Individuals with type 2 diabetes mellitus (T2DM) are at increased risk of coronary artery disease (CAD). The C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene is associated with elevated plasma levels of homocysteine. The association of the MTHFR gene and the level of homocysteine with development of CAD has been studied in various population groups, including patients with T2DM, but the results have been variable. In practice, plasma homocysteine may be ordered as part of a screen for people with CAD or stroke, or who are at high risk for CAD or stroke but no other known risk factors. Testing of C677T polymorphism with or without elevated homocysteine is not recommended and has no clinical utility.

by Prof. Bakri Saeed and Dr Nisreen Mohammed

Type 2 diabetes mellitus and coronary artery disease
Type 2 diabetes mellitus (T2DM) is a major health problem throughout the world. It is a polygenic and multifactorial disease that is a major risk factor for cardiovascular disease. Cardiovascular disease (CVD) comprises coronary artery disease (CAD), also referred to as coronary heart disease (CHD), or ischemic heart disease (IHD), and cerebrovascular disease.

CAD due to atherosclerosis is a cause of significant morbidity and mortality, and is the leading cause of death worldwide. There are several risk factors for CAD. The well-stablished risk factors for CAD include diabetes mellitus, hypertension, smoking and dyslipidemia. There is growing interest in emerging risk factors for improved understanding of the mechanisms that underline cardiovascular disorders and CAD.

T2DM increases the risk for CAD by 2–4-fold compared to people without diabetes. CVD accounts for about 70% of deaths in people with diabetes. Identification and management of risk factors for CAD is an important aspect of management of diabetes mellitus.
Hyperhomocysteinemia and MTHFR polymorphism
Homocysteine is a sulfur-containing amino acid formed from demethylation of methionine. Methionine is the precursor to S-adenosyl methionine (SAMe) and is one of the essential amino acids. SAMe is a major methyl donor and is involved in numerous biological reactions. Homocysteine is metabolized by either remethylation to methionine or transsulfuration to cystathionine. The former reaction is catalysed by the vitamin B12-dependent methionine synthase. The latter reaction is catalysed by the enzyme cystathionine beta-synthase, which requires vitamin B6.

The methyl donor in the remethylation of homocysteine to methionine is 5-methyltetrahydrofolate. The 5,10-methylene-tetrahydrofolate reductase (MTHFR) enzyme catalyses the reduction of 5,10-methylene-tetrahydrofolate to 5-methyltetrahydrofolate. The enzyme requires B2 (riboflavin) as a cofactor (Fig. 1).

Therefore, hyperhomocysteinemia can result from reduced activity of the enzymes involved in homocysteine metabolism or from deficiency of the vitamins which are needed as cofactors in homocysteine metabolic reactions: folate, vitamin B6 and vitamin B12.

Several mutations in the MTHFR gene have been identified and some of them affect the activity of the enzyme. The commonest MTHFR gene mutation is a cytosine-to-thymidine substitution at nucleotide 677 (C677T), which changes alanine into valine, resulting in a thermolabile enzyme with impaired enzymatic activity and leading to hyperhomocysteinemia.

There are two copies of each gene. Therefore, an individual can be homozygous for the mutated gene or can be heterozygous, having one copy of the C677T variant and one normal copy. The C677T homozygous variant enzyme is thermolabile and demonstrates 70% reduced enzyme activity in vitro. The heterozygous C677T MTHFR enzyme has 35% reduced activity in vitro.

Worldwide, the frequency of MTHFR gene mutations varies among racial and ethnic groups, in Africa MTHFR gene polymorphism is markedly low (below 10%) for the C677T allele. In the European and Asian population, estimates of 18.6% and 20.8% were reported [1].

Association with CAD
In recent years hyperhomocysteinemia has been implicated as a risk factor for CAD, independent of other known risk factors. The primary mechanism by which homocysteine promotes atherosclerosis is by impairing endothelial function, which initiates the chain of events resulting in atherosclerotic plaque formation.

Numerous studies looked into the possible association between MTHFR genotypes and plasma homocysteine levels and the incidence of different MTHFR genotypes and hyperhomocysteinemia in CAD patients [2–5]. The results of these studies have been controversial. Several studies have shown the link between the MTHFR C677T gene polymorphism and the risk for CAD but many other studies failed to show association between MTHFR genotypes and plasma homocysteine levels and their role in CAD.

Previous studies in T2DM patients were also controversial. MTHFR polymorphism and hyperhomocysteinemia were shown to be predictors of cardiovascular events among diabetic patients [6, 7], whereas other studies failed to show a role for MTHFR polymorphic variants and homocysteine in increasing susceptibility to cardiovascular disease [8, 9].

Our study
We recently screened 226 consecutive patients with T2DM, <60 years of age, diagnosed according to WHO criteria. Of these, 113 had CAD confirmed by angiography and electrocardiography (ECG) and 113 had no evidence of CAD [10]. PCR and restriction fragment length polymorphism (RFLP) using Hinf1 restriction enzyme were used to determine MTHFR genotypes.

In our study, the T allele had a significant effect on homocysteine level (P value <0.05) and showed strong association with CAD among T2DM patients (odds ratio 6.2, P <0.0001).

Our study indicates that the C677T polymorphism of the MTHFR gene is associated with hyperhomocysteinemia, and the two are independently associated with the presence of CAD in patients with T2DM.

Reasons for controversy
The outcome of these numerous studies and meta-analysis remained contradictory. There was no agreement on the association between MTHFR genotypes and plasma homocysteine levels or the incidence of different MTHFR genotypes and hyperhomocysteinemia in CAD patients.

Plasma homocysteine levels are dependent on interacting nutritional and genetic factors. Some studies suggested that people homozygous for MTHFR C667T polymorphism tend to have hyperhomocysteinemia in the context of low folic acid levels. Supplementation with the vitamins involved in homocysteine metabolism was found to lower plasma homocysteine levels.

Therefore, geographic heterogeneity, nutritional and environmental factors could affect the relationship between MTHFR genotypes and CVD risk in different populations.

Practical points
Homocysteine may be ordered as part of a screen for people with or at high risk of CAD or stroke, especially if there is family history of CAD or stroke but no other known risk factors, such as diabetes, smoking, hypertension, or dyslipidemia. Routine screening of homocysteine, like that of cholesterol, has not been recommended.

Plasma homocysteine concentration may be elevated in B12 and folate deficiency and its measurement has been suggested to give an early indicator of deficiency.

In new-born testing, greatly increased concentrations of homocysteine in the urine and blood suggests a diagnosis of homocystinuria and indicates the need for confirmation of the cause of raised levels.

Most laboratories report normal homocysteine levels in the blood between 5 and 15 µmol/L. Any measurement above 15 µmol/L is considered high.

However, it should be noted that normal levels will vary between ethnic groups and populations. Homocysteine levels increase with age, are lower in pregnancy and are influenced by drugs. These factors should be taken into consideration when interpreting results.

Testing of C677T polymorphism with or without elevated homocysteine is not recommended in patients with CAD or other diseases where MTHFR variants have been implicated, such as thrombophilia or recurrent pregnancy loss.
References
1. Schneider JA, Rees DC, Liu YT, Clegg JB. Worldwide distribution of a common methylenetetrahydrofolate reductase mutation. Am J Hum Genet 1998; 62: 1258–1260.
2. Chehadeh SWEH, Jelinek HF, Al Mahmeed WA, Tay GK, Odama UO, Elghazali GE, et al. Relationship between MTHFR C677T and A1298C gene polymorphisms and complications of type 2 diabetes mellitus in an Emirati population. Meta gene 2016; 9: 70–75.
3. Bickel C, Schnabel R, Zengin E, Lubos E, Rupprecht H, Lackner K, et al. Homocysteine concentration in coronary artery disease: Influence of three common single nucleotide polymorphisms. Nutr Metab Cardiovascular Dis 2017; 27(2): 168–175.
4. Yilmaz H, Isbir S, Agachan B, Ergen A, Farsak B, Isbir T. C677T mutation of methylenetetrahydrofolate reductase gene and serum homocysteine levels in Turkish patients with coronary artery disease. Cell Biochem Funct 2006; 24(1): 87–90.
5. Meisel C, Cascorbi I, Gerloff T, Stangl V, Laule M, Müller JM, et al. Identification of six methylenetetrahydrofolate reductase (MTHFR) genotypes resulting from common polymorphisms: impact on plasma homocysteine levels and development of coronary artery disease. Atherosclerosis 2001; 154(3): 651–658.
6. Lewis SJ, Ebrahim S, Smith GD. Meta-analysis of MTHFR 677C→T polymorphism and coronary heart disease: does totality of evidence support causal role for homocysteine and preventive potential of folate? BMJ 2005; 331(7524): 1053–1058.
7. Bennouar N, Allami A, Azeddoug H, Bendris A, Laraqui A, El Jaffali A, et al. Thermolabile methylenetetrahydrofolate reductase C677T polymorphism and homocysteine are risk factors for coronary artery disease in Moroccan population. J Biomed Biotechnol 2007(1); 80687.
8. Bahadır A, Eroz R, Türker Y. Does the MTHFR C677T gene polymorphism indicate cardiovascular disease risk in type 2 diabetes mellitus patients? Anatolian J Cardiol 2015; 15(7): 524–530.
9. Rahimi Z, Nomani H, Mozafari H, Vaisi-Raygani A, Madani H, Malek-Khosravi S, et al. Factor V G1691A, prothrombin G20210A and methylenetetrahydrofolate reductase polymorphism C677T are not associated with coronary artery disease and type 2 diabetes mellitus in western Iran. Blood Coagul Fibrinolysis 2009; 20(4): 252–256.
10. Mohammed NO, Ali IA, Elamin BK and Saeed BO. The association of methylenetetrahydrofolate reductase gene polymorphism and hyperhomocysteinaemia with coronary artery disease in Sudanese patients with type 2 diabetes. Poster at Focus 2017, Association of Clinical Biochemistry annual meeting.

The authors
Bakri Osman Saeed*¹ PhD, MD, FRCPath, FRCP; Nisreen Osman Mohamed² PhD
¹Faculty of Medicine, Sudan International University, Khartoum, Sudan
²Ahfad Centre for Science and Technology, Ahfad University for Women, Khartoum, Sudan
*Corresponding author
E-mail: saeedbakri@hotmail.com

Clostridium difficile is a leading cause of nosocomial diarrhoea and one of the most common healthcare-associated infections. A dramatic worldwide increase in the incidence of C. difficile infection has occurred over the past two decades with the emergence of hypervirulent strains. Accurate and timely laboratory diagnosis of C. difficile infection is fundamental to ensure patients receive appropriate treatment and proper counter-infection measures are put in place. However, the availability of many commercial tests with different C. difficile targets contributes to uncertainty and controversy around the optimal diagnostic algorithm.

by Dr Guilherme Grossi Lopes Cançado, Prof. Rodrigo Otávio Silveira Silva and Prof. Eduardo Garcia Vilela

Introduction
Clostridium difficile is a major cause of healthcare-associated diarrhoea, and is linked to significant morbidity, economic burden and even mortality. Disagreement between diagnostic tests is an ongoing barrier to clinical decision-making and epidemiological surveillance. With the increasing number and severity of Clostridium difficile infections (CDI) after the emergence of the epidemic BI/NAP1/027 strain in the 2000s, there has been a renewed interest in optimizing the laboratory diagnosis of this infection.

Testing for C. difficile
Cytotoxin neutralization, toxigenic culture and toxin enzyme immunoassay
The two most accurate methods for CDI diagnosis are cytotoxin neutralization (CTN) and toxigenic culture (TC). CTN has long been used as a reference method for C. difficile detection, although different protocols have been proposed. Basically, this technique consists of inoculating a filtrate of a stool suspension into a cell culture (Vero, Hep2, fibroblasts, CHO or HeLa cells) and observing a cytopathic effect (such as cell rounding) as a consequence of disruption of the cell cytoskeleton 24–48 h later. CDI confirmation is obtained by the addition of a specific antiserum directed against C. difficile or against C. sordellii, neutralizing the toxin effects. Studies performed in the last 10 years, however, demonstrated that the sensitivities of CTN protocols range between 60 and 86%, when compared with toxigenic culture. Although culture was found to be the most sensitive method for detecting C. difficile in feces, it is not very specific owing to the possibility of isolating non-toxigenic strains. In order to overcome this issue, it should always be combined with a toxin detection method, such as enzyme immunoassay (EIA), direct cytopathic effect on cell lines or the identification of toxin-related genes by PCR. This notwithstanding, both techniques are time consuming, laborious and require trained personnel, reasons why they are not frequently used in daily practice [1].

In this context, commercial rapid EIA and DNA-based tests are currently the most widely used tools for CDI diagnosis. EIA for toxin detection is fast, cheap, easy to perform and does not require technical training or special equipment: characteristics which have favored its use in low income countries. However, several studies have reported low detection sensitivities of different commercial kits (approximately 50–70%), making toxin EIAs inadequate as standalone tests [2]. Some authors have suggested the analysis of sequential samples from the same patient, but this practice did not significantly increase the positive predictive value of the test and may even amplify the rate of false positives. Furthermore, toxin tests can be falsely negative, which can be due to previous antibiotic treatment, pre-analytic toxin degradation or sampling error (e.g., ileus, fecal dilution). Consequently, toxin EIA may be sufficient and cost-effective as a screening test in clinical settings where there is a low prevalence of CDI, but not for epidemic scenarios.

Detection of glutamate dehydrogenase
Another method that has been used to diagnose CDI is the detection of glutamate dehydrogenase (GDH), a metabolic enzyme constitutionally produced almost exclusively by C. difficile, which is significantly more sensitive than toxin A and B EIAs. We have recently shown that GDH rapid immunoassay presents 100% sensitivity and negative predictive value, compared with the culture-based method, in accordance with order authors [3]. Cheng et al. have advocated the use of GDH in order to improve the diagnostic capacity and control of potential outbreaks of CDI in developing countries, as this test is five to ten times cheaper than molecular assays [4]. In fact, we have recently reported a significant increase in CDI treatment rates in a university hospital of Brazil (from 53.8% to 100%) simply after replacing the toxin EIA by the rapid GDH immunoassay as a screening test [3]. This finding supports the use of GDH in countries with limited economic resources, as it is simple to perform and, unlike PCR, requires no special facilities or personnel qualifications that might restrict its use. Although sensitive, GDH lacks specificity, because it only indicates the presence of the microorganism, instead of toxin production. In this context, a concern with the overdiagnosis and overtreatment of patients with diarrhoea and C. difficile colonization may arise. A negative test can almost rule out CDI and avoid the need for more expensive toxin testing, but a positive GDH assay should preferably be followed by toxin-based diagnostic methods before one can come to a safe conclusion on infection. Asymptomatic carriage of C. difficile occurs in 5–15% of healthy adults, but may be as high as 90% in newborns and healthy infants, and up to 51% in residents in long-term care facilities [5]. In this way, the incorporation of a two-step strategy, including a sensitive organism-based test for screening, such as GDH, followed by a toxin test for confirmation of clinically significant disease, is a reasonable approach. Combined tests including GDH and toxin detection in one easy-to-use cartridge have been recently developed, but several authors have demonstrated limited sensitivity of the toxin component. In our study, the toxin component presented an even lower sensitivity than conventional toxin EIA (50% vs 58%), compared with toxigenic culture [3]. In this way, GDH+/toxin− samples would still have to be submitted to a third test (in a multistep algorithm) to rule out infection, increasing time to diagnosis and health costs. However, some studies have shown that, even without treatment, patients with toxin-negative stool specimens have shorter diarrhoea duration than those with toxin-positive stool specimens. These findings may suggest a limited need for CDI treatment for GDH-positive patients and toxin-negative stool specimens [6].

Molecular testing and rapid diagnosis
In 2009, facing an epidemic of CDI due to hypervirulent C. difficile strains, the US FDA cleared the first commercial molecular test for rapid CDI diagnosis [7]. Shortly afterwards, hospitals in North America and Europe began switching to DNA-based testing strategies as a method of choice for the diagnosis of C. difficile. Nucleic acid amplification testing (NAAT), including rapid testing PCR and loop-mediated isothermal amplification (LAMP), can detect the tcdA/tcdB genes (regulate toxin A/B production) or the tcdC gene (a negative regulator of toxin A and B production) and identify the presence of toxigenic C. difficile in a single step. It has a higher sensitivity (90–95%) and specificity (95–96%) than toxin EIAs and has a rapid turnaround time, but requires specialized equipment and personnel. It is worth noting that the Gene-Xpert^® system can also simultaneously indicate the presence of the potentially ‘hypervirulent’ ribotype 027 strain, giving important epidemiological information. Nonetheless, although a PCR assay can identify toxin genes, it cannot detect the presence of toxin. Several studies have shown that toxin−/C. difficile+ patients present shorter duration of symptoms and better outcomes than toxin+/C. difficile+ individuals, demonstrating that this subpopulation may have either mild CDI or colonization, and even not need to be treated. In this way, some authors have questioned the use molecular tests as standalone tests because of the high likelihood of overdiagnosis and overtreatment [8]. However, there may be a role for identifying carriers to prevent transmission and this issue should be better addressed in future studies.

C. difficile testing protocol
Using reliable and rapid diagnostic tests, such as NAAT, practitioners could offer appropriate treatment earlier, thereby sparing patients a time-consuming evaluation and unnecessary antibiotic therapy and its complications. Despite worldwide advances in analytical technology, transport systems and computerization, many laboratories in developing countries have difficulties in improving turnaround times and diagnostic capability. The optimal diagnostic algorithm for CDI is yet to be adequately defined and may vary according to the underlying clinical and laboratory circumstances. We believe that the decision about which test(s) to use is determined by a combination of what is practical and feasible in a specific setting. In Brazil, for example, where only toxin EIA was available for CDI diagnosis until 2015, the introduction of the GDH test resulted in a dramatic increase in C. difficile treatment rates [3]. All of the reference methods, CTN, toxigenic culture, or PCR, require advanced infrastructure and expensive testing. Smaller, community-based hospitals, where much of C. difficile testing is currently performed, may not have the financial means to establish these methods nor have the staff to perform the time-consuming, highly complex assays. In this way, using GDH–toxin A/B assays may be an adequate option for diagnostic algorithms in developing countries, whereas molecular techniques, toxigenic culture or CTN may be reserved for discordant samples (Figure 1). Future studies should focus on developing simple diagnostic approaches to accurately distinguish active infection from mere colonization.

References
1. Delmée M. Laboratory diagnosis of Clostridium difficile disease. Clin Microbiol Infect 2001; 7(8): 411–416.
2. Silva RO, Vilela EG, Neves MS, Lobato FC. Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil. Rev Soc Bras Med Trop 2014; 47(4): 447–450.
3. Cançado GGL, Silva ROS, Nader AP, Lobato FCF, Vilela EG. Impact of simultaneous glutamate dehydrogenase (GDH) and toxin A/B rapid immunoassay on Clostridium difficile diagnosis and treatment in hospitalized patients with antibiotic-associated diarrhea in a university hospital of Brazil. J Gastroenterol Hepatol 2017; doi: 10.1111/jgh.13901 [Epub ahead of print].
4. Cheng JW, Xiao M, Kudinha T, Xu ZP, Sun LY, Hou X, Zhang L, Fan X, Kong F, Xu YC. The role of glutamate gehydrogenase (GDH) testing assay in the diagnosis of Clostridium difficile infections: a high sensitive screening test and an essential step in the proposed laboratory diagnosis workflow for developing countries like China. PLoS One 2015; 10(12): e0144604.
5. Furuya-Kanamori L, Marquess J, Yakob L, Riley TV, Paterson DL, Foster NF, Huber CA, Clements AC. Asymptomatic Clostridium difficile colonization: epidemiology and clinical implications. BMC Infect Dis 2015; 15: 516.
6. Yuhashi K, Yagihara Y, Misawa Y, Sato T, Saito R, Okugawa S, Moriya K. Diagnosing Clostridium difficile-associated diarrhea using enzyme immunoassay: the clinical significance of toxin negativity in glutamate dehydrogenase-positive patients. Infect Drug Resist 2016; 9: 93–99.
7. Polage CR, Turkiewicz JV, Cohen SH. The never-ending struggle with laboratory testing for Clostridium difficile infection. J Comp Eff Res 2016; 5(2): 113–116.
8. Polage CR, Gyorke CE, Kennedy MA, Leslie JL, Chin DL, Wang S, Nguyen HH, Huang B, Tang YW, Lee LW, Kim K, Taylor S, Romano PS, Panacek EA, Goodell PB, Solnick JV, Cohen SH. Overdiagnosis of Clostridium difficile infection in the molecular test era. JAMA Intern Med 2015; 175(11): 1792–1801.

The authors
Guilherme Grossi Lopes Cançado*^1,2 MD, Rodrigo Otávio Silveira Silva³ PhD, and Eduardo Garcia Vilela¹ MD, PhD
¹Instituto Alfa de Gastroenterologia,
Federal University of Minas Gerais, Brazil.
²Department of Gastroenterology,
Hospital da Polícia Militar de Minas Gerais, Brazil.
³Veterinary School, Federal University of Minas Gerais, Brazil.

*Corresponding author
E-mail: guilhermegrossi@terra.com.br