Viral Nucleic Acid Extraction Research Articles

High frequency of co-infection between SARS-CoV-2 and respiratory Adenoviruses in the Pediatric population in Hamadan, Iran.

The presence of other respiratory pathogens in patients with SARS-CoV-2 infection has been described as a striking feature. However, data on adenovirus co-infection rates and clinical impacts in COVID-19 patients is limited. The purpose of this study is to compare the prevalence of respiratory adenoviruses in children under 15 years of age in Positive and Negative SARS-CoV-2 patients. From September 2020 to January 2021, nasopharyngeal swabs were obtained from 280 patients below 15 years old with influenza-like infection symptoms suspected to be COVID-19 and referred to hospitals in Hamadan province. Nucleic acid was extracted using a High Pure Viral Nucleic acid extraction kit for both viral RNA and DNA. Reverse transcription real-time PCR for detecting SARS-CoV-2 and Real-time PCR for Human Adenoviruses were used. Out of 280 examined samples, 11.7% tested positive for AdV, of which 18 samples originated from the SARS-CoV-2 positive group and 15 samples were from the SARS-CoV-2 negative group. Of 18 co-infected samples, which were categorized in three different ranges of age including, 0-5, 6-10, and 11-15 years old were 11, 4, and 3 patients respectively. Also, 14 patients were hospitalized. Compared with AdV-positive patients, children with Co-infection with SARS CoV-2 had lower levels of white blood cell (WBC) count while erythrocyte sedimentation rate (ESR) and C-reaction protein (CRP) had increased levels. We report a substantial burden of AdV co-infection in pediatric COVID-19 patients. This study revealed most AdV infections lead to hospitalization and change in paraclinical parameters.

Cytomegalovirus Reactivation in Allogenic Haematopoietic Stem Cell Transplant Patients with Haploidentical Versus Fully Human Leukocyte Antigen Matched Donor

Objective: To determine CMV reactivation in hematopoietic stem cell transplant patients with haploidentical versus fully HLA matched donors Methodology: Retrospective Observational study was conducted at Armed Forces Bone Marrow Transplant Centre (AFBMTC) between March 2019 and October 2022. We enrolled a total of 60 consecutive patients.CMV DNA PCR analysis was carried out every week from day +14 to day +100 in order to monitor CMV reactivation during the 100-day post-transplantation period. The SaMag viral nucleic acid extraction kit (Sacace Biotechnologies, Como, Italy) was used to extract the DNA from two millilitres of blood collected in EDTA tubes. The kit's lower limit of detection for blood and serum was 5 copies/mL, while for other body fluids it was 500 copies/mL. An Applied Biosystems 7500 real-time PCR machine (Thermo Fisher Scientific, Waltham, MA) was used to conduct the PCR analysis. Result: The age range from 0.6 to 53 years ago was a median of 15±10.6. 39 patients had CMV reactivation (65%). With a range of 13 to 66 days, the median time to CMV DNAemia was 19.25 days.. Only two (3.3%) of the 39 patients received ganciclovir, while 26 (43.3%) received valganciclovir. Patients with aplastic anaemia had the highest rate of CMV infection (41%), followed by those with ALL (20.5%), AML (12.8%), and primary immunodeficiency (10.2%). A statistically significant connection between the underlying diagnosis and CMV infection. CMV infection was higher in haplo identical transplant recipients (66.6%) than in completely matched recipients (33.3%) and was statistically significantly. Sadly, 14 people passed away, yielding a mortality rate of 23.3%. Primary graft failure was the most frequent cause of mortality, resulting in neutropenic sepsis in 6 patients (42.85%), but 3 of these patients also had CMV reactivation. The rate of overall survival (OS) was found to be 76.7%. The rate of disease-free survival (DFS) was estimated at 71.7%. Notably, neither OS nor DFS in our analysis showed a statistically significant connection with CMV infection. Conclusion: The findings demonstrated a statistically significant association between haploidentical transplantation and CMV reactivation, with higher rates observed in the haploidentical group compared to fully matched recipients. Various factors, including underlying diagnosis, conditioning regimen, and GVHD prophylaxis, were also found to be correlated with CMV reactivation. However, while CMV reactivation was associated with specific factors, it did not impact overall survival or disease-free survival in the study population.

CRISPR/Cas12a-Assisted isothermal amplification for rapid and specific diagnosis of respiratory virus on an microfluidic platform

Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, H1N1, H3N2, IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.

Co-infection of respiratory pathogens in SARS-CoV-2 positive patients at North West India, Rajasthan

PurposeInfection due to SARS-CoV-2 shows wide spectrum of disease from asymptomatic to severe disease and death. Coinfection of SARS-CoV-2 with other respiratory pathogens may affect the severity of disease and its outcome. Identification of other respiratory pathogens may help to initiate proper management and avoid unnecessary complications. Materials and methodsTotal 250 SARS-COV-2 positive patients admitted in S.M.S hospitalwere included in study. Throat and nasopharyngeal swabs samples were collected in Viral Transport Medium (VTM) and nucleic acid extraction was done by automated EasyMag extractor and tested for 20 respiratory viruses and two bacteria by real time PCR. ResultsOut of 250 SARS CoV2 positive samples, 176 (70%) were positive for other respiratory pathogens also. The highest co-infection was due to HCoVOC43 (32.8%) virus followed by bacterial co-infection with S. pneumoniae (14.8%). Six (2.4%) patients with co-infection were on ventilator with age >65yr and three (1.2%) died during treatment. All three cases were found to have other co-morbid diseases like; asthma, Parkinson's and hypertension. ConclusionHigh number of patients were found to have coinfection with other viruses and bacteria, timely identification and providing specific treatment to these patients can help improve outcome.

Comparative Analysis of the Effect of Different Nucleic Acid Extraction Methods on SARS-CoV-2 Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction Results

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of Coronavirus diseases-2019 (COVID-19) disease. Rapid and accurate detection of the virus is vital to prevent transmission and effectively manage the pandemic. The gold standard diagnostic method for this agent is the real-time reverse transcription polymerase chain reaction (qrRT-PCR) test conducted on respiratory tract samples and one of the most critical steps affecting the sensitivity of this test is the nucleic acid extraction stage. However, restrictive factors such as reagent supply and storage conditions limit the testing capacity. Therefore, innovative and cost-effective alternatives are needed to speed up testing and minimize pre-processing steps. The aim of this study was to evaluate the impact and applicability of different methods to enhance the efficiency of the nucleic acid extraction stage in the SARS-CoV-2 qrRT-PCR test. As an alternative to the routinely used viral nucleic acid extraction buffer (vNAT), the modified vNAT method (MvNAT), which includes centrifugation, the R1-R2 kit and the heat treatment (HT) method, was applied to 118 respiratory tract samples. Samples determined with threshold cycle values of (Cq) of ≤ 35 (n= 10), > 35 (n= 42), indeterminate (n= 56) in routine results and negative controls (n= 10) were included in the study. The RNA quantities obtained after extraction for each method were measured and recorded using a spectrophotometric measurement device. All samples were processed using the SARS-CoV-2 qrRT-PCR kit targeting the RdRp region. The results were statistically analyzed using unpaired and paired t-tests and results with a p-value of < 0.05 were considered statistically significant. Excluding negative control samples, while the standard method yielded a Cq value of 48.1% (mean Cq value (Cqmean)= 39.5 ± 6.9) for a total of 108 samples, the MvNAT method produced a Cq value of 11.1% (Cqmean= 38.4 ± 5.2), the R1-R2 kit yielded 14.8% (Cqmean= 35.9 ± 7.1) and HT method resulted in 25% (Cqmean= 31.4 ± 6.3). When the variability in target gene Cq values was analyzed in all samples compared to the standard method, the HT method significantly provided lower Cq values (n= 16; p= 0.007; paired t-test) while the MvNAT method and R1-R2 kit yielded higher Cq values (n= 6; p= 0.025, n= 11; p= 0.004; paired t-test). Sensitivity rates were MvNAT= 31.6%, R1-R2= 57.9%, HT= 84.2%, with 100% specificity for all three methods. The HT method demonstrated a positive extraction efficiency because it is fast, easy and not dependent on reagents. Although this method provided lower Cq values than the standard method, especially in samples with a high viral load, it should be considered that it also has the potential to yield false-negative results in samples with Cq> 35. With this study, it was concluded that the extraction phase of the SARS-CoV-2 qrRT-PCR test can be carried out using various methods that do not require kits or reagents, such as the HT method. However, it is believed that multicenter studies involving a larger number of samples are necessary to standardize the test and assess the possibility of false negatives.

Retrospective testing for the presence of monkeypox virus in a high-risk population from February–June 2022 in Alberta, Canada

A multi-country outbreak of monkeypox virus (MPXV) infections was identified by the World Health Organization in May 2022. The western Canadian province of Alberta identified its first case of MPXV in a returning traveller on June 2, 2022. We undertook a retrospective testing exercise to evaluate whether MPXV may have been circulating in the province earlier. Skin (genital and non-genital) and mucosal lesion swabs submitted for herpes simplex virus (HSV)/varicella zoster virus (VZV)/syphilis testing from male patients attending sexually-transmitted infection clinics across the province of Alberta from January 28 to May 30, 2022 were retrieved from storage. The population tested was selected based on the epidemiology of the current 2022 multi-country MPXV outbreak. Samples underwent viral nucleic acid extraction and testing for the presence of Orthopoxvirus DNA using a commercial real-time polymerase chain reaction (PCR) kit. A total of 392 samples (representing 341 unique individuals of median age 31 years) were retrieved. Of them, 349 (89.0%) samples were submitted for HSV/VZV/syphilis testing, 13 (3.3%) for HSV/VZV only, and 30 (7.7%) for syphilis PCR only. None of the 392 samples tested were found to be positive for Orthopoxvirus DNA. The results of this study indicate that circulation of MPXV in a higher-risk population in Alberta, prior to the first case, was less likely. We recommend that other provinces/territories review their local epidemiology, context and resources prior to conducting similar studies.

Rapid Extraction of Viral Nucleic Acids Using Rotating Blade Lysis and Magnetic Beads.

The complex and lengthy protocol of current viral nucleic acid extraction processes limits their use outside laboratory settings. Here, we describe a rapid and reliable method for extracting nucleic acids from viral samples using a rotating blade and magnetic beads. The viral membrane can be instantly lysed using a high-speed rotating blade, and nucleic acids can be immediately isolated using a silica magnetic surface. The process was completed within 60 s by this method. Routine washing and eluting processes were subsequently conducted within 5 min. The results achieved by this method were comparable to those of a commercially available method. When the blade-based lysis and magnetic bead adsorption processes were performed separately, the RNA recovery rate was very low, and the Ct value was delayed compared to simultaneous lysis and RNA adsorption. Overall, this method not only dramatically shortens the conventional extraction time but also allows for its convenient use outside the laboratory, such as at remote field sites and for point-of-care testing.

Preliminary Study on the Possibility to Detect Virus Nucleic Acids in Post-Mortem Blood Samples.

In many forensic cases, the medical records of the deceased are not available at the time of the autopsy; therefore, no information about the deceased's state of health, including any infectious diseases contracted during life, is accessible. The detection of some of the principal viral infections, such as hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1), could contribute to determining causes of death and interesting applications could be found in medico-legal practice, such as occupational risk assessment. To date, accurate and sensitive serological and molecular assays capable of detecting these viruses have been validated on biological samples taken from living beings, while their efficiency on forensic post-mortem biological samples has yet to be thoroughly assessed. To further this aim, this study evaluated whether the nucleic acid amplification techniques (NAATs) for the detection of viral genomes that are applied in clinical settings can be used, with the same success rate, for these latter samples. Manual viral nucleic acid extraction processes and fully-automated amplification-based detection techniques developed in-house were evaluated on blood samples taken during the routine autopsies of 21 cadavers performed 2 to 9 days after death. Information on HBV, HCV, and HIV-1 seropositive status was previously known for only four of these cadavers. Using automated quantitative real-time PCR (qPCR) and qualitative PCR (end-point) analyses, it was possible to confirm the presence of viral genomes in the four post-mortem whole blood samples with previously reported specific serological positivity. In addition, the genomes of HCV and/or HIV-1 genomes were detected in three other blood samples with unknown serological status at the time of autopsy. Therefore, our findings suggest that molecular assays may detect the presence of viral genomes in forensic post-mortem blood samples up to five days after death. This provides an additional means of investigation that can contribute to the determination of the deceased's cause of death.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Mutational Pattern in the Fourth Pandemic Phase in Greece.

The aim of this study is to investigate the circulating variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Athens and from rural areas in Greece during July and August 2021. We also present a rapid review of literature regarding significant SARS-CoV-2 mutations and their impact on public health. A total of 2500 nasopharyngeal swab specimens were collected from suspected COVID-19 cases (definition by WHO 2021b). Viral nucleic acid extraction was implemented using an automatic extractor and the RNA recovered underwent qRT-PCR in order to characterize the specimens as positive or negative for SARS-CoV-2. The positive specimens were then used to identify specific Spike gene mutations and characterize the emerging SARS-CoV-2 variants. For this step, various kits were utilized. From the 2500 clinical specimens, 220 were tested positive for SARS-CoV-2 indicating a prevalence of 8.8% among suspected cases. The RT-PCR Ct (Cycle threshold) Value ranged from 19 to 25 which corresponds to medium to high copy numbers of the virus in the positive samples. From the 220 positive specimens 148 (67.3%) were from Athens and 72 (32.7%) from Greek rural areas. As far as the Spike mutations investigated: N501Y appeared in all the samples, D614G mutation appeared in 212 (96.4%) samples with a prevalence of 87.2% in Athens and 98.6% in the countryside, E484K had a prevalence of 10.8% and 12.5% in Athens and the rural areas, respectively. K417N was found in 18 (12.2%) samples from Athens and four (5.6%) from the countryside, P681H was present in 51 (34.5%) Athenian specimens and 14 (19.4%) specimens from rural areas, HV69-70 was carried in 32.4% and 19.4% of the samples from Athens and the countryside, respectively. P681R had a prevalence of 87.2% in Athens and 98.6% in rural areas, and none of the specimens carried the L452R mutation. 62 (28.2%) samples carried the N501Y, P681H, D614G and HV69-70 mutations simultaneously and the corresponding variant was characterized as the Alpha (UK) variant (B 1.1.7). Only six (2.7%) samples from the center of Athens had the N501Y, E484K, K417N and D614G mutations simultaneously and the virus responsible was characterized as the Beta (South African) variant (B 1.351). Our study explored the SARS-CoV-2 variants using RT-PCR in a representative cohort of samples collected from Greece in July and August 2021. The prevalent mutations identified were N501Y (100%), D614G (96.4%), P681R (90.1%) and the variants identified were the Delta (90.1%), Alpha (28.2%) and Beta (2.7%).

Genomic and Biological Characterization of Ralstonia solanacearum Inovirus Brazil 1, an Inovirus that Alters the Pathogenicity of the Phytopathogen Ralstonia pseudosolanacearum.

Filamentous bacteriophages contain a single-stranded DNA genome and have a peculiar lifestyle, since they do not cause host cell lysis, but establish a persistent association with the host, often causing behavioral changes, with effects on bacterial ecology. Over the years, a gradual reduction in the incidence of bacterial wilt has been observed in some fields from Brazil. This event, which has been associated with the loss of pathogenicity of Rasltonia spp. isolates due to infection by filamentous viruses of the inovirus group, is widely reported for Ralstonia spp. Asian isolates infected by inoviruses. In an attempt to elucidate which factors are associated with the phenomenon reported in Brazil, we investigated one isolate of R. solanacearum (UB-2014), with unusual characteristics for R. solanacearum, obtained from eggplant with mild wilt symptoms. To verify if the presence of filamentous bacteriophage was related to this phenotype, we performed viral purification and nucleic acid extraction. The phage genome was sequenced, and phylogenetic analyses demonstrated that the virus belongs to the family Inoviridae and was named as Ralstonia solanacerarum inovirus Brazil 1 (RSIBR1). RSIBR1 was transmitted to R. pseudosolanacearum GMI1000, and the virus-infected GMI1000 (GMI1000 VI) isolate showed alterations in phenotypic characteristics, as well as loss of pathogenicity, similarly to that observed in R. solanacearum isolate UB-2014. The presence of virus-infected UB-2014 and GMI1000 VI plants without symptoms, after 3months, confirms that the infected isolates can colonize the plant without causing disease, which demonstrates that the phage infection changed the behavior of these pathogens.

BK Virus Induced Haemorrhagic Cystitis in Haematopoietic Stem Cell Transplant Setting — a 10 Year Retrospective Case Control Study

INTRODUCTIONBK virus (BKV) is a human polyomavirus that usually causes asymptomatic or mild respiratory illness in childhood and thereafter remains latent in renal epithelial cells. With decreased cell-mediated immunity, reactivation of BKV causing haemorrhagic cystitis (HC) is a well-known complication of haematopoietic stem cell transplantation (HSCT) that is associated with increased morbidity, mortality, longer hospital stays and increased hospital costs. This analysis was done to ascertain etiological risk factors, incidence and outcomes of BKV induced HC following HSCT. METHODSIn this retrospective case control study of patients undergoing HSCT in the Department of Haematology at CMC Vellore between 2007 and 2016, we compared patients who developed BKV induced HC with those without BKV infection [controls] matched for age, diagnosis, conditioning regimen and donor source. Data was collected from individual medical records and databases maintained in the Departments of Haematology and Virology. The BKV PCR was designed as a real time PCR that targets the VP1 region of the BKV genome. Viral nucleic acid extraction was performed as recommended on the urine samples of these patients using the QIAamp DNA blood Mini kit (Qiagen, GMBH, Germany). Real-time PCR detection was performed as described previously. RESULTSThere were 1276 (120 haploidentical) transplants that were performed between 2007 to 2016. Two hundred and sixty two (20.5%) patients had HC and BKV qualitative PCR was performed for 189 patients, of which 105 were positive. Among the cases, males were more affected and the median age was 20 years. The most common indication for transplant was aplastic anemia (31.4%), followed by acute myeloid (19.0%) and lymphoblastic (15.2%) leukemia. Predominant conditioning regimens were myeloablative conditioning (57.1%) with reduced intensity conditioning being least common (8.6%). In the patients with HC, Grade 3 HC was most commonly (57.1%) seen and the median time to development of HC was 35 (range 0-858) days. Specific treatment with cidofovir was given to only 5 patients, surgical intervention in 2 patients and foscarnet in 1 patient. Acute GVHD was seen in 53.35%. HLA matched sibling (53.3%) donors, followed by 8 or 9/10 matched unrelated (16.2%) donors were most frequent. Survival was significantly lower in the cases (42.9% vs 61%, p=0.013). Protective effect of non myeloablative conditioning, residual disease at time of transplant, lower CD34 dose, presence of acute GVHD and chronic GVHD, reactivation of CMV and presence of bacterial urinary tract infections (UTI) were significant factors on univariate analysis. Multivariate logistic regression confirmed presence of acute GVHD, bacterial UTI and residual disease at time of transplant as significant risk factors for BK viruria and disease. CONCLUSIONBKV HC was seen in 8% of HSCT patients with mortality of 57.1%. Presence of acute GVHD, bacterial UTI and residual disease at time of transplant emerged as significant risk factors. DisclosuresSrivastava:Bayer Healthcare, Shire, Novo Nordisk, Roche Genentech, LFB: Other: Educational grants / Advisory Board / Grants Review / Data Monitoring Committee, Research Funding.

A Multiplex One-Step RT-qPCR Protocol to Detect SARS-CoV-2 in NP/OP Swabs and Saliva.

Since December 2019, SARS‐CoV‐2 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, https://coronavirus.jhu.edu/map.html). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal/oropharyngeal swabs (NP/OP) and saliva are two specimen types that are most often analyzed to detect SARS‐CoV‐2 by molecular tests that detect viral RNA or by antigen/antibody tests that detect viral proteins and/or the host immune response against the virus. Compared to antigen/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre‐analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS‐CoV‐2 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost‐effective multiplex reverse‐transcription real‐time PCR assay that can be used to detect SARS‐CoV‐2 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS‐CoV‐2 by RT‐qPCR. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Manual viral nucleic acid extraction from NP/OP swabs collected in different media, and from saliva Alternate Protocol 1: Low‐throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1‐14 samples) Alternate Protocol 2: High‐throughput automated extraction on the Kingfisher Flex machine (1‐96 samples) Basic Protocol 2: Multiplex RT‐qPCR protocol to detect SARS‐CoV‐2 Alternate Protocol 3: Multiplex one‐step RT‐qPCR protocol to detect SARS‐CoV‐2 with S and E gene probes labeled with the same fluorochrome

Rapid and sensitive detection of SARS-CoV-2 RNA using the Simplexa™ COVID-19 direct assay.

So far, one of the major drawbacks of the available molecular assays for the diagnosis of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) is the need for viral nucleic acid extraction from clinical specimens. The aim of this study was to evaluate the performances of a newly designed real-time RT-PCR (Simplexa™ COVID-19 Direct assay), that is established with an all-in-one reagent mix and no separate extraction required. The lower limit of detection (LOD) for both target genes resulted the same: 3.2 (CI: 2.9-3.8) log10 cp/mL and 0.40 (CI: 0.2-1.5) TCID50/mL for S gene while 3.2 log10 (CI: 2.9-3.7) log10 cp/mL and 0.4 (CI: 0.2-1.3) TCID50/mL for ORF1ab. The LOD obtained with extracted viral RNA for both S gene or ORF1ab was 2.7 log10 cp/mL. Crossreactive analysis performed in 20 nasopharyngeal swabs confirmed a 100% of clinical specificity of the assay. Clinical performances of Simplexa™ COVID-19 Direct assay were assessed in 278 nasopharyngeal swabs tested in parallel with Corman's method. Concordance analysis showed an "almost perfect" agreement in SARS-CoV-2 RNA detection between the two assays, being κ = 0.938; SE = 0.021; 95% CI = 0.896-0.980. The high sensitivity and specificity of this new assay indicate that it is promising for laboratory diagnosis, enabling highspeed detection in just over one hour, which is significantly faster than the up to five hours currently required by traditional extraction followed by amplification technologies, thus allowing prompt decision making regarding isolation of infected patients.

Investigation of Anti-HCV S/CO Value in Detecting Viremia in Patients with Hepatitis C Virus Infection

Anti-HCV and HCV RNA tests are used in laboratory diagnosis of hepatitis C virus (HCV) infections. False positive results are frequently observed in anti-HCV tests used as screening tests in societies with low prevalence of HCV. The HCV RNA test, which is a confirmatory test, is not performed in every laboratory because it is a high-cost and high-tech test, which can lead to delay in the diagnosis and treatment of patients. In this study, it was aimed to obtain an optimal anti-HCV S/CO value in our laboratory for demonstrating true antibody positivity and viremia in patients by analyzing the relationship between anti-HCV, alanine aminotransferase (ALT) and HCV RNA using retrospective data. Between July 2014 and July 2017, 754.190 anti-HCV tests were performed. Patients aged 18 years or older who were reactive with anti-HCV and those with simultaneous HCV RNA and ALT prompts were included in the study. The second generation CMIA (Abbott, USA) method was used for anti-HCV detection. For quantitative HCV RNA analysis, viral nucleic acid extraction was performed with the QIAsymphony SP/AS (Qiagen, Germany) using the QIAsymphony DSP Virus/Pathogen Midi Kit; and PCR was performed by Rotor-Gene Q (Qiagen, Germany) using Artus HCV QS-RGQ kit. ARCHITECT c and AEROSET systems (Abbott, USA) were used for ALT measurement. HCV genotype determination (622 cases) was performed using GenoSen's HCV Genotyping 1/2/3/4 RG qualitative real time PCR kit (Corbett Research, Australia) and GEN-C 2.0 Reverse Hybridization Strip Assay (NLM Diagnostics, Italy) kit at different periods covered by our study. The optimal threshold value for the relationship between anti-HCV, ALT and HCV RNA was selected based on ROC analysis. Statistical significance was accepted as p<0.05. Of the anti-HCV test results, 10.679 were found to be reactive. 1754 data of 1290 cases with anti-HCV reactivity who were simultaneously tested for HCV RNA and ALT in the same serum were evaluated. Of these, 742 (42%) were found to be HCV RNA positive and 1012 (58%) were found to be HCV RNA negative. ALT and anti-HCV levels of those who were positive for HCV RNA were significantly higher than those with negative HCV RNA (p= 0.001). The threshold point for anti-HCV S/CO according to HCV RNA was found to be 7.13 (sensitivity of 97.4%, specificity of 50.3%, positive predictive value 58.9%, negative predictive value 96.4%), and the cut-off point for ALT was found to be 27.5 IU/L (sensitivity of 77.6%, specificity of 80.8%). For HCV RNA positivity, the area under the ROC curve for anti-HCV and ALT was significantly higher than 0.5 (p= 0.001). No statistically significant difference was found between HCV genotypes in terms of ALT and anti-HCV levels. By using our new threshold in the laboratory workflow, the need to verify with HCV RNA can be reduced, especially in some patients who have been screened for antiHCV for screening purposes. Anti-HCV values below 7.13 S/CO, considering the high negative predictive value of this threshold; a false positive result in a patient presenting for screening can be predicted without waiting for the HCV RNA result. In anti-HCV reactivities determined above 7.13, the possibility of absence of viremia should be considered due to the low positive predictive value.

Comparison of five viral nucleic acid extraction kits for the efficient extraction of viral DNA and RNA from cell-free samples

Comparison of five viral nucleic acid extraction kits for the efficient extraction of viral DNA and RNA from cell-free samples

Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples

BackgroundNumerous protocols for viral enrichment and genome amplification have been created. However, the direct identification of viral genomes from clinical specimens using next-generation sequencing (NGS) still has its challenges. As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS, it is still valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly.ResultsIn this study, we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits: QIAamp Viral RNA Mini Kit (VRMK), QIAamp MinElute Virus Spin Kit (MVSK), RNeasy Mini Kit (RMK), and RNeasy Plus Micro Kit (RPMK), using a mixed respiratory clinical sample without any pre-treatment. This sample contained an adenovirus (ADV), influenza virus A (Flu A), human parainfluenza virus 3 (PIV3), human coronavirus OC43 (OC43), and human metapneumovirus (HMPV). The quantity and quality of the viral extracts were significantly different among these kits. The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK. The MVSK had the lowest Ct values for ADV and PIV3. The RMK revealed the lowest detectability for HMPV and PIV3. The most effective rate of NGS data at 67.47% was observed with the RPMK. The other three kits ranged between 12.1–26.79% effectiveness rates for the NGS data. Most importantly, compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK. The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV, while the RMK revealed the best extraction efficiency by NGS analysis.ConclusionsThe evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis. The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples. In addition, viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus. Our results highlighted the importance of nucleic acid extraction kit selection, which has a major impact on the yield and number of viral reads by NGS analysis. Therefore, the choice of extraction method for a given viral pathogen needs to be carefully considered.

Molecular status of Human Immunodeficiency Virus, Hepatitis B virus, and Hepatitis C virus among injecting drug male commercial sex workers in Surakarta, Indonesia

Male commercial sex workers are one of the high-risk community for blood-borne viruses. However, there are no data concerning the molecular status of Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV) circulated among male commercial sex workers with injecting drug habits in Surakarta, Indonesia. Blood samples obtained from injecting drug male commercial sex workers in Surakarta were examined for HIV antibodies, HBsAg, and HCV antibodies, respectively, by immunological assays. Blood samples were also subjected to viral nucleic acid extraction and molecular detection of HIV, HBV, and HCV by nested (RT) PCRs. The PCR products were purified from agarose gels, and the nucleotide sequences were retrieved and molecular analyzed. HIV, HBV, and HCV were detected in 29.4% (10/34), 17.6% (6/34), and 52.9% (18/34), respectively. HIV CRF01_AE and B were found to be circulating in the community. HBV genotype B3 was predominated, followed by C1. HCV genotype 1a was predominated, followed by 1c, 3a, 1b, and 4a. HIV, HBV, and HCV were found circulating in the male commercial sex workers with injecting drug habits in Surakarta, Indonesia.

Genotyping the human papillomavirus infection in Iranian women referred to Shahid Motahari Hospital, in Urmia, with real-time polymerase chain reaction techniques

Objective(s): Human papillomavirus (HPV) is in viridae papillomavirus family. These viruses have been classified based on the DNA sequences. At least 120 types of these viruses have been identified. Different types of HPV genotypes are associated with lower genital tract infection. This study aimed to identify the genotypes of HPV that cause vaginal infections in women with infection. Materials and Methods: This cross-sectional study was performed on eighty patients with vaginal infection. Extracting viral DNA was performed automatically using MagCore Nucleic Acid Extractor (made in Taiwan) and MagCor® Viral Nucleic Acid Extraction kit (Cartridge code 202) made by MogCore Co. Taiwan, and genotyping the samples was carried out using the real-time polymerase chain reaction (PCR) technique in the Light Cycler 96 System (made in Germany) and real-quality relative quantification-HPV infection high-risk (HR)/low-risk (LR) Multiplex Kit made by AB Analitica® Co. Italy. Results: Among the 80 samples, 30 people (37.5%) were positive for infection with HPV; evaluating the positive genotypes, it was found that of these, 16 people (53.4%) were infected with LR genotypes of HPV, seven patients (23.3%) were infected with only HR HPV genotype, and seven people (23.3%) were infected with both LR and HR genotypes of HPV (coinfection). Conclusion: Considering that HPV infection is asymptomatic, and it has a high prevalence in Iran, early diagnosis and prevention of progressing the infection can prevent the malignancies of the uterus. Molecular techniques, particularly real-time PCR, are reliable methods for detecting the HPV infection seven with quite low viral loads.

Alu and Poet Ankyrine Detection and Quantization in Cell Free Dna of Cancer Patients

In this study, specimens of plasma were collected from 96 from patient diagnosed with cancer from Al Karama teaching hospital. Also plasma collected from 25 healthy individual as control. Cell-free DNA (cfDNA) was extracted from 1ml plasma using high pure viral Nucleic Acid Kit as an alternative to high expensive cfDNA kit. cfDNA extracted according to the manufacturer’s instructions, but without the use of carrier RNA. This represent the first recorded method of using viral nucleic acid extraction kit for collection of cell free DNA. Results indicate that all cancer samples show significant increase of cfDNA in compare with control (p 0.01). and this dramatic increase in DNA concentration provide good indicator about health condition. DNA integrity have been checked depending on amplification of Arthrobacter luteus (Alu sequence). Both small (115) and large (247) large Alu stretches have amplified using RT PCR using SYBR green dye. Results shows that integrity of DNA extracted from patients and control are suitable for any further molecular investigation, and short Alu repeats are much less abundant in health individual in compare with cancer patient of all cancer types under study. Depending on these results one can conclude that almost all DNA obtained from normal patients is finely fragmented due to apoptosis action and that’s why give very little positive. results in compare with corresponding cancer patient which release large DNA fragments resulted from necrosis and NK/Tc cells activity, which is amplifiable more efficiently. In this work another molecular study conducted to investigate the cancer specific sequences. Since we deals with different cancers type in this study, two types of prostate, ovary, testis expressed protein (POTE Ankyrine) were tested 2α and 2β. Results shows that almost all healthy group were negative for specific POTE test while cancer patient samples are positive for POTE specific amplification. This results are promising since it could be developed to be a building block of cfDNA cancer tests.

Optimization and evaluation of a method to detect adenoviruses in river water

Adenoviruses are often implicated in recreational water disease outbreaks but existing methods for their detection perform poorly within these matrices. In this study, small volume (100mL) concentration was used to identify processes that promoted recovery of adenovirus from river water. Several alternative secondary concentration techniques were investigated and compared to the baseline method consisting of primary concentration via filtration, followed by celite mediated secondary concentration. The alternative secondary concentrations included multiple filter elutions, soaking the filter for 15min prior to elution and concentration using pre-treated celite (river water, 1.5% and 3% milk) instead of a filter. Modifications of the viral nucleic acid extraction technique were also evaluated. Concentration using pre-treated celite and a modified extraction technique (10min boil and a 1h ProK incubation at 37°C) recovered significantly higher levels of adenovirus (P=0.001) than other methods tested. This optimized method increased recovery of spiked adenovirus (57±27%) compared to baseline method performance (4±3%) indicating that use of pre-treated celite as opposed to filtration significantly improves recovery. Application of the optimized concentration method to larger volume (1L) of river water resulted in similar recoveries (42±19%) underlying the utility of this method to detect adenovirus from environmental samples.