Dear Editor,

I would like to respond to the commentary written by Dr. R. Balfour Sartor entitled, "Does Mycobacterium avium subspecies paratuberculosis cause Crohn's disease?” The answer to this rhetorical question is yes, and I would like to explain why we now have sufficient information to conclude that Mycobacterium avium paratuberculosis (Map) is a major cause of Crohn's disease.

First, I will address the cons Dr. Sartor includes in table 1 of his commentary which lists arguments for and against a Map causation of Crohn's disease.

1) Differences in clinical and pathological responses in Johne's and Crohn's diseases. This argument is not logical. Map causes a different clinical and pathologic state in sheep and cattle.[1] A related mycobacterium, mycobacterium tuberculosis, causes disease with a differing clinical and pathologic presentation in mice, rabbits, monkeys and humans.[2] Yet this variety of interspecies disease presentations is not an argument against a common pathogen for these diseases.

2) Lack of epidemiological support of transmissible infection. Studies have shown that the incidence and prevalence of Crohn's disease has steadily increased in the past 50 years.[3-5] This increase mirrors the corresponding increasing prevalence of Map induced Johne's disease in dairy cattle. Most observers accept that this alarming increase is not likely genetic but rather environmental in origin.

3) No evidence of transmission to humans in contact with animals infected with Map. Map is not efficiently transmitted by proximity to infected animals. It is most efficiently transmitted by consumption of contaminated food or water.

4) Genotypes of Crohn’s disease and bovine Map isolates not similar. The evidence in this area is conflicting. However, at least two studies show that the isolates are similar.[6-7]

5) Variability in detection of Map by PCR and serological testing. There has certainly been variability in the reported rates of detection of Map in Crohn's disease. These differences are largely due to significant methodological differences among studies. For example, some negative studies have used formalin fixed paraffin embedded tissue.[8-9] There is evidence in the literature that formalin fixation causes

fragmentation of DNA with resultant poor amplification of PCR primers.10 Ryan and others showed that when studying the same formalin fixed paraffin embedded samples of tissue, use of PCR systems which amplified small sequences of the IS900 gene detected Map much more reliably than PCR systems which amplified large sequences of this gene.[11]

6) No evidence of mycobacterial cell wall by histochemical staining. I agree with this point, but it is not evidence against a Map causation of the disease. Using in situ hybridization, Sechi and others demonstrated the DNA of the Map organism in human tissue but could not demonstrate a cell wall. The organism most likely exists in a cell wall deficient or spheroplast form in human tissue.[12]

7) No worsening of Crohn's disease with immunosuppressive agents or HIV infection. This argument does not hold for a related mycobacterial infection and therefore cannot be used as evidence against a Map causation in Crohn’s disease. Leprosy is universally understood to be caused by another slow growing mycobacterium and it does not worsen with steroid therapy. In fact, steroids are used therapeutically in some cases of leprosy.[13-14]

8) No documented cell mediated responses to Map in patients with Crohn's disease. The studies which have been performed in this area could not rule out the possibility of anergy.[15]

9) No therapeutic response to traditional antimycobacterial antibiotics. This argument is nonsensical. Most of the studies reported to date have used antibiotics which are not effective against Map in vitro.[16-17] Why then would we expect the antibiotics which were used in these studies to be effective in vivo?

Many of us are exposed to low levels of viable Mycobacterium avium paratuberculosis on a regular basis. Map survives pasteurization [18] and three studies have shown that it may be cultured from retail supplies of pasteurized milk.[19-21] Thus Map joins a long list of pathogens which may be transmitted through milk and the diseases which have been transmitted by milk include tuberculosis, bovine tuberculosis, salmonellosis, brucellosis, Q fever, listeriosis, yersiniosis and toxigenic e.coli.

The most challenging evidence for skeptics that this bacterium is pathogenic is the fact that many patients with Crohn's disease are bacteremic with Map.[22] In medicine we are taught that persistent septicemia is a pathologic state that causes disease. The leaky bowel theory has been proposed as the explanation for the growth of Map in the blood of Crohn's disease. If this explanation is tenable, why are e.coli and candida which are more prevalent in the fecal stream not cultured from the blood of these patients? These patients can clear e.coli and candida but they cannot clear Map, i.e., Map is pathogenic in these patients. Colonization of diseased bowel is also an unlikely explanation considering the fact that there are patients who are bacteremic with Map who have very little gross endoscopic evidence of Crohn’s disease (personal communication from Dr. Saleh Naser). The bacteria were cultured from the Buffy coat in Naser's study and Map thus joins other pathogenic mycobacteria which are intracellular within macrophages. The others include mycobacterium tuberculosis and mycobacterium leprae.[23] A blood- borne mycobacterial infection nicely explains the extraintestinal manifestations of this disease[24] and the presence of granulomas (which are not satisfactorily explained by other theories).[25] The skeptics are no longer refuting the evidence that Map is in the tissue and/or blood of Crohn's patients. However, now they posit some unlikely role for Map rather than understanding that it is a pathogen in a susceptible host.

I agree with Dr. Sartor that clearance of Map infection with resolution of the patient's Crohn's disease would be excellent evidence that Map is causative of the disease. However, to obtain this evidence under current conditions will be a nearly impossible task. Unless patients carefully avoid dairy products, drink only Map free water and consume Map free beef, they are at continual risk of contracting the infection. Until Map is removed from the food chain and water supplies, attempts to clear the bacterium with antibiotics will be difficult and in many cases futile. Future antibiotic trials should include the recently developed diarylquinoline compound which has been found to have activity against most mycobacteria.[26]

I agree with Dr. Sartor that additional Map related studies are necessary to further elucidate the role of this bacterium in Crohn’s disease. Such studies should include a Map feeding and inoculation study in primates to see whether we can induce Crohn's disease in primates who are infected with the organism. Additional studies could include in vitro studies of the differences in various subjects’ macrophages or neutrophils to kill Map.

I disagree with Dr. Sartor that additional studies will resolve this classic scientific revolution. The only study which could resolve this controversy would be a Map feeding study in a human population. Of course, this study could never be performed.

Because this controversy can not be resolved in short order, we must use the available information to carry out proper public health measures. We need government mandated programs for the testing and culling of Map infected animals from the beef and dairy cattle herds, improved methods of pasteurization, and we should eliminate Map from drinking water (Map is resistant to chlorination). This task is daunting but a similar task has been carried out effectively in the control and elimination of tuberculosis from dairy herds. The cost will be high but will be offset by the economic benefits for the agricultural industry which annually faces large losses due to Johne's disease. In the United States alone, losses to the cattle industry have been estimated at $1.5 billion per year.[27] More importantly, we will save potential future Map infected Crohn's patients from contracting what we now know is a preventable disease.

References

1. Chiodini, R.J., Van Kruiningen, H.J., and Merkal, R.S. Ruminant paratuberculosis (Johne’s disease): the current status and future prospects. Cornell Vet. 1984; 74: 218-62.

2. Capuano, S.V., Croix, D.A., Pawar, S., et al. Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human m. tuberculosis infection. Infect Immun 2003; 71: 5831-44.

3. Loftus, E.V., Silverstein, M.D., Sandborn, W.J., et al. Crohn’s disease in Olmsted County, Minnesota, 1940-1993: incidence, prevalence and survival. Gastroenterology 1998; 114: 1161—8.

4. Tsironi, E., Feakins, R.M., Roberts, C.S.J., et al. Incidence of inflammatory bowel disease is rising and abdominal tuberculosis is falling in Bangladeshis in East London, United Kingdom. American Journal of Gastroenterology 2004; 99: 1749-55.

5. Desai, H.G. and Gupte, P.A. Increasing incidence of Crohn’s disease in India: is it related to improved sanitation? Indian Journal of Gastroenterology 2005;24: 23-4.

6. Overduin, P., Schouls, L., Roholl, P. et al. Use of multilocus variable-number tandem-repeat analysis for typing mycobacterium avium subsp. Paratuberculosis. J Clin Microbiol 2004; 42: 5022-28.

7. Ghadiali, A.H., Strother, M., Naser S.A., et al. Mycobacterium avium subsp. Paratuberculosis strains isolated from Crohn’s disease patients and animal species exhibit similar polymorphic locus patterns. J Clinical Microbiol 2004; 42: 5345-5348.

8. Fujita, H., Yoshinobu, E., Ishige, I., et al. Quantitative analysis of bacterial DNA from mycobacteria spp., bacteroides vulgatus, and escheria coli in tissue samples from patients with inflammatory bowel disease. J Gastroenterol 2002; 37: 509-16.

9. Baksh, F.K., Finkelstein, S.D., Ariyanayagam-Baksh, S.M., et al. Mod Pathol 2004; 17, 1289-94.

10. Goelz,S.E., Hamilton S.R. and Vogelstein, B. Purification of DNA from a formalin fixed and paraffin embedded human tissue. Biochem Biophys Res Commun 1985; 130: 118-26.

11. Ryan, P., Bennett, M.W., Aarons, S., et al. PCR detection of mycobacterium paratuberculosis in Crohn’s disease granulomas isolated by laser capture microdissection. Gut 2002; 51: 665-70.

12. Sechi, L.A., Manuela, M., Francesco, T. et al. Identification of mycobacterium avium subsp. Paratuberculosis in biopsy specimens from patients with Crohn’s disease identified by in situ hybridization. J Clin Microbiol 2001; 39: 4514-17.

13. Marlowe, S.N., Hawksworth, R.A., Butlin, C.R., et al. Clinical outcomes in a randomized controlled study comparing azathioprine and prednisolone versus prednisolone alone in the treatment of severe leprosy type 1 reactions in Nepal. Trans R Soc Trop Med Hyg 2004; 98: 602-9.

14. Richardus, J.H., Withigton, S.G., Anderson, A.M. et al. Adverse events of standardized regimens of corticosteroids for prophylaxis and treatment of nerve function impairment in leprosy: results from the ‘TRIPOD’ trials. Leprosy Review 2003; 74: 319-27.

15. Ibbotson, J.P., Lowes, J.R., Chahal, H. et al. Mucosal cell- mediated immunity to mycobacterial, enterobacterial and other microbial antigens in inflammatory bowel disease. Clin Exp Immunol 1992; 87: 224- 30.

16. Hermon-Taylor, J. The causation of Crohn’s disease and treatment with antimicrobial drugs. It J. Gastroenterol Hepatol 1998; 30: 607-10.

17. Rastogi, Nalin, Goh, K.S. and Labrousse, V. Activity of clarithromycin compared with those of other drugs against mycobacterium paratuberculosis and further enhancement of its extracellular and intracellular activities by ethambutol. Antimicrobl Agents Chemother 1992; 36: 2843-46.

18. Grant, I.R., Ball, H.J. and Rowe, M.T. Effect of higher pasteurization temperatures, and longer holding times at 72 C, on the inactivation of mycobacterium paratuberculosis in milk. Lett Applied Microbiol 1999; 28: 461-65.

19. Grant, I.R., Ball, H.J. and Rowe, M.T. Incidence of mycobacterium paratuberculosis in bulk raw and commercially pasteurized cow’s milk from approved dairy processing establishments in the United Kingdom. Appl Environ Microbiol 2002; 68: 2428-35.

20. Ellingson, J.L.E., Anderson, J.L., Koziczkowski, J.J., et al. Detection of viable mycobacterium avium subsp. Paratuberculosis in retail pasteurized whole milk by two culture methods and PCR. J Food Prot 2005; 68: 966-72.

21. Ayele, W.Y., Svastova, P., Roubal, P. et al. Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow’s milk in the Czech Republic. Appl Environ Microbiol 2005; 71: 1210-14.

22. Naser S.A., Ghobrial, G., Romero, C., et al. Culture of mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn’s disease. Lancet 2004; 364: 1039-44.

23. Harrison’s Principles of Internal Medicine, 15th Edition, McGraw- Hill 2001; 1024 and1035.

24. Greenstein, A.J. et al. The extraintestinal manifestations of Crohn’s disease and ulcerative colitis: a study of 700 patients. Medicine 1976; 55:401.

25. Robbins Pathologic Basis of Disease, 6th Edition, Saunders 1999; 816.

26. Andries, K., Verhasselt, P., Guillemont, J., et al. A diarylquinoline drug active on the ATP synthase of mycobacterium tuberculosis. Science 2005; 307: 223-7.

27. Stabel, J.R., Johne’s disease: a hidden threat. J Dairy Sci 1998; 81: 283-88.

Dear Editor,

The recent paper by Thomas and colleagues[1] in the May 2005 issue of Gut provides further support for an involvement of increased colonic deoxycholic acid formation and absorption in cholesterol gallstone pathogenesis. This paper and others in the series[2-4] from Professor R. Hermon Dowling’s laboratory at Guy’s Hospital, London, was ably placed in perspective with the accompanying comprehensive commentary[5] by Dr. Alan F. Hofmann. He summarized critically the decade-long body of work from the Dowling group and others on the connection between slow intestinal propulsion and the biliary tract in octreotide-treated acromegaly. The salient findings of Dowling’s work may have relevance perhaps to other subsets of gallstone-prone individuals.

Acromegalic patients treated with octreotide display dysfunctional gallbladder motility and sluggish intestinal transit rates. Surprisingly, neither Thomas and colleagues[1] nor Hofmann[5] considered that slower small in addition to slower large intestinal transit times may have important pro-lithogenic consequences per se, in that small intestinal hypomotility may augment biliary cholesterol levels by promoting increased cholesterol absorption from the small intestine. Octreotide does not appear to have any appreciable effect on plasma lipoprotein catabolism and “reverse” cholesterol transport or hepatic cholesterol biosynthesis,[6] hence it is unlikely to promote biliary cholesterol hypersecretion from lipoprotein cholesterol or de novo biosynthesis.

We propose therefore, that heightened cholesterol absorption from the small intestine could provide an important source of the sterol for biliary cholesterol hypersecretion. Because octreotide abolishes cholecystokinin (CCK) release from the small intestine,[7,8] attacks of clinical, presumtively cholelithogenic cholecystitis and cholangitis, well documented in patients with use of this long-acting somatostatin analogue, have customarily been attributed solely to gallbladder hypomotility.[9] However, the loss of post-prandinal CCK release via octreotide,[9] is likely to have an additional pathophysiologic effect in humans. Not only is the contractile response of the gallbladder abolished, but we showed recently that small intestinal propulsion is also slowed significantly in CCK-1 (previously known as CCK- A) receptor knockout mice.[10] Hence by inference, a physiological function of diurnal CCK levels in the healthy mouse and presumably human is to accelerate small intestinal motility, in addition to contracting the gallbladder and relaxing Oddi’s sphincter.

Any degree of disruption of the physiologic motors of the small intestine extends the residence time of cholesterol-rich mixed bile salt micelles in the intestinal lumen. This allows more time for monomeric diffusion and cholesterol absorption by small intestinal enterocytes from the cholesterol-rich mixed micellar reservoir within the chyme. As a result, any deficit in CCK release will induce significant increases in fractional cholesterol absorption from the upper small intestine as we have shown in the CCK-1 receptor knockout mouse compared to wild-type mice.[10] Not surprisingly, there are reports in the literature that a dysfunctional CCK-1 receptor may cause cholesterol gallstones in humans.[11,12] Because the CCK-1 receptor plays such a crucial role in the physiological regulation of gallbladder and small intestinal motility, it is likely that in addition to gallbladder hypomotility, gallstone patients with a dysfunctional CCK-1 receptor also display increased absorption of dietary cholesterol from the dysmotile small intestine.

In his commentary[5] Dr. Hofmann made the point that in several large clinical studies,[13-15] investigators failed to find a strong correlation between biliary deoxycholate levels and cholesterol saturation of bile. This fact alone should prompt an alternative or additional explanation for cholesterol gallstones by the occurence of slow transit constipation in humans. Perhaps the putative increase in cholesterol absorption from the gut may be the “missing link” in the augmented gallstone risk from diminished intestinal motility from any cause. As evidenced by human and animal studies, deoxycholate does not promote cholesterol absorption from the small intestine as efficiently as does its conjugated cholate precursor.[16,17] Moreover, from a physical chemical perspective, higher levels of deoxycholate conjugates in bile may actually counteract augmented cholesterol absorption for reasons of small intestinal hypomotility.

The points we make here are not offered to diminish the putative role of deoxycholic acid in cholesterol gallstone pathogenesis, for which there is abundant evidence from the work of the Dowling laboratory.[1-4] Nonetheless, we argue here that increased fractional absorption of cholesterol from a hypomotile small intestine may result in delivery of more cholesterol to the liver in chylomicron remnants, and hence may play a crucial role in hepatic cholesterol hypersecretion and biliary lithogenicity.[18] Consequently, in the setting of a hypomotile gallbladder, lithogenic bile will have a greatly prolonged residence time, facilitating phase separation of cholesterol crystals and their agglomerate into gallstones.

Two decades ago, Ponz de Leon and colleagues[19] showed that metaclopramide acceleration of small intestinal transit in humans significantly decreased intestinal cholesterol absorption, however they failed to show, in the opposite experiment, that slowing of small intestinal transit with atropine augmented cholesterol absorption. Clearly, the small intestinal dysmotility and cholesterol absorption issues raised here need to be proven in humans but this unique group of patients affords an easily testable cohort.[10] A salutary result would afford an intellectually satisfying explanation for the profound lithogenicity of bile not otherwise accounted for in this group of patients.

Sincerely yours,

Martin C. Carey, M.D., D.Sc.
Department of Medicine
Brigham & Women’s Hospital
Harvard Medical School
Boston, MA 02115, USA

David Q.-H. Wang, M.D., Ph.D.
Department of Medicine
Beth Israel Deaconess Medical Center
Harvard Medical School
Boston, MA 02215, USA

References

1. Thomas LA, Veysey MJ, Murphy GM, et al. Octreotide induced prolongation of colonic transit increases faecal anaerobic bacteria, bile acid metabolising enzymes, and serum deoxycholic acid in patients with acromegaly. Gut 2005;54:630-5.

2. Hussaini SH, Murphy GM, Kennedy C, et al. The role of bile composition and physical chemistry in the pathogenesis of octreotide-associated gallbladder stones. Gastroenterology 1994;107:1503-13.

3. Veysey MJ, Thomas LA, Mallet AI, et al. Prolonged large bowel transit increases serum deoxycholic acid: a risk factor for octreotide induced gallstones. Gut 1999;44:675-81.

4. Thomas LA, Veysey MJ, Bathgate T, et al. Mechanism for the transit- induced increase in colonic deoxycholic acid formation in cholesterol cholelithiasis. Gastroenterology 2000;119:806-15.

5. Hofmann AF. Increased deoxycholic acid absorption and gall stones in acromegalic patients treated with octreotide: more evidence for a connection between slow transit constipation and gall stones. Gut 2005;54:575-8.

6. Olivecrona H, Ericsson S, Angelin B. Growth hormone treatment does not alter biliary lipid metabolism in healthy adult men. J Clin Endocrinol Metab 1995;80:1113-7.

7. Lembcke B, Creutzfeldt W, Schleser S, et al. Effect of the somatostatin analogue sandostatin (SMS 201-995) on gastrointestinal, pancreatic and biliary function and hormone release in normal men. Digestion 1987;36:108- 24.

8. Milenov K, Rakovska A, Kalfin R. Effects of cholecystokinine on the gallbladder motility: interaction with somatostatin and vasoactive intestinal peptide. Acta Physiol Pharmacol Bulg 1995;21:67-76.

9. Fisher RS, Rock E, Levin G, Malmud L. Effects of somatostatin on gallbladder emptying. Gastroenterology 1987;92:885-90.

10. Wang DQ-H, Schmitz F, Kopin AS, Carey MC. Targeted disruption of the murine cholecystokinin-1 receptor promotes intestinal cholesterol absorption and susceptibility to cholesterol cholelithiasis. J Clin Invest 2004;114:521-8.

11. Miller LJ, Holicky EL, Ulrich CD, Wieben ED. Abnormal processing of the human cholecystokinin receptor gene in association with gallstones and obesity. Gastroenterology 1995;109:1375-80.

12. Miyasaka K, Takata Y, Funakoshi A. Association of cholecystokinin A receptor gene polymorphism with cholelithiasis and the molecular mechanisms of this polymorphism. J Gastroenterol 2002;37(Suppl 14):102-6.

13. Gustafsson U, Sahlin S, Einarsson C. Biliary lipid composition in patients with cholesterol and pigment gallstones and gallstone-free subjects: deoxycholic acid does not contribute to formation of cholesterol gallstones. Eur J Clin Invest 2000;30:1099-106.

14. LaRusso NF, Szczepanik PA, Hofmann AF. Effect of deoxycholic acid ingestion on bile acid metabolism and biliary lipid secretion in normal subjects. Gastroenterology 1977;72:132-40.

15. Jüngst D, Muller I, Kullak-Ublick GA, et al. Deoxycholic acid is not related to lithogenic factors in gallbladder bile. J Lab Clin Med 1999;133:370-7.

16. Sama C, LaRusso NF. Effect of deoxycholic, chenodeoxycholic, and cholic acids on intestinal absorption of cholesterol in humans. Mayo Clin Proc 1982;57:44-50.

17. Wang DQ-H, Tazuma S, Cohen DE, Carey MC. Feeding natural hydrophilic bile acids inhibits intestinal cholesterol absorption: studies in the gallstone-susceptible mouse. Am J Physiol 2003;285:G494-502.

18. Wang DQ-H, Zhang L, Wang HH. High cholesterol absorption efficiency and rapid biliary secretion of chylomicron remnant cholesterol enhance cholelithogenesis in gallstone-susceptible mice. Biochim Biophys Acta 2005;1733:90-9.

19. Ponz de Leon M, Iori R, Barbolini G, et al. Influence of small-bowel transit time on dietary cholesterol absorption in human beings. N Engl J Med 1982;307:102-3.

Correspondence to: Dr. Martin C. Carey
Brigham & Women’s Hospital
Department of Medicine
Gastroenterology Division, Thorn 1430
Boston, MA 02115, USA
Email: mccarey@rics.bwh.harvard.edu

Conflict of Interest: None declared

Dear Editor,

We read with great interest the article by Bartolotti et al. (Gut2005;54:852-857) regarding the shift in the predominance of hepatitis C genotype in 373 paediatric patients investigated between 1990 and 2002. The distribution of genotypes showed a major prevalence of HCV genotype 1, recorded in about 60% of children. The genotype 2, 3 and 4 were observed in 17, 15 and 5% of patients respectively. However, the study after 1990 described a declining prevalence of genotypes 1b and 2 and by an increasing proportion of genotype 3 and 4.

We have conducted an analogous genotype determination in 1095 adults patients analyzed between 2000 to 2004. Subjects were been screening previously for anti-HCV antibodies for different reason, such as exposure to major risk factors, evidence of increased ALT, pregnancy and surgical procedures. Consequently, this population group was characterized by a rather homogenous representation of all age groups and included individuals independently of source of infection and liver disease. All patients were infected with HCV, as demonstrated by HCV-RNA detection obtained by a Polymerase Chain Reaction (PCR) assay, COBAS System Amplicor HCV monitor version 2.0 (Roche Diagnostics Systems Inc. Branchburg, NJ, USA). HCV genotyping/subtyping was done with a line-probe assay (INNO-LiPA HCV II, Innogenetics, Ghent, Belgium). The Simmonds classification was used.[1]

The study population included 555 males and 540 females, in the age range of 15-102 years. 44 (4.0%) out of 1095 patients were younger than 30 years old; 179 (16.3% ) aged between 31 and 40; 189 (17.3%) between 41 and 50; 165 (15.1%) between 51 and 60; 304 (27.8%) between 61 and 70 and 190 (17.4%) between 71 and 80. Finally, 24 (2.3%) samples were obtained by patients older than 80 years. All the 1095 HCV-RNA positive samples included in the study were genotyped, and 1063 (97.1%) were also subtyped. The distribution of genotypes showed a major prevalence of HCV genotype 1 (49,3%). Three hundred seventy-one (33.9%) were infected with type 2 and 136 (12.4%) were suffering from genotype 3. HCV-4 was found in 52 (4.7%) persons. Type 5 could be detected only in one (0.1%) patient. The remaining 9 (0.8%) individuals were infected with more genotypes. The distribution of subtypes showed a major prevalence (39.7%) of subtype 1b. Three hundred thirty-one (30.2%) individuals were infected with subtypes 2a/2c and 122 (11.1%) were suffering from subtype 3a. Seventy-two (6.6%) samples were positive for serotype 1a. The serotype 2a and 4c/4d, were determinate only in 32 (2.9%) 36 (3.3%) patients, respectively. The subtypes 3b and 1a/1b were unusually recorded, (12 and 8 subjects respectively). Exceptional results appeared to be the infections with other subtypes and the co-infections (1-2 patients).

The data of subtype distribution according to age group demonstrates a notable variation in the profiles of the subtypes (Table). Subtype 1a was frequently detectable in young patients, was rarely found in old subjects and tended to disappear in the last age groups (p <0.0001). Subtype 1b appears to be more prevalent in individuals aged ≤30 years compared to patients 31-40 and 41-50 years old ( p 0.013). However, subjects in the age group older than 50 (near 50%) years had most frequent infections with subtypes 1b compared with patients 31-40 and 41-50 years old (p <0.0001). The profile of co-infection 2a/2c showed an evident and progressive increase of the prevalence with the increment of age (p <0.0001). Subtype 3a was described more frequently in the second and third age group compared with the first one (p 0.007). However, prevalence of this subtype declined according to increment of age and became irrelevant in the last age group(p <0.0001). An analogous profile was described for co-infection 4c/4d (p <0.0001).

Our data was in accordance with the predominance of genotype 1, and in particular of subtype 1b, observed previously in Italy and in most countries of Europe and of the Word. With respect to the genotype 2, almost completely represented by subtype 2a/2c, and for genotype 3 our results showed data analogous to those reported from other Italian and European studies. In our study genotype 4 was found in 4.1% of patients. HCV genotype 4 appears to be prevalent in North Africa and Middle East .[2-3] However, recent papers showed an high prevalence (4.5%) of HCV genotype 4 in Southern and Central Italy too.[4]

Moreover, we have evaluated the shift in the predominance of HCV subtypes in age groups. Recently, this tendency for a shift in the prevalence of HCV subtypes was noted in Italian and international study.[5-6] A recent model of HCV spread, based on the coalescent theory, which showed that the circulation of subtype 1a continues to increase whereas the extent of infection with subtypes 1b tends to decrease. This is in line with our results on subtype 1a and probably with the epidemic behaviour of subtype 3a and co-infection 4c/4d.On the other hand, the high prevalence of subtype 1b that was observed in our patients younger than 30 years appears to suggest that the increase in rate of this variant has not yet achieved a plateau in Italy. Analogous data were obtained in an other Italian study conducted recently in North-East Italy.[6]

In conclusion, our study has shown a shift of the epidemiological picture of HCV infection, with the diffusion of subtypes 1a, 3a and 4c/4d. These epidemiological changes reported in our patients reflected the shift in pattern of HCV genotypes described by Bartolotti (Gut2005;54:852-857) in children. These subtypes, prevalent in young adults, may represent the source of future infections transmitted mainly by injection drug use, perinatal, and sexual transmission, and it is expected that these subtypes will be distributed evenly in all cohorts.

References

1. 1 Simmonds P, McOmish F, Yap PL et al. Classification of hepatitis C virus into six major genotypes and series of subtypes by phylogenetic analysis of the NS-5 region. J Gen Virol 1993; 74: 2391-9.

2. Zein NN. Clinical significance of hepatitis C virus genotypes. Clin Microb Rew 2000; 13: 223-35.

3. Maertens G, Stuyver L. Genotypes and genetic variation of hepatitis C virus. In: Harrison TJ, Zuckerman AJ, editors. The Molecular Medicine of Viral Hepatitis. Wiley and Sons Ltd. Chichester, England, 1997: 183-233.

4. Matera G, Lamberti A, Quirino A et al. Changes in the prevalence of hepatitis C virus (HCV) genotype 4 in Calabria, Southern Italy. Diagn Microbiol Infect Dis 2002; 42: 169-73.

5. Ross RS, Viazov S, Renzing-Koholer K, Roggendorf M.. Changes in the epidemiology of hepatitis C infection in Germany: shift in the predominance of hepatitis C subtypes. J Med Virol 2000; 60: 122-5.

6. Dal Molin G, Ansaldi F, Biagi C et al. Changing molecular Epidemiology of hepatitis C virus infection in Northeast Italy. J Med Virol 2002; 68: 352-6.

Table: Prevalence of most frequent HCV-subtype in the different age groups

Dear Editor,

We read with interest the case report by Buchel et al., published on the July issue.[1] The authors report a patient with portal hypertension, nodular regenerative hyperplasia and portal thrombosis that presented an avascular hip necrosis after a liver transplantation (OLT). The authors suggest that the whole clinical picture might stem from hyperhomocysteinaemia (high Hcy) and 677C->T heterozigosity for the common gene of methylen-tetrahydrofolate reductase (MTHFR).

Although the history is suggestive of this hypothesis, high Hcy might also be a secondary phenomenon. It is not clear whether high Hcy was demonstrated in a sample obtained before – as suggested in Table – or after OLT – as suggested in the text. In both cases Hcy might be elevated as a consequence of either liver disease or drug treatment.

Liver disease per se raises Hcy, due to the multiple metabolic problems generated by a failing liver, including low folate levels, altered transsulphuration/transmethylation pathway and decreased renal function. High Hcy is present in 50% of liver disease patients, and values as high as 30 µmol/L are not uncommon in end-stage disease [2], independently of MTHFR polymorphism.

After OLT a specific effect of tacrolimus on Hcy metabolism and concentration is well documented. Both calcineurin inhibitors (cyclosporine and tacrolimus) are known to interfere with the folate- dependent remethylation of Hcy, thus raising plasma levels to values in the pathological range. The problem is well documented in patients following with renal [3], cardiac [4] and liver transplant [2]. In a recent analysis of 230 patients submitted to OLT we found 26 cases with fasting Hcy >= 30 µmol/L in the late post-transplant phase, independently of MTHFR polymorphism.[5]

Also the relationship between high Hcy and MTHFR polymorphism deserves a comment. The MTHFR gene independently and unfavourably influences homocysteine metabolism, but high Hcy levels are mainly observed in subjects with low folate levels [6], indicating a low phenotypic expression. Therefore, the presence of the genetic variant, mainly in its heterozygous form, might be an occasional phenomenon without any clinical significance. In conclusion, although the clinical history of the patients raises the suggested pathogenic role of Hcy and genetic predisposition, no clues can be made on the “egg-chicken” sequence, particularly in a patient with positive markers for hepatitis B infection.

For sure, an adequate folate intake or pharmacological supplementation may overcome the genetic predisposition to high Hcy, but may also decrease Hcy independently of any gene defect, and remains a suitable therapeutic option to prevent additional vascular problems.

References

1. Buchel O, Roskams T, Van Damme B, et al. Nodular regenerative hyperplasia, portal vein thrombosis, and avascular hip necrosis due to hyperhomocysteinaemia. Gut 2005;54:1021-3.

2. Bosy-Westphal A, Ruschmeyer M, Czech N, et al. Determinants of hyperhomocysteinemia in patients with chronic liver disease and after orthotopic liver transplantation. Am J Clin Nutr 2003;77:1269-77.

3. Fernandez-Miranda C, Gomez P, Diaz-Rubio P, et al. Plasma homocysteine levels in renal transplanted patients on cyclosporine or tacrolimus therapy: effect of treatment with folic acid. Clin Transplant 2000;14:110-4.

4. Cole DE, Ross HJ, Evrovski J, et al. Correlation between total homocysteine and cyclosporine concentrations in cardiac transplant recipients. Clin Chem 1998;44:2307-12.

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