One-step Reverse Transcription Research Articles

Investigation of Variants In SARS-CoV-2 Infections after Three Doses of COVID-19 Vaccine

Abstract
 Objective: Our study focused on retrospectively assessing variant of concern, specified by the World Health Organization (WHO), with one-step reverse transcription and real-time polymerase chain reaction (RT-PCR) test in SARS-CoV-2 positive patients after three doses of attenuated COVID-19 vaccine.
 Methods: 8.520 samples transported with viral nucleic acid buffer (vNAT) tubes between June 2021 and January 31, 2022, were tested and included in the study. All the patients whose samples were included in our research had 3 doses of CoronaVac (Sinovac Life Science Co, Ltd, Beijing, China). Gender distribution was 4686 (55%) female and 3834 ( 45%) males. Variant specific genome regions only found in B.1.351, P.1 and B.1.1.7 as well as ORF1ab and N gene regions are investigated by the Bio-Speedy® Emerging Plus kit (Bioeksen AR-GE Technologies, Turkey) used to identify the variants in the study.
 Results: All 8.520 samples were SARS-CoV-2 RT-PCR positive. Our study detected alpha and delta variants in 1460 (17.14%) and 3570 (41.9%) patients respectively. 2570 (30.16%) patients did not have any variants according to test results. It was observed that the spread of beta, gamma and other suspicious variants remained at relatively low rates.
 Conclusion: The delta variant became dominant from July until to the end of the year. Declining delta variant rates and increasing cases of suspected variants towards the beginning of December 2021 suggest the omicron variant. Therefore, molecular surveillance studies that are planned to take epidemiological data into consideration and to examine the prevalence and gene-based analysis of local and worldwide variants are required.

A Newly Emerging Serotype A Strain in Foot-and-Mouth Disease Virus with Higher Severity and Mortality in Buffalo than in Cattle Calves in North Egypt.

A severe foot-and-mouth disease (FMD) epidemic struck several Egyptian provinces recently, causing significant losses among animals even in vaccinated farms. This study indicated the existence of the newly emerging foot-and-mouth disease virus (FMDV) and first investigated its effect on the Egyptian water buffalo (Bubalus bubalis) and cattle calves in the Beheira province, north Egypt. Twenty tongue epithelial samples from diseased calves in five infected farms were randomly collected, prepared, and propagated using baby hamster kidney-21 (BHK-21) cells. Whole genomic RNA was extracted from the cells of the third passage. A FMDV genome was detected and serotyped using one-step reverse transcription polymerase chain reactions (RT-PCRs). Nucleotide sequencing of the purified serotype-specific PCR bands was performed, and a maximum likelihood phylogenetic tree based on 600 base pairs of VP1 was constructed. The results identified FMDV, serotype A in all infected samples, whereas the serotypes O and SAT2 were negative. The obtained 20 sequences were identical to each other and similar to the newly reported strain in Egypt that belongs to the Europe-South America (Euro-SA) topotype. The epidemiological and clinical parameters associated with such a strain were fully recorded by veterinarians and analyzed in a single infected farm including 70 cattle and buffalo calves. It caused higher peracute mortalities in buffalo (25.7%; 95% CI: 13-43) than in cattle (8.6%; 95% CI: 2-24) calves. Severe clinical signs such as dullness, hypothermia, bradycardia, and cardiac arrhythmia were common to both except in fatal cases, whereas hyperthermia and respiratory signs were prevalent in cattle calves. In conclusion, we first characterized the newly emerging FMDV in the calves of Beheira as more fatal and severe in buffalo than in cattle calves.

Development of a One-Step Real-Time TaqMan Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay for the Detection of the Novel Variant Infectious Bursal Disease Virus (nVarIBDV) Circulating in China.

The novel variant IBDV (nVarIBDV, especially genotype A2dB1) mainly affects broilers in China. It causes an infection characterized by the atrophy of the bursa, a decrease in the level of lymphocytes, proliferation of fibrous tissue around the follicle, and severe atrophy of the follicle in the bursa. Poultry vaccinated with live IBDV vaccines do not have the challenge present with bursa atrophy, which is misdiagnosed for nVarIBDV because of the lack of other gross clinical symptoms. The present study sought to explore the potential and reliability of the real-time TaqMan analysis method for the detection and discrimination of the nVarIBDV genotype from that of the non-nVarIBDV, especially in live vaccine strains. This method will help monitor vaccinated poultry to control and manage infection with the nVarIBDV IBDVs. The nucleotide polymorphism in the 5'-UTR region and the vp5/vp2 overlapping region of the segment A sequences of IBDV were used to establish a one-step real-time TaqMan reverse transcription polymerase chain reaction (RT-PCR) method in this study. The results showed that the method accurately distinguished the nVarIBDV and non-nVarIBDV strains (especially live vaccine strains), and there were no cross-reactions with the infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), Mycoplasma gallisepticum (M. gallisepticum), Mycoplasma synoviae (M. synoviae), and IBDV-negative field samples. The method showed a linear dynamic range between 102 and 107 DNA copies/reaction, with an average R2 of 0.99 and an efficiency of 93% for nVarIBDV and an average R2 of 1.00 and an efficiency of 94% for non-nVarIBDV. The method was also used for the detection of 84 clinical bursae of chickens vaccinated with the live vaccine. The results showed that this method accurately distinguished the nVarIBDV and non-nVarIBDV strains (vaccine strains), compared with a strategy based on the sequence analysis of HVRs at the vp2 gene or the reverse transcription PCR (RT-PCR) for the vp5 gene. These findings showed that this one-step real-time TaqMan RT-PCR method provides a rapid, sensitive, specific, and simple approach for detection of infections caused by nVarIBDV and is a useful clinical diagnostic tool for identifying and distinguishing nVarIBDV from non-nVarIBDV, especially live vaccine strains.

Fully integrated sample-in-answer-out platform for viral detection using digital reverse transcription recombinase polymerase amplification (dRT-RPA)

Recombinase polymerase amplification (RPA) is one of the most promising diagnostic methods for pathogen detection, owing to the simplified isothermal amplification technique. Using one-step digital reverse transcription RPA (dRT-RPA) to detect viral RNA provides a fast diagnosis and absolute quantification. Here, we present a chip that purifies, digitalizes, and detects viral RNA of SARS-CoV-2 in a fully automated and sensitive manner. The chip purifies the RNA using the surface charge concept of magnet bead-RNA binding, then mixes the RNA with the amplification reagents, digitalizes the amplification mixture, and performs dRT-RPA. RNA-bead complex is transported among purification buffers that are separated by an oil phase. For reagent manipulation and mixing, a magnetic valve system is integrated on the chip, where an external magnet controls the reagent direction and time of addition. Besides, a novel vacuum system is suggested to drive and regulate the reagents into two fluid systems simultaneously in ∼2 min. We also developed a cost-effective way to perform fluorescent detection for dRT-RPA on chip by using EvaGreen® dye. With integrated heating and optical detection system, the on-chip dRT-RPA presents a sample-to-answer detection platform for absolute viral RNA quantitation in 37 min and a sensitivity as low as 10 RNA copies/μL. Hence, this platform is expected to be a useful tool for accurate and automated diagnosis of infectious diseases.

Development and clinical validation of a one-step pentaplex real-time reverse transcription PCR assay for detection of hepatitis virus B, C, E, Treponema pallidum, and a human housekeeping gene

BackgroundWith the safety of blood transfusion being a major public health concern, the development of a rapid, sensitive, specific, and cost-effective multiplex PCR assay for simultaneous detection of hepatitis B virus(HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), and Treponema pallidum(T. pallidum) in blood is crucial.MethodsFive primer pairs and probes were designed towards conserved regions of target genes and used to establish a one-step pentaplex real-time reverse transcription PCR(qRT-PCR) assay for simultaneous detection of HBV, HCV, HEV, T. pallidum, and RNase P(housekeeping gene), providing sample quality check. The clinical performance of the assay was further determined with 2400 blood samples from blood donors and patients in Zhejiang province, and compared the results with commercial singleplex qPCR and serological assays.ResultsThe 95% limit of detection(LOD) of HBV, HCV, HEV, and T. pallidum were 7.11 copies/µL, 7.65 copies/µL, 8.45 copies/µL, and 9.06 copies/µL, respectively. Moreover, the assay has good specificity and precision. Compared to the singleplex qPCR assay, the novel assay for detecting HBV, HCV, HEV, and T. pallidum presented 100% clinical sensitivity, specificity, and consistency. Several discrepant results between serological and pentaplex qRT-PCR assays were found. Of 2400 blood samples, there were 2(0.08%) HBsAg positive samples, 3(0.13%) anti-HCV positive samples, 29(1.21%) IgM anti-HEV positive samples and 6(0.25%) anti-T. pallidum positive samples proven negative in nucleic acid detection. 1(0.04%) HBV DNA positive sample and 1(0.04%) HEV RNA positive sample were detected negative by serological testing.ConclusionsThe developed pentaplex qRT-PCR is the first assay on simultaneous, sensitive, specific, and reproducible detection of HBV, HCV, HEV, T. pallidum, and RNase P in a single tube. It could detect pathogens in blood during the window period of infection and is a good tool for effectively screening blood donors and early clinical diagnosis.

Potential use of high-resolution melting analyses for SARS-CoV-2 genomic surveillance

The pandemic caused by COVID-19 and the emergence of new variants of SARS-CoV-2 have generated clinical and epidemiological impacts on a global scale. The use of strategies for monitoring viral circulation and identifying mutations in genomic regions involved in host interaction are important measures to mitigate viral dissemination and reduce its likely complications on population health. In this context, the objective of this work was to explore the potential of high-resolution melting (HRM) analysis combined with one-step real-time reverse transcription PCR in a closed-tube system, as a fast and convenient method of screening for SARS-CoV-2 mutations with possible implications on host-pathogen interactions. The HRM analyses allowed the distinction of the Gamma, Zeta, Alpha, Delta, and Omicron variants against the predecessors (B.1.1.28, B.1.1.33) of occurrence in Brazil. It is concluded that the molecular tool standardized here has the potential to optimize the genomic surveillance of SARS-CoV-2, and could be adapted for genomic surveillance of other pathogens, due to its ability to detect, prior to sequencing, samples suggestive of new variants, selecting them more assertively and earlier for whole genome sequencing when compared to random screening.

Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA.

Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID.

The First Nationwide Surveillance of Severe Fever with Thrombocytopenia Syndrome in Ruminants and Wildlife in Taiwan.

Since the first discovery of severe fever with thrombocytopenia syndrome virus (SFTSV) in China in 2009, SFTSV has rapidly spread through other Asian countries, including Japan, Korea, Vietnam and Pakistan, in chronological order. Taiwan reported its first discovery of SFTSV in sheep and humans in 2020. However, the prevalence of SFTSV in domestic and wildlife animals and the geographic distribution of the virus within the island remain unknown. A total of 1324 animal samples, including 803 domestic ruminants, 521 wildlife animals and 47 tick pools, were collected from March 2021 to December 2022 from 12 counties and one terrestrial island. The viral RNA was detected by a one-step real-time reverse transcription polymerase chain reaction (RT-PCR). Overall, 29.9% (240/803) of ruminants showed positive SFTSV RNA. Sheep had the highest viral RNA prevalence of 60% (30/50), followed by beef cattle at 28.4% (44/155), goats at 28.3% (47/166), and dairy cows at 27.5% (119/432). The bovine as a total of dairy cow and beef cattle was 27.8% (163/587). The viral RNA prevalence in ticks (predominantly Rhipicephalus microplus) was similar to those of ruminants at 27.7% (13/47), but wild animals exhibited a much lower prevalence at 1.3% (7/521). Geographically the distribution of positivity was quite even, being 33%, 29.1%, 27.5% and 37.5% for northern, central, southern and eastern Taiwan, respectively. Statistically, the positive rate of beef cattle in the central region (55.6%) and dairy cattle in the eastern region (40.6%) were significantly higher than the other regions; and the prevalence in Autumn (September-November) was significantly higher than in the other seasons (p < 0.001). The nationwide study herein revealed for the first time the wide distribution and high prevalence of SFTSV in both domestic animals and ticks in Taiwan. Considering the high mortality rate in humans, surveillance of other animal species, particularly those in close contact with humans, and instigation of protective measures for farmers, veterinarians, and especially older populations visiting or living near farms or rural areas should be prioritized.

Malachite Green-Based Detection of SARS-CoV-2 by One-Step Reverse Transcription Loop-Mediated Isothermal Amplification

The pandemic of severe acute respiratory syndrome 2 (SARS-CoV-2) revealed the necessity of diagnosis of the infected people to prevent the prevalence infection cycle. Many commercial pathogen diagnosis methods are based on the detection of genomic materials. Isothermal amplification methods such as loop-mediated-isothermal amplification (LAMP) are the method of choice in these cases. Reverse transcription steps are efficiently coupled to LAMP for the detection of pathogens with genomic RNAs such as SARS-CoV-2. Many detection systems for LAMP include fluorescent readout systems. Although such systems result in desirable limits of detection, the need for special instrumentation is the main dispute of such systems to become real point of care assays. In contrast, colorimetric detection methods would reduce costs and improve the applicability of the system. In this study one-step reverse transcription-LAMP reaction was established that enables visual detection of the SARS-CoV-2 genome. Nasopharyngeal RNA samples were first validated by reverse transcription quantitative polymerase chain reaction and then subjected to RT-LAMP. To lower the cost associated with the readout system equipment, malachite green (MG) was used. The color change of MG to blue allowed visual detection of the virus. Firstly, experiments were set up as two-step RT-LAMP reaction to identify the best primer sets. In addition, MG concentration was optimized with the significant colorimetric signal for the positive samples. Next, a one-step colorimetric method was developed for the detection of SARS-CoV-2 based on MG color shift in 2 h.

Development and Validation of Rapid Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification for Detection of Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) is a high-priority zoonotic pathogen with the ability to cause massive loss during its outbreak within a very short period of time. Lack of a highly sensitive, instant reading diagnostic method for RVFV, which is more suitable for on-site testing, is a big gap that needs to be addressed. The aim of this study was to develop a novel one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the rapid detection of RVFV. To achieve this, the selected RVFV M segment nucleotide sequences were aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE) software in MEGA11 version 11.0.11 program to identify conserved regions. A 211 pb sequence was identified and six different primers to amplify it were designed using NEB LAMP Primer design tool version 1.1.0. The specificity of the designed primers was tested using primer BLAST, and a primer set, specific to RVFV and able to form a loop, was selected. In this study, we developed a single-tube test based on calorimetric RT-LAMP that enabled the visual detection of RVFV within 30 minutes at 65°C. Diagnostic sensitivity and specificity of the newly developed kit were compared with RVFV qRT-PCR, using total RNA samples extracted from 118 blood samples. The colorimetric RT-LAMP assay had a sensitivity of 98.36% and a specificity of 96.49%. The developed RT-LAMP was found to be tenfold more sensitive compared to the RVFV qRT-PCR assay commonly used in the confirmatory diagnosis of RVFV.

Experimental Protocol for Detecting SARS-CoV-2 in Screenings and Grit Samples of Wastewater Treatment Plants

Introduction: Although various liquid, solid, and gaseous streams of wastewater treatment plants (WWTPs) have been analyzed in many studies for the presence of SARS-CoV-2 RNA, no study was found to sample and detect SARS-CoV-2 RNA in screenings and grit samples separated from primary treatment units of WWTP. Hence, this study aims to provide an experimental protocol for sampling and extracting SARS-CoV-2 RNA from screenings and grits separated from WWTPs. Materials and Methods: First, sampling was conducted to extract SARS-CoV-2 RNA from screenings and grit samples. After sample processing and viral RNA extraction, SARS-CoV-2 RNA detection was performed by one-step reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results: Based on the results, SARS-CoV-2 RNA was successfully extracted from screenings and grit samples of the studied WWTP with concentrations of (1.54 –3.9 × 104) and (0.8 – 2.3 × 104) genomic copies/L, respectively. Conclusion: Considering the successfully isolation and detection of SARS-CoV-2 viral RNA in solid phase samples of WWTP, this method can be applied for extracting SARS-CoV-2 RNA and maybe other viruses from the screenings and grit samples of WWTPs in related studies.

Genetic diversity of porcine reproductive and respiratory syndrome virus and evaluation of three one-step real-time RT-PCR assays in Korea

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV.MethodsIn this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays.ResultsA total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated.ConclusionThe results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.

Establishment of a one-step reverse transcription recombinase polymerase amplification assay for the detection of potato virus S

Potato virus S (PVS) is a noteworthy threat to the propagation of healthy seed potatoes. Accurate and speedy detection is critical for effective PVS management. In the present study, an isothermal-based one-step reverse transcription-recombinase polymerase amplification (RT-RPA) approach was developed to detect PVS infection in potato leaves and tubers. A primer set based on the coat protein gene successfully amplified a 158 bp product out of three primer sets examined. The amplification reaction took less than 30 min to complete with no account of cross-reactivity with major potato viruses. Additionally, amplification of RT-RPA products was performed on the heating system and/or water bath at 38–42 °C. The results of sensitivity analysis revealed that one-step RT-RPA has shown 100 times higher sensitivity than routine RT-PCR for the detection of PVS in infected leaves. Furthermore, ten times higher sensitivity of RT-RPA was observed in infected tubers. The methodology was simplified further by the use of template RNA extracted using a cellular disc paper-based extraction method that detected the PVS more effectively than purified total RNA. PVS was detected in 175 samples (leaves and tubers each) of several potato varieties using this innovative technique. To our acquaintance, this is the first report of one-step RT-RPA using a basic RNA extract derived through cellular disc paper that is significantly sensitive and precise for PVS detection in potatoes. The advantages of one-step RT-RPA in terms of proficiency, robustness, and the availability of a highly pure RNA template make it an attractive choice for seed accreditation, resistance breeding, and field inspections.

Detection of Cucumber green mottle mosaic virus in low-concentration virus-infected seeds by improved one-step pre-amplification RT-qPCR

BackgroundSeeds were an important medium for long-distance transmission of plant viruses. Therefore, appropriate, more sensitive methods for detecting low concentrations of virus-infected in seeds were crucial to ensure the quality of seed lots. In this study, we have developed a one-step pre-amplification reverse transcription quantitative PCR (RT-qPCR) assay based on the TaqMan technology to detect Cucumber green mottle mosaic virus (CGMMV) in zucchini seeds.ResultSeed powder samples with simulated CGMMV-infected at a low concentration were prepared (the mass ratio 1:900 and 1:1000), and their uniformity were verified using one-step pre-amplification RT-qPCR. We used one-step pre-amplification RT-qPCR to detect CGMMV in low-concentration virus-infected seeds and compared this method with universal RT-qPCR and double antibody sandwich–enzyme-linked immunosorbent (DAS–ELISA) assay, the main methods used for virus detection in seeds. The minimum limit of detection (LOD) of the improved one-step pre-amplification RT-qPCR assays for simulated CGMMV-infected seeds in large lots seeds samples were 0.1%.ConclusionsOne-step pre-amplification RT-qPCR assays could reliably and stably detected a single CGMMV-infected seed in 1000 seeds and demonstrated a higher detection sensitivity than universal RT-qPCR (infected seeds versus healthy seeds 1:900) and DAS–ELISA assay (infected seeds versus healthy seeds 1:500). Our improved one-step pre-amplification RT-qPCR assay have proved to be very suitable for the analysis of large seed lots.Graphical

COVID-19 pandemic and kidney disease in children: literature review and own regional observations

Introduction. Currently, the coronavirus infection pandemic caused by the SARS-CoV-2 virus continues around the world. Research data from domestic and foreign authors indicate that the kidneys are a target organ for a new infection, lesions vary from proteinuria and hematuria to acute kidney injury.Aim of the study – to determine the frequency and nature of kidney damage in children with confirmed coronavirus infection.Materials and methods. A retrospective and prospective analysis of cases of confirmed COVID-19 infection in children (n = 441) admitted to the Samara Regional Children’s Infectious Diseases Hospital from March 2020 to July 2021 was carried out. SARSCoV-2 RNA was detected in all patients by a one-step reverse transcription reaction combined with a polymerase chain reaction. The changes in the kidneys that occurred in 57 children were studied. The research results were processed using the Statistica 7.0 software (StatSoft, USA).Results. The involvement of the kidneys in the infectious process was detected in every 8 children with COVID-19 (12.9%), more often in the form of isolated urinary syndrome, the detection rate of which correlated with the severity of the course of coronavirus infection: in severe cases, proteinuria was detected in 31.6% of patients, hematuria – in 21%, acute kidney injury – in 10.5%, diabetic nephropathy – in 5.3%. Kidney damage was combined with damage to the respiratory and gastrointestinal tract, characterized by rapid recovery of urine output and azotemia parameters without special renal therapy. A clinical case of the onset of nephrotic syndrome that developed 2 weeks after suffering a coronavirus infection is described.Conclusions. Children with COVID-19 require kidney function monitoring for early detection and correction in case of impairment. Patients with isolated urinary syndrome in the acute period require long-term observation in order to detect latent renal pathology.

A novel technology for the one-step detection of prune dwarf virus: Colorimetric reverse transcription loop-mediated isothermal amplification assay

A one-step colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to target the coat protein region of genomic RNA of prune dwarf virus (PDV). WarmStart 2X Master Mix from NEB (New England Biolabs) was used for naked eye observation of positive LAMP reactions, where the changing in color from pink to yellowish indicates a positive result. The detection sensitivity of the new technique was 10-fold higher than that of conventional PCR. The assay's specificity was evaluated against the nucleic acids of potential hosts of PDV, prunus necrotic ringspot virus (PNRSV), and apple mosaic virus (ApMV) from the Ilarvirus genus. The novel RT-LAMP test showed high sensitivity in distinguishing PDV and genome of potential hosts and other Ilarvirus genus members. The RT-LAMP test was proven to be reliable for diagnosis of PDV in infected sweet cherry samples from the field. The specific, sensitive, and quick RT-LAMP test described in this study can be used in laboratories and for PDV surveillance in the field. To best of our knowledge, this is the first report of RT-LAMP detection for PDV.

Aedes aegypti abundance, larval indices and risk for dengue virus transmission in Kinondoni district, Tanzania

BackgroundTanzania has experienced periodic dengue outbreaks with increased incidence since 2010. However, there is limited information on vector dynamics and transmission risk in most parts of the country. This study was conducted to determine Aedes mosquito abundance, larval indices and dengue virus infection rate as risk indicators for DENV transmission in Kinondoni district, Dar es Salaam, Tanzania.MethodsA cross-sectional study was conducted in three wards of Kinondoni district in Tanzania between December 2019 and January 2020. In each ward, three streets were randomly selected for adult and immature mosquito sampling. The adult mosquitoes were collected using Mosquito Magnet traps, while mosquito larvae and pupae were inspected in water-holding containers in the selected household compounds. The detection of dengue virus (DENV) in female Aedes mosquitoes was done using a one-step reverse transcription–polymerase chain reaction (RT–PCR) method.ResultsOf the 1416 adult female mosquitoes collected, Ae. aegypti accounted for 16.8% (n = 238). A total of 333 water-holding containers were inspected and 201 (60.4%) had at least an Aedes larvae or pupae. Water-holding containers supporting the breeding of Aedes larvae and pupae included discarded car tires, flowerpots and small and large plastic containers. The overall House Index, Container Index and Breteau Index were 55.1%, 60.4% and 114.2, respectively. None of the 763 female Aedes mosquitoes tested by RT–PCR was found to be infected with DENV.ConclusionThe presence and abundance Ae. aegypti mosquitoes and the large proportion of water-holding containers infested with the mosquito larvae and pupae put residents of Kinondoni district at high risk of DENV transmission. Our findings emphasize the need for continuous mosquito vector surveillance and control to prevent the possibility of future DENV outbreaks in Tanzania.

A laboratory-based study of COVID-19 in Casablanca, Morocco.

Given the spread of coronavirus disease 2019 (COVID-19) and its impact on human health, laboratory confirmation of diagnosis is essential. This study examined the contribution of laboratory diagnosis to the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the diagnosis of COVID-19, taking into account patient risk of exposure to SARS-CoV-2, clinical symptoms and comorbidities. A cross-sectional, laboratory-based study was carried out from 1 April 2020 to 30 April 2020 at the National Reference Laboratory in Morocco using nasopharyngeal samples from patients admitted to the Cheikh Khalifa International University Hospital or other hospitals in Casablanca. A one-step reverse transcription real-time polymerase chain reaction (RT-PCR) was used to detect the presence of the SARS-CoV-2 genome. A national epidemiological investigation form was used to analyze patient exposure risk, clinical symptoms and comorbidities. A total of 793 samples from 375 patients were analyzed and 1150 RT-PCR tests were conducted; 116 patients (30.93%) were COVID-19 positive. Travel to a risk zone, contact with a confirmed COVID-19 case and contact with a person who had been in a risk zone were significantly associated with being positive for COVID-19. Fever and cough were the main symptoms; 7.76 % of positive patients were asymptomatic. This is the first laboratory-based study in Morocco for the diagnosis of COVID-19. Laboratory diagnosis of COVID-19 by RTPCR associated with knowledge of exposure risk factors and clinical symptoms and comorbidities remains essential for clinicians for early, appropriate medical management COVID-19 patients.

A one-step reverse transcription recombinase polymerase amplification assay for lateral flow-based visual detection of PVY

Potato virus Y (PVY) is an abundant and damaging virus which reduces crop yield and marketability. Accurate detection of this economically important virus both in-field and in seed potatoes is considered essential in the control of PVY spread. Current detection methods are focused on immunodetection and PCR-based methods, however, identification of PVY through isothermal amplification is a promising avenue for developing accessible, on-site diagnostics with quick turnaround times. In this work, a rapid recombinase polymerase amplification assay was developed which could readily amplify PVY nucleic acids with good sensitivity and specificity. Additionally, this assay was shown to be capable of amplification directly from RNA in a one-step amplification process, without the need for prior reverse transcription. The assay was coupled with lateral flow technology to provide a rapid visual confirmation of amplification. This nucleic-acid lateral flow immunoassay could feasibly be employed in-field, or at any location where testing is required, to aid in the detection and control of PVY.

Epidemiological characteristics and genetic diversity of norovirus infections among outpatient children with diarrhea under 5\xa0years of age in Beijing, China, 2011\u20132018

BackgroundHuman noroviruses are the leading cause of sporadic cases and outbreaks of viral acute gastroenteritis in all age groups worldwide.MethodsEpidemiological data and fecal specimens were collected between January 2011 and December 2018 from 4911 children < 5 years of age with diarrhea in three districts of Beijing. From 2011 to 2013, One-Step Reverse Transcription Polymerase Chain Reaction (RT-PCR) was used to detect noroviruses, and from January 2014 to December 2018, norovirus GI and GII were screened using duplex quantitative real-time RT-PCR (qRT-PCR). One-Step RT-PCR and RT-seminested PCR were performed to amplify the RNA-dependent polymerase and capsid genes of noroviruses in positive sample. Amplified products were sequenced directly; norovirus was typed using the online Norovirus Genotyping Tool v2.0 and phylogenetic analyses were conducted using MEGA-X.ResultsFrom 2011 to 2018, noroviruses were detected in 16.5% of specimens from children with diarrhea. The highest prevalence was observed in children aged 12 to 23 months (22.4%, 319/1421), followed by children aged 6 to 11 months (17.6%, 253/1441). The highest prevalence of norovirus infections occurred in autumn followed by winter, spring, and summer. From 2011 to 2018, the most prevalent dual types (genotype and polymerase type) were GII.4 Sydney[P31] (51.6%, 239/463), followed by GII.3[P12] (24.0%, 111/463), GII.4 2006b[P4 2006b] (7.3%, 34/463), GII.2[P16] (5.0%, 23/463), GII.17[P17] (2.6%, 12/463) and GII.6[P7] (2.6%, 12/463). GII.4 2006b[P4 2006b] predominated in 2011 and 2012. GII.4 Sydney[P31] predominated from 2013 to 2018. In total, 15 genotypes, 15 P-types and 19 dual types were detected in this study, reflecting the genetic diversity.ConclusionsThere were significant epidemiological characteristics and genetic diversity among outpatient children with norovirus infections < 5 years of age in Beijing from 2011 to 2018. These characteristics differ from those of norovirus outbreaks in Beijing. The complete genome sequences of each genotype are needed to better understand norovirus evolutionary mechanisms.