Limited utility of plasma elafin as a biomarker for skin graft-versus-host disease following allogeneic stem cell transplantation.

BACKGROUND: Acute cutaneous graft-versus-host disease (acGVHD) following haematopoietic stem cell transplant (HSCT) is common but difficult to distinguish from other causes of rash. Plasma elafin has been proposed as a diagnostic and prognostic biomarker of skin GVHD. AIM: To evaluate the role of plasma elafin as a biomarker in acGVHD in an Indian population. METHODS: Plasma elafin was evaluated in a prospective study of HSCT recipients, conducted over 2 years, taking measurements at baseline and at onset of skin rash after HSCT. Patients were categorized into those with GVHD rash, those with non-GVHD rash and those with no rash and the three groups were compared. RESULTS: Two hundred and sixty-one patients with a median age of 16 years (range 1-61 years) and a male predominance (175 : 86 M/F) underwent HSCT during the study period: 56 patients in the GVHD group, 49 in the non-GVHD group and 156 in the no-rash group. The median baseline elafin was similar in all three groups. At the onset of rash, median elafin level was similar between GVHD and non-GVHD rash (34 549 vs. 32 077 pg/mL; P = 0.58) and between GVHD and no rash (34 549 vs. 26 197 pg/mL; P = 0.08). A rise in elafin from baseline was significantly different between GVHD and no rash (P < 0.001) but not between GVHD and non-GVHD rash (P = 0.44). CONCLUSION: The utility of plasma elafin as a biomarker of skin GVHD is very limited. Plasma elafin, although elevated in cutaneous GVHD, is not helpful in distinguishing between GVHD rash and other causes of rash following HSCT.

Utility of tissue elafin as an immunohistochemical marker for diagnosis of acute skin graft-versus-host disease: a pilot study.

BACKGROUND: The skin is the most common organ involved in acute graft-versus-host disease (GvHD). Because histopathology has limited utility in ruling out clinical mimics of acute skin GvHD, more accurate diagnostic techniques are required. AIM: To evaluate the utility of elafin expression in skin by immunohistochemistry (IHC) for accurate diagnosis of acute skin GvHD. METHODS: Consecutive allogeneic haematopoietic stem cell transplant (HSCT) recipients during a 6-month period who developed rash within the first 100 days post-transplant were recruited. Skin biopsies were taken on the day the rash developed. IHC for epidermal elafin was performed and interpreted by a pathologist blinded to the histopathological diagnosis. Staining of ≥ 50% of epidermis was considered positive. Final diagnosis of the rash was assigned using clinical features supported by histopathology. The accuracy of elafin IHC in predicting the final diagnosis of acute GvHD was evaluated. RESULTS: In total, 23 patients (20 male, 3 female; median age 16 years, range 3-53 years) with 27 episodes of skin rash were recruited. Skin rash post-HSCT occurred at a median of 20 days (range 5-45 days). A diagnosis of GvHD was made in 16 episodes (59.26%) while the remaining 11 episodes (40.74%) were judged to be non-GvHD rash. Elafin IHC was positive in all patients with GvHD. Of the 11 episodes of non-GvHD rash, elafin was negative in 8. Thus, the sensitivity and specificity of elafin IHC for predicting acute skin GvHD was 100% and 75%, respectively. CONCLUSION: Tissue elafin is a useful immunohistochemical marker for acute skin GvHD. However, larger studies are needed to validate these results.

Expression of cytokine-mRNA in peripheral blood mononuclear cell of human immunodeficiency virus-1 subtype C infected individuals with opportunistic viral infections from India (South).

Human immunodeficiency virus (HIV) disease progression is associated with a marked change in the level of plasma cytokines. The study reported here investigated the level of mRNA expression of different cytokines: Tumour necrosis factor-alpha (TNF-α), interferon (INF)-gamma, interleukin-10 (IL-10) and IL-21 in the peripheral blood mononuclear cell among the antiretroviral therapy naive subtype C HIV-1 infected individuals and normal healthy controls by real time polymerase chain reaction. The mRNA expressions of all the 4 cytokines in HIV-1 infected individuals were significantly higher compared to healthy controls (P value range 0.0004-0.01). The mean level of IL-10, INF-gamma and TNF-α were higher in HIV infected individuals with low CD4 counts (<300 cells/µl). The IL-10 expression showed a significant negative correlation with CD4 counts (r=-0.25, P=0.04) while IL-21 showed a positive correlation with CD4 counts (r=0.26, P=0.03). There was a significant negative correlation between the cytomegalovirus (CMV) viral load and IL-21 expression. Cytokine levels by mRNA detection avoids the inherent problem of measuring plasma level and this study also provide information on the cytokine levels and CD4+ T cell level among HIV-1 subtype C infected individuals with opportunistic viral infections like CMV.

Generalized pustular psoriasis following allogeneic stem cell transplantation.

Development of psoriasis following allogeneic stem cell transplantation (SCT) is rare, and has been described once previously, following SCT from a sibling donor with psoriasis. This condition should be differentiated from psoriasiform graft-versus-host disease (GvHD) by histopathology. We describe a 9-year-old boy who developed generalized pustular psoriasis 2 months after allogeneic SCT from an HLA-identical sibling donor with no history of psoriasis. Diagnosis was confirmed by clinical features and multiple skin biopsies, which helped to exclude GvHD. The skin lesions responded well to treatment with acitretin. Psoriasis should be considered in the differential diagnosis of skin rash following SCT.

Significance of Epstein-Barr virus (HHV-4) and CMV (HHV-5) infection among subtype-C human immunodeficiency virus-infected individuals.

PURPOSE: Opportunistic viral infections are one of the major causes of morbidity and mortality in HIV infection and their molecular detection in the whole blood could be a useful diagnostic tool. OBJECTIVE: The frequency of opportunistic DNA virus infections among HIV-1-infected individuals using multiplex real-time PCR assays was studied. MATERIALS AND METHODS: The subjects were in two groups; group 1: Having CD4 counts<100 cells/µl (n=118) and the group 2: counts>350 cells/µl (n=173). Individuals were classified by WHO clinical staging system. Samples from 70 healthy individuals were tested as controls. In-house qualitative multiplex real-time PCR was standardised and whole blood samples from 291 were tested, followed by quantitative real-time PCR for positives. In a proportion of samples genotypes of Epstein-Barr virus (EBV) and CMV were determined. RESULTS: The two major viral infections observed were EBV and CMV. The univariate analysis of CMV load showed significant association with cryptococcal meningitis, oral hairy leukoplakia (OHL), CMV retinitis, CD4 counts and WHO staging (P<0.05) while the multivariate analysis showed an association with OHL (P=0.02) and WHO staging (P=0.05). Univariate analysis showed an association of EBV load with CD4 counts and WHO staging (P<0.05) and multivariate analysis had association only with CD4 counts. The CMV load was significantly associated with elevated SGPT and SGOT level (P<0.05) while the EBV had only with SGOT. CONCLUSION: This study showed an association of EBV and CMV load with CD4+ T cell counts, WHO staging and elevated liver enzymes. These viral infections can accelerate HIV disease and multiplex real-time PCR can be used for the early detection. Genotype 1 and 2 of EBV and genotype gB1 and gB2 of CMV were the prevalent in the HIV-1 subtype C-infected south Indians.

Successful treatment of verruca vulgaris with Thuja occidentalis in a renal allograft recipient.

Human papillomavirus-driven verruca vulgaris infection is common in solid organ transplant recipients and increases the risk for squamous cell carcinoma. The available treatment modalities have limited response. We report a renal allograft recipient who presented with multiple warts not responding to cryotherapy and radiosurgery with one turning malignant, needing amputation of the finger. An extract from Thuja occidentalis (White cedar tree) cured the resistant warts on the other fingers, leaving only superficial scars and without affecting allograft function. We have reviewed the pharmacological and clinical properties of T. occidentalis.

Human herpes virus-8 infections among subjects with human immunodeficiency virus infection and normal healthy individuals in India.

Human herpes virus-8 (HHV-8) is etiologically associated with Kaposi's sarcoma. There is insufficient information on the epidemiology of HHV-8 infection from India. Blood samples from 87 human immunodeficiency virus (HIV)-infected individuals and 84 normal healthy blood donors were tested for the HHV-8 IgG antibodies. Further, a total of 309 whole blood samples from treatment-naïve HIV-1-infected individuals and from 70 normal healthy individuals were also collected and tested for HHV-8 DNA. The seroprevalence of HHV-8 was 4.7% in the South Indian population. There was no significant difference in the seroprevalence of HHV-8 in the HIV-infected and uninfected patients. None of the 379 samples tested were positive for HHV-8 DNA. Our study revealed a very low exposure of the South Indian patient population to HHV-8 and multicentric epidemiological studies are needed to understand the prevalence of HHV-8 in different regions of India and to confirm any gender-specific differences in seroprevalence.

Atypical mucocutaneous involvement with Leishmania donovani.

Mucocutaneous leishmaniasis has rarely been reported from India. The usual causative organisms of this infection are Leishmania braziliensis and L. tropica. Another species, L. donovani, which usually causes visceral leishmaniasis, has recently been reported to cause mucocutaneous disease in a few patients from Sri Lanka. We report two patients who had undiagnosed chronic skin lesions for several years. Skin biopsies revealed Leishmania and the species was characterized as L. donovani in both patients. There was considerable improvement in the skin lesions following treatment with liposomal amphotericin B.

The frequency of HIV-I drug resistance mutations among treatment-naive individuals at a tertiary care centre in south India.

Antiretroviral treatment (ART) use in India requires information on baseline drug resistance mutations and polymorphisms in the protease (Pr) and reverse transcriptase (RT) genes of HIV-1 strains from treatment-naïve individuals. We report resistance predictor mutations and polymorphisms in the Pr and the RT sequence of non-clade B HIV-1 strains from ART naïve individuals. The genotypic resistance assay was done on 93 treatment-naïve individuals. The sequences were analysed by Stanford HIV drug resistance data for genotypic drug resistance analysis and REGA HIV-1 subtyping tool. Phylogenetic tree was generated with MEGA 4 for quality control. Ninety-two strains belonged to clade C and one to clade A (A1). Amino acid substitutions were seen at positions associated with drug resistance in Pr gene--10, 24, 74 (each 3%) and position 82 (11%). Substitutions were seen at positions 41 (1%), 100 (1%), 101 (6%), 103 (2%), 179 (6%) and 181 (1%) of the RT sequence known to confer drug resistance in clade B. Polymorphisms in HIV-1 pol gene among treatment-naïve individuals were similar when compared with previous data. One strain each had Y181C substitution, T74S and E35G substitutions in the Pr and one had A98G, K101R and L210FL substitutions in RT.

Detection of opportunistic DNA viral infections by multiplex PCR among HIV infected individuals receiving care at a tertiary care hospital in South India.

PURPOSE: Opportunistic viral infections cause increased morbidity and mortality among human immunodeficiency virus (HIV) infected individuals, especially those who are not on antiretroviral treatment. Early diagnosis of these opportunistic viruses will be able to reduce the risk of disease progression with appropriate intervention. MATERIALS AND METHODS: Multiplex PCR was attempted to detect the opportunistic herpes viruses (HSV-1, HSV-2, VZV, EBV, and CMV), adenovirus and polyoma viruses (JC and BK) in three cocktails of PCR reactions. Subsequently, all the viruses detected were quantitated by testing using monoplex real time PCR. Whole blood samples collected between 2006 and 2007 from 68 treatment naïve HIV-1 infected and 30 normal healthy individuals were tested for these eight viruses. Among the 68 HIV-1 infected individuals 35 had CD4+ T cell count less than or equal to 200 while the other 33 had greater than 200 CD4+ T cells. RESULTS: Among the 68 HIV-1 infected individuals, 49 (72%) were positive for EBV, 5 (7%) samples were positive for CMV. All the five CMV positive individuals had CD4+ T cell count of less than or equal to 200 cells/microL. The mean EBV load among the individuals with a CD4+ T cells of less than or equal to 200 cells/microL was 3.88 log(10) while among those with greater than 200 CD4+ T cells it was 3.75 log(10) . The mean CMV load was 6.98 log(10). Three samples were positive for both CMV & EBV. None of the samples was positive for HSV-1, HSV-2, VZV, Adenovirus, JC and BK viruses. CONCLUSIONS: In our study, multiplex PCR based detection system was found useful in detecting opportunistic viruses in HIV infected individuals. Though EBV is the most prevalent opportunistic viral infection among HIV infected individuals, there was no significant association between EBV load, CD4+ T cell counts and HIV-1 virus load. CMV was seen in HIV infected individuals with low CD4+ T cell counts (less than 200 cells/microL).

Drug resistant mutations detected by genotypic drug resistance testing in patients failing therapy in clade C HIV-1 infected individuals from India.

PURPOSE: There has been an increase in the number of individuals administered antiretroviral therapy (ART) in India but treatment outcome is hampered by increasing development of drug resistance. Previous reports from India have shown M184V as the commonest mutation in treated individuals. However, there is no evidence for any protease mutations in these reports. This study was done to observe the common/unique mutational patterns observed in reverse transcriptase (RT) and protease (Pr) genes of clade C HIV-1 strains from individuals showing treatment failure in India. MATERIALS AND METHODS: The assay was done by sequencing the Pr and RT genes of the HIV-1 strains from 18 individuals failing ART. Analysis was carried out using Stanford HIV drug resistance database (SHDB). The sequences were also submitted to the calibrated population resistance tool of SHDB and Rega HIV-1 sub typing tool. Phylogenetic analysis and quality control were performed with Mega 4. RESULTS: Among the 20 strains, 19 showed resistance to both nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), one strain to NNRTIs and five strains showed protease inhibitors (PI) resistance and 3-class resistance. The most common mutation conferring NRTI resistance was M184V (90%) while K103N (45%) was the most common mutation conferring NNRTI resistance. The M46I mutation was seen in 20% of the Pr sequences. CONCLUSION: Resistance testing to check the prevalence of drug resistance mutations that arise following failure of the first line regimen to establish guidelines for second line regimens in India is a must. Studies are needed to confirm if mutation patterns that arise among clade C following failure of ART are the same as for clade B strains.

Evidence for lower CD4+ T cell and higher viral load in asymptomatic HIV-1 infected individuals of India: implications for therapy initiation.

PURPOSE: We have earlier documented that the south Indian population had lower CD4 counts. The aim of this study was to investigate a previous suggestion on a new CD4+ T cell cut off and association with HIV-1 RNA levels for decision on anti retroviral therapy in India (south). METHODS: We evaluated a new methodology i.e., artus real-time PCR and CD4+ T cell count by Guava EasyCD4 system. From 146 HIV infected individuals seen at a tertiary care centre, blood was collected for CD4+ T cell and HIV-1 RNA estimation. RESULTS: The receiver operating characteristic curve cut off value for the CD4 counts to distinguish between CDC clinical categories A and B was 243 cells/microL, and to distinguish B and C was 153 cells/microL. The RNA level that differentiated CDC A and B was 327473 RNA copies/mL, while for CDC B and C was 688543 copies/mL. There was a significant negative correlation (r = -0.55, P + T cell counts in HIV infected individuals. CONCLUSIONS: A majority with CD4 counts of 201-350 cells/microL in our population had higher viral load than the treatment threshold suggested by the International AIDS society and the above two methodologies are useful in monitoring HIV infections.

Genotypic resistance profile of HIV-1 protease gene: a preliminary report from Vellore, south India.

HIV-1 subtypes other than B are responsible for most new HIV infections worldwide; virus sequence data for drug resistance is described only from a limited number of non-B subtype HIV-1. This study is on mutations and polymorphisms of HIV-1 protease gene that can predict drug resistance in subtype C. The genotypic resistance assay was carried out on 38 HIV-1 strains with their plasma RNA and in nine, the proviral protease gene was sequenced. The treatment naïve strains showed minor resistance mutations, there were no major resistance mutations in the protease gene. We suggest the use of resistance testing to monitor individuals on therapy and also before initiation of therapy, gathering more sequence information for a data bank of Indian strains.

Chemokine profile among human immunodeficiency virus-1 (HIV-1) infected individuals from southern India.

BACKGROUND & OBJECTIVE: Individuals infected with HIV-1 have higher levels of chemokine producing cells compared to uninfected individuals. It is important to know the changes in chemokine levels associated with rate of progression of disease. There is a paucity of information on the plasma chemokines in HIV-1 infected individuals from India. We therefore carried out this study to estimate the levels of three chemokines namely macrophage inflammatory protein alpha (MIP1alpha), MIP1beta and RANTES, in relation to disease status in HIV-1 infected individuals and compared with uninfected individuals. METHODS: RANTES and MIP1alpha were estimated using ELISA in 114 HIV-1 infected and 30 controls, whereas MIP1beta was estimated in 101 HIV infected individuals only and 30 controls. The values were compared to the T cell subsets, HIV-1 viral loads and plasma cytokines (interferon gamma and interleukin-10). RESULTS: Compared to controls the mean MIP1alpha and RANTES level among the HIV-1 infected individuals was higher while MIP1beta level was lower in HIV infected individuals except CDC C groups. There was a significant positive correlation for MIP1á with HIV-1 viral load and IFNgamma, for MIP1alpha with viral load and IL10. There was a significant negative correlation between MIP1alpha with CD4 count and CD4: CD8 ratio and MIP1beta with CD4 count and CD8 count. There was a negativecorrelation between RANTES values and CD8 per cent. INTERPRETATION & CONCLUSION: In conclusion, our study showed a significantly higher level of beta chemokines in south Indian HIV-1 infected individuals compared to controls. These beta chemokines may have the inhibitory effect on HIV-1 only during the initial period and with the progression of disease this inhibitory effect wanes as shown by the positive correlation of beta chemokines with HIV-1 viral load.

Clinicopathologic profile of normocomplementemic and hypocomplementemic urticarial vasculitis: a study from South India.

BACKGROUND: This study aims to study the clinical and histopathological characteristics of hypocomplementemic and normocomplementemic urticarial vasculitis (HUVS and NUV) among dermatology clinic attendees in a tertiary care hospital in South India. PATIENTS AND METHODS: A prospective study was conducted in the dermatology department from February 2003 to May 2004. Seventy-five patients met the inclusion criteria for UV. Sixty-eight patients in whom complement levels were available were classified into either NUV or HUVS groups. Clinical features, laboratory parameters and histological features were compared, and the significance of differences was established using Pearson's Chi-squared test. RESULTS: There was a female preponderance among patients with HUVS. Wheals > 24 h were seen in 90% of patients, and in 54.4% of patients, the wheals were partially blanching or non-blanching. Angioedema was more prevalent in patients with NUV than HUVS (44.4% vs. 21.4%). Systemic involvement was seen in 64.3% of patients with HUVS and 44.4% of patients with NUV. Fever, ANA positivity and systemic lupus erythematosus (SLE) were significantly associated with HUVS. In most cases of UV, a provoking factor could not be identified. Neutrophilic small vessel vasculitis was seen in 42.9% of patients with HUVS and 16.6% patients with NUV. Direct immunofluorescence test showing immunoreactants at the dermo-epidermal junction were present in 60% of patients with HUVS and 33.3% patients with NUV. CONCLUSION: The clinical features of Indian patients with UV were similar to those reported from the West. Fever, ANA positivity and SLE were significantly associated with HUVS.

Investigation of apoptotic markers among human immunodeficiency virus (HIV-1) infected individuals.

BACKGROUND & OBJECTIVES: Apoptosis causes a decline in the counts of uninfected bystander CD4+ T cells in HIV infection. The rate of disease progression of HIV infection is considered to be faster in the developing countries. The present study was carried out to investigate certain markers for apoptosis in immunopathogensis of disease in HIV infected south Indian population. METHODS: Soluble Fas (sFas) antigen and Fas ligand levels in plasma samples from 39 antiretroviral treatment naïve patients was estimated and compared with T cell subsets and HIV-1 viral load. RESULTS: The mean sFas antigen levels among controls and the CDC A, B and C clinical stages were 2.77, 3.08, 3.26 and 3.28 ng /ml respectively, higher though not significantly among HIV-1 infected individuals compared to controls. The mean sFas ligand levels in CDC A, B and C stages were 0.138, 0.125 and 0.117 ng/ml respectively were higher (P<0.001) than controls (0.073 ng/ml) and positively correlated with total lymphocyte % (r=0.43, P =0.007). sFas antigen levels were negatively correlated with total WBC count (r=-0.34, P=0.04), CD4% (r=-0.4, P=0.01) and CD4:CD8 ratio (r=-0.37, P=0.02). There was an increase in plasma levels of sFas antigen and Fas ligand over time in asymptomatics. INTERPRETATION & CONCLUSION: The high levels of sFas antigen and Fas ligand seen in HIV infected individuals suggest increased activation and apoptosis of T cells, due to constant stimulation of the immune system by inter-current infections of HIV infected individuals in south India.

Usefulness of alternate prognostic serum and plasma markers for antiretroviral therapy for human immunodeficiency virus type 1 infection.

In developing countries, the usability of peripheral blood constituents that are low-cost alternatives to CD4-positive (CD4+) T-cell and human immunodeficiency virus type 1 (HIV-1) RNA estimation should be evaluated as prognostic markers. The aim of our study was to investigate the use of plasma levels of dehydroepiandrosterone sulfate (DHEAS), albumin, and C-reactive protein (CRP) as alternate prognostic markers for antiretroviral treatment (ART) response in place of HIV-1 load measurements. Paired blood samples were collected from 30 HIV-infected individuals before and after initiation of ART, 13 HIV-infected individuals before and after completion of antituberculosis therapy (ATT), and 10 HIV-infected individuals not on either ATT or ART. Because of the nonavailability of samples, the CRP estimation was done for samples from only 19, 9, and 8 individuals in groups 1, 2, and 3, respectively. The measurements of all three markers, i.e., DHEAS, albumin, and CRP, were carried out with commercial assays. The differences in the albumin levels before and after ART or ATT were significant (P < 0.05), while the differences in DHEAS and CRP levels were not significant (P > 0.05). When levels of DHEAS among the individuals who were followed up were analyzed, 13 (44.8%) in the ART group and 9 (69%) in the ATT group showed an increase following treatment. Prior to treatment of HIV-infected individuals, there was a significant positive correlation of CD4+ T-cell counts and a negative correlation of viral load with albumin and DHEAS levels (P < 0.01). Among the three plasma markers we tested, plasma albumin and, to some extent, DHEAS show promise as prognostic markers in monitoring HIV infection.

Comparison of Microcapillary Cytometry Technology and Flow Cytometry for CD4+ and CD8+ T-Cell Estimation.

An alternative technology for the estimation of T cells based on a microcapillary technique (Guava Technologies, Hayward, CA) was compared to FACSCount (Becton Dickinson, San Jose, CA). Samples from 51 human immunodeficiency virus-infected and 21 healthy individuals were tested. The correlation (r) of the two systems for CD4(+) T cells was 0.994, and the coefficient of variation was 6.5%, establishing equable performance between the two technologies.

Role of S-100 staining in differentiating leprosy from other granulomatous diseases of the skin.

Since Mycobacterium leprae are rarely demonstrable in the tuberculoid spectrum of leprosy, a confirmatory diagnosis of leprosy can be made on the basis of finding active destruction of cutaneous nerves by granulomatous inflammation in a skin biopsy. Immunoperoxidase staining for S-100 protein, which is a marker for Schwann cells, was used to delineate nerves in lesional skin biopsies of 25 patients with tuberculoid and borderline tuberculoid leprosy as well as 15 controls with nonleprous granulomatous inflammation. Four different patterns of nerve damage were observed: infiltrated, fragmented, absent, and intact. All of the nonleprous granulomatous dermatoses showed only intact nerves, either inside or outside the granuloma, and so S-100 staining can be used to rule out leprosy.