cost-effective way to guide decisions on resource mobilization. Pilot studies have been carried out, but large-scale data covering multiple countries endemic for skin NTDs are lacking. Within the LAMP4YAWS project, we collected integrated data on the burden of multiple skin NTDs., Methods: From March 2021 to March 2023, integrated case searches for yaws alongside other skin conditions were performed in endemic health districts of yaws in Cameroon, Côte d'Ivoire, and Ghana. Integrated activities included training, social mobilization and active case detection. Initial screening involved a brief clinical examination of participants to determine if any skin conditions were suspected. Cases of skin NTDs were then referred to a health facility for appropriate management., Results: Overall 61,080 individuals screened, 11,387 (18.6%) had skin lesions. The majority of individuals (>90%) examined were children aged 15 years old and under. The proportion of serologically confirmed yaws cases was 8.6% (18/210) in Cameroon, 6.8% (84/1232) in Côte d'Ivoire, and 26.8% (440/1643) in Ghana. Other skin conditions based on clinical examination included: scabies, Buruli ulcer, leprosy, lymphatic filariasis (lymphoedema and hydrocele), tungiasis, and fungal infections. The most common conditions were scabies and superficial fungal infections. In Cameroon, scabies and superficial fungal infections accounted for 5.1% (214/4204) and 88.7% (3730/4204) respectively, 25.2% (1285/5095) and 50.4% (2567/5095) in Côte d'Ivoire. In Ghana, 20% (419/2090) of individuals had scabies but superficial fungal infections were not routinely recorded and were reported in only 1.3% (28/2090). Other skin NTDs were less common across all three countries., Conclusion: This study confirms that integrated screening allows simultaneous detection of multiple skin NTDs, maximising use of scarce resources., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tchatchouang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Digital PCR (dPCR) is a powerful method for highly sensitive and precise quantification of nucleic acids. However, designing and optimizing new multiplex dPCR assays using target sequence specific probes remains cumbersome, since fluorescent signals must be optimized for every new target panel. As a solution, we established a generic fluorogenic 6-plex reporter set, based on mediator probe technology, that decouples target detection from signal generation. This generic reporter set is compatible with different target panels and thus provides already optimized fluorescence signals from the start of new assay development. Generic reporters showed high population separability in a colorimetric 6-plex mediator probe dPCR, due to their tailored fluorophore and quencher selection. These reporters were further tested using different KRAS , NRAS and BRAF single-nucleotide polymorphisms (SNP), which are frequent point mutation targets in liquid biopsy. We specifically quantified SNP targets in our multiplex approach down to 0.4 copies per microliter (cp/µL) reaction mix, equaling 10 copies per reaction, on a wild-type background of 400 cp/µL for each, equaling 0.1% variant allele frequencies. We also demonstrated the design of an alternative generic reporter set from scratch in order to give detailed step-by-step guidance on how to systematically establish and optimize novel generic reporter sets. Those generic reporter sets can be customized for various digital PCR platforms or target panels with different degrees of multiplexing.

Yaws, caused by Treponema pallidum ssp. pertenue, remains a significant public health concern in tropical regions of West Africa and the South Pacific, primarily affecting children in remote areas with limited access to hygiene and sanitation. In this study, conducted in three endemic countries of West Africa where yaws remains a significant public health concern (Ghana, Cameroon, and Côte d'Ivoire), we aimed to assess the knowledge, attitudes, and practices related to yaws among community members, community health workers (CHWs), and traditional healers. The study revealed variations in the perception of causes of yaws among community members: the majority or participants in Ghana attributed yaws to germs (60.2%); in Cameroon the most reported form of transmission was contact with or drinking infected water sources (44.6%); and in Côte d'Ivoire both of these answers were also the most prevalent (60.3% germs and 93.% water sources). A substantial proportion of participants in Côte d'Ivoire also associated yaws with witchcraft and divine punishment (44.8%). Only a small proportion of individuals in Ghana and Côte d'Ivoire correctly identified contact with an infected person as a form of transmission (11.9% and 20.7%, respectively) and less than half in Cameroon (42.6%), although more than 98% of all participants reported avoidance behaviours towards yaws infected people due to fear of getting infected. Most participants expressed a preference for seeking care at hospitals (49.2%, 60.6%, 86.2%) or health care professionals including doctors and nurses (58.5%, 41,5% and 17.2%) if they were diagnosed with yaws, although a quarter of participants in Côte d'Ivoire also sought support from traditional healers. The CHWs interviewed were generally well-trained on yaws causes and treatment options, although they often reported low availability of treatment and diagnostic tests for yaws. Our findings underscore the need for community education, awareness campaigns, ongoing CHW training, and improved access to yaws treatment and diagnostic resources. The data also suggest that collaboration with traditional healers, who usually hold a highly esteemed position in the society, such as giving training on yaws causes and transmission or exchanging knowledge on treatment options, could be beneficial in certain regions, particularly in Côte d'Ivoire., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Beiras et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Digital PCRs (dPCRs) are widely used methods for the detection and quantification of rare abundant sequences relevant to fields such as liquid biopsy or oncology. In order to increase the information content and save valuable sample materials, there is a significant need for digital multiplexing methods that are easy to establish, analyse, and interpret, and ideally allow the usage of existing lab equipment. Herein, we present a novel reporter emission multiplexing approach for the digital PCR method (REM-dPCR), which meets these requirements. It further increases the multiplexing capacity of commercial dPCR devices. For example, we present a stepwise increase in multiplexing degrees from a monochrome two-plex assay in one detection channel to a six-plex REM-dPCR assay in a three-color dPCR device for KRAS / BRAF single nucleotide polymorphism (SNP) target sequences. The guidelines for the REM-dPCR design are presented, and the process from duplex to six-plex assay establishment, taking into account the target sequence-dependent effects on assay performance, is discussed. Furthermore, the assay-specific, sensitive and precise quantification of different fractions of KRAS mutant and wild-type DNA sequences in different ratios is demonstrated. To increase the device capacitance and the degree of multiplexing, the REM-dPCR uses the advantage of n target-independent reporter molecules in combination with target sequence-specific mediator probes. Different reporter types are labelled with fluorophores of different signal intensities but not necessarily different emission spectra. This leads to the generation of n independent single-positive populations in the dataspace, created by k detection channels, whereby n > k and n ≥ 2. By usage of target-independent but population-specific reporter types, a fixed set of six optimized signalling molecules could be defined. This reporter set enables the robust generation and precise differentiation of multiple fluorescence signals in dPCRs and can be transferred to new target panels. The set which enables stable signal generation and differentiation in a specified device would allow easy transfer to new target panels.

We demonstrate detection and quantification of bacterial load with a novel microfluidic one-pot wash-free fluorescence in situ hybridization (FISH) assay in droplets. The method offers minimal manual workload by only requiring mixing of the sample with reagents and loading it into a microfluidic cartridge. By centrifugal microfluidic step emulsification, our method partitioned the sample into 210 pL (73 µm in diameter) droplets for bacterial encapsulation followed by in situ permeabilization, hybridization, and signal detection. Employing locked nucleic acid (LNA)/DNA molecular beacons (LNA/DNA MBs) and NaCl-urea based hybridization buffer, the assay was characterized with Escherichia coli , Klebsiella pneumonia , and Proteus mirabilis . The assay performed with single-cell sensitivity, a 4-log dynamic range from a lower limit of quantification (LLOQ) at ~3 × 10 3 bacteria/mL to an upper limit of quantification (ULOQ) at ~3 × 10 7 bacteria/mL, anda linearity R 2 = 0.976. The total time-to-results for detection and quantification was around 1.5 hours.

Introduction: Yaws, caused by the bacterium Treponema pallidum subsp. pertenue, is a neglected tropical disease targeted for eradication by 2030. Improved diagnostics will be essential to meet this goal. Diagnosis of yaws has relied heavily on clinical and serological tools. However, the presence of coendemic cutaneous skin ulcer diseases, such as lesions caused by Haemophilus ducreyi ( HD ), means these techniques do not provide a reliable diagnosis. Thus, new diagnostic tools are needed. Molecular tools such as PCR are ideal, but often expensive as they require trained technicians and laboratory facilities, which are often not available to national yaws programmes., Methods and Analysis: The LAMP4yaws project is a cross-sectional, observational, diagnostic accuracy study of a combined Treponema pallidum ( TP ) and HD loop mediated isothermal amplification (TPHD-LAMP) test performed under real world conditions in three endemic countries in West Africa. Individuals with serologically confirmed yaws will be recruited in Cameroon, Côte d'Ivoire and Ghana. Each participant will provide paired swabs, one of which will be sent to the respective national reference laboratory for yaws quantitative PCR and the other will be tested for both TP and HD using the TPHD-LAMP test at local district laboratories. Sensitivity and specificity of the TPHD-LAMP test will be calculated against the reference standard qPCR. We will also assess the acceptability, feasibility and cost-effectiveness of the test. We anticipate that results from this study will support the adoption of the TPHD-LAMP test for use in global yaws eradication efforts., Ethics and Dissemination: We have received ethical approval from all relevant institutional and national ethical committees. All participants, or their parents or guardians, must provide written informed consent prior to study enrolment. Study results will be published in an open access journal and disseminated with partners and the World Health Organization., Trial Registration Number: NCT04753788., Competing Interests: Competing interests: BLH, GCB, ST, LAB, KOH, LB, NB, TC, TH, SL, CR, IA, AS, MSKS, JPNN, PA, AT, ENT, DKA, SNK, SE, KWA, EMHE, SK, OM and MM declare no competing interests. SF, MB and EL are employees of Mast Diagnostics GmbH, which produce and sell the TPHD LAMP kits and products. A patent covering the mediator displacement probe technique a component of the TPHD-LAMP assay described in the paper has been applied for by the University of Freiburg and Hahn-Schickard., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood. Minimal residual disease (MRD) monitoring is an important prognostic factor for ALL treatment response and patient stratification. MRD monitoring uses personalized real-time PCR to measure the amount of cancer cells among normal cells. Due to clonal tumor evolution or secondary rearrangement processes, MRD markers can disappear during treatment, leading to false-negative MRD results and wrong decision-making in personalized treatments. Therefore, monitoring of multiple MRD markers per patient is required. For the first time, the authors present personalized multiplex mediator probe PCR (MP PCR) for MRD monitoring in ALL. These assays can precisely quantify more MRD markers in less sample material. Therefore, clinical outcomes will be less affected by clonal tumor evolution. Personalized duplex MP PCR assays were developed for different genomic MRD markers, including immunoglobulin/T-cell receptor gene rearrangements, gene fusions, and gene deletions. One duplex assay was successfully applied in a prospective patient case and compared with hydrolysis probes. Moreover, the authors increased the multiplex level from duplex to 4-plex and still met the EuroMRD requirements for reliable quantification. In addition, the authors' MRD-MP design guidelines and multiplex workflow facilitate and accelerate MP PCR assay development. This helps the standardization of personal diagnostics., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types ( KRAS and BRAF ) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex KRAS G13D (9.82 copies/mL) and 4-plex BRAF V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R 2 ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring.

Multiplexing of analyses is essential to reduce sample and reagent consumption in applications with large target panels. In applications such as cancer diagnostics, the required degree of multiplexing often exceeds the number of available fluorescence channels in polymerase chain reaction (PCR) devices. The combination of photobleaching-sensitive and photobleaching-resistant fluorophores of the same color can boost the degree of multiplexing by a factor of 2 per channel. The only additional hardware required to create virtual fluorescence color channels is a low-cost light-emitting diode (LED) setup for selective photobleaching. Here, we present an assay concept for fluorescence color multiplexing in up to 10 channels (five standard channels plus five virtual channels) using the mediator probe PCR with universal reporter (UR) fluorogenic oligonucleotides. We evaluate the photobleaching characteristic of 21 URs, which cover the whole spectral range from blue to crimson. This comprehensive UR data set is employed to demonstrate the use of three virtual channels in addition to the three standard channels of a commercial dPCR device (blue, green, and red) targeting cancer-associated point mutations ( KRAS G12D and G12V). Moreover, a LOD (limit of detection) analysis of this assay confirms the high sensitivity of the multiplexing method ( KRAS G12D: 16 DNA copies/reaction in the standard red channel and KRAS G12V: nine DNA copies/reaction in the virtual red channel). Based on the presented data set, optimal fluorogenic reporter combinations can be easily selected for the application-specific creation of virtual channels, enabling a high degree of multiplexing at low optical and technical effort.

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.

We present an automated point-of-care testing (POCT) system for rapid detection of species- and resistance markers in methicillin-resistant Staphylococcus aureus (MRSA) at the level of single cells, directly from nasal swab samples. Our novel system allows clear differentiation between MRSA, methicillin-sensitive S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS), which is not the case for currently used real-time quantitative PCR based systems. On top, the novel approach outcompetes the culture-based methods in terms of its short time-to-result (1 h vs. up to 60 h) and reduces manual labor. The walk-away test is fully automated on the centrifugal microfluidic LabDisk platform. The LabDisk cartridge comprises the unit operations swab-uptake, reagent pre-storage, distribution of the sample into 20 000 droplets, specific enzymatic lysis of Staphylococcus spp. and recombinase polymerase amplification (RPA) of species (vicK) - and resistance (mecA) -markers. LabDisk actuation, incubation and multi-channel fluorescence detection is demonstrated with a clinical isolate and spiked nasal swab samples down to a limit of detection (LOD) of 3 ± 0.3 CFU μl-1 for MRSA. The novel approach of the digital single cell detection is suggested to improve hospital admission screening, timely decision making, and goal-oriented antibiotic therapy. The implementation of a higher degree of multiplexing is required to translate the results into clinical practice.

We present a versatile tool for the generation of monodisperse water-in-fluorinated-oil droplets in standard reaction tubes by centrifugal step emulsification. The microfluidic cartridge is designed as an insert into a standard 2 mL reaction tube and can be processed in standard laboratory centrifuges. It allows for droplet generation and subsequent transfer for any downstream analysis or further use, does not need any specialized device, and manufacturing is simple because it consists of two parts only: A structured substrate and a sealing foil. The design of the structured substrate is compatible to injection molding to allow manufacturing at large scale. Droplets are generated in fluorinated oil and collected in the reaction tube for subsequent analysis. For sample sizes up to 100 µL with a viscosity range of 1 mPa·s-4 mPa·s, we demonstrate stable droplet generation and transfer of more than 6 × 10 5 monodisperse droplets (droplet diameter 66 µm ± 3 µm, CV ≤ 4%) in less than 10 min. With two application examples, a digital droplet polymerase chain reaction (ddPCR) and digital droplet loop mediated isothermal amplification (ddLAMP), we demonstrate the compatibility of the droplet production for two main amplification techniques. Both applications show a high degree of linearity (ddPCR: R 2 ≥ 0.994; ddLAMP: R 2 ≥ 0.998), which demonstrates that the cartridge and the droplet generation method do not compromise assay performance.

Driven by the vision of robust and portable, yet sensitive DNA detection systems for point-of-need applications, the development of electrochemical DNA sensing principles has been of high interest. Many different principles have been developed and these are regularly reviewed. However, the maturity of electrochemical principles and their ability to produce competitive real-world applications is rarely assessed. In this review, general electrochemical DNA sensing principles are briefly introduced and categorized into heterogeneous vs. homogeneous approaches, and then the subcategories label-free vs. labeled and reagent-less vs. reagent-dependent principles. We then focus on reviewing the electrochemical sensing principles implemented in DNA detection systems, which are commercially available or close to market entry, considering the complete analysis process, automation and the field of application. This allows us to outline and discuss which principles have proved suitable for which kinds of applications, as well as the stage of integration and automation. Examples from all the identified categories of electrochemical DNA sensing principles have found application in commercial detection systems or advanced prototypes. Various applications have already been demonstrated, ranging from on-site skin care testing, to food safety to the most frequent in vitro diagnostic tests, partially conducted in automated sample-to-answer devices. Our review is intended to enable researchers in areas related to electrochemistry, biochemistry or microfluidics to assess the commercial state of the art of electrochemical nucleic acid testing, and the interdisciplinary challenges for further improvements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Inkjet technology as a maskless, direct-writing technology offers the potential for structured deposition of functional materials for the realization of electrodes for, e.g., sensing applications. In this work, electrodes were realized by inkjet-printing of commercial nanoparticle gold ink on planar substrates and, for the first time, onto the 2.5D surfaces of a 0.5 mm-deep microfluidic chamber produced in cyclic olefin copolymer (COC). The challenges of a poor wetting behavior and a low process temperature of the COC used were solved by a pretreatment with oxygen plasma and the combination of thermal (130 °C for 1 h) and photonic (955 mJ/cm²) steps for sintering. By performing the photonic curing, the resistance could be reduced by about 50% to 22.7 µΩ cm. The printed gold structures were mechanically stable (optimal cross-cut value) and porous (roughness factors between 8.6 and 24.4 for 3 and 9 inkjet-printed layers, respectively). Thiolated DNA probes were immobilized throughout the porous structure without the necessity of a surface activation step. Hybridization of labeled DNA probes resulted in specific signals comparable to signals on commercial screen-printed electrodes and could be reproduced after regeneration. The process described may facilitate the integration of electrodes in 2.5D lab-on-a-chip systems.

Yaws, a neglected tropical disease caused by the bacterium Treponema pallidum subspecies pertenue, manifests as ulcerative skin lesions. Nucleic acid amplification tests, like loop-mediated isothermal amplification (LAMP), are versatile tools to distinguish yaws from infections that cause similar skin lesions, primarily Haemophilus ducreyi. We developed a novel molecular test to simultaneously detect T. pallidum and H. ducreyi based on mediator displacement LAMP. We validated the T. pallidum and H. ducreyi LAMP (TPHD-LAMP) by testing 293 clinical samples from patients with yaws-like lesions. Compared with quantitative PCR, the TPHD-LAMP demonstrated high sensitivity and specificity for T. pallidum (84.7% sensitivity, 95.7% specificity) and H. ducreyi (91.6% sensitivity, 84.8% specificity). This novel assay provided rapid molecular confirmation of T. pallidum and H. ducreyi DNA and might be suitable for use at the point of care. TPHD-LAMP could support yaws eradication by improving access to molecular diagnostic tests at the district hospital level.

Cryphonectria hypovirus 1 (CHV-1) infects the chestnut blight fungus Cryphonectria parasitica and acts as a biological control agent against this harmful tree disease. In this study, we screened the recently characterized C. parasitica population in Eurasian Georgia for the presence of CHV-1. We found 62 CHV-1 infected C. parasitica isolates (9.3%) among a total of 664 isolates sampled in 14 locations across Georgia. The prevalence of CHV-1 at the different locations ranged from 0% in the eastern part of the country to 29% in the western part. Sequencing of two specific regions of the viral genome one each in ORFA and ORFB revealed a unique CHV-1 subtype in Georgia. This subtype has a recombinant pattern combining the ORFA region from the subtype F2 and the ORFB region from subtype D. All 62 viral strains belonged to this Georgian CHV-1 subtype (subtype G). The CHV-1 subtype G strongly reduced the parasitic growth of C. parasitica isolates from Georgia, with a more severe effect on the European genepool compared to the Georgian genepool. The CHV-1 subtype detected in Georgia provides a valuable candidate for biological control applications in the Caucasus region ., Competing Interests: The authors declare no conflict of interest.

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

The analysis of a multiple-sequence alignment (MSA) with correlation methods identifies pairs of residue positions whose occupation with amino acids changes in a concerted manner. It is plausible to assume that positions that are part of many such correlation pairs are important for protein function or stability. We have used the algorithm H2r to identify positions k in the MSAs of the enzymes anthranilate phosphoribosyl transferase (AnPRT) and indole-3-glycerol phosphate synthase (IGPS) that show a high conn(k) value, i.e., a large number of significant correlations in which k is involved. The importance of the identified residues was experimentally validated by performing mutagenesis studies with sAnPRT and sIGPS from the archaeon Sulfolobus solfataricus. For sAnPRT, five H2r mutant proteins were generated by replacing nonconserved residues with alanine or the prevalent residue of the MSA. As a control, five residues with conn(k) values of zero were chosen randomly and replaced with alanine. The catalytic activities and conformational stabilities of the H2r and control mutant proteins were analyzed by steady-state enzyme kinetics and thermal unfolding studies. Compared to wild-type sAnPRT, the catalytic efficiencies (k(cat)/K(M)) were largely unaltered. In contrast, the apparent thermal unfolding temperature (T(M)(app)) was lowered in most proteins. Remarkably, the strongest observed destabilization (ΔT(M)(app) = 14 °C) was caused by the V284A exchange, which pertains to the position with the highest correlation signal [conn(k) = 11]. For sIGPS, six H2r mutant and four control proteins with alanine exchanges were generated and characterized. The k(cat)/K(M) values of four H2r mutant proteins were reduced between 13- and 120-fold, and their T(M)(app) values were decreased by up to 5 °C. For the sIGPS control proteins, the observed activity and stability decreases were much less severe. Our findings demonstrate that positions with high conn(k) values have an increased probability of being important for enzyme function or stability.