India contributes to the majority of the malaria burden of Southeast Asia globally (2%), though it has made significant gains in malaria control and eradication as evident by World Health Organisation (WHO) World Malaria Report 2020 with decline of 17.6% cases in the 2018–2019. 45.47% malaria cases in India are reported from Odisha, Chhattisgarh, Jharkhand, Meghalaya and Madhya Pradesh states and majority of the cases (70.54%) were classified as Plasmodium falciparum. The five human Plasmodium species are P. falciparum, P. ovale (2 species), P. vivax and P. malariae. The clinical presentation of malaria varies from fever with non-specific mild symptoms to potentially lethal severe malaria. As per WHO 2015, severe falciparum malaria presents clinically with one or more of the following features: impaired consciousness (Glasgow coma score<11), prostration, multiple convulsions (>2 episodes in 24 hours), acidosis, hypoglycaemia (plasma glucose < 40 mg/dL), severe anemia, renal impairment, jaundice (serum/ plasma bilirubin >3 mg/dl), pulmonary oedema, significant bleeding, shock (> 80 mm Hg systolic in adult with impaired perfusion) and hyperparasitemia (parasitaemia >10%). Severe anemia (<7gm/100 ml /<20% hematocrit) is one of the main clinical presentations of complicated malaria. The underlying etiology of severe anemia include obligate lysis of infected and uninfected red cells during erythrocytic phase. The other underlying mechanisms include splenic sequestration, dyserythropoiesis, bone marrow suppression, inadequate compensatory responses to anemia and loss of anti-complementary defence of red cell membrane CD-55 and CD-59. Recent insights regarding the pathogenesis of malaria anemia has highlighted the role of humoral and cellular autoimmune response particularly anti-phosphatidyl serine antibodies produced by atypical memory B cells[3,4,5].
In spite of major contribution to malaria globally from India, there is paucity of literature from India as well as world regarding the autoantibody-mediated destruction of red cells in malaria due to autoimmune hemolytic anemia (AIHA)[6,7,8,9,10,11,12,13]. We hereby present a case of complicated Plasmodium falciparum malaria with concomitant warm autoimmune hemolytic anemia detected after meticulous transfusion reaction workup in the patient.
A 31-year-old male patient presented in the emergency room with chief complaints of high-grade fever, vertigo, headache and easy fatiguability. Patient had a single episode of epistaxis and rectal bleed. No previous history of diabetes, hypertension or any other chronic illness was elicited. On general physical examination, patient had pallor with no evidence of organomegaly and lymphadenopathy, however, ultrasound abdomen showed mild hepatomegaly (span 17 cm), splenomegaly (14.3 cm) with mild ascites.
A blood requisition form for 3 units of Fresh Frozen Plasma (FFP) along with the EDTA and plain vial sample was received in the Department of Immunohematology and Blood Transfusion. The patient developed itching, rise in body temperature (>1°C from baseline) and rash during the transfusion of the 3rd unit (Blood group - A Rh positive). The transfusion was immediately stopped and injection avil and hydrocortisone were given. The transfusion reaction form along with the post transfusion sample and the FFP unit were received in our department for transfusion reaction workup.
The blood group of the pre- and post-transfusion sample and returned FFP unit was reconfirmed as A Rh D positive and minor Coombs crossmatch was compatible. Indirect Antiglobulin Test (IAT) antibody screen of the pre transfusion sample was negative. Direct Antiglobulin Test (DAT) of post transfusion sample was positive (1+), DAT of pre- transfusion sample though, not routinely performed as part of transfusion reaction workup along with auto-control to rule out immune hemolysis in the patient. DAT and auto control was positive (1+). Further, on DAT screen with monospecific Anti Human Globulin (AHG), red cells were coated with IgG (1+). Elution studies were performed using the DAT positive red cells by using the commercially available acid elution kit (Diacidel, BIORAD) [Figure 1]. The eluate was tested with the commercial antibody identification 11-cell panel (Diacell, BIORAD) and showed a panaggluttination reaction with non-reactive supernatant control.
In view of suspected immune hemolytic anemia, complete blood counts along with peripheral smear examination were advised and it revealed moderate anemia (Hb-7.2g/dL, reticulocytosis (corrected reticulocyte count 4%) and thrombocytopenia (platelet count - 24000/mm3, manual platelet count - 40000/ mm3). Peripheral smear examination showed many crescent shaped gametocytes, single, multiple ring forms and accole forms of Plasmodium falciparum species along with occasional spherocytes (parasitemia - 12%) [Figure 2].
Screening test for Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency was negative. Biochemical investigations showed raised levels of serum LDH (1024 IU/L (range 230–460), raised SGOT (114 U/L), SGPT (92 U/L), normal total bilirubin 1.22mg/ dl (D-0.54, I-0.68) and hypoalbuminemia levels (2.90 g/dl). Coagulation studies were within normal limits. Patient serology for dengue (IgG, IgM ELISA, NS-1 antigen), Typhidot (S. typhi O, H and S. paratyphi AH, BH <40), viral markers, SARS-COV-2 RT-PCR were negative. Stool for occult blood was negative. Microscopic examination of urine was within normal limits with no evidence of hemoglobinuria and hemosiderinuria. The rapid malaria card antigen test (RMAT) based on malarial antigen detection Histidine Rich Protein-2 (HRP-2) and pLDH was positive which confirmed the presence of Plasmodium falciparum species. Thus, based upon the above hematological, biochemical and immunohematological findings, a diagnosis of warm autoimmune hemolytic anemia in complicated malaria was suggested. Patient was administered intravenous bolus of artesunate with dosage of 2.5 mg/kg body weight.
The next day patient developed severe hypotension (50/80 mmHg), had complaints of fresh streaks of blood in sputum and subse quently developed altered sensorium. The arterial blood gas analysis showed evidence of me tabolic acidosis and was started on noradrenaline at the rate of 2 ml/h. Simultaneously, intravenous methylprednisolone 1 mg/kg/day was started for five days, followed by oral route and dose tapering. As multiple packed red blood cells units put up for the patient were incompatible, 1 unit of least incompatible (1+) A Rh positive packed red cells were reserved for the patient to be transfused under steroid cover but as the hematological parameters improved the unit was not transfused. Patient was given 1.2 mg/kg IV daily for 3 days followed by oral 80 mg artemether once daily for 3 days along with IV antibiotics and plenty of oral fluids. The patient was followed up closely clinically, hematologically and for biochemical parameters for delayed post artesunate hemolysis till day 9 (Table 1).
There was drop in hemoglobin levels on day 1 and day 2 which improved by day 4 and slight increase in DAT strength (2+) on day 2. There was no evidence of post artesunate induced delayed hemolysis on close follow up based on DAT strength and hematological indices. After 9 days, the patient was discharged, afebrile, well oriented with improved hemoglobin levels, platelet count and liver function tests however, the DAT was positive on day 9 (1+). The parasitemia was reduced on peripheral smear (<5%). Oral Prednisolone was tapered over six weeks and stopped.
Anemia is a common clinical manifestation of malaria, however D’Acremont V et al. reported thrombocytopenia as the most common hematological alterations (60%), followed by hyperbilirubinemia (40%), anemia (30%), and elevated hepatic aminotransferase levels (25%). In our case, hematological evaluation revealed bicytopenia (anemia and thrombocytopenia). The suggested mechanism of thrombocytopenia may be through peripheral destruction and excessive removal of platelets by splenic pooling as well as platelet consumption by the process of disseminated intravascular coagulation (DIC).
There is a paucity of literature regarding the autoimmune basis of anemia in malaria with a few case reports from India[6,7,8,9,10] and a single case report each from Korea and Canada that have highlighted the DAT positivity and established the autoimmune basis of anemia in Plasmodium falciparum and Plasmodium vivax. Taneja S et al. has reported a case of DAT negative AIHA in 6 month old infant after 10 days of treatment of Plasmodium vivax. Sonali R et al. and Ghosh A et al. from India have reported IgG + C3d coating on the red cells on DAT screen. Drouin J et al. has categorized the IgG subclass as IgG 1. Christine A. facer have reported IgG in Gambian children with Plasmodium falciparum with nearly 50% positivity with C3d and highlighted the fact that Coombs positivity is due to erythrocyte sensitization from passive attachment of circulating complement-fixing malaria antigen-antibody complexes and has no correlation with the level of parasitaemia.
These findings were consistent with our case which showed DAT and autocontrol positivity with presence of IgG coating the red cells and reactive at 37°C in coomb’s phase resulting in destruction of infected and non-parasitized red cells and drop in hemoglobin levels on day 2.
Majority of studies have reported panagglutination reaction in IAT as well as in the eluate. IAT antibody screen panel was negative in our patient and eluate showed pan-agglutination. Autoantibody specificity has been reported as autoanti-E and autoanti-I in adult patient of P. falciparum after 4th day of starting the therapy by Chamnanchanunt S et al.. This discordance may be due to low titers of autoantibody in the patients serum.
Elevated liver enzymes indicate the hepatic phase of the parasite and has positive correlation with moderate to high parasitaemia in falciparum malaria as studied previously (p < 0.0001). This finding was concordant with our case findings.
The patients hematological and clinical condition improved with anti-malarials and steroids as autoimmune hemolysis seems to be short lived. As per literature, the sensitized red cells persist for about six weeks as evident in our case as the patient had positive DAT (1+) at the time of discharge. Lee SH et al. has emphasized the role of lowered recognition threshold for splenic clearance of these immunoglobulins which leads to lower DAT positivity in patients of malaria and hence underreporting of these cases.
This case highlights the fact that in patients of malaria with persistent anemia or sudden drop in hemoglobin levels, autoimmune hemolysis should be ruled out by careful re-evaluation of hematological, biochemical and immunohematological parameters. Even after successful anti-parasitic treatment one should be vigilant about signs of autoimmune hemolysis as it can complicate the course of severe malaria and pose a significant burden on health expenditure. Prompt diagnosis and treatment with corticosteroids and antimalarials are cornerstones of management in these patients. In case of life saving conditions due to severe anemia, least incompatible blood units can be transfused under steroid cover and strict clinican supervision. A clinico-hematological follow up for post artesunate delayed hemolysis is required as serological DAT positivity can persist for six weeks.
The patient was informed and free consent was obtained.
Conflict of interest:
We thank our blood Bank Technicians, Ms. Kumari Sanju and Ms. Anjali for their technical support and help.
1. HFW World Malaria
2. World Health Organization. Guidelines for the treatment of malaria
. 2015Accessed on January 01, 20243rd ed. World Health Organization:313 https://apps.who.int/iris/handle/10665/162441
3. Phillips RE, Looareesuwan S, Warrell DA, Lee SH, Karbwang J, Warrell MJ, et al The importance of anemia in cerebral and uncomplicated falciparum malaria
: role of complications, dyserythropoiesis and iron sequestration Q J Med. 1986;58:305–23
4. Rivera-Correa J, Rodriguez A. Autoimmune Anemia in Malaria
Trends Parasitol. 2020;36(2):91–97
5. Stouti JA, Odindo AO, Owuor BO, Mibei EK, Opollo Mo, Waitumbi Jn. Loss of red blood cell complement regulatory proteins and susceptibility to severe malarial anemia J Infect Dis. 2003;187:522–25
6. Acharya S, Verma A, Singh D, Gupta V, Mahajan S. A case of Plasmodium vivax malaria
associated with severe autoimmune hemolytic anaemia Ann Trop Med Public Health. 2012;5:133
7. Sharma V, Samant R, Hegde A, Bhaja K. Autoimmune Hemolysis in Malaria
: A Report of Three Cases J Assoc Physicians India. 2012;60:129–31
8. Sonani R, Bhatnagar N, Maitrey G. Autoimmune hemolytic anemia in a patient with Malaria
Asian J Transfus Sci. 2013;7:151–2
9. Taneja S, Agarwal N. Autoimmune haemolytic anaemia associated with P. viax malaria
Trop Doct. 2019;49(1):54–55
10. Ghosh A, Sharma S, Choudhury J. Autoimmune Hemolytic Anemia in Plasmodium vivax Malaria
Indian J Pediatr. 2017;84(6):483–84
11. Chamnanchanunt S, Thungthong P, Kudsood S, Somwong W, Hirunmassuwan M. Autoimmune hemolytic anemia and autoantibodies in a patient with Plasmodium falciparum
infection: report of a rare case and review of the literature Asian Biomedicine. 2017;11(5):427–32
12. Lee SW, Lee SE, Chung BH, Hwang TJ, Shin HS. A case of Plasmodium vivax malaria
associated with autoimmune hemolytic anemia Infect Chemother. 2008;40:63–66
13. Drouin J, Rock G, Jolly E. Plasmodium falciparum malaria
mimicking autoimmune hemolytic anemia during pregnancy Can Med Assoc J. 1985;132
14. D’Acremont V, Landry P, Mueller I, Pecoud A, Genton B. Clinical and laboratory predictors of imported malaria
in an outpatient setting: an aid to medical decision making in returning travellers with fever Am J Trop Med Hyg. 2002;66:481–486
15. Facer CA. Direct Coombs antiglobulin reactions in Gambian children with Plasmodium falciparum malaria
. II. Specificity of erythrocyte-bound IgG Clin Exp Immunol. 1980;39(2):279–88
16. Al-Salahy M, Shnawa B, Abed G, Mandour A, Al-Ezzi A. Parasitaemia and its relation to hematological parameters and liver function among patients malaria
in Abs, Hajjah, Northwest Yemen Interdiscip Perspect Infect Dis. 2016;2016:5954394
17. Facer CA. Direct antiglobulin reactions in Gambian children with P. falciparum malaria
: I. Expression of IgG subclass determinants and genetic markers and association with anaemia Clin Exp Immunol. 1980;41:81–90
18. Lee SH, Looareesuwan S, Wattanagoon Y, Ho M, Wuthiekanun V, Vilaiwanna N, et al Antibody dependent red cell removal during P. falciparum malaria
: The clearance of red cells sensitised with IgG anti D Br J Haematol. 1989;73:396–402