Effective point-of-care testing (POCT) is reliant on optimal specimen collection, quality assured testing, and expedited return of results. Many of the POCT are designed to be used with fingerstick ...capillary blood to simplify the blood collection burden. However, fingerstick blood collection has inherent errors in sampling. An evaluation of the use of capillary and venous blood with CD4 POCT was conducted.
Three different specimen collection methods were evaluated for compatibility using the Alere Pima CD4 assay at 5 HIV/AIDS healthcare sites in Dar es Salaam, Tanzania. At each site, whole blood specimens were collected from enrolled patients by venipuncture and fingerstick. Pima CD4 testing was performed at site of collection on venipuncture specimens (Venous) and fingerstick blood directly applied to a Pima CD4 cartridge (Capillary-Direct) and collected into an EDTA microtube (Capillary-Microtube). Venous blood was also tested at the laboratory by the reference CD4 method and Pima for comparison analysis.
All three specimen collection methods were successfully collected by healthcare workers for use with the Pima CD4 assay. When compared to the reference CD4 method, Pima CD4 testing with the Capillary-Microtube method performed similarly to Venous, while Pima CD4 counts with the Capillary-Direct method were slightly more biased (-20 cells/μL) and variable (-229 to +189 cells/μL limit of agreement). Even though all three collection methods had similar invalid Pima testing rates (10.5%, 9.8%, and 8.3% for Capillary-Direct, Capillary-Microtube, and Venous respectively), the ability to perform repeat testing with Capillary-Microtube and Venous specimens increased the likelihood of acquiring a valid CD4 result with the Pima assay.
Capillary blood, either directly applied to Pima CD4 cartridges or collected in an EDTA microtube, and venous blood are suitable specimens for Pima CD4 testing. The advantages of capillary blood collection in an EDTA microtube are that it uses fingerstick collection which mimics venous blood and allows extra testing without additional blood collection.
Point-of-care (POC) CD4 testing can improve access to treatment by enabling decentralization and reducing patient loss-to-follow-up. As new POC CD4 technologies become available, their performance ...should be assessed before widespread deployment. This study reports the findings of five independent evaluations of the PointCare NOW CD4 system.
Evaluations were conducted in Southern Africa (Mozambique, South Africa) and North America (Canada, USA). 492 blood samples (55 from HIV-negative blood donors and 437 from HIV-infected patients, including 20 children aged between 12 and 59 months) were tested with both the PointCare NOW and reference flow cytometry instruments. Assessment of bias, precision and levels of clinical misclassification for absolute and percent CD4 count was conducted.
PointCare NOW significantly overestimated CD4 absolute counts with a mean relative bias of +35.0%. Bias was greater in samples with CD4 counts below ≤ 350 cells/µl (+51.3%) than in the CD4 >350 cells/µl stratum (15.1%). Bias in CD4% had a similar trend with an overall relative mean bias of +25.6% and a larger bias for low CD4 stratum (+40.2%) than the higher CD4 stratum (+5.8%). Relative bias for CD4% in children was -6.8%. In terms of repeatability, PointCare NOW had a coefficient of variation of 11%. Using a threshold of 350 cells/µl, only 47% of patients who qualified for antiretroviral therapy with reference CD4 testing, would have been eligible for treatment with PointCare NOW test results. This was 39% using a 200 cells/µl threshold. Agreement with infant samples was higher, with 90% qualifying at a 25% eligibility threshold.
The performance of the PointCare NOW instrument for absolute and percent CD4 enumeration was inadequate for HIV clinical management in adults. In children, the small sample size was not large enough to draw a conclusion. This study also highlights the importance of independent evaluation of new diagnostic technology platforms before deployment.
We evaluated the protective role of passively transferred circulating antibodies in protecting non-human primates against experimental rotavirus infection. Pooled sera with rotavirus-specific IgG ...titers that were either high (1:10,000), intermediate (1:300), or negative (<1:25) were infused i.v. into naïve pigtailed macaques (ages 3-6 months). Rotavirus-specific IgG could be detected in the sera at 18 h in all animals infused with antibody-containing serum, and fecal IgG titers could be detected only in animals given high-titer pooled sera. When orally challenged with 106fluorescent-forming units of a simian rotavirus strain, YK-1, at 18 h after serum transfer, control animals shed virus starting 1-3 days after challenge and continued to shed virus at high titers for 6-8 days, whereas passively immunized macaques did not shed virus or had delayed shedding at low titers for only a limited time. The observation that passively transferred antibodies can suppress or delay viral infection in rotavirus-challenged pigtailed macaques has important implications for the design and testing of parenteral candidate rotavirus vaccines.
The SA NHLS is the sole owner of the PLG CD4 patent, and receives royalties related to licensing agreement with BC Miami. (2012) Correction: Performance of the PointCare NOW System for CD4 Counting ...in HIV Patients Based on Five Independent Evaluations.
Quality assurance (QA) is a systematic process to monitor and improve clinical laboratory practices. The fundamental components of a laboratory QA program include providing a functional and safe ...laboratory environment, trained and competent personnel, maintained equipment, adequate supplies and reagents, testing of appropriate specimens, internal monitoring of quality, accurate reporting, and external quality assessments. These components are necessary to provide accurate and precise CD4 T-cell counts, an essential test to evaluate start of and monitor effectiveness of antiretroviral therapy for HIV-infected patients. In recent years, CD4 testing has expanded dramatically in resource-limited settings. Information on a CD4 QA program as described in this article will provide guidelines not only for clinical laboratory staff but also for managers of programs responsible for supporting CD4 testing. All agencies involved in implementing CD4 testing must understand the needs of the laboratory and provide advocacy, guidance, and financial support to established CD4 testing sites and programs. This article describes and explains the procedures that must be put in place to provide reliable CD4 determinations in a variety of settings.
During infection of a new host, the first surfaces encountered by herpes simplex viruses are the apical membranes of epithelial cells of mucosal surfaces. These cells are highly polarized, and the ...protein composition of their apical and basolateral membranes are very different, so that different viral entry pathways have evolved for each surface. To determine whether the viral glycoprotein G (gG) is specifically required for efficient infection of a particular surface of polarized cells, apical and basal surfaces were infected with wild-type virus or a gG deletion mutant. After infection of polarized cells in culture, the gG-virus was deficient in infection of apical surfaces but was able to infect cells through basal membranes, replicate, and spread into surrounding cells. The gG-dependent step in apical infection was a stage beyond attachment. After in vivo infection of apical surfaces of epithelial cells of nonscarified mouse corneas, infection by glycoprotein C-or gG-virus was considerably reduced as compared with that observed after infection with wild-type virus. In contrast, when corneas were scarified, allowing virus access to other cell surfaces, the gG and glycoprotein C deletion mutants infected eyes as efficiently as wild-type viruses. A secondary mutation allowing infection of apical surfaces by gG-virus arose readily during passage of the virus in nonpolarized cells, indicating that either the gG-dependent step of apical infection can be bypassed or that another viral protein can acquire the same function.
To effectively analyze the requirements for protection to rotavirus infection, a reliable animal model that reasonably mimics infection and disease in humans is needed. A requirement for an effective ...animal model is the availability of appropriate rotavirus stocks for challenge.
A new simian rotavirus, designated YK-1, was isolated from a 2-year-old immunodeficient pigtailed macaque with chronic diarrhea. YK-1 was distinguishable by electropherotype from the other simian rotavirus strains, SA11 and RRV. One variant of YK-1, clone 311, which was isolated after adaptation and plaque purification in cell cultures, displayed an unusual RNA electropherotype with an abnormally migrating gene 11 segment. Sequence analysis demonstrated a genetic rearrangement that involved a partial duplication of the gene 11 ORF encoding NSP5. YK-1 was identified as a Group A rotavirus belonging to subgroup 1. To further characterize the YK-1 strain, the genes encoding VP4, VP7, and NSP4 were sequenced. Analysis of VP4 and VP7 gene fragments suggests that this strain is a G3P3 rotavirus and is closely related to the simian rotavirus strain RRV. Serotype analysis also identified YK-1 as a G3 rotavirus. The NSP4 genotype of YK-1 is C, the same genotype as RRV.
This newly isolated rotavirus, YK-1, is being used to establish a nonhuman primate model for studying the infectivity, immunity, and pathogenesis of rotavirus and for evaluating candidate rotavirus vaccines.
Peptide exchange in MHC molecules Jensen, Peter E.; Weber, Dominique A.; Thayer, Wesley R ...
Immunological reviews,
December 1999, Letnik:
172, Številka:
1
Journal Article
Recenzirano
Major histocompatibihty complex (MHC)‐encoded glycoproteins bind peptide antigens through non‐covalent interactions to generate complexes that are displayed on tbe surface of antigen‐presenting cells ...(APC) for recognition by T ceils, Peptide‐binding site occupancy is necessary for stable assembly of newly synthesized MHC proteins and export from the endoplasmic reticulum (ER), The MHC class II antigen‐processing pathway provides a mechanism for presentation of peptides generated in the endosomal pathway of APC, The chaperone protein, invariant chain, includes a surrogate peptide that stahilizes newly synthesized class II molecules during transport to endosomal compartments. The invariant chain‐derived peptide must be replaced through a peptide exchange reaction that is promoted by acidic pH and the MHC‐encoded co‐factor HLA‐DM, Peptide exchange reactions are not required for presentation of antigens by MHC class I molecules because they bind antigens during initial assembly in the ER, However, exchange reactions may play an important role in editing the repertoire of peptides presented by both class II and class I molecules, thus influencing the specificity of immunity and tolerance.
Experimental rotavirus infection was investigated in pigtailed macaques to study the infectivity, immunity, and pathogenesis of rotavirus. A challenge virus, YK‐1, was administered intragastrically ...to four seronegative macaques (age: 11–16 months). Although none of the monkeys developed diarrhea, an active infection occurred with high titers of rotavirus antigen detected in stools 2–10 days after challenge. These animals developed rotavirus‐specific antibody responses similar to those seen following primary exposure to rotavirus. YK‐1 was then inoculated in four seropositive macaques (age: 14–16 months). All animals shed viral antigen in their stool, but the titers and duration were significantly less when compared to seronegative macaques. When rechallenged 28 days after initial YK‐1 challenge, the macaques demonstrated significant protection against reinfection. All seropositive animals developed a rise in rotavirus‐specific serum and fecal antibodies during YK‐1 challenge and rechallenge. To independently assess the role of age and preexisting IgG titers to rotavirus, a 4‐month‐old seronegative and 6‐month‐old seropositive macaque were inoculated with YK‐1. The seronegative macaque shed high titers of virus for 9 days, while the seropositive macaque shed only 3 days and in low titer. These data suggest that a primate model of rotavirus infection using the YK‐1 strain may be useful in examining the immune response and protection from infection in pigtailed macaques and indicate that levels and duration of shedding may provide a good measure of protection from natural infection and from that induced by oral or parenteral vaccines. J. Med. Virol. 75:616–625, 2005. Published 2005 Wiley‐Liss, Inc.
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