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

This presentation summarizes the US national cooperative trials for AIDS treatment and some specific efforts in the treatment of viral infections. The underlying principles behind clinical efforts in the control of the human immunodeficiency virus are described to date. In addition, concerns are voiced regarding the oversimplification of treatment issues presented in the public policy area; some will clearly require responses from interested and informed clinical scientists.

A Personal View of Efforts in Treatment of Human Immunodeficiency Virus Infection in 1988

Interferons have now been used in both prophylaxis and treatment of a number of human viral infections. The major action has been as a prophylactic for sites within the body that are not yet involved by disease. Such a prophylactic effect can be obtained early in the treatment of acute viral infection or even during chronic viral disease. Both local and systemic prophylaxis have been achieved with regard to both respiratory and herpesviral illness. In addition, Dane particle suppression can be achieved consistently with dosages of 10 6 units or greater daily to patients with chronic hepatitis B virus infection. In certain cases with prolonged therapy there can be permanent eradication. With leucocyte-derived material of approximately 10 6 or 10 7 units per milligram protein, the major side effects have been an initial febrile response, fatigue, malaise, marrow suppression, and inhibition of hair growth. So far, side effects have been rapidly reversible on lowering of dosage. Present studies with the use of lymphoblastoid interferon and bacterial-derived interferon employ materials of significantly greater specific activity. Such experience suggests that the same general side effects that were limiting with leucocyte interferon are present with interferon produced from recombinant DNA by bacterial as well as with lymphoblastoid interferon.

Interferons as antivirals in man.

SummaryTreatment of mice with the interferon inducer polyinosinic:polycytidylic acid failed to inhibit multiplication of Mycobacterium leprae in the mouse foot pad. This same treatment has recently been shown to inhibit multiplication in vivo of several intra-and extracellular pathogenic microorganisms.

Failure of an interferon inducer to inhibit multiplication of Mycobacterium leprae.

Because of the close coupling of the replication of animal viruses and host cell functions, successful antiviral therapy has demanded agents specifically directed against virus infected cells. In addition, the spread of virus within an infected individual involves a wide variety of cells in which antiviral drugs may or may not act with similar efficacy. Recent studies (1) have demonstrated that host defense to viral infection involves a multitiered series of interrelated, but yet independent responses, which act on virus infected cells as well as on extracellular virus to diminish spread of the agent within the host. Various host defenses act both at the portal of entry and in the target organs with relative efficacy which differs from one virus to another -- or even between strains. In addition to local action of IgA antibody and macrophages, systemic IgM and IgG antibodies and antibody-dependent cellular cytotoxic mechanisms come into play, together with thymus-derived lymphocyte cytotoxicity and lymphoidne production. Such host factors are clearly critical in dictating the pathogenesis of infection of any particular virus. The proper function of all of these events obviously depends on a complex series of host biosynthetic and differentiation steps.

Success of Antiviral Therapy Involves Cooperative Immune Responses

To what extent is interferon fulfilling its promise as a “broad-spectrum” antiviral agent? Current research suggests that stimulation of host interferon by inducers, rather than direct administration, will be the method by which interferon's properties are exploited. The therapeutic effectiveness of induction has already been demonstrated in animal models; limited clinical trials are not far off.

Thomas C. Merigan

Papers

A Personal View of Efforts in Treatment of Human Immunodeficiency Virus Infection in 1988

Interferons as antivirals in man.

Failure of an interferon inducer to inhibit multiplication of Mycobacterium leprae.

Success of Antiviral Therapy Involves Cooperative Immune Responses

Interferon and Interferon Inducers: The Clinical Outlook