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Should haemoglobin A1c be used for the diagnosis of diabetes mellitus in South Africa?

Abstract Diabetes is an important medical problem in sub-Saharan Africa. It has traditionally been diagnosed by means of fasting plasma glucose, random blood glucose or an oral glucose tolerance test. Each of these has limitations and, in 2009, an expert committee of the American Diabetes Association recommended using the haemoglobin A1c (HbA1c) to diagnose diabetes mellitus.1 The aim of this paper is to analyse the advantages and disadvantages of using HbA1c as a diagnostic method for diabetes, and its applicability in the South African setting.


Introduction
Over the past two decades, type 2 diabetes has emerged as an important medical problem in sub-Saharan Africa. Estimates by the International Diabetes Federation suggest that the largest increase in the prevalence of diabetes is expected to occur in the developing world, including Africa. However, there is a paucity of prevalence data from South Africa and the rates published in the few studies available vary depending on the populations studied, as well as the method used in the diagnosis.
Before 2009, the only acceptable tests for the diagnosis of diabetes were based on blood glucose values: either a fasting blood glucose (FPG) of at least 7 mmol/l or a random blood glucose of at least 11.1 mmol/l in a patient with symptoms of diabetes, or a blood glucose of at least 11.1 mmol/l two hours after an oral glucose tolerance test (OGTT). In 2009 an expert committee of the American Diabetes Association (ADA) recommended the use of HbA 1c to diagnose diabetes. 1 In 2010 the ADA adopted the proposal as part of the diagnostic criteria of diabetes, using a cut-off of equal to or greater than 6.5%. They modified the criteria for impaired fasting glucose and impaired glucose tolerance to include individuals with an HbA 1c ranging from 5.7 to 6.4%, and recommended that the diagnosis should be confirmed with a repeat HbA 1c test, unless there are clinical symptoms and blood glucose levels equal to or greater than 11.1 mmol/l. Their recommendations also state that the HbA 1c must be measured in an accredited laboratory and that the different methods for diagnosing diabetes should not be used together, because of the lack of agreement between them. 2 In 2009 the World Health Organization (WHO) conducted a systematic review of the use of HbA 1c to diagnose diabetes, and made similar recommendations. They recommend that the HbA 1c be used for the diagnosis of diabetes using a cutoff value of 6.5%, provided that stringent quality assurance tests are in place and assays are standardised to criteria aligned to international reference values, and that there are no conditions present which preclude the accurate measurement of HbA 1c . 3 The WHO also recommended that long-term prospective studies are carried out in various ethnic groups to establish the precise glucose and HbA 1c levels predictive of microvascular and macrovascular complications. The WHO recommendation states that in an asymptomatic individual diagnosis should be confirmed with an additional test. It also cautions against the use of point-of-care devices, except where no other technology is available, and provided that stringent quality assurance programmes are in place.
The aim of this paper is to analyse the advantages and disadvantages of using HbA 1c to diagnose diabetes in a multiracial society such as South Africa Evidence for the use of HbA 1c Glycated haemoglobin reflects the average endogenous exposure to glucose, including postprandial spikes, and has low intra-individual variability, particularly in individuals without diabetes. It is these characteristics that may Should haemoglobin A 1c be used for the diagnosis of diabetes mellitus in South Africa?
contribute to its apparent superiority over fasting glucose in risk stratification for cardiovascular disease.
The evidence for the use of HbA 1c for diagnosing diabetes is based on the examination of three cross-sectional epidemiological studies that included an Egyptian population (n = 1018), a Pima Indian group (n = 960) and the United States National Health and Nutrition Survey (NHANES, n = 2821). Each of these studies assessed FPG, two-hour glucose and HbA 1c , and also measured the prevalence of retinopathy as assessed by fundus photography or direct ophthalmoscopy. The HbA 1c values at which retinopathy increased in prevalence were between 6 and 7% and were similar among the three groups. 1 It is important to note that, in at least two of these studies, the populations studied were homogenous. A systematic review of nine studies performed in Asian or European populations compared HbA 1c with FPG for the diagnosis of diabetes and used WHO criteria as the gold standard. They reported that HbA 1c and FPG were equally effective tools for the diagnosis of diabetes. An HbA 1c equal to or greater than 6.1% had a sensitivity of 78 to 81% and a specificity of 79 to 84% for the diagnosis of type 2 diabetes. When they used an FPG value of 6.1 mmol/l as cut-off point, the sensitivity ranged between 48 and 64% and the specificity ranged from 94 to 98%. 4 In a population-based study from Singapore, the investigators showed that increasing HbA 1c was associated with all microvascular complications, and that the optimal cut-off point for detecting mild and moderate retinopathy was between 6.6% and 7% respectively, with the prevalence of retinopathy being less than 1% below these cut-off points. 5 The Atherosclerosis Risk in Communities (ARIC) study is a community-based prospective cohort of middle-aged adults from the USA. In this study, data from more than 14 000 black and white adults demonstrated that increasing HbA 1c levels were associated with increasing values of FPG, but were more strongly associated with risk of cardiovascular disease and death from any cause compared with FPG. 6 Comparison of HbA 1c with other methods of diagnosing diabetes

Oral glucose tolerance test
OGTT is recognised as the gold standard in the diagnosis of diabetes and is a sensitive marker of impaired glucose tolerance. However, it requires stringent conditions, such as the ingestion of at least 150 g of carbohydrate per day for at least three days prior to the test, overnight fasting of at least 10 hours, and preventing the patient from walking around during the test. 7 These conditions not only influence the test results, but often make the test impractical. The OGTT also shows much greater intra-individual variability (16.7%) than the FPG or HbA 1c . 8 It is because of these limitations that the ADA recommended FPG as the preferred glucose-based diagnostic test for diabetes. 9 Fasting plasma glucose Small increases in blood glucose substantially increase the risk of diabetes, but glucose measurement is affected by several preanalytical and analytical variables that make it difficult to apply these epidemiological principles to individual patients. Patients are seen by their physicians at any time of day and in an uncertain state of fasting. Fasting glucose levels show significant diurnal variation, which could impact on the diagnosis of diabetes. 10 To reduce glycolysis, glucose is collected in fluoride tubes, after which it should be placed on ice and the plasma should be separated from the cells promptly. 11 In spite of these precautions, the rates of decrease in glucose in the first hour after collection in tubes with and without fluoride are virtually identical, and the decrease in glucose after two hours in a fluoride tube can exceed 0.5 mmol/l. 12 Another problem with the use of FPG alone for the diagnosis of diabetes is that, in epidemiological surveys, it results in a lower disease prevalence compared to the use of the OGTT, and it does not pick up those individuals with impaired glucose tolerance. Motala et al investigated diabetes and other disorders of glycaemia in rural KwaZulu-Natal and showed that if FPG alone was used, the prevalence of diabetes would be 36% lower than when a full OGTT was performed. If an OGTT was not performed, none of those subjects with impaired glucose tolerance would be detected. 13 Soma and Rheeder noted in a group of 120 subjects admitted for elective coronary angiography that nine out of 14 subjects would not have been detected as being diabetic if only an FPG level had been measured, as opposed to a full OGTT. 14 Plasma is the preferred sample type for glucose measurement in the laboratory and there is variability when sample types differ. Whole-blood glucose concentrations are lower than plasma concentrations because of the lower water content of red blood cells, while plasma glucose concentrations have been reported to be lower, higher or the same as those in serum. [15][16][17][18] HbA 1c , on the other hand, does not require an overnight fast and it does not require morning blood collection. HbA 1c is also reported to be relatively stable and shows less intraindividual variability than FPG or two-hour glucose levels. This was illustrated by Selvin et al, who looked at short-term inter-individual variability of FPG and two-hour glucose and HbA 1c in 685 participants without diagnosed diabetes, and showed high variability of both FPG and two-hour glucose levels relative to HbA 1c levels. 8 The case against HbA 1c Despite its greater clinical convenience, preanalytical and biological stability, and assay standardisation, HbA 1c has several limitations that preclude its use for the diagnosis of diabetes in South Africa at this time.

Methods of measuring HbA 1c
Laboratories use a wide variety of analytical techniques to separate and then quantify HbA 1c , based on differences in either molecular structure or molecular charge.
HbA 1c is less positively charged at neutral pH than pyridoxylated normal adult human haemoglobin (HbAo), and this is the basis for the high-performance liquid chromatography (HPLC) method. Spectrophotometric analysis of the various eluants provides the percentage of each haemoglobin species in the sample. This method is rapid and has very good precision. In South Africa it is utilised in some private laboratories, as well as in some of the large academic hospitals. The separation of total glycated haemoglobin or HbA 1c based on molecular structure can be achieved either by immunoassay or by boronate affinity chromatography. Immunoassays in particular may be affected by haemoglobinopathies. Several South African laboratories use immunoassay-based methods to separate glycated haemoglobin from the nonglycated haemoglobin (Cummins R, Fedler C, Zemlin A, personal communication).
Owing to simplicity of use, small sample volume requirements and portability, there is interest in the use of point-of-care devices for the measurement of HbA 1c . While these are acceptable for the monitoring of glycaemic control in diabetic patients, the ADA cautions that pointof-care devices should not be used for the diagnosis of diabetes. An evaluation of eight NGSP (previously the National Glycohemoglobin Standardization Program) -certified HbA 1c point-of-care instruments showed that only two of these instruments met the acceptance criteria of having a coefficient of variation (cv) of less than 3%, and all the instruments showed significant differences in analytical performance between different reagent lot numbers. 19 In addition, point-of-care tests cost more than laboratorybased methods (George J, personal observation). There is no objective information concerning their performance in the hands of those who use them, and proficiency testing might not be carried out.

Standardisation and quality assurance
The ADA endorsement of HbA 1c for the diagnosis of diabetes is based partly on the fact that assays are now highly standardised. 20  All methods used by the major laboratories in South Africa are NGSP certified. This means that, for a panel of 40 samples analysed in duplicate under ideal conditions, the 95% confidence interval of the difference between the method being tested and an NGSP reference laboratory would be within ± 0.75% HbA 1c . Method certification is performed by the manufacturer. Laboratories can obtain documentation from the manufacturer on their system's performance and traceability to DCCT, and should do so, but this does not guarantee accuracy and precision in the clinical laboratory. Laboratories should therefore participate in external proficiency testing programmes using wholeblood quality control material. There is no national quality assurance programme for HbA 1c in South Africa at this time, nor are there data on methodologies currently in use, or on participation and performance in proficiency testing. Until laboratories here demonstrate that their assays are aligned to international reference values and proper quality assurance criteria are adhered to, and that these laboratories are accredited as stipulated by both the ADA and the WHO, HbA 1c should not be used for the diagnosis of diabetes.

Ethnicity
A number of studies have shown that the cut-off of 6.5%, as proposed by the ADA, is not optimal for the diagnosis of diabetes in some ethnic groups. In an Indian population a cut-off equal to or greater than 6.1% was shown to have better diagnostic performance than a cut-off of 6.5%. 22 It appears that people of African descent have higher levels of glycated haemoglobin across the full spectrum of glycaemia. 23 Analysis of data from over 2 000 nondiabetic individuals in the NHANES III population showed that the HbA 1c level increased with age and with ethnicity. In non-Hispanic blacks, and using HbA 1c levels of 6.5 to 6.9%, 87% of those over the age of 64 years would not have diabetes by FPG/OGTT criteria. 24 Jorgensen et al assessed whether ethnicity modified the association between glucose levels and HbA 1c among Greenland Inuit, Inuit immigrants in Denmark and the general Danish population. 25 They looked at OGTT and HbA 1c . All three groups had an increase in prevalence when HbA 1c was used for diagnosis. However, this was most dramatic for the Greenland Inuit, with a jump from 11.2% (OGTT) to 31.7% (HbA 1c ). Another drawback of the use of HbA 1c at this time is that studies comparing FPG-and HbA 1c -based diagnosis often produce discordant results. Analyses of data from nondiabetic Koreans showed that 31.6% of the subjects would have been classified as having diabetes based on FPG, while only 23.5% would have been classified as having diabetes using HbA 1c value of 6.5%. 26 What these studies tell us is that, in some population groups, using FPG to diagnose diabetes results in an increased prevalence when compared to the use of HbA 1c , while the opposite is observed in other populations. The selection of a diagnostic threshold is usually made by means of the receiver operating characteristics curve (ROC) approach, followed by its validation in the target population using likelihood ratios. This evidence is lacking for South Africa.
In addition to ethnicity, other factors that affect HbA 1c levels include age, anaemia, human immunodeficiency virus (HIV) status, renal failure, erythrocyte biochemistry and haemoglobinopathies.

Ageing
Most data on the use of HbA 1c for the diagnosis of diabetes are based on studies carried out in adults. A cross-sectional analysis of data from the NHANES III group demonstrated a consistent increase in HbA 1c with age. 27 There are limited data from children. One recent study carried out on a multiethnic cohort of children and adolescents suggests that a cut-off value of 6.5% underestimates the prevalence of diabetes in children. 28 Thirty per cent of the population in South Africa is under the age of 15 and 8% is over the age of 60, and the use of the prescribed cut-off of 6.5% may be inaccurate in both these groups. 29 At present, neither the WHO nor the ADA advocates the use of age-appropriate reference ranges.

Renal failure
There are few data on the prevalence of chronic kidney disease in Africa, but the general impression is that it is three to four times more frequent than in more developed countries. 30 Chronic kidney disease may falsely increase or decrease HbA 1c levels. Increased values can arise from increased blood urea levels, which cause the formation of carbamylated haemoglobin and which then results in overestimation of haemoglobin using electrical chargebased assays. 31 These patients often have a shortened red blood cell life span and this would potentially reduce HbA 1c levels. 32

HIV infection
A report from the Women's Interagency HIV study, which looked at FPG and HbA 1c in HIV-infected and noninfected women, showed that HbA 1c levels are slightly lower in HIV-infected women than in noninfected women; this was mainly due to higher mean corpuscular volumes. 22 In the proceedings from the Conference on Retroviruses and Opportunistic Infections 2011, investigators showed that using an HbA 1c cut-off of 6.5% would have diagnosed only nine out of 22 patients not previously known to be diabetic, and that in this small group of subjects the optimal cut-off for diagnosis was 5.8%. Of interest was that the use of antiretroviral drugs showed a variable influence on the test characteristics of HbA 1c , with protease inhibitors leading to an underestimation of HbA 1c levels and non-nucleoside reverse transcriptase inhibitors, particularly efavirenz, overestimating HbA 1c levels. 33 This has major implications for our population, which has a high prevalence of HIV infection, and the use of efavirenz in first-line drug treatment.

Glycation gap and erythrocyte biochemistry
One of the explanations put forward for the observed differences in sensitivity and specificity of Hb A1c as a diagnostic tool is the differences in rates of glycation of haemoglobin. 34 Yudkin et al introduced the terms "high glycators" and "low glycators" to describe individuals with higher or lower HbA 1c levels relative to their glucose. 35 Studies comparing intracellular glycation, as assessed by HbA 1c , with extracellular glycation, as assessed by glycated serum proteins, have shown very strong withinperson correlation between HbA 1c and fructosamine, but substantial between-person variation in the relationship between fructosamine and HbA 1c . 36 This tells us that that there are large inter-individual differences between HbA 1c and glucose. Biochemical differences in glucose transport across red cell membranes, subclinical variation in erythrocyte survival, and inherited differences in glycation rates all contribute to the glycation gap. [37][38][39] Anaemia, iron deficiency and haemoglobinopathies Conditions that alter red cell life span alter HbA 1c concentrations, with conditions that shorten red cell survival, such as haemolysis, decreasing HbA 1c , and those disease states that prolong red cell survival increasing HbA 1c . 37 Sickle-cell haemoglobin (HbS) has a valine for glutamic acid substitution at position 6 of the b chain. HbC is a variant mutation at the same site as the sickle cell mutation. Because the S and C variants are close to the N terminus of the b chain, some immunoassays are affected by the presence of these variants. These variants do affect the ionic charge of the haemoglobin molecule, and this may interfere with ion exchange methods. However, careful inspection of chromatograms should identify the presence of abnormal haemoglobin variants and alternate methods may be used.
No whole-blood method is suitable for the assessment of glycaemic control in patients homozygous for HbS, HbC or HbSC. The prevalence of sickle cell disease in indigenous South Africans is low, but can be very high in populations from the malaria-endemic regions of Africa. 40 Iron deficiency has been shown to increase HbA 1c by up to 2%. 41 This is reversed by iron supplementation. 42 The prevalence of iron deficiency in an urban South African female population despite iron fortification was estimated to be about 10%, 43 while the prevalence in rural populations might be higher, at between 18% and 25%. 44,45 In view of these patient variables it is likely that the use of HbA 1c at a cut-off level of 6.5% will lead to overdiagnosis in the elderly, those with iron deficiency and some rapid glycators, while those with renal failure, haemoglobinopathies and slow glycators will be underdiagnosed.
Given the high prevalence of HIV infection in our population, and the frequency with which subjects with diabetes have other medical illnesses, the likelihood that such factors may alter HbA1c is high. Thus it is imperative that large epidemiological studies be undertaken in such patient groups in South Africa to assess the diagnostic accuracy of HbA 1c measurement.

Costs and availability
The cost of HbA1c is more than double that of plasma glucose, but is cheaper than the cost of the OGTT, which includes the cost of a glucose load and two glucose measurements. However, it is probable that other tests will be required in a significant proportion of cases and this will increase the economic burden on the health system. Currently there are no reliable data on the availability of the HbA 1c test in South Africa.

Conclusion
In conclusion, high HbA 1c levels are clearly associated with incident microvascular complications in cohorts of previously diagnosed diabetics. The available evidence supports the role of HbA 1c as a marker of glycaemic control and as a prognostic marker for future complications. However, there is insufficient evidence from South Africa at this time to recommend it as the method of choice for diagnosis. Before it can be recommended for the diagnosis of diabetes in South Africa, it is important to assess the methodologies currently in use and to ensure that they are reliable and traceable to the DCCT method. It is also important that laboratories are accredited and participate in external proficiency programmes. We should work towards the use of NGSP-certified methods for all diagnostic laboratories, and a national quality control programme for HbA 1c measurement.
Appropriate cut-off levels for the population should be determined, based on the association between HbA 1c , FPG, two-hour glucose levels and microvascular complications. Furthermore, the effects of common diseases, such as HIV and malaria, and the effects of antiretroviral therapy on the utility of HbA 1c for the diagnosis of diabetes must be assessed.
Wild et al estimated that, in 2000, more than seven million people in sub-Saharan Africa had diabetes, with a projected increase to over 17 million by 2030. These projections do not take into account the effect of urbanisation and associated increases in rates of obesity across much of urban Africa. 46 Prospective evaluations should be carried out to assess the clinical and economic consequences of modification of the current diagnostic criteria. In the meantime, we should ensure that the majority of the population has access to plasma glucose and the OGTT for the diagnosis of diabetes.
Presently, the HbA 1c should continue to be used as a means of monitoring diabetes control in South Africa, but not for its diagnosis.