Influence of euthanasia, the intensity of inflammatory lesion and viral load in the laboratory diagnosis of rabies in cattle

2021 ◽  
Vol 41 ◽  
Author(s):  
Claudia S. Wisser ◽  
Marcélia E.S. Fernandes ◽  
Elaine Melchioretto ◽  
Daiane Ogliari ◽  
Aldo Gava ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Load ◽  
Inflammatory Lesion ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Clinical Evolution ◽  
Early Stages ◽  
Polymerase Chain

ABSTRACT: This research reports the use of different diagnostic tests in cattle, naturally infected by Rabies lyssavirus (RABV), and correlates the positivity of the tests with the clinical moment of euthanasia, the intensity of the inflammatory lesion and viral load. It also highlights the possibility of euthanasia in early stages of the disease as a way to improve animal welfare. For that, samples of 34 bovine brains were collected for analysis, preserved in 10% buffered formaline and refrigerated with subsequent freezing. The samples were subjected to direct immunofluorescence antibody technique (DFAT) tests, viral isolation in cell culture (VICC), histopathology with hematoxylin and eosin staining (HE), immunohistochemistry (IHC), Shorr stainied neural tissue smears (DSS), Reverse transcription polymerase chain reaction (RT-PCR) and polymerase chain reaction by quantitative reverse transcriptase (qRT-PCR). The areas used for analysis were the cerebellum, parietal telencephalon and thalamus. Samples with Negri bodies (NBs) or immunostaining in at least one of the analyzed areas were considered positive. For the study of the intensity of histological lesions, the lesions were classified into grades 0, 1, 2 and 3 and the positivity of the test in the presence or absence of NBs in one of the three areas analyzed. To verify the influence of the disease clinical evolution, 4-four groups of analysis were created according to the animal’s clinical status at moment of the euthanasia, being: M1 = animal euthanized while standing, M2 = euthanized when in sternal recumbence, M3 = euthanized when in lateral recumbence, M4 = animal with natural death. Of the 34 brains evaluated, IHC was positive in 100% of cases, DFAT was positive in 97.05% of them, and in this negative sample the presence of RABV was confirmed by VICC. NBs ere seen in 88.23% of the cases, and the DSS test was positive in 82.35% of them. All diagnostic techniques showed positive cases in all groups analyzed. Each case was positive in at least two diagnostic methods. All cases that contained NBs were positive for rabies in the other tests. In this study, it was observed that the variables analyzed (intensity of injury and clinical evolution at the moment of euthanasia) had an influence only on HE and DSS techniques, which are based on NB research to form the diagnosis, but did not interfere with the effectiveness of the diagnosis performed by detecting the viral antigen performed by DFAT and IHC. All isolated RABV samples included in the present study have a genetic lineage characteristic of hematophagous bats Desmodus rotundus. The evaluation of qRT-PCR showed that the amount of virus did not interfere in the positivity of the tests. This work shows that IHC and DFAT are safe diagnostic techniques. They are capable of detecting RABV even in euthanized animals in the early stages of clinical evolution with mild intensities of histological lesions.

scholarly journals Cattle rabies: the effect of clinical evolution, viral genetic lineage, and viral load on the severity of histological lesions

2020 ◽  
Vol 40 (4) ◽  
pp. 227-233
Author(s):  
Claudia S. Wisser ◽  
André Thaler Neto ◽  
Helena B.C.R. Batista ◽  
Enio Mori ◽  
Maria E.R. Chierato ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Fluorescent Antibody ◽  
Inflammatory Lesion ◽  
Genetic Lineage ◽  
Fluorescent Antibody Technique ◽  
Chain Reaction ◽  
Antibody Technique ◽  
Qrt Pcr ◽  
Polymerase Chain

ABSTRACT: Our objective was the characterization and staging of histological lesions in different anatomical sites of the central nervous system (CNS) of rabid cattle. The severity of the lesions was compared with the clinical stages of the disease, the variants of viral isolates, and with the load of virus. Thirty-one spontaneously affected rabid cattle the state of Santa Catarina underwent clinical follow-up and were eventually necropsied. CNS tissues were sampled and submitted to direct fluorescent antibody technique (DFAT), immunohistochemistry (IHC), routine histopathology with hematoxylin and eosin stain (HE), reverse transcriptase polymerase chain reaction (RT-PCR), and polymerase chain reaction in quantitative reverse transcriptase in real time (qRT-PCR). Affected cattle were allotted in four groups according to their clinical stage when euthanized: G1, euthanized while standing; G2, euthanized when in sternal recumbence; G3, euthanized when in lateral recumbence; and G4, affected cattle with natural death. In order to evaluate the degree of severity of the lesions and the presence of Negri bodies (NBs), the brain was sectioned at 9 sites. Additionally, spinal cord and trigeminal ganglion sections were examined. The intensity of the lesions was graded as either absent, mild, moderate, or marked, and the presence or absence of the NBs was noted. Histological lesions were characterized by lymphocytic and monocytic meningoencephalitis with NBs in 28 cases. In all analyzed groups, intensities of histological lesions ranging from mild to severe were observed. Brain regions with the highest inflammatory lesion intensity were the medulla at the level of obex, followed by the colliculus and thalamus. NBs were observed in a higher percentage in the cerebellum, followed by medulla at the obex level, striatum complex, and frontal telencephalon. The duration of the clinical course of the disease did not influence the intensity of the inflammatory lesion, but it did influence the presence of NBs, with a higher percentage of these inclusions in cattle that died naturally than in euthanized cattle. All isolated rhabdovirus included in this study were genetically compatible with samples from hematophagous bats Desmodus rotundus. The evaluation by qRT-PCR did not demonstrate a correlation between lesion intensity and the amount of virus.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Performance of the Innova SARS-CoV-2 antigen rapid lateral flow test in the Liverpool asymptomatic testing pilot: population based cohort study

BMJ ◽  
2021 ◽  
pp. n1637 ◽  
Author(s):  
Marta García-Fiñana ◽  
David M Hughes ◽  
Christopher P Cheyne ◽  
Girvan Burnside ◽  
Mark Stockbridge ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Load ◽  
Predictive Value ◽  
High Viral Load ◽  
Test Results ◽  
Chain Reaction ◽  
Viral Loads ◽  
Flow Test ◽  
Polymerase Chain ◽  
Lateral Flow Test

Abstract Objective To assess the performance of the SARS-CoV-2 antigen rapid lateral flow test (LFT) versus polymerase chain reaction testing in the asymptomatic general population attending testing centres. Design Observational cohort study. Setting Community LFT pilot at covid-19 testing sites in Liverpool, UK. Participants 5869 asymptomatic adults (≥18 years) voluntarily attending one of 48 testing sites during 6-29 November 2020. Interventions Participants were tested using both an Innova LFT and a quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) test based on supervised self-administered swabbing at testing sites. Main outcome measures Sensitivity, specificity, and predictive values of LFT compared with RT-qPCR in an epidemic steady state of covid-19 among adults with no classic symptoms of the disease. Results Of 5869 test results, 22 (0.4%) LFT results and 343 (5.8%) RT-qPCR results were void (that is, when the control line fails to appear within 30 minutes). Excluding the void results, the LFT versus RT-qPCR showed a sensitivity of 40.0% (95% confidence interval 28.5% to 52.4%; 28/70), specificity of 99.9% (99.8% to 99.99%; 5431/5434), positive predictive value of 90.3% (74.2% to 98.0%; 28/31), and negative predictive value of 99.2% (99.0% to 99.4%; 5431/5473). When the void samples were assumed to be negative, a sensitivity was observed for LFT of 37.8% (26.8% to 49.9%; 28/74), specificity of 99.6% (99.4% to 99.8%; 5431/5452), positive predictive value of 84.8% (68.1% to 94.9%; 28/33), and negative predictive value of 93.4% (92.7% to 94.0%; 5431/5814). The sensitivity in participants with an RT-qPCR cycle threshold (Ct) of <18.3 (approximate viral loads >10 6 RNA copies/mL) was 90.9% (58.7% to 99.8%; 10/11), a Ct of <24.4 (>10 4 RNA copies/mL) was 69.4% (51.9% to 83.7%; 25/36), and a Ct of >24.4 (<10 4 RNA copies/mL) was 9.7% (1.9% to 23.7%; 3/34). LFT is likely to detect at least three fifths and at most 998 in every 1000 people with a positive RT-qPCR test result with high viral load. Conclusions The Innova LFT can be useful for identifying infections among adults who report no symptoms of covid-19, particularly those with high viral load who are more likely to infect others. The number of asymptomatic adults with lower Ct (indicating higher viral load) missed by LFT, although small, should be considered when using single LFT in high consequence settings. Clear and accurate communication with the public about how to interpret test results is important, given the chance of missing some cases, even at high viral loads. Further research is needed to understand how infectiousness is reflected in the viral antigen shedding detected by LFT versus the viral loads approximated by RT-qPCR.

Comparison of Droplet Digital Polymerase Chain Reaction (ddPCR) and Real-Time Quantitative Polymerase Chain Reaction (qPCR) in Detecting Neonatal Invasive Fungal Infections

2021 ◽  
Vol 11 (3) ◽  
pp. 373-379
Author(s):  
Huitao Li ◽  
Xueyu Chen ◽  
Xiaomei Qiu ◽  
Weimin Huang ◽  
Chuanzhong Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Fungal Isolation ◽  
Blood Samples ◽  
Fungal Strains ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Invasive fungal infection (IFI) is the leading cause of death in neonatal patients, yet the diagnosis of IFI remains a major challenge. At present, most IFI laboratory diagnostic methods are based on classical, but limited, methods such as fungal isolation and culture and histopathological examination. Recently, quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) technology have been adopted to quantify nucleic-acid identification. In this study, we established qPCR and ddPCR assays for IFI diagnosis and quantification. qPCR and ddPCR were carried out using identical primers and probe for the amplification of 18S rRNA. Assay results for three fungal strains were positive, whereas ten non-fungal strains had negative results, indicating 100% specificity for both ddPCR and qPCR methods. Genomic DNA of Candida albicans was tested after a serial dilution to compare the sensitivity of the two PCR methods. The limit of detection of ddPCR was 3.2 copies/L, which was a ten-fold increase compared with that of the qPCR method (32 copies/L). Blood samples from 127 patients with high-risk factors and clinical symptoms for IFI were collected from a NICU in Shenzhen, China, and analyzed using qPCR and ddPCR. Thirty-four blood samples from neonates had a proven or probable diagnosis of IFI, and 25 of these were positive by qPCR, whereas 30 were positive by ddPCR. Among the 93 blood samples from neonates who had a possible IFI or no IFI, 24 were positive using qPCR, and 7 were positive using ddPCR. In conclusion, ddPCR is a rapid and accurate pan-fungal detection method and provides a promising prospect for IFI clinical screening.

scholarly journals UTILITY OF ANTIGEN DETECTION TEST AND POLYMERASE CHAIN REACTION IN THE DIFFERENTIATION OF TUBERCULOUS AND NON-TUBERCULOUS MYCOBACTERIA

2017 ◽  
Vol 10 (6) ◽  
pp. 289
Author(s):  
Yogita Singh ◽  
Raji Vasanth ◽  
Shrikala Baliga ◽  
Dhanashree B
Keyword(s):  
Polymerase Chain Reaction ◽  
False Negative ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Chain Reaction ◽  
College Hospital ◽  
Medical College ◽  
Negative Results ◽  
Polymerase Chain ◽  
Mycobacterium Spp

Objectives: Cultivation and identification of mycobacteria to species level remains difficult and time-consuming. Hence, easy and rapid diagnostic methods are necessary for the differentiation of Mycobacterium tuberculosis (MTB) from non-tuberculous mycobacteria (NTM). The present study aims to detect and differentiate MTB from NTM isolated from clinical samples by immunochromatographic test (ICT) and polymerase chain reaction (PCR). Methods: Over a period of 1 year, clinical samples (n=496) received from suspected cases of TB, at the Department of Microbiology, Kasturba Medical College Hospital, Mangalore were cultured to isolate Mycobacterium spp. Identification of all the isolates was done by conventional biochemical technique, ICT, and PCR. Results: Among the 496 samples processed, 49 (9.87%) were acid-fast bacilli smear positive and 59 (11.89%) samples showed the growth of Mycobacterium spp. Among these, 10 were rapid growers, 49 were slow-growing mycobacteria, out of which 30 were MTB as identified by conventional biochemical reaction. Out of 59 Mycobacterial isolates subjected to ICT for the detection of MPT 64 antigen, only 28 were identified as MTB. However, all the 30 isolates were correctly identified as MTB by PCR. Conclusion: Hence, PCR is essential for rapid differentiation of non-tuberculous Mycobacterium from MTB. False negative results seen with immunochromatographic MPT 64 antigen assay could be due to mutations within the mpt64 gene. Further studies are necessary to characterize these PCR-positive and immunochromatographic assay negative MTB isolates.

scholarly journals Comparative Study of Immune-Diagnostic Tools with Polymerase Chain Reaction in Sub-Clinical Leishmaniasis Isolates

2011 ◽  
Vol 12 (1) ◽  
pp. 34-39 ◽  
Author(s):  
Nazar M Abdalla
Keyword(s):  
Polymerase Chain Reaction ◽  
Skin Test ◽  
Diagnostic Methods ◽  
Diagnostic Tools ◽  
Enzyme Linked Immunosorbent Assay ◽  
Clinical Form ◽  
Chain Reaction ◽  
Leishmanin Skin Test ◽  
Wide Range ◽  
Polymerase Chain

Objective: This study aimed to identify cases of leishmaniasis in the Nuba Mountain area, which is situated in a unique geographical site located in the centre of Sudanese leishmania belt. Wide range of investigations are available for detection of leishmania cases, but still the most reliable and easy test used as screening and epidemiological tool in field studies needs to be evaluated. The most commonly used conventional diagnostic methods direct microscopy and culture have some drawbacks in diagnosing subclinical cases of leishmaniasis. Materials and methods: In this study, comparative properties of various immune-diagnostic tools with Polymerase Chain Reaction used in sub-clinical leishmaniasis isolates were explored. The immune-diagnostic tools involved in this study include- Leishmanin Skin Test (LST), Enzyme Linked Immunosorbent Assay (ELISA) and Direct Agglutination Test (DAT). The study was conducted in the Green Valley village (Rashad Province, South Kordofan State) with a population of 332. Most of the villagers presented with sub-clinical form of leishmaniasis with minor symptoms and signs without the features found in clinical form of visceral leishmaniasis such as fever, diarrhoea, epistaxis, enlarged lymph nodes, spleen and liver. In this study we collected demographic, clinical and epidemiological data using special questionnaire. Leishmanin skin test (LST), ELISA, DAT and PCR for parasite DNA detection were used. Result: The final positive cases detected by PCR were 32 out of 332 belong to L. donovani species. The final positive cases detected by LST were 51.2% of the total population under study, while 11 out of the 37 tested samples were positive by ELISA. All of the 332 villagers showed negative readings by DAT with exception of three individuals who were positive with very high titers. Conclusion: DNA etxtraction and amplification with primers can be a good screening tool in subclinical leishmaniasis isolates. Keyword: Sub-clinical; Leishmaniasis; Leishmanin Skin Test; ELISA; DAT; PCR. DOI: 10.3329/jom.v12i1.5422J Medicine 2011; 12 : 34-39

scholarly journals TRC4 Gene Based PCR Assay in Diagnosis of Tuberculous Meningitis

2017 ◽  
Vol 8 (2) ◽  
pp. 19-22
Author(s):  
Abu Naser Ibne Sattar ◽  
Sanjida Khondakar Setu ◽  
Ahmed Abu Saleh ◽  
Sharmeen Ahmed
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Manifestation ◽  
Tuberculous Meningitis ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Early Stages ◽  
Lowenstein Jensen ◽  
Polymerase Chain ◽  
Tb Pcr ◽  
Is6110 Element

The prevalence of Tuberculous meningitis remains largely underestimated due to nonspecific clinical manifestation at early stages. A total of 20 CSF samples were studied from clinically suspected cases of tuberculous meningitis. All the samples were processed for ZN staining, TB culture on LJ media and TB-PCR using IS6110 and TRC4 primers by standard protocols. Of the total 20 CSF samples, 12 cases were diagnosed as TB meningitis. Most of the tuberculous meningitis cases were found positive by PCR using TRC4 primers (83.33%) followed by IS6110 primer (66.67%) and culture on L-J media(8.33%). None were found positive by ZN staining. TB-PCR usingTRC4 primer showed higher positivity than using IS6110 in detecting tuberculous meningitis, since some strains of MTB may lack the IS6110 element in their genome. PCR assay using TRC4 primer is superior in diagnosing tuberculous meningitis. This study was aimed to evaluate the diagnostic potentials of CSF polymerase chain reaction (PCR) by using TRC4 and IS6110 primers. Further the results were also compared with culture on Lowenstein-Jensen (LJ) media and AFB smear by Zeihl-Nelson (ZN) staining.Bangladesh J Med Microbiol 2014; 08 (02): 19-22

scholarly journals Polymerase Chain Reaction and blood culture for diagnosis of canine sepsis

2018 ◽  
Vol 48 (6) ◽  
Author(s):  
Marcelo Marques da Silveira ◽  
Stéfhano Luis Cândido ◽  
Karin Rinaldi dos Santos ◽  
Maerle Oliveira Maia ◽  
Roberto Lopes de Souza ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Culture ◽  
Diagnostic Value ◽  
Turnaround Time ◽  
Diagnostic Techniques ◽  
Pcr Analysis ◽  
Chain Reaction ◽  
Suspected Sepsis ◽  
Fungal Dna ◽  
Polymerase Chain

ABSTRACT: Sepsis is characterized by the presence of organ dysfunction secondary to the dysregulated systemic inflammatory response associated with an infection, and has high mortality rates. Traditional diagnostic techniques based on non-microbiological isolation are time-consuming and may delay treatment. Thus, this study aimed to compare bacterial and fungal broad-range polymerase chain reaction (PCR) and blood culture for diagnosis of sepsis in dogs. Blood samples from 88 dogs with suspected sepsis were analyzed by blood culture, and PCR to detect bacterial and fungal DNA. On blood culture, 20 (22.7%) samples tested positive for bacterial isolates; however, none tested positive for fungi. Through PCR analysis, bacterial DNA was detected in 46 (52.3%) animals, whereas fungal DNA was present in one (1.1%) sample. Our results showed that PCR-based testing has important diagnostic value for canine blood infections because it has a shorter turnaround time and higher sensitivity than traditional blood culture.

scholarly journals COVID-19, A New Outlook on Pandemic Symptoms and Diagnostic Approach

2022 ◽  
Vol 02 (01) ◽  
Author(s):  
María Fernanda Calderón Hernádez ◽  
Keyword(s):  
Diabetes Mellitus ◽  
Risk Factors ◽  
Polymerase Chain Reaction ◽  
Latin America ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Specificity And Sensitivity ◽  
Polymerase Chain ◽  
Effective Diagnosis ◽  
Age And Sex

Background: The main objective of this research is to learn the symptoms that occur in this pathology, since we are currently still fighting COVID-19, because of this, it is important to keep us informed about the different diagnostic methods available, which help us reach an earlier and more effective diagnosis. Various articles have been compiled to identify as soon as possible the active cases and thus reduce the number of infections. Materials and methods: This research was conducted on the basis of scientific articles and books, related to COVID-19. Methods: This research was conducted based on 15 scientific articles and 3 books, related to COVID-19. Results: The most important risk factors are diabetes mellitus, hypertension, obesity, age and sex. The most common symptoms in Latin America are dry cough, fatigue, sore throat, and fever. The preferred diagnostic test for COVID-19 is the polymerase chain reaction for its specificity and sensitivity Conclusions: As a conclusion, the main objective of the research was achieved, which is to inform the reader about the most relevant symptoms of SARS-CoV-2 in order to improve the identification of suspected cases. Furthermore, we compare various diagnostic methods that exist to date and determine that PCR is the most specific and sensitive.

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