An Observational Study of the Effect of Hemoglobinopathy, Alpha Thalassemia and Hemoglobin E on P. Vivax Parasitemia
Biochemistry Program, Department of Biochemistry, Faculty of Medicine,
Chulalongkorn University, Bangkok 10330, Thailand.
2 Biomedical Sciences Program, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand.
3 Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
4 Department of Tropical Hygiene and Clinical Malaria Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
5 Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
Received: February 3, 2018
Accepted: August 23, 2017
Mediterr J Hematol Infect Dis 2018, 10(1): e2018015 DOI 10.4084/MJHID.2018.015
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Background: The protective effect of α-thalassemia, a common hematological disorder in Southeast Asia, against Plasmodium falciparum malaria has been well established. However, there is much less understanding of the effect of α-thalassemia against P. vivax. Here, we aimed to investigate the proportion of α-thalassemia including the impact of α-thalassemia and HbE on the parasitemia of P. vivax in Southeast Asian malaria patients in Thailand.
Methods: A total of 210 malaria patients, admitted to the Hospital for Tropical Diseases, Thailand during 2011-2012, consisting of 159 Myanmeses, 13 Karens, 26 Thais, 3 Mons, 3 Laotians, and 6 Cambodians were recruited. Plasmodium spp. and parasite densities were determined. Group of deletion mutation (--SEA, -α3.7, -α4.2deletion) and substitution mutation (HbCS and HbE) were genotyped using multiplex gap-PCR and PCR-RFLP, respectively.
Results: In our malaria patients, 17/210 homozygous and 74/210 heterozygous -α3.7 deletion were found. Only 3/210 heterozygous -α4.2 and 2/210 heterozygous--SEA deletion were detected. HbE is frequently found with 6/210 homozygotes and 35/210 heterozygotes. The most common thalassemia allele frequencies in Myanmar population were -α3.7 deletion (0.282), followed by HbE (0.101), HbCS (0.013), -α4.2 deletion (0.009), and --SEA deletion (0.003). Only density of P. vivax in α-thalassemia trait patients (-α3.7/-α3.7, --SEA/αα, -α3.7/-α4.2) but not in silent α-thalassemia (-α3.7/αα, -α4.2/αα, ααCS/αα) were significantly higher compared with non-α-thalassemia patients (p=0.027). HbE did not affect P. vivax parasitemia. The density of P. falciparum significantly increased in heterozygous HbE patients (p=0.046).
Conclusions: Alpha-thalassemia trait is associated with high levels of P. vivax parasitemia in malaria patients in Southeast Asia.
Materials and Methods
Identify Plasmodium spp. infection and parasite density. All blood samples from finger pricks were Giemsa stained for thick and thin blood films. Blood smears were tested every 12 hours from initiation of treatment until they were negative on two consecutive occasions; after that, blood smears were daily tested until patients were discharged. Parasite densities (asexual parasite/microliter of blood) were examined by counting the number per 200 leukocytes (thick film) or per 1,000 erythrocytes (thin film). In interpretation the Plasmodium spp., blood smear films were read under microscope by an independent parasitologist at the Hospital for Tropical Diseases. The species was confirmed by polymerase chain reaction (PCR)-based analysis.
Measurement of G6PD activity. G6PD activity assays were performed prior to treatment and weekly repeated until patients were discharged. Quantitative test for G6PD activity was performed using G6PD kit assay (Trinity Biotech, Bray, County Wicklow, Ireland), which measured NADPH production at wavelength of 340 nm. All samples were run parallel with positive and negative control. Hemoglobin for calculation of G6PD activity was measured using Hb201 (HemoCue, Sweden). G6PD activity <1.5 IU/g Hb classified as G6PD deficient was excluded from the study. Leftover blood samples were kept at -20°C for molecular typing.
Detection of α-thalassemia. Genomic DNA was extracted from peripheral blood using phenol-chloroform method. Alpha-globin gene variants including α-thalassemia trait (--SEA deletion) and silent α-thalassemia (-α3.7, -α4.2 deletion) were investigated by multiplex gap-polymerase chain reaction (multiplex gap-PCR). HbCS and HbE were genotyped using PCR-restriction fragment length polymorphism (PCR-RFLP).[28-29]
Statistical analysis. All statistical analyses were performed using the SPSS version 22.0. The main outcomes of interest were parasite densities of P. falciparum and P. vivax malaria before treatment. Parasite density that was not normally distributed was log-transformed prior to analysis. Parasitemia of α-thalassemia and HbE patients were compared with that of non-thalassemia (HbA) using unpaired T-test. In all statistical analyses, significance levels were set at the 95% confidence interval (CI) (P<0.05).
In this study, 17 homozygous and 74 heterozygous -α3.7 deletion were found among 210 patients, while only three heterozygous -α4.2 and two heterozygous --SEA deletion were detected.
HbE was also highly prevalent, with six homozygotes and 35 heterozygotes. For HbCS, five heterozygous were detected (Table 1). The Myanmese was the major ethnic group in this study accounting for 75% of all patients. Among these, the proportion of α- thalassemia was 48.4% (77/159), including 45.9% (73/159) of -α3.7 deletion, 1.8% (3/159) -α4.2 deletion, 0.6% (1/159) --SEA deletion, and 2.5% (4/159) ααCS whereas HbE was 20.8% (29/159). Allele frequencies were calculated for the major population. The most common was -α3.7 deletion (0.282), followed by HbE (0.101), HbCS (0.013), -α4.2 deletion (0.009), and --SEA deletion (0.003) (Table 1). Thalassemia and hemoglobinopathies were not found in 3 patients with P. malariae and mixed infection patients.
|Table 1. Proportion of thalassemia and hemoglobinopathies in malaria patients, divided by Plasmodium spp. infection and ethnicity.|
Association of α-globin gene dosage, HbE, and parasitemia. To assess the effect of α-globin gene presence and HbE genotype on the parasitemia of P. vivax and P. falciparum, the number of parasites in the blood of α-thalassemia and HbE genotypes were compared with that of non-thalassemia (HbA). The results found that P. vivax density in patients with α-thalassemia trait (-α3.7/-α3.7, --SEA/αα, -α3.7/-α4.2) was 4.21±0.32 log10 value/μl, which was significantly higher than HbA patients (3.89±0.71 log10 value/μl) (p=0.027) (Table 2). Whereas, P. vivax parasitemia was not significantly different in patients who depleted only one α-globin gene or had silent α-thalassemia (-α3.7/αα, -α4.2/αα, ααCS/αα) (3.94±0.66 log10 value/μl) (p=0.707) (Table 2). Nevertheless, HbH patient (-α3.7/--SEA) had low level of P. vivax parasitemia compared with HbA (2.08 log10 value/μl). However, there was no significant effect of the number of alpha globin gene deletions on P. falciparum parasitemia.
However, significant increases of P. falciparum density in heterozygous HbE patients was detected (4.45±0.66 log10 value/μl) (p=0.046) (Table 2). On the other hand, P. falciparum parasitemia was reduced in homozygous HbE patient (3.00 log10 value/μl) (Table 2). Nevertheless, this study could not find the effect of HbE on P. vivax parasitemia.
|Table 2. Association between α-globin gene dosage, HbE genotype and number of Plasmodium vivax and Plasmodium falciparum parasitemia.|
The overall frequencies of α-thalassemia and HbE in Myanmar villagers living in malaria-endemic regions of Myanmar were 37.5% (343/916) and 20.3% (186/916), respectively. Our study is comparable to a previous study and may reflect real prevalence. From our finding and the report of Than support α-thalassemia especially -α3.7 deletion and HbE are highly frequent in both malarial and non-malarial infected Myanmar populations. While it is difficult to demonstrate the protective effect of α- thalassemia and HbE when conducting a study only in malaria patients, our findings of high prevalence of thalassemia traits among malaria patients supports the conclusion that malaria infection risk is not reduced in people with α-thalassemia and HbE. In line with this finding, an increased frequency of uncomplicated malaria was found in people with α+-thalassemia in the Vanuatu study. The high prevalence of α-thalassemia and HbE in Southeast Asia remains unexplained.
In contrast to the Haldane hypothesis, where α-thalassemia is expected to protect from malaria, we observed higher levels of P. vivax parasitemia among people with α-thalassemia trait. Similarly, a study in Papua New Guinea also showed higher P. vivax parasitemia (but not P. falciparum) in α+-thalassemia heterozygous and homozygous children. In addition, the study in Kenya also showed that α+-thalassaemia neither protected against symptomatic malaria nor reduced parasitemia. However, α+-thalassaemia appeared to reduce the rate of severe anemia in falciparum malaria and had lower hospitalization. The contrasting effects may be explained by the lack of P. vivax in African population, while both P. vivax and P. falciparum are prevalent in Southeast Asian region.
Despite the dosage effect of P. vivax density where two alpha gene deletions have higher levels of parasitemia than one gene deletion, the single case of HbH (3 genes deletion) had an unexpectedly lower rather than higher level of parasitemia. We could not make a meaningful conclusion from this one case as it could have occurred by chance. It was possible that this patient was referred early, so parasitemia was still low. It is hypothesized that people with α-thalassemia have more baseline erythropoiesis, resulting in a high proportion of reticulocytes which is the susceptible stage for P. vivax infection. This hypothesis, however, is unlikely as there is no evidence of reticulocytosis in people with α+-thalassemia heterozygous.
Our results showed increased parasitemia of P. falciparum in heterozygous HbE, but also a decrease in one single case of homozygote. Our finding is in line with a previous study in Myanmar population. In vitro studies reveal conflicting results. Nagel et al. demonstrated impairment of the growth of P. falciparum in homozygous HbE, but an average growth in heterozygous HbE. Whereas, Chotivanich et al. found in vitro a reduction in RBC invasion in HbAE heterozygotes, associated with a 4-fold increase in the selectivity index compared the other hemoglobin types studied and in particular the EE homozygotes suggesting that in heterozygote individuals with AE hemoglobin, only a quarter of the RBC population can be invaded by P. falciparum, so parasitemia could remain low. Parasitemia of P. vivax in HbE patients had been previously observed but did not reach significant difference. The effect of HbE on P. vivax parasitemia was not found in this study. Nevertheless, O'Donnell and colleagues showed that HbE patients might be more susceptible for malaria infection, especially P. vivax because their malarial antibodies were significantly increased than non-thalassemia children, which reflected in their clinical severity. Although limited by a small number of patients, one strength of our study is that G6PD deficiency was excluded, which has been well known to confer protection against vivax malaria.
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