A microfluidic biosensor architecture for the rapid detection of COVID-19

The lack of enough diagnostic capacity to detect severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has been one of the major challenges in the control the 2019 COVID pandemic;this led to significant delay in prompt treatment of COVID-19 patients or accurately estimate disease situation. Current methods for the diagnosis of SARS-COV-2 infection on clinical specimens (e.g. nasal swabs) include polymerase chain reaction (PCR) based methods, such as real-time reverse transcription (rRT) PCR, real-time reverse transcription loop-mediated isothermal amplification (rRT-LAMP), and immunoassay based methods, such as rapid antigen test (RAT). These conventional PCR methods excel in sensitivity and specificity but require a laboratory setting and typically take up to six hours to obtain the results whereas RAT has a low sensitivity (typically at least 3000 TCID50/ml) although with the results with 15 mins. We have developed a robust micro-electro-mechanical system (MEMS) based impedance biosensor fit for rapid and accurate detection of SARS-COV-2 of clinical samples in the field with minimal training. The biosensor consisted of three regions that enabled concentrating, trapping, and sensing the virus present in low quantities with high selectivity and sensitivity in 40 minutes using an electrode coated with a specific SARS-COV-2 antibody cross-linker mixture. Changes in the impedance value due to the binding of the SARS-COV-2 antigen to the antibody will indicate positive or negative result. The testing results showed that the biosensor's limit of detection (LoD) for detection of inactivated SARS-COV-2 antigen in phosphate buffer saline (PBS) was as low as 50 TCID50/ml. The biosensor specificity was confirmed using the influenza virus while the selectivity was confirmed using influenza polyclonal sera. Overall, the results showed that the biosensor is able to detect SARS-COV-2 in clinical samples (swabs) in 40 min with a sensitivity of 26 TCID50/ml.

Evolution of the newest diagnostic methods for COVID-19: a Chinese perspective. / ä¸­å›½è§†è§’ä¸‹çš„æ–°å† è‚ºç‚Žæœ€æ–°è¯Šæ–­æ–¹æ³•æ¼”è¿›.

Coronavirus disease 2019 (COVID-19) has continued to spread globally since late 2019, representing a formidable challenge to the world's healthcare systems, wreaking havoc, and spreading rapidly through human contact. With fever, fatigue, and a persistent dry cough being the hallmark symptoms, this disease threatened to destabilize the delicate balance of our global community. Rapid and accurate diagnosis of COVID-19 is a prerequisite for understanding the number of confirmed cases in the world or a region, and an important factor in epidemic assessment and the development of control measures. It also plays a crucial role in ensuring that patients receive the appropriate medical treatment, leading to optimal patient care. Reverse transcription-polymerase chain reaction (RT-PCR) technology is currently the most mature method for detecting viral nucleic acids, but it has many drawbacks. Meanwhile, a variety of COVID-19 detection methods, including molecular biological diagnostic, immunodiagnostic, imaging, and artificial intelligence methods have been developed and applied in clinical practice to meet diverse scenarios and needs. These methods can help clinicians diagnose and treat COVID-19 patients. This review describes the variety of such methods used in China, providing an important reference in the field of the clinical diagnosis of COVID-19.

Low-Cost, Real-Time Polymerase Chain Reaction System with Integrated RNA Extraction.

Rapid, easy-to-use, and low-cost systems for biological sample testing are important for point-of-care diagnostics and various other health applications. The recent pandemic of Coronavirus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. In general, sensitive testing methods require genetic material extraction from the specimen. Unfortunately, current commercially available extraction kits are expensive and involve time-consuming and laborious extraction procedures. To overcome the difficulties associated with common extraction methods, we propose a simple enzymatic assay for the nucleic acid extraction step using heat mediation to improve the polymerase chain reaction (PCR) reaction sensitivity. Our protocol was tested on Human Coronavirus 229E (HCoV-229E) as an example, which comes from the large coronaviridae family of viruses that affect birds, amphibians, and mammals, of which SARS-CoV-2 is a member. The proposed assay was performed using a low-cost, custom-made, real-time PCR system that incorporates thermal cycling and fluorescence detection. It had fully customizable reaction settings to allow versatile biological sample testing for various applications, including point-of-care medical diagnosis, food and water quality testing, and emergency health situations. Our results show that heat-mediated RNA extraction is a viable extraction method when compared to commercial extraction kits. Further, our study showed that extraction has a direct impact on purified laboratory samples of HCoV-229E, but no direct impact on infected human cells. This is clinically relevant, as it allows us to circumvent the extraction step on clinical samples when using PCR.

Tweets and PCR Test-based Analysis and Prediction of Social Response to a Future Pandemic. A Case Study

The COVID-19 pandemic has greatly affected our society badly. It has been a subject of discussion since 2019 due to the increased prevalence of social media and its extensive use, and it has been a source of tension, fear, and disappointment for people all over the world. In this research, we took data from COVID-19 tweets from 10 different regions from July 25, 2020, to August 29, 2020. Using the well-known word embedding technique count-vectorizer, we experimented with different machine learning classifiers on data to train deep neural networks to improve the accuracy of predicted opinions with a low elapsed time. In addition, we collected PCR results from these regions for the same time interval. We compared the opinions in the form of positive or negative responses with the results of the PCR tests per million people. With the help of the results, We figured out a real-time international measure to detect these regions' behaviors for any future pandemic. If we know how a region thinks about an upcoming pandemic, then we can predict the region's real-time behavior for the particular pandemic. This would happen if we had past case studies to compare, like in our proposed research. Copyrights © 2023 The Institute of Electronics and Information Engineers.

Designing a sustainable-resilient-responsive supply chain network considering uncertainty in the COVID-19 era.

Effective supply chain management is crucial for economic growth, and sustainability is becoming a key consideration for large companies. COVID-19 has presented significant challenges to supply chains, making PCR testing a vital product during the pandemic. It detects the presence of the virus if you are infected at the time and detects fragments of the virus even after you are no longer infected. This paper proposes a multi-objective mathematical linear model to optimize a sustainable, resilient, and responsive supply chain for PCR diagnostic tests. The model aims to minimize costs, negative societal impact caused by shortages, and environmental impact, using a scenario-based approach with stochastic programming. The model is validated by investigating a real-life case study in one of Iran's high-risk supply chain areas. The proposed model is solved using the revised multi-choice goal programming method. Lastly, sensitivity analyses based on effective parameters are conducted to analyze the behavior of the developed Mixed-Integer Linear Programming. According to the results, not only is the model capable of balancing three objective functions, but it is also capable of providing resilient and responsive networks. To enhance the design of the supply chain network, this paper has considered various COVID-19 variants and their infectious rates, in contrast to prior studies that did not consider the variations in demand and societal impact exhibited by different virus variants.

Emerging technologies for COVID-19, diagnosis, prevention, and management

Virus-related respiratory epidemics have been the most common cause of infectious disease worldwide for the past century. SARS-CoV-2 has been a threat to humanity all across the world from the beginning of 2020. The COVID-19 pandemic can only be controlled by identifying and isolating the new cases, thereby interrupting community transmission. Policymakers, health care providers, and interdisciplinary scientists must collaborate to ensure pandemic preparedness utilizing cutting-edge technology. Nonetheless, the development of novel nanotherapeutic and vaccination techniques is critical. To address these issues, further research about the SARS-CoV-2 structure as well as the precise immunological response it elicits in the human body is needed. © 2023 Elsevier Inc. All rights reserved.

Detection and Diagnosis Technologies for SARS-CoV-2

Detection and diagnosis platforms play key roles in early warning, outbreak control and exit strategy for any pandemic, and they are especially pertinent for the Coronavirus disease 2019 (COVID-19) pandemic. The challenges posed by the speed and extent of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) spread around the globe also offered unprecedented opportunities for the development and deployment of novel strategies and products - not only vaccines and therapeutics, but also diagnostics. This chapter provides a brief summary of the vast array of molecular, serological, cell-based and other diagnostic tools for the specific detection of SARS-CoV-2 infections and immune responses. The focus is on the principles and applications of each platform, while detailed protocols can be found in the cited references. © 2023 by World Scientific Publishing Co. Pte. Ltd.

Ultrafast Nucleic Acid Detection Equipment with Silicon-Based Microfluidic Chip.

Recently, infectious diseases, such as COVID-19, monkeypox, and Ebola, are plaguing human beings. Rapid and accurate diagnosis methods are required to preclude the spread of diseases. In this paper, an ultrafast polymerase chain reaction (PCR) equipment is designed to detect virus. The equipment consists of a silicon-based PCR chip, a thermocycling module, an optical detection module, and a control module. Silicon-based chip, with its thermal and fluid design, is used to improve detection efficiency. A thermoelectric cooler (TEC), together with a computer-controlled proportional-integral-derivative (PID) controller, is applied to accelerate the thermal cycle. A maximum of four samples can be tested simultaneously on the chip. Two kinds of fluorescent molecules can be detected by optical detection module. The equipment can detect viruses with 40 PCR amplification cycles in 5 min. The equipment is portable, easily operated, and low equipment cost, which shows great potential in epidemic prevention.

Single nucleotide polymorphism in Angiotensin-converting enzyme-2 gene as a risk for COVID-19 among Iraqi people

Angiotensin-converting enzyme-2 (ACE-2) is an essential element in the renin-angiotensin RAS system and plays a key role in coronavirus entrance to the human body and attachment to the cell. Variation in ACE-2 may increase the capability of coronavirus to binding with human tissues and lead to variation in disease severity among patients. For this reason, this study aimed to take some SNPs in different COVID-19 patient cases to show the possible role of Angiotensin-Converting Enzyme-2 (ACE2) polymorphism in people that could have severe infections caused by SARS-CoV-2 by detecting different single nucleotide polymorphisms SNPs on this gene by PCR-sequencing. This cross-sectional study includes 100 diagnosed COVID-19 patients during a period between November and December 2021. The result revealed that three SNPs rs1463669655, rs746202722, and rs201035388 located on ACE-2 (GRCh38.p13) gene did not associate with the severity of COVID-19 disease because all patients have either the wildtype or the heteterotype allele as the following 0 % GG, 100 % AG, and 0% AA as the genotype distribution in severe cases, and the genotype distribution in non-severe cases was 0 % GG, 100 %AG, and 0 % AA and 100 % AA, 0 % AG, and 0% GG as the genotype distribution in severe cases, and the genotype distribution in non-severe cases was 100 % AA, 0 %AG, and 0 % GG and 100 % GG, 0 % GA, and 0% AA as the genotype distribution in severe cases, and the genotype distribution in non-severe cases was 100 % GG, 0 %GA, and 0 % AA for these three SNPs respectively. The ACE-2 angiotensin-converting enzyme gene was studied because of its active participation in the entry of the Coronavirus into the human body and its binding to alveolar cells and concluded that three SNPs rs1463669655, rs746202722, and rs201035388 located on ACE-2(GRCh38.p13) gene did not associate with the severity of COVID-19 disease. © Author (s). Publishing rights @ ANSF.

Isolation and Molecular Diagnosis of Aspergillus Species Associated with Corona Virus Disease and their Sensitivity to Some Antifungal in Patients of Wasit Governorate

Background: Respiratory infections caused by fungal pathogens present a growing global health concern and are a major cause of death in immunocompromised patients. Worryingly, corona virus disease-19 (COVID-19) resulting in acute respiratory distress syndrome has been shown to predispose some patients to airborne fungal co-infections. Material and methods: One hundred sample include nasal;throat swab and sputum sample samples were collected from patients suffering from Corona virus disease underlying different disease. This study was recorded from October 2021 to the end of April 2022, including the criteria, patients of all age groups in isolation wards in Al-Zahra Teaching Hospital in wait Governorate. Samples were cultured in sabered agar and incubated for 10 days at 25°C under aerobic conditions. The identification of molds was done depended on the shape and color of the fungus on the plate and examined under the microscope and kept for further conformation and detection of the most common virulence factor by polymerase chain reaction (PCR), as well as to measure the sensitivity of these isolates against some antifungal drugs. Results: The total positive samples for molds isolation 17(17%) out of 100 samples were collected from patients suffering from Corona virus. The most isolated species was Aspergillus fumigatus with 9/40 (22.5%), followed by Afflatus was 5/40 (12.5%) and 3/40(7.5%) was Atreus. The results in this study show Gliotoxin gene was detected 8/9 (88.8%) in Fumigates, with PCR product of this gene was approximately 512 bp. While investigate aflatoxin (far) gene was found 5/5 (%100) in Afflatus, with PCR product of this gene was approximately 588bp. And lipase gene was detected by PCR in 2/3 (66.6) samples isolation of A. terries. The PCR amplification of lipase gene was approximately 591 bp. The antifungal drugs Clotrimazole and Econazole had most effect against each fungal spp. in this study. While antifungal drug Miconazole resist by Aspergillus spp., while Nystatin and Ketoconazole were resisted by Aspergillus spy only. The conclusion of this study was showed Aspergillus fumigatus were the most dominant inpatientswithpatients with Coronavirus diseases and Clotrimazole, Ketoconazole were the most antifungal drugs that have a wide effect on fungal infections. © 2022, ResearchTrentz Academy Publishing Education Services. All rights reserved.

Direct SARS-CoV-2 Detection System Utilizing Simple-to-Use Capillary Gel Electrophoresis Sample-to-Result.

We present a Direct SARS-CoV-2 Detection System that achieves sample-to-results in less than two hours in three simple steps. The Detection System includes Direct one-step Reverse Transcription PCR (RT-PCR) reagents (Qexp-MDx kit), a portable thermal cycler (Qamp-mini) with a pre-programmed chip and a simple-to-use Capillary Gel Electrophoresis system (Qsep Series Bio-Fragment Analyzer) with high fluorescence detection sensitivity to solve the problems associated with traditional real-time PCR (qPCR) systems which produces inaccurate test results with high false negative and false positive rates. The proposed simple-to-use detection platform can provide high detection sensitivity (identify less than 20 copies), fast results (less than 120 minutes) and cost-effective results which should be suitable for decentralized testing application of COVID-19.

Liver resection in a patient with persistent positive PCR test for coronavirus disease 2019 (COVID-19): a case report.

BACKGROUND: The perioperative mortality rate is high in patients with coronavirus disease 2019 (COVID-19), and infection control measures for medical care providers must be considered. Therefore, the timing for surgery in patients recovering from COVID-19 is difficult. CASE PRESENTATION: A 65-year-old man was admitted to a hospital with a diagnosis of moderate COVID-19. He was transferred to our hospital because of risk factors, including heavy smoking history, type 2 diabetes mellitus, and obesity (BMI 34). Vital signs on admission were a temperature of 36.1 °C, oxygen saturation > 95% at rest, and 94% on exertion with 3 L/min of oxygen. Chest computed tomography (CT) showed bilateral ground-glass opacities, predominantly in the lower lungs. Contrast-enhanced abdominal CT incidentally revealed a liver tumor with a diameter of 80 mm adjacent to the middle hepatic vein, which was diagnosed as hepatocellular carcinoma (HCC). After being administered baricitinib, remdesivir, dexamethasone, and heparin, the patient's COVID-19 pneumonia improved, his oxygen demand resolved, and he was discharged on day 13. Furthermore, the patient was initially scheduled for hepatectomy 8 weeks after the onset of COVID-19 following a discussion with the infection control team. However, 8 weeks after the onset of illness, a polymerase chain reaction (PCR) test was performed on nasopharyngeal swab fluid, which was observed to be positive. The positive results persisted till 10 and 11 weeks after onset. Both Ct values were high (≥ 31) out of 45 cycles, with no subjective symptoms. Since we determined that he was no longer contagious, surgery was performed 12 weeks after the onset of COVID-19. Notably, medical staff wearing personal protective equipment performed extended anatomical resection of the liver segment 8 ventral area in a negative-pressure room. The patient had a good postoperative course, with no major complications, including respiratory complications, and was discharged on postoperative day 14. Finally, none of the staff members was infected with COVID-19. CONCLUSIONS: We reported a case regarding the timing of surgery on a patient with persistently positive PCR test results after COVID-19, along with a literature review.

A review of thermal impact of surface acoustic waves on microlitre droplets in medical applications

The surface acoustic waves (SAW) propagate inside the microdroplets resulting in kinetic and thermal impacts. The kinetic drives fluid particles inside the droplet while thermal impact increases the liquid’s temperature. This paper provides a comprehensive review of the research investigations related to internal kinetics and heating inside the microdroplet caused by the acoustic waves. The main factors that affect the kinetics and convection heat transfer are the piezoelectric materials, shape of the interdigital transducer (IDT) and mode of acoustic waves. Internal streaming (kinetic) leads to particle mixing, particle manipulation, cell sorting, cell patterning, cell separation, measuring the concentration of immunoglobulin and so forth. The effect of changing the mode of waves and the shape of IDT on the relevant applications are presented. Internal convection heat transfer is important where heating of the liquid is essential for many applications such as monitoring blood coagulation in the human plasma and an acoustic tweezer for particle trapping. Experimental methods developed by researchers to realise uniform temperature with constant heating and cooling cycles are also discussed. Such methods are widely used in the polymerase chain reaction (PCR) to detect COVID-19 infection. The heating of the droplet can be efficiently controlled by changing the input power and by varying the duty factor. © The Author(s) 2022.

Guidance for SARS-CoV-2 RNA-Based Molecular Assay Analytical Performance Evaluations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is frequently diagnosed through detection of viral RNA using nucleic acid amplification testing (NAAT) assays that are usually used in centralized settings. Following the publication of the SARS-CoV-2 genetic sequence, multiple diagnostic assays were launched in 2020. These assays require evaluation beyond manufacturer self-reported performance to determine whether they are suitable for use, meet country acceptance criteria, and are compatible with existing in-country platforms. In order to meet the demand for testing services, rapid yet robust assay performance evaluations are required. In our setting, these evaluation protocols required the use of residual patient specimens and reference materials, as typical clinical trials are time-consuming and limited by cost and the cyclical nature of SARS-CoV-2 infection. This protocol is designed to assist in the rapid and robust evaluation of nucleic acid-based assays for the detection of SARS-CoV-2 using limited specimens, reference materials, and test kits. While it is specific for RNA-based assays, it can be adapted for fully automated analyses. The preparation and processing of evaluation panels is described, followed by methods for analytical precision analysis and data visualization. Assay robustness and scalability are briefly discussed as these can be critical for implementation. This protocol is designed to be flexible and alternative options are provided throughout the text where possible.

Sensitivity and efficiency of RNA sample pooling for real-time quantitative polymerase chain reaction testing for SARS-CoV-2

Background: In spite of the worth of pool testing in public health, data on the sensitivity and efficiency of real-time quantitative polymerase chain reaction (RT-qPCR) pool testing for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in middle and low-income countries are limited. Methods: We mixed single specimens of extracted RNA positive for the SARS-CoV-2 envelope (E) gene by RT-qPCR with negative specimens, in pools of 4 (n=89), 8 (n=92), 16 (n=102), and 32 (n=105) specimens each. We estimated the average change in cycle threshold (Ct) for each pool size and added it to the Ct values of the first 1,350 tests in our lab, to obtain dilution-corrected Ct values. We estimated pool sensitivity as the proportion of samples with dilution-corrected Ct >40, and used it in simulations of the efficiency (tests used/true case detected) of binary split pool testing. Results: We tested 388 pools. Average Ct changes were 2.21, 2.51, 3.27, and 3.94 cycles, for pools of 4, 8, 16, and 32 specimens, respectively. Corresponding pool tests sensitivities were 91.1%, 89.6%, 85.8% and 82.5%. Pool testing was substantially more efficient than individual testing. For prevalence of 0.5% to 2.0%, the efficiency of pools of ≥8 specimens was 30% to 280% higher, and the number of people tested was 4.4 to 13.9 times higher than those of individual testing. Conclusions: Binary split pool testing substantially increases the number of people tested and the number of true cases detected per test used. This strategy is key to curtail the transmission of SARS-CoV-2, by increasing efficiency in the identification and isolation of symptomatic and asymptomatic infected individuals. © 2022 Journal of Innovation Management. All rights reserved.

Rapid SARS-CoV-2 Virus Enrichment and RNA Extraction for Efficient Diagnostic Screening of Pooled Nasopharyngeal or Saliva Samples for Dilutions Up to 1:100.

As COVID-19 transmission control measures are gradually being lifted, a sensitive and rapid diagnostic method for large-scale screening could prove essential for monitoring population infection rates. However, many rapid workflows for SARS-CoV-2 detection and diagnosis are not amenable to the analysis of large-volume samples. Previously, our group demonstrated a technique for SARS-CoV-2 nanoparticle-facilitated enrichment and enzymatic lysis from clinical samples in under 10 min. Here, this sample preparation strategy was applied to pooled samples originating from nasopharyngeal (NP) swabs eluted in viral transport medium (VTM) and saliva samples diluted up to 1:100. This preparation method was coupled with conventional RT-PCR on gold-standard instrumentation for proof-of-concept. Additionally, real-time PCR analysis was conducted using an in-house, ultra-rapid real-time microfluidic instrument paired with an experimentally optimized rapid protocol. Following pooling and extraction from clinical samples, average cycle threshold (CT) values from resultant eluates generally increased as the pooling dilution factor increased; further, results from a double-blind study demonstrated 100% concordance with clinical values. In addition, preliminary data obtained from amplification of eluates prepared by this technique and analyzed using our portable, ultra-rapid real-time microfluidic PCR amplification instrument showed progress toward a streamlined method for rapid SARS-CoV-2 analysis from pooled samples.

Epidemiology of SARS--CoV--2 with Implications of Reinfection Update After 01 Year of Ongoing Pandemic:: Cross Sectional Study From Tertiary Care Hospital From Southern Region of Pakistan

Objective: To share the epidemiological, clinical and laboratory -based evidence of severe acute respiratory syndrome Corona Virus-2 with focus on the cases of re-infection;an update after one year of the ongoing pandemic. Study Design: Prospective observational study. Place and Duration of Study: Department of Pathology, in collaboration with Department of Medicine, Combined Military Hospital, Malir, from Mar 2020 to Feb 2021. Methodology: Total 5190 nasopharyngeal swabs were collected and transported to the laboratory in viral transport media for severe acute respiratory syndrome Corona Virus-2, from all symptomatic patients with a history of exposure/traveling from endemic areas and those requiring admission in hospital and were screened for COVID-19 as per hospital standing protocols. Results: 561(10.8%) patients were PCR positive for severe acute respiratory syndrome Corona Virus-2. The mean age of patients was 39.45±31.9 years and a majority of patients were males 426 (76%). The most common symptoms were fever and dry cough followed by myalgia and shortness of breath. 37 (9%) patients died due to the severity of the illness. Total 6 (1.46%) cases of laboratory-confirmed reinfection of severe acute respiratory syndrome Corona Virus-2 were reported. 2(33%) cases of reinfection were observed in health care workers, mortality was seen in a single patient associated with old age and comorbidities. Conclusion: In our study, the severity of the disease was directly related to the age of patients and underlying comorbidities. Reinfection was associated with increased viral load and exposure to the infected environment. © 2022, Army Medical College. All rights reserved.

Recent advances in nucleic acid analysis and detection with microfluidic and nanofluidics

Microfluidic- and nanofluidics-based nucleic acid sensing and analysis have become of interest to the public, especially during the current COVID pandemic. In this chapter, we provide a comprehensive review of recent research dedicated to the advances of nucleic acid analysis and detection including various polymerase chain reaction platforms, isothermal target amplification methods, and emerging amplification-free methods, such as optofluidics sensing, electrochemical sensing, thermal sensing, and advanced microscopy for label-free DNA/RNA analysis. The future advancement and prospects of nucleic acid analysis are also discussed. © 2022 Elsevier B.V. All rights reserved.

Screening for Viral Nucleic Acids in the Cerebrospinal Fluid of Dogs With Central Nervous System Inflammation.

Central nervous system (CNS) inflammation is a common cause of neurological dysfunction in dogs. Most dogs with CNS inflammation are diagnosed with presumptive autoimmune disease. A smaller number are diagnosed with an infectious etiology. Additionally, at necropsy, a subset of dogs with CNS inflammation do not fit previously described patterns of autoimmune disease and an infectious cause is not readily identifiable. Because viral infection is a common cause of meningoencephalitis in people, we hypothesize that a subset of dogs presented with CNS inflammation have an occult viral infection either as a direct cause of CNS inflammation or a trigger for autoimmunity. The goal of this research was to screen cerebrospinal fluid from a large number dogs with CNS inflammation for occult viral infection. One hundred seventy-two dogs with neurological dysfunction and cerebrospinal fluid (CSF) pleocytosis were identified. Of these, 42 had meningoencephalitis of unknown origin, six had steroid-responsive meningitis-arteritis, one had eosinophilic meningoencephalitis, five had documented infection, 21 had and undetermined diagnosis, and 97 had a diagnosis not consistent with primary inflammatory disease of the CNS (e.g., neoplasia). CSF samples were subsequently screened with broadly reactive PCR for eight viral groups: adenovirus, bunyavirus, coronavirus, enterovirus, flavivirus, herpesvirus, paramyxovirus, and parechovirus. No viral nucleic acids were detected from 168 cases screened for eight viral groups, which does not support occult viral infection as a cause of CNS inflammation in dogs. La Crosse virus (LACV) nucleic acids were detected from four cases in Georgia. Subclinical infection was supported in two of these cases but LACV could not be ruled-out as a cause of infection in the other two cases, suggesting further research is warranted to determine if LACV is an occult cause of CNS inflammation in dogs.

Assessment of Cost-Efficient Thermocycler Prototype for Polymerase Chain Reaction and Loop-Mediated Isothermal Amplification

DNA amplification-based diagnostic is the most accurate method among others, especially during the COVID-19 pandemic. Thus, increasing the global demand for instrumentation andamplification reagents locally, hence increasing import. It is a worrying state in terms of logisticsand the future domestic market. An effort for domestic production is a must. Previously a costefficientthermocycler prototype using Raspberry Pi and Phyton coding is constructed.Thermocycler prototype flow measurement and heat distribution have previously been tested. Thisresearch aims to compare thermocycler prototypes and commercial for in two types of DNAamplification reactions, polymerase chain reaction (PCR) and loop-mediated isothermalamplification (LAMP). PCR is the most and more common method than LAMP, with the maindifference of PCR require thermal cycling and LAMP operate in isothermal conditions. LAMP has aquicker reaction time and operates at a lower temperature. DNA pol with high strand displacementactivity is used for LAMP, in this research Bsm pol is used for LAMP and Taq pol for PCR. Since theprototype thermocycler is designed to be as simple and inexpensive as possible for ease ofmanufacture and accessibility for every layer of society. Hence, its heat control and stability are notas good as a commercial thermocycler, with huge temperature fluctuation resonance from its setpoint.That causes prototype incapability of performing PCR, no DNA band at 250-500 bp range ingel electrophoresis. However, the prototype is capable of performing LAMP, existing <100 bp DNAgradient band in gel electrophoresis. The prototype is also capable of performing LAMP below itsprotocol temperature and time separately, 62°C and 40 minutes compared to the protocol of 66°Cand 60 minutes © 2021,International Journal of Technology. All Rights Reserved.