Challenges in Implementing HIV Load Testing in South Africa

Transcription

1 SUPPLEMENT ARTICLE Challenges in Implementing HIV Load Testing in South Africa Wendy S. Stevens 1 and Theresa M. Marshall 2 1 Department of Molecular Medicine and Haematology and 2 National Institute for Communicable Diseases, National Health Laboratory Service and University of the Witwatersrand, Johannesburg, South Africa The South African antiretroviral treatment guidelines recommend the use of human immunodeficiency virus type 1 (HIV-1) load testing for patient monitoring and, in particular, to assist in switching to second-line treatment regimens. There are significant challenges to implementing HIV load testing on the scale that is required in South Africa. To put this in context, 560,000 HIV-infected individuals are receiving antiretroviral therapy, and program recommendations include viral load testing twice per year. Currently, a 3-tiered laboratory infrastructure exists with tertiary facilities and, to some extent, secondary laboratories able to implement quantitative HIV nucleic acid testing. Challenges include high sample volumes, transportation logistics from remote sites, costs, phlebotomy in children, a national skills shortage, and sample throughput of technology platforms. Several approaches are thus being explored simultaneously: (1) the feasibility of establishing higher throughput and more automated central laboratories; (2) improvement of current sample collection, transportation, and storage techniques; (3) alternative viral load technologies, including flow-based marker screening approaches to reduce testing volumes, and (4) point-of-care viral load testing strategies for clinics. The development of appropriate solutions for each laboratory tier in South Africa will require close collaboration between researchers in the field and partners in industry. Sub-Saharan Africa currently bears the burden of the human immunodeficiency virus (HIV) epidemic, accounting for 67% of the total 33.2 million infected individuals [1]. South Africa is a middle-income country with greater resource and regulatory capacity than many countries in the region [2]. Despite this, the epidemic has not been brought under control, and South Africa accounts for at least 8% of global infections, with an estimated 5.7 million adults and children having HIV infection [3]. Approximately 18.8% of the adult population aged years are affected, with a dis- Potential conflicts of interest: none reported. Financial support: National Health Laboratory Service, South Africa. Supplement sponsorship: This article is part of a supplement entitled Need for Point-of-Care HIV Molecular Diagnostic Technologies in Resource-Limited Settings, which is based on the workshop Novel Technologies in Rapid HIV-1 Viral Detection and was sponsored by the Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services. Reprints or correspondence: Dr W. Stevens, Molecular Medicine and Haematology, University of the Witwatersrand, 7 York Rd, Parktown, Johannesburg, South Africa (wendy.stevens@nhls.ac.za). The Journal of Infectious Diseases 2010; 201(S1):S78 S by the Infectious Diseases Society of America. All rights reserved /2010/20108S1-0012$15.00 DOI: / proportionate burden of infection being borne by women. High rates of mother-to-child transmission continue to fuel the pediatric epidemic, and it is estimated that 300,000 children are infected and may require initiation of antiretroviral therapy (ART) [4]. AIDS is a major cause of mortality and was responsible for 1300,000 deaths in 2005 [5]. The response to the HIV epidemic in South Africa has been clouded by the inequities of the past, maldistribution of health care resources between the private and public sectors, high levels of poverty and unemployment, and delays in initiation of an ART program until 2004 [6]. It has been estimated that these delays cost 3.8 million person-years during [7]. To complicate matters further, there is substantial national geographic variation in the distribution of HIV disease [8], and the response to the epidemic by provincial health authorities varies considerably. ART has also been complicated by high rates of coinfection with tuberculosis and hepatitis B virus. NATIONAL ART PLAN The national response to HIV infection was included in a national operational plan developed in 2003 [6]. S78 JID 2010:201 (Suppl 1) Stevens and Marshall

2 The South African National Strategic Plan has also recently been updated for the period [3]. ART guidelines were published in 2004 [6]. These were modified from the World Health Organization (WHO) treatment guidelines for resource-poor settings from 2003 [9]. Concern has recently been expressed that these guidelines have not been updated in South Africa according to revised WHO guidelines from 2006 [10]. Current treatment guidelines in South Africa advocate therapy initiation at a CD4 cell count!200 cells/ml or at WHO stage IV, with an AIDS defining illness [6]. Significant pressure is mounting to change this cutoff value to!350 cells/ml, on the basis of experience related to the reduction in the incidence of opportunistic infections, such as tuberculosis, observed at this level. A change to this cutoff value would contribute to a significant increase in patients requiring treatment and would consequently increase the numbers of viral load assays performed. Clinical follow-up is quarterly, with CD4 cell count and viral load testing recommended at baseline and every 6 months after therapy initiation [6]. In accordance with national treatment guidelines, viral load testing is used to determine treatment failure with a repeated threshold HIV RNA level of copies/ml being used to determine a treatment switch. There is no requirement for viral load monitoring in the second-line treatment regimen, with no defined third-line treatment regimens available. The use of viral load assays has enabled prolongation of the first-line treatment regimen by using a targeted adherence strategy based on viral load results [11]. As of August 2008, 560,883 individuals initiated ART in South Africa [12]. More than 50,000 children were estimated to be receiving ART a substantial increase from!3000 in February Recent work conducted locally (National Institutes of Health funded Children with HIV Early Antiretroviral Therapy study) revealed that early diagnosis and treatment reduced early infant mortality by 76% and HIV progression by 75%; these figures are likely to change future policy and to result in significant expansion of the pediatric treatment program [13]. Despite these massive strides in improving access to ART, it was estimated in February 2007 that only 36% of the original target of the national operational plan of 2003 was reached [14]. Of note, the availability of viral load testing resulted in very little attention being paid by clinicians to CD4 cell counts [15]. To maintain the current number of individuals in the program translates into a requirement of at least 1.2 million viral load assays annually, without the major anticipated increases. NATIONAL HEALTH LABORATORY SERVICE (NHLS) Laboratory testing in the public sector is currently the jurisdiction of a single NHLS of which the mandate it is to provide affordable, accessible, and efficient laboratory services [16, 17]. The NHLS currently has 265 laboratories nationally and employs 6380 individuals (see Figure 1 for distribution of laboratories). There is a 3-tier laboratory system consisting of tertiary reference laboratories (accounting for 21% of the laboratories), secondary facilities (10%), and primary health care laboratories (69%). Tertiary care laboratories are capable of implementing sophisticated virological assays, handle large volumes of work, and possess appropriate technical resources, including electricity, freezer capacity, dedicated laboratory space, skilled personnel, training programs, and quality control. Medium-tier laboratories do not have these specialized facilities, and thus, suitable assays for these environments are generally less complex techniques that provide medium or low throughput processing. Primary care laboratories or clinic facilities can cope with conducting technically nondemanding assays, such as rapid and point-of-care tests. Laboratory facilities in South Africa are unevenly distributed throughout the country. Highvolume reference laboratories are located mainly at the industrial, urban centers; medium-tier laboratories are affiliated to the provincial, secondary hospitals that are found in the urban and periurban areas. Low-tier laboratories are affiliated with the primary health care hospitals located in the rural areas (Table 1). IMPLEMENTATION OF LABORATORY MONITORING PROGRAM Implementation of highly active antiretroviral therapy under the national roll-out program in South Africa raised the awareness that access to more-affordable laboratory tests for monitoring of therapy was essential. Despite the substantial decreases in the cost of ART and the assays required to monitor patients receiving ART, costs of laboratory tests add significantly to the overall cost of individual patient s treatment [18]. In the context of the incredibly large number of individuals requiring therapy in South Africa, laboratory investigations for monitoring HIV infection and AIDS continues to represent a significant financial challenge for the country. This mirrors the situation in numerous laboratory environments in other developing countries. NATIONAL CD4 CELL COUNT TESTING PROGRAM A rapid expansion of CD4 cell count testing was undertaken in response to the national treatment plan. In 2004, there were 4 laboratories conducting centralized, flow cytometric based CD4 cell count testing; in 2008, this number expanded to 61 centers, conducting 250,000 CD4 cell count tests per month (Figure 2) [19]. All testing is conducted using the Pan- Leucogated CD4 platform (Beckman Coulter), which allows for improved stability of assays, greater affordability, and standardization [20]. The national CD4 cell count external quality Challenges of HIV Load Testing JID 2010:201 (Suppl 1) S79

3 Figure 1. Distribution of laboratories of the National Health Laboratory Service in South Africa. Black dots represent laboratory sites, and large circles represent major laboratory centers. Reprinted with permission from the National Health Laboratory Service.

4 assurance program monitors performance across all national sites, with excellent performance achieved across the country (standard deviation,!1.2; coefficient of variation,!8%) [19]. There is awareness that a significant number of individuals are lost to follow-up in the health care systems, not returning for their CD4 cell count result, and thus, there is an ongoing need for development of a rapid point-of-care CD4 cell count assay. NATIONAL VIRAL LOAD TESTING PROGRAM Figure 2. Annual number of CD4 cell count assays conducted in the public sector in South Africa, April 2004 September 2008 Viral load testing has been significantly more challenging to implement because of the skill required to conduct testing, the cost of commercial assays, and the cold chain required for transportation of samples. Challenges have not been any different from those faced in other resource-poor settings in Africa [21]. Perhaps the significant difference is the sheer volume of viral load tests that need to be performed monthly to support the national treatment program. Currently, 17 laboratories perform viral load testing in 8 provinces nationally; plans were to expand to 20 laboratories in 9 provinces by the end of May 2009, but no expansion has yet been undertaken. Testing has expanded from 34,000 viral load assays annually to 1,625,236 in 2008 (January to September) (Figure 3). Currently, 120,000 viral load assays are performed each month in the NHLS. Of note, the NHLS is currently performing 4-fold more viral load assays per month than were performed during the entire first 9 months of the program and as many CD4 cell count tests per month as were performed during the first 9 months of the program, reflecting the increase in the number of individuals accessing care in the health care services in South Africa. The commercially available viral load technologies described in this Supplement in Stevens et al [22] have been evaluated in South Africa before and at various times during the national roll-out program. The in-house real-time assay developed locally has largely been abandoned because of the difficulties in ordering reagents, standardizing procedures, and training staff without supplier support. The selection of testing was conducted by a stringent procurement tender process focusing largely on total cost of assay and ease of implementation. A process of prolonged consultation that included various stakeholders from the national Department of Health, various academics (both clinicians and laboratory staff), and with nonprofit organizations, such as the Clinton Foundation, was undertaken. The anticipated sample volumes were very high, and thus, a low price per reportable result was negotiated. All equipment costs were included in the cost per test, enabling equipment leasing that obviated huge capital outlay. Because of the technical skills available at several tertiary reference laboratories, a process of centralization of laboratory services was selected. Other factors that required consideration included (1) turnaround time of the assay, which was stipulated by clinicians at 72 h; (2) time taken to conduct the assay; and (3) sample transportation needs. The tender process resulted in the selection of the biomérieux NucliSENS EasyQ assay (version 1.1) [23]. This real-time assay combines nucleic acid sequence based amplification technology with detection by molecular beacons. Testing was implemented using the semi-automated biomérieux mini-mag extraction system. This extraction procedure reflects a combination of BOOM chemistry and magnetic silica particles [23]. CHALLENGES ENCOUNTERED IN A VIRAL LOAD PROGRAM Sample volumes. There have been numerous challenges, including the requirement for harmonization and standardization of testing practices across the country, to facilitate cost savings. At program initiation, there were few centers with the requisite skills to conduct large-scale molecular testing. Volumes of test- Table 1. Assays Considered for Viral Load Testing and the Appropriate Laboratory Tier at the Time of Implementation of an Antiretroviral Therapy Program Laboratory type Tertiary reference Secondary (medium-tier) Primary care Assays NAT strategies (Roche Amplicor [COBAS and Taqman] and bdna, NucliSENS EasyQ); Abbott RealTime in-house realtime PCR options; flow-based options a May do NAT; Exavir load assay; flow-based options a POC technologies a or sample preparation for referral NOTE. NAT, nucleic acid testing; PCR, polymerase chain reaction; POC, point of care. a Not available for implementation at time of program initiation (in research and development). Challenges of HIV Load Testing JID 2010:201 (Suppl 1) S81

5 Figure 3. Annual number of HIV load assays conducted in the public sector in South Africa, April 2004 September 2008 ing far exceeded predictions by the national government in the first few months, thus placing huge strain on centers conducting viral load testing. The rapid scale-up of testing meant that automation was required faster than had been anticipated at initiation of the tender process and that the mini-mag methodology was not appropriate for the required volumes of testing. This assay was replaced by the NucliSENS easymag system of extraction (biomérieux) [24]. This instrument has exactly the same extraction chemistry but automates the labor-intensive NucliSENS minimag washing steps, thereby significantly reducing hands-on time. Staff resources and training. Maintaining adequate staffing levels has been a recurrent challenge, with a massive shortage of skilled personnel and a high turnover of technical staff in the country. The situation is not dissimilar to that experienced in ART clinics [25]. Inadequate practical or theoretical training in molecular techniques is provided at technical training institutions, thus placing a huge training burden on senior staff in the laboratory services. Viral load testing, particularly after automation is implemented, is repetitive, requiring great effort to avoid boredom among staff. Ongoing professional development is thus critical to ensure staff retention. Additional training becomes particularly problematic, however, in laboratories with high staff shortages and huge sample volumes. As with clinic health professionals, laboratory staff are prone to experience burn-out because of the increased processing volumes experienced at each facility. Of note, these programs do not only involve a massive increase in testing, such as viral load and CD4 cell count measurement, but also a significant increase in safety testing (eg, hemoglobin and alanine transaminase levels), thus affecting all laboratory disciplines. Infant diagnosis of HIV infection with use of DNA-based nucleic acid testing performed in the same laboratories is an additional burden. Method validation. Detailed method validation was an important component of the program initiation, and because a test was selected in an area where there was limited local and international experience, validation contributed to the work load in the initiation phase of the program. A valuable lesson was learned with respect to the need for significant field validation of assay performance before implementation. Few international guidelines are available for describing appropriate method validation of HIV load assays; this is currently being addressed by organizations, such as the World Health Organization and the Centers for Disease Control and Prevention, and groups, such as the Forum for HIV Collaborative Research. Recently, Stevens et al [26] provided recommendations for method validation and the appropriate selection of CD4 cell count testing. The following phases were established for the method validation process: (1) background review and (2) professional installation of equipment, appropriate staff training and device familiarization, development of standard operating procedures, consideration of sample selection (including appropriate matrix and sample preparation), and the development of records for performance monitoring. Local samples needed to be evaluated to ensure adequate performance for detection of HIV subtype C, the most prevalent subtype in South Africa [27]. The following factors were deemed to be critical components of the analysis: (1) precision, (2) accuracy, (3) evaluation of quality control samples, and (4) reportable range and linearity. These validations were described with regard to use of the mini-mag extraction process [23] and use of automated easymag extraction [23, 24]. Both assays were validated against the laboratory reference assay (the Roche COBAS Ampliprep and the Roche COBAS Amplicor combination) at implementation. PERFORMANCE OF THE NUCLISENS EASYQ ASSAY HIV RNA levels (for HIV-1 subtype C) quantitated by the NucliSENS EasyQ assay correlated significantly with those quantitated by the Roche COBAS Amplicor Monitor assay (r, 0.874; P!.001) [23]. Assay comparison was possible by converting Roche values from RNA copies per milliliter to international units per milliliter by multiplying the Roche value by Reproducibility of the NucliSENS EasyQ assay in the log 6 IU/mL range yielded coefficient of variation of 1.3% 2.84% for 2 well-trained technologists. The high sample volumes and the labor-intensive nature of the mini-mag extraction made this approach impractical, necessitating the implementation and validation of the easymag systems. In the study using the easymag analyzer, a significant correlation existed between the log values of the easymag and EasyQ HIV-1 method and the COBAS AmpliPrep and Amplicor method [24]. This finding was confirmed by the analysis using methods of agreement (the percentage similarity model and Bland-Altman analysis). The percentage of similarity model revealed a mean observed similarity of 96% and a mean percentage difference ( standard deviation) of 3.8% 5.0%, with overall good agreement with the Amplicor assay (percentage of similarity coefficient of variation, 5.2%). For Bland-Altman, limits of agreement (1.31; 0.55) and the mean difference (0.38) were within the bound- S82 JID 2010:201 (Suppl 1) Stevens and Marshall

6 aries of clinically acceptable differences between reportable results of the assays. Advantages of the easymag and EasyQ analyzer combination included a fairly wide dynamic range, and the throughput of samples was greatly improved using the easymag and EasyQ HIV-1 system. One hundred forty-four samples could easily be processed within 6 h with use of 1 easymag and 1 EasyQ instrument. A similar output using the Roche Ampliprep and Amplicor combination required 1 Ampliprep and 3 Amplicor analyzers. Concerns that require ongoing evaluation include the frequent occurrence of gelation of the samples during transportation in the lysis buffer provided by biomérieux and the high percentage of invalid samples flagged by the EasyQ analyzer (mean, 4% 7%). In addition, contamination is particularly easy with this assay, necessitating designated areas for extraction and for amplification and detection and vigilant monitoring. Several modifications were made to the assay and standard operating procedures as the program progressed; these were (1) the inclusion of negative controls that are not present in kit reagents and are essential to identify contamination early, (2) the addition of positive controls with known copy numbers (PeliSpy HIV-RNA external quality control; Acrometrix), (3) environmental monitoring for contamination, (4) continuous monitoring of numbers of invalid samples, and (5) interfacing of the instrumentation to the National Health Laboratory Information System to reduce the potential for transcription errors. Suppliers in the region have had similar problems with regard to staffing and are frequently unable to provide adequate technical support to laboratories in the country. CLINIC SITE DIFFICULTIES Great difficulties were experienced initially by treating clinicians with respect to interpretation of viral load results at program initiation. There was a lack of understanding that results needed to be interpreted in the log scale and that acceptable technical differences between assays may well be in the region of log copies/ml [28]. This was compounded by the fact that the assay implemented had reportable units in IU/mL, which was different from previously reported RNA copies/ml. The transportation of samples from many clinic sites remains a significant challenge. Transportation of samples on ice at 2 8 C, as recommended with the lysis buffer from biomérieux, has been very difficult to implement efficiently across the country. Clinics do not have facilities for centrifugation, and the addition of sample to the lysis buffer is therefore conducted in regional laboratories. The chain of custody and quality assurance of this process is very difficult to monitor from an analytical laboratory level, because it involves phlebotomists at the site and the regional laboratory and receiving staff at the analytical facility and at the processing laboratory. For this reason, many sites have switched to using the plasma preparation tubes, which require a fewer number of manual intervention steps. The predominant subtype in South Africa remains HIV-1 subtype C (prevalence, 97%) [23], and thus far, the bulk of cases appear to be detected using the NucliSENS EasyQ assay. Cases have been identified that involve HIV enzyme linked immunosorbent assay positive samples and low CD4 cell counts but no amplification using the NucliSENS EasyQ assay; these cases are being investigated further. The addition of an assay that targets an alternative region of the genome, such as the pol region (either Bayer bdna or Abbott RealTime), is used to repeat testing for the problem cases. In addition, laboratory interpretation of results can be extremely difficult because of the lack of clinical information that accompanies the samples. There is currently no link between laboratory information systems and hospital systems detailing clinical information. In general, this is a weakness of the national program, in which all laboratory results can be collected and audited but cannot be matched to clinical outcomes, except for at a few clinics conducting research programs. Mining this laboratory system has, however, provided useful data for program monitoring by the provincial health authorities [15]. CONCLUSIONS Ongoing research and development of novel technological approaches, validation of commercially available assays, and operational research on the best ways to deliver results to support the program are ongoing challenges for academic laboratories in South Africa. Perhaps the feature that distinguishes South Africa from many other developing country is the ability to drive such research programs. Therefore, several approaches are being explored simultaneously: (1) the feasibility of establishing higher throughput and more automated central laboratories; (2) the improvement of sample collection, transportation, and storage techniques; (3) alternative viral load technologies, including flow-based marker screening approaches to reduce testing volumes; and (4) point-of-care viral load testing strategies for clinics. In an attempt to address automation issues, other assays have been evaluated, such as the Roche COBAS Ampliprep, COBAS Taqman 48, and docked Taqman 96, as well as the Abbott m2000sp and RealTime analyzer combination. Local research and development is also under way for a semiquantitative point-of-care viral load assay. The latter approach has included proof of concept for detection of HIV RNA by a lateral flow dipstick by using a novel isothermal amplification technique. This would have several applications in South Africa, including (1) monitoring adherence and therapy switch for patients receiving ART in real time; (2) diagnosis of acute HIV infection at clinics where patients at high risk Challenges of HIV Load Testing JID 2010:201 (Suppl 1) S83

7 receive care, such as those for patients with sexually transmitted infections; and (3) diagnosis of HIV infection in infants. In summary, lessons learned from this large-scale implementation have assisted a number of laboratories with implementation of viral load testing in sub-saharan Africa. As rapid advances in technology platforms and assays continue, this process is likely to be ever-changing, with ongoing challenges. References 1. UNAIDS/WHO. AIDS Epidemic Update en/knowledgecentre/hivdata/epiupdate/epiupdarchive/2007/default.asp. Accessed 1 March United States President Emergency Plan for AIDS Relief 2008 Country Profile: South Africa. Accessed 1 March Department of Health. HIV and AIDS and STI Strategic Plan for South Africa Accessed 20 February Meyers T, Moultrie H, Naidoo K, Cotton M, Eley B, Sherman G. Challenges to pediatric HIV care and treatment in South Africa. J Infect Dis 2007; 196(Suppl 3):S474 S Groenewald P, Nannan N, Bourne D, Laubscher R, Bradshaw D. Identifying deaths from AIDS in South Africa. AIDS 2005; 19: Department of Health. National Antiretroviral Treatment guidelines, Accessed November Chigwedere P, Seague GR 3rd, Gruskin S, Lee TH, Essex M. Estimating the lost benefits of antiretroviral drug use in South Africa. J Acquir Immune Defic Syndr 2008; 49: Shaikh N, Abdullah F, Lombard CJ, Smit L, Bradshaw D, Makubalo L. Masking through averages intraprovincial heterogeneity in HIV prevalence within the Western Cape. S Afr Med J 2006; 96: Report of the WHO/UNAIDS International Consensus Meeting on Technical and Operational Recommendations for Emergency Scalingup of Antiretroviral Therapy in Resource-Limited Settings: emergency scale-up of antiretroviral therapy in resource-limited settings: technical and operational recommendations to achieve 3 by 5, Lusaka, Zambia. Geneva: World Health Organization, Beck EJ, Vitoria M, Mandalia S, Crowley S, Gilks CF, Souteyrand Y. National adult antiretroviral therapy guidelines in resource limited countries: concordance with 2003 WHO guidelines? AIDS 2006; 20: Orrell C, Harling G, Lawn SD, et al. Conservation of first-line antiretroviral treatment regimen where therapeutic options are limited. Antivir Ther 2007; 12: National comprehensive HIV and AIDS treatment plan statistics: August 2008 Report. Health information, evaluation and research division, Department of Health. Accessed 1 March Violari A, Cotton MF, Gibb DM, et al. Early antiretroviral therapy and mortality among HIV-infected infants. N Engl J Med 2008; 359: Joint Civil Society Monitoring Forum. South African Antiretroviral Programme Treatment Gap. Accessed November Boulle A, Bock P, Osler M, et al. Antiretroviral therapy and early mortality in South Africa. Bull World Health Organ 2008; 86: National Health Laboratory Services. Accessed November NHLS Act. Government Gazette CT 2000; Glencross DK, Scott L, Stevens G, Mendelow BV, Stevens W. The challenge of laboratory monitoring of HIV. S Afr Med J 2002; 92: Glencross DK, Janossy G, Coetzee LM, et al. Large-scale affordable Panleucogated CD4(+) testing with proactive internal and external quality assessment: In support of the South African national comprehensive care, treatment and management programme for HIV and AIDS. Cytometry B Clin Cytom 2008; 74(Suppl 1):S40 S Glencross D, Scott L, Jani IV, Barnett D, Janossy G. CD45-assisted PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration. Cytometry 2002; 50: Calmy A, Ford N, Hirschel B, et al. HIV viral load monitoring in resource-limited regions: optional or necessary? Clin Infect Dis 2007; 44: Stevens WS, Scott LE, Crowe SM. Quantifying HIV for monitoring antiretroviral therapy in resource-poor settings. J Infect Dis 2010; 201(Suppl 1):S16 S26 (in this supplement). 23. Stevens W, Wiggill T, Horsfield P, Coetzee L, Scott LE. Evaluation of the NucliSens EasyQ assay in HIV-1-infected individuals in South Africa. J Virol Methods 2005; 124: Stevens W, Horsfield P, Scott L. Evaluation of the performance of the automated NucliSENS easymag and EasyQ systems versus the Roche AmpliPrep-AMPLICOR combination for high-throughput monitoring of human immunodeficiency virus load. J Clin Microbiol 2007;45: Tobi P, George G, Schmidt E, Renton A. Antiretroviral treatment and the health workforce in South Africa: how have ART workers been affected by scaling up? Trop Med Int Health 2008; 13: Stevens W, Gelman R, Glencross D, Scott L, Crowe S, Spira T. Evaluation of CD4 enumeration technology for the monitoring of HIV disease. Nature Rev Microbiol 2008; 6:S29 S Van Harmelen J, Van der Ryst E, Loubser A, et al. A predominantly HIV type 1 subtype C-restricted epidemic in South African urban populations. AIDS Res Hum Retroviruses 1999; 15: Martin D. Appropriate laboratory monitoring of HIV. S Afr Med J 2000; 90: S84 JID 2010:201 (Suppl 1) Stevens and Marshall