INTRODUCTION

Malaria has wreaked havoc on mankind since ancient times and remains a priority public health problem in tropical and subtropical regions of the world. There are currently five species of parasites of the genus Plasmodium that cause disease in humans, which are transmitted by the bite of mosquitoes of the genus Anopheles, with Plasmodium vivax (Pv) and Plasmodium falciparum (Pf) being the most dangerous and frequent ¹.

In 2020, there were 241 million malaria cases and 627,000 deaths worldwide, with an increase of 14 million cases and 69,000 deaths from the 2019 figures ²; with a sharp increase in malaria cases and deaths globally, despite the effort deployed by malaria control programs to achieve malaria elimination in endemic countries, which were affected by the interruptions in health services during the COVID-19 pandemic ³.

In Peru, malaria permanently threatens the health of the population, mainly in the department of Loreto (capital, Iquitos) where 90% of cases are concentrated, with a predominance of Pv followed by Pf ^{4 - 6}. This department is classified as an endemic region with low transmission among rural and peri-urban communities, although there are high foci of transmission in some parts of Loreto ⁷. The clinical characteristics of malaria depend on the species and range from short febrile episodes to severe systemic complications and death ^{3 , 8}.

The change in leukocyte parameters is the most common complication found in malarial patients and plays an important role in the clinical severity of the disease and mortality ⁹. Malaria with severe organ failure or blood abnormalities is usually associated with Pf ^{3 , 10}. In addition to severe anemia, there are other alterations such as thrombocytopenia or reduction in the number of platelets, leukocytosis or leukopenia, hemoglobinuria, cardiovascular collapse and shock, acute kidney injury, metabolic acidosis, and hypoglycemia ^{11 - 13}. In contrast, mild malaria, with benign forms, is related to Pv, but is also capable of producing severe anemia, shock, lung injury, acute renal failure, and cerebral malaria ^{12 , 13}.

Early diagnosis is important for prompt and timely treatment and disease management ^{1 - 3}. However, knowledge of the hematological characteristics of malaria is limited to Pf cases from African and Asian regions ^{14 , 15}, and little or nothing is known about the leukocyte characteristics of Pv in endemic populations of Loreto; its relationship as a tool for hematological diagnosis could allow a more adequate management of the possible complications of malaria. This study evaluated the variation of hematological profiles before, during and after treatment of patients infected with uncomplicated Plasmodium vivax (Pv) and P. falciparum (Pf) malaria in a population of the Loreto region, comparing it with healthy individuals from the same area for its potential use as predictors of disease progression and treatment efficacy.

MATERIALS AND METHODS

RESULTS

Between the years 2010 and 2012, 425 participants aged from 10 to 44 years were evaluated; 236 were female and 189 were male.

All participants were confirmed to have malaria by microscopy, which resulted in 93 (21.9%) positive for Pv, 34 (8.0%) positive for Pf and 298 (70.1%) negative for malaria. All microscopy-positive results and 99 participants randomly selected from the 298 microscopy-negative were evaluated by PCR to confirm the results.

As shown in Table 1, the baseline hematological indicators such as hematocrit, blood cell count (RGB), banded neutrophils, segmented neutrophils, eosinophils, and platelets were statistically different (p < 0.001) among participants infected with uncomplicated malaria compared to the negative group (participants without malaria). Monocytes and lymphocytes in the studied groups were not statistically different from each other.

For visit 1 (day 0 of the study), analyses of the generalized linear model with identity linkage adjusted for age and gender conducted to quantify the differences between participants infected with P. vivax and P. falciparum (Figure 1) compared to the negative group (Table 2), showed that participants infected with Pv and Pf obtained low percentages for hematocrit (Pv: -2.3%, Pf: -2.7%), RGB (Pv: -0.6%, Pf: -0.5%), eosinophils (Pv: -3.6%, Pf: -3.2%) and platelets (Pv: -2.8%, Pf: -2.6% x 104 platelets/mm³) compared to the negative group (participants without malaria). On the contrary, malaria-infected participants obtained high percentages of banded neutrophils (Pv: 2.1%, Pf: 3.7%) compared to the negative group. We found a different pattern only in segmented neutrophils, where only those infected with Pv had a higher percentage (2.7%) compared to Pf and the negative group.

Figure 1 Hematological profiles by day of visit and Plasmodium species. 

The analysis of variations in the hematologic profile over time compares visit 2, day 7 (during treatment) and visit 3, day 28 (after treatment) with visit 1, day 0 and Plasmodium species. An increasing and decreasing continuous linear relationship between time and outcome was found in model 1 (Table 3), for platelets and ablated neutrophils, respectively. For platelets, those positive for Pv and Pf had a daily increase of 0.11 x 10⁴ platelets/mm³. Pf positives showed a daily decrease of 0.10% of banded neutrophils, whereas Pv infections had a daily decrease of 0.1% less than that observed for Pf infections.

With model 2 (Table 4), we found a nonlinear relationship between time and outcome for eosinophils, hematocrit and segmented neutrophils with no difference between Plasmodium species. In Pf and Pv infections, eosinophils had an initial mean increase of 5.9% at visit 2, day 7 (during treatment) and a reduced increase of 2.6% at visit 3, day 28 (after treatment) when compared to the initial measurement (visit 1, day 0, before treatment). This is why results show a decline without achieving normalization. In contrast, hematocrit and segmented neutrophils showed an average decrease of 3.2% and 3.1% respectively at visit 2, day 7 (during treatment), but no difference was found at visit 3, day 28 (after treatment) compared to the baseline (visit 1, day 0, before treatment), thus achieving normalization of the values. In this model, only banded neutrophils from Pv-infected participants were different at visit 2 with a 1.9% decrease, lower than in Pf-infected participants, but not at day 28 (after treatment).

DISCUSSION

In this study we found that patients infected with Pv and Pf had lower infection indicators such as hematocrit, WBC, eosinophils, and platelets, in contrast, segmented neutrophils and banded neutrophils had higher values. We found variations of the hematological profile over time (before, during and after treatment) in both Plasmodium species, with a daily decrease in ablated neutrophils and a daily increase in platelets. In addition, regarding the differences by Plasmodium species over time, the Pv-infected participants showed a lower percentage of banded neutrophils compared to the Pf-infected participants.

Our study is one of the first reports in the Loreto region that demonstrates the variations in hematological profiles over time (before, during and after treatment) in patients infected with uncomplicated malaria according to Plasmodium species (Pv or Pf), with hematological indicators such as hematocrit, WBC, banded neutrophils, segmented neutrophils, eosinophils, and platelets being statistically different (p < 0.001) from the negative group (participants without malaria).

The elimination of malaria remains a challenge in malaria endemic countries, because it can be a fatal disease and about half of the world’s population is at risk of contracting it ², which is why, in 2015, WHO launched the Global Technical Strategy for Malaria 2016-2030, highlighting the urgent need to increase interventions, improve preventive measures, diagnostic testing, treatment and surveillance of the disease. In Peru, malaria cases mostly occur in the Amazon, mainly in the Loreto region, with more than 85% of the country’s cases ⁵, and with the presence of the two most dangerous species (Pv and Pf), making it a major public health concern.

Malaria is known to produce hematological abnormalities including anemia and thrombocytopenia due to Pf and Pv, which are considered a hallmark of malaria ^{11 , 19}. Hematologic changes are the most common complications encountered in malaria patients and have been correlated with mortality and prognosis in malaria-infected patients ^{9 , 19}. Evaluating the white blood cell count and its differentials are among the most basic and primary studies done in patients presenting with fever of short duration. Likewise, neutrophils are essential to fight the disease mainly when the patient presents acute fever, which is expected to decrease after the patient is treated ^{20 , 21}.

We found that white blood cell levels were lower in Pv and Pf infected patients, which is similar to other studies conducted in endemic areas ^{19 , 22}. Unlike another study in the Amazon (Brazil) ²³, we found an increase in the segmented neutrophil count in malaria-infected patients compared to controls, although it was not as marked as what was described by Kini and Chandrashekhar ¹⁹. However, WBC levels were low in infected persons, and did not show leukopenia (<4000 cells/µL), as reported by McKenzie et al. ²⁴ in a previous study in Iquitos, Peru. It has been described that the WBC count decreases at the same time as fever begins and infection becomes detectable by microscopy ¹⁸. In addition, lower white blood cell counts in infected persons are characteristic at the onset of a blood infection ²⁵.

Likewise, thrombocytopenia is one of the most persistent symptoms in severe malaria and manifests in varying percentages with different severity degrees; it has also been reported as a sensitive marker for the diagnosis of malaria in acute febrile illness ^{26 , 27}. In this study thrombocytopenia was found to be associated with Plasmodium infections, and was not related to prognostic severity.

Although neutrophil levels were high in Pv infections, banded neutrophils showed higher levels in Pf infections, similar to those reported in studies from India ²⁸ where the ratio of counts was uninfected < Pv < Pf. These high levels in Pf infections could be due to the large inflammatory response caused by the parasites. Banded neutrophils are considered acute phase markers with a tendency to increase on the first day of blood infection ²³.

The high number of eosinophils in the negative group (9%) is common in the Peruvian Amazon and may be explained by the high prevalence of soil-transmitted helminth infections ²⁹ and protozoa (47%) ³⁰, an aspect to be considered for future studies in order to avoid confounding factors.

It is necessary to recognize some limitations of our study, first the age of the data (2010-2012) and the small number of diagnosed Plasmodium infections, which did not allow a broader analysis of the differences in the levels and composition of leukocytes in these two Plasmodium species. Another limitation was the absence of a clinical variable such as duration of symptoms, comorbidities (HIV, malnutrition, etc.) or the presence of pregnancy, which are associated with hematological changes. Despite these limitations, this study has allowed us to find that hematological variations do exist in patients infected with Plasmodium sp. in the Peruvian Amazon.

In conclusion, malaria infections produced by Pv and Pf, at the onset of infection (before treatment) show a decrease of the hematocrit, eosinophils, and platelets, with an increase in banded neutrophils, showing that these hematological parameters vary over time during and after treatment; platelets increased and banded neutrophils decreased. Differences between Plasmodium species over time showed a marked daily decrease in banded neutrophils in Pv-infected patients compared to Pf. These results demonstrate that the hematological profile in uncomplicated malaria-positive participants varies over time during and after treatment, and is an indicator of disease progression that can help in the therapeutic surveillance of Plasmodium-infected patients. Future studies should measure hemoglobin levels to avoid confusion and clarify the role of rapid diagnosis for active case detection with the onset of anemia and the study of hematologic profiles in patients who develop severe malaria.