scholarly journals Development and Evaluation of a Real-Time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA To Diagnose Tuberculosis in Multiple Animal Species

2007 ◽  
Vol 14 (12) ◽  
pp. 1563-1571 ◽  
Author(s):  
Noel P. Harrington ◽  
Om P. Surujballi ◽  
W. Ray Waters ◽  
John F. Prescott
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Reverse Transcription ◽  
Skin Testing ◽  
Gamma Interferon ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sequence Information ◽  
Tuberculin Skin Testing ◽  
Reverse Transcription Pcr ◽  
Ifn Γ

ABSTRACT Tuberculosis of free-ranging and captive wildlife, including species implicated in the maintenance and transmission of Mycobacterium bovis, is a difficult disease to diagnose and control. Historically, diagnosis of tuberculosis has relied largely upon assays of cell-mediated immunity (CMI), such as tuberculin skin testing. This approach, however, is problematic or impractical for use with many wildlife species. Increasingly, in vitro diagnostic tests, including gamma interferon (IFN-γ)-based assays, are replacing or complementing skin testing of cattle and humans. Analogous assays are unavailable for most wildlife because of a lack of species-specific immunological reagents. This report describes the development and validation of a whole-blood assay to quantify antigen-specific IFN-γ mRNA expression by quantitative real-time reverse transcription-PCR. Oligonucleotide primers and probes were designed and tested for reactivity towards several susceptible species of interest with respect to tuberculosis infection. The assay was subsequently optimized to quantify the IFN-γ mRNA expression in elk and red deer (Cervus elaphus) and was evaluated for its ability to detect mycobacterial antigen-specific responses of experimentally tuberculosis-infected animals. The assay was a simple, rapid, and sensitive measure of antigen-specific CMI. The IFN-γ mRNA responses correlated well with IFN-γ protein production and showed performance in determining an animal's infection status superior to that of either lymphocyte proliferation or IFN-γ protein enzyme-linked immunosorbent assay methods. An additional advantage is the ease with which the assay can be modified to reliably quantify IFN-γ expression by using consensus sequences of closely related species or of other species for which IFN-γ sequence information is available.

scholarly journals Development of a Human Gamma Interferon Enzyme Immunoassay and Comparison with Tuberculin Skin Testing for Detection ofMycobacterium tuberculosis Infection

1998 ◽  
Vol 5 (4) ◽  
pp. 531-536 ◽  
Author(s):  
Nuket Desem ◽  
Stephen L. Jones
Keyword(s):  
Enzyme Immunoassay ◽  
Skin Test ◽  
Skin Testing ◽  
Gamma Interferon ◽  
Tuberculin Skin Testing ◽  
In Vitro Test ◽  
Detection Range ◽  
Ifn Γ

ABSTRACT A sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-γ) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-γ per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-γ. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-γs. Cross-reactions with other human cytokines or with IFN-γs derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-γ in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-γ were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-γ responsiveness in vitro. These results support the further study of the blood culture–IFN-γ EIA system as an alternative to skin testing for the detection of human TB infection.

scholarly journals Detection and Typing of Human Pathogenic Hantaviruses by Real-Time Reverse Transcription-PCR and Pyrosequencing

2007 ◽  
Vol 53 (11) ◽  
pp. 1899-1905 ◽  
Author(s):  
Marit Kramski ◽  
Helga Meisel ◽  
Boris Klempa ◽  
Detlev H Krüger ◽  
Georg Pauli ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hantaan Virus ◽  
Hantavirus Infection ◽  
Sequence Information ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Reverse Transcription Pcr ◽  
Pcr Assays

Abstract Background: Because the clinical course of human infections with hantaviruses can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is based mainly on serologic assays, and the detection of hantaviral RNA by the commonly used reverse transcription (RT)-PCR is difficult because of high sequence diversity of hantaviruses and low viral loads in clinical specimens. Methods: We developed 5 real-time RT-PCR assays, 3 of which are specific for the individual European hantaviruses Dobrava, Puumala, or Tula virus. Two additional assays detect the Asian species Hantaan virus together with Seoul virus and the American species Andes virus together with Sin Nombre virus. Pyrosequencing was established to provide characteristic sequence information of the amplified hantavirus for confirmation of the RT-PCR results or for a more detailed virus typing. Results: The real-time RT-PCR assays were specific for the respective hantavirus species and optimized to run on 2 different platforms, the LightCycler and the ABI 7900/7500. Each assay showed a detection limit of 10 copies of a plasmid containing the RT-PCR target region, and pyrosequencing was possible with 10 to 100 copies per reaction. With this assay, viral genome could be detected in 16 of 552 (2.5%) specimens of suspected hantavirus infections of humans and mice. Conclusions: The new assays detect, differentiate, and quantify hantaviruses in clinical specimens from humans and from their natural hosts and may be useful for in vitro studies of hantaviruses.

scholarly journals Quantitative Real-Time Reverse Transcription-PCR Analysis Reveals Stable and Prolonged Neurotoxin Cluster Gene Activity in a Clostridium botulinum Type E Strain at Refrigeration Temperature

2008 ◽  
Vol 74 (19) ◽  
pp. 6132-6137 ◽  
Author(s):  
Ying Chen ◽  
Hannu Korkeala ◽  
Jere Lind�n ◽  
Miia Lindstr�m
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Expression Levels ◽  
Reverse Transcription Pcr ◽  
Two Temperatures ◽  
Group Ii

ABSTRACT The relative expression levels of six botulinum neurotoxin cluster genes in a group II Clostridium botulinum type E strain grown at 10 or 30�C were investigated using quantitative real-time reverse transcription-PCR. An enzyme-linked immunosorbent assay was used to confirm neurotoxin expression. Distinct mRNA and toxin production patterns were observed at the two temperatures. The average relative mRNA levels at 10�C were higher than (ntnh and p47), similar to (botE), or lower than (orfx1, orfx2, orfx3) those at 30�C. The maximum botE expression levels and average neurotoxin levels at 10�C were 45 to 65% of those at 30�C. The relative mRNA levels at 10�C declined generally slowly within 8 days, as opposed to the rapid decline observed at 30�C within 24 h. Distinct expression patterns of the six genes at the two temperatures suggest that the type E neurotoxin cluster genes are transcribed as two tricistronic operons at 30�C, whereas at 10�C monocistronic (botE or orfx1 alone) and bicistronic (ntnh-p47 and orfx2-orfx3) transcription may dominate. Thus, type E botulinum neurotoxin production may be involved with various temperature-dependent regulatory events. In light of group II C. botulinum type E being a dangerous food-borne pathogen, these findings may be important in terms of the safety of refrigerated packaged foods of extended durability.

scholarly journals Diagnostic Implications of Antigen-Induced Gamma Interferon Production by Blood Leukocytes from Mycobacterium bovis-Infected Reindeer (Rangifer tarandus)

2006 ◽  
Vol 13 (1) ◽  
pp. 37-44 ◽  
Author(s):  
W. R. Waters ◽  
M. V. Palmer ◽  
R. E. Slaughter ◽  
S. L. Jones ◽  
J. E. Pitzer ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Skin Testing ◽  
The United States ◽  
Gamma Interferon ◽  
Tuberculin Skin Testing ◽  
Blood Leukocytes ◽  
Vitro Assay ◽  
Apparent Lack ◽  
Ifn Γ

ABSTRACT The only approved method of tuberculosis (TB) surveillance of reindeer within the United States is tuberculin skin testing; however, skin testing has an apparent lack of specificity, since numerous reindeer are classified as reactors, yet Mycobacterium bovis is not isolated from tissues upon necropsy. The objective of this study was to evaluate the ability of an in vitro assay (the Cervigam assay) to detect gamma interferon (IFN-γ) produced by blood leukocytes in response to mycobacterial antigens from M. bovis-infected reindeer. Thirteen male reindeer ∼9 months of age were inoculated with 105 CFU M. bovis in their tonsillar crypts. Stimulation of whole-blood cultures with a mitogen resulted in significant production of IFN-γ compared to that by nonstimulated samples. Responses by infected reindeer to M. bovis purified protein derivative (PPD) were as much as 3.5-fold higher than those by noninfected reindeer (n = 4). Despite differences in responses to PPD by the two groups, reindeer within the noninfected group had responses of >0.1 change in optical density (ΔOD) (a level generally considered positive) to PPD. Mean responses by infected reindeer to a rESAT-6-CFP-10 fusion protein (Mycobacterium tuberculosis complex specific) were as much as 20-fold higher than respective responses by noninfected reindeer at all time points. Additionally, responses by 3/4 noninfected reindeer were <0.1 ΔOD (considered negative) at each time point. To further evaluate the specificity of the assay, samples were collected from reindeer in a TB-free herd. All reindeer had responses to mitogen; however, only 1 of 38 had a response to PPD, and none of the reindeer responded to rESAT-6-CFP-10. Together, these findings indicate that IFN-γ-based tests may prove useful for TB surveillance of reindeer.

scholarly journals Comparison of Two Gamma Interferon Release Assays and Tuberculin Skin Testing for Tuberculosis Screening in a Cohort of Patients with Rheumatic Diseases Starting Anti-Tumor Necrosis Factor Therapy

2011 ◽  
Vol 18 (12) ◽  
pp. 2102-2108 ◽  
Author(s):  
Dimitrios Vassilopoulos ◽  
Stamatoula Tsikrika ◽  
Chrisoula Hatzara ◽  
Varvara Podia ◽  
Anna Kandili ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Rheumatic Diseases ◽  
Tumor Necrosis ◽  
Skin Testing ◽  
Gamma Interferon ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tuberculin Skin Testing ◽  
Tuberculosis Screening ◽  
Ltbi Screening ◽  
Necrosis Factor

ABSTRACTGamma interferon release assays (IGRAs) are increasingly used for latentMycobacterium tuberculosisinfection (LTBI) screening in patients with rheumatic diseases starting anti-tumor necrosis factor (anti-TNF) therapies. We compared the performances of two IGRAs, an enzyme-linked immunospot release assay (T-SPOT.TB) and an enzyme-linked immunosorbent assay (QuantiFERON-TB Gold In Tube [QFT-GIT]), to that of tuberculin skin testing (TST) for LTBI screening of 157 consecutive rheumatic patients starting anti-TNF therapies. Among 155 patients with valid results, 58 (37%) were positive by TST, 39 (25%) by T-SPOT.TB assay, and 32 (21%) by QFT-GIT assay. IGRAs were associated more strongly with at least one risk factor for tuberculosis (TB) than TST. Risk factors for a positive assay included chest X-ray findings of old TB (TST), advanced age (both IGRAs), origin from a country with a high TB prevalence, and a positive TST (T-SPOT.TB assay). Steroid use was negatively associated with a positive QFT-GIT assay. The agreement rate between IGRAs was 81% (kappa rate = 0.47), which was much higher than that observed between an IGRA and TST. If positivity by either TST or an IGRA was required for LTBI diagnosis, then the rate of LTBI would have been 46 to 47%, while if an IGRA was performed only for TST-positive patients, the respective rate would have been 11 to 17%. In conclusion, IGRAs appear to correlate better with TB risk than TST and should be included in TB screening of patients starting anti-TNF therapies. In view of the high risk of TB in these patients, a combination of one IGRA and TST is probably more appropriate for LTBI diagnosis.

scholarly journals Real-Time Reverse Transcription-PCR Quantification of Cytokine mRNA Expression in Golden Syrian Hamster Infected with Leishmania infantum and Treated with a New Amphotericin B Formulation

2006 ◽  
Vol 50 (4) ◽  
pp. 1195-1201 ◽  
Author(s):  
S. Rama Iñiguez ◽  
M. A. Dea-Ayuela ◽  
J. A. Sanchez-Brunete ◽  
J. J. Torrado ◽  
J. M. Alunda ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Reverse Transcription ◽  
Syrian Hamster ◽  
Leishmania Infantum ◽  
Cytokine Mrna ◽  
Golden Syrian Hamster ◽  
Cytokine Mrna Expression ◽  
Reverse Transcription Pcr ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT A real-time quantitative reverse transcription-PCR assay was developed for the quantification of cytokine mRNA expression in the golden Syrian hamster Mesocricetus auratus infected with Leishmania infantum and treated with amphotericin B (AMB) formulated in microspheres made of human serum albumin (HSA). Treatment was administered intravenously on days 69, 71, and 73 postinfection (p.i.) with 107 metacyclic promastigotes, at doses of 2 and 40 mg/kg of AMB. High infection levels were recorded for untreated animals by day 76 p.i., with parasite loads always about 2 log10 per gram higher in the liver than in the spleen. Treatment was highly effective with both doses, but at 40 mg/kg, almost complete parasite elimination was achieved. mRNA expression of gamma interferon (IFN-γ) and, to a lesser extent, tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β) in spleen cells was up-regulated in most animals of the untreated group. The mRNA expression of interleukin-4 was strongly down-regulated in untreated as well as treated infected animals. Treatment with the lower dose of AMB-HSA down-regulated the mRNA expression of IFN-γ and TNF-α, with no effect on the deactivating cytokine TGF-β. In contrast, treatment with the higher dose (40 mg/kg) of the formulation caused moderate up-regulation of IFN-γ and TNF-α and strong suppression of TGF-β. Treatment of noninfected animals did not alter the cytokine expression pattern with regard to untreated controls. Our results suggest that treatment of L. infantum-infected Syrian hamsters with highly effective nontoxic doses of AMB-HSA causes deactivation of the anti-inflammatory cytokine TGF-β, which in turn results in up-regulation of the Th1 cytokines IFN-γ and TNF-α.

scholarly journals Ο ρόλος της παχυσαρκίας στην ανοσολογική απάντηση ασθενών με σύνδρομο σήψης

2015 ◽  
Author(s):  
Αναστασία Κολυβά
Keyword(s):  
Antioxidant Capacity ◽  
Real Time ◽  
Immunosorbent Assay ◽  
Reverse Transcription ◽  
Total Antioxidant Capacity ◽  
Thiobarbituric Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thiobarbituric Acid Reactive Substances ◽  
Reverse Transcription Pcr ◽  
Total Antioxidant

Σκοπός: Η σήψη αποτελεί μια από τις σημαντικότερες αιτίες νοσηλείας και θνησιμότητας στον ανεπτυγμένο κόσμο, όπου σχεδόν τα δύο - τρίτα του πληθυσμού υποφέρουν από παχυσαρκία. Σαν αποτέλεσμα, η συνύπαρξη των δύο αυτών καταστάσεων έχει γίνει όλο και συχνότερη στην κλινική πράξη και ένας συνεχώς αυξανόμενος αριθμός κλινικών μελετών προσπαθεί να προσεγγίσει την πιθανή επίδραση της παχυσαρκίας στην νοσηρότητα και θνησιμότητα των ασθενών με σήψη, με έως τώρα αντιφατικά αποτελέσματα. Σκοπός της παρούσας μελέτης είναι να διερευνήσει τον τρόπο με τον οποίο η παχυσαρκία επηρεάζει την ανοσιακή απάντηση των σηπτικών ασθενών, εκτιμώντας τον αριθμό και την κατάσταση ενεργοποίησης των μακροφάγων του λιπώδους ιστού, τα επίπεδα του TNFα στον ορό και στον λιπώδη ιστό και δείκτες οξειδωτικού stress στο πλάσμα.Ασθενείς και Μέθοδοι: Μελετήθηκαν 106 ασθενείς, οι οποίοι χωρίστηκαν σε τέσσερις ομάδες (Ελέγχου n=26, Παχυσαρκίας n=27, Σήψης n=27, Σήψης & Παχυσαρκίας n=26). Ο αριθμός των μακροφάγων στο υποδόριο και ενδοκοιλιακό λίπος και οι υπότυποί τους (M1 και M2) αναγνωρίστηκαν με ανοσοϊστοχημική τεχνική υπό μικροσκόπηση. Τα επίπεδα του TNFα mRNA στο υποδόριο και ενδοκοιλιακό λίπος μετρήθηκαν με real-time reverse transcription-PCR. Στον ορό τα επίπεδα του TNFα μετρήθηκαν με sandwich enzyme-linked immunosorbent assay (ELISA). Το οξειδωτικό stress στο πλάσμα εκτιμήθηκε χρησιμοποιώντας επιλεγμένους βιοδείκτες [TBARS (thiobarbituric acid-reactive substances), Πρωτεϊνικά Καρβονύλια, TAC (total antioxidant capacity)].Αποτελέσματα: Παρατηρήθηκε ότι η σήψη αυξάνει τον ολικό αριθμό και τον Μ2 υπότυπο των μακροφάγων στο ενδοκοιλιακό λίπος, ενώ η παχυσαρκία δεν φάνηκε να επηρεάζει τη συγκέντρωση των μακροφάγων στο λίπος. Η παχυσαρκία βρέθηκε ότι αυξάνει τα επίπεδα του TNFα mRNA (P<0.05) στο ενδοκοιλιακό λίπος καθώς επίσης και τα επίπεδα των TBARS (P<0.001) και Πρωτεϊνικών Καρβονυλίων (P<0.001) στο πλάσμα των σηπτικών ασθενών. Τα επίπεδα της TAC στο πλάσμα βρέθηκε ότι μειώνονται και τα επίπεδα TNFα στον ορό ότι αυξάνονται με τη σήψη, ενώ δεν επηρεάζονταν από την παχυσαρκία. Συμπεράσματα: Η παχυσαρκία σχετίζεται με αυξημένη παραγωγή TNFα στον λιπώδη ιστό και αύξηση του οξειδωτικού stress, προάγοντας την προ-φλεγμονώδη απάντηση στους σηπτικούς ασθενείς.

Quantification of G250 mRNA expression in renal epithelial neoplasms by real-time reverse transcription-PCR of dissected tissue from paraffin sections

Pathology ◽  
2003 ◽  
Vol 35 (6) ◽  
pp. 513-517 ◽  
Author(s):  
Tarek A. Bismar ◽  
Fernando J. Bianco ◽  
Hongquan Zhang ◽  
Xingli Li ◽  
Fazlul H. Sarkar ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Reverse Transcription ◽  
Paraffin Sections ◽  
Reverse Transcription Pcr ◽  
Epithelial Neoplasms
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