|Year : 2019 | Volume
| Issue : 1 | Page : 53-60
Hematological features in malarial infection and their variations with parasite density: A retrospective analysis of 6-year data in an Indian city
Prabu Anandhan Motchan1, Priya Subashchandrabose1, Madhu Basavegowda2, Anita Suryanarayan1
1 Department of Pathology, MMetropolis Healthcare Limited –Chennai Laboratory (formerly Lister Metropolis Laboratory), Chennai, Tamil Nadu, India
2 Department of Community Medicine, JSS Medical College, Mysore, Karnataka, India
|Date of Web Publication||18-Feb-2019|
Dr. Prabu Anandhan Motchan
Metropolis Healthcare Limited –Chennai Laboratory (formerly Lister Metropolis Laboratory), #3, Jagannathan Road, Nungambakkam, Chennai - 600 034, Tamil Nadu
Source of Support: None, Conflict of Interest: None
BACKGROUND: Considering that physicians routinely perform complete blood counts (CBCs) for patients with fever in India, it is useful in comparing these counts across malaria positive and negative cases, as well as the variation of these features with parasite density.
OBJECTIVES: This study aims to compare hematological characteristics in malarial positive and negative patients, compare parasite density with hematological characteristics, estimate sensitivity and specificity of hematological characteristics as indicators of malarial infection, and compare platelet distribution width (PDW) and plateletcrit (PCT) in positive cases with negative cases.
MATERIALS AND METHODS: We performed a retrospective analysis of 6-year data in a pathology laboratory to compare CBC of malaria-positive (n = 299) versus malaria-negative blood (n = 287) samples. PDW and PCT tests were available for some samples (n = 100) for a subanalysis. Parasite density, determined by scoring (1+ to 4+) in quantitative buffy coat-malarial parasite detected cases (n = 279), was compared with hematological features.
RESULTS: The hematology of malarial infection was characterized by bicytopenia or pancytopenia (P < 0.001, odds ratio [OR] = 11.14) of which thrombocytopenia was the most prominent component (P < 0.001, OR = 37.94), followed by anemia due to reduction in red blood cell (RBC) count effecting a decrease in packed cell volume (PCV) (P < 0.003, OR = 2.13) with an elevated red cell distribution width (P < 0.025, OR = 1.78). Higher parasite density was associated with increased incidence of anemia and severe thrombocytopenia. PDW was elevated (P < 0.001, OR = 6.93) and PCT was reduced (P < 0.001, OR = 123.64) in positive cases.
CONCLUSIONS: Thrombocytopenia with reduction in PCV or reduced RBC count is a distinguishing feature of malaria.
Keywords: Malaria, packed cell volume, pancytopenia, parasite density, thrombocytopenia
|How to cite this article:|
Motchan PA, Subashchandrabose P, Basavegowda M, Suryanarayan A. Hematological features in malarial infection and their variations with parasite density: A retrospective analysis of 6-year data in an Indian city. Int J Health Allied Sci 2019;8:53-60
|How to cite this URL:|
Motchan PA, Subashchandrabose P, Basavegowda M, Suryanarayan A. Hematological features in malarial infection and their variations with parasite density: A retrospective analysis of 6-year data in an Indian city. Int J Health Allied Sci [serial online] 2019 [cited 2019 Jul 17];8:53-60. Available from: http://www.ijhas.in/text.asp?2019/8/1/53/252452
| Introduction|| |
Malaria parasites, given their ability to invade erythrocytes, can affect changes in hematological parameters of the host. Anemia in malaria-positive patients has been documented as a distinctive feature.,,, Thrombocytopenia, another frequent complication in patients with malaria, was initially reported in the 1960s with documented cases attributed to either Plasmodium vivax or Plasmodium falciparum infection. The feature of low platelet count, which defines thrombocytopenia, may be inversely related to malarial parasite density,, while others negate this relationship.,,
Detection of malaria infection is usually by peripheral smear, and parasite density is reported either as the percentage of infected red blood cells (RBCs) or as parasites per white blood cell (WBC) count multiplied by the number of WBCs per microliter of blood. Therefore, it is worthwhile comparing the quantitative buffy coat-malarial parasite (QBC-MP) score of malaria-positive patients with their respective hematological features. QBC-MP test for diagnosing malaria parasite is reported to have a sensitivity of 97.77% and specificity of 99.73% in a laboratory setting compared to thick and thin peripheral blood smears (PBSs). This test also gives a reliable estimate of parasite density as the average number of parasites was observed in 20 fields. The basis of the QBC-MP method is that as malarial parasites within the RBC mature, they decrease the buoyant density of RBCs, and when centrifuged in microhematocrit tubes coated with acridine orange dye, the infected RBCs are positioned between packed RBCs and buffy coat. However, it has been noted that evaluation of hematological changes as clues for diagnosing malaria is more desirable with a larger sample size which lends greater validity.,,
Initially, it was believed that in malaria-infected patients, platelets were being consumed in intravascular coagulation as abnormal coagulation tests were noted., However, these findings could not be confirmed by others.,, Subsequent studies hypothesized that platelets were altered immunologically before removal by the reticuloendothelial system at an excessive rate. Furthermore, evidence of antimalarials responsible for causing thrombocytopenia is absent.,
A retrospective study, in New York, on patients who had traveled to malaria endemic countries noted that thrombocytopenia was useful in predicting malaria, with a sensitivity of 100% and specificity of 86%, lending utility to physicians who initially missed the diagnosis of malaria in 80% of cases. In comparison, a study in India observed that thrombocytopenia had a sensitivity of 60% and a specificity of 88% in predicting malaria. A hospital-based study of 172 malaria-positive patients in a group of 723 patients with fever noted a significant reduction in hemoglobin, platelet count, total leukocyte count (TLC), and an increase in red cell distribution width (RDW) in the malaria-positive patients and so suggested that these hematological changes could be used to predict malaria.
This study, therefore, seeks to (i) compare hematological characteristics in malaria-positive and malaria-negative cases, (ii) assess the association between parasite density and various hematological characteristics, (iii) estimate sensitivity and specificity of hematological characteristics as indicators of malarial infection, and (iv) assess platelet distribution width (PDW) and plateletcrit (PCT) in malaria-positive cases and compare them with malaria-negative cases.
To fulfill these objectives, this study was conducted using data from 2011 to 2016 collected in a laboratory receiving specimens from various localities of Chennai, capital city of Tamil Nadu state, India, with all laboratory analyses performed at a single diagnostic facility.
| Materials and Methods|| |
As per data from the Tamil Nadu Health and Family Welfare Department, it is observed that incidence of malarial infection is reducing in the state. Chennai is endemic for malaria and reports the highest proportion of cases in Tamil Nadu.
Various hematology reports performed of malaria-positive cases from the year 2011 to 2016 were collected. These reports were retrieved from electronic records maintained by Metropolis Healthcare Limited, Nungambakkam, Chennai. This is a stand-alone laboratory receiving specimens from clinics in various localities of Chennai. Cases were detected either by QBC-MP or PBS methods. In this laboratory, blood counts are performed using a fully automated cell counter (Coulter® LH780, Beckman Coulter Inc., California). Patient data were irreversibly anonymized to maintain confidentiality, and ethical approval was obtained.
[Figure 1] is an algorithm for the study flow and the final recruitment number of samples for data analyses based on the inclusion and exclusion criteria. Initially, reports of all patients irrespective of gender and age who were QBC-MP or PBS positive for malarial parasite and had either a complete blood count (CBC) or a few components of a CBC were included in the study. A total of 14675 QBC-MP and 2370 PBS samples for malaria detection were reported from 2011 to 2016. Cases with concomitant dengue or Leptospira positive reports were excluded from the study. Of these, 490 samples were QBC-MP positive while 57 were PBS positive for malaria. Of the 490 QBC-MP-positive reports, 279 met the inclusion criteria. Of these 279 samples who met the inclusion criteria, 260 had CBCs, 16 had only platelet counts, 2 had only hemoglobin, and 1 had only total and differential counts (DC). This resulted in the sample size differing for each hematological characteristic. 211 QBC-MP reports did not have any other hematological investigations. Of the 57 smear-positive hematological investigations, 20 had CBCs and were included in the study. Then, 279 QBC-MP-positive reports were utilized for studying changes in hematological characteristics with variations in QBC-MP grade. A total of 299 malaria-positive cases (both QBC-MP and smear positive) were utilized for evaluating changes in hematological characteristics as predictors of malaria. A total of 287 malaria-negative cases were built by stratified random sampling for each year from 2011 to 2016. Cases with positive dengue, Leptospira, raised erythrocyte sedimentation rate, or C-reactive protein were excluded from the study.
|Figure 1: Flowchart depicting blood samples investigated for malaria and other hematological parameters|
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One part of the study involved grouping the 279 QBC-MP-positive reports according to the grade and then comparing their hematological characteristics. QBC-MP grade is the average number of malarial parasites observed in 20 fields when the microhematocrit tube is viewed under fluorescent microscopy. The assigned grades are 1+: <1parasite/field, 2+: 1–10/field, 3+: 11–100/field, and 4+: >100/field. In this study, reports with QBC-MP 1+ and 2+ were combined into a single group referred to as "low parasite density" while QBC-MP 3+ and 4+ were grouped together as "high parasite density." Subsequently, hematological characteristics of 299 malaria-positive reports and 287 malaria-negative reports were compared. Another analysis was performed as regards PCT and PDW for 21 available malaria-positive patients, and these were compared with 79 malaria-negative cases with the same. This was because PCT and PDW tests were only introduced in this laboratory after June 2016, resulting in limited sample availability for our study.
The variables examined in this study were hemoglobin, RBC count, packed cell volume (PCV), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), MCH concentration (MCHC), red cell distribution width (RDW), platelet count, mean platelet volume (MPV), TLC, DCs, PCT, and PDW. Specific hematological tests were defined as either normal or abnormal using the appropriate reference range for age and gender presented in textbook Dacie and Lewis Practical Haematology.
Data were stored in a Microsoft Excel® spreadsheet. Statistical analysis was performed using SPSS for Windows version 23 (IBM©, Chicago, IL, USA). Percentages were calculated for nominal and ordinal data such as normal or altered hematological tests for different QBC-MP grades and for malaria-positive and malaria-negative groups. Analysis was done by binary logistic regression, initially univariate followed up with stepwise multivariate logistic regression. Odds ratios (ORs) were reported. P = 0.05 or less was considered significant. Sensitivity, specificity, likelihood ratio, positive predictive values (PPVs), and negative predictive values (NPVs) of hematological tests for predicting malaria were also calculated, using an online module.
| Results|| |
Demographic and hematological profile
A total of 299 malaria-positive patients were identified in the final analysis. Of these, 268 (89.6%) were 13 years and above, 5 (1.7%) were 1 year old, 13 (4.3%) were between 2 and 6 years, and another 13 (4.3%) were between 7 and 12 years old. Mean age was 34.4 ± 18.5 years. As per gender distribution, 194 patients were male (64.9%) with a male-to-female ratio of 9:5. Of the total 299 positive cases, 290 were infected with P. vivax, two with P. falciparum, and seven infected with both P. vivax and P. falciparum. [Table 1] provides hematological characteristics of malaria-positive versus malaria-negative cases.
|Table 1: Comparison of hematological characteristics of malaria-positive and malaria-negative cases|
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In univariate analysis, incidence of reduced hemoglobin, reduced RBC count, reduced PCV, elevated RDW, thrombocytopenia, elevated MPV, as well as leukopenia, neutrophilia, and monocytopenia, respective for age and gender, were found to be significantly higher (P < 0.05) in the malaria-positive group. The other hematological characteristics were not significantly different between malaria-positive and malaria-negative groups (P > 0.05). In 100 cases with 21 malaria positive and 79 malaria negative, elevated PDW criteria were present in 61.9% of malaria-positive versus 19% in malaria-negative cases, and this comparison was statistically significant (P < 0.001). Reduced PCT criteria respective for age were present in 95.2% of malaria-positive versus 13.9% in malaria-negative cases (P < 0.001).
All variables significant at 0.25 and below in the univariate analysis except for PDW and PCT [Table 1] were tested in a multivariate analysis. The variables that were significant in the stepwise method are shown below in [Table 2].
In stepwise multivariate analysis, only reduced PCV, elevated RDW, and thrombocytopenia were significant. The OR and intervals are given in [Table 2].
An interaction effect between thrombocytopenia and reduced RBC count (P < 0.001, OR: 12.61) as well as leukopenia (P < 0.001, OR: 6.697) prompted a further analysis. Separately, the malaria-positive and malaria-negative cases were identified as having pancytopenia (below normal RBC, leukopenia, and thrombocytopenia) or bicytopenia (any two of the features of pancytopenia) and then compared in [Table 3]. This was not included with the initial analysis as bicytopenia or pancytopenia would be collinear with thrombocytopenia.
In another subanalysis, it was noted that incidence of reduced MCV was significantly higher (26.9%) in malaria-positive cases with elevated RDW as against 11.9% in malaria-positive cases with normal RDW (P = 0.003). Furthermore, it was noted that within the malaria-positive group, incidence of elevated MPV was greater in thrombocytopenic malaria-positive cases compared to nonthrombocytopenic malaria-positive cases (P < 0.05).
Hematological characteristics versus parasite density (quantitative buffy coat-malarial parasite score)
Parasite density defined by QBC-MP scoring was assessed against laboratory characteristics, and these data are summarized in [Table 4]. In univariate analysis, incidence of anemia, reduced MCV, reduced MCH and thrombocytopenia as well as neutrophilia and monocytopenia, respective for age and gender, was significantly higher (P < 0.05) in cases with higher parasite density. Characteristics such as RBC count, MCHC, MPV, TLC, lymphocyte count, eosinophil count, and basophil count were not differential between low and high scores (P > 0.05) while tests such as PCV and RDW, although not significant, were still indicative of a trend.
|Table 4: Variations in hematological characteristics with parasite density|
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However, after stepwise multivariate logistic regression, only increased incidence of thrombocytopenia (P = 0.005 OR: 2.589, confidence interval [CI]: 1.330, 5.041) and reduced hemoglobin (P = 0.003 OR = 2.284 CI: 1.336, 3.902) was significantly associated with higher parasite density.
Another significant observation was that 20% of high parasite density cases had platelet counts below 50,000/cumm compared to 10.4% of low parasite density cases, and conversely, only 14.1% of high parasite density cases had platelet above 150,000 compared to 28.3% of low parasite density cases (P = 0.005).
Sensitivity and specificity
Thrombocytopenia as a test for malaria diagnosis has the highest sensitivity of 82.43% (95% CI: 77.61, 86.59), specificity of 89.55% (95% CI: 85.41–92.84), positive likelihood ratio of 7.89, negative likelihood ratio of 0.20, PPV of 89%, and NPV of 90%. However, thrombocytopenia with reduced PCV had the highest specificity of 95.12 (95% CI: 91.95, 97.31). Others are given in [Table 5].
|Table 5: Sensitivity and specificity of potential indicators for malaria infection|
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| Discussion|| |
Malaria infection was associated with incidence of reduced PCV for age group and gender in this study. This reduced PCV observed was associated with reduced RBC count (P < 0.001, OR: 2.97), and this interaction was stronger than that between PCV and hemoglobin (P < 0.001, OR: 2.22). An observation from our study was that higher parasite density was associated with increased incidence of reduced hemoglobin. It is evident thus that in the malaria positive patients, RBC count reduction leads to PCV reduction which subsequently causes reduction in hemoglobin level. This is in contrast to dengue hemorrhagic fever in which an increase in PCV may be noted. However, it is well established that anemia is widely prevalent in the Indian population, which is also consistent with its incidence in 31.7% of the malaria-negative cases in this study. But to note, malaria patients are more prone to excessive hemolysis and reduced RBC production which leads to anemia., Malaria infection was associated with elevated RDW in this study, which has also been noted by another study. Elevated RDW may arise from RBCs enlarged due to infection with malarial parasites. Although a correlation between elevated RDW and macrocytosis has been reported previously, we could not confirm this but instead found malaria-positive cases with elevated RDW had a significantly higher incidence of reduced MCV. This could be due to the high prevalence of iron-deficiency anemia in the Indian population, which results in microcytic and hypochromic RBCs.
As expected, incidence of thrombocytopenia in this study was associated with malaria infection. However, in contrast to other studies,,, we are in agreement with some, that higher parasite density is associated with increased incidence of thrombocytopenia. In this study, a significantly greater percentage of individuals with higher parasite density had platelet counts <50,000/cumm. Thrombocytopenic malaria-positive patients had significantly higher incidence of elevated MPV which corroborates with a study, which explained the increase in platelet size as a compensation for reduced number of platelets in the periphery. In our study, P. vivax was the predominant infecting species (97%), with P. falciparum and mixed infections accounting for the remaining cases. Therefore, we did not compare the hematological variations with regard to species of the parasite. Other studies, however, have reported thrombocytopenia in both P. vivax malaria and P. falciparum malaria with no significant difference in mean platelet counts of both groups.,
Two studies, reported leukopenia and monocytopenia in malaria-infected patients. In our study after the multivariate analysis, we observed that these were insignificant when compared to thrombocytopenia. However, in a separate analysis, when RBC count, total leucocyte count, and platelets are seen in the context of pancytopenia or bicytopenia, we report a strong association between bicytopenia or pancytopenia and malarial infection. This was strengthened by the strong association between thrombocytopenia and reduced RBC count, leukopenia, and monocytopenia.
Malaria infection was associated with incidence of increased PDW and reduced PCT in our study, and this may be attributed to platelet anisocytosis. Elevated MPV and PDW have been also observed in patients with idiopathic thrombocytopenic purpura. In relation to sensitivity and specificity, among all the tests, thrombocytopenia had highest sensitivity of 82.43% and specificity of 89.55% with likelihood ratio of 7.89. However, in practice, it will not be very specific, considering that dengue and leptospirosis infections are also present with thrombocytopenia. This is why we excluded dengue and leptospirosis cases from this study. With this exclusion, thrombocytopenia with reduced PCV as a test for malaria diagnosis gave a higher specificity of 95.12%.
| Conclusions|| |
The hematology of malarial infection is characterized by bicytopenia or pancytopenia of which thrombocytopenia is the most prominent component (with the highest sensitivity), followed by anemia due to reduction in RBC count effecting a decrease in PCV with an elevated RDW. There is a higher incidence of leukopenia as well among malaria-positive patients with a higher incidence of monocytopenia and neutrophilia. Higher parasite density is associated with an increased incidence of anemia, increased incidence of thrombocytopenia, and severe thrombocytopenia. Finally, since thrombocytopenia is common to both dengue and malaria (both prevalent in this part of the world), thrombocytopenia with reduction in PCV or reduced RBC count seen in the latter could serve as a distinguishing feature from the former, which is characterized by a rise in PCV. This is very relevant clinically. Monocytopenia and neutrophilia are features seen in malarial infection that can help in this regard too, considering that dengue infection has neutropenia instead. These hematological features can help physicians suspect dengue infection, considering that the laboratory diagnosis of malaria using thick and thin blood smears demands experience of the technicians, which may be wanting sometimes. Furthermore, the more sensitive and specific test QBC-MP is not available in all centers and may not be ordered for economically weaker patients.
We could not exclude patients with iron-deficiency anemia from the sample for want of serum iron investigations.
It would be interesting to compare the hematological features of malaria- and dengue-positive cases, especially PCV, monocyte, and neutrophil counts, as a follow-up of this study which is of clinical relevance.
The authors express immense gratitude to Prof. Tilakavati Karupaiah (National University of Malaysia) and Associate Prof. Karuthan Chinna (University of Malaya) for their very valuable guidance and inputs.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]