Case Report: Visceral Leishmaniasis-associated Hemophagocytic Lymphohistiocytosis in Adults: A Case Series and Literature Review

ABSTRACT.

Hemophagocytic lymphohistiocytosis (HLH) is a rare and fatal complication of visceral leishmaniasis (VL). To provide a basis for early and correct diagnosis and to improve prognosis in the future, we describe a case series of VL-associated HLH in adults in our center in the past decade after review of all reported cases of adult VL-associated HLH in English through May 2022. In our case series, a total of 111 patients were diagnosed with VL. Among these patients, only six cases were diagnosed with VL-associated HLH. All patients tested positive for serology. Leishmania was detected for the first time by bone marrow aspiration (BMA) in three of the six patients and in the other three patients after three or four BMAs. It took more than 1 month from onset to diagnosis of VL for all the six cases, and the longest time was 6 months. Five of the six patients recovered after receiving sodium stibogluconate. VL-associated HLH is rare but potentially life-threatening in adults and predisposes to early delays in diagnosis. However, diagnostic techniques are not complicated or difficult, so it is more important to consider that it is not recognized by physicians. Although guidelines recommend liposomal amphotericin B as the most effective therapy, our experience suggests that sodium stibogluconate can be an alternative option when liposomal amphotericin B is unavailable or unaffordable.

INTRODUCTION

Leishmaniasis, also known as kala-azar, is a vector-borne disease caused by Leishmania infection. Leishmania is transmitted between mammalian hosts by female phlebotomine sandflies. There are three main clinical syndromes: cutaneous, mucosal, and visceral leishmaniasis. Visceral leishmaniasis (VL) is the systemic form, which may be life-threatening if appropriate treatment is not given in time.¹ The global incidence of new VL cases was between 50,000 and 90,000 in 2017, with 2,000 and 3,000 deaths.¹ However, leishmaniasis was cited as one of the neglected tropical diseases more than a decade ago due to a lack of resource location for development in diagnostics and treatment options.² VL is endemic in more than 60 countries, but more than 90% of cases were reported from only seven counties (Brazil, Ethiopia, India, Kenya, Somalia, South Sudan, and Sudan).³

VL is caused by disseminated infection of Leishmania parasites throughout the reticuloendothelial system. Typical clinical features include persistent irregular fever, splenomegaly, hepatomegaly, pancytopenia, hypergammaglobulinemia, and weight loss. Many infectious diseases have these manifestations in common, which may lead to misdiagnosis at the early stage. Prolonged VL may lead to severe complications. Hemophagocytic lymphohistiocytosis (HLH) is a rare and fatal complication of visceral leishmaniasis. HLH is a syndrome of excessive inflammation and tissue damage due to abnormal immune activation and excessive inflammation, and occurs less frequently in adults. HLH can be secondary to any serious infection. Epstein-Barr virus (EBV) infection is considered the most frequent trigger. VL is a rare trigger that usually occurs in immunosuppressed patients. VL and HLH have overlapping clinical features, such as fever, hepatosplenomegaly, and cytopenia, which leads to misdiagnoses. For this reason, early diagnosis of VL-associated HLH is critical but challenging. When chronically infected with Leishmania, it leads to the activation of macrophages. The persistent activation of macrophages leads to an excessive cytokine storm that triggers hemophagocytic syndrome,^4–6 which may lead to multiorgan failure and high mortality.⁷

Although there are several reports on HLH in VL,^8–10 there are rare reports in English from China. Moreover, clinicians’ knowledge of VL diagnosis and treatment is lacking, and their diagnostic ability is weak in nonendemic areas. Therefore, the summary of this case series and literature review will help strengthen clinicians’ knowledge of VL and its rare and serious complication HLH; improve diagnostic timeliness; achieve early diagnosis, early reporting, and early treatment of VL; and improve patient prognosis.

CASE REPORTS

Cases were screened from adult inpatients diagnosed with VL at West China Hospital (WCH) of Sichuan University, China, from January 1, 2008, to August 31, 2018. WCH is not located at or near the VL-endemic area. Included patients were those who met the criteria of clinical practice guidelines by the Infectious Diseases Society of America and the American Society of Tropical Medicine and Hygiene.¹¹ Namely, rapid diagnostic tests (RDTs) and serologic testing for antileishmanial antibody-recombinant K39 antigen (rK39) were positive and confirmed by visualization of the amastigote in bone marrow aspirate (BMA) histopathological examination. HLH was diagnosed according to the HLH 2004 guideline by the Histiocyte Society.¹²

A total of 111 patients were diagnosed with VL. Among these patients, only six cases were diagnosed with VL-associated HLH. Interestingly, all the VL-associated HLH patients were male in this study. HIV, hepatitis C virus, hepatitis B virus, EBV, and syphilis infections were excluded in all six patients. The main clinical features, treatments, and outcomes of the six VL-associated HLH cases are summarized in Table 1. Typical Leishmania and hemophagocytic cells were found in the BMA smear and bone marrow biopsy (Figure 1). All patients tested positive for serology (rK39 RDTs). Leishmania was detected for the first time by BMA in three of the six patients and in the other three patients after three or four BMAs. It took more than 1 month from onset to diagnosis of VL for all the six cases, and the longest time was 6 months. Five of the six patients recovered after receiving sodium stibogluconate (S SGL). Case 2 was serious and dying when he was admitted to hospital. After receiving short-term specific therapy, this patient and his family refused all treatments, and he died after half month post-discharge. All the five surviving patients had no posttherapy relapses.

Table 1.

Summary of our patients and all reported adult patients with visceral leishmaniasis-associated hemophagocytosis lymphohistiocytosis

Ref.	Age (y)	Sex	Country	Endemic region	Fever	Hepato/ splenomegaly	Pancytopenia	Triglycerides > 265mg/dL	Fibrinogenemia < 1.5 g/L	Hemophagocytosis in BMA	Low NK cell activity	Ferritin > 500 ng/mL	Soluble CD25 > 2,400 U/L	Immunocompromised host	Diagnosis delay (weeks)	Diagnosis made by	Therapy	Outcome (days)
Our cases 2022	58	M	China	+	+	+	+	−	+	+	NL	+	+	−	4	1st BMA/serology	VP16 + CS + S SGL	Alive
	58	M		+	+	+	+	−	+	+	NL	+	NL	−	16	1st BMA/serology	S SGL	Died (15)
	38	M		+	+	+	+	+	−	+	NL	+	+	−	24	1st BMA/serology	CS + S SGL	Alive
	43	M		+	+	+	+	−	+	+	NL	+	+	−	16	BMA/serology	CS + S SGL	Alive
	47	M		+	+	+	+	+	+	+	−	+	+	−	4	BMA/serology	S SGL	Alive
	27	M		+	+	+	+	−	−	+	−	+	+	−	4	BMA/serology	CS + S SGL	Alive
Dujardin et al., 2021	25	M	France	+	+	+	+	−	+	+	NL	+	NL	+ (HIV)	2	1st BMB/serology	LAmB	Alive
Cruz et al., 2021	26	M	Brazil	+	+	+	+	NL	+	+	NL	NL	NL	−	6	1st BMA/serology/NNN culture+	LAmB	Died (2)
	20	F		+	+	+	+	−	NL	+	NL	+	NL	−	4	Serology/NNN culture+	CS + IVIG + LAmB	Alive
	41	F		+	+	+	+	NL	NL	+	NL	+	NL	−	3	1st BMA/NNN culture+	LAmB + CS	Alive
Kooli et al., 2021	25	M	Tunisia	−	+	+	+	NL	NL	+	NL	+	NL	+ (HIV)	NL	1st BMA/BM PCR+	S SGL	Alive
Badiola et al., 2021	27	F	Spain	+	+	+	+	−	NL	+	NL	+	NL	−	94	BM PCR+/NNN culture+	CS + IVIG + CsA + VP-16 + Tocilizumab + LAmB	Alive
	72	M		+	+	+	+	−	NL	+	NL	+	NL	−	1	BM PCR+/NNN culture+	CS + CsA + IVIG + LAmB	Died (NL)
	46	M		+	+	+	+	−	NL	+	NL	+	NL	+ (HIV)	6	BM PCR+	CS + IVIG + LAmB	Died (4)
	43	M		+	+	+	+	+	NL	+	NL	+	NL	−	9	BM PCR+	CS + LAmB	Alive
	67	M		+	+	+	+	−	−	+	NL	+	NL	−	5	BM PCR+	CS + IVIG + LAmB	Alive
	47	F		+	+	+	+	+	+	+	NL	+	NL	−	9	BM PCR+	CS + IVIG + LAmB	Alive
Diamantidis et al., 2020	82	F	Greece	−	+	−	+	+	+	+	NL	+	NL	−	3	1st BMA/BM PCR+	CS + IVIG + LAmB	Alive
Alkhaldy et al., 2020	48	F	SaudiArabia	−	+	+	+	NL	NL	+	NL	+	NL	−	24	1st BMA	LAmB + CS	Alive
Kumari et al., 2020	28	M	India	−	+	+	+	+	NL	NL	NL	+	NL	−	4	BMA	LAmB	Alive
	40	F		−	+	+	+	+	NL	NL	NL	+	NL	−	24	BMA	Ampho B + LAmB	Alive
	26	M		−	+	+	+	+	NL	NL	NL	+	NL	−	18	BMA	LAmB	Alive
	35	M		−	+	+	+	+	NL	NL	NL	+	NL	−	17	BMA	Ampho B + LAmB	Alive
	61	M		−	+	+	+	+	NL	NL	NL	+	NL	−	12	BMA	LAmB	Alive
	34	F		−	+	+	+	+	NL	NL	NL	+	NL	−	24	BMA	LAmB	Died (60)
	45	M		−	+	+	+	+	NL	NL	NL	+	NL	−	20	BMA	LAmB	Alive
Vanhinsbergh et al., 2019	53	M	England	−	+	+	+	−	NL	−	NL	+	+	−	24	Serology	LAmB	Alive
Theodosiou et al., 2019	62	F	England	−	+	+	+	NL	+	+	NL	+	NL	−	2	BM PCR+/serology	CS + VP16 + LAmB	Alive
Machelart et al., 2019	76	M	France	+	+	+	+	+	NL	+	NL	+	NL	−	NL	Spleen biopsy/ serology/BM PCR+	CS + VP16 + CsA + LAmB	Alive
Singh et al., 2019	40	M	India	+	+	+	+	+	NL	NL	NL	+	NL	−	4	Serology	Ampho B + Miltefosine	Alive
George et al., 2019	27	M	India	+	+	+	+	NL	+	+	NL	+	NL	−	17	1st BMA	LAmB + CS	Died (NL)
Mahendiran et al., 2018	23	M	England	+	+	+	+	+	NL	−	NL	+	NL	−	1	Serology/BM PCR+	Ampho B + Miltefosine + CS	Alive
Panda et al., 2016	25	F	India	+	+	+	+	+	NL	NL	NL	+	NL	−	20	1st BMA/ serology	LAmB	Alive
Raina et al., 2017	40	M	–	−	+	+	+	+	NL	+	NL	+	NL	−	4	1st BMA	LAmB	Alive
Rios-Fernandez et al., 2016	27	F	Spain	+	+	+	+	+	NL	−	NL	+	NL	−	132	BMB	CsA+CS+ VP16 + adalimumab+ miltefosine+S SGL+ tocilizuma	Alive
Raina et al., 2016	40	M	India	−	+	+	+	+	NL	+	NL	+	NL	−	4	1st BMA	Ampho B	Alive
Ranjan et al., 2016	53	F	India	−	+	+	+	NL	+	NL	NL	+	NL	−	12	1st BMA	Ampho B	Alive
	30	M		+	+	+	+	+	−	+	+	+	NL	−	4	Serology	LAmB+ CS	Alive
Comont et al., 2015	64	M	France	+	+	+	+	−	+	+	NL	+	NL	+ (RDD)	3	Serology/BM PCR+	LAmB+CS+VP16 + pentamidin	Alive
Pawar et al., 2015	30	F	India	−	+	+	+	+	NL	−	NL	+	NL	−	2	Serology	LAmB + CS	Alive
Merelli et al., 2015	22	M	Italy	−	+	+	+	NL	NL	NL	+	+	NL	−	4	1st BMA	LAmB	Alive
Watkins et al., 2014	73	M	USA	+	+	+	+	−	+	+	NL	+	NL	−	3	1st BMA/serology	LAmB	Alive
Molto et al., 2010	60	M	Spain	−	+	+	+	+	NL	−	NL	+	NL	+ (anti-TNFα)	2	1st BMA	LAmB	Alive
Martin et al., 2009	18	M	India	−	+	+	+	+	+	+	NL	+	NL	+ (CGD)	16	1st BMA/serology	LAmB + IFNγ	Died (25)
Patel et al., 2009	35	M	India	−	+	+	+	+	NL	−	NL	+	NL	+ (HIV)	1	1st BMA	Ampho B	Alive
Rajagopala et al., 2008	23	M	India	−	+	+	+	−	+	+	+	+	NL	−	8	Serology	Ampho B + IVIG	Alive
Kilani et al., 2006	34	M	Tunisia	−	+	+	+	+	+	+	NL	NL	NL	−	4	Peripheral blood culture	None	Died (2)
Fiteni et al., 2004	23	F	Spain	−	+	−	+	−	−	+	NL	NL	NL	+(Harada’s)	1	1st BMA	S SGL + LAmB	Alive
Kontopoulou et al., 2002	33	F	Greece	−	+	+	+	NL	NL	+	NL	NL	NL	−	1	Serology	IVIG + LAmB	Alive
	39	M		−	+	+	+	NL	NL	+	NL	NL	NL	−	4	1st BMA/serology	LAmB	Alive
Bhutani et al., 2002	28	M	India	−	+	+	+	NL	NL	−	NL	NL	NL	−	2	Serology/NNN culture+	Ampho B	Alive
Cortés et al., 1997	59	M	Spain	−	+	+	+	NL	NL	+	NL	NL	NL	−	4	1st BMA/serology/NNN culture+	CS + Glucantamine + IVIG	Died (30)
Bessis et al., 1993	38	M	France	−	+	+	+	NL	NL	+	NL	NL	NL	−	4	BMA	Specific therapy	Alive
Rodriguezcuartero et al., 1991	33	M	Spain	−	+	+	+	NL	NL	+	NL	NL	NL	−	16	1st BMA	Glucantamine	Alive
Matzner et al., 1979	22	M	Israel	+	+	+	+	NL	NL	+	NL	NL	NL	−	20	BMA	S SGL	Alive

Ampho B = amphotericin desoxycholate; BM PCR = polymerase chain reaction performed on bone marrow; BMA = bone marrow aspiration; BMB = bone marrow biopsy; CGD = chronic granulomatous disease; CS = corticosteroids; CsA = cyclosporine A; F = female; IVIG = intravenous immunoglobin; LAmB = liposomal amphotericin B; M = male; NL = not listed; NNN = Nicolle–Novy–McNeal; RDD = Rosai-Dorfman disease; S SGL = sodium stibogluconate; TNFα = tumor necrosis factor-alpha; VP16 = etoposide.

Figure 1.

Typical leishmania and hemophagocytic cells in the bone marrow aspirate and biopsy. A. Bone marrow aspirate showing Leishmania (black arrow) (case 3, Wright-Giemsa stain, 1000x). B. Bone marrow aspirate showing a small amount of Leishmania (black arrow) (case 4, Wright-Giemsa stain, 1000x). C. Bone marrow aspirate showing monocytes (red arrow) engulfing leukocyte precursors (black arrow) (case 6, Wright-Giemsa stain, 1000x). D. Bone marrow biopsy showing Leishmania (black arrow) (case 3, Hematoxylin-eosin stain, 400x). E. Bone marrow biopsy showing Leishmania (black arrow) (case 3, Hematoxylin-eosin stain, 1000x magnification with oil). F. Abdominal CT scan showing the obvious splenomegaly (red arrow) (case 3). The yellow line in the lower right corner of the photomicrographs A, B, C, D and E is 10 µm.

LITERATURE SEARCH AND STUDY SELECTION

Two of the authors independently conducted a systematic search of English literature through May 2022 using the terms “visceral leishmaniasis,” “kala-azar,” “hemophagocytosis,” and “hemophagocytic lymphohistiocytosis” in the PubMed, Embase, and Web of Science databases. Only those articles that fulfilled the criteria for VL-associated HLH in adults (≥ 18 years old) were included for analysis. Both abstracts and full text articles, where available, were reviewed. Data regarding appearing in endemic regions or not, clinical features, time of diagnosis delay, treatment, course, and outcomes were extracted in a predesigned data extraction form.

RESULTS

We reviewed all reported cases of adult VL-associated HLH in English through May 2022. Our review yielded 34 articles of VL-associated HLH in adults with a total of 49 patients^10,13–45 (Table 1). Males accounted for 69.4% of cases of VL-associated HLH. The median age was 35 years (20–82 years). A total of 40.8% of cases came from or had visited endemic areas in recent months or years. All patients had clinical manifestations characterized by fever, hepatosplenomegaly, and pancytopenia. BMA established the presence of hemophagocytosis in 81.6% of cases. Notably, the first BMA often failed to establish the presence of Leishmania and was negative for Leishmania in 59.2% of cases. Repeated bone marrow assessments increased the diagnostic yield. In six cases with a negative marrow, serological tests-rK39 RDTs were positive in all patients. Meanwhile, 83.7% of patients were apparently immunocompetent. A total of 71.4% of patients were treated with liposomal amphotericin B (LAmB), and 18.4% were treated with amphotericin deoxycholate. Only two patients were treated with S SGL monotherapy. The mean delay in diagnosis of the reported cases was 13 weeks (range: 1 week–33 months). After receiving specific therapy, 81.6% of patients returned to normal. A total of 18.4% of patients died of VL-HLH, mainly due to infection, bleeding, and multiple organ failure.

DISCUSSION

Leishmaniasis is considered was determined to be a neglected tropical disease more than a decade ago.² The mortality rates of leishmaniasis are various in different areas and different hospitals. West China Hospital of Sichuan University is known as one of the best hospitals with high medical quality in China, especially in southwest China. Although patients with VL-associated HLH were not diagnosed before they came to our hospital, after arriving at our center, our doctors had experience in diagnosing the disease, and the treatment was relatively timely, so the mortality rate was reduced. In general, specific treatment of the trigger cause of HLH may eliminate the abnormal immune activation that caused it in the first place.^46,47 Prognosis appears to be better in VL-associated HLH cases that are identified and promptly treated with specific therapy (LAmB or S SGL).^10,48,49 In one deceased patient in our case series, the diagnosis of VL-associated HLH was made when the disease had progressed to an advanced stage, and specific treatment was not available in a timely and effective manner. The mortality rate was indeed low in the cases we reviewed (18.4%) because they all received specific treatment after diagnosis despite delayed diagnosis. This is also partly because some patients with VL-associated HLH are not diagnosed with the disease until after death, so the reported mortality rate may be lower than the actual mortality rate.

As is well known, VL occurs mainly in children and immunocompromised patients and is relatively rare in adults. In this article, we review all adult VL-associated HLH to date. Leishmaniasis continues to be endemic in some specific regions of China. The reported VL cases are mainly from specific areas of Xinjiang, Gansu, and Sichuan Provinces, which account for more than 95% of all VL cases in China.³ All six cases in our center were visited or lived in one of the endemic areas. The case reports we reviewed also showed that 40.8% had come from or visited the endemic area in the past few months or years. The percentage of male patients was 69.4%. Meanwhile, our center’s study showed that all VL-associated HLH patients were male. The greater prevalence of VL-associated HLH in men may simply be due to the high prevalence of VL in men, and more studies are needed in the future to explain this phenomenon. Additionally, it is not easy to diagnose VL at the early stage, even in endemic areas, because VL and HLH have overlapping clinical features, such as fever, hepatosplenomegaly, and cytopenia. Prolonged infection may finally induce HLH. For the six cases in our center, it took 1 to 6 months from onset to diagnosis of VL. Additionally, the case reports we reviewed have shown a mean delay in the diagnosis of VL-associated HLH of 13 weeks (ranging from 1 week to 33 months). This may be one reason that most physicians lack sufficient knowledge of VL and HLH, especially those who live outside epidemic areas. This may lead to confusion due to the overlap of clinical symptoms of VL with other infectious diseases and VL with HLH.⁵⁰ In this situation, diagnostic tests play a critical role in the correct diagnosis.

As in our own cases and the cases we reviewed, the first BMA does not commonly detect the presence of amastigote forms in the reticuloendothelial system at onset due to the pauci-microbial nature of this disease or the lack of experience of pathologists in VL diagnosis. Therefore, in cases of strong clinical suspicion of HLH associated with VL, repeated BMAs and repeated examinations of BMAs by experienced physicians are needed. Although BMA is an invasive procedure, it is a highly feasible and acceptable to our patients. BMA is less sensitive but safer and is the preferred source of diagnostic samples.¹¹ To diagnose the disease, we had three patients who underwent two or three BMAs without reluctance. Of course, there are also studies reporting that a second BMA may not be necessary if the correct technique is used for a prolonged slide examination.^51,52 Spleen aspiration does have a high diagnostic yield, but it has higher risk (e.g., life-threatening hemorrhages⁵³) compared with BMA.¹¹ Therefore, spleen puncture is generally not performed unless necessary. In our included literature, only one patient underwent splenic aspiration. Spleen aspiration is not performed in most patients. RDTs for rK39 are one of the most commonly used tests for the diagnosis of Leishmania infection. The sensitivity of rK39 RDTs may be reported to be as high as 97% (95% CI 90–100%).¹¹ All the six patients in our center were given rK39 RDTs and received positive results. In addition, of the six cases with negative BMA results who underwent serological tests (rK39 RDTs) in previous case reports, all patients (100%) had positive rK39 RDT results of diagnostic significance. Therefore, we strongly recommend early administration of rK39 RDTs when VL is clinically suspected, regardless of the results of BMAs.

Agents against leishmaniasis include amphotericin B, pentavalent antimonial drugs, paromomycin, and miltefosine.¹ LAmB has the highest therapeutic efficacy and the most favorable safety profile.⁵⁴ However, LAmB is not easily available and is too expensive to be affordable for most Chinese patients. S SGL is the only medication offered free for VL therapy by the China Center for Disease Control, and then it is used as the first-line choice. LAmB is used for only a few patients who have failed S SGL therapy or who are resistant to S SGL in China. Five of the six patients in our center received S SGL treatment and recovered. There were no significant S SGL–related adverse effects. The most commonly used medication in previous case reports was LAmB, accounting for 71.4%. Only two patients received S SGL monotherapy, and all recovered without any obvious adverse effects.

In conclusion, VL-associated HLH is rare but potentially life-threatening in adults. The diagnosis may be difficult. VL should be considered in patients with clinical manifestations of fever, hepatosplenomegaly, and pancytopenia; epidemiologic history should be pursued, and rK39 RDTs as well as BMAs should be performed. For BMAs of patients with suspected VL-associated HLH, clinicians should promptly contact pathologists to emphasize the possibility of this disease and avoid missed diagnoses. Repeated BMAs are required if necessary. Although guidelines recommend LAmB as the most effective therapy, our experience suggests that S SGL can be an alternative option when LAmB is unavailable or unaffordable.