scholarly journals Magnesium Functions as Superior Co-factor for Measuring Reverse Transcriptase Activity of HIV-1, HIV-2, and SIV

2020 ◽  
Vol 2 (4) ◽  
Author(s):  
Salequl Islam ◽  
Mohammad Ali Moni ◽  
Atsushi Tanaka ◽  
Hiroo Hoshino
Keyword(s):  
Reverse Transcriptase ◽  
Cell Line ◽  
Indirect Immunofluorescence ◽  
Dna Detection ◽  
Immunofluorescence Assay ◽  
Indirect Immunofluorescence Assay ◽  
Detection Techniques ◽  
Proviral Dna ◽  
Infected Cells ◽  
Hiv 1

This study compared different detection methods of human/simian immunodeficiency virus (HIV/SIV) infections in the cell line systems; notably, i) Indirect immunofluorescence assay (IFA), ii) integrated proviral DNA detection, iii) detection of syncytia, iv) measurement of reverse transcriptase (RT) activity. RTs of various retroviruses require cations, including Mg2+, Mn2+, Ni2+, and Cu2+, for their enzyme-activities. The study further compared the roles of Mg2+ and Mn2+ as cofactors for RT activities of freshly harvested HIV-1, HIV-2, and SIV. The NP-2/CD4/coreceptor cells were seeded for overnight and infected with viral inoculums at a multiplicity of infection (MOI) 1.0. The cells were passaged regularly in a 2-3 days interval and maintained up to 2 weeks.  Infected cells were detected by indirect immunofluorescence assay (IFA). Multinucleated giant cells (MGC) in syncytia were quantified by Giemsa-staining. Proviral DNA was detected by PCR, and reverse transcriptase (RT) activity was measured.  Two different cations, Mg2+ and Mn2+ were used as cofactors for RT assay. We found all the strains of HIV-1, HV-2 and SIV to infection in the cell line conveniently. IFA had identified all the viral infections in the infected cells. Proviral DNA detection, syncytia formation was observed in the infected cells. We found a better performance of Mg2+ as cofactor over Mn2+ in RT assay for HIV-1, HIV-2, SIV. Different four detection techniques of HIV/SIV infections show high level of agreement in the NP-2-based cell line system. Mg2+ remains a better cofactor for RT.

scholarly journals Efficiency of indirect immunofluorescence assay as a confirmatory test for the diagnosis of human retrovirus infection (HIV-1 and HTLV-I/II) in different at risk populations

1999 ◽  
Vol 41 (3) ◽  
pp. 159-164 ◽  
Author(s):  
René GASTALDELLO ◽  
Sandra GALLEGO ◽  
María Beatriz ISA ◽  
Silvia NATES ◽  
Silvia MEDEOT
Keyword(s):  
Sensitivity And Specificity ◽  
Indirect Immunofluorescence ◽  
Immunofluorescence Assay ◽  
Indirect Immunofluorescence Assay ◽  
Antibody Detection ◽  
Confirmatory Test ◽  
Kappa Index ◽  
Predictive Values ◽  
Persistently Infected ◽  
Hiv 1

We compared the indirect immunofluorescence assay (IFA) with Western blot (Wb) as a confirmatory method to detect antibodies anti retrovirus (HIV-1 and HTLV-I/II). Positive and negative HIV-1 and HTLV-I/II serum samples from different risk populations were studied. Sensitivity, specificity, positive, negative predictive and kappa index values were assayed, to assess the IFA efficiency versus Wb. The following cell lines were used as a source of viral antigens: H9 ( HTLV-III b); MT-2 and MT-4 (persistently infected with HTLV-I) and MO-T (persistently infected with HTLV-II). Sensitivity and specificity rates for HIV-1 were 96.80% and 98.60% respectively, while predictive positive and negative values were 99.50% and 92.00% respectively. No differences were found in HIV IFA performance between the various populations studied. As for IFA HTLV system, the sensitivity and specificity values were 97.91% and 100% respectively with positive and negative predictive values of 100% and 97.92%. Moreover, the sensitivity of the IFA for HTLV-I/II proved to be higher when the samples were tested simultaneously against both antigens (HTLV-I-MT-2 and HTLV-II-MO-T). The overall IFA efficiency for HIV-1 and HTLV-I/II-MT-2 antibody detection probed to be very satisfactory with an excellent correlation with Wb (Kappa indexes 0.93 and 0.98 respectively). These results confirmed that the IFA is a sensitive and specific alternative method for the confirmatory diagnosis of HIV-1 and HTLV-I/II infection in populations at different levels of risk to acquire the infection and suggest that IFA could be included in the serologic diagnostic algorithm.

scholarly journals The antinuclear antibody HEp-2 indirect immunofluorescence assay: a survey of laboratory performance, pattern recognition and interpretation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anne E. Tebo ◽  
Robert L. Schmidt ◽  
Kamran Kadkhoda ◽  
Lisa K. Peterson ◽  
Edward K. L. Chan ◽  
...  
Keyword(s):  
Indirect Immunofluorescence ◽  
Clinical Laboratory ◽  
Immunofluorescence Assay ◽  
Indirect Immunofluorescence Assay ◽  
Medical Laboratory ◽  
Nuclear Staining ◽  
International Consensus ◽  
In Vitro Diagnostic ◽  
Traditional Pattern

Abstract Background To evaluate the interpretation and reporting of antinuclear antibodies (ANA) by indirect immunofluorescence assay (IFA) using HEp-2 substrates based on common practice and guidance by the International Consensus on ANA patterns (ICAP). Method Participants included two groups [16 clinical laboratories (CL) and 8 in vitro diagnostic manufacturers (IVD)] recruited via an email sent to the Association of Medical Laboratory Immunologists (AMLI) membership. Twelve (n = 12) pre-qualified specimens were distributed to participants for testing, interpretation and reporting HEp-2 IFA. Results obtained were analyzed for accuracy with the intended and consensus response for three main categorical patterns (nuclear, cytoplasmic and mitotic), common patterns and ICAP report nomenclatures. The distributions of antibody titers of specimens were also compared. Results Laboratories differed in the categorical patterns reported; 8 reporting all patterns, 3 reporting only nuclear patterns and 5 reporting nuclear patterns with various combinations of other patterns. For all participants, accuracy with the intended response for the categorical nuclear pattern was excellent at 99% [95% confidence interval (CI): 97–100%] compared to 78% [95% CI 67–88%] for the cytoplasmic, and 93% [95% CI 86%–100%] for mitotic patterns. The accuracy was 13% greater for the common nomenclature [87%, 95% CI 82–90%] compared to the ICAP nomenclature [74%, 95% CI 68–79%] for all participants. Participants reporting all three main categories demonstrated better performances compared to those reporting 2 or less categorical patterns. The average accuracies varied between participant groups, however, with the lowest and most variable performances for cytoplasmic pattern specimens. The reported titers for all specimens varied, with the least variability for nuclear patterns and most titer variability associated with cytoplasmic patterns. Conclusions Our study demonstrated significant accuracy for all participants in identifying the categorical nuclear staining as well as traditional pattern assignments for nuclear patterns. However, there was less consistency in reporting cytoplasmic and mitotic patterns, with implications for assigning competencies and training for clinical laboratory personnel.

scholarly journals Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists

2020 ◽  
Vol 58 (9) ◽  
pp. 1489-1497 ◽  
Author(s):  
Lisa K. Peterson ◽  
Anne E. Tebo ◽  
Mark H. Wener ◽  
Susan S. Copple ◽  
Marvin J. Fritzler
Keyword(s):  
Indirect Immunofluorescence ◽  
Antinuclear Antibodies ◽  
Current Practice ◽  
Clinical Laboratory ◽  
Immunofluorescence Assay ◽  
Indirect Immunofluorescence Assay ◽  
Medical Laboratory ◽  
International Consensus ◽  
Clinical Laboratories ◽  
Reading Interpretation

AbstractBackgroundThe indirect immunofluorescence assay (IFA) using HEp-2 cell substrates is the preferred method by some for detecting antinuclear antibodies (ANA) as it demonstrates a number of characteristic staining patterns that reflect the cellular components bound as well as semi-quantitative results. Lack of harmonized nomenclature for HEp-2 IFA patterns, subjectivity in interpretation and variability in the number of patterns reported by different laboratories pose significant harmonization challenges. The main objectives of this study were to assess current practice in laboratory assessment of HEp-2 IFA, identify gaps and define strategies to improve reading, interpretation and reporting.MethodsWe developed and administered a 24-item survey based on four domains: educational and professional background of participants, current practice of HEp-2 IFA testing and training, gap assessment and the perceived value of International Consensus on Antinuclear Antibody Patterns (ICAP) and other factors in HEp-2 IFA assessment. The Association of Medical Laboratory Immunologists (AMLI) and American Society for Clinical Pathology administered the survey from April 1 to June 30, 2018, to members involved in ANA testing. This report summarizes the survey results and discussion from a dry workshop held during the 2019 AMLI annual meeting.ResultsOne hundred and seventy-nine (n = 179) responses were obtained where a significant number were clinical laboratory scientists (46%), laboratory directors (24%), supervisors (13%) or others (17%). A majority of respondents agreed on the need to standardize nomenclature and reporting of HEp-2 IFA results. About 55% were aware of the ICAP initiative; however, among those aware, a significant majority thought its guidance on HEp-2 IFA nomenclature and reporting is of value to clinical laboratories. To improve ICAP awareness and further enhance HEp-2 IFA assessment, increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials.ConclusionsBased on these suggestions, future efforts to optimize HEp-2 IFA reading, interpretation and reporting would benefit from more hands-on training of laboratory personnel as well as continuous collaboration between professional organizations, in vitro diagnostic manufacturers and clinical laboratories.

Multidrug-Resistant HIV-1 RNA and Proviral DNA Variants Harboring New Dipeptide Insertions in the Reverse Transcriptase pol Gene

2002 ◽  
Vol 29 (1) ◽  
pp. 102-104 ◽  
Author(s):  
Laurent Andréoletti ◽  
Laurence Weiss ◽  
Ali Si-Mohamed ◽  
Christophe Piketty ◽  
Thierry Prazuck ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Multidrug Resistant ◽  
Proviral Dna ◽  
Dna Variants ◽  
Hiv 1

scholarly journals A case of human monocytic ehrlichiosis in Serbia

2014 ◽  
Vol 142 (1-2) ◽  
pp. 79-82 ◽  
Author(s):  
Bogdan Arsic ◽  
Ana Gligic ◽  
Elizabeta Ristanovic ◽  
Branislav Lako ◽  
Aleksandar Potkonjak ◽  
...  
Keyword(s):  
Indirect Immunofluorescence ◽  
Clinical Picture ◽  
Peripheral Blood ◽  
Immunofluorescence Assay ◽  
High Fever ◽  
Tick Bite ◽  
Indirect Immunofluorescence Assay ◽  
Ehrlichia Chaffeensis ◽  
Serum Samples ◽  
History Of

Introduction. Ehrlichiosis is a bacterial zoonosis transmitted by hematophagous arthropods - ticks. In humans, it occurs as monocytic, granulocytic, and ewingii ehrlichiosis. Pathological process is based on parasitic presence of Ehrlichia organisms within peripheral blood cells - monocytes and granulocytes. Case Outline. Fifty-two year old patient was admitted to hospital due to high fever of over 40?C that lasted two days, accompanied with chills, muscle aches, malaise, loss of appetite, headache, confusion, breathing difficulties, and mild dry cough. The history suggested tick bite that occurred seven days before the onset of disease. Doxycycline was introduced and administered for 14 days, causing the disease to subside. Indirect immunofluorescence assay was used to analyze three serum samples obtained from this patient for Ehrlichia chaffeensis antibodies, and peripheral blood smear was evaluated for the presence of Ehrlichia and Ehrlichia aggregation into morulae. Conclusion. Ehrlichiosis should be considered in each case where there is a history of tick bite together with the clinical picture (high fever, chills, muscle aches, headache, generalized weakness and malaise, and possible maculopapular rash). The presence of Ehrlichia chaffeensis antibodies was confirmed in a patient with the history of tick bite, appropriate clinical picture and indirect immunofluorescence assay. This confirmed the presence of human monocytotropic ehrlichiosis, a disease that is uncommonly identified in our country.

scholarly journals A Novel Engineered Single-Chain Antibody Fragment for Targeting Pediatric Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-36
Author(s):  
Sara M.A. Mohamed ◽  
Keith Sia ◽  
Karl-Heinz Friedrich ◽  
Andreas Wohlmann ◽  
Savvas Savvides ◽  
...  
Keyword(s):  
Indirect Immunofluorescence ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Statistical Significance ◽  
Immunofluorescence Assay ◽  
Laser Scanning Microscopy ◽  
Indirect Immunofluorescence Assay ◽  
Single Chain ◽  
Genetic Characteristics ◽  
Significant Difference

Introduction: Philadelphia-like acute lymphoblastic leukaemia (Ph-like ALL) is a high-risk ALL subtype characterized by an inferior survival rate and chemotherapeutic drug resistance (Tasian et al, Blood 130: 2064-2072, 2017). Around 50% of Ph-like ALL cases harbour gene rearrangements leading to the overexpression of cytokine receptor-like factor 2 (CRLF2) (Loh et al, Blood 121: 485-488, 2013). CRLF2 (also known as thymic stromal lymphopoietin receptor, TSLPR) heterodimerizes with the interleukin-7 receptor alpha (IL-7Rα) subunit to form the functional TSLPR. Upon TSLP binding, CRLF2 activates the JAK/STAT signalling pathway, leading to enhanced proliferation and survival of leukemia cells resulting in poor outcomes in patients (Harvey et al, Blood 115: 5312-5321, 2010). The extracellular overexpression of CRLF2 and association with poor prognosis suggest the translational value of designing precision-based therapeutics targeting CRLF2 in Ph-like ALL. Although conventional immunotherapy using full-sized antibodies is a promising treatment strategy that can improve treatment efficiency and minimize off-target toxicity, their clinical translation is challenging due to the high production cost and large size affecting targeting efficiency (Holliger et al, Nat Biotech 23: 1126-1136, 2005). Herein, we validated a novel single-chain variable fragment against CRLF2 (CRLF2-ScFv) for targeting Ph-like ALL cells. Methods: A CRLF2-rearranged Ph-like ALL cell line (MHH-CALL-4) was lentivirally transduced with a CRLF2-targeting shRNA driven by an inducible promoter, enabling the inducible knockdown of CRLF2. CRLF2 expression in MHH-CALL-4 cells after shRNA induction (KD-CALL-4) was evaluated using fluorescence-activated cell sorting (FACS). The cellular association of the CRLF2-ScFv was determined in MHH-CALL-4 and KD-CALL-4 at 4° and 37°C using an indirect immunofluorescence assay. Confocal laser scanning microscopy was used to visualize and compare the cellular association of CRLF2-ScFv in MHH-CALL-4 and KD-CALL-4. The cellular association of CRLF2-ScFv was also investigated ex vivo using a small panel of Ph-like and non-Ph-like ALL patient-derived xenografts (PDXs), representing similar immunophenotype and genetic characteristics to their original disease subtypes (Liem et al, Blood 103: 3905-3914, 2004), and peripheral blood mononuclear cells (PBMCs) to investigate the non-selective association. CRLF2 expression in MHH-CALL-4 and Ph-like ALL PDX cells was quantified using indirect immunofluorescence assay. The downstream impact of CRLF2-ScFv on STAT5 phosphorylation (pSTAT5) was determined by FACS either with or without TSLP stimulation. The statistical significance was tested using Unpaired unequal variances t-test or one-way ANOVA followed by multiple comparisons test. Statistical significance was considered when P ≤ 0.05. All experiments were performed in triplicate. Results: KD-CALL-4 showed a 75% reduction in CRLF2 expression compared with MHH-CALL-4 cells (P = 0.0009). CRLF2-ScFv exhibited a 94% higher association with MHH-CALL-4 compared with KD-CALL-4 cells at 37°C (P = 0.0013). The association of CRLF2-ScFv with MHH-CALL-4 cells was reduced by 75% at the non-proliferating state of cells at 4°C compared to 37°C (P = 0.006). Orthogonally viewed confocal microscopy images showed 82% higher intracellular uptake of CRLF2-ScFv in MHH-CALL-4 compared to KD-CALL-4 cells (P = 0.0003). CRLF2-ScFv showed >80% higher association with a Ph-like PDX sample compared with a control CRLF2low PDX and PBMCs (P < 0.001). Of note, a Ph-like ALL PDX exhibited only one-third of the association with CRLF2-ScFv compared with MHH-CALL-4 cells (P = 0.04), corresponding to the significant difference in CRLF2 surface expression (P = 0.01). CRLF2-ScFv significantly reduced pSTAT5 expression in MHH-CALL-4 cells (P = 0.05) and prevented TSLP-induced STAT5 phosphorylation (P = 0.01), suggesting competition with the TSLP binding site. Conclusion: CRLF2-ScFv is a selective targeting moiety for CRLF2 with a significant internalization potential and receptor antagonistic effect, highlighting the therapeutic implications for targeting Ph-like ALL. Keywords: CRLF2, ScFv, STAT5 phosphorylation, Patient-Derived Xenografts. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document