Factors that inhibit the amplification of nucleic acids by PCR are present with target DNAs from many sources. The inhibitors generally act at one or more of three essential points in the reaction in the following ways: they interfere with the cell lysis necessary for extraction of DNA, they interfere by nucleic acid degradation or capture, and they inhibit polymerase activity for amplification of target DNA. Although a wide range of inhibitors is reported, the identities and modes of action of many remain unclear. These effects may have important implications for clinical and public health investigations, especially if the investigations involve food and environmental screening. Common inhibitors include various components of body fluids and reagents encountered in clinical and forensic science (e.g., hemoglobin, urea, and heparin), food constituents (e.g., organic and phenolic compounds, glycogen, fats, and Ca 21 ), and environmental compounds (e.g., phenolic compounds, humic acids, and heavy metals). Other, more widespread inhibitors include constituents of bacterial cells, nontarget DNA and contaminants, and laboratory items such as pollen, glove powder, laboratory plasticware, and cellulose. This review discusses the findings of many studies related to clinical, food, and environmental microbiology, including approaches that have been used to overcome inhibition and facilitate amplification for detection and typing. Few areas of biological science remain untouched by the invention of PCR (34, 81, 99). Other methods for amplifying nucleic acids (72, 123), such as Qb replicase (18), ligase chain reaction (13, 128), single-stranded sequence replication (17, 47), strand displacement amplification (126, 127), and nucleic acid sequence-based amplification (23, 122), have been described, but these methods have received less attention. Problems sometimes occur with PCR, however (124). Despite early indications of great sensitivity, the sensitivity of PCR may be a negative aspect of the procedure, since the most commonly reported problem is false-positive results due to cross-contamination (98, 124). This problem can be overcome by UV irradiation (100), with sodium hypochlorite (92), and by photochemical or enzymic methods (25, 36, 40, 78). One problem that is less discussed is reaction inhibition. This may be total or partial and can manifest itself as complete reaction failure or as reduced sensitivity of detection. In some cases, inhibition may be the cause of false-negative reactions, since few workers incorporate internal controls in each reaction tube. Early evidence of exquisite sensitivity with mammalian cells (53) involving detection of a single molecule of DNA from a hair was not reproduced when PCR was applied to many microbial (and some mammalian) situations, where poor sensitivity, specificity, and reproducibility have been reported (16, 82, 86, 129, 132, 134). There may also be potentially important effects in PCR typing reactions (121), and difficulties can occur in post-PCR manipulation (61). Although systematic study of inhibition has seldom been the focus of published investigations, many workers have reported these effects in the course of other studies (12, 19, 21, 124, 129, 132, 133). Considering the prevalence of this problem, it is surprising that few systematic and mechanistic studies of PCR inhibition have been reported. Rossen et al. (97) contributed the most comprehensive study of PCR inhibition, identifying inhibitory factors in foods, bacterial culture media, and various chemical compounds. These inhibitory factors included organic and inorganic chemicals, detergents, antibiotics, buffers, enzymes, polysaccharides, fats, and proteins. This review lists and discusses inhibitors and methods that can overcome the attenuation of amplification in clinical, food, and environmental microbiology. It is beyond the scope of this paper to discuss in detail the various physical, enzymic, and chemical methods used in the extraction, purification, and quantitation of nucleic acids. Those methods are presented and discussed in commercial literature and elsewhere (14, 95, 96, 106, 134).

https://aem.asm.org/content/aem/63/10/3741.full.pdf

Inhibition and facilitation of nucleic acid amplification.

We evaluated three cellular and five serologic markers that are affected by infection with the human immunodeficiency virus type 1 (HIV-1) for their ability to predict the progression to clinical acquired immunodeficiency syndrome (AIDS). The cellular markers were the number of CD4+ T cells, the number of CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells. The serologic markers were the serum levels of neopterin (a product of stimulated macrophages), beta 2-microglobulin, soluble interleukin-2 receptors, IgA, and HIV p24 antigen. We evaluated the usefulness of these measures as markers of the progression to AIDS prospectively, over four years, in a cohort of 395 HIV-seropositive homosexual men who were initially free of AIDS. CD4+ T cells (expressed as an absolute number, a percentage of lymphocytes, or a ratio of CD4+ to CD8+ T cells) were the best single predictor of the progression to AIDS, but the serum neopterin and beta 2-microglobulin levels each had nearly as much predictive power. The neopterin level appeared to be a slightly better predictor than the beta 2-microglobulin level. The levels of IgA, interleukin-2 receptors, and p24 antigen had less predictive value. A stepwise multivariate analysis indicated that the best predictors, in descending order, were CD4+ T cells (the percentage of lymphocytes or the CD4+: CD8+ ratio), the serum level of neopterin or beta 2-microglobulin, the level of IgA, that of interleukin-2 receptors, and that of p24 antigen. The last three markers had little additional predictive power beyond that of the first two. We conclude that of the eight markers studied, progression to AIDS was predicted most accurately by the level of CD4+ T cells in combination with the serum level of either neopterin or beta 2-microglobulin. At least one of these two serum markers, which reflect immune activation, should be used along with measurement of CD4+ T cells in disease-classification schemes and in the evaluation of responses to therapy.

https://www.nejm.org/doi/pdf/10.1056/NEJM199001183220305

The prognostic value of cellular and serologic markers in infection with human immunodeficiency virus type 1.

Objective: To investigate the relation between detection of syncytium-inducing (SI), human immunodeficiency virus type 1 (HIV-1) variants, rate of CD4+ cell decline, and clinical progression. Desig...

Prognostic Value of HIV-1 Syncytium-Inducing Phenotype for Rate of CD4+ Cell Depletion and Progression to AIDS

Background. Primary infection with the human immunodeficiency virus (HIV-1) frequently causes an acute, self-limited viral syndrome. To examine the relations among viral replication, the immune response of the host, and clinical illness during this initial phase of infection, we undertook a quantitative, molecular, and biologic analysis of infectious HIV-1 in the blood and plasma of three patients with symptomatic primary infection and of a sexual partner of one of them. Methods. During an eight-week period of primary infection, HIV-1 was cultured frequently in dilutions of plasma and peripheral-blood mononuclear cells (PBMC), and levels of HIV-1 antigen and antibody were determined sequentially by enzyme-linked immunosorbent assay and immunoblotting. Replication-competent HIV-1 proviruses were cloned and characterized biologically. Results. Six to 15 days after the onset of symptoms, high titers of infectious HIV-1 (from 10 to 103 tissue-culture—infective doses per milliliter of plasma) and vira...

High titers of cytopathic virus in plasma of patients with symptomatic primary HIV-1 infection.

We determined incidence and future projections of dementia after AIDS onset in 492 homosexual men with AIDS in the Baltimore/Los Angeles sites of the Multicenter AIDS Cohort Study, 64 of whom developed dementia. We studied various risk factors for dementia, including demographic and clinical features, medical history, markers of immune status before AIDS, and zidovudine use. During the first 2 years after AIDS, HIV dementia developed at an annual rate of 7%. Overall, 15% of the cohort followed through death developed dementia. The median survival after dementia was 6.0 months. Using a proportional hazards model, risk factors for more rapid development of dementia were lower hemoglobin (relative hazard, 0.59 per additional 2 g/dl; p = 0.0005) and body mass index (relative hazard, 0.64 per additional 5 kg/m2; p = 0.05) 1 to 6 months before AIDS, more constitutional symptoms 7 to 12 months before AIDS (relative hazard, 1.68 per additional symptom, p = 0.005), and older age at AIDS onset (relative hazard, 1.60 per decade older; p = 0.009). In a multivariate model, pre-AIDS hemoglobin remained the most significant predictor of dementia. There were no significant risks defined from demographic characteristics, specific AIDS-defining illnesses, zidovudine use before AIDS, or CD4+ lymphocyte count before AIDS. We project that 12 months after the first AIDS diagnosis, 7.1% of survivors will have dementia. The observed association between anemia, low weight, constitutional symptoms, and dementia suggests a role for cytokines inducing both systemic and neurologic disease.

Dementia in AIDS patients: Incidence and risk factors

The three year actuarial progression rate to the acquired immune deficiency syndrome (AIDS) in a cohort of men in San Francisco who were seropositive for the human immuno-deficiency virus (HIV) was 22%. An additional 26 (19%) developed AIDS related conditions. β 2 Microglobulin concentration, packed cell volume, HIV p24 antigenaemia, and the proportion and number of T4 lymphocytes each independently predicted progression to AIDS. β 2 Microglobulin was the most powerful predictor. The 111 subjects tested who were normal by all predictors (40%) had a three year progression rate of 7%, and the 68 subjects who were abnormal by two or more predictors (24%) had a progression rate of 57%. Two thirds of all men who progressed to AIDS were in the last group. The median T4 lymphocyte count in subjects who did not progress to AIDS fell from 626 × 10 6 to 327 × 10 6 /1. HIV p24 antigenaemia developed in 7% of the subjects per year. The proportion who were abnormal by two or more predictive variables rose to 41%. At three years an estimated two thirds of the seropositive subjects showed clinical AIDS, an AIDS related condition, or laboratory results that were highly predictive of AIDS. It is concluded from the observed rates and the distribution of predictive variables at three years that half of the men who were seropositive for HIV will develop AIDS by six years after the start of the study, and three quarters will develop AIDS or an AIDS related condition.

https://www.bmj.com/content/bmj/296/6624/745.full.pdf

Seropositivity for HIV and the development of AIDS or AIDS related condition: three year follow up of the San Francisco General Hospital cohort.

Significant changes in HIV antigen level in the serum of patients treated with azidothymidine.

Background. The p24 antigen of human immunodeficiency virus type 1 (HIV-1) is sometimes detected before antibody (anti-HIV-1) is detectable in the serum of recently infected persons. This has led to the consideration of p24-antigen testing for routine screening of blood donors. Methods. To estimate how many HIV-infected seronegative donors would be identified if p24-antigen screening was introduced, we tested selected donations from a repository of 200,000 serum samples from voluntary donors that was established in late 1984 and early 1985. The 8597 serum samples selected for p24-antigen screening were chosen because their donors had demographic characteristics known to be associated with a high prevalence of seropositivity. Results. The prevalence of anti-HIV-1 antibodies in the 1984–1985 serum samples selected for p24-antigen screening was 1.54 percent — more than 100 times the 0.012 percent prevalence in present-day donations in the United States. The antigen was detected in 15 of 132 serum sa...

Screening of Selected Male Blood Donors for p24 Antigen of Human Immunodeficiency Virus Type 1

During a 1-year period of study at two plasma collection centers, 7 of 35 000 plasma donors seroconverted to the human immunodeficiency virus (HIV) and had stored plasma samples that predated or antedated the seroconversion period. From each donor, three to eight plasma samples that had been collected at 2- to 7-day intervals were tested for IgG and IgM antibodies to HIV with enzyme immunoassays, Western blot testing, and radioimmunoprecipitation assays. The presence of an HIV viremic phase was demonstrated by the infectivity of plasma on normal, phytohemagglutinin-stimulated peripheral blood mononuclear cells and by the detection of HIV antigen. In 5 of these donors, HIV antigen was detected prior to or simultaneously with IgG to HIV; these HIV-antigen—positive samples overlapped an IgM immune response. The disappearance of detectable HIV antigen, and to a lesser extent plasma infectivity, was concurrent with the development of an IgG immune response. Although the improved sensitivity of a recombinant DNA-derived anti-HIV screening assay shortened the "window period" between initial HIV infection and antibody detection, HIV antigen and plasma HIV viremia were the only markers of HIV infection for several days in 2 donors. These results demonstrate that HIV plasma viremia and antigenemia occur prior to seroconversion in healthy plasma donors. ( JAMA . 1989;262:64-69)

Markers of HIV infection prior to IgG antibody seropositivity

Redefining the HIV-infectious window period in the chimpanzee model: evidence to suggest that viral nucleic acid testing can prevent blood-borne transmission.

Jean-Pierre Allain

Papers

Seropositivity for HIV and the development of AIDS or AIDS related condition: three year follow up of the San Francisco General Hospital cohort.

Significant changes in HIV antigen level in the serum of patients treated with azidothymidine.

Screening of Selected Male Blood Donors for p24 Antigen of Human Immunodeficiency Virus Type 1

Markers of HIV infection prior to IgG antibody seropositivity

Redefining the HIV-infectious window period in the chimpanzee model: evidence to suggest that viral nucleic acid testing can prevent blood-borne transmission.