Genetic red cell disorders and severity of falciparum malaria in Myanmar.

Genetic red cell disorders and severity of falciparum malaria in Myanmar Myint Oo,' Tin-Shwe,2 Marlar-Than,3 & W.J. O'Sullivan4 A hospital-based survey was undertaken to investigate the relationship between the incidence and severity of malaria infection and various red cell disorders in Myanmar. The mean parasitaemia levels of patients with ac- or,-thalassaemia trait or with severe glucose-6-phosphate dehydrogenase (G6PD) deficiency were lower than those of individuals with normal haemoglobin AA or with heterozygous haemoglobin E. The double genetic defect of thalassaemia trait and severe G6PD deficiency appear- ed to confer some degree of protection against malaria. Introduction The high mortality caused by falciparum malaria over many centuries has created significant selective pressure on human populations in malaria-endemic areas, which is believed to be responsible for the prev- alence of deleterious genetic traits such as sickle-cell anaemia, thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency (1-4). A combination of factors has seen the resur- gence of malaria as a major disease throughout the world. Recent experience in Myanmar emphasizes the scope of the problem; it is estimated that during the malaria season (May-August), up to 40% of the total population is infected with Plasmodium fal- ciparum (5). In 1991, for example, approximately 200 000 people required hospitalization for malaria, and 4000 of the confirmed cases, were fatal. Despite the importance of malaria, no systematic study of the relationship between its incidence and various red cell disorders in Myanmar has been reported. Myanmar represents a particularly complex situation; in addition to the high frequency of malar- ia, there is a wide range of ethnic groups and a high I Deputy Director, Parasitology Research Division, Department of Medical Research, Ministry of Health, Yangon, Myanmar. 2 Senior Scientist, Clinical Research Division, Department of Medical Research, Ministry of Health, Yangon, Myanmar. 3Defence Services General Hospital (1), Ministry of Defence, Yangon, Myanmar. 4Professor, Faculty of Biological and Behavioural Sciences, The University of New South Wales, Sydney 2052, Australia. Requests for reprints should be sent to this author. Reprint No. 5645 overall incidence of red cell disorders, which vary between ethnic groups. For example, the prevalence of haemoglobin E (HbE) ranges from approximately 20% among Bamar, Shan and Mon ethnic groups to only 1% among the Chin ethnic group (6). There is also a high incidence of G6PD deficiency (hemi- zygous) among Bamar, with the incidence among Kayin and Mon groups being up to 16% in some areas (7, 8). The overall incidence of 3-thalassaemia trait is about 4% (9) and of a-thalassaemia trait, 10% (10). As a first step towards obtaining a more rigorous overview of the relationship between red cell disor- ders and the incidence of malaria, we describe here the results of an extensive hospital-based survey on the relationship between falciparum malaria and genetic red cell abnormalities in Myanmar. Materials and methods Study subjects A total of 383 patients with severe falciparum mala- ria were selected from Thayarwady Township Hospi- tal and the Defence Services General Hospital, Min- galadon, Yangon. The hospitals are 100 km apart and patients were recruited on an individual basis. All patients were adult males, aged 19-45 years. Ethni- cally all were Bamar (Burmese). The following groups were excluded: under-15-year-olds; females; other ethnic groups (e.g., Shan, Kayin, Kachin, Chin, Mon, and Rakhine); and individuals who had re- ceived antimalarial treatment in the previous 7 days. Bulletin of the World Health Organization, 1995, 73 (5): 659-665 © World Health Organization 1995 659 Myint-Oo et al. Severity of malaria The WHO criteria for defining the severity of the manifestations of falciparum malaria (11) were used, as outlined below. * Category I: Essentially uncomplicated falciparum malaria, with parasitaemia <5%. * Category II: "Hyperparasitaemia" cases, with more than 5% of red blood cells parasitized (or more than 250 000 parasites per p1 of blood), but no other severe manifestation of malaria. * Category III: Patients with impaired conscious- ness: IlIla: Impaired consciousness but rousable (Glas- gow coma score >9/15, and no other severe man- ifestation); and IlIb: Cerebral malaria (unrousable coma); (Glas- gow coma score <9/15, with or without other severe manifestations such as renal failure, se- vere anaemia, jaundice, etc.). Parasite count Both thick and thin peripheral blood smears were Giemsa-stained. The number of asexual parasites per 400 white blood cells was multiplied by 20 to give a quantitative count per pl. Sample collection and analysis Samples of venous blood (5-8 ml) from infected patients were collected in heparinized tubes on the day of admission. Acid-citrate-dextrose (ACD) was added (1:4) to any samples that had to be transport- ed. Estimation of haemoglobin, erythrocyte counts (using a colorimeter),a packed cell volume (PCV), and preliminary screening tests for G6PD deficiency (methaemoglobin reduction test) (12) were carried out immediately after the blood samples were collec- ted in the hospitals. Other procedures were carried out at the central laboratory, Department of Medical Research, Yangon. G6PD variants and genotypes were assessed using starch gel electrophoresis (12) or agarose gel electrophoresis (Myint-Oo, W.J. O'Sullivan, unpublished results, 1993). Red cell indices, haemoglobin AA (HbAA) measurements, cellulose acetate electrophoresis (13), and starch gel electrophoresis (14) were carried out routinely on all samples. Screening for abnormal haemoglobins and thalassaemia traits was performed using standard methods (15). a Lange, Model LP1, Bruno Lange GmbH, Berlin, Germany. Classification of thalassaemia was according to the protocols described by Modell & Berdoukas (16). The criteria for a-thalassaemia were as follows: HbAA, normal; haemoglobin F (HbF), normal; mean corpuscular haemoglobin, (MCH) <25 pg; at least two abnormalities in erythrocyte morphology; serum iron level, .60 ,ug/100 ml; increased osmotic resis- tance in 0.35-0.40% buffered saline; and absence of HbE. The following criteria for 65-thalassaemia were used: HbAA raised (>4.0%); HbF normal or raised (>2.5%); 25 pg <MCH<27 pg; at least two abnormalities in erythrocyte morphology; serum iron level >60 pg/lOO ml; increased osmotic resis- tance in 0.35-0.40% buffered saline; and absence of HbE. No a-thalassaemia/l-thalassaemia double heterozygotes were found in this study; however, DNA analysis was not available. Results Two parameters were measured in the study-the degree of clinical severity and the level of parasitae- mia. These parameters were related to the nature of the infected cells. The results shown in Table 1 illustrate the rela- tionship between haemoglobin variants and clinical severity of malaria in the 383 study subjects. No major differences between the different groups were observed. There is an indication of a lesser degree of clinical severity for patients with ,-trait, and prob- ably for a-trait and homozygous HbE (HbEE), though the number of patients with HbEE were too few for the results to be convincing. However, a sta- tistical analysis (Student's t test) to compare the inci- dence rates of patients with various red cell genetic abnormalities classified as categories Illa and IlIb indicated that patients with a-trait and ,-trait had significantly lower incidences in category IlIb (P <0.01). One patient with n-trait was classified as category IlIb. The relationship between the level of parasitae- mia and haemoglobin variants is illustrated in Fig. 1. Attention is drawn to the very high parasitaemias observed in some subjects, particularly those with HbAA and heterozygous HbE (HbAE). Mean parasi- taemia levels were as follows: 3.05 ± 0.51 for HbAA (normal); 5.29 ± 1.29 for HbAE (P >0.05); 3.30 ± 0.91 for HbEE (P >0.05); 1.77 ± 0.32 for a-trait (P <0.01); 5.01 ± 1.54 for ,B-trait (P >0.05); and 1.98 ± 0.59 for 613-trait (P <0.01). Thus there were no sta- tistically significant differences between mean para- sitaemia levels in HbAA, HbAE, HbEE, and 1-thalas- saemia-trait erythrocytes. This result was unexpected but it should be noted that parasitaemia levels can sometimes reach up to 40% in Myanmar. However, 660 WHO Bulletin OMS. Vol 73 1995 Malaria and red cell genetics in Myanmar Table 1: Relationship between incidence of erythrocyte variants and clinical severity of ma- lariaa No. in clinical category: Variant II Ilia Illb Total Haemoglobin AA 79 (56.4)b 82 (56.2) 29C (56.9) 33 (71.7) 223 (58.2) Haemoglobin AE 25 (17.9) 30 (20.5) 11C (21.6) 12 (26.1) 78 (20.4) Haemoglobin EE 5 (3.6) 3 (2.1) oc 0 8 (2.1) Thalassaemia a-Trait 10 (7.1) 11 (7.5) 4 (7.8) 0 25 (6.5) ,8-Trait 13 (9.3) 15 (10.3) 4 (7.8) 0 32 (8.4) 85-Trait 8 (5.7) 5 (3.4) 3 (5.9) 1 (2.2) 17 (4.4) Total 140 146 51 46 383 a Based on statistical analysis (Student's t test), patients with a-trait, 5-trait and 65-trait had significantly lower incidences (P<0.01). Values for other individuals were not significant. b Figures in parentheses are percentages of the total for each category. there is an indication of a trend to slightly lower parasitaemias with a- and 68-thalassaemia traits (P <0.01). A similar study on the relationship between G6PD deficiency and clinical severity of malaria is shown in Fig. 2. The variants tested were G6PD normal (GdB+ variant, enzyme activity, 3.24 ± 0.41 IU/gHb), G6PD mild deficiency (GdB+ variant, enzyme activity 1.87 ± 0.34 IU/gHb), and G6PD severe deficiency (GdMYanmar variant, G6PD activity 0.14 ± 0.03 IU/gHb) (Myint-Oo, unpublished results, 1993). All three types of G6PD status were found among patients with severe malaria except that three cases with GdMYanmar could be allocated to category Illa, though there were none in category IlIb. The mean parasitaemia level of the GdMYanmar variant (1.94 ± 0.89) was significantly (P <0.01) lower than those of the other two types (GdB+: 3.69 ± 0.47 and GdB-: 4.42 ± 2.21). It should be noted that G6PD levels are affected by changes in red cell turnover in acute malaria, and inferences of genotypes can only be tentative in the absence of DNA analysis of G6PD alleles. A high incidence of double genetic defects was detected. Of the 383 patients studied. 25 (6.5%) car- ried more than one defective gene, either a combina- tion of HbE and G6PD deficiency or a combination of a43-thalassaemia traits and G6PD deficiency. However, no a-thalassaemia/p-thalassaemia double heterozygotes were detected. Discussion The survey was designed to provide basic informa- tion on two separate but related topics with respect to the relationship of red cell disorders and malaria in Myanmar: the effect of the disorders on parasitae- mia levels, and their effect on the clinical severity of malaria. While significant trends in the degree of parasitaemia were observed, this was only partially reflected in the degree of clinical response to infec- tion. In only a few situations did the genetic disor- ders appear to result in protection against the disease state. A summary of the distribution of subjects with various red cell genetic defects (a combination of haemoglobin variants, thalassaemias and G6PD vari- ants) and clinical severity of falciparum malaria is given in Table 2. There was some evidence for a degree of protection for relatively smaller numbers of subjects with HbEE, a- and f-thalassaemia traits, and severe G6PD deficiency in category IlIb in par- ticular. Of all individuals with double genetic defects, only one with HbAE and mild G6PD defi- ciency was in category IlIb. However, X2 test analy- sis of the data (Table 3) indicated no significant cor- relation between the clinical categories and red cell genetic defects (either G6PD-deficient variants or thalassaemia traits). On the other hand, combination of the results for subjects with a- and P-thalassaemia traits did indicate a significant relationship between clinical severity of malaria and thalassaemia traits genetics (Table 4). The first in-vitro study on P. falciparum in HbE- containing erythrocytes was carried out by Nagel et al., who demonstrated a moderate decrease in growth in HbEE cells, but normal growth in HbAE cells (17). Another study from Thailand appeared to con- tradict this result (18). The apparent conflict between these two studies was resolved when Vernes et al. found diminished parasite growth both in homo- zygote HbEE cells and heterozygote HbAE cells, although the effect was small in the latter (19). On WHO Bulletin OMS. Vol 73 1995 661 Myint-Oo et al. Fig. 1. Scattergram showing the relationship between Plasmodium falciparum parasitaemia and haemoglobi- nopathies (% parasitaemias are plotted on a semi-log scale; each dot represents a single patient; standard errors are indicated). (AA = haemoglobin A2; AE = hetero- zygous haemoglobin E; EE = homozygous haemoglobin E). 100 50 20 .t 10 cc I-1 cc E cn co cca- 0- 0.5 223 78 8 25 32 17 cases cases cases cases cases cases OU.-,- :0 * 00 * *- *1 .1 * *--* I** 4:::"4:::." *-.: :-- *i *00 *0 . _. (a C.) 0 6 z * 0 *ok. * 0 * * 0 * *0: * 0 Fig. 2. The relationship between G6PD deficiency and categories (ref. 11) of clinical severity of malaria (GdB+ = G6PD normal; GdB = G6PD mild deficiency; GdMyanmar = G6PD. severe deficiency (<5% of normal enzyme activity)). II I dia Clinical category 0.2 F 0.1 AA AE Haemoglobinopathy the other hand, Kruatrachue et al. also reported that HbE and thalassaemia appear to confer no advantage in respect to parasite rate, parasite density, and mor- tality of falciparum malaria in Thai children (20, 21). It was also observed that the parasitaemia levels did not correlate with the clinical severity of malaria. In the present study, individuals with severe G6PD deficiency appeared to suffer less clinical fal- WHO 95335 ciparum malaria than those with normal or mildly deficient G6PD, although the numbers involved were too small to be statistically significant. However, a significant reduction in the mean parasitaemia level was also observed for individuals with severe G6PD deficiency. Since the deficiency state includes a wide range of variants and erythrocyte enzyme status, the relationship between this genetic disorder and falcip- arum malaria needs to be further explored. Nagel has pointed out that, although mortality from P. falcipar- um infection is relatively low, selection might still operate through an indirect mechanism (22). Individuals with double genetic defects (either a-thalassaemia trait with GdB- or GdMyanmar, or thalassaemia trait with GdB- or GdMyanmar) were not observed in category IIIb (i.e., they did not reach the unrousable coma stage, or die), although this finding was not statistically significant owing to the small sample size. Of the 383 cases with falciparum malar- WHO Bulletin OMS. Vol 73 1995 7 . 0 : * 0 0 0 . * 0 T *-0 662 Malaria and red cell genetics in Myanmar Table 2: Relationship between overall red cell genetic defects and clinical severity of falcip- arum malaria No. in clinical category: Class Genesa Normal AA = GdB+ AE + GdB+ EE + GdB+ AA + GdB AA + GdMyanmar a-thal + GdB, .-thal + GdB+ P8-thal + GdB+ Total AE + GdB a-thal + GdB a-thal + GdMyanmar P-thal + GdB ,B-thal + GdMYan,ar P6-thal + GdMyanmar Total II Illa Illb 67 70 21 27b 22 29 11 11c 5 3 0 0 6 8 6 6 6 4 2 0 5 8 2 0 9 13 3 0 6 4 3 59 69 27 18 3 1 1 1 0 4 2 1 1 1 3 2 1 0 0 1 0 0 0 0 0 1 14 7 3 a AA = haemoglobin AA; AE = heterozygous haemoglobin E; EE = homozygous haemoglobin E; a-thal = a-thalassaemia trait; P-thal = 1-thalassaemia trait; P6-thal = PS-thalassaemia trait; GdB+ = G6PD normal; GdE = G6PD mild deficiency (10-60% of normal enzyme activity); GdMyanmar= G6PD severe deficiency (less than 5% of normal enzyme activity). b Three patients from this group died. c One patient from this group died. Table 3: Frequency of red cell variants and severity of malaria No. in clinical category: 11 G6PD variantsa GdB+ GdB- GdMyanmar Total 114 11 15 140 (1 17.Q)b (13.5) (9.5) 127 11 8 146 llla (122.0) (14.1) (9.9) 40 (42.6) 8 (4.9) 3 (3.5) 51 Illb Total 39 (38.9) 7 (4.4) 0 (3.1) 46 320 37 26 383 Combined defectsc Normal Single defect Double defect Total 67 (67.6) 59 (63.2) 14 (9.1) 140 70 (70.5) 69 (65.9) 7 (9.5) 146 21 (24.6) 27 (23.0) 3 (3.3) 51 27 (22.2) 18 (20.8) 1 (3.0) 46 185 173 25 383 a x2 test = 10.6 (P >0.10; not significant). b Figures in parentheses are the expected frequency. c X2 test = 7.73 (P >0.10; not significant). WHO Bulletin OMS. Vol 73 1995 Total 185 73 8 26 12 15 25 14 173 5 3 7 3 4 3 25 1 1 663 Myint-Oo et al. Table 4: Statistical analysis of subjects with thalas- saemia traitsa Clinical category (X2 test): Trait Illa Illb aX 4 (3.3)b 0 (3.0) f3 4 (4.3) 0 (3.8) a Data for ax- and ,- thalassaemia traits were pooled because of the small numbers. x2 Test = (6.8 - 0)2/6.8; P <0.01 (1 degree of freedom). b Figures in parentheses are the expected frequency (under the null hypothesis that haemoglobin trait and clinical category are not associated), e.g., 3.3 = (25 x 51)/383. ia, the four who died with cerebral malaria had nor- mal genes or single genetic defects. The main objective of this study was to look for evidence that, within the Myanmar population, some of the erythrocyte disorders present conferred signif- icant protection against malaria, such as that obser- ved for sickle-cell anaemia (HbS) (23) and Mela- nesian ovalocytes (24, 25). While no definitive evidence was found, some trends indicative of protection against severe falciparum malaria were observed. Acknowledgements This work was funded by the WHO Regional Office for South-East Asia, New Delhi, India. We thank Dr Aung-Than-Batu for his valuable guidance and support throughout the study. Resume& Relation entre les troubles genetiques des erythrocytes et la gravite du paludisme a falciparum au Myanmar Des 6chantillons de sang veineux ont et6 prelev6s chez quatre groupes de malades entrant dans diverses cat6gories de paludisme a falciparum grave et complique: 140 sujets de la cat6gorie 1, 146 de la cat6gorie 11, 51 de la cat6gorie lila et 46 de la categorie Illb. Les examens suivants ont 6te realises afin d'6tudier les divers types d'ano- malies genetiques des erythrocytes: morphologie des 6rythrocytes; h6moglobine globulaire moyen- ne; electrophorese de I'hemoglobine; h6moglobine AA (HbAA), h6moglobine F (HbF), fer s6rique; fragilit6 osmotique; glucose-6-phosphate d6shy- drog6nase (G6PD); 6lectrophorese de la G6PD. Des num6rations parasitaires ont 6t6 faites sur frottis minces r6alises le jour de I'admission et colores au Giemsa. La parasit6mie moyenne chez les sujets por- teurs d'h6moglobine AA et d'hemoglobine E het6- rozygote 6tait sensiblement plus elevee que chez les sujets porteurs des genes des traits thalass6- miques a ou 63 (p <0,01). La parasit6mie moyen- ne chez les sujets atteints d'un d6ficit s6vere en G6PD etait 6galement, de fa9on significative, plus faible que chez les sujets ayant soit une G6PD normale soit un d6ficit leger en G6PD (p <0,01). Les sujets pr6sentant le double d6faut g6n6tique d'un trait thalass6mique et d'une forme grave de d6ficit en G6PD semblaient poss6der une meil- leure protection contre le paludisme grave que les sujets normaux ou ceux pr6sentant un de- faut gen6tique unique. Cependant, les r6sultats n'etaient pas statistiquement significatifs, si l'on excepte le fait qu'une analyse par x2 indique un certain degr6 de protection chez un petit groupe rassemblant les porteurs des traits thalassemi- ques a etf. References 1. Motulsky AG. Metabolic polymorphisms and the role of infectious diseases in human evolution. Human biology, 1960: 32: 28-63. 2. Allison AC. Population genetics of abnormal hae- moglobins and glucose-6-phosphate dehydrogenase deficiency. In: Jonxis JHP, ed. Abnormal haemoglo- bins in Africa. Oxford, Blackwell, 1965: 365-383. 3. Luzzatto L et al. Genetic variation in the host and adaptive phenomena in Plasmodium falciparum infection. In: Evered D, Whelan J, eds. Malaria and the red cell. London, Pitman, 1983: 139-173 (Ciba Foundation Symposium 94). 4. Golenser J et al. Inhibitory effect of a fava bean component in the in vitro development of Plasmo- dium falciparum in normal and glucose-6-phosphate dehydrogenase deficient erythrocytes. 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