Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments.

Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement.

Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

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The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

Background and Methods National surveillance data show recent, marked reductions in morbidity and mortality associated with the acquired immunodeficiency syndrome (AIDS). To evaluate these declines, we analyzed data on 1255 patients, each of whom had at least one CD4+ count below 100 cells per cubic millimeter, who were seen at nine clinics specializing in the treatment of human immunodeficiency virus (HIV) infection in eight U.S. cities from January 1994 through June 1997. Results Mortality among the patients declined from 29.4 per 100 person-years in 1995 to 8.8 per 100 person-years in the second quarter of 1997. There were reductions in mortality regardless of sex, race, age, and risk factors for transmission of HIV. The incidence of any of three major opportunistic infections (Pneumocystis carinii pneumonia, Mycobacterium avium complex disease, and cytomegalovirus retinitis) declined from 21.9 per 100 person-years in 1994 to 3.7 per 100 person-years by mid-1997. In a failure-rate model, increases in t...

Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators.

This invention provides methods of monitoring the expression levels of a multiplicity of genes. The methods involve hybridizing a nucleic acid sample to a high density array of oligonucleotide probes where the high density array contains oligonucleotide probes complementary to subsequences of target nucleic acids in the nucleic acid sample. In one embodiment, the method involves providing a pool of target nucleic acids comprising RNA transcripts of one or more target genes, or nucleic acids derived from the RNA transcripts, hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on surface, where the array comprising more than 100 different oligonucleotides and each different oligonucleotide is localized in a predetermined region of the surface, the density of the different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm2, and the oligonucleotide probes are complementary to the RNA transcripts or nucleic acids derived from the RNA transcripts; and quantifying the hybridized nucleic acids in the array.

Expression monitoring by hybridization to high density oligonucleotide arrays

SUMMARY The availability of an increasing number of antiretroviral agents and the rapid evolution of new information has introduced extraordinary complexity into the treatment of HIV-infected persons. In 1996, the Department of Health and Human Services and the Henry J. Kaiser Family Foundation convened the Panel on Clinical Practices for the Treatment of HIV to develop guidelines for the clinical management of HIV-infected adults and adolescents. This report recommends that care should be supervised by an expert, and makes recommendations for laboratory monitoring including plasma HIV RNA, CD4 cell counts and HIV drug resistance testing. The report also provides guidelines for antiretroviral therapy, including when to start treatment, what drugs to initiate, when to change therapy, and therapeutic options when changing therapy. Special considerations are provided for adolescents and pregnant women. As with treatment of other chronic conditions, therapeutic decisions require a mutual understanding between the patient and the health care provider regarding the benefits and risks of treatment. Antiretroviral regimens are complex, have major side effects, pose difficulty with adherence, and carry serious potential consequences from the development of viral resistance due to non-adherence to the drug regimen or suboptimal levels of antiretroviral agents. Patient education and involvement in therapeutic

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Guidelines for the use of antiretroviral agents in HIV-infected adults and adolescents

The following CDC staff members prepared this report: National Center for Infectious Diseases Division of HIV/AIDS Kenneth G. Castro, M.D. John W. Ward, M.D. Laurence Slutsker, M.D., M.P.H. James W. Buehler, M.D. Harold W. Jaffe, M.D. Ruth L. Berkelman, M.D. Office of the Director Associate Director for HIV/AIDS James W. Curran, M.D., M.P.H. 1993 Revised Classification System for HIV Infection and Expanded Surveillance Case Definition for AIDS Among Adolescents and Adults Summary CDC has revised the classification system for HIV infection to emphasize the clinical importance of the CD4+ T-lymphocyte count in the categorization of HIV-related clinical conditions. This classification system replaces the system published by CDC in 1986 (1) and is primarily intended for use in public health practice. Consistent with the 1993 revised classification system, CDC has also expanded the AIDS surveillance case definition to include all HIV-infected persons who have less than 200 CD4+ T-lymphocytes/uL, or a CD4+ T-lymphocyte percentage of total lymphocytes of less than 14. This expansion includes the addition of three clinical conditions

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1993 Revised Classification System for HIV Infection and Expanded Surveillance Case Definition for AIDS Among Adolescents and Adults

Summary
After heating at 37° in minimal Eagle's medium, the alternating polyriboadenylic-polyribouridylic acid [poly r(A-U)] became much more active in producing cellular resistance to virus infection and interferon production in four different cell lines (human skin fibroblasts, mouse embryo fibroblasts, mouse L 929 cells and rabbit kidney (RK 13) cells). The thermal activating effect was neutralized by pre-treatment of the cells with DEAE-dextran, which increased the antiviral activity of unheated and decreased the activity of heated poly r(A-U) to the same level. Under all conditions tested, the degree of cellular resistance to virus infection closely paralleled the amounts of interferon produced suggesting that interferon production is responsible for the antiviral resistance produced by poly r(A-U).

Thermal activation of the antiviral activity of synthetic polyribonucleotides: influence of DEAE-dextran in various cell cultures

Antivirals with clinical potential

AIDS Clinical Trials Group (ACTG) 246/946 was a double-blinded, randomized, controlled trial of HIV-1 MN rgp160 ImmunoAG vaccine in HIV-infected patients with CD4+ T cell counts ≥500 and 200-400/mm3. The main objectives were to study the safety and immunogenicity of this vaccine and to study the persistence of the immune responses after vaccination over a longer period of time. Fifteen patients with CD4+ T cell counts of ≥500/mm3 were enrolled in the ACTG 246 study. ACTG 246 patients received a monthly injection of vaccine or control for 6 months and then injections every 2 months. After completion of this study, seven new patients with CD4+ T cell counts of 200-400/mm3 entered into the ACTG 946 study. These study patients received highly active antiretroviral therapy (HAART) (ritonavir, didanosine, and stavudine) for 9 weeks to stabilize their viral load and then each patient received a monthly injection of vaccine or control substance for 6 months with HAART. The study of these two relatively small popu...

Effect of Therapeutic Immunization with HIV Type 1 Recombinant Glycoprotein 160 ImmunoAG Vaccine in HIV-Infected Individuals with CD4+ T Cell Counts of ≥500 and 200-400/mm3 (AIDS Clinical Trials Group Study 246/946)

Three infants with congenital rubella syndrome were given human leukocyte (alpha) interferon at doses of 2 x 10(5) to 7 X 10(5) U/kg per day for 10 days. A transient decrease in pharyngeal virus excretion was observed with treatment. No significant side effects were associated with the administration of human leukocyte interferon to these infants.

Alpha interferon administration to infants with congenital rubella.

SummaryHomogeneous monolayers of human fibroblasts were infected with Newcastle disease virus (NDV) and 1 hr later treated with the synthetic RNA polycytidylate: polyinosinate (poly C:I) The induction of interferon by poly C:I and NDV could be distinguished on the basis of differences in (1) the kinetics of interferon production, and (2) the sensitivity to inhibitors of RNA and protein synthesis The results indicated that both poly C:I and NDV were fully able to induce the production of interferon in these dually stimulated monolayers The release of interferon induced by poly C:I appeared to be terminated shortly before the appearance of viral interferon in the extracellular medium Our findings suggest that termination of the nonviral interferon response did not involve direct inactivation of interferon It appears that a inhibitor substance blocked the continued formation of nonviral interferon, without significantly affecting the induction of viral interferon

Thomas C. Merigan

Papers

Thermal activation of the antiviral activity of synthetic polyribonucleotides: influence of DEAE-dextran in various cell cultures

Antivirals with clinical potential

Effect of Therapeutic Immunization with HIV Type 1 Recombinant Glycoprotein 160 ImmunoAG Vaccine in HIV-Infected Individuals with CD4+ T Cell Counts of ≥500 and 200-400/mm3 (AIDS Clinical Trials Group Study 246/946)

Alpha interferon administration to infants with congenital rubella.

Simultaneous Viral and Non-Viral Interferon Production in Human Cell Cultures