Quantification of human polyomavirus JC virus load in urine and blood samples of healthy tribal populations of North-Eastern part of West Bengal,India

Background: Human polyomavirus JC (JCV) is a widespread human virus with profound pathogenic potential. A study was undertaken to quantify JCV load in urine and peripheral blood samples of immunocompetent, apparently healthy tribal individuals of North-Eastern part of West Bengal, India for the first time. Materials and Methods: One hundred and thirteen samples of urine or blood were collected from different tribal groups of this region. For the quantitative estimation of the viral load in each sample, real-time polymerase chain reaction method using the SYBR Green dye was employed. Results: The viral load estimated was found in the range between 3.5 × 10² and 2.12 × 10⁶ copies/ml of samples having a mean and median viral copy numbers of 8.67 × 10⁵ and 9.19 × 10⁵ copies/ml of sample respectively. Conclusion: The mean viral DNA load in urine samples of the studied immunocompetent population was found to be higher than that found in a study conducted in the USA, but lower than similar groups of Italy and healthy adult women in the USA. However when compared with median values of viral DNA loads in urine samples of immunocompetent human subjects of Kuwait, Portugal, and Switzerland the observed viral DNA load was found to be substantially higher.     

Viable SARS-CoV-2 in various specimens from COVID-19 patients

ObjectivesThe aim was to determine whether various clinical specimens obtained from COVID-19 patients contain the infectious virus.MethodsTo demonstrate whether various clinical specimens contain the viable virus, we collected naso/oropharyngeal swabs and saliva, urine and stool samples from five COVID-19 patients and performed a quantitative polymerase chain reaction (qPCR) to assess viral load. Specimens positive with qPCR were subjected to virus isolation in Vero cells. We also used urine and stool samples to intranasally inoculate ferrets and evaluated the virus titres in nasal washes on 2, 4, 6 and 8 days post infection.ResultsSARS-CoV-2 RNA was detected in all naso/oropharyngeal swabs and saliva, urine and stool samples collected between days 8 and 30 of the clinical course. Notably, viral loads in urine, saliva and stool samples were almost equal to or higher than those in naso/oropharyngeal swabs (urine 1.08 ± 0.16–2.09 ± 0.85 log₁₀ copies/mL, saliva 1.07 ± 0.34–1.65 ± 0.46 log₁₀ copies/mL, stool 1.17 ± 0.32 log₁₀ copies/mL, naso/oropharyngeal swabs 1.18 ± 0.12–1.34 ± 0.30 log₁₀ copies/mL). Further, viable SARS-CoV-2 was isolated from naso/oropharyngeal swabs and saliva of COVID-19 patients, as well as nasal washes of ferrets inoculated with patient urine or stool.DiscussionViable SARS-CoV-2 was demonstrated in saliva, urine and stool samples from COVID-19 patients up to days 11–15 of the clinical course. This result suggests that viable SARS-CoV-2 can be secreted in various clinical samples and respiratory specimens.     

Human papillomavirus DNA detected in peripheral blood samples from healthy Australian male blood donors

Recent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In light of these findings, DNA extracted from PBMCs from healthy blood donors were examined in order to determine how common HPV DNA is in blood of healthy individuals. Blood samples were collected from 180 healthy male blood donors (18–76 years old) through the Australian Red Cross Blood Services. Genomic DNA was extracted and specimens were tested for HPV DNA by PCR using a broad range primer pair. Positive samples were HPV‐type determined by cloning and sequencing. HPV DNA was found in 8.3% (15/180) of the blood donors. A wide variety of different HPV types were isolated from the PBMCs; belonging to the cutaneous beta and gamma papillomavirus genera and mucosal alpha papillomaviruses. High‐risk HPV types that are linked to cancer development were detected in 1.7% (3/180) of the PBMCs. Blood was also collected from a healthy HPV‐positive 44‐year‐old male on four different occasions in order to determine which blood cell fractions harbor HPV. PBMCs treated with trypsin were negative for HPV, while non‐trypsinized PBMCs were HPV‐positive. This suggests that the HPV in blood is attached to the outside of blood cells via a protein‐containing moiety. HPV was also isolated in the B cells, dendritic cells, NK cells, and neutrophils. To conclude, HPV present in PBMCs could represent a reservoir of virus and a potential new route of transmission. J. Med. Virol. 81:1792–1796, 2009. © 2009 Wiley‐Liss, Inc.     

Parvovirus B19 seroprevalence,viral load,and genotype characterization in volunteer blood donors from southern Brazil

Usually transmitted via respiratory droplets, parvovirus B19 (B19V) can also be acquired by blood transfusion especially because of viral persistence, resistance to blood treatment procedures, and high viral load during the early infection phase. This is particularly problematic in immunocompromised or anemic patients where the infection can have a severe outcome. As B19V DNA was detected in blood donations from South Brazil during a viral metagenomic survey performed by Next-Generation Sequencing, the objective of this retrospective study was to evaluate the seroprevalence, B19V DNA presence and circulating genotypes in a Hospital Blood Transfusion Service in Santa Maria city in South Brazil (Rio Grande do Sul state). Among 480 volunteer blood donors, 53.9% (n = 258 of 479) were anti-B19V IgG-positive, and 9 (1.9%) plasma samples presented B19V DNA. In almost all cases (n = 7 of 9, 77.8%), B19V DNA load was accompanied by the presence of anti-B19V IgG suggesting a persistent infection. The sequencing of the strains demonstrated that all belong to genotype 1 which is the most prevalent worldwide. The analysis of the recipient information of the positive for B19V DNA units revealed no related posttransfusion adverse effects. Our results demonstrate for the first time, B19V seroprevalence, viral load, and genotypes among blood donors from South Brazil and give a light for the circulation and impact of this B19V in this part of the country.     

Diagnostic strategies for SARS-CoV-2 infection and interpretation of microbiological results

BackgroundTo face the current COVID-19 pandemic, diagnostic tools are essential. It is recommended to use real-time RT-PCR for RNA viruses in order (a) to perform a rapid and accurate diagnostic, (b) to guide patient care and management and (c) to guide epidemiological strategies. Further studies are warranted to define the role of serological diagnosis and a possible correlation between serological response and prognosis.ObjectivesThe aim was to guide clinical microbiologists in the use of these diagnostic tests and clinicians in the interpretation of their results.SourcesA search of literature was performed through PubMed and Google Scholar using the keywords SARS-CoV-2, SARS-CoV-2 molecular diagnosis, SARS-CoV-2 immune response, SARS-CoV-2 serology/antibody testing, coronavirus diagnosis.ContentThe present review discusses performances, limitations and use of current and future diagnostic tests for SARS-CoV-2.ImplicationsReal-time RT-PCR remains the reference method for diagnosis of SARS-CoV-2 infection. On the other hand, notwithstanding its varying sensitivity according to the time of infection, serology represents a valid asset (a) to try to solve possible discrepancies between a highly suggestive clinical and radiological presentation and negative RT-PCR, (b) to solve discrepancies between different PCR assays and (c) for epidemiological purposes.     

Quantitative detection of hepatitis C virus RNA in urine of patients with chronic hepatitis C using a novel real-time PCR assay

Hepatitis C virus (HCV) RNA can be detected in body fluids such as urine. However, because of deficiencies in established isolation and detection methods, the actual prevalence and form of HCV RNA in the urine of patients with hepatitis C remain unclear. To more sensitively and accurately measure urine HCV RNA levels, a novel real-time PCR assay with a modified isolation method and short amplicon designed for short HCV RNA fragments was developed in this study. The limit of detection, precision, linearity, and specificity of the assay was evaluated and demonstrated high-quality performance. The prevalence of HCV RNA in the urine of viremic patients infected with HCV was 60% (36/60), as determined by a 62-bp assay. The HCV RNA detection rate and concentration were much lower with a 157-bp assay, and were undetectable with 222- and 304-bp assays. With the 62-bp assay, patients with detectable urine HCV RNA had significantly higher plasma HCV RNA levels ( P < 0.001), and plasma and urine concentrations were significantly positively correlated ( R ² = 0.708, P < 0.001). The method not only increased the detection rate of urine HCV RNA but also revealed the presence of short HCV RNA fragments in urine, indicating that urine from CHC patients with normal kidney function should not be infectious. In addition, it raised the possibility of urinary HCV RNA as a potential noninvasive marker for therapeutic monitoring of patients with hepatitis C.     

Development of multiplex real-time quantitative PCR for simultaneous detection of Chlamydia trachomatis,Mycoplasma hominis,Ureaplasma urealyticum,and Mycoplasma genitalium in infertile women

PurposeSexually Transmitted Diseases (STDs) can cause sterility and many other problems for women planning pregnancy. Currently, almost 340 million people worldwide suffer from Sexually Transmitted Infections (STIs). This study made attempts to quickly identify STDs' most critical infectious agents using dedicated primers and probes.MethodsThe present study was done on the cervical samples of 200 infertile women. After extracting the total DNA of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium, quantitative methods were employed to determine the rate of target bacteria using multiplex real-time PCR.ResultsThe multiplex qPCR showed the rates of 47%, 16%, 46%, and 16.5% for Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium in infertile women, respectively. In some patients, there were co-infections with two or three bacteria. The diagnostic approach used in our research could be employed as an alternative detection tool to identify the four most common STD-associated bacterial agents while detecting mixed infections.ConclusionsInfertile women with no biological problems could have their genital tract checked using this newly designed identification technique and get proper treatment for their infections as quickly as possible.     

Immunohistochemical,molecular, and pathomorphological study of liver biopsy specimens during chronic hepatitis C

We carried out a comparative study of replication markers of hepatitis C virus (HCV RNA) in some biological substrates and NS3-antigen in liver biopsy specimens. It was found that 82% liver specimens contained RNA HCV, in 44% cases HCV RNA was present in the serum, mononuclear blood cells, and liver. The presence of NS3-antigen in hepatocytes can be considered as a structural marker of HCV replication, which is confirmed by positive correlation with the results of PCR for viral RNA in tissue specimens. In most cases the presence of HCV replication markers did not correlate with activity of the infectious process assessed by biochemical and histological tests.     

Animal models for SARS-CoV-2 and SARS-CoV-1 pathogenesis,transmission and therapeutic evaluation

There is a critical need to develop animal models to alleviate vaccine and drug development difficulties against zoonotic viral infections. The coronavirus family, which includes severe acute respiratory syndrome coronavirus 1 and severe acute respiratory syndrome coronavirus 2, crossed the species barrier and infected humans, causing a global outbreak in the 21^st century. Because humans do not have pre-existing immunity against these viral infections and with ethics governing clinical trials, animal models are therefore being used in clinical studies to facilitate drug discovery and testing efficacy of vaccines. The ideal animal models should reflect the viral replication, clinical signs, and pathological responses observed in humans. Different animal species should be tested to establish an appropriate animal model to study the disease pathology, transmission and evaluation of novel vaccine and drug candidates to treat coronavirus disease 2019. In this context, the present review summarizes the recent progress in developing animal models for these two pathogenic viruses and highlights the utility of these models in studying SARS-associated coronavirus diseases.     

SARS-CoV-2 and natural infection in animals

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the novel coronavirus disease (COVID-19) pandemic, which has caused serious challenges for public health systems worldwide. Due to the close relationship between animals and humans, confirmed transmission from humans to numerous animal species has been reported. Understanding the cross-species transmission of SARS-CoV-2 and the infection and transmission dynamics of SARS-CoV-2 in different animals is crucial to control COVID-19 and protect animal health. In this review, the possible animal origins of SARS-CoV-2 and animal species naturally susceptible to SARS-CoV-2 infection are discussed. Furthermore, this review categorizes the SARS-CoV-2 susceptible animals by families, so as to better understand the relationship between SARS-CoV-2 and animals.     

Investigation of an Outbreak of Varicella in Chandigarh,North India,Using a Real-time Polymerase Chain Reaction Approach

Outbreaks of varicella are reported when susceptible population accumulates. This study reports a chickenpox outbreak in Burail in August 2014, wherein 20 laboratory-confirmed cases were identified by the detection of varicella zoster virus (VZV) DNA and VZV IgM antibodies. The viral load between vesicular swabs and serum samples from 8 patients with active lesions was found to have good correlation and further also related with disease severity. Real-time polymerase chain reaction can be useful for early diagnosis of an outbreak and vesicular swab can be used as a less invasive sample for assessing the disease severity.     

A rapid and reliable PCR method for genotyping the ABO blood group. II: A2 and O2 alleles

PCR permits direct genotyping of individuals at the ABO locus. Several methods have been reported for genotyping ABO that rely on differentiating the A, B, and O alleles at specific base substitutions. However, the O allele as defined by serology comprises at least two alleles (O¹ and O²) at the molecular level, and most current ABO genotyping methods only take into account the O¹ allele. Determining the presence of the O² allele is critical, as this not-infrequent allele would be mistyped as an A or a B allele by standard PCR typing methods. Furthermore, none of the methods to date distinguish between the A¹ and A² alleles, even though 10% of all white persons are blood group A². We have developed a method for genotyping the ABO locus that takes the O² and A² alleles into account. Typing for A² and O² by diagnostic restriction enzyme digestion is a sensitive, nonradioactive assay that provides a convenient method useful for forensic and paternity testing and for clarifying anomalous serological results. © 1996 Wiley-Liss, Inc.