Detection Cell-Free DNA (cfDNA) Using Nested-PCR as a Diagnosis Tool for Human Fascioliasis Infection

2020 ◽  
Author(s):  
Mojgan ARYAEIPOUR ◽  
Bahram KAZEMI ◽  
Arezoo BOZORGOMID ◽  
Mahdi MOHEBALI ◽  
Hakim AZIZI ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Deoxyribonucleic Acid ◽  
Urine Samples ◽  
Parasitic Diseases ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Serum Samples ◽  
Human Fascioliasis ◽  
Stool Samples ◽  
Human Stool

Background: We aimed to detect Fasciola specific deoxyribonucleic acid (DNA) by nested-PCR assay on human stool and urine samples and compare the results with the respective ELISA diagnostic assay. Methods: Overall, 206 clinically suspected cases of fascioliasis were enrolled in the study. Blood samples were collected from all the patients, and serum samples were isolated. ELISA assay, using Fasciola somatic antigen (SA), was carried out to detect anti Fasciola antibodies for the collected sera. DNA was randomly extracted from 25 stool and 10 urine samples of seropositive individuals and was evaluated by conventional PCR and nested PCR methods. The nested-PCR results were confirmed by sequencing the 430 bp region of ribosomal ITSI gene. Stool and urine samples from patients with different parasitic diseases and 25 stool samples from healthy individuals served as controls. Urine samples were collected from 10 healthy controls as well. Results: Fascioliasis was detected by ELISA in 24.8% of the individuals. Of these, 25 seropositive patients were randomly assigned to the study. Fasciola DNA was identified in the stool samples of 96% of seropositive patients by nested PCR but ova of Fasciola was detected by parasitology methods in only 20% of seropositive cases. Fasciola DNA was identified in 90% of the urine samples by nested PCR. No cross-reactions were observed with other parasites. Conclusion: Detection of cfDNA in stool and urine samples has high accuracy and thus can be used for the diagnosis of Fasciola infection in human.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

2012 ◽  
Vol 61 (1) ◽  
pp. 183-186 ◽  
Author(s):  
Xian-Quan Cai ◽  
Hai-Qiong Yu ◽  
Jian-Shan Bai ◽  
Jian-Dong Tang ◽  
Xu-Chu Hu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clonorchis Sinensis ◽  
Pcr Assay ◽  
Stool Samples ◽  
Human Stool

scholarly journals Outbreak of caprine abortion by Toxoplasma gondii in Midwest Brazil

2011 ◽  
Vol 31 (11) ◽  
pp. 933-937 ◽  
Author(s):  
Flávio Henrique Bravim Caldeira ◽  
Daniel Guimarães Ubiali ◽  
Isabela de Godoy ◽  
Valéria Dutra ◽  
Daniel Moura de Aguiar ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Brucella Abortus ◽  
Nested Pcr ◽  
Neospora Caninum ◽  
Pcr Assay ◽  
Serum Samples ◽  
Pathology Laboratory ◽  
Mato Grosso ◽  
Veterinary Pathology ◽  
Gross Lesions

An outbreak of abortion by Toxoplasma gondii in goats on a farm in the Brazilian Midwest is reported. Gross lesions were not observed in seven aborted fetuses submitted to the Veterinary Pathology Laboratory, Federal University of Mato Grosso, for necropsy investigation. The main histologic lesions were mononuclear cell pneumonia and necrotizing encephalitis in varying degrees of intensity. PCR for Brucella abortus and Neospora caninum and aerobic cultures were negative in all cases. Antibody titles against T. gondii varying from 1:1024 to 1:32.768 were detected in serum samples from four aborted goats. Nested-PCR assay for T. gondii were positive in brain samples of all cases submitted. These findings indicate that T. gondii infection should be considered in the diagnosis of abortion in goats in Midwest Brazil.

scholarly journals Nested PCR Assay for Detection of Granulocytic Ehrlichiae

1998 ◽  
Vol 36 (4) ◽  
pp. 1090-1095 ◽  
Author(s):  
Robert F. Massung ◽  
Kim Slater ◽  
Jessica H. Owens ◽  
William L. Nicholson ◽  
Thomas N. Mather ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Limit Of Detection ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Serum Samples ◽  
Blood Specimens ◽  
The 16S Rrna Gene ◽  
Edta Blood

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted fromIxodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.

scholarly journals Diagnosis of canine leptospirosis: evaluation of two PCR assays in comparison with the microagglutination test

2019 ◽  
Vol 39 (4) ◽  
pp. 255-262
Author(s):  
Paula L. Martin ◽  
Nestor O. Stanchi ◽  
Bibiana F. Brihuega ◽  
Estela Bonzo ◽  
Lucía Galli ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Urine Samples ◽  
Clinical Samples ◽  
Conventional Pcr ◽  
Serum Samples ◽  
Presumptive Diagnosis ◽  
Microagglutination Test ◽  
Pcr Assays

ABSTRACT: Canine leptospirosis is definitely diagnosed by demonstrating seroconversion in paired serum samples from the acute and convalescent period by the microagglutination test (MAT). However, the application of a polymerase chain reaction (PCR) assay can provide earlier confirmation of suspected cases. The objective of this study was to evaluate two PCR assays used in diagnosis of human leptospirosis (lipL32 real-time PCR and rrs conventional PCR) in cultured microorganisms and experimentally contaminated samples (whole blood, serum, urine), and investigate their applicability in clinical samples from dogs with presumptive diagnosis of leptospirosis by using the MAT as a reference. The analytical sensitivity of the lipL32 real-time PCR was 1 genome equivalent per reaction, whereas that for the rrs conventional PCR was 10 genome equivalents per reaction. Both assays amplified the pathogenic strains but were negative when evaluating the DNA of other microorganisms that may be present in clinical samples. The lipL32 real-time PCR detected 100 bacteria/mL in whole blood samples, 1000 bacteria/mL in serum samples and 10 bacteria/mL in urine samples, whereas the rrs conventional PCR detected 1000 bacteria/mL in whole blood and serum samples and 100 bacteria/mL in urine samples. Seven out of the 51 samples from dogs with presumptive diagnosis of leptospirosis were considered as confirmed cases. ThelipL32 real-time PCR detected positive results in six of the seven confirmed cases, whereas the rrs conventional PCR detected four. The PCR assays evaluated proved to be useful diagnostic tools in the confirmation of canine leptospirosis when used together with the MAT.

Conventional PCR for molecular diagnosis of human strongyloidiasis

Parasitology ◽  
2014 ◽  
Vol 141 (5) ◽  
pp. 716-721 ◽  
Author(s):  
R. B. SITTA ◽  
F. M. MALTA ◽  
J. R. PINHO ◽  
P. P. CHIEFFI ◽  
R. C. B. GRYSCHEK ◽  
...  
Keyword(s):  
Molecular Diagnosis ◽  
Agar Plate ◽  
Strongyloides Stercoralis ◽  
Conventional Polymerase Chain Reaction ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Culture Methods ◽  
Specific Primers ◽  
Stool Samples ◽  
Species Specific

SUMMARYStrongyloidiasis is frequently asymptomatic and diagnosis of latent infection is difficult due to limitations of current parasitological and serological methods. This study aimed to verify the use of conventional polymerase chain reaction (PCR) assay for molecular diagnosis of Strongyloides stercoralis infection. Fresh stool samples were obtained from 103 individuals: 33 S. stercoralis positive, 30 positive for other parasites and 40 negative for parasitological methods. These samples were examined by the Lutz, Rugai and agar plate culture methods and conventional PCR assay. Two sets of primers (S. stercoralis species-specific and genus-specific sets), located in the 18S ribosomal RNA gene, were used for PCR. Of the 33 samples positive for S. stercoralis by parasitological methods, 28 (84·8%) were also detected by PCR assay using species-specific primers and 26 (78·8%) using genus-specific primers. Among the stool samples negative by parasitological methods, seven (17·5%) were positive by PCR using species-specific primers and two (5·0%) using genus-specific primers. In conclusion, the conventional PCR assay described in this study using a species-specific primer pair provided a molecular method for S. stercoralis diagnosis in human stool samples.

scholarly journals Experimental Model of Progressive Disseminated Trichosporonosis in Mice with Latent Trichosporonemia

2000 ◽  
Vol 38 (9) ◽  
pp. 3260-3266 ◽  
Author(s):  
Eiji Yamagata ◽  
Perparim Kamberi ◽  
Yuriko Yamakami ◽  
Atsuro Hashimoto ◽  
Masaru Nasu
Keyword(s):  
Nested Pcr ◽  
Critical Period ◽  
Survival Rates ◽  
Myelogenous Leukemia ◽  
Pcr Assay ◽  
Serum Samples ◽  
Significant Difference ◽  
Acute Myelogenous ◽  
Underlying Mechanisms ◽  
Immunocompromised Hosts

Trichosporon asahii and Trichosporon mucoides are the most common strains of fungi that cause disseminated trichosporonosis, a severe opportunistic infection in immunocompromised hosts. We have previously established a nested PCR assay using serum samples for detection of both strains. Here we describe a new experimental animal model for investigating the underlying mechanisms of disseminated trichosporonosis. T. asahii (OMU239, a clinical isolate from a patient with acute myelogenous leukemia) and 8-week-old ICR male mice were used in all experiments. A suspension of T. asahii (3 × 106 CFU/animal) was injected into the caudal vein of each mouse after immunosuppression with cyclophosphamide (200 mg/kg of body weight/day for 2 days) and prednisolone (30 mg/kg/day for 1 day). Mice were then divided into four subgroups (R0, R1, R2, and R3) based on the time of reimmunosuppression. The latter was performed using the same drugs 1 week (group R1), 2 weeks (group R2), and 3 weeks (group R3) after fungal infection. Reimmunosuppression was not performed in group R0. The 5-week-survival rates of mice after T. asahiiinfection were 0% for group R1, 50% for group R2, 80% for group R3, and 80% for group R0. There was a significant difference in the survival rates between group R1 and either group R0 or R3 (P < 0.05). Fungal clearance in peripheral blood and various organs of group R1 and R2 was delayed relative to that of group R0 but was similar to the control in group R3 in spite of reimmunosuppression. Our results suggest that the critical period for the development of disseminated trichosporonosis in our model is shorter than 3 weeks after T. asahii infection. We concluded that mice during this critical period were in a state of latent trichosporonemia. Comparison of the survival rates suggests that the nested PCR assay was more useful than blood culture and glucuronoxylomannan antigen assay in the detection of this latent trichosporonemia.

scholarly journals PCR Detection of DNA Specific forTrichosporon Species in Serum of Patients with Disseminated Trichosporonosis

1999 ◽  
Vol 37 (3) ◽  
pp. 694-699 ◽  
Author(s):  
Hiroyuki Nagai ◽  
Yuriko Yamakami ◽  
Atsuro Hashimoto ◽  
Issei Tokimatsu ◽  
Masaru Nasu
Keyword(s):  
Early Diagnosis ◽  
Nested Pcr ◽  
Response To Therapy ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Serum Samples ◽  
Target Dna ◽  
Specific Fragment ◽  
26S Rrna

Deep-seated trichosporonosis is a lethal opportunistic infection that disseminates rapidly and widely in immunocompromised patients, and early diagnosis is crucial for the treatment of this infection. We developed a novel nested-PCR assay that detects DNA specific for clinically important strains of Trichosporon in serum samples from patients with disseminated trichosporonosis. In this assay, two sets of oligonucleotide primers were derived from the sequence of 26S rRNA genes of Trichosporon asahii. The specific fragment was amplified from T. asahii and T. mucoides, but not from other microorganisms, including some other basidiomycetous fungi (Cryptococcus, Malassezia,Rhodotorula, and Sporobolomyces). Target DNA was detected by the nested PCR with as little as 5 fg of the extracted DNA of T. asahii. In a study using 11 clinical samples, the specific fragment was detected by the nested PCR in 64% (7 of 11) of sera from patients with histologically diagnosed disseminated trichosporonosis, while glucuronoxylomannan antigen was detected in only 54% (6 of 11) of the samples. Our new nested-PCR assay using serum samples can be performed repeatedly throughout the course of the disease. In addition, not only can it be used for early diagnosis of trichosporonosis, but it may also be beneficial for monitoring its progress or response to therapy.

scholarly journals Clinical Evaluation of a New PCR Assay for Detection of Coxiella burnetii in Human Serum Samples

1998 ◽  
Vol 36 (1) ◽  
pp. 77-80 ◽  
Author(s):  
G. Q. Zhang ◽  
Sa V. Nguyen ◽  
H. To ◽  
M. Ogawa ◽  
A. Hotta ◽  
...  
Keyword(s):  
Human Serum ◽  
Coxiella Burnetii ◽  
Nested Pcr ◽  
Restriction Enzymes ◽  
Pcr Assay ◽  
Serum Samples ◽  
Gene Encoding ◽  
Human Serum Samples ◽  
Positive Serum ◽  
Pcr Method

A nested PCR method was developed for the detection ofCoxiella burnetii in human serum samples. Two pairs of oligonucleotide primers were designed to amplify a 438-bp fragment of the com1 gene encoding a 27-kDa outer membrane protein ofC. burnetii. The primers amplified the predicted fragments of 21 various strains of C. burnetii but did not react with DNA samples from other microorganisms. The 438-bp amplification products could be digested with restriction enzymes SspI and SalI. The utility of the nested PCR was evaluated by testing human serum samples. The com1 gene fragment was amplified from 135 (87%) of 155 indirect immunofluorescence test (IF)-positive serum samples and from 11 (11%) of 100 IF-negative serum samples. The nested PCR with primers targeted to the com1gene appeared to be a sensitive, specific, and useful method for the detection of C. burnetii in serum samples.

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