Pathology Section DOI : 10.7860/JCDR/2018/35968.11989
Year : 2018 | Month : Sep | Volume : 12 | Issue : 09 Page : EC06 - EC09

Haematological Alterations in Initially Diagnosed and Relapse/Recurrent Cases of Malaria: A Comparative Study

Deepti Arora1, Sanjay Kaushik2, Sheenam Azad3, Anupreet Kaur4, Sanjeev Kishore5

1 Assistant Professor, Department of Pathology, Shri Guru Ram Rai Institute of Medical and Health Sciences, Dehradun, Uttarakhand, India.
2 Associate Professor, Department of Pathology, Shri Guru Ram Rai Institute of Medical and Health Sciences, Dehradun, Uttarakhand, India.
3 Professor, Department of Pathology, Shri Guru Ram Rai Institute of Medical and Health Sciences, Dehradun, Uttarakhand, India.
4 Resident, Department of Pathology, Shri Guru Ram Rai Institute of Medical and Health Sciences, Dehradun, Uttarakhand, India.
5 Professor, Department of Pathology, Shri Guru Ram Rai Institute of Medical and Health Sciences, Dehradun, Uttarakhand, India.


NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Sanjay Kaushik, Associate Professor, Department of Pathology, Shri Mahant Indresh Hospital and Shri Guru Ram Rai, Institute of Medical and Health Sciences (SGRRIM and HS), Patel Nagar, Dehradun-248001, Uttarakhand, India.
E-mail: skaushikjnmc@gmail.com
Abstract

Introduction

Malaria is endemic in India with an estimated 70-100 million cases each year. An alteration of haematological profile is one of the hallmarks in patients with malaria. A variety of haematological alterations like progressively increasing anaemia, leucocytosis or leucopenia, thrombocytopenia have been reported.

Aim

To evaluate and compare the effect of initially diagnosed cases and cases of relapse/ recurrence of malaria on haematological parameters and their correlation with Parasitic Index (PI).

Materials and Methods

This prospective study included 183 patients of initially diagnosed malaria cases and 64 follow-up cases of malaria. The diagnosis of malaria was confirmed on peripheral blood film and/or malaria antigen test. Complete blood counts were performed using automated Sysmex XN-1000. MP antigen test was done by Alere bioline SD malaria antigen kit. The Pearson’s Chi-square test was used for comparison of the patient’s data for association between distribution of severe anaemia in initially diagnosed cases of malaria and in cases with relapse/recurrence in relation to PI. The association of Grade III & IV thrombocytopenia in initially diagnosed cases of malaria and in cases with relapse/recurrence in relation to PI was evaluated using the paired Student’s t-test.

Results

Out of 183 initially diagnosed malaria cases, 163 cases had P. vivax and 20 cases had P. falciparum. All the cases of P. vivax and 75% of cases of P. falciparum had anaemia. All the cases had thrombocytopenia. Among 64 follow-up cases, 57 had P. vivax and 7 cases had P. falciparum infection. Approximately 50% cases of P. vivax and 40% cases of P. falciparum had normal Hb levels. Normal platelet counts were observed in 14% of P. vivax and 28.5% of P. falciparum cases. Majority of the cases (≅ 85%) of both the groups had PI ≤10.

Conclusion

Anaemia and thrombocytopenia are frequent findings in malaria with severity of thrombocytopenia related to PI. Thrombocytopenia is a constant finding in initially diagnosed cases of malaria but in cases of relapse/recurrence platelet counts may be normal.

How to cite this article

Deepti Arora, Sanjay Kaushik, Sheenam Azad, Anupreet Kaur, Sanjeev Kishore, .HAEMATOLOGICAL ALTERATIONS IN INITIALLY DIAGNOSED AND RELAPSE/RECURRENT CASES OF MALARIA: A COMPARATIVE STUDY.Journal of Clinical and Diagnostic Research [serial online]2018 Sep[cited:2019 May 23] 09 EC06 - EC09
 Available from
http://www.jcdr.net//back_issues.asp

Introduction

Amongst the numerous infectious diseases known to man, malaria is considered to be a menace in most of the developing countries since long. This disease is endemic in India with an estimated 70-100 million cases each year. Malaria is a protozoan disease caused by the Plasmodium species. There are five species of Plasmodium viz., P. falciparum, P. ovale, P. vivax, P. malariae, P. knowlesi that are known to cause malaria in humans [1-3].

Despite extensive worldwide efforts towards reduction of malarial transmission, it remains the most serious and widespread protozoan disease. In tropical countries malaria is considered as the main differential diagnosis of acute febrile illness. An alteration of haematological profile is one of the hallmarks in patients with malaria. A prompt and accurate diagnosis is the key to effective management and reduction in morbidity as well as mortality [1].

Microscopic examination is the established method and gold standard for the laboratory confirmation of malaria that requires technical expertise and repeated smear examinations [1].

Being the parasite of blood it induces a wide array of haematological changes. A variety of haematological alterations like progressively increasing anaemia, leucocytosis or leucopenia, thrombocytopenia and rarely Disseminated Intravascular Coagulation (DIC) have been reported with various species of Plasmodium [1,3-7].

So much work has already been done on haematological alterations in cases of malaria with emphasis on newly diagnosed cases only [8-17]. There is paucity of data in literature on haematological derangements seen in cases of relapse/recurrence. Despite extensive search no study in the literature comparing the haematological alterations in initially diagnosed cases of malaria and cases of relapse/recurrence was found. It is strongly felt that the knowledge of haematological changes in cases of malaria relapse/recurrence will further aid in diagnosis and appropriate treatment planning. With this background the present study was undertaken to evaluate and compare haematological alterations in initially diagnosed cases of malaria and cases of relapse/recurrence.

Materials and Methods

This was a prospective cohort study carried out in the department of Pathology at Shri Guru Ram Rai Institute of Medical and Health Sciences, Dehradun. An informed written consent was taken from patients and the study was approved by ethical committee of the institute (SGRR/REC/54/13). The study was conducted for consecutive three years from Jan 2014 to Dec 2016 and all indoor as well as outdoor cases diagnosed with Plasmodium infection by peripheral blood smear examination and/ or rapid antigen test were included in the study. Relevant history, clinical findings and routine haematological investigations were recorded on a pre-designed proforma.

A total of 183 cases were taken during the study period. In all the cases Haemoglobin, Total leucocyte count and Platelet count were done by automated method using Sysmex XN-1000. Platelet count was also done manually to check for EDTA induced pseudothrombocytopenia and to look for giant platelets which may falsely result in low platelet count [18,19]. All the cases of Plasmodium infection were sub-categorised based on haemoglobin levels, TLC and platelet count as follows [18,20,21]:

Normal Hb: 12 gm/dL and above, Mild Anaemia: 10–12gm/dL, Moderate Anaemia: 7 – 10gm/dL, Severe Anaemia: < 7gm/dL

Normal leucocyte count: 4000 – 11000/L, Leucopenia < 4000/L, Leucocytosis > 11000/L.

Normal platelet count: 1,00,000/cumm and Above, Grade I thrombocytopenia: 75000 – 1,00,000/cumm, Grade II thrombocytopenia: 50,000–75000/cumm, Grade III thrombocytopenia: 10,000 - 50,000/cumm, Grade IV thrombocytopenia: 10000/cumm and below.

MP antigen test was done by Alere bioline SD malaria antigen kit. Peripheral blood smears were prepared, stained with leishman stain and examined. PI was calculated as number of parasitized Red Blood Cells (RBCs) per 1000 (RBCs). Asexual forms of parasites i.e., ring form; trophozoite and schizont were included to calculate PI. The parasitic densities were graded as 0-5%, 6-10%, 11-15%, 16-20% and 21% & above [9].

Further, a subset of patients whose symptoms subsided after taking complete treatment of malarial infection presented again within 14 days with complaints of fever, were considered as relapse/recurrence and studied. The same haematological parameters and PI were observed and results were compared with the previous group.

Statistical Analysis

The Pearson’s Chi-square test was used for comparison of the patient data for association between distribution of severe anaemia in initially diagnosed cases of malaria and in cases with relapse/recurrence in relation to PI.

The association of Grade III & IV thrombocytopenia in initially diagnosed cases of malaria and in cases with relapse/recurrence in relation to PI was evaluated using the paired Student’s t-test.

Adjustments were made using backward stepwise logistic regression and included all variables. All tests were 2-tailed. Significance was declared at p <0.05.

All statistical tests were conducted using Statistical Package for the Social Sciences (SPSS) for Windows version 21.0 (SPSS Inc., Chicago, IL). Descriptive analysis was used, and the data was represented in the form of tables. The tables were created in Microsoft excel and Microsoft word.

Results

The study included 183 cases of initially diagnosed Plasmodium infection, of which 163 cases had P. vivax infection and 20 cases had P. falciparum infection. This group included 105 males and 78 females with a male to female ratio of 1.34:1. The mean age of these patients were 29.7 with the highest proportion in 21-30 years age group. All the cases of P. vivax and 75% of cases of P. falciparum had anaemia. Total leucocyte count was normal in more than 50% cases (P. vivax-68.1% and P. falciparum-50%). All the cases had thrombocytopenia although majority (> 60% of P. vivax and 50% of P. falciparum) had grade I thrombocytopenia. The PI was ≤10 in majority of cases (85%) [Table/Fig-1].

Haemoglobin level, Total leucocyte count, Platelet count and Parasitic index of cases initially diagnosed with malarial infection

P. VivaxP. Falciparum
HaemoglobinInitially Diagnosedn=163 (%)#Initially Diagnosedn=20 (%)#
Normal0 (0)05 (25)
Mild anaemia113 (69.3)03 (15)
Moderate anaemia28 (17.1)05 (25)
Severe anaemia22 (13.4)07 (35)
Total leucocyte count
Normal111 (68.1)10 (50)
Leucopenia42 (25.7)04 (20)
Leucocytosis10 (6.1)06 (30)
Platelet count
Normal0 (0)0 (0)
Grade I Thrombocytopenia99 (60.7)11 (55)
Grade II Thrombocytopenia24 (14.7)2 (10)
Grade III Thrombocytopenia36 (22.1)6 (30)
Grade IV Thrombocytopenia4 (2.4)1 (5)
Parasitic index
0-5107 (65.6)7 (35)
6-1035 (21.4)10 (50)
11-1513 (7.9)1 (5)
16-203 (1.8)2 (10)
21 and above5 (3.1)0 (0)

#figure in parenthesis indicate percentage


A total of 64 cases presented with relapse/ recurrence. Out of these, 57 had P. vivax infection and seven cases had P. falciparum infection. This group had male to female ratio of 1.28:1 with 36 males and 28 females. The mean age was 29.9 with maximum cases in 21-30 years age group. Approximately 50% cases of P. vivax and 40% cases of P. falciparum had normal Hb levels. Both the infections had almost similar number of cases with normal leucocyte counts (around 55%) and PI ≤10 (≅85%). While majority of cases in this group had thrombocytopenia, in some cases platelet counts were normal (P. vivax 14% and P. falciparum 28.5%) [Table/Fig-2].

Haemoglobin level, Total leucocyte count, Platelet count and Parasitic index of relapse/ recurrence cases of malarial infection

P. VivaxP. Falciparum
HaemoglobinRelapse/ Recurrencen=57 (%)Relapse/ Recurrencen=7 (%)
Normal30 (52.6)03 (42.8)
Mild anaemia5 (8.7)01 (14.2)
Moderate anaemia15 (26.3)02 (28.5)
Severe anaemia7 (12.2)01 (14.2)
Total leucocyte count
Normal31 (54.38)04 (57.14)
Leucopenia15 (26.31)02 (28.57)
Leucocytosis11 (19.2)01 (14.28)
Platelet count
Normal8 (14)02 (28.5)
Grade I Thrombocytopenia20 (35.1)02 (28.5)
Grade II Thrombocytopenia11 (19.3)01 (14.2)
Grade III Thrombocytopenia17 (29.8)02 (28.5)
Grade IV Thrombocytopenia1 (1.7)(0)
Parasitic index
0-540 (70.17)3 (42.8)
6-108 (14.03)3 (42.8)
11-157 (12.28)1 (14.2)
16-202 (3.5)0
21 and above00

#figure in parenthesis indicate percentage


Both the groups were compared to correlate the severity of anaemia and thrombocytopenia with PI. The results are shown in [Table/Fig-3,4]. It was observed that there is no significant influence of parasitic densities on severity of anaemia. Grade III and Grade IV thrombocytopenia showed statistically significant correlation with parasitic densities in both initially diagnosed and relapse/recurrence cases.

Distribution of severe anaemia in initially diagnosed cases of malaria and in cases with relapse/recurrence in relation to parasitic index.

Parasitic Index(PER 1000 RBC)AnaemiaInitial Casesn=183 (%)Relapse/ Recurrencen=64 (%)Pearson Chi-Squarep-value(2-sided)
0-5%Severe17 (14.9)n=11405 (11.6)n=4311.2500.259
6-10%Severe07 (15.5)n=4502 (18.2)n=11
11-15%Severe01 (7.1)n=140 (0)n=8
16-20%Severe01 (20)n=501 (50)n=2
21% & aboveSevere03 (60)n=50 (0)n=0

Distribution of Grade III & IV thrombocytopenia in initially diagnosed cases of malaria and in cases with relapse/recurrence in relation to parasitic index

Parasitic Index(per 1000 RBC)ThrombocytopeniaInitial Casesn=183 (%)Relapse/Recurrencen=64 (%)Mean±SDp-value
0-5%Grade III + IV12 (10.5)n=1142 (4.6)n=435.4±2.70.011
6-10%Grade III + IV15 (33.3)n=4510 (90.9)n=11
11-15%Grade III + IV10 (71.4)n=1406 (75)n=8
16-20%Grade III + IV05 (100)n=502 (100)n=2
21%% and aboveGrade III + IV05 (100)n=50 (0)n=0

*p-value calculated using paired students t-test


Discussion

Malaria causes numerous haematological alterations of which thrombocytopenia and anaemia are the most common. This is so characteristic of malaria that in some places, it is used as an indicator of malaria in patients presenting with fever. There are many studies which indicate that in addition to category of malaria, precise haematological changes may vary with nutritional status, demographic factors, immune status and haemoglobinopathy [9-11].

In the present study, incidence of P. vivax was more than P. falciparum in both the groups which is in concordance with some previous studies as well [6,8]. However, some of the studies reported higher incidence of P. falciparum which could be due to different demographic and geographic profile [11-13].

In the initially diagnosed cases, majority had anaemia (96.7%), although it was mild in P. vivax (69.3%) and moderate to severe in P. falciparum (60%). Similar results have been observed in previous studies which reported presence of anaemia in 81.2%-94.4% cases [14-16, 22]. In contrast, a study conducted by Richard MW et al., in London, observed anaemia in only 15% [23]. There is a wide variation in anaemia due to malaria infection depending upon the geographical location of the study. In developing countries, majority of the population have iron and folate deficiency due to inadequate dietary intake along with other bacterial and parasitic infections which could also contribute to anaemia.

Changes in WBC are less definite in malaria and there was a wide variation seen amongst various studies. Majority of the initially diagnosed cases in the present study had normal leucocyte count which correlated well with other studies [11,15,17]. Amongst those with abnormal count, it was observed that leucopenia was more common in P. vivax (25.7%) whereas in P. falciparum infection, there were more cases of leucocytosis (30%). The results were in concordance with study done by Abro AH et al., which reported that leucocytosis was more common in P. falciparum infection [11]. Akhtar S et al., in their study reported that 11.11% of P. vivax cases had leucopenia as compared to 7.69% cases of P. falciparum infection though cases with leucocytosis were almost similar in both the infections [15].

A study by Maina RN et al., concluded that thrombocytopenia as the strongest predictor of malaria [24]. In another study, low platelet counts were more consistently associated with P. falciparum infection rather than P. vivax [25]. But in our study, thrombocytopenia was present in all the cases of malaria in the initially diagnosed group irrespective of the species of Plasmodium. However, the incidence of thrombocytopenia was higher (100%) as compared to previous studies [15,22,25]. In the present study, severe thrombocytopenia was a rare finding in P. vivax infection (2.4%) similar to the study conducted by Robinson P et al., [26].

Khatib Y et al., reported 95.41% cases of P vivax and 81.57% of P falciparum having PI <5 [27]. In the present study, 65.6% cases of P. vivax and 35% cases of P. falciparum had PI <5 though, majority (≅85%) had P.I < 10.

In the present study it was observed that anaemia and thrombocytopenia were not a consistent finding in the relapse group. Normal Hb levels were reported in 52.6% of P. vivax and 42.8% of P. falciparum cases and normal platelet counts were observed in 14% of P. vivax and 28.5% of P. falciparum cases. The possible explanation is that the patients in relapse group have already taken treatment within 15 days for malaria; this may have contributed to normal Hb and platelet counts.

Thus, patients having prior history of malaria even after taking treatment presented again with fever should be investigated keeping malaria also as a possible diagnosis in mind. Malaria antigen test should be performed in cases of suspicion of malaria and peripheral smear should be thoroughly inspected for malarial parasite.

Further, on analysing the influence of parasitic densities on severity of anaemia and thrombocytopenia in both the groups, it was observed that there was not much relation between PI and severity of anaemia as there are numerous factors affecting anaemia in developing country like ours. However, thrombocytopenia is strongly related to PI.

Limitation

The present study was a single centre study with a relatively small sample size, which might limit statistical relevance. Further studies with a longer duration of follow-up and larger sample size may give more clinically relevant results.

Conclusion

Anaemia is a common finding in malaria, although in developing country like ours, anaemia can be multifactorial and is not much related to the parasitic densities. Thrombocytopenia is a consistent finding in initially diagnosed cases of malaria but cases of relapse/recurrence may present with normal platelet counts. Severity of thrombocytopenia is definitely related to PI. For low PI, severity of thrombocytopenia increases in initially diagnosed cases but is variable with cases of relapse/recurrence of malaria; however with high PI thrombocytopenia is more marked in initially diagnosed cases of malaria. Therefore, a close watch should be maintained on the platelet count in all cases of malaria to prevent and manage haemorrhagic episodes.

#figure in parenthesis indicate percentage#figure in parenthesis indicate percentage*p-value calculated using paired students t-test

References

References[1]Gupta NK, Bansal SB, Jain UC, Sahare K, Study of thrombocytopenia in patients of malaria Trop Parasitol 2013 3(1):58-61.

[2]Hussain MM, Sohail M, Abhishek K, Raziuddin M, Investigation on Plasmodium falciparum and Plasmodium vivax infection influencing host haematological factors in tribal dominant and malaria endemic population of Jharkhand Saudi J Biol Sci 2013 20:195-203.

[3]Tilak V, Jain R, Pancytopenia–a clinico-haematologic analysis of 77 cases Indian J Pathol Microbiol 1999 42(4):399-404.

[4]Dhungat MP, Dhungat PP, Thrombocytopenia in patients of malaria – correlation with type of malaria and it’s clinical significance Online International Interdisciplinary Research Journal 2013 3(2):21-26.

[5]Gill MK, Makkar M, Bhat S, Kaur T, Jain K, Dhir G, Thrombocytopenia in malaria and its correlation with different types of malaria Ann Trop Med Public Health 2013 6(2):197-200.

[6]Jairajpuri ZS, Rana S, Hassan MJ, Nabi F, Jetley S, An analysis of haematological parameters as a diagnostic test for malaria in patients with acute febrile illness: an institutional experience Oman Med J 2014 29(1):12-17.

[7]Patel A, Jain S, Patel B, Modi B, Haematological changes in P. Falciparum & P.Vivax Malaria Natl J Med Res 2013 3(2):130-33.

[8]Rajkumar A, Rao S, Sundaram S, Clinical outcome in malaria - reiterating the role of parasitic index Indian J Clin Prac 2012 22(9):450-53.

[9]Quintero JP, Siqueira AM, Tobon A, Blair S, Moreno A, Arevalo-Herrera M, Malaria-related anaemia: a Latin American perspective Mem Inst Oswaldo Cruz 2011 106(Suppl 1):91-104.

[10]Sumbele IU, Sama SO, Kimbi HK, Taiwe GS, Malaria, moderate to severe anaemia, and malarial anaemia in children at presentation to hospital in the mount cameroon area: a cross-sectional study Anaemia 2016 2016:5725634

[11]Abro AH, Ustadi AM, Younis NJ, Abdou AS, Hamed DA, Saleh AA, Malaria and haematological changes Pak J Med Sci 2008 24(2):287-91.

[12]Wasnik PN, Manohar TP, Humaney NR, Salkar HR, Study of clinical profile of falciparum malaria in a tertiary referral centre in Central India J Assoc Physicians India 2012 60:33-36.

[13]Kotepui M, Piwkham D, PhunPhuech B, Phiwklam N, Chupeerach C, Duangmano S, Effects of malaria parasite density on blood cell parameters PLoS One 2015 10(3):e0121057

[14]Biswas R, Sengupta G, Mundle M, A controlled study on haemograms of malaria patients in Calcutta Indian J Malariol 1999 36(1-2):42-48.

[15]Akhtar S, Gumashta R, Mahore S, Maimoon S, Haematological changes in malaria: A comparative study J Pharm Bio Sci 2012 2(4):15-19.

[16]Malhotra Bhatia A study of clinical and hematological manifestations of malaria Indian J Haematol Blood Transfus 1997 15(8):40-44.

[17]Sundar BS, Rao VG, Prasad D, Sankaraiah K, Reddy RS, Vinodh BP, Hematological and biochemical alterations in malaria patients with clinical correlation in a tertiary care hospital Int J BiolMed Res 2013 4(2):3139-42.

[18]Lonial S, Waller EK, Richardson PG, Jagannath S, Orlowski RZ, Giver CR, Risk factors and kinetics of thrombocytopenia associated with bortezomib for relapsed, refractory multiple myeloma Blood 2005 106(12):3777-84.

[19]Izak M, Bussel JB, Management of thrombocytopenia F1000Prime Rep 2014 6:45

[20]Bivalkar NY, Wingkar KC, Joshi AG, Jagtap S, Assessment of severity & types of anaemia during pregnancy in rural population in western Maharashtra Indian J Basic Applied Med Res 2014 4(1):160-63.

[21]Asadollahi K, Hastings IM, Beeching NJ, Gill GV, Asadollahi P, Leukocytosis as an alarming sign for mortality in patients hospitalized in general wards Iran J Med Sci 2011 36(1):45-49.

[22]Sharma SK, Das RK, Das BK, Das PK, Haematological and coagulation profile in acute falciparum malaria J Assoc Physicians India 1992 40(9):581-83.

[23]Richards MW, Behrens RH, Doherty JF, Short report: hematologic changes in acute, imported Plasmodium falciparum malaria Am J Trop Med Hyg 1998 59(6):859

[24]Maina RN, Walsh D, Gaddy C, Hongo G, Waitumbi J, Otieno L, Impact of Plasmodium falciparum infection on haematological parameters in children living in Western Kenya Malar J 2010 9(Suppl 3):S4

[25]Haroon H, Fazel PA, Naeem M, Mobin A, Naqvi AH, Makki K, Hide and seek: hematological aspects of malaria - a developing country perspective J Infect Dev Ctries 2013 7(3):273-79.

[26]Robinson P, Jenney AW, Tachado M, Yung A, Manitta J, Taylor K, Imported malaria treated in Melbourne, Australia: epidemiology and clinical features in 246 patients J Travel Med 2001 8(2):76-81.

[27]Khatib Y, Patel R, Sequeira K, Agrawal G, Chikhale N, Hematological and biochemical alterations in malaria and their correlation with Parasitic Index IOSR J Pharm 2015 5(9):53-56.