Clinical significance of cytomegalovirus infection in patients with inflammatory bowel disease

Abstract

Cytomegalovirus (CMV) infection is common in humans. The virus then enters a “latency phase” and can reactivate to different stimuli such as immunosuppression. The clinical significance of CMV infection in inflammatory bowel disease is different in Crohn’s disease (CD) and ulcerative colitis (UC). CMV does not interfere in the clinical course of CD. However, CMV reactivation is frequent in severe or steroid-resistant UC. It is not known whether the virus exacerbates the disease or simply appears as a bystander of a severe disease. Different methods are used to diagnose CMV colitis. Diagnosis is classically based on histopathological identification of viral-infected cells or CMV antigens in biopsied tissues using haematoxylin-eosin or immunohistochemistry, other tests on blood or tissue samples are currently being investigated. Polymerase chain reaction performed in colonic mucosa has a high sensitivity and a positive result could be associated with a worse prognosis disease; further studies are needed to determine the most appropriate strategy with positive CMV-DNA in colonic mucosa. Specific endoscopic features have not been described in active UC and CMV infection. CMV colitis is usually treated with ganciclovir for several weeks, there are different opinions about whether or not to stop immunosuppressive therapy. Other antiviral drugs may be used. Multicenter controlled studies would needed to determine which subgroup of UC patients would benefit from early antiviral treatment.

Key Words: Cytomegalovirus; Inflammatory bowel disease; Crohn’s disease; Ulcerative colitis; Hematoxylin and eosin; Immunohistochemical; Polymerase chain reaction; Ganciclovir; Infectious colitis

NATURAL HISTORY OF CYTOMEGALOVIRUS INFECTION

Cytomegalovirus (CMV) is a member of the Herpesviridae family which contains a double-stranded DNA. It often causes primary infection in humans, and later persists lifelong in a latent stage. In different situations of immunosuppression [birth, human immunodeficiency virus (HIV) infection, use of immunosuppressant therapy or chemotherapy] the virus can reactivate[1,2] and cause disease, but this may also occur in immunocompetent hosts.

In CMV infection, CMV antigens or antibodies can be detected in blood, whereas CMV disease symptoms generally appear in a target organ[3]. The prevalence of CMV infection is high, ranging from 30%-100%[4-7], depending on age and race of population tested[8-10].

Primary CMV infection in immunocompetent individuals is usually asymptomatic, and rarely manifests itself as a mononucleosis-type disease similar to that caused by Epstein-Barr virus, consisting of fever, fatigue and cervical lymphadenopathy. Subsequently, the virus enters a latency phase in endothelial cells, macrophages or granulocyte stem cells[11-13]. Virus can reactivate in the healthy adult, this new activation being typically asymptomatic; however, this delicate balance can be disrupted in patients whose immune response is compromised, which may lead to development of symptoms in different organs, such as CMV colitis[13]. The use of highly active antiretroviral therapy in patients with HIV has decreased significantly CMV disease in these patients[14]; on the other hand, induced immunodeficiency in situations such as solid organ transplant or inflammatory bowel disease (IBD) sometimes allows CMV disease to develop, with significant morbidity and even mortality[15].

Antibody response to CMV infection reflects this relapsing-remitting pattern. In primary infection, an early increase of specific IgM antibodies occurs, which can be detected in the first week of infection[16], showing a sensitivity of 100% and a specificity of 99%[17]. Over the next 3-6 mo immunoglobulin M (IgM) antibodies fall to undetectable levels, although some may persist for up to 12-24 mo. Persistence of IgM antibodies could be related to concomitant immunosuppression[18]. Reactivation can cause again a rising IgM titers[17,19]. Immunoglobulin G (IgG) antibody production occurs within a few weeks of IgM increase[16]. When patients develop specific CMV IgG antibodies, they are considered “seropositives”. In the reactivation of CMV, IgG antibody levels do not change, so that this situation can only be differentiated by the detection of an increase in IgM antibody titers that does not always occur. Thus, serology has a limited value in the diagnosis of reactivation[10]. Seronegative patients are not at risk for CMV disease, unless primoinfection occurs.

Gastrointestinal tract is a common site of CMV disease, with preference the esophagus and the colonic mucosa (especially at the right colon). The pouch mucosa behaves in a similar way to colon mucosa[20-22]. Clinical presentation of CMV colitis begins with watery diarrhoea that may be progress to rectal bleeding, abdominal pain, fatigue and fever. Other presentations such as fever of unknown origin, megacolon or digestive hemorrhage, are also possible.

It is unknown whether the virus remains in colon after a primary infection or if it disappears spontaneously[23]. A recent study[24] demonstrated persistence of colonic CMV [using polymerase chain reaction (PCR) method], following an acute ulcerative colitis (UC) flare-up despite remission of symptoms. The virus may disappear or not from the colon after an acute UC flare-up, but if it persists is detected only by PCR DNA, without displaying inflammatory changes in colonic mucosa or CMV antigens in infected colonic cells.

There have been reports of CMV colitis in immunocompetent hosts[25,26], especially in elderly patients with comorbidities in whom the disease followed a severe course with high mortality rate. Also, rare cases of CMV colitis preceding IBD have been reported[27].

RELATIONSHIP BETWEEN CMV AND IBD

The association between CMV and IBD was described long ago. The first case report dates from 1961, when Powell et al[28] described a patient with UC and cytomegalic inclusion disease. Since then, questions remain about the role of CMV in these patients: does CMV reactivation exacerbate the disease in patients with established IBD or is reactivation a consequence of IBD activity and its treatment with CMV acting as an innocent bystander[29-31]? Interpretation of existing results is limited because most studies are small and retrospective, different diagnostic methods of CMV detection are available and even different classifications are used for the severity of concomitant IBD[3].

CMV colitis occurs in “seropositive” patients with IBD. CMV does not appear to interfere with the clinical evolution of Crohn’s disease (CD), and its involvement in UC is debated, especially in severe flare-ups.

DIAGNOSTIC TECHNIQUES

Diagnostic techniques for CMV infection

Serology: Serology is useful to determine previous viral exposure and identify patients at risk, as only seropositive patients (positive IgG antibodies in blood) may develop CMV disease[30,31].

Antigenemia assay: It detects viral protein pp65 produced in peripheral blood polymorphonuclear leukocytes. It is a simple and rapid technique, with a sensitivity of 60%-100% and a specificity of 83%-100%[32,33]. However, it does not differentiate between latent infection and active disease[23], no association exists with virus reactivation in the intestinal mucosa[31,34], and false negatives can occur in neutropenic patients[35].

PCR DNA amplification assay in blood: This test is supplanting antigenemia. Sensitivity ranges 65%-100% and specificity ranges 40%-92%[32,36,37]. This diagnostic method cannot differentiate between latency and activity states, so it is necessary to determine a cut-off above which active infection is diagnosed in patients with IBD[30,35], as well as in solid organ transplantation, where a viremia > 1000 copies/100 000 leukocytes indicates symptomatic CMV infection[38]. This method can be used both to detect disease and to monitor treatment response; a slow or absent decline in DNA levels after treatment could be an early indicator of drug resistance[39], and positive result after therapy indicates continuing treatment[40]. Most studies in patients with IBD have reported a correlation between identification of CMV by PCR in blood and detection colonic CMV by haematoxylin and eosin (HE) or immunohistochemistry (IHC)[34,41-43].

PCR assay in stool: A qualitative and quantitative PCR assay for CMV DNA has been performed on human faecal specimens from immunocompromised patients[44,45].

Diagnostic techniques for CMV colitis

Histological diagnosis is considered the “gold standard” for diagnosing CMV disease in the gastrointestinal tract[46-51]. Several methods are available.

Histology by HE: Typically reveals cytomegalic cells that are 2 or 4 fold larger than normal cells, containing basophilic intranuclear inclusions in eccentric location surrounded by a clear halo, giving it an “owl’s eye” appearence. Cells show a thickened nuclear membrane and smaller granular intracytoplasmic inclusions. Colonic biopsies should be taken from inflamed mucosa near or within the ulcer. This method has very high specificity (92%-100%), but its main problem is a poor sensitivity (10%-87%), making the diagnosis requires many samples and a trained pathologist[46,47]. Anecdotally cytomegalic cells have been described in colon biopsies from normal mucosa in healthy individuals[48,49].

IHC in colon biopsies: It involves the identification of CMV antigens in infected cells. It has a higher sensitivity than HE (78%-93%)[50,51]. Other techniques, however, do not rely on histology.

PCR DNA amplification assay in colon mucosa: PCR DNA amplification assay in colon mucosa has the greatest accuracy for virus detection[29,52,53] and may be used as a qualitative or quantitative method. PCR DNA levels in the colon are not related to viremia levels measured in blood[31,43,54,55]. Some studies detected prevalences greater than 30% in IBD patients[56], although the significance of a positive result in absence of histological signs of CMV infection is unclear. Very few studies have shown a correlation between histology (HE/IHC) and PCR results[31,43]. This suggests that detection of low DNA levels could determine latent infection and requires a cut-off level of viremia to distinguish infection from disease[30]. In other words, a positive result in colon does not necessarily reflect the involvement of CMV in UC flare-ups. A recent study from France suggests a cut-off of > 250 copies/mg for CMV disease[54] with a sensitivity of 100% and a specificity of 66%. To avoid false positives, and awaiting further studies, this determination should be performed only in patients with active UC refractory to conventional treatment.

Viral culture: Viral culture was previously regarded as the gold standard in CMV detection. Culture has a sensitivity of 45%-78% and a very high specificity (89%-100%). The virus is placed in a fibroblast tissue culture and diagnosis is made once the virus causes cytopathic changes in fibroblasts. The problem is that the result takes days to weeks[10], so this method is not used in clinical practice[57].

Typical endoscopic findings of CMV colitis: Typical endoscopic findings of CMV colitis are microerosions, deep ulcers and pseudotumoral lesions[58]. Most studies in patients with IBD, specifically in active UC, have not found specific endoscopic features[43,54,59,60]. Anecdotal studies describe some characteristic endoscopic (large, irregular, punched-out or longitudinal ulcers) with virus[61]. Discrepancy can be explained by the different criteria used to define CMV infection or disease.

In conclusion, different methods of detection of CMV have different sensitivities and specificities. In the setting of a severe UC flare-up refractory to conventional treatment, colonic IHC or PCR in blood should be performed, PCR presents a good correlation with the results of HE and IHC[30]. Serology and antigenemia only are useful as negative predictors for CMV infection, since they do not correlate with actual CMV colitis. The role of PCR positivity in colon remains to be determined, it is not known how to interpret its positivity in the absence of histological changes. However, recent studies imply positivity of this method in the disease prognosis[54]. Current European guidelines[62] recommend the use of tissue PCR or IHC to detect CMV in patients with UC resistant to immunomodulators, whereas older American guidelines[63] recommend performing sigmoidoscopic biopsy and viral culture in refractory cases of UC.

CMV PREVALENCE IN IBD

Overall prevalence of CMV is unknown in IBD patients. Most of studies have been carried out using a selected patient group and using different diagnostic methods, so that the available data include a wide range of prevalences.

CMV prevalence in CD

Seropositivity in CD patients is as high as in other populations (70%)[64,65], however CMV disease is rare in CD, making the virus an unlikely etiological factor in the novo development of IBD[27,64,66,67].

In most surveys, IHC did not detect CMV in CD patients, and PCR in tissue or stool samples found a very small frequency (< 5% of patients)[27,66-69], except in one study that used a highly sensitive PCR (detection < 10 copies of DNA) which detected CMV in 66% of individuals with CD and in 29% of controls, showing no association between CMV DNA and disease activity, suggesting the authors that small quantities of viral DNA are not clinically relevant[29].

Recently some theories have been published that explain these findings based on small studies[70], so results should be interpreted with caution. Tumor necrosis factor-α (TNFα) would be significantly associated with CMV infection or reactivation in IBD. However interferon-γ (IFNγ), which is produced from CD4 + T cells[71], could suppress CMV reactivation. CD is considered a Th1-type inflammatory process with high expression of IFNγ, which does no help the virus reactivation and may explain the different prevalence of CMV disease in UC and CD[70].

CMV prevalence in UC

Prevalence of CMV infection is about 70%[31], a similar percentage to that of the general population.

Patients with inactive or mild-moderate UC did not show an increased risk of CMV colitis, using HE and IHC[27,31,41] or IHC in colectomy patients undergoing surgery for dysplasia or cancer[72].

The most extensive literature is on severe and/or steroid-refractory colitis. These two terms are used interchangeably in studies and are not defined clearly, so the results are difficult to interpret[30].

Severe colitis: The combination of serological tests and rectal biopsies found a CMV disease prevalence of around 20%[42,73]. Risk factors that have been identified are female gender, older age, pancolonic disease with active inflammation at histology and azathioprine therapy[60,72,73]. According only to antigenemia, prevalence increased to 34%[60], while using HE or IHC in colonic mucosa prevalence decreased to 3%[74].

Severe steroid-resistant colitis: In retrospective studies the prevalence according to HE was 0.5%[75]. The prevalence of CMV increased by combining HE and IHC with antigenemia (20%-40%)[31,41,72,76,77]. The prevalence using blood PCR was 60%[43], whereas PCR results in colon were lower (38%)[78]. There is a poor correlation between peripheral and colonic viral load, this can be explained by theory proposed by Criscuoli et al[24,42] about a specific genotype, which possibly has a particular colonic tropism and pathogenic character, whilst other genotypes are not pathogenic.

Urgent colectomy for colitis: A higher prevalence of CMV is expected in these patients because the greater severity of their disease, but the prevalence using HE or IHC ranged between 11.5% and 27%[77,79-82], which is similar to the previous groups.

Experimental studies have identified three factors that influence the reactivation of CMV infection in active colitis: (1) increased cell proliferation in inflamed tissue with ulcers that attract CMV; (2) inherent impaired natural killer cell activity presented by patients with IBD[83,84]; and (3) use of immunosuppressive drugs[31,42,69]. The third factor is controversial[73-75,80,85], since use of steroids may be either a risk factor or a surrogate marker of severity disease[41]. In vitro data suggest that steroids and cyclosporine could support the replication of CMV[76,86,87]. Infliximab treatment has not been associated with increased risk of CMV reactivation in IBD patients[66]. Moreover, TNFα has been identified as an activator of CMV, and thus infliximab (IFX) could promote viral latency, by lowering the levels of this cytokine[88,89]. This suggests that IFX could be used in patients with severe UC flare and CMV reactivation. However, some cases of tacrolimus-refractory UC that were treated with IFX did not have a favourable outcome[90].

CMV reaches the mucosa by way of monocytes and then colonize the colonic cells, acquiring particular affinity for the inflammatory sites, probably due to the presence of pro-inflammatory cytokines such as IFNγ and TNFα produced by macrophages and T-cells in active UC[91-94]. CMV avoids immune recognition of macrophages and uses immune specific functions to reactivate and spread[95]. Recent studies suggested CMV can appear only in inflamed tissue and is not found in healthy tissue[54].

Hommes et al[94] proposed the following sequence to explain the pathophysiology of CMV disease in patients with active UC: (1) initiation phase, mucosal inflammatory response induces expression of cytokines and chemokines which activates latently infected cells and the migration of monocytes and dendritic cells into the inflamed mucosa; (2) reactivation phase, in which infected monocytes differentiate into tissue macrophages and dendritic cells; and (3) consolidation phase, during which the virus causes an active replication predominantly in endothelial cells that likely exacerbates inflammation. The virus in the first and second phase has not led to histological damage and can only be detected by PCR-DNA. It would be appropriate to detect CMV in these phases, so the optimal treatment would be administered at an early stage before complicating the disease. They do not consider CMV as an innocent bystander but rather involved in resistance to immunosuppressive treatment.

In the same line, other authors suggest that the positivity of CMV DNA in the colonic mucosa in patients with refractory UC indicates uncontrolled intestinal inflammation, suggesting a change in immunosuppressive therapy[96].

Coincidental detection of primary CMV infection at the first appearance of IBD has been reported before[66,97], primary CMV infection in IBD patients without immunosupression[98,99], and even disseminated CMV infection in CD[100].

PROGNOSIS OF CMV COLITIS IN IBD

As discussed above, it is unclear whether CMV superinfection leads to a more aggressive course of pre-existing inflammatory disease[29,30].

Referring only to UC, case-reports and retrospective series[29,42,55,68,69] suggested that CMV may trigger a steroid-refractory flare-up and worsen disease prognosis[76,77,80,85,101], associated increased risk of toxic megacolon[81] and surgical intervention[31,77,82,102,103]. This hypothesis is supported by two prospective studies[41,54]. The second, a recent French study, relates positivity of CMV (PCR in colon > 250 copies/mg) to treatment resistance (steroids and other three line of treatment), demonstrating that early initiation of antiviral therapy in these cases delays treatment resistance and improves prognosis[54].

However, other studies[3,27,34,43,72] that analyzed prognosis in patients with severe UC refractory to conventional treatments did not find increased disease severity nor increased rate of colectomy in CMV positive patients.

These conclusions are limited by differences in quality of existing studies.

TREATMENT OF CMV COLITIS IN IBD

It is difficult to draw conclusions about the role of antiviral therapy based on the available evidence[23], which is of varying quality.

Most authors recommend the use of antivirals[30,31,41,43,75] in steroid-refractory UC flare-up and CMV positive patients. Treatment can significantly decrease the mortality rate and need for surgery. However, some studies show that treatment of CMV does not alter disease course[80], and has even been shown the transience of positivity of serological tests (antigenemia, PCR) in patients with severe UC, but which as discussed earlier has little correlation with PCR determination in colon[34,94].

As CMV reactivation impact in clinical outcome is not yet elucidated, a rational approach would be to determine the CMV serology (IgG) when prescribing steroids in UC[31]. If steroid-refractoriness develops, rectal biopsies should be conducted only in seropositive patients, who are the ones who are at risk of developing CMV disease[31]. A detection of CMV (HE, IHC or PCR) in the inflamed mucosa should imply that standard therapies alone will not control intestinal inflammation, and make mandatory to consider alternative therapies[43], including CMV treatment[30]. If detection methods are not readily available, empiric therapy of IgG positive individuals with steroid-refractory disease is probably justified.

An original study recommended only antiviral treatment in severe UC with large ulcers (> 5 mm) and positive PCR in colon, all patients with positive PCR and no large ulcers in endoscopy responded to conventional therapy[104].

CMV colitis is typically treated with ganciclovir, although other antivirals have been used as foscarnet, valganciclovir and cidofovir[105-107]. Ganciclovir has a poor oral bioavailability so initially it has to be administered intravenously[105,106]. The recommended dosage is 5 mg/kg twice daily for at least 3 wk[107]. After some days of initial treatment the patient can switched to oral ganciclovir (1 g three times daily), although its value is uncertain. Treatment benefits must outweigh the risks associated with the medications used, as this drug can trigger significant adverse effects such as bone marrow suppression, particularly neutropenia[105]. Other side effects include headaches, somnolence, psychosis, elevated transaminases, fever and rash[105]. Foscarnet can be used when ganciclovir is contraindicated (90 mg/kg intravenously twice daily for 3-6 wk)[107]. Its main adverse effect is nephrotoxicity.

Response rate to antiviral therapy is 72% (range 50% to 83%)[31,41,43,75], but these data should be interpreted with caution because “response” was not uniformly defined in these studies and patients were treated concomitantly with immunosuppressants[30], although some authors decreased the dose of immunosuppressive drug[75]. Other authors consider to increase immunosuppressive therapies (other than corticosteroids) if CMV-DNA in colonic mucosa is detected, because a positive finding of CMV-DNA in inflamed colonic mucosa of patients with refractory UC suggests uncontrolled intestinal inflammation[96].

Antiviral treatment should be considerate in disseminated disease[108] which has very poor prognosis.

ECCO guides[62] and American College of Gastroenterology[63] recommend treatment when CMV is detected in colon tissue. Immunosuppressive therapy is stopped only in case of severe systemic CMV reactivation[62].

A multicenter randomized controlled trial with a large number of patients, using homogeneous diagnostic methods, including endoscopic evaluation, would be required to determine in which subgroup of patients early antiviral treatment would be useful[23,96].