scholarly journals Diagnosis and molecular typing of rabies virus in samples stored in inadequate conditions

2014 ◽  
Vol 8 (08) ◽  
pp. 1016-1021 ◽  
Author(s):  
Fernando J Beltran ◽  
Federico Gury Dohmen ◽  
Horacio Del Pietro ◽  
Daniel M Cisterna
Keyword(s):  
Rabies Virus ◽  
Molecular Typing ◽  
Diagnostic Tests ◽  
Limit Of Detection ◽  
Antibody Test ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Nucleoprotein Gene ◽  
Three Samples

Introduction: The exposure of nervous tissue samples to high temperatures affects the sensitivity of rabies virus diagnostic tests, causing degradation of the viral structure. This study evaluated reverse transcriptase polymerase chain reaction (RT-PCR) for the diagnosis and molecular characterization of brain tissue samples in an advanced state of decomposition and poorly conserved viral isolates by comparing it with routine diagnostic tests. Methodology: A panel of three canine brain samples exposed to controlled decomposition for 7, 15, 30, and 120 days were evaluated using fluorescence antibody test (FAT), mouse inoculation test (MIT), and RT-PCR. In addition, 14 isolates of rabies variants, representing the largest circulation in Argentina, preserved in inadequate cooling for six to eight years were analyzed. Molecular typing of strains was performed using a 159-nucleotide region corresponding to the nucleoprotein gene. Results: The three samples analyzed were positive by RT-PCR at all the decomposition times evaluated, in contrast to results observed with FAT and MIT, which rapidly became negative. In addition, 100% of the inadequately preserved samples were characterized molecularly. The limit of detection of RT-PCR was 0.5 MICDL50/0.03 mL. Conclusion: RT-PCR can be useful for rabies diagnosis and typing of putrefying samples or rabies isolates stored in inadequate conditions.

scholarly journals Antigenic and genetic characterization of the first rabies virus isolated from the bat Eumops perotis in Brazil

2008 ◽  
Vol 50 (2) ◽  
pp. 95-99 ◽  
Author(s):  
Juliana Galera Castilho ◽  
Flávia Marchizeli Canello ◽  
Karin Corrêa Scheffer ◽  
Samira Maria Achkar ◽  
Maria Luiza Carrieri ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Antibody Test ◽  
Nucleoprotein Gene ◽  
Adult Mice ◽  
Vampire Bats ◽  
The City ◽  
Immunofluorescence Antibody Test ◽  
Immunofluorescence Antibody

Although the main transmitters of rabies in Brazil are dogs and vampire bats, the role of other species such as insectivorous and frugivorous bats deserves special attention, as the rabies virus has been isolated from 36 bat species. This study describes the first isolation of the rabies virus from the insectivorous bat Eumops perotis. The infected animal was found in the city of Ribeirão Preto, São Paulo. The virus was identified by immunofluorescence antibody test (FAT) in central nervous system (CNS) samples, and the isolation was carried out in N2A cell culture and adult mice. The sample was submitted to antigenic typing using a panel of monoclonal antibodies (CDC/Atlanta/USA). The DNA sequence of the nucleoprotein gene located between nucleotides 102 and 1385 was aligned with homologous sequences from GenBank using the CLUSTAL/W method, and the alignment was used to build a neighbor-joining distance-based phylogenetic tree with the K-2-P model. CNS was negative by FAT, and only one mouse died after inoculation with a suspension from the bat's CNS. Antigenic typing gave a result that was not compatible with the patterns defined by the panel. Phylogenetic analysis showed that the virus isolated segregated into the same cluster related to other viruses isolated from insectivorous bats belonging to genus Nyctinomops ssp. (98.8% nucleotide identity with each other).

scholarly journals Molecular epidemiological analysis of wild animal rabies isolates from India

2019 ◽  
Vol 12 (3) ◽  
pp. 352-357 ◽  
Author(s):  
Gundallhalli Bayyappa Manjunatha Reddy ◽  
Rajendra Singh ◽  
Karam Pal Singh ◽  
Anil Kumar Sharma ◽  
Sobharani Vineetha ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Reservoir Host ◽  
Wild Animals ◽  
Rt Pcr ◽  
Epidemiological Analysis ◽  
Nucleoprotein Gene ◽  
Single Cluster

Aim: This study was conducted to know the genetic variability of rabies viruses (RVs) from wild animals in India. Materials and Methods: A total of 20 rabies suspected brain samples of wild animals from different states of India were included in the study. The samples were subjected for direct fluorescent antibody test (dFAT), reverse transcription polymerase chain reaction (RT-PCR), and quantitative reverse transcriptase real-time PCR (RT-qPCR). The phylogenetic analysis of partial nucleoprotein gene sequences was performed. Results: Of 20 samples, 11, 10, and 12 cases were found positive by dFAT, RT-PCR, and RT-qPCR, respectively. Phylogenetic analysis showed that all Indian wild RVs isolates belonged to classical genotype 1 of Lyssavirus and were closely related to Arctic/Arctic-like single cluster indicating the possibility of a spillover of rabies among different species. Conclusion: The results indicated the circulation of similar RVs in sylvatic and urban cycles in India. However, understanding the role of wild animals as reservoir host needs to be studied in India.

scholarly journals Characterization of handmade Nepali paper as a platform for paper analytical device to determine anti-diabetic drug

2021 ◽  
Author(s):  
Ram Bhattarai ◽  
Sanam Pudasaini ◽  
Mukesh Sah ◽  
Bhanu Neupane ◽  
Basant Giri
Keyword(s):  
Limit Of Detection ◽  
Low Cost ◽  
Water Contact ◽  
Regulatory Standards ◽  
Three Samples ◽  
Limit Of Quantitation ◽  
Metformin Concentration ◽  
Analytical Device

The COVID-19 pandemic has highlighted the need of eco-friendly and locally or distributed manufacturing of diagnostic and safety products. Here, we characterized five handmade papers for their potential application to make paper analytical device (PADs). The handmade papers were made from locally available plant fiber using eco-friendly method. Thickness, grammage, and apparent density of the paper samples ranged from 198 μm to 314 μm, 49 g/m2 to 117.8 g/m2, and 0.23 to 0.39 g/cm3, respectively. Moisture content, water filtration and wicking speed ranged from 5.2% to 7.1%, 35.7 to 156.7, and 0.062 to 0.124 mms-1, respectively. Further, water contact angle and porosity ranged from 76˚ to 112˚ and 79% to 83%, respectively. The best paper sample one was chosen to fabricate PADs which were used for the determination of metformin. The metformin assay on PADs followed linear range from 0.0625 to 0.5 mg/mL. The assay had limit of detection and limit of quantitation of 0.05 mg/mL and 0.18 mg/mL respectively. The new method was used to test metformin samples (n=20) collected from local pharmacies. The average amount of metformin concentration in samples was 465.6 ± 15.1mg/tablet. Three samples did not meet the regulatory standards. When compared with spectrophotometric method, PADs assay correctly predicted 18 out of 20 samples. The PADs assay on handmade paper may provide a low-cost and easy-to-use system to screening the quality of drugs and other point-of-need applications.

scholarly journals Digoxigenin-labeled probe for rabies virus nucleoprotein gene detection

2006 ◽  
Vol 39 (2) ◽  
pp. 159-162 ◽  
Author(s):  
Pedro Carnieli Junior ◽  
Armando Moraes Ventura ◽  
Edison Luiz Durigon
Keyword(s):  
Rabies Virus ◽  
Diagnostic Techniques ◽  
N Gene ◽  
Rt Pcr ◽  
Gene Detection ◽  
Nucleoprotein Gene ◽  
Polymerase Chain ◽  
Labeled Probe ◽  
The Cost ◽  
Rabies Diagnosis

A digoxigenin-labeled probe was produced from the Pasteur virus strain for the detection of the rabies virus N gene. The probe hybridization was performed from amplified N gene obtained by reverse transcription polymerase chain reaction and the results by RT-PCR and hybridization showed 100% agreement. The hybridization, when carried out in products amplified by RT-PCR, increases the sensitivity of this technique even more and confers specificity to the diagnosis. The technique described in this work will be useful in rabies diagnosis laboratories, once the cost is compatible with traditional rabies diagnostic techniques.

scholarly journals Molecular characterization of nucleoprotein gene of rabies virus from Maharashtra, India

2016 ◽  
Vol 62 (2) ◽  
pp. 105 ◽  
Author(s):  
P Charan ◽  
S Mehta ◽  
R Dahake ◽  
S Mukherjee ◽  
A Chowdhary
Keyword(s):  
Molecular Characterization ◽  
Rabies Virus ◽  
Nucleoprotein Gene

scholarly journals Phylogenetic relationships of a rabies virus isolate in Costa Rica

2021 ◽  
Vol 14 (1) ◽  
pp. e3713
Author(s):  
Luis Castro Rodríguez ◽  
Bernal León ◽  
Lisbeth Ramírez Carvajal
Keyword(s):  
Costa Rica ◽  
Rabies Virus ◽  
Complete Genome ◽  
Phylogenetic Analyses ◽  
Gene Trees ◽  
Sylvatic Cycle ◽  
Tissue Samples ◽  
Genomic Libraries ◽  
Nucleoprotein Gene ◽  
Complete Genomes

Introduction: The sylvatic cycle of rabies is a significant sanitary burden in Central America. The Costa Rican government monitors cases since 1985 and infections from bats are still reported for wild animals, livestock, and humans, generating a need of further pathogen characterization in the region. Objective: To compare rabies phylogenetic analyses from complete genomes with nucleoprotein gene studies. Methods: For the phylogenetic analyses we used four rabies tissue samples collected in 2018, and generated complete genomes by Next-Generation sequencing (NGS). We also extracted RNA from tissues of confirmed cases and generated ssDNA using several primers. Double-stranded DNA was generated and used to generate genomic libraries. Results: We describe, for the first-time, the complete genome of four sequences of the rabies virus isolated in Costa Rica in 2018. Complete genome trees resembled the topology of nucleoprotein gene trees. All isolates were related to Desmodus rotundus. One sample group into Lineage (L)2, and the remaining samples group in L1, matched previous reports from regional rabies viruses. Conclusion: Our method produces valid viral assemblies from clinical specimens without target enrichment or viral isolation. 

scholarly journals Evaluation of a rapid immunochromatographic diagnostic test (RIDT) for diagnosis of rabies in samples from Argentina

2018 ◽  
Vol 12 (06) ◽  
pp. 415-421 ◽  
Author(s):  
Federico Gury Dohmen ◽  
Esteban Kovacs ◽  
Natalia Elizabeth Prestrera ◽  
Fernando Javier Beltrán
Keyword(s):  
Diagnostic Test ◽  
Rabies Virus ◽  
Limit Of Detection ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Field Conditions ◽  
Clinical Samples ◽  
Viral Origin ◽  
Kappa Test ◽  
Specificity And Sensitivity

Introduction: Rabies is a globally widespread zoonosis of viral origin that causes fatal encephalitis in humans and animals. In countries where rabies is endemic and there is a lack of well-equipped diagnostic laboratories, a rapid immunochromatographic diagnostic test (RIDT) for detection of rabies could be an indispensable tool. In this study we evaluated the limit of detection, as well as specificity and sensitivity of RIDT, compared to the standard fluorescent antibody test (FAT). Methodology: A total of 174 samples were diagnosed by both RIDT and FAT. Fresh clinical samples, poorly conserved samples and brains in advanced state of decomposition generated under laboratory conditions were used to resemble field conditions. The sensitivity of RIDT was evaluated with CVS fixed strain of rabies virus (RABV), previously titrated in 21-day old albino mice and compared with the Reverse Transcription – Polimerase Chain Reaction (RT-PCR) technique in parallel. Additionally, the Mouse Inoculation Test (MIT) was used to perform the antigenic characterization of Rabies virus variants. Results: The limit of detection of RIDT was 100 LD50 / 0.03 mL and its performance, as compared to that of FAT, showed a sensitivity of 97.96%, a specificity of 100% and a concordance by the Kappa test of 0.98 with 95% CI. Conclusions: RIDT provides results comparable to those of FAT and this test can be considered as an appropriate method under the field conditions, even in samples that are not suitable for FAT due to their state of decomposition.

scholarly journals Importation of canid rabies in a horse relocated from Zimbabwe to South Africa : research communication

2005 ◽  
Vol 72 (1) ◽  
Author(s):  
C.T. Sabeta ◽  
J.L. Randles
Keyword(s):  
South Africa ◽  
Rabies Virus ◽  
Fluorescent Antibody ◽  
Clinical Signs ◽  
Antibody Test ◽  
Domestic Dog ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Training Centre ◽  
Polymerase Chain

In July 2003 a 2-year-old Thoroughbred colt was imported from Harare, Zimbabwe to the Ashburton Training Centre, Pietermaritzburg, South Africa. Five months after importation, the colt presented with clinical signs suggestive of rabies: it was uncoordinated, showed muscle tremors and was biting at itself. Brain tissue was submitted for analysis and the clinical diagnosis was confirmed by the fluorescent antibody test and reverse-transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis of the nucleotide sequence of the cytoplasmic domain of the glycoprotein and the G-L intergenic region of the rabies virus confirmed it to be an infection with a canid rabies virus, originating from an area in Zimbabwe endemic for the domestic dog (Canis familiaris) and side-striped jackal (Canis adustus) rabies.

scholarly journals Reconciling epidemiological models with misclassified case-counts for SARS-CoV-2 with seroprevalence surveys: A case study in Delhi, India

2020 ◽  
Author(s):  
Rupam Bhattacharyya ◽  
Ritwik Bhaduri ◽  
Ritoban Kundu ◽  
Maxwell Salvatore ◽  
Bhramar Mukherjee
Keyword(s):  
Diagnostic Tests ◽  
False Negative ◽  
False Negative Rate ◽  
Antibody Test ◽  
Population Based ◽  
National Capital Region ◽  
Rt Pcr ◽  
High False Negative Rate ◽  
True Number ◽  
National Capital

Underreporting of COVID-19 cases and deaths is a hindrance to correctly modeling and monitoring the pandemic. This is primarily due to limited testing, lack of reporting infrastructure and a large number of asymptomatic infections. In addition, diagnostic tests (RT-PCR tests for detecting current infection) and serological antibody tests for IgG (to assess past infections) are imperfect. In particular, the diagnostic tests have a high false negative rate. Epidemiologic models with a latent compartment for unascertained infections like the Susceptible-Exposed-Infected-Removed (SEIR) models can provide predictions for unreported cases and deaths under certain assumptions. Typically, the number of unascertained cases is unobserved and thus we cannot validate these estimates for a real study except for simulation studies. Population-based seroprevalence studies can provide a rough estimate of the total number of infections and help us check epidemiologic model projections. In this paper, we develop a method to account for high false negative rates in RT-PCR in an extension to the classic SEIR model. We apply this method to Delhi, the national capital region of India, with a population of 19.8 million and a COVID-19 hotspot of the country, obtaining estimates of underreporting factor for cases at 34-53 times and that for deaths at 8-13 times. Based on a recently released serological survey for Delhi with an estimated 22.86% seroprevalence, we compute adjusted estimates of the true number of infections reported by the survey (after accounting for misclassification of the antibody test results) which is largely consistent with the model outputs, yielding an underreporting factor for cases from 30-42. Together with the model and the serosurvey, this implies approximately 96-98% cases in Delhi remained unreported and whereas only 109,140 cases were reported on July 10, the true number of infections varied somewhere between 4.4-4.6 million across different estimates. While repeated serological monitoring is resource intensive, model-based adjustments, run with the most up to date data, can provide a viable option to keep track of the unreported cases and deaths and gauge the true extent of transmission of this insidious virus.

scholarly journals In-house modification and improvement of the CDC real-time PCR diagnostic assay for SARS-CoV-2 detection.

2020 ◽  
Author(s):  
Srirupa Das ◽  
Candice Dowell-Martino ◽  
Lisa Arrigo ◽  
Paul N. Fiedler ◽  
Sandra Lobo
Keyword(s):  
Real Time ◽  
Health Care Providers ◽  
Real Time Pcr ◽  
Diagnostic Tests ◽  
Limit Of Detection ◽  
World Health ◽  
Diagnostic Assay ◽  
Care Providers ◽  
Rt Pcr ◽  
The World

The world is currently facing an unprecedented pandemic caused by the novel coronavirus SARS-CoV-2 (COVID-19) which was first reported in late 2019 by China to the World Health Organization (WHO). The containment strategy for COVID-19, which has non-specific flu-like symptoms and where upwards of 80% of the affected has either mild or no symptoms, is critically centered upon diagnostic testing, tracking and isolation. Thus, the development of specific and sensitive diagnostic tests for COVID-19 is key towards the first successful step of disease management. Public health organizations like the WHO and the US-based Centers for Disease Control and Prevention (CDC) have developed real-time PCR (RT-PCR) based diagnostic tests to aid in the detection of acute infection. In this study we sought to modify the CDC RT-PCR diagnostic assay protocol to increase its sensitivity and to make the assay directly portable to health care providers in a community-based hospital setting. A number of modifications to the original protocol were tested. Increasing the RT-PCR annealing temperature by 7°C to 62°C was associated with the most significant improvement in sensitivity, wherein the cycle-threshold (Ct) value for the N2 assay was reduced by ~3 units, in effect both reducing the overall number of inconclusive results and yielding N1/N2 assays to have similar Ct values. The limit of detection of the modified assay was also improved (0.86 RNA copies/μl for both nCoV 2019_N1/N2 assays) compared to the CDC RT-PCR diagnostic assay (1 and 3.16 RNA copies/μl for nCoV 2019_N1 and N2 assay, respectively). Using this modification, there was no significant effect on SARS-CoV-2 detection rate when viral RNA extraction was performed either manually or through an automated extraction method. We believe this modified protocol allows for more sensitive detection of the virus which in turn will be useful for pandemic management.

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