Novel detection of parvovirus B19 DNA & IgM antibodies in patients with non-occlusive gangrene of stomach & bowel.
Gangrene of intestine due to non-occlusive bowel infarction (NOBI) is a rare and distinct clinical entity found by many surgeons globally and in India with an estimated incidence of 0.45 per cent of all emergency surgeries and 1 per cent of all laparotomies (1). Gangrene of stomach is still rarer and that too due to non-occlusive ischaemia is perhaps unknown. Ischaemic gangrene of the bowel is a dramatic clinical event, often posing great difficulty in the diagnosis at the time of admission but usually results in to a massive small bowel gangrene leading to high mortality (60-80%) (1,2). Besides rarity, its etiologies too remains to be known. Vascular endothelial cell can be directly infected by several viruses like dengue, herpes viruses, measles, mumps, HTLV-1, HIV, marburg, ebola and hantaan viruses and that theses viruses can cause vasculitis3. Vasculitis in turn can cause decreased gut perfusion leading to severance of blood supply to the bowel causing ischemia, infarction followed by gangrene of the bowel. In case of gangrene due to occlusive bowel infarction (OBI) arterial pulsations are not felt in mesenteric vessels at laparotomy and venous thrombus in mesenteric venules including superior mesenteric vein. These can be seen in (a) mechanical obstructions of the bowel occluding its blood supply such as in strangulated hernia, volvulus, adhesions, malignancies etc (4-8) or (b) thromboembolic disorders (9). In contrast, gangrene due to non-occlusive bowel infarction (NOBI) (10-12) arterial pulsations can be felt until terminal arcade of mesenteric vessels and there is no mechanical obstruction to blood flow nor any venous thrombosis in the mesenteric venules or superior mesenteric vein on laparotomy.
The etiology or etiological factors causing NOBI are unknown. Non-specific microbial infections (13) were incriminated in a few and no specific cause could be identified in most studies (14,15). Some authors have suggested an immunological basis for NOBI (15). In these reported cases small bowel is the affected organ. However, there are not many reports of idiopathic acute gastric necrosis in newborns except in one HIV positive case (14,16,17) but not of overt gangrene. We came across a case of ischemic non-occlusive gangrene of the proximal stomach in 2003 where no obvious cause or factors leading to the etiology could be found. Vascular endothelial cells also have receptor for human parvovirus B19 (B19) and which can be directly infected by viruses3 and have recently been reported to be associated with vasculitis. Probability of B19 induced gangrene was thought off and investigated. Surprisingly, the patient's serum tested positive for parvovirus B19 specific IgM antibodies as well as for B19 DNA by PCR. Therefore, a prospective study was taken up to investigate B19 and other possible etiological agents or factors which might cause vasculitis leading to bowel gangrene.
Material & Methods
Patients: Twelve cases of gangrene of stomach or bowel (8 males and 4 female; median age 40 yr) presented with slow onset of pain abdomen, malaise, body ache, low-grade fever and loose motions to Sanjay Gandhi Post Gradate Institute of Medical Sciences, Lucknow. Symptoms were more intense than usually encountered. Careful physical examination, detailed laboratory investigations including plain X-Ray of chest and abdomen, ultrasonography and contrast enhanced computerized tomography (CECT) of the abdomen were done and documented. Diagnosis of gangrene was established on laparotomy. Most of the patients presented as midnight emergencies hence were enrolled whenever convenient between 2003 to 2009. Moreover, since the place of study is a tertiary care super speciality institute only cases having difficulty in diagnosis were referred.
All the patients were operated surgically, studied clinically and investigated for probable viruses known to cause vasculitis with special reference to B19. Additionally, immuno-haematological markers and histopathologic examination of the resected bowel tissues were done to look for the pathologic features like granulomatous diseases, malignancy and to document gangrene.
Surgical case definitions: Laparotomy done in all the patients showed characteristic picture of bowel gangrene with lusterless bluish-violet bowel. The extent of gangrene and viable healthy portions of the bowel were delineated. Depending on the presence or absence of mesenteric pulsations and thrombus in the proximal mesenteric vessels and omentum, 8 cases had gangrene due to non-occlusive bowel infarction (NOBI) while four cases were grouped in to the occlusive bowel infarction (OBI) respectively (Table I). Patients were blinded to the laboratory.
Controls: For the presence or absence of B19 DNA (n=36) in resected bowel tissues or archieved biosy tissues comprising of 10 antral (suspected of Helicobacter pylori infection) and 17 colonic biopsies (ulcerative colitis) and 09 (celiac disease) were taken. While normal control sera for finding frequency of anti-B19 IgM antibodies comprised of 127 kidney donors having no history of recent B19 infection i.e. rash, anaemia or arthropathy.
Virological, immuno-haematological studies: At the time of first presentation of bowel gangrene blood samples were collected for all patients aseptically for B19 serology and IgM antibodies against viruses associated with vasculitis and for auto-antibodies. Anti-B19 IgM antibodies were estimated in the sera by in-house ELISA (after checker-board titrations) as described previously (18) as well as by commercial ELISA (IBL Hamberg, Germany) and by Western Blot (R-Biopharm, Germany). Tests were performed according to manufacturer's instructions. Anti-B19 IgG ELISA was standardized (in-house) similarly as for IgM ELISA and cut-off optical density (O.D.) were taken as equal to or more than mean + 2 SD of OD value obtained from analysing sera of 1000 voluntary blood donors (authors unpublished data). Additionally, IgM antibodies were tested against HTLV-I (General Biologicals, Taiwan), HIV-1+2 (Biorad, France; Biomeurix, Netherland; Trinity Biotech, Irland), CMV (Biomeurix, Netherland), HSV1+2 (Biorad, France), mumps virus (Novatech, Germany) and Mycobacterium tuberculosis (Anda TB, France) using ELISA kits. Further coagulation profiles, anti-nuclear and anti-smooth muscle antibodies were investigated.
Bacterial (BACTEC) and fungal cultures: Blood and resected bowel tissues from cases were cultured for aerobic and anaerobic bacteria by BACTEC and fungi on Sabaraud's biphasic media and identified as per standard procedures.
DNA extraction and PCR for parvovirus B19: DNA was extracted in sera from all 12 cases of gangrene of the bowel and in pooled sera (pool of 10 in each) from 127 kidney donors (normal control) by QIA amp Ultra Sens kit (Qiagen, Germany) and from resected bowel tissues of cases as well as controls using QIA amp Mini kit (tissue protocol). The PCR was performed in PCR Works Station (Clean spot, Coy 14500 Michigan, USA; WHO) using forward primer sequences (5'-3') B1- GGTTGATTATGTGTGGG-(nt 3187-3206) and reverse primer B2.--ACT GAAGTCAT GCT TGG-(nt 3558-3539) from VP1 and VP2 common region as described previously (18). Sterile distilled water was used as negative control while cloned and purified VP1 and VP2 portion of B19 DNA (from a case of erythema infectiosum) was used as a positive control (donated by Dr Y Matsunaga, NIID, Tokyo, Japan). Similarly a nested--PCR was done on sera as well as on resected and archieved bowel tissues using set of primers from VP1 unique regions19 of B19 namely, primer B3- TGTGTGTTGTGTGCA AC- (nt 2193-2209) and primer B4- CAAACTTCCTTGAAAATG- (nt3119-3235). One microliter of PCR products were then subjected to second round of amplificationusing internal primers B5CAA AAGCATGTGGAGTGAGG-(nt2229-2245) and B6-GTGCTG TCAGTAACC TG TAC- (nt 30653082). Measures to prevent contamination in PCR reactions as recommended were carefully observed. On agarose gel electrophoresis and ethidium bromide staining PCR amplicons after 35 cycles (Perkin Elmer, GeneAmp 9600,USA) were of 372 bp while in nested-PCR amplicons of 942 bp in the first round and 853 bp after second round of amplification respectively were regarded as B19 DNA positive. For confirmation of results extraction of DNA from sera and resected tissues, PCR and nested-PCR were all done twice. Few samples were subjected to PCR individually without positive DNA control to rule out contamination in PCR reactions and for re-confirmation.
Histopathological examination: All 12 resected bowel tissues were examined grossly and after haematoxylin and eosin/trichrome staining of microtome cutsections.
The physical characteristics of patients are shown in Table I. On the basis of clinical findings and CECT bowel gangrene was suspected that was confirmed on laparotomic findings. At surgery, (gross examination) in all the eight NOBI patients the mesentery was not gangrenous, arterial pulsations were present over the proximal arcades and there was no evidence of clots in the major veins. Among eight cases of NOBI (Table II) one had distal stomach gangrene while five cases had distal ileal gangrene and the rest two had massive small bowel gangrene with or without involvement of proximal colon. In contrast, in four cases thrombus were detected in superior mesenteric vein hence categorized as OBI. The affected bowel portions were grossly gangrenous (Table II) and with no features suggestive of gas gangrene which is a separate entity.
Depending on the level of contamination and impending septicemia, resection of gangrenous portion of bowel with exteriorisation of the proximal and distal end was done in all the cases of gangrene involving small bowel. After three months, restoration of continuity could be done only in four patients who survived. Three patients of stomach gangrene underwent either partial or total gastrectomies with differing anastomosis (Table II). After completion of the procedures, each of the specimen were cut open and examined thoroughly for mucosal changes, arterial and venous patency. The resected bowel tissues were then sent for histopathological examination which showed characteristic features suggestive of ischaemic gangrene. In cases of stomach gangrene mucosa was lost (Fig.1a), gastric glands were healthy denoting that no other pathology were observed in the mucosa (Fig.1b) blood vessels had mucous plug (Fig.1c) besides infiltration of inflammatory cells suggestive of vasculitis. Similarly in cases with gangrene of the intestines mucosa was denuded (Fig. 2a) without any evidence of granulomatous pathology or neoplasm. Attached mesentery showed evidence of tissue edema with acute inflammatory features. In the skin biopsy, in one patient (case no.5) done about a year prior to present condition was suggestive of necrotizing vasculitis (Fig. 2b). Haematologically, NOBI cases were anaemic and more importantly reticulocytopenic seen in five of seven NOBI cases tested had slightly raised total leukocyte count except in one (Table I). Biochemical findings showed that all had normal blood sugar, liver function, renal function tests and coagulation profile. Immunologically, anti-nuclear and anti-smooth muscle and antibodies were negative. Microbiologically, blood culture showed no growth when cultured for aerobic, anaerobic bacteria and fungi in all the cases except case no 5, which had Pseudomonas aeruginosa. Peritoneal fluids in perforated cases and resected tissue in most cases grew E. coli and Enterococcus faecalis (Table I). The cases, were scattered in time and place.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Virologically B19 DNA was detected in serum samples in 6 of the 8 cases of non-occlusive bowel gangrene by PCR with persistence of B19 DNA for one month in the first case (Table III; Fig. 3a). Nested-PCR detected B19 genome in all 8 cases (Fig. 3b) (data not shown for last two patients of NOBI (nos 7 & 8). Additionally, in three of them (no. 2, 5 and 6) showed B19 DNA in resected tissues by nestedPCR only (data not shown) and their sera had antiB19 IgM and IgG antibodies by ELISA (by in-house and IBL Hamberg ELISA kit) and had anti-B19 IgM antibodies in sera by Western-blot (data not shown). In the remaining NOBI cases three had anti-B19 IgG only and the remaining two were negative for both. In contrast in four cases of occlusive bowel gangrene (OBI) had no evidence of active B19 infection and they lacked both B19 DNA and IgM but two cases had IgG antibody (case no. 9 & 11). In controls no B19 DNA was detected in any of the 127 normal control pooled sera nor in the 36 bowel biopsies tissues while anti-B19 IgM and IgG antibodies were detected in two (1.6 %) and 56 (44%) of 127 normal kidney donors respectively. All NOBI cases lacked IgM antibodies against pathogens which may be associated with vasculitis such as HTLV-I, HIV-1+2, CMV, HSV-1+2, mumps viruses and M. tuberculosis by ELISA.
[FIGURE 3 OMITTED]
Clinically, only one patient who was diabetic had reappearance of gangrene at the stomal site of the small bowel (no. 4) and within six hours of surgery required further resection. He developed postoperative ventilator associated respiratory infections and succumbed to these after prolonged period of illness. Two patients (nos 3 and 8) with massive small bowel gangrene died due to short bowel syndrome; while patients no. 6 died due to sepsis and pneumonia (Table II). All but one of them were from rural areas (except no. 6) while none had any addiction except patients number four who was a tobacco chewer. Medical risk factors that could explain a possibility of embolic pathology in non-occlusive gangrene were not observed with the exception of a solitary case. In contrast, both patients of occlusive gangrene (OBI) had these risk factors (Table I) but otherwise had normal laboratory parameters. There were no surgical factors like adhesions or mechanical obstructions occluding blood supply which could result in NOBI. On follow up, four patients each of surviving NOBI and OBI were without any related problem.
Bowel gangrene primarily due to vascular afflictions are very rarely seen may be due to acute mesenteric ischaemia (AMI), a vascular emergency where diagnosis is often difficult and overlooked and delay in diagnosis results in grave outcome. AMI primarily consists of four pathologic process like arterial embolism, arterial thrombosis, non-occlusive mesenteric ischemia (results in NOBI) and venus thrombosis (MVT). They have similar clinical presentation but potentially fatal pathologic endpoint, the bowel gangrene (10). Non-occlusive mesenteric ischemia described in sixth centuries is defined as diffuse intestinal ischemia that often results in intestinal gangrene in the presence of a patent arterial trunk and that results in decreased blood flow to the bowel causing cellular injury. In the present study, we encountered 12 cases of bowel gangrene of which 8 were due to NOBI confirmed by laparotomy and histopathological features suggesting ischaemic gangrene. The NOBI patients had no obvious cause or risk factors such as reduced cardiac output, renal disease, diabetes mellitus or drugs but had extensive necrotic bowel gangrene requiring massive surgical resection (Table II). Four of the 8 NOBI patients died (50%), the high mortality due to their late presentation (Table II).
Microbial etiology of NOBI remains obscure and so is the site of occlusion. Association of B19 infection with vasculitis and thrombotic microangiopathy (3) besides localized microvascular thrombi (20) formation further supports our observations. Characteristic finding of vasculitis in biopsy material of involved bowel was highly suggestive in our cases and we did find fibrin thrombus in the lumen of blood vessels supplying the stomach with infiltration of inflammatory cells. Evidences favouring B19 infection in eight NOBI cases were presence of reticulocytopenia (in 5 of 7 cases tested), anemia (severe in one cases) and more importantly finding of free DNA in serum. The incidence of B19 in sera has been estimated in voluntary blood donors by PCR and it ranged from 1: 3,000 to 1: 30,000 and by nested-PCR in 1:167 (0.6%) donors (21) hence we did not find B19 DNA in control sera. However, three cases of NOBI had B19 DNA in tissues also with anti-B19 IgM and IgG antibodies in sera by ELISA (tested thrice) and Western blot. B19 DNA was not found in the archieved control tissues. Histopathologic features were highly suggestive of ischemic necrosis with no evidence of granuloma or other pathology. Further, one patient (no. 5) had necrotizing vasculitis of the skin (a year ago), known to be associated with B19 virus (Fig. 1c). NOBI patients had neither IgM antibodies to other putative agents causing vasculitis (20) nor had any thrombotic states or auto-antibodies. B19 endothelial cell parasitism by B19 virus has been reported to accelerates endothelial cell apoptosis, neoantigenicity and secondary antibody formation (22) as seen in a 38 yr male who developed intestinal and cutaneous infarctions. B19 viremia and localization of B19 RNA transcripts to vascular endothelium were uncovered and a diagnosis of catastrophic endothelial cell injury was attributed to B19 infection. The patient showed significant improvement with IVIG treatment. Further owing to the ability of B19 to infect endothelial cells it can cause severe multi-organ endothelial cell injury (22). Antibody defect documented in chronic B19 infection (23) has been suggested to be due to a qualitative
abnormality in antibody binding to the virus. Finkel et al (24) also described three patients who had atypical serological responses to the B19 infection, although none had any immunodeficiency disorder. Treatment with corticosteroids and cyclophosphamide did not control vasculitis in contrast IVIG therapy led to rapid improvement of the systemic vascultis manifestations, clearing of the chronic B19 infection, and long-term remission. These observations suggest an etiological relation between B19 infection and systemic necrotising vasculitis in these patients and indicate a potentially curative role for IVIG in such disorders. Inability of patients to develop an anti-B19 IgM antibody response or the viral infection preceded the clinical illness by a sufficient interval so as to allow the acute IgM antibody response to vane or the disease results from reactivation of prior infection or reinfection has been reported (25). This explains lack of IgM antibodies in our five cases of NOBI. However, there was no history of rash, arthralgia or arthropathy in any case. B19 infections in our cases are likely to be acute or acute on a chronic infection because of the presence of DNAemia which persisted for one month in first case who had also suffered from necrotizing vasculitis of the skin. Since anemia was mild, no blood transfusion was given which rules out the possibility of transfusion transmitted B19 infection.
Parvovirus B19 has wide clinical spectrum (26,27) which may still be incomplete (24). In India, B19 is endemic and its seroprevalence among 1000 adult voluntary blood donors was found to be 39.9 per cent (Kishore J, Srivatava M, Choudhury N, unpublished data). Infections due to B19 are occurring in India and we reported the first global case of B19 induced amegakaryocytic thrombocytopenia (28) and again first global case of myositis in a child with erythma infectiosum (29) besides B19 causing pure red cell aplasia (18) and juvenile rheumatoid arthritis (30).
Direct evidence of organ involvement was seen as sub-acute massive necrotizing myocarditis by canine parvovirus type-2 infection in a mongrel puppy (31). Gastro-intestinal involvement like enteritis by animal parvoviruses has been described including death of a wild wolf (31,32). In another study in cats and dogs infected with animal parvoviruses had intestinal lesions characteristic of parvovirus infection with parvoviral DNA in all 10 dogs and in nine of the 10 cats; besides detection of antigen of the virus in seven of 10 canine and eight of 10 feline intestines (33). Interestingly, beneficial effects occurred with interferon-omega treatment in canine parvovirus enteritis (34). Moreover, PCR amplifications of genomic DNA from two different regions of a virus further confirm its identity and also indicates presence of B19 genome. Viral load in sera appears to be low in two patients as they were negative by PCR but positive by nested-PCR. The lower limit of B19 DNA detection in sera by PCR was determined previously to be 2.4 virions x [10.sup.3]/ml (35) hence remaining four patients of NOBI had higher viral load. Rigorous measures were taken to avoid amplicon and sample to sample contamination and the entire procedure of DNA extraction, PCR and nested PCR was repeated with sterile distilled water as negative templates. Owing to lack of an animal model for B19, Koch's postulates could not be fulfilled. However, Koch-Henle postulates incorporating molecular evidences were met in all NOBI cases (36). Preventive or therapeutic use of I.V.I.G. to reduce viraemia and prevent further progress or recurrence in NOBI cases remains unknown in want of guidelines and unproven etiology. At present, early diagnosis of gangrene and surgical resection is the mainstay of management and even massive resections were compatible with good quality of life (37) while therapy with prostaglandin E1 may increase survival (38). Hence B19 infection may be considered in cases of non-occlusive ischemic gangrene of bowel and studied more closely by serological and molecular tools in larger prospective studies to unveil its true role and clinical significance.
This study was supported in part by a grant of Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India.
Received March 16, 2009
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Reprint requests: Dr Janak Kishore, Professor, Division of Virology, Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow 226 014, UP, India e-mail: email@example.com
Janak Kishore, Nihar R. Dash *, Rajan Saxena * & Narender Krishnani **
Departments of Microbiology, * Gastro-Intestinal Surgery & ** Pathology, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow, India
Table I. Physical characteristics and laboratory profiles in patients with gangrene of the bowel Case Age in Medical risk Hb TLC No. yr/sex factors Duration g % x [10.sup.3] 1 16/M Nil 5 days 11.2 12 2 40/M Nil 7 days 10.8 14 3 42/M Testicular 3 days 6.6 7 Tumor 4 59/M DM 4 days 10.2 11.8 5 40/F RA 7 days 12.4 12 6 55/F Nil 5 days 9.6 13.6 7 12/F Nil 4 days 12 12 8 20/M Nil 6 days 10.8 15.1 9 78/M DM+OMI 2 days 10 12.2 10 22/F Corro.str. 2 days 9.2 16.6 Esophagus 11 38/M Doudenal 5 11.2 9.6 Ulcer 12 50/M Carcinoma stomach 7 9.6 10.3 Case Reticulocytes Tissue No. % organism 1 ND E. coli Enterococcus faecalis 2 0 E. coli Entero faecium 3 0 Entero faecalis C. frenudii 4 0 E. coli Entero faecalis 5 0 E. coli Entero faecalis 6 0 E. coli Entero faecalis 7 1 E. coli 8 1 Anaerobic cocci Entero faecalis 9 4 E. coli E. faecalis 10 2 E. coli Enterobactor spp 11 ND Pseudomonas aeruginosa, Staph. aureus 12 ND E. coli 1 to 8, non occlusive bowel gangrene cases; 9-12, occlusive bowel gangrene cases DM, diabetes mellitus; OMI, old myocardial Infarction; Duration, duration of symptom to surgery; Hb, haemoglobin; TLC, total leucocyte count; Retic, reticulocyte count; ND, not done; Corro. str. eso, corrosive stricture esophagus Table II. Operative findings and follow-up Patient Organ No. involved Surgical procedure 1 Proximal stomach Proximal gastrectomy 2 Terminal ileum one foot Two stage procedure 3 Near total small bowel Resection and exteriorisation 4 Terminal ileum right Two stage procedure colon with perforation 5 Terminal ileum one foot Two stage procedure 6 Jejunum to mid transverse Resection and colon exteriorosation 7 Distal ileum Two stage procedure 8 Entire small bowel Resection-exteriorisation 9 Right colon Two stage procedure 10 Stomach antrum Antrectomy with feeding jejunostomy 11 Doudenal performation Segmental ileal resection, small bowel fistula Ileco-coecal anastomosis 12 Stomach body & antrum Total gastrectomy Roux Y- esophageal jejunostomy Patient Post-operative Follow-up No. complications (up to 1 yr) 1 Anastomotic Leak, repaired Uneventful 2 Nil Uneventful 3 Short gut Syndrome Death 4 Ventilator Associated Death pneumonia 5 nil Uneventful 6 Continued sepsis Death 7 Wound infection abscess Uneventful 8 Short bowel syndrome Died 9 Nil Uneventful 10 Retrogastric abscess Gastro-jejunostomy 11 Hypoxia, needed ventilator Haemodynamic instability Uneventful 12 Multiple site peritoneal Uneventful collections Two stage procedure- Resection with exteriorisation followed by restoration of continuity after 3 months Table III. Parvovirus B19 antibodies and DNA in 12 cases of gangrene of bowel Parvovirus B19 antibodies S. ELISA Western-Blot No. IgM IgG IgM 1. -ve +ve -ve 2. +ve +ve +ve 3. -ve -ve -ve 4. -ve -ve -ve 5. +ve +ve +ve 6. +ve +ve +ve 7. -ve +ve -ve 8. -ve +ve -ve 9. -ve +ve -ve 10. -ve -ve -ve 11. -ve +ve -ve 12. -ve -ve -ve Parvovirus B19 DNA Resected bowel Serum Tissues S. No. PCR Nested-PCR Nested-PCR 1. +ve * +ve -ve 2. +ve +ve +ve 3. +ve +ve -ve 4. +ve +ve -ve 5. -ve +ve +ve 6. -ve +ve +ve 7. +ve +ve -ve 8. +ve +ve -ve 9. -ve -ve -ve 10. -ve -ve -ve 11. -ve -ve -ve 12. -ve -ve -ve * repeat sera collected after one month
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|Author:||Kishore, Janak; Dash, Nihar R.; Saxena, Rajan; Krishnani, Narender|
|Publication:||Indian Journal of Medical Research|
|Date:||May 1, 2010|
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