Low-Cost Technologies for HIV in Resource-Poor Settings
Low-Cost Technologies for HIV in Resource-Poor Settings
Low-cost monitoring continues to be an issue of importance in resource-poor settings. Investigators across the globe are searching for less expensive ways to diagnose HIV infection and to measure CD4+ cell counts and viral load, although most clinicians in resource-poor countries think that measurement of viral load will never be cost-effective and that it is not essential for management.
Rapid Antibody Tests
The best assays currently in use in resource-poor countries are the HIV rapid antibody tests such as the Abbott Determine and the UniGold assays. These tests use a simple finger stick and provide a result within 15 minutes, with the benefits that nonclinicians can perform the test, no venipuncture (with its attendant risk of needle stick injuries) is involved, and patients can see the result for themselves.
Ziyambi and colleagues evaluated the performance of rapid HIV antibody tests when used by laboratory or nonlaboratory staff. Table 1 shows the results achieved by the laboratory technologists.
Table 1. Comparison of Rapid HIV Antibody Tests
The tests were then conducted at 2 clinical sites, with or without supervision by a technologist. Specimens were also sent for confirmatory enzyme immunoassay (EIA) and Western blot testing. There were 3791 consecutive visits. Results are shown in Table 2.
Table 2. Performance of Rapid HIV Antibody Tests Performed With or Without Supervision
Uptake of testing also increased from 77% to 97% when they switched from EIA to rapid testing. Thus, once again the feasibility of rapid testing was demonstrated.
Calero and colleagues in the United States tested an algorithm in which 2 simultaneous rapid tests were initially used to test a sample. If results were both nonreactive, the sample was considered negative. If both test results were positive, the result was confirmed with a third rapid test; if that result was positive as well, the sample was considered positive for HIV. If the results of the first 2 tests were discordant, then the third test was regarded as a tie-breaker. A total of 416 HIV-uninfected and 24 HIV-infected specimens from the United States were tested to validate this algorithm. There were no false-positive results and no false-negative results using this system with any combination of the Multispot HIV-1/2, Ora Quick, and Hema-Strip HIV-1/2 tests. For international sites, however, the use of 3 separate tests adds to the cost.
Clinical Markers of HIV Disease Stage
Turning to the monitoring of HIV-infected patients, possible clinical markers were described by Mekonnen and colleagues, who are conducting an ongoing cohort study in Ethiopia, a collaboration with the Ethiopian Health and Nutrition Institute and the Division of Public Health and Environment Amsterdam, The Netherlands. The investigators found that simple markers such as anemia, lymphocyte count < 1500 cells/mm, low body mass index, and HIV symptoms were independent predictors of short-term survival. Using the presence of at least 1 of these markers as a criterion to initiate antiretroviral therapy, 126 patients would have begun treatment compared with 128 patients if international guidelines were used. Of the 116 who would have met both sets of criteria for starting treatment, 78% would have started therapy at the same time by either method.
Alternatives to CD4+ Cell Count
Several groups described the use of total lymphocyte count (TLC) as a replacement for CD4+ cell count in untreated patients, and Robin Wood's group in Cape Town, South Africa, also described the use of TLC to monitor highly active antiretroviral therapy (HAART). This group has previously shown that a TLC < 1250 cells/mm correlates well with a CD4+ cell count < 200 cells/mm. In their new report on 266 patients each evaluated at 5 time points, they described a significant correlation between all the pairs of changes in TLC and changes in CD4+ cell count (R = 0.61; P < .01). Jacobson and colleagues at the University of California, San Francisco, compared the sensitivity and specificity of the absolute lymphocyte count (ALC) and the TLC, here defined as ALC plus large lymphocytes. They selected 1 pair of results from 2044 patients and generated Receiver Operator Characteristic Curves. An ALC < 1750 cells/mm and a TLC < 1900 cells/mm had maximal sensitivity of 74% and specificity of 73% as predictors of a CD4+ cell count < 350 cells/mm.
Less Expensive Techniques for Measuring CD4+ Cell Count
With regard to specific methodologies for measuring CD4+ cell count, Sherring and colleagues in Ottawa, Canada, presented data on Beckman Coulter's Coulter Manual CD4 Kit and Dynal's Dynabeads CD4 method. The results are presented in Table 3. These tests are too expensive and involve too many steps for my taste. As an alternative, with a high-volume contract, Becton, Dickinson and Company will pay for the BD FACSCount System machine and the maintenance contract with a cost of reagents of $11/assay and fewer steps.
Table 3. Comparison of 2 Methods for CD4+ Cell Counting
The same group working with George Janossy described a modified commercially available Luminex 100 4-parameter flow cytometer. By removing unnecessary optical parts and using a 12-volt rechargeable battery and a red diode laser, they were able to cheaply quantify CD45+/CD4+ and CD45+/CD8+ cells.
Ilesh Jani, Debbie Glencross, George Janossy, Howard Shapiro, Peter Mugyenyi, and others have embarked on a wonderful project to decrease the cost of CD4+ cell count monitoring, as described at http://www.AffordCD4.com. They have a multifront program aimed at examining each part of the process with a critical eye. They have introduced generic reagents for CD4, CD8, and CD45 and are giving away these monoclonal antibodies. The group showed data comparing absolute CD4+ cell counts using a single-tube, 2-color CD45+/CD4+ protocol vs the standard 3-tube, 3-color assay. Both assays were performed on a volumetric single platform cytometer with a throughput of 200 samples/day, with accurate reporting of 16 parameters including white blood cell count and differential. This group finds that the panLeucogating method using a dual-platform system and the Dynabeads methods using microscopy both cost $10, and the panLeucogating on volumetry method costs less than $5.
A note of caution was sounded regarding the use of CD4+ cell counts and the criteria for initiation of therapy in the US guidelines. Mehendale and coworkers at Johns Hopkins, Baltimore, Maryland, reported on 46 drug-naive recent seroconverters who were monitored for 720 days in Pune, India. They found that despite a median viral load set point of only 28,729 copies/mL, the annual decline in CD4+ cell count was 120 cells/mm per year, which is greater than that reported among US seroconverters. Thus, any algorithm for treatment using even standard assays must be validated in these settings.
Virologic Assays
Rodriguez and colleagues are hot on the chase of affordable and portable assays for all the necessary laboratory measures. Using microchip technology in which agarose microbeads covalently coated with molecules to detect HIV antibodies, HIV RNA, CD4, and p24 antigen are placed in the wells of stamp-sized microchips, they presented data on the detection of the antibodies and the antigen. Stay tuned for more.
Withum and associates evaluated an ultrasensitive p24 antigen assay for use in the diagnosis of pediatric HIV infection. The method involves heat denaturation of the sample combined with signal amplification using the PerkinElmer tyramide signal amplification (TSA) ELAST system. A total of 91 children born to HIV-infected women in North Carolina and 74 specimens from babies born to HIV-infected women from around the United States were tested. The combined results showed remarkable sensitivity and 100% specificity (Table 4), making this a much less expensive method for diagnosis of perinatal infection in the United States ($8 per test, vs $50-$70 for polymerase chain reaction testing). However, it is not clear whether the same method will be useful for the HIV clades that are prevalent in Africa and elsewhere.
Table 4. Evaluation of an Ultrasensitive p24 Antigen Assay
Conclusion
Although progress is being made in the search for more affordable testing methods for use in resource-poor regions, it is important that lack of technology does not prevent the introduction of treatment. For instance, most successful tuberculosis programs in resource-poor countries do not monitor liver enzyme levels. As Joep Lange said, "There is actually very little evidence that laboratory monitoring prevents mortality due to drug toxicity, but there is an awful lot of evidence that not treating symptomatic HIV infection is lethal, and usually not in a nice way; why are we always more concerned about doing harm than about not doing good?"
References
Low-cost monitoring continues to be an issue of importance in resource-poor settings. Investigators across the globe are searching for less expensive ways to diagnose HIV infection and to measure CD4+ cell counts and viral load, although most clinicians in resource-poor countries think that measurement of viral load will never be cost-effective and that it is not essential for management.
Rapid Antibody Tests
The best assays currently in use in resource-poor countries are the HIV rapid antibody tests such as the Abbott Determine and the UniGold assays. These tests use a simple finger stick and provide a result within 15 minutes, with the benefits that nonclinicians can perform the test, no venipuncture (with its attendant risk of needle stick injuries) is involved, and patients can see the result for themselves.
Ziyambi and colleagues evaluated the performance of rapid HIV antibody tests when used by laboratory or nonlaboratory staff. Table 1 shows the results achieved by the laboratory technologists.
Table 1. Comparison of Rapid HIV Antibody Tests
| Kit | Sensitivity (n = 512) | Specificity (n = 512) |
|---|---|---|
| Capillus | 100% | 100% |
| UniGold | 100% | 100% |
| Abbott Determine | 100% | 100% |
| Virocheck | 98.5% | 93.5% |
The tests were then conducted at 2 clinical sites, with or without supervision by a technologist. Specimens were also sent for confirmatory enzyme immunoassay (EIA) and Western blot testing. There were 3791 consecutive visits. Results are shown in Table 2.
Table 2. Performance of Rapid HIV Antibody Tests Performed With or Without Supervision
| Determine | UniGold | |||
|---|---|---|---|---|
| Sensitivity | Specificity | Sensitivity | Specificity | |
| Counselors with supervision | 100% | 99.8% | 97% | 100% |
| Counselors without supervision | 100% | 99.9% | 98.2% | 99.9% |
Uptake of testing also increased from 77% to 97% when they switched from EIA to rapid testing. Thus, once again the feasibility of rapid testing was demonstrated.
Calero and colleagues in the United States tested an algorithm in which 2 simultaneous rapid tests were initially used to test a sample. If results were both nonreactive, the sample was considered negative. If both test results were positive, the result was confirmed with a third rapid test; if that result was positive as well, the sample was considered positive for HIV. If the results of the first 2 tests were discordant, then the third test was regarded as a tie-breaker. A total of 416 HIV-uninfected and 24 HIV-infected specimens from the United States were tested to validate this algorithm. There were no false-positive results and no false-negative results using this system with any combination of the Multispot HIV-1/2, Ora Quick, and Hema-Strip HIV-1/2 tests. For international sites, however, the use of 3 separate tests adds to the cost.
Clinical Markers of HIV Disease Stage
Turning to the monitoring of HIV-infected patients, possible clinical markers were described by Mekonnen and colleagues, who are conducting an ongoing cohort study in Ethiopia, a collaboration with the Ethiopian Health and Nutrition Institute and the Division of Public Health and Environment Amsterdam, The Netherlands. The investigators found that simple markers such as anemia, lymphocyte count < 1500 cells/mm, low body mass index, and HIV symptoms were independent predictors of short-term survival. Using the presence of at least 1 of these markers as a criterion to initiate antiretroviral therapy, 126 patients would have begun treatment compared with 128 patients if international guidelines were used. Of the 116 who would have met both sets of criteria for starting treatment, 78% would have started therapy at the same time by either method.
Alternatives to CD4+ Cell Count
Several groups described the use of total lymphocyte count (TLC) as a replacement for CD4+ cell count in untreated patients, and Robin Wood's group in Cape Town, South Africa, also described the use of TLC to monitor highly active antiretroviral therapy (HAART). This group has previously shown that a TLC < 1250 cells/mm correlates well with a CD4+ cell count < 200 cells/mm. In their new report on 266 patients each evaluated at 5 time points, they described a significant correlation between all the pairs of changes in TLC and changes in CD4+ cell count (R = 0.61; P < .01). Jacobson and colleagues at the University of California, San Francisco, compared the sensitivity and specificity of the absolute lymphocyte count (ALC) and the TLC, here defined as ALC plus large lymphocytes. They selected 1 pair of results from 2044 patients and generated Receiver Operator Characteristic Curves. An ALC < 1750 cells/mm and a TLC < 1900 cells/mm had maximal sensitivity of 74% and specificity of 73% as predictors of a CD4+ cell count < 350 cells/mm.
Less Expensive Techniques for Measuring CD4+ Cell Count
With regard to specific methodologies for measuring CD4+ cell count, Sherring and colleagues in Ottawa, Canada, presented data on Beckman Coulter's Coulter Manual CD4 Kit and Dynal's Dynabeads CD4 method. The results are presented in Table 3. These tests are too expensive and involve too many steps for my taste. As an alternative, with a high-volume contract, Becton, Dickinson and Company will pay for the BD FACSCount System machine and the maintenance contract with a cost of reagents of $11/assay and fewer steps.
Table 3. Comparison of 2 Methods for CD4+ Cell Counting
| Test | Time for Assay (minutes) | Number of Steps for Assay | Cost per Test | CD4+ Cell Count by Specified Assay (cells/mm) |
CD4+ Cell Count by Flow Cytometry (cells/mm) |
|---|---|---|---|---|---|
| Coulter Manual CD4 Kit | 10 | 12 | $13 | 1116 + 122 | 904 + 50 |
| Dynabeads CD4 | 40 | 28 | $7 | 1006 + 252 | 904 + 50 |
The same group working with George Janossy described a modified commercially available Luminex 100 4-parameter flow cytometer. By removing unnecessary optical parts and using a 12-volt rechargeable battery and a red diode laser, they were able to cheaply quantify CD45+/CD4+ and CD45+/CD8+ cells.
Ilesh Jani, Debbie Glencross, George Janossy, Howard Shapiro, Peter Mugyenyi, and others have embarked on a wonderful project to decrease the cost of CD4+ cell count monitoring, as described at http://www.AffordCD4.com. They have a multifront program aimed at examining each part of the process with a critical eye. They have introduced generic reagents for CD4, CD8, and CD45 and are giving away these monoclonal antibodies. The group showed data comparing absolute CD4+ cell counts using a single-tube, 2-color CD45+/CD4+ protocol vs the standard 3-tube, 3-color assay. Both assays were performed on a volumetric single platform cytometer with a throughput of 200 samples/day, with accurate reporting of 16 parameters including white blood cell count and differential. This group finds that the panLeucogating method using a dual-platform system and the Dynabeads methods using microscopy both cost $10, and the panLeucogating on volumetry method costs less than $5.
A note of caution was sounded regarding the use of CD4+ cell counts and the criteria for initiation of therapy in the US guidelines. Mehendale and coworkers at Johns Hopkins, Baltimore, Maryland, reported on 46 drug-naive recent seroconverters who were monitored for 720 days in Pune, India. They found that despite a median viral load set point of only 28,729 copies/mL, the annual decline in CD4+ cell count was 120 cells/mm per year, which is greater than that reported among US seroconverters. Thus, any algorithm for treatment using even standard assays must be validated in these settings.
Virologic Assays
Rodriguez and colleagues are hot on the chase of affordable and portable assays for all the necessary laboratory measures. Using microchip technology in which agarose microbeads covalently coated with molecules to detect HIV antibodies, HIV RNA, CD4, and p24 antigen are placed in the wells of stamp-sized microchips, they presented data on the detection of the antibodies and the antigen. Stay tuned for more.
Withum and associates evaluated an ultrasensitive p24 antigen assay for use in the diagnosis of pediatric HIV infection. The method involves heat denaturation of the sample combined with signal amplification using the PerkinElmer tyramide signal amplification (TSA) ELAST system. A total of 91 children born to HIV-infected women in North Carolina and 74 specimens from babies born to HIV-infected women from around the United States were tested. The combined results showed remarkable sensitivity and 100% specificity (Table 4), making this a much less expensive method for diagnosis of perinatal infection in the United States ($8 per test, vs $50-$70 for polymerase chain reaction testing). However, it is not clear whether the same method will be useful for the HIV clades that are prevalent in Africa and elsewhere.
Table 4. Evaluation of an Ultrasensitive p24 Antigen Assay
| Result by Ultrasensitive p24 assay | Infant Infection Status | |
|---|---|---|
| Infected | Uninfected | |
| Positive | 94 | 0 |
| Negative | 3 | 68 |
Although progress is being made in the search for more affordable testing methods for use in resource-poor regions, it is important that lack of technology does not prevent the introduction of treatment. For instance, most successful tuberculosis programs in resource-poor countries do not monitor liver enzyme levels. As Joep Lange said, "There is actually very little evidence that laboratory monitoring prevents mortality due to drug toxicity, but there is an awful lot of evidence that not treating symptomatic HIV infection is lethal, and usually not in a nice way; why are we always more concerned about doing harm than about not doing good?"
References
Stakteas SCP, Tanuri A, Rayfield M, Vergara A, Samo-Gudo J, Barreto A. Laboratory and field evaluation of the performance of simple and rapid HIV tests in Mozambique. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract MoPeB3104.
Ziyambi Z, Osewe P, Taruberekera N. Evaluation of the performance of non-laboratory staff in the use of simple rapid HIV antibody assays at New Start voluntary counseling and testing (VCT) centers. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract MoPeB3110.
Calero EK, Malia JA, Sawyer R, et al. Rapid HIV-1 diagnostic algorithms for use in HIV infection screening. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract MoPeB3091.
Mekonnen Y, Dukers N, Sanders E, et al. Simple predictive markers of death in HIV-1 infected Ethiopians and their relevance in the decision of initiation of antiretroviral therapy (ART). Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract TuPeB4669.
Badri M, Wood R. Role of total lymphocyte count for monitoring HAART in a resource-constrained setting. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract MoPeB3112.
Post FA, Wood R, Maartens G. CD4 and total lymphocyte counts as predictors of HIV disease progression. QJM. 1996;89:505-508.
Jacobson MA, Liu L, Khayam-Bashi H, Deeks S, Hinger C, Kahn J. Absolute or total lymphocyte count as a marker for initiating highly active antiretroviral therapy. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract MoPeB3100.
Sherring A, Bergeron M, Mandy F. Comparison of two alternative CD4 T-cell enumerating methods suitable for laboratories with low volume immunophenotyping in resource-poor settings. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract MoPeB3119.
Mandy F, Bese A, Barnabe P, Solajic Z, Bergeron M, Janossy G. A compact, affordable and portable CD4 T-cell machine. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract MoPeB3243.
Janossy G, Jani IV, Bradley NJ, Pitfield T, Glencross DK. New concepts in affordable CD4+ T cell enumeration for resource-poor settings. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract MoPeB3220.
Tugume SB, Otim TW, Atwiine D, et al. Inexpensive CD4 lymphocyte subset estimation by dual platform (DP) CD45 assisted panleucogating method using afford monoclonal antibodies. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract DB10658.
Janossy G, Jani I, Bradley NJ, Pittfield T, et al. Affordable CD4 T cell counts by flow cytometry III. CD45 gating for volumetric analysis. Clin Diag Lab Immunol. 2002.In press.
Jani IV, Janossy G, Igbal A, et al Affordable CD4 T cell counts by flow cytometry II. The use of fixed whole blood in resource poor settings. J Immunol Methods. 2001;257:145-154.
Glencross DK, Scott LE, Jani IV, Barnett, D, Janossy G. CD45 assisted pan-leucogating for accurate, cost effective dual platform CD4 T cell enumeration Cytometry. 2002;50:69-77.
Mehendale SM, Risbud AR, Bollinger RC. Rapid disease progression in human immunodeficiency virus type 1 infected seroconverters in Pune, India. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract TuPeC4707.
Rodriguez WR, Christodoulides N, Ali M. Development of affordable and portable HIV and CD4 diagnostic tests using microchips. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract WeOrB1343.
Withum DG, Fiscus SA, Respess RA, et al. Evaluation of an ultrasensitive p24 antigen assay (UPTA) for use in the diagnosis of pediatric HIV-1 infection. Program and abstracts of the XIV International AIDS Conference; July 7-12, 2002; Barcelona, Spain. Abstract ThPeB7227.
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