A loop-mediated isothermal amplification test for yaws: a multi-country diagnostic accuracy evaluation.

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.

A rapid economical multiplex PCR-RFLP method for molecular detection and genotyping of Giardia duodenalis clinical isolates.

Giardia duodenalis is a common cause of diarrheal illness in regions with limited resources. The demand for rapid and cost-effective detection and genotyping methods in large-scale epidemiological studies and clinical diagnostics is imperative. Hence, we developed a multiplex PCR-RFLP technique targeting the tpi gene of G. duodenalis. The assay successfully screened G. duodenalis positive clinical samples (6.33 %; 36/565). It was also able to categorize the isolates into assemblages A (41.66 %; 13/36) and B (58.33 %; 23/36), as well as into subassemblages: AI (13.8 %; 5/36), AII (27.77 %; 10/36), BIII (36.11 %; 15/36) and BIV (22.22 %; 8/36). High diagnostic sensitivity (94.2 %), specificity (100 %) and accuracy (97.1 %) of the PCR assay were obtained, indicating its reliability for diagnosing giardiasis. Notably, the assay demonstrated close concordance with microscopy (κ=0.85) and reference PCR (κ=0.98) results. The optimized method offers a cost-effective and rapid approach for G. duodenalis detection and genotyping, convenient for epidemiological studies and clinical diagnostics.

Cost-Effectiveness Analysis of Molecular Testing for Indeterminate Thyroid Nodules in Nova Scotia.

BACKGROUND: The objective of the following retrospective review was to perform a cost-effectiveness analysis of the use of molecular testing of indeterminate thyroid nodules compared to current management practices in Nova Scotia, Canada. METHODS: All cases of cytologically indeterminate thyroid nodules from January 1st, 2014 to December 31st, 2018 were reviewed. All interventions related to an indeterminate thyroid nodule were recorded. Patients were excluded if less than 18 years old if no further information regarding medical management was electronically available beyond the diagnosis of an indeterminate thyroid nodule, history of radiation, or previous thyroid surgery prior to diagnosis of an indeterminate thyroid nodule in the remaining lobe. Microcosting was performed to determine the cost of all relevant interventions including repeat fine needle aspiration biopsy, ultrasound, thyroid surgery(s), and molecular testing. Institution-specific transition state probabilities were calculated and used to build a cost-effectiveness analysis model. Model output was an incremental cost-effectiveness ratio, defined as the ratio of cost difference to effectiveness difference between routine molecular testing and the current management strategy, yielding cost per surgery avoided. RESULTS: The mean effectiveness of the current management of indeterminate thyroid nodules in Nova Scotia based on the American Thyroid Association guidelines is 64% at a mean cost of $6431, while the simulated mean effectiveness of routine molecular testing is 89% at a mean cost of $8414. Differences in management strategies generated an incremental cost-effectiveness ratio of $7876 per surgery avoided. CONCLUSION: Routine molecular testing is the more effective strategy for the appropriate management of indeterminate thyroid nodules; however, it comes at a higher mean cost compared to the current management strategy. As the cost of molecular testing continues to decrease, and the cost of OR resources continues to rise, molecular testing is likely to become the optimal strategy in Nova Scotia.

An Ultra-Compact and Low-Cost LAMP-Based Virus Detection Device.

Timely and accurate detection of viruses is crucial for infection diagnosis and treatment. However, it remains a challenge to develop a portable device that meets the requirement of being portable, powerless, user-friendly, reusable, and low-cost. This work reports a compact ∅30 × 48 mm portable powerless isothermal amplification detection device (material cost ∼$1 USD) relying on LAMP (Loop-Mediated Isothermal Amplification). We have proposed chromatographic-strip-based microporous permeation technology which can precisely control the water flow rate to regulate the exothermic reaction. This powerless heating combined with phase-change materials can maintain a constant temperature between 50 and 70 °C for a duration of up to 49.8 min. Compared with the conventional methods, it avoids the use of an additional insulation layer for heat preservation, greatly reducing the size and cost. We have also deployed a color card and a corresponding algorithm to facilitate color recognition, data analysis, and storage using a mobile phone. The experimental results demonstrate that our device exhibits the same limit of detection (LOD) as the ProFlex PCR for SARS-CoV-2 pseudovirus samples, with that for both being 103 copies/µL, verifying its effectiveness and reliability. This work offers a timely, low-cost, and easy way for respiratory infectious disease detection, which could provide support in curbing virus transmission and protecting the health of humans and animals, especially in remote mountainous areas without access to electricity or trained professionals.

[Molecular diagnosis of bacteriaemia: benefits of using the FilmArray® BCID2 sepsis panel in a third level hospital]. / Diagnóstico molecular de bacteriemias: beneficios del empleo del panel de sepsis BCID2 de Filmarray® en un hospital de tercer nivel.

INTRODUCTION: Delay in initiating appropriate antimicrobial therapy prolongs hospitalization, increases in-hospital mortality, and raises economic costs. Currently, the identification and susceptibility testing of bacteria in positive blood cultures require a considerable amount of time. The objective of this study was to assess the impact of the BCID2 FilmArray® (FA) panel on the timing of appropriate antimicrobial therapy and potential antimicrobial costs. METHODS: This is a retrospective observational study focused on positive blood cultures in hospitalized patients. FA processing was conducted concurrently with routine sample processing. Changes in antibiotic treatments based on FA results were evaluated, and the reduction in antimicrobial therapy duration and associated cost savings were calculated. RESULTS: Eighty-seven bacteremia episodes were analysed. In 42 (48%) of them antimicrobial therapy was de-escalated to narrower spectrum agents, while in 7 (8%) therapy was escalated to broader spectrum antimicrobials. Additionally, in 8 (9%) antimicrobials were switched without changing spectrum and in 30 (34%) no changes were made based on FA results. Antimicrobial changes were made 2.3 days faster than with routine sample processing resulting in calculated potential savings of US$ 7408. CONCLUSION: The implementation of FA facilitated a faster administration of appropriate antimicrobial therapy, leading to a reduction in the duration of broadspectrum empirical antimicrobial therapy and subsequent economic savings.

Introducción: Los retrasos en el tratamiento antimicrobiano adecuado de las bacteriemias prolongan la estadía hospitalaria, aumentan la mortalidad e incrementan los costos. Aún hoy en día se requiere un tiempo considerable para obtener la identificación y antibiograma de los microorganismos en los hemocultivos positivos. El objetivo fue evaluar el impacto de la implementación del panel BCID2 de FilmArray® (FA) sobre el tiempo de inicio de tratamientos antimicrobianos adecuados y sobre los costos potenciales de los mismos. Métodos: Estudio observacional retrospectivo de los hemocultivos positivos de pacientes hospitalizados, procesados por FA y por metodología tradicional. Se evaluaron los cambios de antimicrobianos en base a los resultados del FA. Se calcularon los días de reducción de tratamiento antimicrobiano y el ahorro potencial en el uso de los mismos, teniendo en cuenta también los costos del FA. Resultados: Se analizaron 87 episodios de bacteriemia. En 42 (48.3%) de ellos se desescaló el tratamiento a antimicrobianos de menor espectro, en 7 (8%) se escaló a antimicrobianos de mayor espectro, en 8 (9.2%) se cambió el antimicrobiano sin variar el espectro y en 30 (34.5%) no se realizaron cambios con los resultados del FA. Los cambios de antimicrobianos se realizaron en promedio 2.3 días más rápido que con los métodos convencionales. Se calculó un ahorro potencial de US$ 7408. Conclusión: La implementación del panel BCID2 de FilmArray® permitió adecuar los tratamientos antimicrobianos más rápidamente acortando la duración de los tratamientos empíricos de amplio espectro, lo cual resultó costo-efectivo.

Economic Impact of Whole Genome Sequencing and Whole Transcriptome Sequencing Versus Routine Diagnostic Molecular Testing to Stratify Patients with B-Cell Acute Lymphoblastic Leukemia.

Whole genome and whole transcriptome sequencing (WGTS) can accurately distinguish B-cell acute lymphoblastic leukemia (B-ALL) genomic subtypes. However, whether this is economically viable remains unclear. This study compared the direct costs and molecular subtype classification yield using different testing strategies for WGTS in adolescent and young adult/adult patients with B-ALL. These approaches were: (1) combined BCR::ABL1 by fluorescence in situ hybridization (FISH) + WGTS for all patients; and (2) sequential BCR::ABL1 FISH + WGTS contingent on initial BCR::ABL1 FISH test outcome. The cost of routine diagnostic testing was estimated using Medicare or hospital fees, and the additional cost of WGTS was evaluated from the health care provider perspective using time-driven activity-based costing with resource identification elicited from experts. Molecular subtype classification yield data were derived from literature sources. Parameter uncertainty was assessed through deterministic sensitivity analysis; additional scenario analyses were performed. The total per patient cost of WGTS was $4319 (all costs reported in US dollars); consumables accounted for 74% of the overall cost, primarily driven by sequencing-related consumables. The incremental cost per additional patient categorized into molecular subtype was $8498 for combined BCR::ABL1 FISH + WGTS for all patients and $5656 for initial BCR::ABL1 FISH + WGTS for select patients compared with routine diagnostic testing. A reduction in the consumable costs of WGTS or an increase in the yield of molecular subtype classification is favorable.

An affordable detection system based on RT-LAMP and DNA-nanoprobes for avian metapneumovirus.

Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming. KEY POINTS: •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity. •Fifty field samples have been visualized using DNA-nanoprobe validation. •The developed system is a reliable, fast, and cost-effective option for POCT.

Abbott realtime MTB assay for detecting Mycobacterium tuberculosis complex in respiratory specimens: a cost-benefit analysis.

PURPOSE: Molecular screening for Mycobacterium tuberculosis (MTB) can lead to rapid empirical treatment inception and reduce hospitalization time and complementary diagnostic tests. However, in low-prevalence settings, the cost-benefit balance remains controversial due to the high cost. METHODS: We used a Markov model to perform an economic analysis to evaluate the profit after implementing molecular MTB screening (Period B) compared with conventional culture testing (Period A) in respiratory samples from 7,452 consecutive subjects with presumed tuberculosis (TB). RESULTS: The proportion of positivity was comparable between both periods (P > 0.05), with a total of 2.16 and 1.78 samples/patient requested in periods A and B, respectively (P < 0.001). The mean length of hospital stay was 8.66 days (95%CI: 7.63-9.70) in Period B and 11.51 days (95%CI: 10.15-12.87) in Period A (P = 0.001). The healthcare costs associated with diagnosing patients with presumed TB were reduced by €717.95 per patient with PCR screening. The probability of remaining hospitalized and the need for a greater number of outpatient specialty care visits were the variables with the most weight in the model. CONCLUSION: Employing PCR as an MTB screening method in a low-prevalence setting may increase the profits to the system.

Application of LAMP assay for detection of carbapenem-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii complex in ICU admitted sepsis patients: A rapid and cost-effective diagnostic tool.

Carbapenem-resistant significant members of Acinetobacter calcoaceticus-Acinetobacter baumannii (CR-SM-ACB) complex have emerged as an important cause of sepsis, especially in ICUs. This study demonstrates the application of loop-mediated-isothermal-amplification (LAMP) assay for detection of CR-SM-ACB-complex from patients with sepsis. Whole-blood and culture-broths(CB) collected from patients with culture-positive sepsis were subjected to LAMP and compared with PCR, and RealAmp. Vitek-2 system and conventional PCR results were used as confirmatory references. The sensitivity and specificity of LAMP(97 % & 100 %) and RealAmp(100 % & 100 %) for detection of CR-SM-ACB-complex from CB were better than PCR(87 % & 100 %). Diagnostic accuracy of LAMP, RealAmp, and PCR for detection of SM-ACB-complex from CB was 98.5 %, 100 %, and 88.5 % respectively. Turnaround time of Culture, LAMP, PCR, and RealAmp was 28-53, 6-20, 9-23, and 6-20hours, respectively. LAMP is a simple, inexpensive tool that can be applied directly to positive CB and may be customized to detect emerging pathogens and locally-prevalent resistance genes and to optimize antimicrobial use.

Cost analyses for malaria molecular diagnosis for research planners in India and beyond.

BACKGROUND: Malaria elimination mandates early and accurate diagnosis of infection. Although malaria diagnosis is programmatically dependent on microscopy/RDTs, molecular diagnosis has much better diagnostic accuracy. Higher cost of molecular diagnoses is a recognized challenge for use at the point of care. Because funding is always a recognized constraint, we performed financial cost-analyses of available molecular platforms for better utilization of available budget. METHODS: Two strategies were applied to deduce the cost per sample. Strategy 1 included recurring components (RC) in minimum pack size, and biologist's time whereas strategy 2 included only RC and non-recurring components and costs are calculated for sample sizes (1-1,000,000) to infer the sample size effect. RESULTS: Spin column-based manual DNA extraction (US$ 3.93 per sample) is the lowest-cost method, followed by magnetic bead-based automated, semi-automated, and PCI-based manual method. Further, DNA extraction cost per sample via spin column-based manual method and semi-automated method decreases with an increase in sample size up to 10,000. Real-time PCRs are ~ 2-fold more economical than conventional PCR, regardless of sample size. CONCLUSIONS: This study is the first for malaria to estimate systematic molecular diagnosis financial costs. Kit-based and automated methods may replace conventional DNA extraction and amplification methods for a frugal high-throughput diagnosis.

Sample-to-result molecular diagnostic platforms and their suitability for infectious disease testing in low- and middle-income countries.

INTRODUCTION: Diagnostics are an essential, undervalued part of the health-care system. For many diseases, molecular diagnostics are the gold standard, but are not easy to implement in Low- and Middle-Income Countries (LMIC). Sample-to-result (S2R) platforms combining all procedures in a closed system could offer a solution. In this paper, we investigated their suitability for implementation in LMIC. AREAS COVERED: A scorecard was used to evaluate different platforms on a range of parameters. Most platforms scored fairly on the platform itself, ease-of-use and test consumables; however, shortcomings were identified in cost, distribution and test panels tailored to LMIC needs. The diagnostic coverage for common infectious diseases was found to have a wider coverage in high-income countries (HIC) than LMIC. A literature study showed that in LMIC, these platforms are mainly used as diagnostic tools or evaluation of diagnostic performance, with a minority assessing the operational characteristics or the clinical utility. In this narrative review, we identified various points for adaptation of S2R platforms to LMIC conditions. EXPERT OPINION: For S2R platforms to be suitable for implementation in LMIC some modifications by the manufacturers could be considered. Furthermore, strengthening health systems and digitalization are vital; as are smaller, cheaper, faster, and sustainable technologies.

Low-cost conventional PCR techniques enable simultaneous detection of bacterial sexually transmitted infections with enhanced sensitivity and specificity.

PURPOSE: Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) and Mycoplasma hominis (MH), the three most common treatable bacterial sexually transmitted infections (STIs) worldwide can lead to many complications if remain untreated. Screening of high-risk population with highly sensitive methods will lead to significant improvement in patient outcomes and will prevent downward transmission. The advantages of Polymerase chain reaction (PCR) based assay are not only high sensitivity and specificity, but also detection of multiple organisms in a single reaction which reduce the result turn-around time. The aim of the present study was to evaluate the feasibility of a multiplex PCR assay method targeting 16S rRNA gene for simultaneous detection of NG, CT and MH infection along with their trend and occurrence among high-risk population in Assam, Northeast India. METHODS: A cross-sectional study was undertaken, where a total of 200 randomly selected patients from high-risk population were included. After validation of singleplex PCR, Multiplex PCR (M-PCR) was performed along with the traditional culture method for NG. RESULTS & CONCLUSION: The overall agreement of M-PCR with singleplex PCR was very high (100%). The occurrence of STI was found to be very high (101/200; 50.5%). Furthermore, co-infection was detected in 10/200; 5%) individuals. Infection was more common among young individuals (p < 0.05) and males out-numbered females (p < 0.05). The most common organism detected was CT (42/200; 21%) followed by NG (41/200; 20.5%) and MH (20/200; 10%). The M-PCR assay workflow is simple, cost effective and can be used in routine diagnostic laboratories with basic molecular facilities.

Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Background: Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. Methods: A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Results: Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. Conclusion: The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

Clinical impact and cost-consequence analysis of ePlex® blood culture identification panels for the rapid diagnosis of bloodstream infections: a single-center randomized controlled trial.

To assess clinical impact and perform cost-consequence analysis of the broadest multiplex PCR panels available for the rapid diagnosis of bloodstream infections (BSI). Single-center, randomized controlled trial conducted from June 2019 to February 2021 at a French University hospital with an institutional antimicrobial stewardship program. Primary endpoint was the percentage of patients with optimized antimicrobial treatment 12 h after transmission of positivity and Gram stain results from the first positive BC. This percentage was significantly higher in the multiplex PCR (mPCR) group (90/105 = 85.7% %, CI95% [77.5 ; 91.8] vs. 68/107 = 63.6%, CI95% [53.7 ; 72.6]; p < 10- 3) at interim analysis, resulting in the early termination of the study after the inclusion of 309 patients. For patients not optimized at baseline, the median time to obtain an optimized therapy was much shorter in the mPCR group than in the control group (6.9 h, IQR [2.9; 17.8] vs. 26.4 h, IQR [3.4; 47.5]; p = 0.001). Early optimization of antibiotic therapy resulted in a non-statistically significant decrease in mortality from 12.4 to 8.8% (p = 0.306), with a trend towards a shorter median length of stay (18 vs. 20 days; p = 0.064) and a non-significant reduction in the average cost per patient of €3,065 (p = 0.15). mPCR identified all the bacteria present in 88% of the samples. Despite its higher laboratory cost, the use of multiplex PCR for BSI diagnosis leads to early-optimised therapy, seems cost-effective and could reduce mortality and length of stay. Their impact could probably be improved if implemented 24/7.

Diagnostic outcome of pro bono neurogenetic diagnostic service in Sri Lanka: A wealth creation.

The inherited disease community in Sri Lanka has been widely neglected. This article aimed to present accumulated knowledge in establishing a pro bono cost-effective national, island-wide, free-of-charge molecular diagnostic service, suggesting a model for other developing countries. The project provided 637 molecular diagnostic tests and reports free of charge to a nation with limited resources. We pioneered the implementation of mobile clinics and home visits, where the research team acted as barefoot doctors with the concept of the doctor and the researcher at the patient's doorstep. Establishing pro bono, cost-effective molecular diagnostics is feasible in developing countries with limited resources and state funding through the effort of dedicated postgraduate students. This service could provide an accurate molecular diagnosis of Duchenne muscular dystrophy, Huntington's disease, Spinocerebellar ataxia, and Spinal muscular atrophy, a diagnostic yield of 54% (343/637), of which 43% (147/343) of the patients identified as amenable for available gene therapies. Initiated human resource development by double doctoral degree opportunities with international collaborations. Established a neurobiobank and a national registry in Sri Lanka, a rich and unique repository, wealth creation for translational collaborative research and sharing of information in neurological diseases, as well as a lodestar for aspiring initiatives from other developing countries.

Update on immunohistochemistry in bone and soft tissue tumors: Cost-effectively replacing molecular testing with immunohistochemistry.

Soft tissue tumors form part of a challenging domain in diagnostic pathology owing to their comparative rarity, astonishing histologic diversity, and overlap between entities. Many of these tumors are now known to be defined by highly recurrent, or, in some instances, unique molecular alterations. Insights from gene profiling continue to elucidate the wider molecular landscape of soft tissue tumors; many of these advances have been co-opted by immunohistochemistry (IHC) for diagnostic applications. There now exists a multitude of antibodies serving as surrogate markers of recurrent gene fusions, amplifications, and point mutations, which, in certain settings, can replace the need for more resource and time-intensive cytogenetic and molecular genetic analyses. IHC presents many advantages including rapid turnaround time, cost-effectiveness, and interpretative reproducibility. A sensible application of these immunohistochemical markers complemented by a working knowledge of the molecular pathogenesis of bone and soft tissue tumors permits accurate diagnosis in the majority of cases. In this review, we will outline some of these biomarkers while emphasizing molecular correlates and highlighting interpretative challenges and pitfalls.

Atypical lipomatous tumor/well differentiated liposarcoma and related mimics with updates. When is molecular testing most cost-effective, necessary, and indicated?

The classification and work-up of adipocytic neoplasms remains challenging and sometimes controversial. Since its initial description by Dr. Enterline, the variety of subtypes and morphological appearances considered to represent the spectrum of atypical lipomatous tumor/well differentiated liposarcoma (ALT/WDL) has expanded, resulting in significant morphologic overlap with other entities, including the recently described atypical spindle cell/pleomorphic lipomatous tumor (ASPLT), conventional spindle cell/pleomorphic lipoma (SPL), and so-called "low-grade" forms of dedifferentiated liposarcoma (DL). Nevertheless, the distinction of most examples of ALT/WDL from lipomas/lipoma-like lesions is easily performed on routine histologic examination but can be problematic if the characteristic atypical cells are poorly represented, particularly in small biopsy specimens, obscured by other cellular elements (inflammation), or simply not recognized. The discovery that lipomatous tumors harbor specific and unique karyotypes and molecular events has resulted in ancillary tests that can help provide more accurate diagnoses, especially in less-than-optimal scenarios. Confirmation of MDM2 immunohistochemical over-expression and detection of the MDM2 gene rearrangement via fluorescent in situ hybridization (FISH) have proven particularly reliable and useful. While FISH analysis for MDM2 gene amplification may be helpful for confirming (or excluding) ALT/WDL, it also can lead to overutilization and overdependence. Furthermore, a small subset of otherwise typical ALT/WDL lack MDM2 gene amplification, employing alternative molecular pathways. The recent recognition of ASPLT has introduced a tumor easily mistaken morphologically for ALT/WDL, often exhibiting bizarre and pleomorphic lipoblasts, but lacking the underlying molecular abnormalities and subsequent risk of dedifferentiation. ASPLT also have overlapping features with the better-established SPL but with a greater tendency to locally recur and more frequent involvement of the distal extremities. The precise criteria separating cellular forms of ALT from what some consider "low grade" forms of DL remains controversial and inconsistently applied, even among individual pathologists within institutions. Given their underlying shared cytogenetic abnormality, molecular testing has no utility in this distinction. Herein is a comprehensive historical overview of ALT/WDL, with updates on its distinction from other similar lipomatous tumors and DL, including practical evidence-based criteria for the appropriate cost-effective use of MDM2 testing.

Ferrobotic swarms enable accessible and adaptable automated viral testing.

Expanding our global testing capacity is critical to preventing and containing pandemics1-9. Accordingly, accessible and adaptable automated platforms that in decentralized settings perform nucleic acid amplification tests resource-efficiently are required10-14. Pooled testing can be extremely efficient if the pooling strategy is based on local viral prevalence15-20; however, it requires automation, small sample volume handling and feedback not available in current bulky, capital-intensive liquid handling technologies21-29. Here we use a swarm of millimetre-sized magnets as mobile robotic agents ('ferrobots') for precise and robust handling of magnetized sample droplets and high-fidelity delivery of flexible workflows based on nucleic acid amplification tests to overcome these limitations. Within a palm-sized printed circuit board-based programmable platform, we demonstrated the myriad of laboratory-equivalent operations involved in pooled testing. These operations were guided by an introduced square matrix pooled testing algorithm to identify the samples from infected patients, while maximizing the testing efficiency. We applied this automated technology for the loop-mediated isothermal amplification and detection of the SARS-CoV-2 virus in clinical samples, in which the test results completely matched those obtained off-chip. This technology is easily manufacturable and distributable, and its adoption for viral testing could lead to a 10-300-fold reduction in reagent costs (depending on the viral prevalence) and three orders of magnitude reduction in instrumentation cost. Therefore, it is a promising solution to expand our testing capacity for pandemic preparedness and to reimagine the automated clinical laboratory of the future.

Clinical and economic impact of 'ROS1-testing' strategy compared to a 'no-ROS1-testing' strategy in advanced NSCLC in Spain.

BACKGROUND: Detection of the ROS1 rearrangement is mandatory in patients with advanced or metastatic non-small cell lung cancer (NSCLC) to allow targeted therapy with specific inhibitors. However, in Spanish clinical practice ROS1 determination is not yet fully widespread. The aim of this study is to determine the clinical and economic impact of sequentially testing ROS1 in addition to EGFR and ALK in Spain. METHODS: A joint model (decision-tree and Markov model) was developed to determine the cost-effectiveness of testing ROS1 strategy versus a no-ROS1 testing strategy in Spain. Distribution of ROS1 techniques, rates of testing, positivity, and invalidity of biomarkers included in the analysis (EGFR, ALK, ROS1 and PD-L1) were based on expert opinion and Lungpath real-world database. Treatment allocation depending on the molecular testing results was defined by expert opinion. For each treatment, a 3-states Markov model was developed, where progression-free survival (PFS) and overall survival (OS) curves were parameterized using exponential extrapolations to model transition of patients among health states. Only medical direct costs were included (€ 2021). A lifetime horizon was considered and a discount rate of 3% was applied for both costs and effects. Both deterministic and probabilistic sensitivity analyses were performed to address uncertainty. RESULTS: A target population of 8755 patients with advanced NSCLC (non-squamous or never smokers squamous) entered the model. Over a lifetime horizon, the ROS1 testing scenario produced additional 157.5 life years and 121.3 quality-adjusted life years (QALYs) compared with no-ROS1 testing scenario. Total direct costs were increased up to € 2,244,737 for ROS1 testing scenario. The incremental cost-utility ratio (ICUR) was 18,514 €/QALY. Robustness of the base-case results were confirmed by the sensitivity analysis. CONCLUSIONS: Our study shows that ROS1 testing in addition to EGFR and ALK is a cost-effective strategy compared to no-ROS1 testing, and it generates more than 120 QALYs in Spain over a lifetime horizon. Despite the low prevalence of ROS1 rearrangements in NSCLC patients, the clinical and economic consequences of ROS1 testing should encourage centers to test all advanced or metastatic NSCLC (non-squamous and never-smoker squamous) patients.

End-to-end system for rapid and sensitive early-detection of SARS-CoV-2 for resource-poor and field-test environments using a $51 lab-in-a-backpack.

COVID-19 has exposed stark inequalities between resource-rich and resource-poor countries. International UN- and WHO-led efforts, such as COVAX, have provided SARS-CoV-2 vaccines but half of African countries have less than 2% vaccinated in their population, and only 15 have reached 10% by October 2021, further disadvantaging local economic recovery. Key for this implementation and preventing further mutation and spread is the frequency of voluntary [asymptomatic] testing. It is limited by expensive PCR and LAMP tests, uncomfortable probes deep in the throat or nose, and the availability of hardware to administer in remote locations. There is an urgent need for an inexpensive "end-to-end" system to deliver sensitive and reliable, non-invasive tests in resource-poor and field-test conditions. We introduce a non-invasive saliva-based LAMP colorimetric test kit and a $51 lab-in-a-backpack system that detects as few as 4 viral RNA copies per µL. It consists of eight chemicals, a thermometer, a thermos bottle, two micropipettes and a 1000-4000 rcf electronically operated centrifuge made from recycled computer hard drives (CentriDrive). The centrifuge includes a 3D-printed rotor and a 12 V rechargeable Li-ion battery, and its 12 V standard also allows wiring directly to automobile batteries, to enable field-use of this and other tests in low infrastructure settings. The test takes 90 minutes to process 6 samples and has reagent costs of $3.5 per sample. The non-invasive nature of saliva testing would allow higher penetration of testing and wider adoption of the test across cultures and settings (including refugee camps and disaster zones). The attached graphical procedure would make the test suitable for self-testing at home, performing it in the field, or in mobile testing centers by minimally trained staff.