scholarly journals Design of primers in the molecular detection of Feline Panleukopenia Virus

2021 ◽  
Vol 8 (3) ◽  
pp. 019-029
Author(s):  
Cristóbal Heraldo Carreño ◽  
Carlos Osvaldo Navarro ◽  
María Antonieta Jara
Keyword(s):  
High Mortality ◽  
Elisa Test ◽  
Etiologic Agent ◽  
Vp2 Gene ◽  
Mortality And Morbidity ◽  
Feline Panleukopenia Virus ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Negative Controls ◽  
Positive Controls

Feline Panleukopenia is a disease characterized by a reduction in the number of circulating leukocytes and enteritis with degeneration of the intestinal villi. The etiologic agent, called Feline Panleukopenia virus (FPV), belongs to the Parvoviridae family, is highly contagious and has high mortality and morbidity. Although vaccination of healthy cats is the most effective way to prevent the disease, once the symptoms appear, the treatment is supportive, presenting high mortality in the first days of the disease. FPV positive cats should be hospitalized and isolated for at least 2 weeks to avoid viral transmission. Early detection is usually done with the enzyme-linked immunoadsorption assay (ELISA) test that detects viral antigens in stool samples. As a complementary diagnostic method, in this work it was proposed to implement the Polymerase Chain Reaction (PCR) aimed at the diagnosis of FPV initially in positive samples from two feline vaccines and one canine vaccine. As an approximation to real samples, the commercial vaccines were mixed with feces and blood from a healthy cat. The results showed that the In Silico design was successful strategy based on the VP2 gene sequences of FPV available in GenBank® in conjunction with the sharp visualization of positive controls of the expected size and without amplification. nonspecific or negative controls. The fragments obtained has a nucleotide identity percentage greater than 97% with respect to the information available in Genbank® and corroborates the detection of a VP2 fragment. Thus, the future option of applying the same protocol in a diagnostic way is opened, using samples obtained from suspected patients who are infected with FPV.

scholarly journals Dynamic evolution of canine parvovirus in Thailand

2020 ◽  
Vol 13 (2) ◽  
pp. 245-255
Author(s):  
N. Inthong ◽  
S. Kaewmongkol ◽  
N. Meekhanon ◽  
K. Sirinarumitr ◽  
T. Sirinarumitr
Keyword(s):  
Amino Acids ◽  
Clinical Signs ◽  
Canine Parvovirus ◽  
Specific Primers ◽  
Highly Pathogenic ◽  
Vp2 Gene ◽  
Fecal Samples ◽  
Feline Panleukopenia Virus ◽  
Close Relationship ◽  
Polymerase Chain

Background and Aim: According to the previous study, the circulating canine parvovirus (CPV) in Thailand is 2a and 2b. Nowadays, CPV mutants, including CPV-2c, have been identified in many parts of the world. This study aimed to investigate the genetic diversity of the circulating CPV in Thailand. Materials and Methods: Eighty-five CPV-positive fecal samples were obtained from dogs with either acute hemorrhagic diarrhea or diarrhea. The complete VP2 gene of these samples was amplified using VP2 specific primers and polymerase chain reaction (PCR). The obtained full-length VP2 sequences were analyzed and a phylogenetic tree was constructed. Results: Sixty and 25 CPV-positive fecal samples were collected in 2010 and 2018, respectively. Thirty-four samples were new CPV-2a and 31 samples were new CPV-2b due to amino acids substitution at position 297 (Ser-Ala). In 2018, 5 new CPV-2a, 19 CPV-2c, and 1 feline panleukopenia virus (FPV) were found, but no new CPV-2b was detected. Moreover, most of the CPV in this study had amino acids mutations at positions 324 and 440. The phylogenetic construction demonstrated the close relationship between the current new CPV-2a with the previous CPV-2a reported from Thailand, China, Uruguay, Vietnam, Singapore, and India. Interestingly, the current new CPV-2b in this study was not closely related to the previous CPV-2b reported in Thailand. The CPV-2c in this study was closer to Asian CPV-2c and further from either European or South America CPV-2c. Interestingly, FPV was identified in a diarrhea dog. Conclusion: The evolution of CPV in Thailand is very dynamic. Thus, it is important to monitor for CPV mutants and especially the clinical signs relating to these mutants to conduct surveillance for the emergence of new highly pathogenic CPV in the future.

No Evidence of HIV-1 Infection in Seronegative Hemophiliacs and in Seronegative Partners of Seropositive Hemophiliacs through Polymerase Chain Reaction (PCR) and Anti-NEF Serology

1991 ◽  
Vol 65 (05) ◽  
pp. 478-482 ◽  
Author(s):  
Françoise Ferrer-Le-Coeur ◽  
Martine Mariotti ◽  
Philippe Hivert ◽  
Eric Pascal Satre ◽  
Françoise Bouchardeau ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Low Risk ◽  
Peripheral Blood Mononuclear ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Polymerase Chain ◽  
Negative Controls ◽  
Positive Results ◽  
Hiv 1 ◽  
Positive Controls

SummaryWe prospectively studied a well-characterized cohort including 60 seronegative hemophiliacs or von Willebrand’s disease patients, 6 seronegative female sexual partners of seropositive hemophiliacs, 59 seropositive hemophiliacs or von Willebrand’s disease patients and 2 seropositive partners of seropositive hemophiliacs (used as positive controls), and 117 seronegative low risk individuals (used as negative controls). PCR assay, performed in peripheral blood mononuclear cells using three primer pairs in the gag, pol, LTR regions, showed no positive results in the 60 seronegative patients, in the 6 seronegative partners of seropositive patients and in the 117 seronegative low risk individuals, while PCR was positive with at least one primer pair in 53 (87%) of 61 seropositive patients. Anti-nef serology (Westem-blot) was negative in seronegative patients, in seronegative partners of seropositive patients and positive in 58% out of the seropositive individuals. These results strongly suggest an absence of HIV-1 infection in individuals with a lastingly negative HIV serology.

scholarly journals Comparison of partial and full VP2 gene sequences of parvovirus from domestic cats in Turkey

2019 ◽  
Vol 6 (4) ◽  
Author(s):  
Zeynep Akkutay-Yoldar ◽  
B. Taylan Koç
Keyword(s):  
Full Length ◽  
Chain Reaction ◽  
Vp2 Gene ◽  
Feline Panleukopenia Virus ◽  
Partial Length ◽  
Separate Branch ◽  
Gene Coding ◽  
Polymerase Chain ◽  
Em Gene ◽  
Partial Genome

Parvoviruses are ubiquitous pathogens that cause a fatal disease in cats and are able to mutate for cross-species transmission. Both the feline panleukopenia virus (FPV) and the canine parvovirus (CPV), with their antigenic variants, induce a disease in cats that presents with similar signs. The aim of this study was to determine the presence of parvoviruses in blood and exudate samples from five clinically symptomatic cats (from Ankara, Turkey). The gene coding for the VP2 structural capsid protein of the obtained parvoviruses was amplified by polymerase chain reaction (PCR), purified and partially or nearly full-length sequenced. The maximum likelihood (ML) method was used for molecular characterization. Phylogenetic analysis based on nearly full-length sequencing of the VP2 gene and amino acid arrangement showed that four of the viral strains were closely related and localized in the same FPV cluster. The fifth strain found was located in the same cluster but on a separate branch. Viral field strains were included in the CPV-2 group as determined by partial genome analysis: four fitted in the CPV-2c, and one in a separate clade within the CPV-2b group. To our knowledge, this is the first report that details nearly full-length VP2 gene characterisation in Turkish cats. Overall, nearly full-length VP2 contrasts were more effective to determine the origin of parvovirus strains, than partial length comparisons.

scholarly journals Differential Diagnosis ofEntamoebaspp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Thiago dos Santos Gomes ◽  
Mariana Coimbra Garcia ◽  
Flavia de Souza Cunha ◽  
Heloisa Werneck de Macedo ◽  
José Mauro Peralta ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Intestinal Parasites ◽  
Laboratory Diagnosis ◽  
Sybr Green ◽  
Similar Species ◽  
Green Is ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Negative Controls

Amoebiasis, a disease caused byEntamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of theE. histolytica/E. disparcomplex. Furthermore, morphologically similar species such asEntamoeba hartmannicontribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system forE. histolyticaandE. disparand a single real-time PCR forE. hartmanni. The multiplex protocol detected up to 0.0143 pg ofE. histolyticaDNA and 0.5156 pg ofE. disparDNA, and the average melting temperature (Tm) was 73°C and 70°C, respectively. ForE. hartmanni, theTmwas 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested,E. disparDNA was detected in 37; none exhibitedE. histolyticaDNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however,E. hartmanniDNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

scholarly journals Nanocarriers of Miconazole or Fluconazole: Effects on Three-Species Candida Biofilms and Cytotoxic Effects In Vitro

2021 ◽  
Vol 7 (7) ◽  
pp. 500
Author(s):  
Anne Caroline Morais Caldeirão ◽  
Heitor Ceolin Araujo ◽  
Laís Salomão Arias ◽  
Wilmer Ramírez Carmona ◽  
Gustavo Porangaba Miranda ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Artificial Saliva ◽  
Cytotoxic Effects ◽  
Promising Alternative ◽  
Colony Forming Units ◽  
Mtt Reduction ◽  
Diphenyl Tetrazolium Bromide ◽  
Negative Controls ◽  
Positive Controls

The contribution of different Candida species in oral fungal infections has stimulated the search for more effective therapies. This study assessed the antibiofilm effects of nanocarriers of miconazole (MCZ) or fluconazole (FLZ) on Candida biofilms, and their cytotoxic effects on murine fibroblasts. Three-species biofilms (Candida albicans/Candida glabrata/Candida tropicalis) were formed on 96-well plates, and they were treated with nanocarriers (iron oxide nanoparticles coated with chitosan—“IONPs-CS”) of MCZ or FLZ at 39/78/156 µg/mL; antifungals alone at 156 µg/mL and artificial saliva were tested as positive and negative controls, respectively. Biofilms were analyzed by colony forming units (CFU), biomass, metabolic activity, and structure/viability. The cytotoxicity (L929 cells) of all treatments was determined via 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction assay. Data were submitted to one- or two-way ANOVA, followed by Tukey’s or Fisher LSD’s tests (p < 0.05). IONPs-CS-MCZ at 78 µg/mL promoted similar antibiofilm and cytotoxic effects compared with MCZ at 156 µg/mL. In turn, IONPs-CS-FLZ at 156 µg/mL was overall the most effective FLZ antibiofilm treatment, surpassing the effects of FLZ alone; this nanocarrier was also less cytotoxic compared with FLZ alone. It can be concluded that both nanocarriers are more effective alternatives to fight Candida biofilms compared with their respective positive controls in vitro, being a promising alternative for the treatment of oral fungal infections.

scholarly journals Seasonal Differences in Cyclospora cayetanensis Prevalence in Colombian Indigenous People

2021 ◽  
Vol 9 (3) ◽  
pp. 627
Author(s):  
Hagen Frickmann ◽  
Juliane Alker ◽  
Jessica Hansen ◽  
Juan Carlos Dib ◽  
Andrés Aristizabal ◽  
...  
Keyword(s):  
Indigenous People ◽  
Rainy Season ◽  
Gastrointestinal Symptoms ◽  
Dry Season ◽  
Seasonal Effects ◽  
Chain Reaction ◽  
Resource Limited Settings ◽  
Resource Limited ◽  
Stool Samples ◽  
Polymerase Chain

Fecal-orally transmitted cyclosporiasis is frequent in remote resource-limited settings in Central and South America with poor hygiene conditions. In this study, we aimed at assessing seasonal effects on the epidemiology of colonization or infection with C. cayetanensis in Colombian indigenous people living under very restricted conditions. In the rainy season between July and November and in the dry season between January and April, stool samples from indigenous people with and without gastrointestinal symptoms were collected and screened for C. cayetanensis applying in-house real-time polymerase chain reaction (PCR). In the rainy season and in the dry season, positive PCR results were observed for 11.8% (16/136) and 5.1% (15/292), respectively, with cycle threshold (Ct) values of 30.6 (±3.4) and 34.4 (±1.6), respectively. Despite higher parasite loads in the rainy season, fewer individuals (2/16, 12.5%) reported gastrointestinal symptoms compared to the dry season (6/15, 40%). In conclusion, considerable prevalence of C. cayetanensis in Colombian indigenous people persists in the dry season. Low proportions of gastrointestinal symptoms along with higher parasite loads make colonization likely rather than infection.

scholarly journals Multicenter Evaluation of an ELISA for the Detection of Cryptosporidium spp. Antigen in Clinical Human Stool Samples

2021 ◽  
Vol 9 (2) ◽  
pp. 209
Author(s):  
Romy Razakandrainibe ◽  
Célia Mérat ◽  
Nathalie Kapel ◽  
Marc Sautour ◽  
Karine Guyot ◽  
...  
Keyword(s):  
Large Scale ◽  
Cryptosporidium Oocyst ◽  
Clinical Importance ◽  
Epidemiological Studies ◽  
Elisa Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cryptosporidium Spp ◽  
Stool Samples ◽  
Conventional Microscopy ◽  
Human Stool

Human cryptosporidiosis remains underdiagnosed, and rapid/accurate diagnosis is of clinical importance. Diagnosis of the Cryptosporidium oocyst in stool samples by conventional microscopy is labor-intensive, time-consuming, and requires skillful experience. Thus, we aimed to evaluate the usefulness of a coproantigen enzyme-linked immunosorbent assay (ELISA) test in detecting Cryptosporidium spp. from fecal specimens. For this aim, we evaluated the performances of a commercial ELISA (CoproELISA Cryptosporidium kit, Savyon Diagnostics, Israel) for the detection of Cryptosporidium spp. in random clinical stool samples through a multicenter study. The sensitivity and specificity for coproantigen ELISA were 98.86% and 94.32%, respectively. The coproantigen ELISA results indicate that the simple, rapid, reliable, and standardized immunoassay test is sensitive and specific for routine diagnosis, and may be useful for large-scale epidemiological studies of cryptosporidiosis.

scholarly journals High Frequency of Cryptosporidium hominis Infecting Infants Points to A Potential Anthroponotic Transmission in Maputo, Mozambique

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 293
Author(s):  
Idalécia Cossa-Moiane ◽  
Hermínio Cossa ◽  
Adilson Fernando Loforte Bauhofer ◽  
Jorfélia Chilaúle ◽  
Esperança Lourenço Guimarães ◽  
...  
Keyword(s):  
Public Hospitals ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
P Value ◽  
Cryptosporidium Hominis ◽  
Cryptosporidium Spp ◽  
Pcr Rflp ◽  
Maputo City ◽  
Stool Samples ◽  
Polymerase Chain

Cryptosporidium is one of the most important causes of diarrhea in children less than 2 years of age. In this study, we report the frequency, risk factors and species of Cryptosporidium detected by molecular diagnostic methods in children admitted to two public hospitals in Maputo City, Mozambique. We studied 319 patients under the age of five years who were admitted due to diarrhea between April 2015 and February 2016. Single stool samples were examined for the presence of Cryptosporidium spp. oocysts, microscopically by using a Modified Ziehl–Neelsen (mZN) staining method and by using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) technique using 18S ribosomal RNA gene as a target. Overall, 57.7% (184/319) were males, the median age (Interquartile range, IQR) was 11.0 (7–15) months. Cryptosporidium spp. oocysts were detected in 11.0% (35/319) by microscopy and in 35.4% (68/192) using PCR-RFLP. The most affected age group were children older than two years, [adjusted odds ratio (aOR): 5.861; 95% confidence interval (CI): 1.532–22.417; p-value < 0.05]. Children with illiterate caregivers had higher risk of infection (aOR: 1.688; 95% CI: 1.001–2.845; p-value < 0.05). An anthroponotic species C. hominis was found in 93.0% (27/29) of samples. Our findings demonstrated that cryptosporidiosis in children with diarrhea might be caused by anthroponomic transmission.

scholarly journals 781. C.difficile PCR+/ Toxin EIA- treat or not treat? A clinician survey

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S435-S436
Author(s):  
Sarath G Nath ◽  
Francesca Lee ◽  
Anjali Bararia ◽  
Ank E Nijhawan
Keyword(s):  
Clinical Judgment ◽  
Clinical Care ◽  
Cost Effective ◽  
False Positive Diagnosis ◽  
Missed Diagnosis ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Low Sensitivity ◽  
Survey Responses ◽  
And Training

Abstract Background C.difficile Toxin Polymerase Chain Reaction (C.diff PCR) and C.difficile Toxin Enzyme Immunoassays (toxin EIA) are commonly used tests to diagnose Clostridoides difficile infection (CDI). C.diff PCR cannot differentiate between colonization and infection, leading to a higher false-positive diagnosis of CDI. Toxin EIA has low sensitivity leading to a missed diagnosis of CDI. In patients with C.diff PCR positive(+) and Toxin EIA negative(-), clinical judgment is often needed regarding the decision to treat or not to treat. C.diff cytotoxic assay (CCA), is a more sensitive method to detect the toxin but is time-consuming and not readily available. Methods Between 6/2019 and 12/2019, 83 patients who were admitted to the hospital, met our inclusion criteria (C.diff PCR+/EIA-). Clinicians who cared for these patients were contacted and surveyed with a predesigned questionnaire evaluating the rationale of treatment. Also, a simultaneous medical records review was done to ensure consistency. Along with this C.diff PCR+/EIA- stool samples were sent to ARUP laboratories for CCA. The CCA results were not available for clinicians and did not impact clinical care. Average cost for a CCA assay was $29 Results Demographics of the clinicians were variable (Table 1). Several parameters were considered when making decisions regarding treatment and GI/ID were frequently involved (figure 1). Among the 83 patients, 41(49%) were CCA (+) and 42(51%) were CCA (-). 48 of 83 (58%) patients received treatment for CDI. 25 of 48 (52%) patients who were treated were CCA positive while 23 of 48 (48%) patients were CCA negative. Among the untreated patients, 16/35 (46%) were CCA+ while 19/35(54%) were CCA-. There was no statistically significant correlation between clinical judgment and CCA assay results (p: 0.56 on the Chi test). Demographics of the clinicians Clinician survey responses CDI Treatment and by CCA positivity Conclusion Clinicians regardless of their background and training face challenges with the treatment of C.diff PCR+/EIA- patients. Patient outcomes based on the incorporation of CCA assay into an algorithm for C.diff PCR+/EIA- patients, need to be evaluated. But it has a potential role in stopping unnecessary CDI treatment as well as avoidance of missed treatment opportunities while possibly also being cost-effective. Disclosures Ank E. Nijhawan, MD, MPH, Gilead (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support)

Investigation of Children with Acute Gastroenteritis by Multiplex PCR Method in Central Part of Turkey

2022 ◽  
Author(s):  
Fatih Yılmaz ◽  
Havva Kaya ◽  
Mehmet Özdemir
Keyword(s):  
Multiplex Pcr ◽  
Acute Gastroenteritis ◽  
Hospital Admissions ◽  
Age Groups ◽  
Viral Gastroenteritis ◽  
Viral Agent ◽  
Mortality And Morbidity ◽  
Stool Samples ◽  
Pcr Method ◽  
Norovirus Gii

Abstract Objective Gastroenteritis is a disease that affects all age groups, especially children, and causes high mortality and morbidity in all countries. The most common agents of acute gastroenteritis are viral agents. As a result, millions of diarrhea attacks and hospital admissions occur worldwide every year due to viral gastroenteritis. This study uses the multiplex polymerase chain reaction (PCR) method to investigate the viruses that are the causative agents of viral gastroenteritis in the pediatric patient group in Konya, Turkey. Methods Stool samples of 94 patients aged 0 to 18 years sent from Emergency clinics and Pediatric outpatient clinics, Meram Medical Faculty Hospital Pediatric clinics, Konya Necmettin Erbakan University to Medical Microbiology Laboratory with a diagnosis of gastroenteritis between February and December 2018 were included in the study. Stool samples were stored at –80°C until the time of the analysis. Deoxyribonucleic acid/ribonucleic acid isolation from stool samples was performed with EZ1 Virus Mini Kit v2.0 (Qiagen, Hilden, Germany) using an automatic extraction system (BioRobot EZ1 system, Qiagen). The presence of astrovirus, rotavirus, adenovirus, norovirus (GI, GII), and sapovirus agents was investigated by the multiplex PCR method (Fast Track Diagnostics, Luxembourg) viral gastroenteritis kit. Results Viral gastroenteritis agents were detected in 56.3% of the patients. One viral agent was detected in 47 (50%) of these patients and at least two viral agents in 6 (6.3%) of them. Norovirus GII was detected in 20 (21.2%) of the children included in the study, adenovirus in 13 (13.8%), rotavirus in 11 (12.8%), astrovirus in 11 (11.7%), sapovirus in 4 (4.2%), and norovirus GI in 1 (1.06%). When the distribution of viral agents was examined by months, the most number of agents were observed (21; 35%) in May, followed by April and June (12; 20%). Considering the distribution of the prevalence of the agents by age, it was seen to be mainly between 0 and 12 months (42%). Conclusion Considering that the most common viral agent in our region is norovirus GII, it will be useful to investigate the norovirus that is not routinely examined in children who are admitted to clinics with the complaint of gastroenteritis. It will be appropriate to examine routinely adenovirus, rotavirus, and norovirus in the laboratory, especially in children with diarrhea and vomiting in the winter and spring months.

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