Diagnostic Value of Bone Marrow Cell Morphology in Visceral Leishmaniasis-Associated Hemophagocytic Syndrome of Two Cases


 Background: Visceral leishmaniasis related-hemophagocytic lymphohistiocytosis (VL-HLH) is a hemophagocytic syndrome caused by Leishmania infection. VL-HLH is rare, especially in nonendemic areas where the disease is severe, and mortality rates are high. The key to diagnosing VL-HLH is to find the pathogen; therefore, the Leishmania must be accurately identified for timely clinical treatment.Case presentationWe retrospectively analyzed the clinical data, laboratory examination results and bone marrow cell morphology of two children with VL-HLH diagnosed via bone marrow cell morphology between July 2017 and January 2021 at Kunming Children’s Hospital of Yunnan, China.Two cases suspected of having malignant tumors at other hospitals and who had undergone ineffective long-term treatment were transferred to Kunming Children’s Hospital. They had repeated fevers, pancytopenia, hepatosplenomegaly, hypertriglyceridemia, and hypofibrinogenemia over a long period and met the HLH-2004 standard. Their HLH genetic test results were negative, and primary HLH was excluded. Both children underwent chemotherapy as per the HLH-2004 chemotherapy regimen , but it was ineffective and accompanied by serious infections. We found Leishmania amastigotes in their bone marrow via morphological examination of their bone marrow cells, which showed hemophagocytic cells; thus, the children were diagnosed with VL-HLH. After being transferred to a specialty hospital for treatment, the condition was well-controlled. Conclusion: Morphological examination of the bone marrow cells played an important role in diagnosing VL-HLH. When clinically diagnosing secondary HLH, VL-HLH should be considered in addition to common pathogens, especially in patients for whom HLH-2004 chemotherapy regimens are ineffective. For infants and young children, bone marrow cytology examinations should be performed several times and as early as possible to find the pathogens to reduce potential misdiagnoses.


Background
Hemophagocytic syndrome (HPS), also known as hemophagocytic lymphohistiocytosis (HLH), is divided into primary and secondary forms. The secondary etiology is complex and can be caused by infection, malignant tumors, and autoimmune diseases.Visceral leishmaniasis related-hemophagocytic lymphohistiocytosis (VL-HLH) is very rare in childhood, especially in nonendemic areas. The disease is severe with high mortality rates of up to 100% without early diagnosis and treatment [1]. Therefore, the leishmaniasis-associated pathogen must be rapidly and accurately identi ed for clinical and timely treatment. Here, we report two young patients with VL-HPS diagnosed via bone marrow cell morphology at the Children's Hospital of Kunming, Yunnan, China in the past 5 years.

Case Presentation
Case 1 A 2-year-old girl from Weining, Guizhou, China, presented in July 2017 with a repeated irregular fever lasting 3 months and reaching 39°C-40°C. After the illness, she was hospitalized at a local hospital for 2 months. Her whole blood cells decreased progressively, and she underwent symptomatic and supportive treatment, including meropenem, vancomycin, piperacillin, tazobactam, gamma globulin, methylprednisolone, cefoperazone, sulbactam, Taineng, and a blood transfusion. She continued to have repeated fevers, coughing with sputum, abdominal distension, anorexia, and fatigue. She had a history of mosquito bites and contact with a domestic dog 1 month before onset as well as a history of Epstein-Barr virus-related hemophagocytic syndrome.

Case 2
A 9-month-old girl from Zhaotong City, Yunnan Province, China, who had been living in Zhouqu County, Gansu Province, from July to September 2020 was admitted to our hospital. During this period, the infant had a history of mosquito bites that resolved after 3 months. She had diarrhea for half of December 2020, with a subsequent irregular fever lasting 1 month and reaching 39°C-40°C and decreased blood cells in the peripheral blood. She did not recover at her local hospital and was thus transferred to Kunming Children's Hospital.
Both patients had hepatosplenomegaly, hypertriglyceridemia, and hypo brinogenemia. Tables 1-5 show the relevant results. The results of the hemophagocytic cells in both infants' bone marrow while at the other hospital met the HLH-2004 standard [2]. The results of the primary HPS-related genetic test were negative and ruled out congenital HPS. All results were in accordance with the HPS-2004 chemotherapy regimen [2].
According to HLH-2004 chemotherapy regimen : Dexamethasone: from Day 2 after admission, 10 mg/(m 2 .d) for 2 weeks, 5 mg/(m 2 .d) for 4 days; Etoposide: from Day 6 after admission, twice a week, 5 mg/kg each time, a total of 4 times; Other support treatment: blood and immunoglobulin transfusion,recombinant human granulocyte stimulating factor.However, after standardized HLHassociated chemotherapy, the symptoms did not signi cantly disappear, and the patients still had fevers and severe infection. Both patients' bone marrow cell morphology was re-examined at Kunming Children's Hospital. The pathogens of Kala-azar and Leishmania amastigotes were found, along with hemophagocytic cells; thus, VL-HPS was diagnosed. However, because our hospital lacks the drugs to treat this disease, the patients were transferred to a specialty hospital. Telephone interviews con rmed that after receiving symptomatic medication at the specialty hospital, the patients' conditions quickly improved. Both patients recovered and were discharged.

Pathogenic examination
The results of the examination for related pathogens revealed the kala-azar pathogen/Leishmania amastigotes in the bone marrow of both patients (Figs. 1 and 2).Leishmania amastigotes can be seen inside and outside of phagocytes,Its shape is round and oval, with a diameter of about 2 to 5 um,The cytoplasm is light blue, with a large round nucleus inside, and the nucleus is purplish red.Beside the nucleus, a small, rod-shaped, and darkly colored moving matrix can be seen.The morphological characteristics were consistent with those of Leishmania amastigotes. Hemophagocytic cells were easily seen in the bone marrow of both patients (Figs. 3 and 4.2). One child was infected with the Epstein-Barr virus but tested negative for other pathogens (Table 2). Red blood cell count,×10 12 /L) 4.0-5.5 2.0 3.8 3. Routine blood test, infection index and biochemical examination resultsdecreased signi cantly but remained higher than the normal reference range. Alanine aminotransferase, lactate dehydrogenase and α-hydroxybutyric acid were increased, and albumin was decreased in both children (Table 3).Both children had reductions in whole blood cells or in two lines before treatment. The infection indicators (i.e., highsensitivity C-reactive protein, procalcitonin, and ferritin) were signi cantly increased, and brinogen was signi cantly reduced. After 14 days of treatment per the HLH-2004 chemotherapy regimen, their routine blood indexes changed little, and their infection indexes

Discussion And Conclusions
HPS is a life-threatening disease caused by excessive in ammation and multiple organ dysfunction, resulting in uncontrollable lymphocyte and macrophage activation and proliferation [3]. HLH is divided into primary and secondary forms. Infection is the most common cause of secondary HLH [4]. VL-HLH is very rare in childhood and has a high mortality rate if not diagnosed and treated early [1].
Kala-azar is caused by Leishmania, and sand ies are the main transmission vector. The infectious agents of this disease are mainly the patients and dogs. The disease is transmitted between humans and animals directly or between humans and animals via blood sucking by sand ies [5][6][7]. The disease has obvious regional characteristics. VL is scattered throughout six western provinces in be seen in the bone marrow when many phagocytic cells contain Leishmania amastigotes. At same time these phagocytes may also contain platelets, red blood cells, and white blood cells, and if the laboratory technicians are unfamiliar with Leishmania amastigotes or do not read the results carefully, they may mistake them for platelets. Many reports have found that kala-azar is often misdiagnosed owing to clinicians' and technicians' lack of knowledge of Leishmania amastigotes [10]. The morphologies of Leishmania, Penicillium marneffei, and histoplasma have many similarities and are easily confused. Clinicians and technicians must be familiar with the morphological characteristics of various pathogens and the differences between them. No Leishmania amastigotes were found in either child via bone marrow cell morphology at the previous hospital; thus, the children were misdiagnosed with hematological malignancies. The key to diagnosing these children is to detect the Leishmania amastigotes via bone marrow cell morphology combined with their epidemiological histories. To diagnose HLH secondary to kala-azar, nding the pathogen in the bone marrow is the most reliable diagnostic criterion.
VL-HLH-associated mortality is relatively high. If HLH treatment is ineffective, clinicians should consider whether the HLH is secondary to VL. Both patients in this study underwent 14 days of chemotherapy according to the HLH-2004 chemotherapy regimen, but the chemotherapeutic effect was unsatisfactory, their clinical symptoms did not signi cantly improve, and their liver and kidney functions of did not recover as per the related infection indicators. After the disease was clearly diagnosed, they received symptomatic treatment, and the disease was quickly controlled. Both patients recovered and were discharged from the hospital. When clinically diagnosing HLH, clinicians should actively search for the cause. If standard treatment for HLH is ineffective, detailed epidemiological histories should be taken, bone marrow cytology examinations should be performed quickly, and HLH secondary to VL should be ruled out.
In summary, bone marrow cell morphological examinations play a vital role in diagnosing VL-HLH. When secondary HLH is diagnosed clinically, common pathogens and VL-HLH should both be considered, especially in infants and young children who could not be treated satisfactorily as