Dolly Daniel

Distribution of Hepatitis B Virus Genotypes in Blood Donors and Chronically Infected Patients in a Tertiary Care Hospital in Southern India

Hepatitis B virus (HBV) genotypes differ in their potential for causing disease. Consecutive patients with chronic HBV infection (CHBV) (n=122) and blood donors (n=67) positive for hepatitis B surface antigen and HBV DNA were genotyped using polymerase chain reaction-restriction fragment-length polymorphism. The ratio of male to female subjects was significantly higher in the blood donor group than in the group of patients with CHBV (P=.0004). Among patients with CHBV, genotype D was detected in 57.3%, genotype A was detected in 18%, and genotype C was detected in 11.5%. Only genotypes D and A were detected in blood donors. The difference between the detection rate of genotype C in patients with CHBV and in blood donors was significant (11.5% vs. 0%; P=.009). Patients with CHBV who had genotype C had higher alanine transaminase (ALT) levels than those who had genotype A (P=.044) or genotype D (P=.014). Detection of genotype C in patients with CHBV and the association of genotype C with higher ALT levels may predict that this genotype has a greater potential for causing disease than other genotypes.

Association of HLA and TNF polymorphisms with the outcome of HBV infection in the South Indian population

The role of host genetic factors in the pathogenesis and outcome of hepatitis B virus (HBV) infection is not well known. We assessed the association of HLA and TNF (rs361525, rs1800629, rs1799724, rs1800630 and rs1799964) polymorphisms with HBV outcome in the South Indian population. Association of HLA polymorphism was analyzed in 90 individuals from each group, that is, spontaneous recovery (SR) and chronic-HBV (C-HBV) infection. The role of TNF polymorphisms was evaluated in 150 subjects with SR and 137 patients with C-HBV infection. After adjusting for age and sex, HLA-DRB1*07:01 was strongly associated with chronicity (corrected P-value (pc) <0.005, odds ratio (OR) 3.76, 95% confidence interval (CI) 1.84–7.68). The rs1800630 genotype was associated with HBV outcome in codominant (pc<0.01, OR=1.99, 95% CI 1.30–3.05) and dominant (pc<0.01, OR=2.28, 95% CI 1.35–3.84) analyzing models after adjusting for age and sex. Similarly, the rs1799964 genotype was associated with HBV outcome in codominant (pc=0.01, OR=1.57, 95% CI 1.09–2.27) and dominant (pc<0.01, OR=2.21, 95% CI 1.27–3.83) analyzing models. Haplotype analysis (rs1799964/rs1800630/rs1799724/rs1800629/rs361525) revealed that the CACGG haplotype was strongly associated with C-HBV infection (P=0.0004). Our study suggests that inheritance of HLA and TNF polymorphisms might explain the outcome of HBV infection in the South Indian population.

Bioscavenger therapy for organophosphate poisoning – an open-labeled pilot randomized trial comparing fresh frozen plasma or albumin with saline in acute organophosphate poisoning in humans

Introduction. Traditional treatment of organophosphate poisoning (OP) with oximes has had limited success. Fresh frozen plasma (FFP) or albumin, acting as bioscavengers to mop up free organophosphate, has been recently proposed as a treatment modality. In this pilot open-label, three-arm, randomized controlled study exploring proof of concept, we evaluated if bioscavenger therapy had a role in OP. Patients and methods. Sixty patients with significant poisoning presenting within 12 hours, with suppression of pseudocholinesterase activity to < 1,000 U/L, were randomized to receive FFP (8 bags, 250 mL each over 3 days), 20% human albumin (4 × 100 mL over 3 days), or saline (2,000 mL over 3 days) in addition to atropine and supportive care. Pseudocholinesterase and organophosphate levels were measured pretreatment, post-infusion (Day 2, Day 3), and predischarge and expressed as mean ± standard error. The incidence of intermediate syndrome, need for mechanical ventilation, atropine requirement, and mortality were assessed. Results. Twenty patients received albumin and 19 patients each FFP or saline. FFP increased pseudocholinesterase levels (250 ± 44–1,241 ± 364 U/L) significantly (p = 0.007). Small, nonsignificant increases were observed with saline (160 ± 30–259 ± 78) and albumin (146 ± 18–220 ± 61). Organophosphate levels reduced in all 3 arms; no clear-cut trends were observed. We observed more cases of intermediate syndrome with FFP [10/19 (53%) vs. 5/20 (25%) vs. 5/19 (26%), FFP, albumin, and saline arms (p = 0.15)]. The interventions did not affect ventilatory requirements (14/19 vs. 15/20 vs. 14/19) or prevent delayed intubation. There were no differences in mean (±standard error) atropine requirement (in milligrams) in the first 3 days (536 ± 132 vs. 361 ± 125 vs. 789 ± 334) and duration (in days) of ventilation (10.0 ± 2.1 vs. 7.1 ± 1.5 vs. 7.5 ± 1.5) or hospital stay (12.4 ± 2.2 vs. 9.8 ± 1.4 vs. 9.8 ± 1.6). Two patients developed adverse effects with FFP. Mortality was similar (4/19 vs. 5/20 vs. 2/19, p = 0.6). Conclusions. Despite significant increase in pseudocholinesterase levels with FFP, this pilot study did not demonstrate favorable trends in clinical outcomes with FFP or albumin.

A model for human leukocyte antigen-matched donor-swap transplantation in India.

Background. In developing countries such as India, extending donor-swap transplantation (DSTx) to human leukocyte antigen (HLA)-mismatched patient-donor pairs would increase well-matched living donor kidney transplantation rates, resulting in use of less immunosuppression and less expenses, lower infective morbidity, and better survival. A model for DSTx based on HLA matching is presented. Methods. Consecutive HLA class 1 antigen (A, B) tests of prospective renal allograft recipients and their related donors, performed at a single center in India was analyzed retrospectively using an HLA matching program to determine the proportion of prospective recipients with poorly matched related donors who could have benefited by DSTx based on HLA matching. Results. Over the past 17.5 years, 2,129 prospective renal allograft recipients and 2,890 donors were tested for HLA class I (A and B) antigens. Of the prospective recipients, 33% did not have well-matched donors (defined as blood group compatible and sharing ≥2 of 4 HLA class I antigens). Among such recipients, 19.2% could have found a well-matched donor-swap pair within a year at a single center. This number would increase to 38% if four major national centers were involved with a shared HLA registry. Conclusions. Nearly 40% of prospective recipients without well-matched donors would find a donor-swap pair based on HLA matching within a year, with coordination among four national centers and a shared HLA registry, increasing the well-matched living donor renal transplant rates and improving transplant outcomes. This finding is relevant in the context of Indian government amending the Transplantation of Human Organs Act to encourage DSTx.

Impact of pretransplant splenectomy on patients with β-thalassemia major undergoing a matched-related allogeneic stem cell transplantation

Abstract:  Impact of pretransplant splenectomy in patients with β‐thalassemia major undergoing an allogeneic SCT has never been addressed. Twenty‐seven class III patients (29 transplants) had a pretransplant splenectomy. The outcome of these 29 transplants was compared with 76 transplants in class III who did not have a splenectomy. Patients in the splenectomy group were older (11.7 ± 5.0 vs. 8.5 ± 3.5 yr; p = 0.003) and had a larger liver size (5.7 ± 1.8 vs. 4.4 ± 1.6 cm; p = 0.000). Splenectomized patients had a significantly faster time to ANC >500/mm3 (15.4 ± 5.9 vs. 17.5 ± 4 days; p = 0.002) and platelet >20 000/mm3 (22.5 ± 6.7 vs. 32.5 ± 13.6 days; p = 0.000). The splenectomized group had a significantly reduced requirement of blood transfusion in the first 100 days post‐transplant (5.5 ± 5.1 vs. 7.2 ± 5.4 units; p = 0.017). There were significantly more deaths related to peri‐transplant infections in the post‐splenectomy group (24% vs. 5.3%; p = 0.0001). The graft rejections were comparable between the two groups (20.7% vs. 14.5%; p = 0.55). The incidence of acute and chronic GVHD, late infections, and deaths from RRT was not significantly different between the two groups. The five‐yr EFS (31.0 ± 8.6 vs. 60.8 ± 5.98; p = 0.003) and OS (39.7 ± 9.3 vs. 71.8 ± 5.5; p = 0.002) was significantly worse in the splenectomized group. In conclusion, pretransplant splenectomy among patients with β‐thalassemia major was associated with faster engraftment, reduced transfusion support, a higher incidence of peri‐transplant infection related deaths, and a reduced EFS and OS.

Occurrence of False Positives during Testing for Antibodies to Hepatitis C Virus among Volunteer Blood Donors in India

ABSTRACT The hepatitis C virus antibody statuses of only 11 (21.5%) of 51 initially reactive samples from volunteer blood donors could be confirmed by using additional screening and confirmatory assays; 23 (45%) were negative by all subsequent assays. Seventeen samples (33.3%) gave variable results in the different assays. The core and NS5 antigens were most immunogenic. An algorithm for serological screening of volunteer blood donors in blood banks of developing countries is suggested.

High Frequency of the 1896 Precore Mutation in Patients and Blood Donors with Hepatitis B Virus Infection from the Indian Subcontinent

AbstractAim: Hepatitis B virus (HBV) e antigen (HBeAg)-negative variants are reported to harbor 1896 precore mutants, and predict a worse clinical outcome. The aim of this study was to estimate the incidence of a precore mutation (1896) in both patients with chronic hepatitis B (CH-B) infection and blood donors in a tertiary care hospital in south India. Methods: One hundred and twenty-two consecutive HBV DNA-positive CH-B patients (group I) and 102 HBsAg-positive ‘healthy’ blood donors (group II) were recruited. Samples found to be positive for HBV DNA were further studied. A nested PCR was used for the detection of HBV DNA. The 1896 precore mutation was detected using PCR-restriction fragment length polymorphism (RFLP). Nucleotide sequencing was performed on representative samples to confirm PCR-RFLP findings. The study population was stratified comprising: group IA: 17 HBeAg-positive CH-B patients; group IB: 105 HBeAg-negative CH-B patients; group IIA: 12 HBeAg-positive blood donors; and group IIB: 55 HBeAg-negative blood donors. Results: There was no significant difference in the HBeAg-positive status between groups I and II. Significantly higher levels of alanine transaminase (ALT) were seen in groups IA and IB than in groups IIA and IIB, respectively (p = 0.033; p = 0.004). A significantly higher proportion of CH-B patients (32.7%) were positive for anti-HBc IgM compared with the blood donor groups (10.4%; p = 0.0006). Among the HBeAg-negative subjects, 69% of the CH-B patients and 65% of the blood donors showed evidence of 1896 precore mutant. This infection included the 1896 mutant exclusively or mixed infection involving the 1896 mutant and 1896 wild-type. Discussion: The absence of detectable HBeAg in most of the viremic blood donors and patients emphasizes the need for HBV DNA testing irrespective of HBeAg status. Mixed infection was detected in a higher proportion (42.6%) of CH-B patients than in blood donors (26.8%; p = 0.031). Among those with mixed infection, a significant proportion (44.2%) of CH-B patients, had ALT levels greater than the upper limit of normal (ULN), as compared with the blood donor groups (16.6%; p = 0.036). Conclusions: The majority of CH-B patients and blood donors were negative for HBeAg despite their positive HIV DNA status. About two-thirds of the HBsAg-positive blood donors were viremic. Mixed infection was detected more frequently in CH-B patients and appears to be associated with more pronounced liver damage, as indicated by increased ALT levels.

Low frequency of occult hepatitis B infection in anti-HBc seropositive blood donors: experience from a tertiary care centre in South India.

Dear Sir, Historically, transfusion-associated transmission was the major cause of hepatitis B virus (HBV) disease burden worldwide. Increased awareness of donor screening for hepatitis B surface antigen (HBsAg) has greatly reduced the rate of transmission of HBV. However, blood transfusion in the early acute phase of infection and HBV surface gene variants have justified the need for HBV core antibody (anti-HBc) screening and implementation of nucleic acid testing in donor populations. The residual risk of HBV transmission through blood transfusion is estimated to be 1 in 200,000 to 500,000 and is comparatively higher than that for hepatitis C virus (HCV) and human immunodeficiency virus (HIV)1. Occult infection could be one of the reasons for the relatively high risk of HBV infection. The clinical impact of occult HBV infection among recipients of blood and blood products is not completely known. The majority of anti-HBc positive individuals acquire HBV surface antibody (anti-HBs) through natural clearance of infection or as a result of HBV vaccination. However, an anti-HBs level that clearly precludes the circulation of HBV DNA and disease transmission has not yet been clearly identified. We attempted to address these issues by analysing the seroprevalence of HBV DNA in HBsAg negative, anti-HBc positive healthy blood donors with varying levels of anti-HBs antibody. Blood donors in our tertiary care hospital in south India were recruited in October 2008. Serum samples from these donors were tested for HBsAg, HIV and HCV antibody using Vitros ECI (Ortho-Clinical Diagnostics, Raritan, NJ, USA). A total of 1,300 replacement blood donors negative for these serological markers were investigated for the presence of anti-HBc antibody (Diasorin S.p.A. Saluggia, Italy). All the samples that were confirmed to be anti-HBc positive in Architect Anti-HBc II (Abbott, Weisbaden, Germany) were further tested for anti-HBs levels (AxSYM, Abbott, Weisbaden, Germany) and categorised into three groups according to these levels: anti-HBs negative, anti-HBs 100 mIU/mL. DNA was extracted from 200 μL of plasma using the QIAamp DNA blood MiniKit (Qiagen GmbH, Hilden, Germany) and HBV DNA was quantified using a CE-marked artus® HBV RG real-time polymerase chain reaction (PCR) (Qiagen GmbH, Hilden, Germany) in the Rotor-Gene 3,000 or 6,000 platform (Corbett Research, Mortlake, Australia). The assay targets the 134 bp region of the HBV core gene and the lower limit of detection (LLD) stated by the manufacturer is 20 IU/mL (system 1). Samples were further tested by another sensitive, FDA approved automated nucleic acid system (Abbott RealTime HBV, Weisbaden, Germany) targeting the surface region of the HBV genome. The LLD in this case is 10 IU/mL with a sample input of 500 μL (system 2). The study was approved by the Institutional Review Board of the hospital. The median age of the study donors was 32 (range, 14–65) years and most of the donor were male (89%). Of 1300 samples tested for anti-HBc, 217 (16.7%) were confirmed to be positive. When these anti-HBc positive blood donors were tested for anti-HBs, 86 (39.6%) were anti-HBs negative, 58 (26.7%) had an anti-HBs titre 100 mIU/mL. The available 184 samples from the anti-HBc seropositive blood donors were tested for HBV DNA and all were negative on testing in system 1 (artus® HBV RG PCR). When all these samples were further tested in the automated nucleic acid system 2 (Abbott RealTime HBV), two samples were found to be positive for HBV DNA with a viral load of <10 IU/mL (Figure 1). The overall prevalence of occult HBV in anti-HBc seropositive individuals was thus 1.1%. Figure 1 Flow Chart of the Study. Of the two HBV DNA positive donor samples, the anti-HBs titer of one sample was 63 mIU/mL and the other sample was anti-HBs negative. Previous reports have documented a prevalence of occult HBV of 7.5 to 30% in India2–5. In contrast, here we found a prevalence of 1.1% occult HBV in anti-HBc seropositive healthy blood donors. Such a low rate of occult HBV infection from a region of intermediate endemicity for HBV is noteworthy. The differences in rates of occult HBV seen in this population questions the uniformity of our screening practices. Though most studies have used sensitive nested PCR, HBsAg assays used in the diagnosis of occult HBV are highly varied (Table I). The difference in rates of occult HBV reported across the country may be due to the varying sensitivity of HBsAg assays used for donor screening. Donors may well be HBsAg positive when screened by more sensitive HBsAg assays, hence eliminating them from the category of donors with so-called occult infection. Furthermore, contamination in the nested PCR approach for nucleic acid detection is possible and replicate testing to discriminate true and false positive HBV DNA results is required. Table I Occult HBV infection in Indian anti-HBc seropositve blood donors. In a context of limited resources, anti-HBc can be an affordable marker for diagnosing occult HBV infection. However, there are reports of occult infection in anti-HBc seronegative individuals. There is, therefore, a residual risk of HBV transmission in transfusion settings that employ anti-HBc alone as a supplementary marker for occult HBV screening. Moreover, the use of anti-HBc screening relies on the geographic endemicity of HBV. Screening for anti-HBc and excluding about 20% of anti-HBc positive blood donations without knowing the HBV DNA status will result in a higher discard rate of blood units. Anti-HBs is the antibody protecting against HBV and might serve to neutralise the infectivity of these virions. Earlier studies suggested that blood units with >100 mIU/mL of anti-HBs antibody were safe for transfusion. The two HBV DNA positive donors in our study had anti-HBs titers 100 mIU/mL is not common and may not require further HBV DNA testing. However, different assays used for anti-HBs testing may also account for the variability of anti-HBs titres. Moreover, transfusions cannot be considered solely on anti-HBs levels as there are studies that have shown the presence of HBV DNA in individuals with >100 mIU/mL of anti-HBs antibody. Anti-HBs as a screening assay in blood donors does, therefore, need further evaluation as additional testing of anti-HBs in this setting will add to further cost and delay to the release of blood units. Our attempt to rule out false negative HBV DNA results by using a second real-time system with enhanced sensitivity picked up two samples with a viral load of <10 IU/mL. The estimated LLD (95% detection limit) of this assay as determined by the WHO International Standard for HBV was 1.43 IU/mL (unpublished data). This ensures the reliability of the HBV DNA status reported in this study. The presence of very low HBV DNA levels in blood donors necessitates the need for highly sensitive assays with a LLD of less than 10 IU/mL for the correct diagnosis of occult HBV infection. The lower prevalence of HBV DNA seen in anti-HBc seropositive donors provides indirect evidence that the preponderance of HBV DNA in anti-HBc seronegative donors will be much lower. Although we did not see high rates of occult infection in our setting, nucleic acid testing is still recommended as the residual risk of HBV transmission from healthy donors remains. Follow-up of our two HBV DNA positive blood donations could have helped to understand the clinical significance of these occult infections. In summary, the presence of HBV DNA in anti-HBc seropositive blood donations was low in our setting. The different rates of occult HBV infection reported across the country may be due to the varying sensitivity of HBsAg assays used for donor screening. The implementation of anti-HBc screening in donor populations is limited as excluding isolated anti-HBc blood donations will result in higher discard rates of blood units especially in higher endemicity regions. Nucleic acid testing cannot be exempted as there is a residual risk of HBV transmission in healthy blood donors. Anti-HBc and nucleic acid testing in transfusion settings can be minimised by the use of very sensitive HBsAg screening assays. Larger, multicentre studies are required to understand the actual burden of occult HBV infection in transfusion settings.

Association of mannose-binding lectin polymorphisms and HBV outcome in a South Indian population

Mannose binding lectin (MBL) is an important innate immune system pattern recognition molecule. The MBL gene polymorphisms are reported to play a crucial role in outcome of hepatitis B virus (HBV) infection. In this study, we ascertained the association of MBL genotypes with HBV outcome in a South Indian population. The MBL gene polymorphisms at codons 52, 54 and 57 of exon I, and promoter polymorphisms at −221 were typed by polymerase chain reaction‐sequence specific primer in spontaneously recovered and in chronic HBV group. The allele frequency of codon 52 ‘C’ was significantly higher in chronic HBV group than in the recovered group (98.5% vs. 93.6%; P = 0.003) and codon 52 ‘T’ was significantly higher in recovered group than in the chronic group (6.4% vs. 1.5%; P = 0.003). In multivariate analysis, after adjusting for age, sex and state of origin, codon 52 ‘CC’ and ‘CT’ genotypes were significantly associated with chronicity and recovery respectively [odds ratio (OR), 0.25; 95% confidence interval (CI), 0.08–0.80, P = 0.02] in co‐dominant analyzing models. This was re‐affirmed in analysis performed exclusively on Tamil Nadu subjects (OR, 0.23; 95% CI, 0.06–0.93, P = 0.039). The frequency of low/none haplotype (XY/O) was significantly higher in recovered group than in chronic group (15.6% vs 7.5%) and associated with spontaneous recovery (OR, 2.28; 95% CI, 1.04–4.99, P = 0.035). Our results provide preliminary evidence that inheritance of codon 52 genotypes and XY/O haplotype associated with low MBL level substantially determine the outcome of HBV infection in a sympatrically isolated South Indian population.

Frequency of HIV type 2 infections among blood donor population from India: a 10-year experience.

PURPOSE In India, HIV-2 epidemic is alongside with HIV-1. Blood banks are introducing nucleic acid testing (NAT) for screening. The limitation of NAT systems is the inability to detect HIV-2. MATERIALS AND METHOD An analysis of HIV screening of a blood bank at a tertiary care center from 1998 to 2007 was carried out. RESULTS A total of 175026 donors were screened by serological assays and 789 were reactive for HIV antibody. Only 478 (61%) were confirmed positive by Western blot/immunoblot. There were 465 (97.2%) donations positive for HIV-1, 6 (1.3%) for HIV-2 (monotypic infection) and 7 (1.5%) for HIV-1 and HIV-2 (dual infection). CONCLUSION We show the presence of HIV-2 infection among the blood donors and the need for incorporating HIV-2 detection also in the NAT systems.