Methotrexate is an established treatment for inflammatory bowel disease, however it is commonly only used as second-line therapy due to concerns over side effects. This article reviews the evidence for using methotrexate polyglutamate levels in the management of rheumatoid arthritis and psoriasis in addition to inflammatory bowel disease with a view to optimizing treatment and helping to prevent toxicity.

by Dr E. L. Johnston, Dr S. C. Fong, Dr A. M. Marinaki, Dr M. Arenas-Hernandez and Dr J. D. Sanderson

Introduction
Methotrexate (MTX) is a folate analogue. It was first used in the 1950s to induce remission in childhood leukemias. Since then its clinical benefit has been widely utilized in the treatment of several inflammatory conditions, including rheumatoid arthritis (RA) and psoriasis, and more recently, inflammatory bowel disease (IBD).

Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory conditions affecting the gastrointestinal tract, collectively known as IBD. MTX is not as commonly used in the treatment of IBD as other immune modulators, particularly thiopurines. This centres around concerns regarding toxicity and side effects, although in the RA population MTX is frequently used and is considered safe and effective. Monitoring methotrexate, by means of measuring red-cell methotrexate-polyglutamate (MTX-PG) levels, offers the potential to assess adherence along with optimizing dose. However, MTX-PG levels are currently underused because of conflicting evidence regarding interpretation of levels.

Inflammatory bowel disease and methotrexate
The use of methotrexate as a treatment in IBD was initially postulated in the late 1980s when a small study showed an improvement in disease activity indexes, and some histological improvement in the CD cohort, in patients with refractory IBD [1]. Since then, MTX has increasingly been used as a second-line treatment, particularly in those when thiopurine or anti-TNF therapy has failed or not been tolerated.

The European Crohn’s and Colitis Organisation (ECCO) guidelines on the management of CD [2] advise that methotrexate 25 mg/week can be used to treat active CD as an alternative to thiopurines. This is based on a randomized control trial (RCT) in 1995 [3] that showed a significant benefit in taking 25 mg/week of intramuscular (IM) MTX compared with placebo following withdrawal from steroids (39% vs. 19%). It is commonly prescribed orally which is easier for administration and favoured by patients. However, a small study [4] comparing oral to subcutaneous (SC) MTX showed the bioavailability of the oral preparation was variable, despite folic acid use, and favoured SC delivery.

There have been no large studies comparing thiopurines and methotrexate to treat CD and the largest RCT to date looking at the use of MTX as a concomitant immunosuppressant when combined with infliximab, compared to infliximab as monotherapy, showed no benefit in steroid free remission [5].

The evidence to support MTX use in inducing and maintaining remission in patients with UC is less robust with very few good quality RCTs. These studies have shown no benefit over placebo and, therefore, a recent Cochrane review did not support its use [6]. However, two large international RCTs (METEOR and MERIT-UC) looking at the use of MTX for active UC are ongoing.

When MTX is being considered as a treatment option for IBD there are often concerns over the safety of the drug. MTX use requires careful monitoring, particularly of liver function tests because of the risk of hepatotoxicity. However, a retrospective study of its use in CD found it was safe and well tolerated [7]. The commonest side effect was nausea in 22% (17 patients) with only 10% of patients experiencing abnormal liver function tests, resulting in 6% having to stop MTX.

Methotrexate polyglutamate levels
MTX is taken weekly and is commonly administered orally but can be used SC or IM. Despite a stable dose and route of administration there is significant interpatient variability in clinical response and the prevalence of side effects, which is a major drawback of therapy. It has, therefore, long been hypothesized that measuring MTX drug levels could be both a predictor of drug efficacy and a marker of potential toxicity.

MTX levels peak within hours of oral ingestion and are detectable for less than 24 hours in the serum. Weekly dosing offers no steady-state concentration and, therefore, serum levels are of no clinical benefit. Once in the serum, MTX is transported intracellularly by a reduced folate carrier (RFC) and is changed into a polyglutamated form (MTX-PG₁). Further glutamic acid residues (GLUT) are added resulting in up to seven polyglutamates (MTX-PG_1–7). This is show in Figure 1.

By using high-performance liquid chromatography it is possible to quantify the seven glutamic residue species in red blood cells [8]. This was first used in children with acute lymphoblastic leukemia [9] and has subsequently been found to correlate with disease activity in other chronic inflammatory conditions. However, MTX-PG_6–7 have not previously been detected in RA patients taking MTX [14]; therefore, commonly only MTX-PG_1–5 are measured.

Early data suggested that MTX-PG_1–2 correlated poorly with drug efficacy in RA; however, the total long-chain polyglutamates (MTX-PG_3–5) better reflected the drug effect [8]. MTX-PG₃ is the predominant polyglutamate species in red blood cells and is useful to calculate the total long-chain concentrations [10].

Clinical use of methotrexate polyglutamate levels
MTX is widely prescribed for the treatment of RA. Dervieux et al. [10] first looked at the clinical use of MTX-PG measurements in the RA population. In 108 patients who had been on MTX over 3 months, higher MTX-PG levels were associated with a better clinical response to the drug. In particular, patients with a total MTX-PG_1–5 that was >60 nmol/L were found to have less tender and swollen joints. The same group expanded their cohort and once again showed that patients with MTX-PG_1–5 <60 nmol/L were four times more likely to have a poor response to MTX than those with MTX-PG_1–5 >60 nmol/L [11].

Stamp et al. [12] noted large interpatient variability in MTX-PG levels and set out to identify factors that influence levels. Using univariate analysis they found that increased age, impaired renal function, longer duration of treatment and the use of prednisolone resulted in higher MTX-PG levels, whereas smokers generally had lower MTX-PG levels. In contrast to the studies by Dervieux et al., they also surprisingly found that higher doses of MTX were associated with higher MTX-PG levels and increased disease activity [13]. In addition there was no association between MTX-PG levels and adverse effects.

The same group looked at the timing of MTX-PG blood levels and time to steady state [14]. MTX-PG₁ was detected 1–2 weeks after first ingestion; however, MTX-PG5 was detected after a median of 7 weeks (range 1–28 weeks). In addition the median time for MTX-PG_1–5 to reach steady-state concentration was 27.5 weeks and the median time for MTX-PG_1–5 to become undetectable after the last dose was 15 weeks. This highlights that MTX may take up to 6 months to achieve full clinical benefit, which is important to consider when using the levels to assess compliance or to guide dose alteration.

The main trial to be done outside the field of rheumatology was a 55-patient, prospective study into using MTX-PG levels to assess clinical response and compliance in patients with psoriasis [15]. This found the time to steady state of MTX-PG_1–5 was between 12–24 weeks, and there was no significant correlation between MTX-PG levels and disease activity.

Methotrexate polyglutamate levels and inflammatory bowel disease
There have been only two studies addressing the potential use of MTX-PG levels in IBD. Egan et al. looked at the total levels when addressing the question of the optimal dose of MTX needed to induce remission in steroid-requiring IBD [16]. They found that subcutaneous initial doses of 15 and 25 mg/week in 32 patients were equally efficacious. In this cohort MTX-PG concentration reached a plateau at around 6–8 weeks after the initiation of therapy and no statistical difference was found between the levels across both doses of the drug. In addition the levels did not correlate with active disease or drug toxicity and did not change significantly after change in MTX dose.

A more recent prospective study from Brooks et al. looked specifically at MTX-PG concentrations in 18 patients with IBD that were on stable doses of MTX [8]. MTX-PG were measured on three occasions and compared to disease activity and reports of toxic side effects. MTX-PG were detected in all the patients and there was little variability in the levels over the study period. Similar to the Stamp et al. RA study [13], higher MTX-PG_4&5 were associated with worse disease activity as well as higher toxic effects.

The cohort was small and heterogeneous with different doses of MTX prescribed (median 20 mg/week) and varied administration methods (oral, subcutaneous and via percutaneous endoscopic gastrostomy tube), which is likely to have had a bearing on the results. The data from a similar cohort was presented at Digestive Diseases Week in 2014 [17], which concluded that MTX-PG could be useful in assessing adherence. A non-significant trend showed higher concentrations were associated with active disease, but this may be due to higher doses of MTX being used in those with active disease.

Summary
Methotrexate is an established treatment for IBD. It is an efficacious and well tolerated therapeutic option in CD, particularly when administered SC. More studies are ongoing in the UC population. Measuring MTX-PG levels in RBC has the potential to not only monitor compliance but also correlate with disease activity and toxicity. Two large studies in patients with RA have produced conflicting results but in the small, IBD trials, higher MTX-PG levels, particularly MTX-PG_4&5 correlated with increased disease activity and toxicity. It is important, however, to be aware that MTX-PG are influenced by other factors, particularly age and renal function, and may take up to 6 months to reach steady state.

Future trends and developments
Measuring drug levels plays an important role in the management of patients with IBD, as demonstrated by the monitoring of thioguanine nucleotides in those prescribed azathioprine [18]. Measuring MTX-PG offers an exciting step towards individualizing drug treatment and reducing toxicity in those taking MTX. However, at the moment there is a lack of substantial evidence to support the use of measuring MTX-PG levels in IBD, aside from monitoring compliance [19]. A large, prospective trial is warranted to determine clinical benefit before widespread use in the IBD population is advocated.

References
1. Kozarek RA, Patterson DJ, Gelfand MD, et al. methotrexate induces clinical and histological remission in patients with refractory inflammatory bowel disease. Ann Intern Med. 1989; 110: 353–356.
2. Dignass A, Van Assche G, Lindsay JO, et al. The second European evidence-based consensus on the diagnosis and management of Crohn’s disease: Current management. J Crohn’s Colitis 2010; 4: 28–62.
3. Feagan BG, Rochon J, Fedorak RN, et al. Methotrexate in the treatment of Crohn’s disease. New England Journal of Medicine. 1995; 332: 292–297.
4. Kurnik D, Loebstein R, Fishbein E, et al. Bioavailability of oral vs. subcutaneous low-dose methotrexate in patients with Crohn’s disease. Aliment Pharmacol Ther. 2003; 18(1): 57–63.
5. Feagan BG, McDonald JW, Panaccione R, et al. Methotrexate in combination with infliximab is no more effective than infliximab alone in patients with Crohn’s disease. Gastroenterology 2014; 146(3): 681–688.
6. Chande N, Wang Y, MacDonald JK, et al. Methotrexate for induction of remission in ulcerative colitis. Cochrane Database Syst Rev. 2014; 8.
7. Chande N, Abdelgadir I, Gregor J. The safety and tolerability of methotrexate for treating patients with Crohn’s disease. J Clin Gastroenterol. 2011; 45: 599–601.
8. Brooks A, Begg E, Zhang M, et al. Red blood cell methotrexate polyglutamate concentrations in inflammatory bowel disease. Ther Drug Monit. 2007; 29: 619–625.
9. Lena N, Imbert AM, Brunet P, et al. Kinetics of methotrexate and its metabolites in red blood cells. Cancer Drug Deliv. 1987; 4(2): 119–127.
10. Dervieux T, Furst D, Lein DO, et al. Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis. Arthritis Rheum. 2004; 50(9): 2766–2774.
11. Dervieux T, Furst D, Lein DO, et al. Pharmacogenetic and metabolite measurements are associated with clinical status in patients with rheumatoid arthritis treated with methotrexate: results of a multicentred cross sectional observational study. Ann Rheum Dis. 2005; 64: 1180–1185.
12. Stamp LK, O’Donnell JL, Chapman PT, et al. Determinants of red blood cell methotrexate polyglutamate concentrations in rheumatoid arthritis patients receiving long-term methotrexate treatment. Arthritis Rheum. 2009; 60(8): 2248–2256.
13. Stamp LK, O’Donnell JL, Chapman PT, et al. Methotrexate polyglutamate concentrations are not associated with disease control in rheumatoid arthritis patients receiving long-term methotrexate therapy. Arthritis Rheum. 2010; 62(2): 359–368.
14. Dalrymple JM, Stamp LK, O’Donnell JL, et al. Pharmacokinetics of oral methotrexate in patients with rheumatoid arthritis. Arthritis Rheum. 2008; 58(11): 3299–3308.
15. Woolf RT, West SL, Arenas-Hernandez M, et al. Methotrexate polyglutamates as a marker of patient compliance and clinical response in psoriasis: a single-centre prospective study. Br J Dermatol. 2012; 167: 165–173.
16. Egan LJ, Sandborn WJ, Tremaine WJ, et al. A randomised dose-response and pharmacokinetic study of methotrexate for refractory inflammatory Crohn’s disease and ulcerative colitis. Aliment Pharmacol and Ther. 1999; 13: 1597–1604.
17. Ward MG, Fong S, Nasr I, et al. Higher red blood cell methotrexate polyglutamates correlate with increased disease activity, and are useful in assessing adherence. Abstract presented at Digestive Disease Week 2014.
18. Smith M, Blaker, P, Patel C, et al. The impact of introducing thioguanine nucleotide monitoring into an inflammatory bowel disease clinic. Int J Clin Pract. 2013; 67(2): 161–169.
19. Bruns T, Stallmach A. Drug monitoring in inflammatory bowel disease: helpful of dispensable? Dig Dis. 2009; 27: 394–403.

The authors
Emma L. Johnston¹ MBBS BSc MRCP, Steven C. Fong¹ MBBS MRCP, Anthony M. Marinaki² PhD, Monica Arenas-Hernandez² PhD, Jeremy D. Sanderson*¹ MD FRCP
¹Inflammatory Bowel Disease Centre, Dept of Gastroenterology, Guy’s and St Thomas’ NHS Foundation Trust, London, UK.
²Purine Research Laboratory, Viapath, Guy’s & St. Thomas’ NHS Foundation Trust, London, UK.

*Corresponding author
E-mail: jeremy.sanderson@kcl.ac.uk

Over the last few years, hepatitis C virus (HCV) infection has emerged as one of the most significant causes of chronic liver disease worldwide, with estimated prevalence ranging from 2.2 to 3.0%. Since January 2011, the Infectious Diseases Department of San Raffaele Scientific Institute in Milan carried out a prevention programme called ‘EASY test project’, to diagnose the HCV infection. In these four years a total of 35,000 subjects have been approached to inform them about HCV infection and other sexually transmitted diseases. Of the total eligible volunteers, 6500 (18,6% of contacted subjects) performed HCV tests on saliva and completed the interview in the alternative ‘street lab’. We believe that increasing awareness of these alternative tests among individuals at risk and providers may be an appropriate strategy to increase the number of people who know their serological status and who could be linked to care and engaged in care!

by M.R. Parisi, Dr L. Soldini, Dr G. Vidoni, Dr K. Schlusnus, Dr F. Dorigatti, and Prof. A. Lazzarin

Background
Over the last few years, hepatitis C virus (HCV) infection has emerged as one of the most significant causes of chronic liver disease worldwide, with estimated prevalence ranging from 2.2 to 3.0% (1).

In our country, the proportion of subjects infected with HCV is approximately 2% of the general population with a gradient that increases from the north to the south and the islands and with age (60% of patients with hepatitis C are over 65 years old). It is estimated that about 1 million people in Italy are ill with hepatitis C (2).
As acute HCV infection is usually asymptomatic, early diagnosis is rare. Those people who are developing chronic infection, even though undiagnosed, may suffer serious liver damage. In fact, a significant proportion of HCV infected subjects will ultimately progress to liver cirrhosis and/or hepatocellular carcinoma, making chronic HCV infection a major health problem (3, 4).

Despite the excellent accuracy of the tests currently available for the detection of anti-HCV antibodies (anti-HCV), the delay in reporting the results, the need for specialized equipment for processing the samples and interpreting the results, as well as the need to transfer individuals to sample collection and processing centres, limit their use as screening tools. Serologic points-of-care tests (POCTs) have several advantages, namely that they require little specialized apparatus, can be brought to the individuals who are to be tested and allow diagnosis in as little as a few minutes in different clinical settings (5). These advantages might be translated into increased testing opportunity and, ultimately, identification of more patients who could benefit from antiviral treatment (6). Over the last few years, several tests for rapid detection of anti-HCV have been developed and are currently in use in various countries; however, only recently, the first POCT was approved by the U.S. Food and Drug Administration (7). The investigation of the diagnostic accuracy of POCTs and rapid tests for the detection of anti-HCV is a highly relevant topic. As well as the great importance of the issue in terms of public health, there is a lack of studies evaluating the performance of several of the currently used tests.

EASY test project
Since January 2011, the Infectious Diseases Department of San Raffaele Scientific Institute in Milan carried out a Prevention Program called “EASY test project”, using the new oral test (rapid and non-invasive) the OraQuick® HCV Rapid Antibody Test (OraSure technologies, Inc.) to diagnose the HCV infection. The test is a single-use, immunoassay for the qualitative detection of antibodies to hepatitis C virus (anti-HCV) in oral fluid, fingerstick whole blood, venipuncture whole blood and plasma specimens. The HCV rapid test received the FDA approval for use with oral fluid on 28 June 2010.

The clinical sensitivity and specificity of the OraQuick HCV test using oral fluid were 97.8% (95% confidence interval [CI]) and 100% (95% CI, 98.4-100%), respectively (8). 

The main objective of the project is to evaluate the acceptability of an alternative, free and anonymous HCV test offer, available in different settings (in Points of Care, STDs Prevention clinics and General Practitioner surgeries) (9, 10). Furthermore, contacting the ‘hard-to-reach’ people with this anonymous and free test offer could reduce or stop this public health problem, by making an easy link to healthcare.

Subjects who underwent the test were asked to complete an anonymous questionnaire, through which it has been possible to collect a series of data on risk behaviours of the population tested. The questionnaire was devised with the intention of collecting demographic and risk behaviour data, as well as previous HCV/HIV testing experience, information about sex, drug use, educational level, nationality, general behaviours, use of HIV/HCV prevention services, previous surgical practices, invasive diagnostic practices, dental cares, tattoos or sexually transmitted diseases. Post-test counselling has been provided to all HCV reactive and non-reactive subjects, by the Infectious Diseases Department physicians involved in the study. The test was been carried out by a biologist or a practitioner, following the manufacturer’s procedures.

If the HCV oral test provided a preliminary positive result, a venipuncture was performed immediately for standard test confirmation, supported by the post-test counselling.

The results were received in two working days. At this point, the HCV-positive patient was contacted directly by the infectious diseases specialist for the visit and the diagnostic procedures to define the liver disease status and eventually to start the treatment, according to the guidelines for when HCV viral load and genotype are identified.
In these four years a total of 35,000 subjects have been approached to inform them about HCV infection and other sexually transmitted diseases. Of the total eligible volunteers, 6500 (18.6%) performed HCV tests on saliva and completed the interview in the alternative ‘street lab’. From the questionnaires we know that this initiative has been much appreciated.

Discussion
In recent years, advances in detection technology made available a range of POCTs for different infectious diseases. It is now possible to screen and diagnose those conditions at primary healthcare settings, using minimally invasive tests. In the present study, a new POCT for HCV infection has been performed on oral fluid. The use of oral fluid is an attractive alternative based on the fact that collection of plasma or serum samples requires equipment and training, and is more time consuming.

The FDA-approved OraQuick HCV Rapid Antibody Test (OraSure Technologies) is one of the most studied rapid tests for the diagnosis of HCV infection.

The development of rapid alternative tests for the diagnosis of HCV infection is to facilitate access to testing to reduce the individual risk of disease progression and social costs.

Despite the excellent sensitivity and specificity of third-generation enzyme immunoassays (EIAs), the turnaround time for reporting test results is at least one day, thereby making it difficult to deliver the results to tested individuals at first visit. Rapid tests are formatted such that they do not require complicated instrumentation or testing by skilled technical staff. They potentially generate results within an hour and therefore may be used for point-of-care testing.  Rapid tests are obviously more expensive than conventional immunoassays and are not designed for testing large batches of specimens. However, in no-clinical settings and laboratories that conduct low-volume testing, adoption of rapid oral testing can be cost-effective. CDC guidelines formulated for confirming screening anti-HCV results remain to be refined to accommodate rapid anti-HCV testing. It is important to emphasize that OraQuick HCV test has not been approved for general screening. A positive result of a rapid anti-HCV positive test is indicative of the presence of anti-HCV and, again, does not indicate active infection (11).

We successfully conducted this rapid HCV testing and counselling programme with the goal of spreading the use of saliva test anonymously and free of charge. We aim to facilitate access to testing in alternative settings, in order to understand if the ‘hard-to access’ population would access salivary rapid testing versus the conventional settings.

Increasing awareness of these alternative tests among individuals at risk and providers may be an appropriate strategy to increase the number of people who know their serological status and who could be linked to care and engaged in care!

The recent introduction of rapid oral HCV antibody test could completely change the HCV diagnosis approach by facilitating the possibility of testing millions of people worldwide (in particular in the developing countries).

For these reasons, we hope the oral-fluid based rapid HCV tests could become the ‘gold standard’ to facilitate the HCV screening access and become the standard of care and the basis for the national HCV testing algorithm in many countries with spread HCV epidemic, also in the dental care surgeries.

References
1. Lavanchy D. The global burden of hepatitis C. Liver Int. 2009; 29: 74–81.
2. Istituto Superiore di Sanita (ISS). Available at: www.iss.it.
3. Hoofnagle JH. Hepatitis C: the clinical spectrum of disease. Hepatology 1997; 26: 15S–20S.
4. Hutin Y, Kitler M, Dore G, Perz J, Armstrong G, Dusheiko G, et al. Global burden of disease (GBD) for hepatitis C. J Clin Pharmacol. 2004; 44: 20–29.
5. Ferreira-Gonzales A, Shiffman ML. Use of diagnostic testing for managing hepatitis C virus infection. Semin Liver Dis. 2004; 24: 9–18.
6. Tucker JD, Bien CH, Peeling RW. Point-of-care testing for sexually transmitted infections: recent advances and implications for disease control. Curr Opin Infect Dis. 2013; 26: 73–79.
7. Food and Drug Administration. Available at: http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfClia/detail
8. OraQuick HCV Rapid Antibody Test. Available from:
http://www.fda.gov/MedicalDevices/productsandMedicalProcedures/DeviceApprovalsandClearances/Recently-approved-Devices
9. Parisi MR, Soldini L, Di Perri G, Tiberi S, Lazzarin A, et al. Offer of rapid testing and alternative biological samples as practical tools to implement HIV screening programs. New Microbiol. 2009; 32(4): 391–396.
10. Parisi MR, Soldini L, Vidoni GM, Clemente F, Mabellini C, Belloni T, Nozza S, Brignolo L, Negri S, Rusconi S, Schlusnus K, Dorigatti F, Lazzarin A. Cross-sectional study of community serostatus to highlight undiagnosed HIV infections with oral fluid HIV-1/2 rapid test in non-conventional settings. New Microbiol. 2013; 36(2): 121–132.
11. Center for Disease Control and Prevention. Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. Available from: http://www.cdc.gov/hepatitis/hcv/Management.htm.

The authors
Maria Rita Parisi^*1 MSc, Laura Soldini² MD, Gianmarino Vidoni³ MD, Karin Schlusnus⁴ PhD, Fernanda Dorigatti² MD, Adriano Lazzarin¹ MD
¹Division of Immunology, Transplantation and Infectious Diseases, Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy
²Laboraf Diagnostic and Research OSR S.p.A., San Raffaele Scientific Institute, Milan, Italy
³Prevention Department, Reference Centre for HIV and STDs, Local Public Health Unit, Milan, Italy
⁴ANLAIDS Lombardia Onlus, Milan, Italy

*Corresponding author
E-mail: parisi.mariarita@hsr.it