Evaluation of Three Automated Extraction Systems for the Detection of SARS-CoV-2 from Clinical Respiratory Specimens

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly contagious and causes coronavirus disease 2019 (COVID-19). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is the most accurate and reliable molecular assay to detect active SARS-CoV-2 infection. However, a rapid increase in test subjects has created a global bottleneck in testing capacity. Given that efficient nucleic acid extraction greatly affects reliable and accurate testing results, we compared three extraction platforms: MagNA Pure 96 DNA and Viral NA Small Volume kit on MagNA Pure 96 (Roche, Basel, Switzerland), careGENETM Viral/Pathogen HiFi Nucleic Acid Isolation kit (WELLS BIO Inc., Seoul, Korea) on KingFisher Flex (Thermo Fisher Scientific, Rocklin, CA, USA), and SGRespiTM Pure kit (Seegene Inc., Seoul, Korea) on Maelstrom 9600 (Taiwan Advanced Nanotech Inc., Taoyuan, Taiwan). RNA was extracted from 245 residual respiratory specimens from the different types of samples (i.e., NPS, sputum, and saliva) using three different kits. The 95% limits of detection of median tissue culture infectious dose per milliliter (TCID50/mL) for the MagNA Pure 96, KingFisher Flex, and Maelstrom 9600 were 0.37–3.15 × 101, 0.41–3.62 × 101, and 0.33–1.98 × 101, respectively. The KingFisher Flex platform exhibited 99.2% sensitivity and 100% specificity, whereas Maelstrom 9600 exhibited 98.3–100% sensitivity and 100% specificity. Bland–Altman analysis revealed a 95.2% concordance between MagNA Pure 96 and KingFisher Flex and 95.4% concordance between MagNA Pure 96 and Maelstrom 9600, indicating that all three platforms provided statistically reliable results. This suggests that two modifying platforms, KingFisher Flex and Maelstrom 9600, are accurate and scalable extraction platforms for large-scale SARS-CoV-2 clinical detection and could help the management of COVID-19 patients.

MAVRICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples v2

Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magneticnanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities.

A low-cost, portable, and practical LAMP device for point-of-diagnosis in the field

Transition of rapid, ready-to-use, and low-cost nucleic acid-based detection technologies from laboratories to points of sample collection has drastically accelerated. However, most of these approaches are still incapable of diagnosis starting from sampling, through nucleic acid isolation and detection in the field. Here, we developed a simple, portable, low-cost, colorimetric, and remotely controllable platform for reliable, high-throughput, and rapid diagnosis using loop mediated isothermal amplification (LAMP) assays. It consists of a thermally isolated cup, low-cost electronic components, a polydimethylsiloxane sample well, and a fast prototyped case that covers electronic components. The steady-state temperature error of the system is less than 1%. We performed LAMP, Colony-LAMP, and Colony PCR reactions using the yaiO2 primer set for Escherichia coli and Pseudomonas aeruginosa samples at 65˚C and 30 min. We detected the end-point colorimetric readouts by the naked eye under day light. We confirmed the specificity and sensitivity of our approach using pure genomic DNA and crude bacterial colonies. We benchmarked our Colony-LAMP detection against Colony PCR. The number of samples tested can easily be modified for higher throughput in our system. We strongly believe that our platform can greatly contribute rapid and reliable diagnosis in versatile operational environments.

Direct RT-PCR amplification of SARS-CoV-2 from clinical samples using a concentrated viral lysis-amplification buffer prepared with IGEPAL-630

The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide. Nucleic acid amplification serves as the gold standard method for confirmation of COVID-19 infection. However, challenges faced for diagnostic laboratories from undeveloped countries includes shortage of kits and supplies to purify viral RNA. Therefore, it is urgent to validate alternative nucleic acid isolation methods for SARS-CoV-2. Our results demonstrate that a concentrated viral lysis amplification buffer (vLAB) prepared with the nonionic detergent IGEPAL enables qualitative detection of SARS-CoV-2 by direct Reverse Transcriptase-Polymerase Chain Reaction (dRT-PCR). Furthermore, vLAB was effective in inactivating SARS-CoV-2. Since this method is inexpensive and no RNA purification equipment or additional cDNA synthesis is required, this dRT-PCR with vLAB should be considered as an alternative method for qualitative detection of SARS-CoV-2.

The use of molecular diagnostics of BLV in the study of various biological material

It is known that milk is one of the factors in the transmission of susceptible cattle to retroviral infection, in this case, induced bovine leukemia virus (BLV). On the other hand, one of the most important aspects of studying the biological properties of BLV is the potential ability of the virus to infect xenogenic species of animals, including humans, who consume animal products and are in close contact with cattle. Milk may contain leukocytes with integrated proviral DNA, which produce mature BLV virions and the mature virions themselves, which have come from the bloodstream. The use of molecular diagnostics of this biological material will make it possible to more effectively track contaminated dairy products on farms. However, the detection of BLV proviral DNA in milk is important not only for human health, but also for animals. Since some of the products are used directly on farms, for example for feeding young animals. In regard to this, we have undertaken optimization of the methodology for the study of milk for the presence of proviral DNA of BLV. The article presents the results of the analysis of milk from 5 seropositive cows of the Holstein breed by the method of molecular diagnostics real-time polymerase chain reaction (PCR-RT). When optimizing the method of sample preparation of milk, casein and other impurities that are inhibitors of the amplification reaction were removed, in order to reduce their influence, the samples were centrifuged under various conditions. Several nucleic acid isolation techniques were used to extract proviral DNA. Based on the results of the studies, a positive amplification result was recorded in 1 out of 5 animals. At the same time, this positive result was found in test tubes with complementary DNA (cDNA) samples, it means that RNA was isolated both from the "white ring" fraction with white blood cells and monocytes, and from the sediment cells.

The analysis of the correlations between HBsAg quantitation level, virological markers and histopathological findings in patients with chronic hepatitis B

In recent years, several studies suggested that HBsAg titers in blood samples obtained during Hepatitis B treatment could be used to estimate the treatment outcomes. The present study aims to discuss the correlation between HBsAg quantification levels and the virological, serological and histopathological findings in chronic hepatitis B patients.The study included chronic hepatitis B patients who underwent liver biopsy between 2011 and 2013 at Dicle University, Faculty of Medicine, Infectious Diseases and Clinical Microbiology Clinic.. The patient demographics were recorded (age, gender). Patient AST tests were conducted with the spectrophotometric method. After the DNAs were isolated with AmplipPrep Total Nucleic Acid Isolation Kit, DNA level was determined with the COBAS® Amplip / Cobas® Taqman® HBV test V2.0 for HBV. The patient HBV DNA levels were recorded as IU / ml. HBsAg quantitation was studied with the Access device and the Elisa method.The study was conducted with 53 patients. The mean patient age was 28,73 ± 8.15. Out of the 53 patients, 35 (66%) were male and 18 (34%) were female. The mean patient HBsAg quantitation was 631,42 ± 406.55, fibrosis score was 1,35 ± 0.87, ALT index score was 67,07 ± 53.37, and HAI index score was 4,54 ± 1.55. In the statistical analysis, it was determined that there were negative correlations between the HBsAg DNA level (R: -0,273, P: 0.048) and HBSAG quantitation (R: -0,273, P: 0.048), fibrosis score , ALT (R: -0,477, P: 0.001), and HAI index scores (R: -034, P: 0,013), while there was a positive correlation with the HBeAg positivity (R: 0.477, p: 0.001). There were negative correlations between the HBsAg quantitation level and virological (HBV DNA level), histopathological (fibrosis score, HAI index) findings and a positive correlation with serological (HBeAg positivity) findings. As HBsAg quantitation level increased, fibrosis score and HBV DNA level decreased.

High-Quality Nucleic Acid Isolation from Hard-to-Lyse Bacterial Strains Using PMAP-36, a Broad-Spectrum Antimicrobial Peptide

The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.

Metapneumovirus infection in children

Objective: to study the clinical features of metapneumovirus infection in children of different ages in a hospital. Materials and methods. A retrospective analysis of medical records of 142 patients aged 1 month to 14 years inclusive who were hospitalized in the period from January 2012 to April 2019. Metapneumovirus infection was confirmed by hMPV nucleic acid isolation by PCR in nasopharyngeal smears. Results. Metapneumovirus infection is detected among hospitalized children with acute respiratory viral infections in 4,4% of cases. In the age structure, 72,2% are children under 4 years old, and the maximum incidence rate is among children aged 3 years of life. The leading clinical symptoms are cough in 93,0% of cases and rhinitis in 96,5% of cases.In 88,2% of children, the disease proceeds with an increase in temperature 38 C, including in 34,6% 39,5 C and above. Symptoms of gastrointestinal dysfunction in the form of vomiting and diarrhea develop in 26,1% and 22,5% of children, respectively. 78,2% of patients requiring hospitalization suffer hMPV infection with damage to the lower respiratory tract, including in the form of bronchitis in 85,6% of cases and pneumonia in 14,4% of cases. The disease is complicated by the development of bronchial obstructive syndrome in 38,7% and acute respiratory failure in 22,3% of cases. ARF and BOS are significantly more likely to develop in children of the first 3 years of life 71,0% versus 29,0% in children of the older age group (p = 0.038) and 69,8% against 30,2% (p = 0.007), respectively. In a clinical blood test for hMPV infection, leukopenia and leukocytosis are detected only in 3,5% and 12,7% of cases, respectively, as well as an increase in ESR in 23,9% of children. The level of CRP in the 93,0% of children was less than 20 mg/l. Conclusions. Virological confirmation of metapneumovirus infection in hospitalized children with lower respiratory tract infections contributes to the formation of an adequate therapeutic tactic.