Effect of α+ Thalassemia on the Severity of Plasmodium falciparum Malaria in Different Sickle Cell Genotypes in Indian Adults: A Hospital-Based Study.

There is a paucity of literature on the association of α+-thalassemia, sickle-cell hemoglobin disorders, and malaria in India. This study aimed to understand the effect of α+-thalassemia on the severity of Plasmodium falciparum malaria in adults with respect to sickle-cell genotypes. The study subjects were categorized into 'severe-malaria' and 'uncomplicated-malaria' and age-gender matched 'control' groups. Sickle-cell and α+-thalassemia were investigated in all the recruited subjects. The effect of α+-thalassemia on the severity of malaria was analyzed in HbAA and sickle-cell genotypes (HbAS and HbSS) separately. The prevalence of α+-thalassemia in various groups ranged from 41.5% to 81.8%. The prevalence of α+-thalassemia was lower (OR = 1.64; p = 0.0013) in severe malaria (41.5%) as compared to healthy controls (53.8%) with HbAA genotype. In contrast, in HbAS genotype, the prevalence of α+-thalassemia was higher (OR = 4.11; p = 0.0002) in severe malaria (81.8%) compared to controls (52.2%). In severe malaria with HbAA genotype, there was a significantly higher hemoglobin level and low MCV and MCH level in patients with α+-thalassemia compared to the normal α-globin genotype. Further, the incidence of cerebral malaria, hepatopathy, and mortality was lower in patients (HbAA) with α+-thalassemia as compared to normal α-globin genotype (HbAA). In severe malaria with either HbAS or HbSS genotype, only a few parameters showed statistical differences with respect to α+-thalassemia. Low prevalence of α+-thalassemia in severe malaria with HbAA genotype compared to healthy controls with HbAA genotype indicates the protective effect of α+-thalassemia against severe malaria. However, the high prevalence of α+-thalassemia in patients with HbAS genotype depicts its interference in the protective effect of sickle-cell against severe malaria.

Spectrum of hemoglobin disorders in southern Odisha, India: a hospital based study.

BACKGROUND: Hemoglobin disorders are the leading health concern in the world including India. There is a paucity of literature on the spectrum of hemoglobin disorders in southern districts of Odisha state. This study was undertaken to elucidate the occurrence of different hemoglobin disorders in a tertiary health care facility of Odisha state, India. METHODS: The study cases were suspected patients of all age groups advised for screening of different hemoglobin disorders. Hemoglobin disorders were screened by sickling slide test and high-performance liquid chromatography (HPLC) using the Variant-II hemoglobin testing system as per the manufacturer's guidelines. RESULTS: Over 2 years, 2332 blood samples (including 1102 pediatric and 1230 adult cases) were investigated, out of which, 1380 (59.2%) of cases had abnormal hemoglobin disorders. The most common was sickle cell disorders (48.67%, 1135/2332) followed by ß-thalassemia (11.32%, 264/2332). Some rare variants were detected as hemoglobin D-Punjab, hemoglobin E, hemoglobin Lepore, hereditary persistence of fetal hemoglobin, hemoglobin with high P2 window, hemoglobin with high P3 window etc, Among the cases with abnormal hemoglobin disorders, 744 (53.9%), 545 (39.5%) and, 91 (6.6%) cases were found to have the heterozygous, homozygous and, double heterozygous state. Of the 188 ante-natal cases screened, 31.4% of cases had abnormal hemoglobin variants with sickle cell disorders being the most prevalent one. CONCLUSION: Along with the high occurrence of sickle cell disorders in the study area, some other rare hemoglobin disorders are also prevalent which calls for a large community-based cohort study.

Profiling of 35 Cases of Hb S/Hb E (HBB: c.20A>T/HBB: c.79G>a), Disease and Association with α-Thalassemia and ß-Globin Gene Cluster Haplotypes from Odisha, India.

Hb S/Hb E (HBB: c.20A>T/HBB: c.79G>A) is an uncommon variant of sickle cell disease resulting from coinheritance of Hb S and Hb E. Clinico-hematological and biochemical parameters of 35 cases of Hb S/Hb E disease were studied and compared with 70 matched cases of homozygous sickle cell disease (Hb SS) and Hb S/ß-thalassemia (ß-thal) with IVS-I-5 (G>C) (HBB: c.92+5G>C). The influence of α-thal and that of of ß-globin gene cluster haplotypes among Hb S/Hb E disease was also studied. Statistical analysis was done using GraphPad InStat version 3.06. Of the 35 cases, 20 (57.14%) had a moderate clinical presentation. Mean lactate dehydrogenase (LDH) level, vaso-occlusive crises (VOCs) per year, and annual blood transfusion requirements were significantly lower in Hb S/Hb E cases than in the other two groups. The hemoglobin (Hb) and packed cell volume (PCV) levels were significantly high in Hb S/Hb E cases with α-thal and these cases were associated with microcytic-hypochromic anemia. α-Thalassemia did not influence clinical presentation in Hb S/Hb E cases. The ß-globin gene cluster haplotypes of 70 alleles of Hb S/Hb E revealed an association of five typical haplotypes [Arab-Indian (A-I), Benin, Bantu, Cameroon and Senegal] in 95.71% cases. Hb S/Hb E disease exhibit asymptomatic to moderate phenotypic expression. However, further in-depth studies on Hb S/Hb E will help in reducing the disease burden especially in high-risk countries like India.

Erythrocyte cAMP in Determining Frequency of Acute Pain Episodes in Sickle Cell Disease Patients from Odisha State, India.

Vaso-occlusive crisis (VOC) occurs more frequently during stress in sickle cell disease patients. Epinephrine released during stress increases adhesion of sickled red blood cells (RBCs) to endothelium and to leukocytes, a process mediated through erythrocyte cyclic adenosine monophosphate (cAMP). Increased adhesion of sickled RBCs retards blood flow through the capillaries and promotes vaso-occlusion. Therefore, we examined the association of RBC-cAMP levels with frequency of acute pain episodes in sickle cell disease subjects. Using a case control study design, we measured RBC-cAMP levels, fetal hemoglobin (Hb F), α-thalassemia (α-thal) and other hematological parameters at baseline (sham treated) and after stimulation with epinephrine. The cases consisted of sickle cell disease subjects with three or more acute pain episodes in the last 12 months, and those without a single acute pain episode in the last 12 months were considered as controls. Significantly higher cAMP values were found in cases than the controls, in both sham treated (p < 0.001) and epinephrine treated RBCs (p < 0.001) by Wilcoxon Rank Sum test. However, significant association of cAMP values was observed both on univariate [odds ratio (OR): 4.8, 95% confidence interval (95% CI): 1.51-15.19, p < 0.008) and multivariate logistic regression analyses only in epinephrine treated (OR: 5.07, 95% CI: 1.53-16.82, p < 0.008) but not in sham-treated RBCs. In the covariates, Hb F consistently showed protective effects in univariate as well as in multivariate analyses. Frequent acute pain episodes are associated with higher cAMP levels than those with less frequent pain episodes, only after stimulation with epinephrine but not with baseline level.

Association of fetal hemoglobin level with frequency of acute pain episodes in sickle cell disease (HbS-only phenotype) patients.

BACKGROUND: Sickle cell disease (SCD) is a Mendelian single gene disorder with highly variable phenotypic expression. In the present study, we analyzed the influence of HbF, alpha thalassemia and other hematological indices to determine their association with acute pain episodes. METHOD: This case control study consisted of SCD subjects with HbS phenotype experiencing three or more acute pain episodes in last twelve months (cases) and without any episode of acute pain during last twelve months (controls). Hematological parameters, HbF, and presence of alpha thalassemia were assessed in all subjects. RESULTS: A statistically significant difference between HbF levels (P < 0.025, χ2 test) and alpha thalassemia (P < 0.008, χ2 test) was observed between controls and cases group. Univariate analysis indicated that increased HbF levels > 25% (OR: 0.37, 95% CI: 0.18-0.77, P < 0.008) and presence of alpha thalassemia (OR: 0.53, 95% CI: 0.33-0.85, P < 0.009) provided protection, while multivariate analysis revealed significant protection was attributable only by higher HbF levels (OR: 0.39, 95% CI: 0.17-0.88, P < 0.025). Significantly higher HbF levels were observed only in the 11-20 age group of cases in comparison to controls (Student's t-test, P < 0.001). CONCLUSION: Higher concentrations of HbF are associated with protection against frequent episodes of acute pain crisis in SCD patients.

Comparative study of clinical presentation and hematological indices in hospitalized sickle cell patients with severe Plasmodium falciparum malaria.

BACKGROUND: Sickle-cell-gene has a high frequency in malaria endemic regions. In India, though the prevalence of both sickle-cell-gene and malaria are high, no study has been carried out. This study aims to find out the possible differences in hematological and clinical parameters in severe falciparum malaria with respect to sickle cell genotypes. METHODS: Five hundred fourteen adults with severe falciparum malaria hospitalized in Department of Medicine, Veer Surendra Sai Institute of Medical Sciences and Research, Burla, between August, 2010 to December, 2014 were included and categorized on the basis of sickle cell genotypes. The hematological parameters were compared by one-way-analysis-of-variance and incidence of sub-phenotypes of severe malaria was compared by χ2 test across the groups. RESULTS: Patients with sickle cell anemia (HbSS) and severe falciparum malaria had lower hemoglobin level compared to patients with normal ß-globin genotype (HbAA) and sickle cell trait (HbAS). Most of the hematological parameters were homogeneous in patients with HbAA and HbAS and different from patients with HbSS. Incidence of acute renal failure was low (χ2, 9.91; p, 0.002) and jaundice was high (χ2, 5.20; p, 0.022) in patients with HbSS. No clinical difference was observed in patients with HbAA and HbAS. The mortality was low (χ2, 4.33; p, 0.037) and high (χ2, 10.48; p, 0.001) in patients with HbAS and HbSS respectively compared to patients with HbAA. CONCLUSION: Though sickle-cell-gene protects against falciparum infections, the hematological parameters and sub-phenotypes of severe malaria remain unchanged when the infection progresses to a severe form in patients with HbAA and HbAS. Presence of hemolytic anemia in patients with HbSS shows diverse hematological and clinical phenotypes as compared to others. High mortality in patients with HbSS emphasizes the need for a better preventive approach to save valuable lives.

Study of Seminal Fluid Parameters and Fertility of Male Sickle Cell Disease Patients and Potential Impact of Hydroxyurea Treatment.

INTRODUCTION: Male Sickle cell disease (SCD) patients often have moderate to severe hypogonadism resulting in abnormal seminal fluid parameters due to testicular dysfunction. Hydroxyurea (HU), the only drug found to be effective in preventing morbidity and mortality in sickle cell disease patients has been found to further aggravate the testicular dysfunction. MATERIAL AND METHODS: This was a prospective study done at a tertiary care hospital over 26 months between September 2011 to October 2013. 100 male sickle cell disease patients of age group 15 to 45 years were recruited in the study. We evaluated seminal fluid indices in all patients and the effect of hydroxyurea on seminal fluid parameters. Hydroxyurea was given at low dose of 10mg/kg/day orally to patients with frequent vaso-occlusive crisis and frequent need of blood transfusion. Seminal fluid analysis was done according to WHO criteria before starting hydroxyurea and every 3 months after initiation of hydroxyurea. Patients with abnormal seminal parameters before hydroxyurea therapy were not given hydroxyurea therapy. Patients with abnormal sperm parameters were subjected for FNAC of testis. In sickle cell disease patients with hydroxyurea therapy, who developed abnormal seminal fluid parameters, hydroxyurea was stopped for 3 months and seminal fluid parameters were re-evaluated. Patients who had recovery of seminal indices after hydroxyurea cessation were restarted with hydroxyurea therapy at low dose. RESULTS: Among Sickle cell disease patients without hydroxyurea therapy, 18% of patients developed oligospermia and 4% developed azoospermia. Among sickle cell disease patients with hydroxyurea therapy, 20% of patients developed oligospermia and 10% developed azoospermia. Seminal fluid parameters reverted back to normal after stoppage of hydroxyurea for 3 months in 73% of patients. CONCLUSIONS: Alteration of sperm parameters is seen in a significant number of sickle cell disease patients. Also, alterations of seminal fluid parameters are exacerbated by hydroxyurea treatment even with low dose. Therefore, treatment with hydroxyurea in adolescent and adult male sickle cell disease patients should be preceded by routine assessment of seminal fluid parameters and followed up regularly every 3 months for any change in seminal fluid parameters for evidence of hydroxyurea toxicity.

Association of CD40L gene polymorphism with severe Plasmodium falciparum malaria in Indian population.

BACKGROUND & OBJECTIVES: Many host genetic factors are associated with the disease severity and fatal outcome of falciparum malaria. CD40L gene has been found to be one of the most important factors associated with malaria in African countries. This study was aimed to investigate the possible association of CD40L gene polymorphism in severe falciparum malaria in Indian adults. METHODS: One hundred fifteen adult cases with severe falciparum malaria were included in the study. Two single- nucleotide polymorphisms (SNPs) of CD40L gene, CD40L-726(C/T) and CD40L+220(C/T) were investigated, and the possible association with different clinical sub-phenotypes of severe falciparum malaria were analyzed. RESULTS: Statistically no significant difference was observed in the incidence of CD40L-726C between the patients and control group. The incidence of CD40L+220C allele was found to be significantly higher (OR, 2.25; p = 0.03) in male patients compared to controls but no significant difference was observed in females. Haplotype data showed the susceptibility of -726T/+220C haplotype to severe malaria whereas -726C/+220T was associated with protection against severe malaria. CD40L+220C allele was associated with severe malarial anaemia in males (χ2 = 6.60; p = 0.01). INTERPRETATION & CONCLUSION: CD40L gene polymorphism was found to be associated with severe falciparum malaria in Indian population especially in severe malarial anaemia. CD40L may be considered as a factor of immunity in understanding the pathophysiology of falciparum malaria.

Inheritance of Hereditary Persistence of Fetal Haemoglobin (HPFH) in a Family of Western Odisha, India.

Hereditary persistence of foetal haemoglobin (HPFH) is a rare inherited haemoglobin disorders in India. We encountered five cases of HPFH-3 in heterozygous condition in a single family of western Odisha, India. All the cases had raised % HbF (26.1±3.23%) with pancellular distribution of HbF in erythrocytes. There were no abnormalities found in the red cell indices. All the cases were asymptomatic till date with normal growth and development. Molecular confirmation of this haemoglobin disorders is important for control and prevention of haemoglobinopathies in this region.

Influence of Sickle Cell Gene on the Allelic Diversity at the msp-1 locus of Plasmodium falciparum in Adult Patients with Severe Malaria.

Although several studies have supported that sickle cell trait (HbAS) protects against falciparum malaria, the exact mechanism by which sickle gene confers protection is unclear. Further, there is no information on the influence of the sickle gene on the parasitic diversity of P. falciparum population in severe symptomatic malaria. This study was undertaken to assess the effect of the sickle gene on the parasite densities and diversities in hospitalized adult patients with severe falciparum malaria. The study was carried out in 166 adults hospitalized subjects with severe falciparum malaria at Sickle Cell Clinic and Molecular Biology Laboratory, Veer Surendra Sai Institute of Medical Sciences and Research, Burla, Odisha, India. They were divided into three groups on the basis of hemoglobin variants HbAA (n=104), HbAS (n=30) and HbSS (n=32). The msp-1 loci were genotyped using a PCR-based methodology. The parasite densities were significantly high in HbAA compared to HbAS and HbSS. The multiplicity of infection (MOI) and multi-clonality for msp-1 were significantly low in HbSS and HbAS compared to HbAA. The prevalence of K1 (p<0 .0001) and MAD20 (p=0.0003) alleles were significantly high in HbAA. The RO33 allele was detected at a higher frequency in HbSS and HbAS, compared to K1 and MAD20. Sickle gene was found to reduce both the parasite densities and diversity of P. falciparum in adults with severe malaria.

Low and fixed dose of hydroxyurea is effective and safe in patients with HbSß(+) thalassemia with IVS1-5(G→C) mutation.

BACKGROUND: Despite compelling evidence that hydroxyurea is safe and effective in sickle cell disease, it is prescribed sparingly due to several barriers like knowledge gaps in certain genotypes, apprehension about its safety and toxicity, and limited resources. We undertook this study to find out the efficacy and safety of HU in patients with HbSß(+) -thalassemia with IVS1-5(G→C) mutation. PROCEDURE: We registered 318 patients with HbSß(+) -thalassemia with IVS1-5(G→C) mutation. Of these, 203 were enrolled for hydroxyurea treatment at a low and fixed dose of 10 mg/kg/day. One hundred four patients (Group-I: 37 children and Group-II: 67 adults) with ≥2 years of hydroxyurea treatment were studied. RESULTS: The rate of vaso-occlusive crises, requirement of blood transfusion and rate of hospitalization reduced from 3 to 0.5, 1 to 0 and 1 to 0 in Group-I and 3 to 0, 1 to 0 and 0.5 to 0 in Group-II respectively after HU therapy (P < 0.0001). %HbF level, hemoglobin, MCV and MCH increased significantly, whereas HbS, WBC, platelet count, serum-bilirubin and LDH levels decreased significantly after HU therapy. It has been observed that along with fairly subtle hematological changes following HU therapy, there was a substantial clinical improvement occurred in these patients. Transient myelotoxicity was observed in 4.8%. There was minimal gonadal toxicity without affecting reproductive function. CONCLUSION: In view of easy affordability, better acceptability, minimal toxicity, the need of infrequent monitoring and its potential effectiveness, low and fixed dose of hydroxyurea is suitable for treatment of patients with HbSß(+) -thalassemia in resource poor setting.

Prevalence of deletional alpha thalassemia and sickle gene in a tribal dominated malaria endemic area of eastern India.

Inherited hemoglobin disorders like alpha thalassemia and sickle gene are common in the Indian subcontinent. These disorders in the heterozygous state act as malaria resistance genes and influence the susceptibility to Plasmodium falciparum malaria. There is inadequate knowledge about the epidemiology of these malaria resistance genes in the tribal dominated malaria endemic region of the state of Odisha in eastern India. A cross sectional prevalence study was undertaken in 594 subjects in five tribal populations in this region, namely, Sahara (42.4%), Kutia Kandha (30.0%), Kuda (15.8%), Gond (9.8%), and Oraon (2.0%). Sickling test, Hb electrophoresis, HPLC, and molecular studies were undertaken to diagnose the prevalence of sickle allele, ß -thalassemia allele, and deletional alpha thalassemia. Sickle and ß thalassemia alleles were found in 13.1% and 3.4% of subjects, respectively. Sickle allele was found both in heterozygous (10.1%) and homozygous state (3.03%). The prevalence of alpha thalassemia was 50.84% with an allelic frequency of 0.37. Both α (-3.7) and α (-4.2) alpha thalassemia were detected with an allele frequency of 0.33 and 0.04, respectively. The high prevalence of alpha thalassemia and sickle gene in this population is probably due to selection pressure of endemic malaria in this part of India.

The effect of hydroxyurea on compound heterozygotes for sickle cell-hemoglobin D-Punjab--a single centre experience in eastern India.

BACKGROUND: Although hydroxyurea is the only effective agent for the treatment of sickle cell disease, published experience with this drug is limited to treatment of homozygous sickle cell anemia and HbS/ß thalassemia. The role of hydroxyurea in the treatment of patients with HbSD-Punjab, a rare hemoglobinopathy with phenotypic expression similar to that of sickle cell anemia is unknown. PROCEDURE: Over a period of 10 years, we followed 42 patients with HbSD-Punjab, of which 20 presented with severe clinical manifestations (≥3 episodes of VOC and/or ≥2 units of blood transfusion in the previous 12 months). These 20 patients were enrolled for treatment with hydroxyurea at a dose of 10 mg/kg/day and followed prospectively for a period of 24 months. RESULTS: The frequency of VOC decreased significantly and none of them required blood transfusion while receiving hydroxyurea. The HbF, total hemoglobin, MCV, MCH, and MCHC levels increased significantly, whereas HbS, WBC, platelet count, total serum bilirubin, and LDH levels decreased significantly in all the patients. No short-term drug toxicity was observed. CONCLUSION: This study describes the use of hydroxyurea therapy in patients with HbSD-Punjab. Low dose hydroxyurea (10 mg/kg/day) was found to be effective in reducing the clinical severity in patients with HbSD-Punjab without any short-term toxicity. In view of easy affordability amongst poor patients, widespread acceptability by patients and doctors, the need of infrequent monitoring and its potential effectiveness, low dose hydroxyurea is suitable for treatment of patients with HbSD-Punjab.

Procalcitonin as a biomarker of bacterial infection in sickle cell vaso-occlusive crisis.

Sickle cell anaemia (SCA) patients with vaso-occlusive crisis (VOC) have signs of inflammation and it is often difficult to diagnose a bacterial infection in them. This study was undertaken to evaluate the role of serum procalcitonin (PCT) as a biomarker of bacterial infection in acute sickle cell vaso-occlusive crisis. Hundred homozygous SCA patients were studied at Sickle Cell Clinic and Molecular Biology Laboratory, V.S.S. Medical College, Burla, Odisha, India. All the patients were divided into three categories namely category-A (VOC/ACS with SIRS but without evidence of bacterial infection - 66 patients), category-B (VOC/ACS with SIRS and either proven or suspected bacterial infection - 24 patients) and category-C (SCA patients in steady state without VOC/ACS or SIRS - 10 patients). Complete blood count, C-reactive protein (CRP) estimation and PCT measurement were done in all the patients. There was no significant difference in TLC and CRP values between category-A and B. In category-A, the PCT level was <0.5 ng/mL in 83.3% and 0.5-2 ng/mL in 16.7% of cases. In category-B, all the patients had PCT value >0.5 ng/mL with 87.5% of patients having >2 ng/mL. In category-C, PCT value was <0.5 ng/mL. PCT had a high sensitivity (100%) and negative predictive value (100%) for bacterial infection at a cutoff value of 0.5 ng/mL; whereas the specificity is excellent at a cut-off value of 2 ng/mL. SCA patients with VOC/ACS and SIRS having a PCT level of <0.5 ng/mL have a low probability of bacterial infection whereas PCT value of >2 ng/mL is indicative of bacterial infection necessitating early antimicrobial therapy.

Low dose hydroxyurea is effective in reducing the incidence of painful crisis and frequency of blood transfusion in sickle cell anemia patients from eastern India.

There are several questions pertaining to dosage, duration and potential long-term toxicity of hydroxyurea (HU) therapy. Use of HU is extremely limited in eastern India because of its high cost and apprehension of its toxicities. We undertook this study to assess the clinical, biochemical and hematological efficacy of minimal dose HU (10 mg/kg/day) in 118 sickle cell anemia patients (27 pediatric and 91 adults). The frequency of painful crises reduced significantly in 71.5 and 92.2% in pediatric and adult cases, respectively. Ninety-five percent of the patients became transfusion independent. The baseline Hb F, total hemoglobin (Hb), MCV, MCH and MCHC levels increased significantly, whereas the WBC, platelet count and total serum bilirubin values decreased significantly. This is the first study of minimal dose HU therapy in eastern India that showed impressive improvement in clinical and hematological parameters with minimal toxicity.