Hematological Characteristics of Yemeni Adults and Children with Visceral Leishmaniasis. Could Eosinopenia be a Suspicion Index?
1 Department of Medicine, Faculty of Medicine and Health Sciences, Sana'a University, Sana'a, Yemen
2 Department of Medicine, Hematology Unit, Al-Jomhori Teaching Hospital, Sana'a, Yemen
3 Department of Biochemistry and cytogenetics, Faculty of Medicine and Health Sciences, Sana'a University, Sana'a, Yemen
4 Al-Amana Specialized Laboratories, Sana'a, Yemen
Received: June 26, 2017
Accepted: August 3, 2017
Mediterr J Hematol Infect Dis 2017, 9(1): e2017056 DOI 10.4084/MJHID.2017.056
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Background and objectives:
Delay in the diagnosis of visceral leishmaniasis (VL) particularly in
non-endemic areas is associated with higher mortality. In our
experience, we found that marked bone marrow eosinopenia was a very
frequent accompaniment of VL and might be a useful clue for the
diagnosis, which indicates the opportunity for further morphological
assessment. The aim of this study was to describe the hematological
characteristics including peripheral blood and bone marrow findings of
Yemeni adults and children with VL.
Materials and Methods
The control group included 51 subjects, randomly selected from the records of 207 non-VL patients, 60 years old or younger (considering that the maximum age for patients with VL was 60 years old), who presented with fever, splenomegaly and pancytopenia or bicytopenia during the period of the study between October 2010 and October 2014. Their presenting CBC and WBC differential were taken before any treatment. They were performed by the same machine and in the same way as for all patients including patients with VL. The bone marrow examination was carried out at initial presentation as part of the evaluation of their splenomegaly and cytopenia. Their marrow aspiration Giemsa stained smears were reviewed to determine the eosinophil series percentage and to compare the results with those of patients with VL. They were examined by the same laboratory and clinical hematologist who reviewed the bone marrow smears of patients with VL.
Definitions. Bone marrow eosinopenia: eosinophil series count of less than 0.3% of total marrow myeloid cells calculated as the average number in at least 20 cellular fields examined i.e. at least 20x200= 4000 cells were counted [Normal range of eosinophils on aspirated bone marrow: 0.3-4.0% and the normal mean: 2.2%].
Dyserythropoiesis: Presence of dysplastic changes of erythropoiesis including megaloblastic features, binuclear and polynuclear normoblasts and other dyserythropoietic features (e.g. internuclear bridges, nuclear budding) with a frequency of > 5 per 100 erythroid cells
Hypercellular marrow: a cellularity > 50% in adults and > 80% in children.
Increased lymphocytes (marrow): Lymphocytes > 5% of total non-erythroid cells in adults and > 10% in children.
Increased plasma cells (marrow): Plasma cells > 5% of total non-erythroid cells in both adults and children.
Hemophagocytosis: Presence in the bone marrow of macrophages which phagocytize blood and bone marrow cells including red cells, erythroblasts, other leukocytes and or platelets.
Evaluation of iron stores: decreased marrow iron stores: less than one iron-positive cell, on the average for each x 40 field or absent iron-positive cells; increased marrow iron stores: more than two iron-positive cells for each x 40 field. Decreased marrow sideroblasts: sideroblasts less than 3% of total erythroblasts in the marrow.
Statistical analysis. The data were collected, tabulated and compiled in a computer database. SPSS version 21 was used to analyze data. Frequencies and percentages were used to describe categorical data. Unpaired Independent Samples T test was used to evaluate the comparison between adults and children regarding the means of Hb, PCV, MCV, MCH, WBC, platelet, neutrophil, lymphocyte, monocyte and eosinophil counts. Chi squared test was used to compare the degree of abnormal peripheral blood counts and also the peripheral blood and bone marrow morphological data between adults and children. Unpaired Independent Samples T test was used to evaluate the comparison between Patients with VL and control subjects regarding the means of Hb, WBC, platelet, neutrophil, lymphocyte, monocyte, and eosinophil counts. Chi squared test was used to compare the degree of abnormal peripheral blood counts and bone marrow eosinopenia between VL patients and controls.
Table 1 shows the mean values of the peripheral blood counts of adults and children with VL and table 2 shows the type and degree of abnormal peripheral blood counts.
|Table 1. The mean values of peripheral blood counts of Yemeni adults and children and with visceral leishmaniasis.|
|Table 2. Type and degree of abnormal peripheral blood counts in Yemeni adults and children with visceral leishmaniasis.|
All patients had moderate to severe anemia (Hb range: 4.6-10.4 g/dl) including 16 (32%) patients who had severe anemia; only one patient had Hb > 10 g/dl (10.4 g/dl).
Forty-one (87%) patients had leukopenia, and 42 (89.4%) patients had neutropenia including 34 (72.3%) patients who had significant neutropenia.
Thrombocytopenia was present in 44 (93.6%) patients including 39 (83%) patients who had significant thrombocytopenia. Eosinopenia was present in 42 (89.4%) patients including 27 (57.4%) patients who had absolute eosinopenia.
All patients had either pancytopenia or bicytopenia: 34 (72.3%) and 13 (27.7%) respectively.
The red blood cell morphological characteristics of Yemeni adults and children with visceral leishmaniasis showed that anisocytosis, anisochromia, and microcytic RBCs were the most common red blood cell morphological findings which were present in 30 (63.8%), 25 (53.2%) and 23 (49%) respectively. Ten (21%) patients had poikilocytosis, and five (10%) had tear drop red blood cells.
Table 3 shows the bone marrow morphological characteristics of Yemeni adults and children with visceral leishmaniasis. Regarding bone marrow morphological findings, marked bone marrow eosinopenia was the most common finding which was seen in 44 (93.6%) patients. Forty (85%) patients had hypercellular marrow, and 24 (51%) patients had dyserythropoiesis. Decreased iron stores were present in 27 (57.4%) patients, and 20 (42.6%) had a reduced number of sideroblasts. Only three (6.4 %) patients had increased iron stores. Hemophagocytosis was recognized in two (4.3) patients, and bone marrow plasmacytosis was seen in eight (17%) patients (Figures 1,2,3).
|Table 3. Bone marrow morphological characteristics of Yemeni adults and children with visceral leishmaniasis.|
|Figure 1. Bone marrow aspirate smear showing amastigote forms of Leishmania donovani associated with dyserythropoiesis (Giemsa 100x).|
|Figure 2. Bone marrow aspirate smear showing amastigote forms of Leishmania Donovani inside macrophages associated with frequent lymphocytes and dyserythropoiesis (Giemsa 100x).|
|Figure 3. Bone marrow aspirate smear showing amastigote forms of Leishmania Donovani inside macrophages associated with frequent lymphocytes and dyserythropoiesis (Giemsa 100x).|
The control group included 51 subjects (30 males and 21 females) with the main age (±SD) of 20.71±11.92 years (Range: 0.5-60). The mean values of the peripheral blood counts of control subjects was 7.80±1.75 (g/dl) for Hb, 2703.92±826.55 (x106/L) for WBC, 1069.76±626.50 (x106/L) for neutrophils, 1347.76±630.26 (x106/L) for lymphocytes, 88.10±108.84 (x106/L) for eosinophils, 201.06±197.72 (x106/L) for monocytes and 74.65±62.68 (x109/L) for platelets. Comparison of the mean values of peripheral blood counts between patients with VL and control subjects showed no significant difference in any of the above mean values except that patients had significantly lower eosinophil counts (p value 0.000) and lower lymphocyte count (p value 0.014). Table 4 shows comparison of abnormal peripheral blood counts and bone marrow eosinopenia between Yemeni patients with VL and control subjects. Patients had significantly more peripheral blood eosinopenia and lymphopenia and bone marrow eosinopenia compared to control subjects.
|Table 4. Comparison of abnormal peripheral blood counts and bone marrow eosinopenia between Yemeni patients with visceral leishmaniasis and control subjects.|
Most hematological features including anemia, leukopenia, thrombocytopenia, and pancytopenia are non-specific. Such features are frequent in patients with other infectious diseases, some hematological disorders and some autoimmune collagenous diseases.[7,25-28] Diagnosis of VL is straight forward in endemic areas where the disease is suspected and aided by confirmatory laboratory tests including reliable serological tests. However, in non-endemic areas, the differential diagnosis includes a broad spectrum of diseases as mentioned above and serological diagnosis is not reliable. Finding amastigotes in tissue smears is the most reliable diagnostic test. Splenic aspiration is a risky procedure and is not a usual in non-endemic areas. Bone marrow aspiration remains the safest procedure. However, the sensitivity of such method depends on the time spent examining the smears. Paying careful oriented attention and adequate time searching for the parasites increases the sensitivity to around 100%. However, such a time which may take few hours cannot be paid for all patients presenting with the above hematological features because of the high incidence of the diseases presenting with such features. Finding additional clues may limit the number of cases highly suspected of being VL, which need careful, time-consuming study.
The peripheral blood features of our patients which included anemia, thrombocytopenia, leukopenia, bicytopenia, and pancytopenia are not different from those reported from other studies in Asia, Africa, and Mediterranean region or South America.[4,29] Hypercellular marrow and dyserythropoiesis were also common in our patient group which is similar to that reported in other studies.[30,31] Bone marrow lymphocytosis was also frequent among our patients similar to other studies. Our patients also had significant peripheral blood lymphocytopenia. The presence of peripheral blood lymphopenia in association with bone marrow lymphocytosis has been explained by the notion that lymphocytes migrate to the affected lymphoid tissues to build an inflammatory response and that bone marrow lymphocytosis is a compensatory response that provides lymphocytes to organs affected by the parasite.[32,33] Only one child and one adult of our patients had bone marrow features of hemophagocytosis. Hemophagocytosis was reported to be a rare occurrence in patients with visceral leishmaniasis causing diagnostic dilemma and unusual presentation.[11,34] Our patients showed decreased marrow iron which is consistent with their common finding of microcytic red blood cells. This picture is due to the malnutrition these patients usually have as a consequence of anorexia and is also explained by the fact that the disease affects the poor population predominantly.[25,35] Severe anemia, malnutrition and long duration of illness were shown to be associated with an increased risk of death.[25,36] These issues should be addressed in evaluating and managing patients with VL. The marked bone marrow eosinopenia associated with marked peripheral blood eosinopenia are the characteristics which were most common in our patients with VL and usually are not reported both together in other simulating illnesses. Bone marrow eosinopenia in VL has not been signaled in humans so far. However, it has been reported in symptomatic canine VL as opposed to asymptomatic canine VL and was found to be correlated with peripheral eosinopenia and it has been regarded together with peripheral blood lymphocytopenia as a biomarker of severe disease. Eosinophilic hypoplasia in symptomatic canine VL has been explained by bone marrow dysfunction, which may have contributed to the severe eosinopenia.[37,38] On the other hand, Eosinophil infiltration in the lymph-nodes of mice infected by Leishmania major was found to be influenced by sex and parasitic load and that it reflects ineffective inflammation. The previous studies in humans on hematological manifestations of VL did not evaluate bone marrow eosinopenia as a feature or as a clue to the diagnosis of the disease. A single study reported three of 18 bone marrow aspirates who had prominent marrow eosinophils. However, the authors themselves of the study did not find any report of similar observation in the searched medical literature. We also didn't find other reports of this finding of marrow eosinophilia. Therefore, secondary causes of eosinophilia could not be excluded in these cases. Furthermore, the percentage of those patients with eosinophilia was too small to regard it a significant finding: 3/18 [16%]. Our study also showed that there was no significant difference between adults and children regarding peripheral blood and bone marrow eosinopenia or concerning other hematological features.
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