Application of nested PCR to detectPenicillium marneffeiin serum samples

The avian herpesvirus Marek’s disease virus (MDV) has a worldwide distribution and is responsible for T-lymphoma in chickens. The question as to whether MDV poses a public health hazard to humans was first raised when the virus was isolated in 1967. However, no irrefutable results have been obtained in immunological and virological studies. We used a nested-PCR to detect MDV DNA in human serum samples. A total of 202 serum samples from individuals exposed and not exposed to poultry was tested by nested-PCR for a target sequence located in the MDV gD gene. The assay system was specific and sensitive, making it possible to detect a single copy of the target sequence. Forty-one (20%) of the 202 serum samples tested positive for MDV DNA. The prevalence of MDV DNA was not significantly different in the group exposed to poultry and the group not exposed to poultry. There was also no difference due to age or sex. Alignment of the 41 gD sequences amplified from human sera with eight gD sequences amplified from MDV-infected chicken sera showed a maximum nucleotide divergence of 1·65%. However, four ‘hot-spot’ mutation sites were identified, defining four groups. Interestingly, two groups contained only human MDV-gD sequences. The status of the MDV genome detected in human blood is discussed.

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Outbreak of caprine abortion by Toxoplasma gondii in Midwest Brazil

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2011001100001 ◽

2011 ◽

Vol 31 (11) ◽

pp. 933-937 ◽

Cited By ~ 6

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.

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Detection and Molecular Characterization of Orientia tsutsugamushi from Suspected Scrub Typhus Patients in Mizoram, India

International Journal of Current Microbiology and Applied Sciences ◽

10.20546/ijcmas.2021.1010.061 ◽

2021 ◽

Vol 10 (10) ◽

pp. 514-523

Author(s):

Vanramliana Gabriel Rosangkima ◽

Lalnunnemi Ralte Lalremruata ◽

Christine Vanlalbiakdiki Sailo Hunropuia ◽

Deborah Lalnghakmawii Lalfakzuala Pautu

Keyword(s):

Nested Pcr ◽

Scrub Typhus ◽

Specific Antigen ◽

Serum Samples ◽

Antigen Gene ◽

Molecular Tests ◽

Related Genotype ◽

High Degree ◽

Higher Sensitivity

Serologic and molecular tests were performed for the diagnosis and to detect O. tsutsugamushi genotypes that are circulating in the state of Mizoram, India. Blood samples from scrub typhus-suspected patients were collected from Synod Hospital, Durtlang, Mizoram. Weil-Felix and immunochromatographic test (ICT) were performed from the serum samples. Nested PCR (nPCR) amplification of 47kDa outer membrane protein antigen gene and 56kDa type-specific antigen gene were done from the whole blood. 141/177 (79.66%) and 134/177 (75.7%) cases showed the presence of antibody against scrub typhus by Weil-Felix and ICT assays respectively. 76/177 (42.93%) patients showed the presence of 47kDa OMP antigen gene by nPCR while 55/177 (31.07%) showed the presence of 56kDa TSA gene by nPCR. Phylogenetic analysis of 56kDa TSA gene sequence revealed that Karp-related genotype was the most common genotype in the study area followed by Kato-related genotype. In this study, a high degree of diversity of O. tsutsugamushi was observed similar to the observations reported from other parts of India. Nested PCR of 47kDa OMP antigen gene showed higher sensitivity as compared to nPCR amplification of 56kDa TSA gene suggesting it as the assay of choice for diagnosis of scrub typhus disease.

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Laboratory Diagnosis of Toscana Virus Infection by Enzyme Immunoassay with Recombinant Viral Nucleoprotein

Journal of Clinical Microbiology ◽

10.1128/jcm.37.3.649-652.1999 ◽

1999 ◽

Vol 37 (3) ◽

pp. 649-652 ◽

Cited By ~ 26

Author(s):

Dario Soldateschi ◽

Grazia Maria Dal Maso ◽

Marcello Valassina ◽

Laura Santini ◽

Silvia Bianchi ◽

...

Keyword(s):

Enzyme Immunoassay ◽

Nested Pcr ◽

Immune Status ◽

Laboratory Diagnosis ◽

Summer Season ◽

Immunoglobulin M ◽

Toscana Virus ◽

Serum Samples ◽

Rna Sequences ◽

Specific Serum

A recombinant enzyme immunoassay (rEIA) to detect serum immunoglobulin M (IgM) and IgG to Toscana virus (TOSV) was developed with the aim of establishing a simple and easily available assay for diagnosing acute and/or previous infections. The rEIA, based on the recombinant nucleoprotein of TOSV expressed in Escherichia coli, was evaluated with 97 serum samples collected in an area where TOSV is endemic and compared to an analogous assay based on cell-derived TOSV. Discordant results were resolved by immunoblotting (IB). Twenty-two of these samples, obtained from subjects hospitalized during the summer season with meningitis of suspected TOSV etiology, were further characterized by indirect immunofluorescence and IB, and detection of specific TOSV RNA sequences in the cerebrospinal fluid of these patients was attempted by nested PCR. The results indicated that rEIA was able to diagnose acute TOSV infection by detection of specific serum IgM in all of the subjects with TOSV meningitis confirmed by nested PCR or serology. The overall sensitivity and specificity of rEIA were both 100% for IgM detection and 100 and 96.6%, respectively, for IgG detection. Thus, rEIA appears to be a simple and reliable laboratory test for the diagnosis of acute TOSV infection and for the assessment of immune status.

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Sensitivity of nested PCR toward human cytomegalovirus-DNA in native sera and in extracted DNA of serum samples

Egyptian Academic Journal of Biological Sciences G Microbiology ◽

10.21608/eajbsg.2010.16711 ◽

2010 ◽

Vol 2 (1) ◽

pp. 1-8

Author(s):

Sahar Shoman ◽

Ashraf Tabl ◽

Hussam Ghanem ◽

Mohamed Nabil

Keyword(s):

Human Cytomegalovirus ◽

Nested Pcr ◽

Serum Samples

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Nested PCR Assay for Detection of Granulocytic Ehrlichiae

Journal of Clinical Microbiology ◽

10.1128/jcm.36.4.1090-1095.1998 ◽

1998 ◽

Vol 36 (4) ◽

pp. 1090-1095 ◽

Cited By ~ 237

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.

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

Iranian Journal of Public Health ◽

10.18502/ijph.v49i6.3367 ◽

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.

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Evidence of active herpesvirus 6 (variant-A) infection in patients with lymphadenopathy in Belém, Pará, Brazil

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652003000500008 ◽

2003 ◽

Vol 45 (5) ◽

pp. 283-288 ◽

Cited By ~ 4

Author(s):

Ronaldo B. Freitas ◽

Maria R. Freitas ◽

Alexandre C. Linhares

Keyword(s):

Nested Pcr ◽

Age Groups ◽

Human Herpesvirus ◽

Enzyme Linked Immunosorbent Assay ◽

Active Infection ◽

Serum Samples ◽

Recent Infection ◽

Cervical Lymph Nodes ◽

Duration Of Symptoms ◽

Herpesvirus 6

A total of 323 patients with lymphadenopathy were selected in Belém, Brazil, between January 1996 and December 2001, and screened for the presence of human herpesvirus-6 (HHV-6) IgM- and- IgG antibodies by enzyme-linked immunosorbent assay (ELISA). When seroprevalence is analyzed by gender, similar rates are found for female (60.6%) and male (55.7%) individuals. Seventy-seven (23.8%) patients were HHV-6-IgM-and- IgG-positive (IgM+ subgroup), with positivity rates of 29.7% and 17.7% (p = 0.0007) for female- and male individuals, respectively. Sera from a subgroup (n = 120) of these subjects, with high HHV-6 antibody levels (either IgM+ or IgG+ reactivities), were subsequently processed for the presence of HHV-6 DNA by polymerase chain reaction (PCR)/nested PCR. Active infections (IgM+ and/or IgG+ high levels specific antibodies plus detection of viral DNA) were diagnosed in 20/77 (20.0%) and 8/43 (18.6%); subgroup of the 120 individuals suspected of having HHV-6 suggestive recent infection. All (n = 28) cases of active infection were found to be associated with HHV-6 variant-A (HHV-6A), as detectable by PCR/nested PCR, using variant-specific primer that amplify regions of 195 base pairs (bp) (HHV-6A) and 423 bp (HHV-6B). Rates of HHV-6 DNA detection between female and male patients were similar (p > 0.05) in the IgM+ and IgG+ groups: 20.4% versus 35.7% and 25.0% versus 13.0%, respectively. HHV-6 DNA was detected across < 5 through 41-50-year age-groups for patients whose serum samples were IgM+, with rates ranging from 7.7% (female subjects aged < 5 years) to 80.0% (male, 11-20 years). Among patients whose serological status was IgG+, HHV-6 DNA was detected in < 5, 6-10, 21-30 and > 50 age-groups at rates that ranged from 15.4% (male, < 5 years of age) to 100.0% (female aged 11-20 years). Swelling cervical lymph nodes were the most common sign, accounting for 9 (32.0%) cases in each gender group. Among patients (n = 28) with active infection by HHV-6A variant, duration of symptoms lasted 1-5 days in 35.7% of subjects, whereas in 64.3% of them the disease lasted 6-20 days. Our data suggest that it is worth seeking for HHV-6 infection whenever a patient (infant or adult) presents with lymphadenopathy as a prominent symptom in the course of an acute febrile illness.

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Molecular detection of Ehrlichia canisand Anaplasma platys in dogs in Southern Brazil

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612013000300007 ◽

2013 ◽

Vol 22 (3) ◽

pp. 360-366 ◽

Cited By ~ 15

Author(s):

Camila Serina Lasta ◽

Andrea Pires dos Santos ◽

Joanne Belle Messick ◽

Simone Tostes Oliveira ◽

Alexander Welker Biondo ◽

...

Keyword(s):

Nested Pcr ◽

Chronic Infection ◽

Molecular Detection ◽

Hematological Parameters ◽

Southern Brazil ◽

Anaplasma Platys ◽

Porto Alegre ◽

Serum Samples ◽

Hematological Abnormalities ◽

Dot Elisa

The aims of this study were to determine the occurrence ofAnaplasma platys and Ehrlichia canisinfection in dogs in Porto Alegre, Southern Brazil; and to investigate their association with hematological abnormalities. Serum samples from 196 dogs were first tested using dot-ELISA for antibodies against Anaplasmaspp. and Ehrlichia canis. Peripheral blood samples from 199 dogs were subjected to 16S rRNA nested PCR (nPCR) for A. platysand E. canis, followed by DNA sequencing to ensure pathogen identity. A total of 19/196 samples (9.69%) were positive forAnaplasma spp. using ELISA and 28/199 (14.07%) samples were positive for A. platys by nested PCR. All the dog samples were negative for E. canis, both in anti-E. canisantibody tests and in nested PCR. There were no significant differences in hematological parameters between A. platys-PCR positive and negative dogs and Anaplasma spp. serologically positive dogs, except for basophil counts, which were higher in nPCR-positive dogs. This is the first report showing A. platys presence in dogs in Southern Brazil. In conclusion, hematological parameters may not be sufficient to diagnose A. platys infection in dogs in Southern Brazil, probably due either to low pathogenicity or to chronic infection. On the other hand, E. canis may either have very low occurrence or be absent in dogs in Porto Alegre.

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