BK polyoma viral infection in renal allograft recipients.

BACKGROUND: BK polyoma viral nephropathy (BKVAN) has emerged as a significant cause of renal allograft loss. The literature on BK viral infection from India is scarce. The study was therefore undertaken to evaluate impact of BK polyoma viral (BKV) infection on renal allograft recipients in Indian scenario from a service renal transplantation centre. METHODS: Renal allograft recipients who underwent graft biopsy formed the part of this descriptive cross-sectional study group. The clinicopathological profile of the patients was analysed. The diagnostic modalities employed were histopathology, immunohistochemistry using antibody for Simian virus 40 large T antigen along with real time quantification of the BK viral DNA load in the urine and the serum. RESULTS: One hundred forty seven renal allograft recipients were evaluated. 73.47 percent (108/147) patients presented with graft dysfunction and rest were protocol biopsies. There were 53 cases of rejection related diagnosis, 8 cases of graft pyelonephritis, 64 cases showed normal histology and rest exhibited miscellaneous causes. Nineteen percent (28/147) cases were positive for BKV DNA (viruria 26/147, 17.6% and viraemia 8/147, 5.44%. 3.4 percent (5/147) exhibited histological and immunohistochemical evidence of BKVAN. Nuclear enlargement, smudging and intranuclear inclusions along with plasma cell rich interstitial nephritis were important features observed on histopathology. Concomitant acute rejection was seen in 4/5 cases of BKVAN. All cases of BKVAN exhibited viraemia (> 2500 copies/mL), though cut-off values could not be defined statistically due to small sample size. Positive statistical correlation was observed between use of anti-thymocyte globulin (induction therapy and/or treatment of steroid resistant rejection, Pearson ×(2) value 6.9, P=0.008) and rejection episodes (Pearson ×(2) value 9.8, P = 0.007) with BKV infection. CONCLUSION: BK polyoma nephropathy should be added to the list of differential diagnosis considered for a renal allograft dysfunction. Renal biopsy remains the gold standard for diagnosis supplemented by non-invasive molecular techniques for screening and monitoring of BKV infection.

Human immunodeficiency virus type 1 gp 41 mutations in proviral DNA among antiretroviral treatment-naive individuals from India.

The HIV-1 gp41 has been identified as an important target for the immune response, for the development of antiviral and vaccine strategies, and for epidemiologic studies. This study describes the HIV-1 env gp41 region mutations, associated with enfuvirtide (ENF) resistance, in proviral DNA from PBMCs in antiretroviral treatment-naive individuals from Pune, India. Twenty-one antiretroviral drug-naive chronically HIV-1-infected individuals were enrolled. The study sequences belonged to subtype C (n = 17), subtype A1 (n = 2), and CRF_AE (n = 2). In subtype B-infected individuals, the various HR1 region substitutions in env gp41 that have been associated with ENF resistance include A30V, L33S/T/V, L34M, G36D/E/S/V, I37T/K/V, V38A/M/E/G, Q39R, Q40H, N42T/D, N43D/K/S, L44M, L45M, R46M, L54M, and Q56K/R as well as N126K and S138A in the HR2 region. The study sequences did not reveal any ENF resistance-associated mutations at env gp41 amino acid positions: 36 to 45. The presence of L54M and Q56K in combination is associated with 5-fold reduced sensitivity to inhibition by ENF. The mutation L54M was seen in seven subtype C and two CRF_AE study sequences. Q56K was observed in a subtype A1 sequence. All the study sequences harbored N42S, a natural polymorphism associated with increased susceptibility to ENF. Of the mutations V38A and N140I, known to provide immunologic gain, the latter was observed in four subtype C sequences. This is the first study from India highlighting the presence of certain mutations in Indian subtype C env gp41, which may play a role in the evolution of subtype-specific variations in the resistance to ENF and associated immune response.

Hormone Receptors, Her-2/Neu and Chromosomal Aberrations in Breast Cancer.

BACKGROUND: There is a great deal of disparity in the incidence of breast cancer in rural and urban India on one hand and between India and Western population on the other. METHODS: We analysed steroid receptor status in cases of breast cancer in a small sample of patients in armed forces. Infiltrating duct carcinomas of breast recorded histologically in mastectomy specimens in last two years were accessioned in the present study with reference to patient and tumour characteristics. RESULT: In contrast to the higher rates reported in western literature, only 33 % of the tumours expressed estrogen receptors (ER) and progesterone receptors (PR), of which 24% were ER positive and 30% PR positive. Negative steroid receptor status did not correlate with presence or absence of metastatic nodes, however it was predominant amongst the high grade infiltrating duct carcinomas in this study. Necrosis and lymphovascular invasion demonstrated an inverse relationship with the ER/ PR reactivity. 70% of the node positive cases expressed Her -2/ Neu, reflecting a higher immunoreactivity in this subset of patients. Aneusomy for chromosomes 1, 11 and 17 was common in node positive cases. CONCLUSION: Evaluation of chromosomal aberrations by Fluorescent In Situ Hybridization (FISH) technique correlates well with traditional histological parameters.

Spectrum of Haemoglobinopathies in a Tertiary Care Hospital of Armed Forces.

BACKGROUND: Thalassaemia and other structural haemoglobinopathies are the major genetic disorders prevalent in certain parts of the world including India. This study presents the pattern of haemoglobinopathies amongst the referred patients of anaemia in a two-year period. METHODS: A total of 1032 patients were studied during a two-year period for anaemia investigation. Haematological indices, sickling test and haemoglobin electrophoresis with quantification of the bands was done in all cases. RESULT: Out of 1032 cases, 774 (75%) were normal and 258 (25%) cases had abnormal haemoglobin pattern. Of the 258 abnormal cases, 136 (53%) were males and 122 (47%) were females. Of all cases of anaemia 370 (36%) were microcytic hypochromic, 237 (23%) macrocytic, 151 (15%) were dimorphic and the rest (26%) had normocytic normochromic picture. 82% of microcytic hypochromic anaemias had reduced serum iron and elevated total iron binding capacity (TIBC), whereas 85% had decreased serum ferritin levels. Spectrum of haemoglobinopathies prevalent were ß-Thalassemia trait (17%), followed by sickle cell trait (2.3%). Other haemoglobinopathies in descending order of frequency were sickle cell disease (1.7%), Hb D trait (1%), Hb E trait (0.8%), sickle cell - ß thalassemia, Hb E disease, E - ß thalassemia (0.6% each) and thalassemia major (0.4%). CONCLUSION: This study provides a comprehensive database on the spectrum of haemoglobinopathies in the Armed Forces. It is suggested that detection of HbA2 should be carried out in all the high-risk groups with anaemia.

Renal Transplantation - An Experience of 500 Patients.

BACKGROUND: Renal transplantation is the treatment modality of choice for patients with end stage kidney failure. We present our experience of graft and patient survival of initial 500 renal transplants performed between May 1991 and July 2006, at Army Hospital (R&R). MATERIAL AND METHODS: All patients received triple drug immunosuppression with cyclosporine/tacrolimus, azathioprine/ mycophenolate mofetil and steroids. Patients in high risk group received induction therapy with IL-2 receptor blockers/anti-thymocyte globulin. RESULTS: Majority of the recipients (79%) were males, whereas majority of the donors (59.4%) were females. In the donor profile, 385 (77%) transplants were live related, 108 (21.6 %) were spousal and 7 (1.4%) were cadaveric transplants. Mean age of the donors and recipients was 42.11 ± 11.53 years (range 19-72 years) and 33 ± 9.39 years (range 5-60 years) respectively. Eighty two patients (16.4%) were lost to follow up and the present data on rejections, patients and graft survival pertains to 418 patients. These patients have been followed up for a mean period of 2.63 years (SE, 0.122; median 1.8 years; range 0-13.36 years). Acute rejection episodes occurred in 115 (27.3%) patients and 95% of these could be reversed with steroids/ATG. Sixty eight patients (16%) have died on follow-up. Our one-year, 5 year and 10 year estimated graft survival is 95.4% (SE, 0.01), 80.5% (SE, 0.03) and 53.1% (SE, 0.09) respectively and patient survival at one year is 93.2% (SE, 0.01). The estimated graft and patient survival in our series is 9.83 (95% CI, 8.92-10.73) and 9.80 (8.93-10.67) years respectively. CONCLUSION: This centre's short-term graft survival of 95.4% is comparable to the best centres of the world.

Hypercoagulable State.

BACKGROUND: The hypercoagulable state results from a complex interplay of blood coagulation factors, coagulation-inhibitory factors, platelets and the vascular endothelium. Imbalance of the complex interplay between these factors results in thrombosis often complicated by embolism. The causes of thrombosis are varied and maybe congenital or acquired. The current interest is centered on the congenital deficiency of coagulation inhibitors as there is an increasing awareness of their involvement in thrombosis, especially in the young. METHODS: A total of 42 patients with thrombosis were studied. The most common clinical presentation was deep vein thrombosis. All the cases were evaluated for coagulation inhibitors Antithrombin, resistance to activated protein C, Protein C and Protein S using standard assay kits. RESULTS: Resistance to activated protein C (n=10) was seen to be the commonest cause of thrombophilia. This was followed by deficiency of Antithrombin (n-4), Protein C (n=3) and Protein S (n=2). Majority of our cases were in the third decade of life. CONCLUSION: The identification of the underlying aetiology is important for instituting specific therapy and patient management.

Evaluation of Suspected Monoclonal Gammopathies: Experience in a Tertiary Care Hospital.

BACKGROUND: Monoclonal gammopathies occurs in patients with malignant diseases of plasma cells and lymphocytes and in few benign conditions. The objective of this study was to assess the precision, accuracy and confirmation of monoclonal gammopathies on serum protein electrophoresis (SPE) and the clinical relevance of detection and characterization of M component. METHODS: All samples received for serum electrophoresis in the last 3 years were analysed for data on M band positivity and correlating it with clinical profile of the patients. Immunofixation (IFE), Immunoelectrophoresis (IEP) and IgG, IgM estimation were carried out in few cases. The follow up of cases was done by serial monitoring of SPE and ß2 microglobulin levels. RESULTS: 1155 samples were received during the 3 years period. 282 (24.4%) samples were positive for M component on SPE. Of these, 239 (84.8%) patients had M spike in λ region and 43 patients had M spike in ß region. The mean load of the M protein band in the λ region was 37.8% and in ß region was 35.8%. IgG with κ chain was seen in 40%, IgG with λ chain was seen in 50%, 5% patients each had IgM with κ and IgA with λ light chain. 246 samples (96.5%) had high levels of ß2 microglobulin. Of the 116 cases of multiple myeloma, IgG levels was more commonly raised (5%) as compared to IgA (6.9%) and IgM (5.2%). CONCLUSION: It is recommended that SPE should be performed in patients having unexplained weakness, anaemia, back pain, osteoporosis, osteolytic lesions, fractures, renal insufficiency or recurrent infections.

Real Time-PCR HBV-DNA Analysis: Significance and First Experience in Armed Forces.

BACKGROUND: HBV DNA quantitation is used extensively world wide for the diagnosis and monitoring of treatment of Hepatitis B virus (HBV) infection. However, it has still to be popular in India. The aim of this study was to quantitate HBV - DNA by Real time - PCR method in Hepatitis B and in immuno-compromised patients, to compare the results with HBeAg detection and to monitor the response to therapy of chronic Hepatitis B patients to antivirals. METHODS: Ninety one serum samples of Hepatitis group of patients (all HBsAg positive), 41 samples from immuno-compromised patients (all HBsAg negative) and 49 patients of Chronic Hepatitis B group (all HBsAg positive) were the subjects of this first ever study in Armed Forces. Twenty serum samples from healthy volunteers and non-hepatitis B patients served as negative controls. The amplification detection was carried out in a Rotor-Gene 2000-sequence detector. RESULTS: Amongst Hepatitis B group, 33% (30/91) of the samples were positive for HBV-DNA and 26% (24/91) of samples were positive for HBeAg. In the immuno-compromised group of patients 14.6% (6/11) of samples were positive for HIV-DNA and 9.7% (4/41) were positive for HBeAg. Of the Chronic Hepatitis B patients on treatment, all (100%) were positive by HBV-DNA, whereas 29/49 (59.2%) were positive by HBeAg before treatment. After treatment with antivirals, 06/49 (12.2%) were positive by both tests and 11/49 (22.5%) were positive only by HBV-DNA. 32/49 (65.3%) patients became negative serologically after therapy. CONCLUSION: HBeAg status did not necessarily reflect HBV-DNA level in the serum, as 10/91 (11%) in the Hepatitis B group, 2/41 (4.9%) in the immuno compromised group and 20/49 (40.8%) patients in the Chronic Hepatitis B group were positive for HBV-DNA but negative for HBeAg. HBV-DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV-DNA quantitation and may be started in Armed Forces referral centers in the near future.

Hepatitis C Virus Infection in Haemodialysis Patients: "Wolf in Sheep's Clothing".

BACKGROUND: 170 million people are infected with the Hepatitis C virus (HCV) around the world. Approximately 50%-70% patients infected with HCV develop chronic liver disease. Haemodialysis patients constitute an especially important group with high HCV prevalence. Outbreaks of HCV infection in dialysis units have been documented. Detection of anti-HCV antibodies is a convenient and conventional mode of documentation. However, in this group, it has it's own caveats. METHODS: 48 patients who had undergone or were on haemodialysis (HD) and had undergone a minimum of 15 dialysis sittings were studied. HCV infection was documented both by anti-HCV antibody detection and HCV RNA testing. A comparative evaluation of results by both tests was done. RESULTS: Out of a total of 48 patients, HCV RNA was detected in 38 (79.16%) and anti-HCV antibodies in 13(27.07%). Out of 48 patients 10(20.83%) were negative for both parameters. 22.91% (11/48) of patients were positive for both HCV RNA and anti-HCV antibody. 56.25% (27/48) were HCV RNA positive but anti-HCV antibodies were not detectable in their sera. 2 patients (04.16%) had a positive anti-HCV antibody status despite HCV RNA being negative. In 20.83% (10/48) both parameters were undetectable. CONCLUSION: Chronic liver disease (CLD), particularly due to HCV infection, is a major complication amongst haemodialysis (HD) patients. Without reliable assays for antigenemia and the inability of antibody tests to define viremia in all cases, the detection of viral nucleic acid is necessary for diagnosis of active HCV infection.

Diagnosis of Malaria Infection Using Non Radioactive Malaria Diagnostic System (NOMADS).

BACKGROUND: Malaria remains one of the leading causes of morbidity and mortality. A definitive and early diagnosis remains the biggest challenge world-wide. Light microscopy of blood smears has been the gold standard in diagnosis of malaria for decades. This routine microscopic diagnosis is often unreliable and may not be available at many peripheral health centers. Hence newer diagnostic techniques have been developed based on antigen detection. METHOD: Microscopy and Non-radioactive Malaria Detection System (NOMADS) to diagnose falciparum malaria were compared. Specificity and sensitivity of this technique and applicability of the kit for rapid and reliable malaria diagnosis were evaluated. 2579 samples of blood were processed. Both thick and thin blood smear examination and NOMADS was carried out on each of them. All smear positive samples and highly suspicious clinical cases were also subjected to detection of HRP-2 antigen by ICT Malaria Pf test. RESULTS: The detection rate for malaria on smear examination (both vivax and falciparum) was highest at Dimapur (7.41%), followed by Tezpur (7.13%), Kolkata (7%), Guwahati (6%) and Changsari (3.6%). All centers had greater incidence of falciparum compared to vivax except Kolkata where only vivax was detected. The sensitivity of NOMADS was 0%, 4.8%, 13.5%, 42.9% and 52.8% at Kolkata, Tezpur, Guwahati, Changsari and Dimapur respectively. The specificity of the test ranged between 91.8% at Changsari to 95.9% at Dimapur. The specificity at Tezpur, Kolkata and Guwahati was 92.3%, 94% and 95.3% respectively. CONCLUSION: The study revealed that the test kit developed needs to be standardised as regards calculation of cut off values for each of the test runs and reproductibility of optical density readings. Immuno-Chromatography Test (ICT) is helpful in early diagnosis, management and follow-up of cases of malignant malaria.

Cytokines : as useful Prognostic Markers in Lymphoma Cases.

Cytokines are believed to be involved in the pathogenesis and enhanced expression in patients with Hodgkin's and Non-Hodgkins lymphoma. Based on this phenomenon, a multicentric study was carried out in various lymphoma cases. The diagnosis of lymphoma was made on tissue biopsies and fine needle aspiration cytology (FNAC). Out of a total of 72 cases studied, 45 were of Hodgkin's lymphoma (62.5%) and 27 cases were of Non-Hodgkin's lymphoma (37.5%). Maximum cases of Hodgkin's disease occurred in the age group of 30-40 years and males outnumbered females. Hodgkin's lymphoma cases were predominantly of mixed cellularity histologic type (46.66%) whereas majority cases of Non-Hodgkin's lymphoma were of high grade histologic type (48.14%) with predominance in the age group 51-60 years. In both these type of lymphomas, the IL-2R and IL-6 levels were found to be increased more than four fold (as compared to healthy controls) (p<0.05). The cytokine levels decreased after chemotherapy in patients showing response to therapy. However, there were few conflicting and unreliable trends in the IL-6 levels after chemotherapy where elevated IL-6 levels persisted in patients in clinical remission. Overall, it was seen that both IL-2R and IL-6 can be used as an indicator for assessing prognosis and drug therapy in lymphoma cases. IL-2R was found to be a better prognostic marker than IL-6 in assessing the response of lymphoma patient to chemotherapy, more so in Hodgkin's disease.

Medical arthroscopy--emerging era of rheumatological intervention.

OBJECTIVE: To evaluate the safety and diagnostic value of arthroscopy performed by a rheumatologist. METHODS: Decisions for performing arthroscopy were taken when detailed clinical history-and relevant rheumatological investigations failed to arrive at a definite diagnosis. Arthroscopies were performed under local anesthesia as a daycare procedure. Synovial biopsies taken during procedures were subjected to histopathological examination (HPE). RESULTS: Of the 50 patients enrolled, 39 were males while 11 were females with mean age of 35.5 years. In lower limb oligoarthritis group of patients, three had macroscopic picture of crystal arthropathy, rest of the 29 patients revealed gross picture indicative of non-specific synovitis. While in polyarticular group of eight patients, three had macroscopic picture suggestive of crystal arthropathy (probably polyarticlar gout) while five were indicative of rheumatoid arthritis. In monoarticular disease pattern (n= 10) macroscopic picture findings were as follows- crystal arthropathy-two, tubercular-three, synovial chondromatosis-one and non-specific synovitis-two. HPE of synovium did not correlate in many cases. CONCLUSION: Arthroscopy using a 4 mm scope under local anesthesia in the hands of rheumatologists is a safe daycare procedure. In few cases arthroscopy helped in arriving at a final diagnosis but many patients remained undiagnosed. Both the rheumatologists and the pathologists require further experience in this field.

Renal Transplantation - Calcutta Experience.

31 renal transplant procedures have been performed at this centre. Renal donors were father in 4, mother in 4, brother in 12, sister in 4, brother-in-law in 1 and wives in 6 cases. Median age of recipients and donors was 35.2 years (20-55) and 38.3 years (24-60) respectively. After a mean follow up of 15.7 months (2-40), graft survival was 96.7% and patient survival 90-3%. Three patients (9.6%) required surgical re-exploration, one each for, peri-graft haematoma, arterial kink and graft artery thrombosis. 6 patients (19.3%) required anti rejection therapy with resultant complete normalisation of graft functions. Medical complications noted were post transplant diabetes mellitus in 6 (19.3%), azathihoprine induced bone marrow suppression in 1(3.2%), tuberculosis in 2 (6.4%), post transplant erythrocytosis in 2 (6.4%) and recurrent urinary tract infection (UTI) in one (3.2%) patients. 3 patients (9.6%) died with functioning graft, one due to lung infection and the other due to haemorrahagic pancreatitis and third due to infective endocarditis.

Role of Molecular Typing in Live Related Donor Renal Transplantation.

In renal transplantation, a good HLA-DR match is associated with higher success rate of graft outcome. It is particularly so In high risk recipients. Serological HLA-DR typing is not always easy due to a number of technical problems. In view of this, a comparison of serological and molecular typing was done in our institutions. A total of 64 live related donor patients of renal transplantation were studied. Serological typing was done by conventional methods. Molecular HLA class II typing was done by polymerase chain reaction (PCR) based sequence specific oligonucleotide probe (SSOP) hybridization technique. An overall discrepancy of 19.5% was observed in the DR typing obtained by serology and PCR-SSOP of all the recipients and donors. 14.5% of cases showed discrepancy in the results of only one DR antigen. Serological typing failure was seen in 10.9% of total cases. In 19.5% cases, only one DR antigen was assigned by PCR-SSOP as compared to two antigens by serological methods. Maximum number of discrepancies were seen in DR 2 antigens. There was no appreciable difference of graft survival shown in the patients typed by both methods. However, higher incidence of acute graft rejection episodes were seen in patients with 1 antigen mismatch as compared to zero mismatch. It is concluded that HLA-DR typing should be carried out by molecular methods as these have been found to be more specific and accurate.

USEFULNESS OF EVALUATION OF ANTIMEASLES ANTIBODIES IN PRETERM BABIES.

As per WHO recommendations, measles vaccine is administered at the age of 9 months which is based on studies demonstrating seroconversion (from positive to negative) at this age. However this contention may not hold good in preterm babies since they may have lower initial levels of passively transferred IgG antimeasles antibodies of maternal origin. To explore this possibility, 50 preterm babies (gestational age less than 37 weeks) were studied for antimeasles antibodies. Serum samples were collected at birth and then at 3 months and 5 months of age in all the cases. Antimeasles antibody assay was done in all the serum samples using ELISA kits. At birth 32% of infants were positive for antimeasles antibodies whereas 60% were weakly positive and 8% were negative. At 3 months of age 50% were sero negative, 2% positive and 40% weakly positive. The sero negativity was found to be 98% at 5 months with only 2% remaining positive. Since seroconversion is seen to occur in this vast majority of preterm infants at the age of 5 months, antimeasles vaccine should be administered at this age to this subset of more vulnerable babies.

LIVER RESECTION FOR METASTASES FROM COLORECTAL CANCER.

Five percent of patients with liver secondaries from colorectal carcinoma are potentially resectable and several studies have demonstrated significantly improved survival following resection. Two hundred and ten patients operated for colorectal carcinoma were followed up. Computed tomography confirmed potentially resectable metastasis to the liver in 38. On exploration 18 patients who had 4 or less hepatic metastases and no extrahepatic disease, underwent resection of their secondaries. Fourteen were males and 4 females with a mean age of 43.5 (SD 13.6, range 18-72) years. Ten patients presented with synchronous liver metastasis and 8 had metachronous disease. There was no post-operative mortality. All 18 have been followed up. for a median period of 23.5 (range 12-38) months. Seven patients are alive and well with no evidence of recurrence at a median period of 28 months (survival 39%). Four are alive with local recurrence in the liver. Median time to recurrence was 22 months. Seven patients have died of disseminated disease. The disease free survival at 28 months is 39% and the overall survival 61%. A close follow-up protocol for all patient undergoing curative surgery for colorectal cancer is essential, if such patients are to be selected early.