Alida Harahap

Occult hepatitis B in blood donors in Indonesia: altered antigenicity of the hepatitis B virus surface protein

Background and aimsOccult hepatitis B virus infection (OBI) poses a challenge to the safety of blood donation. The prevalence of OBI is not well documented in Indonesia, although this information in such an endemic country is needed. This study was aimed to evaluate the prevalence of occult hepatitis B in blood donors from two cities of Indonesia, and to study the genetic variation and its effect on the predicted antigenicity of HBsAg.MethodsSerum samples of 309 regular blood donors negative for HBsAg were tested for anti-HBs and anti-HBc. Hepatitis B virus (HBV) DNA isolated from anti-HBc-positive samples were analyzed by polymerase chain reaction, cloned and sequenced. Antigenic properties of identified HBsAg mutants were predicted by calculation of the antigenic index.ResultsOf the 309 HBsAg-negative samples, anti-HBc was positive in 134 (43.4%) and HBV DNA was detected in 25 (8.1%). Seven of the viremic samples had nucleotide substitutions (A521G, A551T, C582T, and A562G) in the S gene, causing amino acid mutations (T123A, M133L, and T143M) in the ‘a’ determinant of HBsAg that resulted in changes in the predicted antigenicity.ConclusionsOBI was detected in blood donors’ samples in Indonesia. Anti-HBc was shown to be a better screening parameter than HBsAg, however, it might result in the loss of donors particularly in endemic countries. HBsAg detection failure in this study might be due to mutations altering the protein antigenicity and/or the low-level carriage of HBV.

Hydrops Fetalis Associated with Homozygosity for Hb Adana [α59(E8)Gly→Asp (α2)]

We describe cases of hydrops fetalis associated with nondeletional α-thalassemia (α-thal), in three unrelated Indonesian families. The genotypes of the fetuses and their parents were generated by DNA sequencing and by a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP)-based method to rapidly identify mutations detected by sequencing. Two of the fetuses had hydrops fetalis and homozygous α59(E8)Gly→Asp (α2), also known as Hb Adana. The third fetus was also suspected to be homozygous for Hb Adana because both parents were carriers of this mutation. This study shows that homozygosity for Hb Adana is associated with hydrops fetalis in the Indonesian population. We discuss this mutation and its various phenotypes including compound heterozygosity with other α-thal mutations and describe a simple approach to genetic testing that will clarify the risk of hydrops fetalis in the offspring of couples carrying this nondeletional mutation.

Molecular basis of β-thalassemia in indonesia: Application to prenatal diagnosis*

Background : To facilitate an effective prevention program, the β-thalassemia mutations in the different ethnic groups in Indonesia were characterized. Methods and Results : The amplification refractory mutation system and artificially created restriction site were used to detect seven known mutations previously described in the Indonesian population. Other mutant alleles were identified by chemical cleavage mismatch, double-stranded sequencing, and Southern blotting. With these methods 78% of β-thalassemia mutant alleles have been detected so far. Thirteen different β-thalassemia mutations were characterized, nine of which had previously been described in the Jakarta population. The most frequent mutation is HbE (29%), followed by IVS1-nt5 (19%), and Cd 35 (8%). The frequencies of the other mutations varied from 4% to less than 1%. Two large gene deletions, Filipino β-deletion and Hb Lepore, were identified in patients from the eastern part of Indonesia. Conclusions : The ethnicity and clinical hematology of cases in the region should be considered in the screening strategy for carriers and antenatal diagnosis of β-thalassemia in Indonesia. Direct sequencing proved to be the appropriate method for detecting the unknown mutations, and Southern blotting had to be used for large deletions.

Alpha thalassaemia in Indonesia: phenotypes and molecular defects.

Alpha thalassaemia is a hereditary blood disorder characterized by haemolytic anaemia. It is caused by mutations in the α-globin genes which lead to a decrease in or absence of α-globin chain production. Since the α-globin chain is the subunit for both fetal (α2γ2) and adult (α2s2) haemoglobin, homozygous a-thalassaemia can cause anaemia in fetuses and adults. Another hereditary blood disorder, s-thalassaemia, is caused by mutations in the s-globin gene. Together, α-and s-thalassaemia are the most common single gene genetic disorders in humans (Weatherall, 1997). These disorders are primarily found in areas where malaria was and may still be endemic.

Assessment of Point-of-Care Diagnostics for G6PD Deficiency in Malaria Endemic Rural Eastern Indonesia

Background Patients infected by Plasmodium vivax or Plasmodium ovale suffer repeated clinical attacks without primaquine therapy against latent stages in liver. Primaquine causes seriously threatening acute hemolytic anemia in patients having inherited glucose-6-phosphate dehydrogenase (G6PD) deficiency. Access to safe primaquine therapy hinges upon the ability to confirm G6PD normal status. CareStart G6PD, a qualitative G6PD rapid diagnostic test (G6PD RDT) intended for use at point-of-care in impoverished rural settings where most malaria patients live, was evaluated. Methodology/Principal Findings This device and the standard qualitative fluorescent spot test (FST) were each compared against the quantitative spectrophotometric assay for G6PD activity as the diagnostic gold standard. The assessment occurred at meso-endemic Panenggo Ede in western Sumba Island in eastern Indonesia, where 610 residents provided venous blood. The G6PD RDT and FST qualitative assessments were performed in the field, whereas the quantitative assay was performed in a research laboratory at Jakarta. The median G6PD activity ≥5 U/gHb was 9.7 U/gHb and was considered 100% of normal activity. The prevalence of G6PD deficiency by quantitative assessment (<5 U/gHb) was 7.2%. Applying 30% of normal G6PD activity as the cut-off for qualitative testing, the sensitivity, specificity, positive predictive value, and negative predictive value for G6PD RDT versus FST among males were as follows: 100%, 98.7%, 89%, and 100% versus 91.7%, 92%, 55%, and 99%; P = 0.49, 0.001, 0.004, and 0.24, respectively. These values among females were: 83%, 92.7%, 17%, and 99.7% versus 100%, 92%, 18%, and 100%; P = 1.0, 0.89, 1.0 and 1.0, respectively. Conclusions/Significance The overall performance of G6PD RDT, especially 100% negative predictive value, demonstrates suitable safety for G6PD screening prior to administering hemolytic drugs like primaquine and many others. Relatively poor diagnostic performance among females due to mosaic G6PD phenotype is an inherent limitation of any current practical screening methodology.

The role of allergic risk and other factors that affect the occurrence of atopic dermatitis in the first 6 months of life.

Background Atopic dermatitis (AD) is a chronic inflammation of the skin that often appears in early childhood. The manifestation is related to the tendency towards T helper 2 cytokine immune responses (interleukin (IL)-4, IL-5). Genetic factors are suggested to play important roles in AD, and it can be transmitted to newborns, increasing their risk of developing allergies. Objective To determine the association between cord-blood cytokine levels (IL-5, interferon (IFN) γ), cord-blood total immunoglobulin E (IgE) level, perinatal environmental exposure, and the risks of allergy as well as the development of AD in the first 6 months of life. Methods A 6-month cohort study with a nested case-control within was conducted on newborns in Jakarta from December 2008 until May 2009. After the umbilical cord blood samples were taken and stored, subjects were followed up monthly until 6 months old. The occurrence of AD and lifestyle or environmental exposures were recorded. The allergic risk was determined using a modified pediatric allergy immunology work groups scoring system based on allergic history (allergic rhinitis, asthma, AD) in the family. The levels of IL-5 and IFN-γ were measured using ELISA and total IgE by CAP system FEIA. Multivariate analysis was used to evaluate risk factors. Results This study was conducted on 226 subjects. The incidence of AD was 16.4%; of those, 59% had low risk allergy, 38.5% moderate, and 2% high risk. AD mostly occurred at the age of 1 month (57%). Cord blood samples were examined in 37 subjects with AD and 51 without AD; of those, 25% showed high levels of total IgE (>1.2 IU/µL), and 51% showed normally-distributed high absorbance IL-5 values (≥0.0715, absolute value was undetected). The increased level of IL-5 was directly proportional to IgE. High absorbance IFN-γ values (≥0.0795, absolute value = 18.681 pg/µL) were observed in 52% of subjects. Conclusion The associations between the risk of allergy in the family, cord-blood total IgE, IL-5, IFN levels, and some perinatal environmental exposure with AD in the first 6 months of life have not been established.

G6PD Deficiency at Sumba in Eastern Indonesia Is Prevalent, Diverse and Severe: Implications for Primaquine Therapy against Relapsing Vivax Malaria

Safe treatment of Plasmodium vivax requires diagnosis of both the infection and status of erythrocytic glucose-6-phosphate dehydrogenase (G6PD) activity because hypnozoitocidal therapy against relapse requires primaquine, which causes a mild to severe acute hemolytic anemia in G6PD deficient patients. Many national malaria control programs recommend primaquine therapy without G6PD screening but with monitoring due to a broad lack of G6PD deficiency screening capacity. The degree of risk in doing so hinges upon the level of residual G6PD activity among the variants present in any given area. We conducted studies on Sumba Island in eastern Indonesia in order to assess the potential threat posed by primaquine therapy without G6PD screening. We sampled 2,033 residents of three separate districts in western Sumba for quantitative G6PD activity and 104 (5.1%) were phenotypically deficient (<4.6U/gHb; median normal 10U/gHb). The villages were in two distinct ecosystems, coastal and inland. A positive correlation occurred between the prevalence of malaria and G6PD deficiency: 5.9% coastal versus inland 0.2% for malaria (P<0.001), and 6.7% and 3.1% for G6PD deficiency (P<0.001) at coastal and inland sites, respectively. The dominant genotypes of G6PD deficiency were Vanua Lava, Viangchan, and Chatham, accounting for 98.5% of the 70 samples genotyped. Subjects expressing the dominant genotypes all had less than 10% of normal enzyme activities and were thus considered severe variants. Blind administration of anti-relapse primaquine therapy at Sumba would likely impose risk of serious harm.

Interaction of Hb adana (HBA2: c.179G>A) with deletional and nondeletional α(+)-thalassemia mutations: diverse hematological and clinical features.

We describe 27 cases of mild-to-severe α-thalassemia (α-thal) syndrome caused by interaction of Hb Adana [α59(E8)Gly→Asp, GGC>GAC (α2)] with deletional and nondeletional α+-thal mutations in Indonesian patients. Hematological profiles and clinical manifestations of all patients were assessed by routine procedures. The genotypes were generated by a multiplex-polymerase chain reaction (m-PCR), PCR-RFLP (restriction fragment length polymorphism)-based method, and DNA sequencing. The α-thal patients who had Hb Adana in combination with the 3.7 kb deletion mostly have mild-to-moderate anemia. In contrast, patients who were compound heterozygotes for Hb Adana and nondeletional mutations, generally showed a more severe anemia and it mostly presented in childhood. Thus, accurate diagnosis of α-thal disorders is not only important for future management of these patients but also for providing proper genetic counseling to the family.

Phenotypic variability of Filipino β°-thalassemia/HbE patients in indonesia

Three Indonesian patients with identical genotypes, each compound heterozygotes for Filipino β°‐thalassemia/HbE, expressed different clinical severities. One patient has mild disease and is transfusion independent, while the other two are severely affected and transfusion dependent. The size of the Filipino β°‐globin gene deletion was confirmed to be 45 kb, resolving conflicting values given in the literature. Neither ameliorating genetic factors such as α‐globin gene deletions or the XmnI restriction site polymorphism at position ‐158 upstream of the Gγ‐globin gene, nor differences in β‐globin gene haplotype, explain the phenotypic variation. These observations have implications for the development of antenatal diagnosis in Indonesia, as at present it is not possible to give an accurate prediction of severity of phenotype for this common genotype. Am. J. Hematol. 62:7–12, 1999.

Rapid screening for the most common β thalassaemia mutations in south east Asia by PCR based restriction fragment length polymorphism analysis (PCR-RFLP)

Editor—The heterogeneity of the molecular lesions which underlie the failure of erythropoietic cells to synthesise normal haemoglobin in β thalassaemia1 is a complicating factor in its molecular diagnosis. However, although more than 100 different mutations have been identified, mostly single base substitutions or small deletions and insertions in the β globin gene, in many populations the bulk of β thalassaemia is caused by a population specific spectrum of only a small number of mutations.1The strategy for the detection of mutations in patients, therefore, normally involves screening in the first instance for a small number of mutations that are the most common for the population concerned. Two of the most commonly used screening procedures are dot blot or reverse dot blot hybridisation2 and the amplification refractory mutation system (ARMS).3 While these procedures are satisfactory in the hands of the more experienced specialised laboratories, the exacting conditions required for the performance of allele specific hybridisation or PCR amplification steps have made them less reproducible in less advanced laboratories.4 5 In south east Asia, where in some regions the frequency of β thalassaemia can be as high as 10%,6 7 many laboratories in medium sized provincial hospitals and universities are suitably …