Polymerase chain reaction in detecting HIV infection among seropositive infants: relation to clinical status and age and to results of other assays.

The polymerase chain reaction (PCR) for human immunodeficiency virus (HIV) was evaluated using coded blood specimens from infants whose clinical status is now known. A micromethod for the efficient isolation of mononuclear cells from small volumes of blood, and definitions of PCR positivity that took into account the number and purity of these mononuclear cells, were established in an attempt to define parameters for quality assurance. Results of HIV culture, p-24 antigen, and HIV-specific IgA obtained on the same specimens were compared to PCR results. PCR had a specificity of 100% among 83 specimens from 50 babies known to be uninfected. Sensitivity among 26 HIV-infected infants older than 3 months was 98% (44 of 45 specimens); the one negative specimen, which had also been culture negative, gave a positive PCR result on the remaining aliquot when tested after decoding. Among infected infants less than 3 months old, which is an age when diagnosis by other assays is most problematic, PCR identified 10 of 10 patients (10 of 11 specimens) including two younger than one month. Viral culture showed the best concordance with PCR; however, in three infants, positive PCR results were observed several months before positive results were observed by viral culture.

Detection of IgM and IgA HIV antibodies after removal of IgG with recombinant protein G.

Indirect assays for IgM and IgA antibodies often lack sensitivity and specificity due to interference from IgG antibodies. To overcome this problem we have developed a simple procedure using recombinant protein G coupled to agarose beads to remove the interfering IgG. A series of HIV seroconversion panels was tested by IgM and IgA immunoblot after protein G treatment in order to evaluate IgG removal and to study appearance of IgM and IgA antibodies in early HIV infection. Protein G treatment removed 99.9% of the IgG and reduced IgG anti-HIV titers of over 1/100,000 to undetectable levels. Both IgM and IgA HIV antibodies were detected as early in seroconversion as were IgG HIV antibodies. IgA HIV antibodies persisted for a longer period of time, reacted with more HIV proteins, and showed more intense staining than IgM HIV antibodies.

Monoclonal antibody testing of lymphocytes after overnight storage.

Often monoclonal antibody testing of lymphocytes is not performed until the day after blood collection for reasons of convenience or due to the need to transport the blood to other facilities. In order to determine whether accurate results can be obtained on the day after collection, we compared results obtained after storage overnight at 4 degrees C or 22 degrees C with results obtained with fresh lymphocytes. Lymphocytes from 24 normal individuals were evaluated with 10 monoclonal antibodies using an immunofluorescence technique with analysis by flow cytofluorometry. There were markedly altered results obtained with lymphocytes separated on the day after collection from whole blood stored at 4 degrees C. Lymphocytes separated from whole blood stored at 22 degrees C showed moderate changes in reactivity with some monoclonal antibodies. Lymphocytes that were separated from fresh blood and then stored at 4 degrees C or 22 degrees C showed results similar to fresh lymphocytes. These results underscore the importance of proper processing of blood samples to avoid misinterpretation of results.

Measurement of the distribution of indium-111 on human plasma proteins using immunoprecipitation.

The distribution of radioactivity on plasma proteins labeled by addition of [111In]oxine to citrated plasma was investigated. Analyses of plasma proteins separated on Sephadex G-200 columns showed that 23-36% of the 111In was associated with proteins with molecular weight greater than 200,000 daltons and the remaining 111In was associated with proteins with molecular weight less than 100,000 daltons, presumably transferrin. Affinity chromatography experiments showed that less than 2% of the radioactivity was associated with albumin. Further identification of the labeled proteins and quantitation of associated radioactivity was performed by precipitating specific proteins with antibodies. These studies showed that the 111In was distributed on transferrin (54-76%), fibrinogen (11-24%), IgM (8-20%), C3 (10-21%), and haptoglobin (3-8%). 111In associated with fibrinogen, IgM, and haptoglobin was over-estimated in some experiments due to binding of 111In-labeled C3 to the antigen-antibody precipitates.

The relationship of granulocyte ATP to chemotactic response during storage.

During the storage of granulocytes, bactericidal activity declines more slowly than does chemotactic response (CTR). Bacterial killing involves increased activity of the hexose monophosphate shunt, oxygen utilization and the generation of toxic products of oxygen. Chemotaxis is probably a contractile process involving myosin and actin filaments and possibly ATP as a source of energy. Thus, maintainance of ATP may be important in granulocyte preservation. During storage at 1 to 6 C of granulocytes collected by continuous and intermittent flow centrifuge leukapheresis, both CTR and ATP decreased approximately 33 percent. Decreases in CTR and ATP were 12 and 10 percent respectively when cells were stored at 20 to 24 C. Further decreases in CTR and ATP occurred between 24 and 48 hours of storage, although levels of both were higher in cells stored at 20 to 24 C compared with those stored at 1 to 6 C. When the results from all storage conditions were combined, the overall coefficient of correlation between CTR and ATP was 0.71 (p less than .05). Although ATP is probably not the only important variable in granulocyte preservation, granulocytes may resemble red blood cells in that a minimal level of ATP may be necessary for adequate function.

Studies of the kinetics of indium-111-labeled granulocytes.

Studies of the in vivo kinetics of granulocytes labeled in vitro with indium-111 were carried out in 10 normal subjects. The granulocyte suspension was prepared with a Ficoll-Hypaque density gradient and labeled with indium-111-oxine. No elution or reutilization of the radioisotope was demonstrable in vitro. The average intravascular recovery of indium-labeled granulocytes was 30% +/- 6, and the t 1/2 was 5.0 +/- 1.6 hr. Normal in vitro function of these cells was demonstrated by bacterial killing and chemotaxis assays. Because indium-111 decays by gamma emission, the fate of in vivo labeled granulocytes can be followed with scintigraphic techniques. Images obtained indicated normal uptake of activity in the liver and spleen. Effective in vivo function of indium-labeled granulocytes was demonstrated in four patients by the localization of radioactivity at sites of inflammation or abscess. Although the intravascular recovery with this method is lower than that reported for some other radioisotope methods, the comparison of abnormal findings with normal values by this technique is probably valid. Indium-labeled granulocytes should prove useful in the study of granulocyte collection, transfusion, histocompatibility, and storage.

Effects of storage of granulocytes on their fate in vivo.

During storage of granulocytes, the chemotactic response deteriorates first and to the greatest extent. Because in vitro assays such as chemataxis may not adequately predict the fate of granulocytes in vivo, we determined the intravascular kinetics and migration into skin windows of indium-111 labeled granulocytes. Granulocytes stored for 24 hours at either 1 to 6 or 20 to 24 degrees C had significantly reduced intravascular recoveries and migration into skin windows. Granulocytes stored for only eight hours at 20 to 24 degrees C had normal intravascular recovery and migration into skin windows. In granulocytes stored for eight hours at 1 to 6 degrees C, the intravascular recovery and migration into skin windows were reduced. Because of the method of calculating migration, this observation suggests that granulocytes which do circulate after storage at 1 to 6 degrees C had decreased ability to migrate into the skin windows. Granulocytes can be stored for up to eight hours at 20 to 24 degrees C without reduction in their ability to circulate and migrate to a site of inflammation. However, since many practical issues in granulocyte storage are not resolved, granulocytes should be transfused as soon as possible after collection.

Effect of leukocyte antibodies on the fate in vivo of indium-111-labeled granulocytes.

The effect of different leukocyte antibodies on the fate in vivo of granulocytes is not known. Thus, the optimum in vitro serologic tests to determine a safe and effective granulocyte transfusion or to diagnose immune destruction of granulocytes in other clinical situations have not been identified. We have studied the effect of granulocyte agglutinating (GA), granulocytotoxic (GC), and lymphocytotoxic (LC) antibodies on the intravascular recovery and half-life (t 1/2) and the extravascular localization of Indium-111-granulocytes in 50 patients. GA antibodies caused reduced granulocyte recovery and t 1/2 in three of three non-neutropenic patients (one with anti-NB1), increased sequestration of cells in the liver, and failure of granulocytes to localize at sites of infection in two of two patients (one with anti-NA1). In contrast, GC antibodies in five patients and LC antibodies in one patient did not cause reduced intravascular recovery or t 1/2 of granulocytes. In nine patients with GC and six patients with LC antibodies, incompatible granulocytes localized at known sites of infection. It appears that GA, but not GC nor LC, antibodies alter the fate in vivo of granulocytes.

IgA and IgM human immunodeficiency virus antibodies in weakly reactive or false-negative blood donors.

Detection of antibodies to the human immunodeficiency virus (HIV) in recently infected donors is crucial to prevent the transmission of HIV infection via blood products. To determine whether specific antibodies of the IgA or IgM class are present as markers of recent infection in donor specimens that have borderline reactivity on routine enzyme immunoassay (EIA) screening, 15 specimens that were positive by immunoblot were tested for IgA and IgM HIV antibodies. All 15 had detectable IgA HIV antibodies, and 14 had IgM HIV antibodies. The 15 specimens were tested further, each by two independent laboratories, with nine licensed EIAs. Two of the nine EIAs found all 15 units positive in both laboratories; seven EIAs found 1 to 5 of the 15 units negative, for a total of 31 false-negative results. The results indicated a difference between the sensitivity of EIA kits using only anti-IgG reagents and of kits using multispecific reagents that react with IgG and other classes of antibody. In a modified procedure, the addition of enzyme-conjugated anti-IgA or anti-IgM to the kit's enzyme-conjugated reagent increased the optical density values of most false-negative specimens to the positive range. It was concluded that licensed kits vary in reactivity with IgA and IgM HIV antibodies and that sensitivity could be increased by improved detection of these classes of antibody.

Newborn screening for congenital Toxoplasma infection: five years experience in Massachusetts, USA.

Since 1986, all Massachusetts newborn filter paper blood specimens, and some from New Hampshire, have been screened for the presence of IgM antibodies to Toxoplasma gondii using an enzyme-linked IgM capture assay. Among approximately 530,000 infants screened, 40 had serologically confirmed congenital toxoplasma infection, and 4 additional infants had borderline serologies, for an overall identification and treatment rate of 1:12,000. False positive results from the newborn's filter paper specimen occurred in 22 infants (1/24,000); these were clarified by tests for IgM and IgG in serum specimens obtained 2-3 weeks later from the infant and mother. The screening program would have failed to detect 3 infants with severe infection who were diagnosed on clinical grounds prenatally or at birth and lacked IgM. No infants with a later diagnosis of congenital toxoplasmosis that was missed by screening are known to our statewide network of pediatric infectious disease consultants. Follow-up studies are in progress to evaluate more completely the sensitivity of the IgM assay in newborns and the efficacy of treatment.

Seroprevalence of human immunodeficiency virus among childbearing women. Estimation by testing samples of blood from newborns.

Attempts to predict the course of the epidemic of acquired immunodeficiency syndrome (AIDS) have been hampered by the lack of an objective, practical way to estimate the prevalence of infection with the human immunodeficiency virus (HIV) in the general population. Testing for the prevalence of HIV infection in women should be a sensitive means to track the epidemic and to study the potential for perinatal transmission. Antibodies in maternal blood are contained in neonatal blood specimens routinely collected on absorbent paper for other purposes, such as screening for phenylketonuria; we therefore tested for HIV antibody in these specimens. Analysis of batches of individually blinded specimens from selected hospitals protected the anonymity of the mothers and babies and was cost efficient. Using the newborn's blood as an indicator of the mother's serologic status, we concluded that 1 of every 476 women (2.1 per 1000) giving birth in Massachusetts was positive for HIV antibody by immunofluorescence assay or enzyme-linked immunosorbent assay, both confirmed by immunoblot (Western blot) testing. The prevalence of HIV infection varied according to the type and location of the maternity hospitals; rates of seropositivity were highest in inner-city hospitals (8.0 per 1000), lower in mixed urban and suburban hospitals (2.5 per 1000), and lowest in suburban and rural hospitals (0.9 per 1000). This method is useful for collecting data needed to plan and evaluate prevention strategies and to predict the health care resources that will be needed to care for women and children who contract AIDS. Because other states have newborn screening programs similar to the Massachusetts program, this approach can be used for national surveillance of AIDS in women.

Sensitivity and specificity of human T-lymphotropic virus (HTLV) types I and II polymerase chain reaction and several serologic assays in screening a population with a high prevalence of HTLV-II.

BACKGROUND: Since 1988, all blood donations in the United States have been screened for antibodies to human T-lymphotropic virus type I (HTLV-I). However, the sensitivity of current serologic tests for the detection of HTLV type II (HTLV-II) antibodies and the diagnostic utility of direct tests for HTLV-I and -II using polymerase chain reaction (PCR) are poorly defined. STUDY DESIGN AND METHODS: Five hundred sixty-nine HTLV-I- or -II-seropositive and 687 age- and sex-matched seronegative samples from a high-risk population at an inner-city emergency department were selected. All samples were tested with four HTLV enzyme immunoassays (EIAs), one Western blot assay and one type-specific Western blot assay, one HTLV type-specific EIA, and a research HTLV-I/II PCR kit. RESULTS: Sensitivity of the various EIAs ranged from 95.1 to 99.5 percent, and specificity ranged from 97.2 to 99.4 percent. PCR performed in duplicate without selective retesting had lower sensitivity (85.1 %) and specificity (88.0%). However, PCR detected 20 (3.2%) HTLV-I-positive and 47 (7.5%) HTLV-II-positive samples among the 627 samples that were negative in all EIAs. The type-specific EIA and PCR assay had the highest rate of concordance in classifying samples as either HTLV-I or II, with the type-specific EIA and type-specific Western blot having the next highest rates of concordance. CONCLUSION: In this sample set from a population at high risk for HTLV-II, screening with HTLV-I/II PCR had lower sensitivity and specificity than that with EIAs. However, 4.1 to 10.8 percent of samples were PCR positive but seronegative for HTLV-I or -II, and their true infection status remains undetermined.

In vitro function and post-transfusion survival of granulocytes collected by continuous-flow centrifugation and by filtration leukapheresis.

The function of granulocytes collected by continuous-flow centrifugation (CFC) and by filtration leukapheresis (FL) was studied in vitro, and the post-transfusion recovery and intravascular survival of these cells was studied by autologous transfusion in normal donors. Granulocytes collected by both FL and CFC leukapheresis (CFCL) functioned normally in the quantitative nitroblue tetrazolium, oxygen consumption, and chemotaxis assays. Bacterial killing was slightly but consistently decreased in FL but not CFCL granulocytes. The post-transfusion recovery of control granulocytes collected by ordinary phlebotomy averaged 52% in eight transfusions, compared with 34% for six CFCL granulocyte concentrates and 16% for six FL concentrates. The intravascular half-times were 3.8 hr for phlebotomy and 3.0 hr for CFCL granulocytes. FL granulocytes had survival curves which were nonlinear and a single half-life could not be calculated. The average half-time 30 min after transfusion was 1.3 hr, and 3 hr after transfusion it was 2.6 hr. Granulocytes collected by FL had a mild impairment of bacterial killing, decreased post-transfusion recovery, and altered intravascular kinetics. None of these abnormalities was found in granulocytes collected by CFCL.

A simple technique for the inactivation of IgM antibodies using dithiothreitol.

This report describes a simple method for inactivating 19S red cell antibodies using the odorless compound dithiothreitol (DTT). 30 sera which contained antiglobulin-active red cell antibodies showed virtually no alteration in activity after DTT treatment, while 30 different sera containing cold-active red cell antibodies showed almost total elimination of activity following DTT treatment. Six sera were separated into IgG and IgM fractions. All red cell activity was eliminated in the IgM fraction, but was unaffected by DTT treatment of the IgG fraction. This method of inactivating IgM antibodies should be of value in investigating cases of hemolytic disease of the newborn and in studying sera containing mixtures of warm and cold red cell antibodies.

Early diagnosis of HIV infection in infants by detection of IgA HIV antibodies.

With the aim of achieving earlier diagnosis of human, immunodeficiency virus (HIV) infection in infants, IgA and IgM HIV antibodies in serum samples from babies born to seropositive mothers were assayed by immunoblot and enzyme-linked immunosorbent assay after removal of IgG with recombinant protein G. 64 samples were from 38 HIV-infected babies with Centers for Disease Control classifications of P1 or P2. Among these infected children IgA HIV antibodies were present in all 23 samples from those older than 12 months, in 12 of 18 samples from babies aged 6-12 months, in 5 of 10 samples from babies aged 3-5 months, and in 2 of 13 from babies under 3 months old. The 6 IgA-negative samples from infants over 6 months were all from infants with severe AIDS and/or hypogammaglobulinaemia. IgA HIV antibodies were present in twice as many samples as IgM HIV antibodies (66% vs 33%). No IgM or IgA HIV antibodies were detected in infants who subsequently seroreverted or in infants born to seronegative mothers. The correlation of the serological results with clinical information on each child suggests that detection of IgA HIV antibodies is an effective method for early diagnosis of HIV-infected infants without signs of infection.

New testing strategy to detect early HIV-1 infection for use in incidence estimates and for clinical and prevention purposes.

CONTEXT: Differentiating individuals with early human immunodeficiency virus 1 (HIV-1) infection from those infected for longer periods is difficult but important for estimating HIV incidence and for purposes of clinical care and prevention. OBJECTIVE: To develop and validate a serologic testing algorithm in which HIV-1-positive persons with reactive test results on a sensitive HIV-1 enzyme immunoassay (EIA) but nonreactive results on a less sensitive (LS) EIA are identified as having early infection. DESIGN: Diagnostic test and testing strategy development, validation, and application. Specimens were tested with both a sensitive HIV-1 EIA (3A11 assay) and a less sensitive modification of the same EIA (3A11-LS assay). SETTINGS AND PARTICIPANTS: For assay development: 104 persons seroconverting to HIV-1 comprising 38 plasma donors, 18 patients of a sexually transmitted disease clinic in Trinidad, and 48 participants in the San Francisco Men's Health Study (SFMHS); 268 men without the acquired immunodeficiency syndrome (AIDS) in the SFMHS who had been infected for at least 2.5 years; and 207 persons with clinical AIDS; for testing strategy validation: 488 men in the SFMHS from 1985 through 1990 and 1275449 repeat blood donors at 3 American Red Cross blood centers from 1993 through 1995; and for HIV-1 incidence estimates: 2717910 first-time blood donors. We retrospectively identified persons eligible for a study of early infection. MAIN OUTCOME MEASURE: Ability to identify early HIV infection. RESULTS: Estimated mean time from being 3A11 reactive/3A11-LS nonreactive to being 3A11 reactive/3A11-LS reactive was 129 days (95% confidence interval [CI], 109-149 days) [corrected]. Our testing strategy accurately diagnosed 95% of persons with early infection; however, 0.4% (1/268) of men with established infection and 2% (5/207) of persons with late-stage AIDS were misdiagnosed as having early HIV-1 infection. Average yearly incidence estimates in SFMHS subjects were 1.5% per year vs observed average incidence of 1.4 per 100 person-years. Incidence in repeat blood donors using the sensitive/less sensitive assay testing strategy was 2.95 per 100000 per year (95% CI, 1.14-6.53/100000) vs observed incidence of 2.60 per 100000 person-years (95% CI, 1.49-4.21/100000). Overall incidence in first-time blood donors was 7.18 per 100000 per year (95% CI, 4.51-11.20/100000) and did not change statistically significantly between 1993 and 1996. Use of the sensitive/less sensitive testing strategy alone would have identified all 17 persons with antibodies to HIV-1 eligible for a study of early HIV-1 infection and would have increased enrollment. CONCLUSIONS: The sensitive/less sensitive testing strategy provides accurate diagnosis of early HIV-1 infection, provides accurate estimates of HIV-1 incidence, can facilitate clinical studies of early HIV-1 infection, and provides information on HIV-1 infection duration for care planning.