Diagnosis of human immunodeficiency virus type 1 infection with different subtypes using rapid tests.

We evaluated six rapid tests for their sensitivity and specificity in diagnosing human immunodeficiency virus type 1 (HIV-1) infection using 241 specimens (172 HIV-1 positive, 69 HIV-1 negative) representing different HIV-1 subtypes (A [n = 40], B [n = 47], C [n = 28], E [n = 42], and F [n = 7]). HIVCHEK, Multispot, RTD and SeroStrip were 100% sensitive and specific. Capillus failed to identify two of eight subtype C specimens (overall sensitivity of 98. 85%), while the SUDS test (the only test approved by the Food and Drug Administration) gave false-positive results for 5 of 69 seronegative specimens (specificity of 93.24%). Our results suggest that although rapid tests perform well in general, it may be prudent to evaluate a rapid test for sensitivity and specificity in a local population prior to its widespread use.

Impact of HIV type 1 subtype variation on viral RNA quantitation.

We evaluated the performance of three HIV-1 RNA quantitation methods (Amplicor HIV-1 MONITOR-1.0, NASBA, and Quantiplex HIV RNA 2.0 [branched DNA (bDNA)]) using plasma specimens (N = 60) from individuals from Asia and Africa infected with one of three HIV-1 subtypes (A, Thai B [B'] or E; N = 20 each). Our results demonstrate that of the 20 subtype A specimens, 19 were quantifiable by the bDNA assay compared with 15 by the MONITOR-1.0 and 13 by NASBA. Of those quantifiable, the mean log10 difference was 0.93 between bDNA and MONITOR-1.0 and 0.46 between bDNA and NASBA. For subtype B' specimens, the correlation among methods was better with only 2 specimens missed by NASBA and 3 by the bDNA assay. However the missed specimens had viral burden near the lower limit (1000 copies/ml) for these assays. For the 20 subtype E specimens, MONITOR-1.0 and NASBA quantified RNA in 17 and 14 specimens, respectively, as compared with 19 specimens quantified by the bDNA assay. The correlation among different assays, especially between bDNA/NASBA and MONITOR-1.0/NASBA, was poor, although the mean log10 difference for subtype E specimens was 0.4 between bDNA and MONITOR-1.0 and only 0.08 between bDNA and NASBA. The addition of a new primer set, designed for non-B HIV-1 subtypes, to the existing MONITOR assay (MONITOR-1.0+) resulted in RNA detection in all 60 specimens and significantly improved the efficiency of quantitation for subtypes A and E. Our data indicate that HIV-1 subtype variation can have a major influence on viral load quantitation by different methods. Periodic evaluation and modification of these quantitative methods may be necessary to ensure reliable quantification of divergent viruses.

Maternal virus load and perinatal human immunodeficiency virus type 1 subtype E transmission, Thailand. Bangkok Collaborative Perinatal HIV Transmission Study Group.

To determine the rate and risk factors for human immunodeficiency virus (HIV)-1 subtype E perinatal transmission, with focus on virus load, pregnant HIV-infected women and their formula-fed infants were followed prospectively in Bangkok. Of 281 infants with known outcome, 68 were infected (transmission rate, 24.2%; 95% confidence interval, 19.3%-29.6%). Transmitting mothers had a 4.3-fold higher median plasma HIV RNA level at delivery than did nontransmitters (P<.001). No transmission occurred at <2000 copies/mL. On multivariate analysis, prematurity (adjusted odds ratio [AOR], 4.5), vaginal delivery (AOR, 2.9), low NK cell percentage (AOR, 2.4), and maternal virus load were associated with transmission. As RNA quintiles increased, the AOR for transmission increased linearly from 4.5 to 24.8. Two-thirds of transmission was attributed to virus load>10,000 copies/mL. Although risk is multifactorial, high maternal virus load at delivery strongly predicts transmission. This may have important implications for interventions designed to reduce perinatal transmission.

Detection of antibodies to human immunodeficiency virus type 1 in oral fluids: a large-scale evaluation of immunoassay performance.

Paired serum and oral-fluid (OF) specimens (n = 4,448) were collected from blood donors and patients attending local sexually transmitted disease clinics in Trinidad and Tobago and the Bahamas and were tested for the presence of human immunodeficiency virus type 1 (HIV-1) antibodies. Sera were tested by Abbott AB HIV-1/HIV-2 (rDNA) enzyme immunoassay (EIA), and positive specimens were confirmed by Cambridge HIV-1 and HIV-2 Western blotting (WB). OF specimens were collected with the OraSure collection device and were tested by Murex GACELISA and by two EIAs from Organon Teknika (the Oral Fluid Vironostika HIV-1 Microelisa System [OTC-L] and the Vironostika HIV-1 Microelisa System [OTC-M]). EIA-reactive OF specimens were confirmed by miniaturized WB (OFWB). GACELISA detected all 474 HIV-1 seropositive specimens (sensitivity, 100%). OTC-L detected 470 positive specimens (sensitivity, 99.2%), while OTC-M detected 468 positive specimens (sensitivity, 98.8%). Specificities ranged from 99.2 to 100% for the three assays. Concordance of OFWB with serum WB was 99.4%, and banding patterns determined by the two methods were similar. The immunoglobulin G (IgG) concentration of OF specimens ranged from 0.21 to 100 microg/ml, with a mean of 17.1 microg/ml. Significant differences in OF IgG concentrations were observed between HIV antibody-positive and HIV antibody-negative persons (31.94 versus 15.28 microg/ml, respectively [P < 0.0001]). These data further confirm the suitability of OF specimens for detection of HIV-1 antibodies. Currently available HIV-1 antibody assays provide sensitivities and specificities with OF specimens comparable to those achieved with serum specimens.

PIP: The use of oral fluid (OF) as a specimen for detecting antibodies to infectious agents has become increasingly popular since the approach was first described in the 1980s. OF is a mixture of saliva, mucosal and bacterial products, and gingival crevicular fluid. 4448 paired serum and OF specimens collected from 4448 blood donors and patients attending 3 sexually transmitted disease clinics in Trinidad and Tobago and the Bahamas were tested for the presence of HIV-1 antibodies. The sera were tested by Abbott AB HIV-1/HIV-2 (rDNA) enzyme immunoassay (EIA), and positive specimens were confirmed by Cambridge HIV-1 and HIV-2 Western blotting (WB). OF specimens were collected using the OraSure collection device and were tested by Murex GACELISA and 2 EIAs from Organon Teknika (OTC-L and OTC-M). EIA-reactive OF specimens were confirmed by miniaturized WB (OFWB). GACELISA detected all 474 HIV-1 seropositive specimens, OTC-L detected 470 positive specimens, and OTC-M detected 468 positive specimens. Specificities were 99.2-100% for the three assays. There was a 99.4% concordance of OFWB with serum WB, and banding patterns determined by the two methods were similar. The immunoglobulin G (IgG) concentration of OF specimens was 0.21-100 mcg/ml, with a mean of 17.1 mcg/ml. Significant differences in OF IgG concentrations were observed between HIV antibody-positive and HIV antibody-negative persons. These data support the suitability of OF specimens for detecting HIV-1 antibodies. Currently available HIV-1 antibody assays provide sensitivities and specificities with OF specimens comparable to those achieved with serum specimens.

Field evaluation of rapid HIV serologic tests for screening and confirming HIV-1 infection in Honduras.

OBJECTIVE: To determine the ability of simple, rapid tests to identify HIV-1 antibody-positive specimens in field settings using the World Health Organization's (WHO) alternative testing strategies. DESIGN: Three-phase evaluation of simple, rapid assays using banked specimens and prospectively collected serum specimens at regional hospitals and rural clinics. METHODS: Seven test (Retrocell, Genie, HIVCHEK, SUDS HIV-1, Testpack, Serodia HIV-1, and HIV-1/2 RTD) were evaluated and results compared with standard enzyme immunoassay (EIA) and Western blot results (phase 1). Further evaluation consisted of prospective testing of routine specimens at regional (phase 2; n = 900) and rural, peripheral laboratories (phase 3; n = 1266) throughout Honduras with selected assays. RESULTS: Sensitivity and specificity were calculated for each assay and combination of assays for each phase to evaluate the effectiveness of the WHO alternative testing strategies. All tests in all phases were > 99% sensitive after correcting for technical errors, with two exceptions (SUDS, phase 1; HIVCHEK, phase 3). In phase 3, where the testing algorithm was diagnostic, several combinations of assays were 100% sensitive and specific using WHO strategy II or III. For the Honduras Ministry of Health, the combination of Retrocell and Genie was found to be equally sensitive, more specific (no indeterminate results), and less expensive than EIA/Western blot. CONCLUSION: Combinations of rapid, simple HIV antibody assays provide sensitivity and specificity performance comparable to EIA/Western blot. Application of these combinations in the WHO alternative testing strategies provides an inexpensive and effective method of determining HIV status. Assay combinations using these strategies can be easily performed in small, rural laboratories and have been implemented in routine HIV screening in Honduras.

PIP: In 1992, the World Health Organization (WHO) introduced 3 HIV testing algorithms designed to provide rapid, accurate results equivalent to those obtained by enzyme immunoassay (EIA) and Western blot but at reduced costs. The capability of the WHO strategy to identify HIV-1 antibodies in field settings was evaluated at regional hospitals and rural clinics in Honduras. In the study's first phase, the results of 7 tests (Retrocell, Genie, HIVCHEK, SUDS HIV-1, Testpack, Serodia HIV-1, and HIV-1/2 RTD) were compared with results for 600 sera previously tested by EIA and Western blot. Phase 2 entailed prospective testing of 900 routine specimens at regional laboratories, while phase 3 screened 1266 specimens at rural, peripheral laboratories. In the first phase of the analysis, 5 assays had a sensitivity of 100%; the remaining 2 were 99.7% and 99.3% sensitive and specificities ranged from 92.8 to 100%. In field settings, sensitivities ranged from 96.4 to 99.3%. Moreover, in the third phase, several combinations of tests were 100% sensitive or specific when the WHO strategy of basing the choice of assay on the purpose of the screening (seroprevalence studies, screening of blood, or patient diagnosis) was employed. The combination of Retrocell and Genie was found to be equally sensitive, more specific, and less expensive than EIA or Western blot.

Oral fluid as a specimen for detection and confirmation of antibodies to human immunodeficiency virus type 1.

Paired serum and oral fluid specimens (n = 287) were collected with the Omni-Sal device and were assayed for the presence of antibodies to human immunodeficiency virus type 1 (HIV-1). Enzyme immunoassays (EIAs)--Abbott 3A11, an Organon Teknika Corporation research-use-only test, and the Murex GACELISA--were used per the manufacturers' inserts or were modified slightly to accommodate the oral fluid specimens. Compared with serum Western blot (immunoblot) results, each EIA had a sensitivity of 100% and the specificities were 89.6% for the Abbott 3A11 EIA, 96.5% for the GACELISA, and 97.8% for the Organon Teknika Corporation EIA. Specificities based on specimens that were repeatedly reactive were 99.3% for all EIAs. A miniaturized Western blot technique used for confirmatory testing of both the serum and oral fluid specimens found 149 of the 287 samples to be HIV-1 antibody positive in both sample types. The Western blot banding patterns observed for the serum and oral fluid specimens were essentially identical. Immunoglobulin G concentrations were determined for all oral fluid specimens and ranged from < 0.5 to > 40.0 micrograms/ml. Immunoglobulin G concentrations did not correlate with the ability of any of the EIAs to detect HIV-1-specific antibody or with the ability of the modified Western blot to detect HIV-1 protein-specific antibodies.

Highly specific V3 peptide enzyme immunoassay for serotyping HIV-1 specimens from Thailand.

OBJECTIVE: To develop and evaluate a simple V3 peptide-based enzyme immunoassay (EIA) for large-scale serotyping of HIV-1 specimens from Thailand. DESIGN: Serologic reactivities with synthetic peptides derived from the V3 loop of gp120 were used for typing HIV-1 specimens. METHODS: Synthetic peptides PND-A and PND-B, derived from the consensus amino-acid sequences of the V3 loop of gp120 from two major genomic variants of HIV-1 in Thailand (A and B), were evaluated in an EIA on 61 Thai HIV-1 sera for which genotypes had been determined by polymerase chain reaction. The peptide EIA was then applied to sera from 188 HIV-1-infected patients, selected in non-random, convenience samples of known risk groups from four geographic regions of Thailand. RESULTS: The sensitivities and specificities of PND-A and PND-B were 86% (30 out of 35) and 96% (25 out of 26) and 92% (24 out of 26) and 94% (33 out of 35), respectively, with 100% predictive values of a monoreactive positive test for both peptides. The assay classified 101 specimens as serotype A, 39 as serotype B, eight as serotype AB (dually reactive), and 40 as untypable (non-reactive). Excluding dual reactors and non-reactors, 92% (77 out of 84) of specimens from patients probably infected by sexual contact were serotype A; conversely, 76% (28 out of 37) of injecting drug users were serotype B. CONCLUSION: The serologic results corroborated previous findings, in a smaller subset of samples, of an apparent segregation of viral subtypes by mode of transmission, suggesting two separate HIV-1 epidemics in Thailand. This peptide EIA could be a valuable epidemiologic tool in determining the dynamics of the rapid spread of HIV-1 in Thailand.

PIP: A simple synthetic enzyme immunoassay (EIA) for serotyping HIV-1 specimens from Thailand, based on gp120 V3 loop peptide, was developed and tested on 188 sera from 4 regions of the country. There are 2 major known gene variants of HIV-1 in Thailand designated genotype A and B. The peptide EIA was tested on 61 sera that had been characterized by polymerase chain reaction and DNA sequencing. The EIA was then tested on 188 sera from high risk groups collected in the northern, northeastern, central and southern regions in mid-1991. The PND-A assay was 86% sensitive and 96% specific; the PND-B assay was 96% sensitive and 92% specific. The EIAs showed 100% predictive values when sera known to be reactive to only HIV A or B were tested. In the series there were also 8 sera reactive to both A and B and 40 not reactive to either variant. Excluding dual and non-reactors, 92% of patients with sexual high risk factors had HIV-1 type A and 76% of those with IV drug use history had type B. The results suggest that 2 HIV-1 epidemics have occurred in Thailand, an initial wave in 1988 among IV drug users and a later wave centered among prostitutes and their clients.

Factors influencing HIV-1 banding patterns in miniaturized western blot testing of dried blood spot specimens.

In the HIV Seroprevalence Survey among Childbearing Women (SCBW), antibodies to human immunodeficiency virus type 1 are detected using enzyme immunoassays (EIA) and Western blot (WB) methods modified to accommodate samples of blood dried on special collection paper. Dried blood spot (DBS) eluates positive by EIA are tested by one of two WB methods, the miniblot technique using equipment from Immunetics Corporation and the PBS Integra assay (pageblot) from Genetic Systems. In this report we compared the performance of the two WB methods. The identity and position of the viral proteins on the WB were identified using monoclonal antibodies and monospecific antisera. The blots differed substantially in their composition and concentration of viral glycoproteins. Performance of the WB assays with DBS elution buffers from different EIA kits was equivalent except for samples eluted in the Abbott buffer, which reduced detection of antibodies to the p31, p51, p55, and p66 viral proteins. Case classification of DBS, positive sera, dilution curve samples, and seroconversion panels was equivalent by both tests in the presence of all elution buffers. Proficiency evaluation panels sent to SCBW participating laboratories over a 3-year period were used to note the differences between the two WB methods in detection of antibodies to the viral glycoproteins.

HIV-1 serologic test results for one million newborn dried-blood specimens: assay performance and implications for screening.

In a population-based national survey conducted in 1988-90, more than one million neonatal dried-blood specimens were tested for maternal antibody to human immunodeficiency virus type 1 (HIV-1). Enzyme immunoassays (EIA) and Western blot tests were performed in 20 state laboratories following standardized procedures. The observed predictive value of a repeatedly reactive EIA results closely coincided with that expected on the basis of manufacturer's estimates of test sensitivity and specificity for dried-blood specimens. Of the 2,845 EIA-reactive specimens tested by Western blot, 1,323 (47%) were positive, 1,270 (45%) were negative, and 252 (9%) were indeterminate. False-positive EIA and indeterminate Western blot results occurred at rates independent of seroprevalence. These data help characterize the results to be expected from screening of similar low-seroprevalence populations and constitute a base line for the detection of systematic testing errors.

Oligomeric nature of transmembrane glycoproteins of HIV-2: procedures for their efficient dissociation and preparation of Western blots for diagnosis.

Western blot (WB) analysis of various strains of HIV-2 indicated that transmembrane glycoprotein (TMP) of HIV-2 exists as trimers. These trimers have molecular weights and electrophoretic mobilities in the region of the major external glycoprotein, gp120, resulting in WB misidentification during diagnosis. A simple and rapid procedure was developed using trichloroacetic acid (TCA) to efficiently dissociate oligomeric forms of the TMP to monomers prior to the preparation of WB. This procedure permitted the unambiguous identification of antibodies to gp120 and to the TMP. Use of HIV-2 WB strips without any oligomeric forms of the TMP demonstrated (1) that cross reactivity of HIV-1-positive specimens on HIV-2 WB was mainly directed to Gag and Pol proteins, with some reactivity to gp36/gp41 TMP, but none to gp120; (2) that these strips can substantially reduce the number of specimens falsely identified as dually (HIV-1 and HIV-2) reactive; and (3) that HIV-2-positive specimens reacted to viral gp120 in a strain-specific manner, demonstrating high antigenic variation in this glycoprotein. It is recommended that this general procedure of viral protein dissociation be used for HIV-2 WB preparation.

Monitoring of filamentous fungal growth by in situ microspectrophotometry, fragmented mycelium absorbance density, and 14C incorporation: alternatives to mycelial dry weight.

Monitoring of filamentous fungal growth by spectrophotometry is generally considered not feasible. This report describes the monitoring of growth of the filamentous fungi Trichophyton mentagrophytes, Rhizopus oryzae, and Sporothrix schenckii in broth by two new spectrophotometric methods and by 14C incorporation from [U-14C]glucose. Microcultures (200 microliter) were prepared in 96-well, flat-bottom microtiter trays, and macrocultures (4 ml) were prepared in glass vials proportionally scaled up from microcultures. Mycelium accumulation in microcultures was measured without terminating the cultures by in situ microspectrophotometry. Accumulation in macrocultures was monitored by uniformly fragmenting the mycelium with a Broeck tissue grinder and by measuring absorbance density in plastic cuvettes with a dual-beam spectrophotometer. Absorbance measurements were found to increase linearly with mycelial weight. In situ absorbance correlated with absorbance density of fragmented mycelium, indicating that both methods monitored growth equivalently. Both defined lag-, exponential-, and stationary-growth phases. Increases in 14C incorporation, absorbance, and mycelial dry weight were kinetically identical for macrocultures and microcultures of T. mentagrophytes. For R. oryzae and S. schenckii, with the exception of R. oryzae growing in microcultures, incorporation of 14C also defined lag, exponential, and stationary growth after selection of the appropriate isotope-specific activity. This incorporation correlated directly with absorbance. We conclude that in situ microspectrophotometry, fragmented mycelium absorbance density, and, to a lesser extent, 14C incorporation can be used to effectively monitor filamentous fungal growth.

Lyophilized microculture susceptibility test for ketoconazole, miconazole, clotrimazole, and griseofulvin against dermatophytes.

A lyophilized microculture antimycotic susceptibility testing system for ketoconazole, miconazole, griseofulvin, and clotrimazole is described. Microculture plates were loaded with 100 microliters of medium and 10 microliters of appropriate concentrations of the four antimycotics and were lyophilized to complete dryness. The lyophilized plates were stored at -70 degrees C or 4 degrees C or in a desiccator at 25 degrees C. Samples from each storage condition were rehydrated at 1, 2, 3, 4, 5, 6, 8, 10, and 12 months and inoculated with Trichophyton mentagrophytes (Robin) Blanchard ATCC 18748. All of the minimal inhibitory concentrations (MICs) generated from the lyophilized microcultures were within one experimental dilution of MICs derived from fresh microcultures. The ability of reconstituted lyophilized microcultures to consistently produce MICs comparable to MICs derived from fresh microcultures was characterized. Nine dermatophyte isolates were tested five times each over a 70-day period. The MICs derived were reproducible and comparable to MICs determined by freshly prepared microculture tests. Lyophilization of freshly prepared antimycotic-containing microcultures does not alter the MIC resolution of the testing system and provides an effective method of storage of prepared antimycotic tests for ketoconazole, miconazole, clotrimazole, and griseofulvin.

Factors affecting griseofulvin susceptibility testing of Trichophyton rubrum in microcultures.

A microculture broth assay system for griseofulvin susceptibility testing of Trichophyton rubrum was further characterized. The effects of mass and number of colony-forming units of a fragmented mycelial inoculum, 5- or 8-day incubation periods, 25 or 32 degrees C incubation temperatures, and the solvents used to dissolve griseofulvin on the minimal inhibitory concentration (MIC) of griseofulvin were determined. An inoculum density with an absorbance of 0.600 at 450 nm ensured successful inoculation of all microcultures. Reduction of the inoculum mass to an absorbance of 0.200 lowered the number of colony-forming units in the inoculum by 60 to 80%. This decreased the efficiency of inoculation but did not alter the resulting MIC. There was no correlation between MIC and the number of colony-forming units used to initiate growth. Neither incubation temperature nor the length of incubation affected the MIC. The use of either acetone or ethanol to solubilize griseofulvin likewise had no effect on the MIC. The mean reproducibility of the MICs determined with the microculture method was 96%.

Antimycotic susceptibility testing of dermatophytes in microcultures with a standardized fragmented mycelial inoculum.

Standardization of a fragmented dermatophyte mycelial inoculum free of conidia for use in a broth microculture antimycotic susceptibility testing system is described. Ten clinical dermatophyte isolates were grown in submerged broth cultures at 35 degrees C. The mycelia were harvested before conidia appeared and were fragmented to 10 to 50-micron segments with a Broeck ground-glass tissue grinder. The density of the fragmented mycelium preparation was found to be adjustable on the basis of its spectrophotometric density expressed as absorbance measured at 450 nm. The minimal absorbance density that produced a 100% inoculation efficiency was determined for each isolate, and from these data an absorbance density of 0.600 was selected for inoculation of microcultures used in antimycotic susceptibility testing. The 0.600-absorbance inoculum of each dermatophyte isolate was tested for its capacity to successfully inoculate antimycotic agent-containing microcultures and generate minimal inhibitory concentrations of griseofulvin, clotrimazole, miconazole, and ketoconazole. The effect of the length of incubation on the minimal inhibitory concentrations was determined. It was concluded that the 0.600-absorbance density fragmented mycelial inoculum assured the rapid and uniform inoculation of the broth microculture antimycotic susceptibility system.