The challenging diagnosis of haemophagocytic lymphohistiocytosis in an HIV-infected patient

Abstract

The differential diagnosis of febrile pancytopenia in the setting of HIV infection can be challenging. The authors report a case of a 34-year-old man with advanced HIV infection (TCD4=8 cells/mm³) and a 2-month history of fever, weight loss and asthaenia. On observation, he was emaciated, hyperthermic and pale, with a haemorrhagic oropharyngeal lesion, penile violaceous lesions and palpable hepatosplenomegaly. Blood tests showed pancytopenia, hyperferritinaemia and hypertriglyceridaemia; imaging confirmed hepatosplenomegaly and a bone marrow biopsy revealed HIV-associated dyshematopoiesis. Biopsy of the aforementioned lesions displayed Kaposi sarcoma; extensive investigation was negative for other opportunistic infections or lymphoproliferative disease. Despite only a posteriori histological documentation, haemophagocytic lymphohistiocytosis (HLH) was considered; chemotherapy and antiretrovirals were started, with excellent response. There was, however, disease relapse requiring an intensification course, with sustained remission at 18-month follow-up. HLH is a rare disease, with non-specific presentation, requiring a high index of suspicion since treatment delay can be fatal.

Background

The differential diagnosis of fever in the setting of immunosuppression associated with HIV infection can be challenging, as it includes numerous opportunistic infections and malignancies. Furthermore, haematological abnormalities such as pancytopenia are common in these patients, either because of HIV-associated dyshematopoiesis or indirect effects of opportunistic infections, drug toxicity (including antiretrovirals) or malnutrition, among other mechanisms.¹

Haemophagocytic lymphohistiocytosis (HLH), also designated haemophagocytic syndrome (HPS), was first described in 1939 by Scott and Robb-Smith.² It is a rare and often life-threatening condition characterised by cytotoxic immune dysregulation leading to uncontrolled hypercytokinaemia, abnormal T lymphocyte and macrophage activation, with subsequent haemophagocytosis in the reticuloendothelial system.^3–5 In infants and young children, HLH is predominantly due to mutations in genes responsible for cytotoxic function of natural killer (NK) cells and T lymphocytes—genetic or primary HLH—whereas, in adults, it is more often associated with infections, malignancies, autoimmune diseases and acquired immunodeficiency states such as HIV/AIDS—acquired or secondary HLH (table 1). Herpesviruses are the most common triggers, but a wide variety of bacterial, fungal and parasitic infections have also been associated with HLH.^{6 7}

Table 1.

Acquired-HLH and associated diseases

Infection
Viral	Herpesviruses (EBV, CMV, HHV-8, HSV), HIV, HTLV, adenovirus, HAV, HBV, HCV, measles, mumps, Rubella, dengue, hantavirus, parvovirus B19, enterovirus, influenza
Bacterial	Staphylococcus aureus, Campylobacter spp, Fusobacterium spp, Mycoplasma spp, Chlamydia spp, Legionella spp, Streptococcus pneumoniae, Salmonella typhi, Rickettsia spp, Brucella spp, Ehrlichia spp, Borrelia burgdorferi
Mycobacterial	M. tuberculosis, M. avium complex, M. kansasii
Fungal	Candida spp, Cryptococcus spp, Pneumocystis spp, Histoplasma spp, Aspergillus spp, Fusarium spp
Parasitic	Plasmodium falciparum, Plasmodium vivax, Toxoplasma spp, Babesia spp, Strongyloides spp, Leishmania spp
Malignancy
Haematologic	T-cell/NK-cell lymphomas, ALCL, ALL, Hodgkin's lymphoma, multiple myeloma, acute erythroid leucaemia
Non-haematologic	Prostate and lung cancer, hepatocellular carcinoma
MAS	Systemic-onset juvenile idiopathic arthritis, Kawasaki disease, systemic lupus erythematosus, seronegative spondyloarthropathies
Acquired immunodeficiency	HIV/AIDS, transplantation, chemotherapy, immunosuppressive treatment

ALCL, anaplastic large-cell lymphoma; ALL, acute lymphocytic leucaemia; CMV, cytomegalovirus; EBV, Epstein-Barr virus; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HHV-8, human herpesvirus 8; HLH, hemophagocytic lymphohistiocytosis; HSV, herpes simplex virus; HTLV, human T-lymphotropic virus; MAS, macrophage activation syndrome; NK, natural killer.

In 2007, the HLH Study Group of the Histiocyte Society revised the diagnostic guidelines for HLH,³ suggesting that five of the following eight criteria are required to make the diagnosis, unless family history or molecular diagnosis is consistent with HLH (box 1): fever, splenomegaly, at least two lineages of cytopenia, hypertriglyceridaemia (≥265 mg/dL) or hypofibrinogenaemia (≤1.5 g/L), haemophagocytosis on histology, low/absent NK-cell activity, hyperferritinaemia (≥500 μg/L) and increased levels of sCD25 (soluble interleukin-2 receptor, a marker of macrophage activity). Other features commonly reported include hepatomegaly, rash, lymphadenopathy and elevated transaminases.

Box 1. Diagnostic criteria for haemophagocytic lymphohistiocytosis (HLH).

1. Molecular diagnosis consistent with HLH (familial disease/known genetic defect)

   Pathological mutations of PRF1, UNC13D, Munc18-2, Rab-27A, STX11, SH2D1A or BIRC4

2. Diagnostic criteria for HLH fulfilled (5 of the 8 criteria listed below)

   1. Initial (1991) diagnostic criteria (to be evaluated in all patients)

      • Fever (>38.5°C)
      • Splenomegaly
      • Cytopenias (affecting ≥2 of 3 lineages in the peripheral blood):

        • Haemoglobin (Hb)<9 g/dL (in infants <4 weeks: Hb<10 g/dL)
        • Platelets <100 000/μL
        • Neutrophils <1 000/μL
      • Hypertriglyceridaemia and/or hypofibrinogenaemia:

        • Fasting triglycerides ≥3.0 mmol/L (ie, ≥265 mg/dL)
        • Fibrinogen ≤1.5 g/L
      • Haemophagocytosis in bone marrow, spleen, lymph nodes
      • No evidence of malignancy
   2. New diagnostic criteria (2004)

      • Decreased or absent natural killer-cell activity (according to local reference)
      • Ferritin ≥500 μg/L
      • sCD25 (ie, soluble interleukin (IL) 2 receptor) ≥2 400 U/mL or ≥4 800 pg/mL

Comments: If haemophagocytic activity is not proven at the time of presentation, further search for haemophagocytic activity is encouraged. If the bone marrow specimen is not conclusive, material may be obtained from other organs. Serial marrow aspirates over time may also be helpful.

The following findings may provide strong supportive evidence for the diagnosis:

•   A.  Spinal fluid pleocytosis (mononuclear cells) and/or elevated spinal fluid protein,

•   B.  Histological picture in the liver, resembling chronic persistent hepatitis (biopsy).

Other abnormal clinical and laboratory findings consistent with the diagnosis are: cerebromeningeal symptoms, lymph node enlargement, jaundice, oedema and skin rash. Hepatic enzyme abnormalities, hypoproteinaemia, hyponatraemia, increased very-low-density lipoprotein, decreased high-density lipoprotein.

The diagnosis can be established if either one listed above is fulfilled. Criteria met by the patient are italicised.

Nevertheless, because of the non-specific, atypical or insidious clinical presentation, with some patients not fulfilling all criteria or developing them only later during the course of the disease, the definitive diagnosis is often difficult and requires a high level of suspicion in order to implement appropriate treatment without delay.

Case presentation

A 34-year-old Caucasian man presented to the emergency room in September 2012, with a 2-month history of fever (mentioning temperatures of 38–39°C, with no characteristic daily pattern), loss of about 15% of total body weight, night sweats, anorexia, asthaenia and a mild productive cough of mucous sputum. He denied dyspnoea, chest pain, dysphagia or odynophagia, abdominal pain, vomiting, diarrhoea, constipation, headache, altered vision, focal strength deficit and symptoms of other organs or systems. The history was remarkable for HIV-1 infection (subtype B, homosexual transmission), diagnosed 10 years before, but without medical follow-up until January 2011, when he was observed in our immunodeficiency clinic. At the time, he was significantly immunosuppressed with a TCD4 lymphocytes count of 102 cells/mm³ (6.4%) and an HIV viral load of 339 154 copies/mL (5.53 log). In light of his immunovirological status, antiretroviral therapy and Pneumocystis jirovecii pneumonia prophylaxis were proposed, which the patient failed to start; he was lost to follow-up for the next 18 months until the current presentation. During this period, he lived in an urban area, and cited a trip to Brazil 9 months earlier, but denied travelling to the countryside or other epidemiologically relevant contacts. He denied regular medication aside from paracetamol SOS, smoking, drinking and drug use. On physical examination, the patient looked emaciated, hyperthermic (39.4°C) and tachycardic, skin and mucous membranes were pale, his oropharynx was hyperaemic with a haemorrhagic lesion in the right posterior amygdalin pillar, violaceous lesions were observed on his penis (figure 1) but he had no other skin rashes, cardiopulmonary auscultation was normal and, on abdominal palpation, painless hepatosplenomegaly was noted, with no palpable peripheral lymphadenopathies. His blood tests showed pancytopenia (haemoglobin 6.6 g/dL, erythrocytes 2.340×10⁹/L, leucocytes 1.200×10⁶/L, neutrophils 610×10⁶/L and platelets 40 000×10⁶/L), elevated C reactive protein (110 mg/L) and liver aminotransferases (aspartate 114 U/L, alanine 103 U/L) (table 2); his chest radiograph was normal. He was given a red blood cell (RBC) transfusion and admitted to our internal medicine ward for aetiological investigation.

Figure 1.

Violaceous penile skin lesions (biopsy compatible with Kaposi sarcoma).

Table 2.

Blood tests at presentation and 18 months after completing chemotherapy

	Presentation	Post-CHT	Normal range
Haemoglobin (g/dL)	6.6	16.7	(13.5–17.5)
Erythrocytes (×109/L)	2.340	5.030	(4.500–6.000)
Reticulocytes (%)	2.51	2	(0.5–1.5)
Lactate dehydrogenase (U/L)	283	209	(<248)
Leucocytes (×106/L)	1.200	4.500	(4.500–11.000)
Neutrophils (×106/L)	610	2.820	(2.000–8.500)
Platelets (×106/L)	40.000	167.000	(150.000–450.000)
C reactive protein (mg/L)	110	0.6	(<5.0)
Fibrinogen (g/L)	4.6	–	2.38–4.98
Aspartate aminotransferase (U/L)	114	32	(<50)
Alanine aminotransferase (U/L)	103	36	(<50)
Triglycerides (mg/dL)	407	198	(≤150)
Ferritin (µg/L)	4258	314	(24–336)
Soluble CD25 (pg/mL)	6130	–	(458–1997)
NK-cell activity	Decreased	–
TCD4 cell count (cells/mm3 and %)	8 (2.3%)	144 (13.6%)	(500–1.330)
TCD8 cell count (cells/mm3 and %)	262 (78.8%)	745 (70.4%)	(190–1.000)
HIV viral load (copies/mL)	1.034.215	<20	(<20)

Diagnostic criteria for HLH are italicised.

CHT, chemotherapy; NK, natural killer; TCD4 and TCD8, T lymphocytes.

Further blood tests revealed high ferritin levels (4528 µg/L), transient and self-limited hepatic cholestasis (γ-glutamyl transferase 248 U/L, alkaline phosphatase 657 U/L), hypertriglyceridaemia (407 mg/dL), as well as immunity deterioration with a TCD4 count of 8 cells/mm³ (2.3%) and an HIV viral load of 1 034 215 copies/mL (6 log). Bearing in mind the numerous differential diagnoses of fever in the immunodeficiency setting, further complementary examinations aimed at excluding opportunistic infections and lymphoproliferative disease were carried out. Serological testing for hepatitis A, B and C virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), parvovirus B19, Treponema pallidum, Brucella, Rickettsia conorii, Aspergillus, Cryptococcus neoformans and Histoplasma capsulatum was negative for acute infection; serum PCR assays for herpes simplex virus 1 (HSV-1), EBV and human herpesvirus 8 (HHV-8) were positive, but viraemia quantification for EBV was negative, while quantification methods for both HSV-1 and HHV-8 were unavailable at our centre. A thoracoabdominopelvic CT displayed marked homogeneous hepatosplenomegaly, but there were no other pathological findings, namely mediastinal, hilar, axillary or abdominopelvic lymphadenopathies (figure 2). A myelogram revealed normal cellularity and was negative for Leishmania. In addition to direct microscopic observation, serological and PCR assays for Leishmania in peripheral blood were negative. The bone marrow biopsy was hypercellular, displaying HIV-associated dyshematopoiesis affecting the three lineages and no evidence of neoplastic infiltration or microorganisms (fungus, acid and alcohol fast bacilli—AAFB, HHV-8) (figure 3A). Considering the patient's history of cough, although self-limited early in the hospital stay, bronchoalveolar lavage was performed but showed no Pneumocystis jirovecii, AAFB or fungus, only non-specific inflammatory findings. A lumbar puncture was also performed, but, similarly, the cerebrospinal fluid (CSF) analysis was negative for syphilis, herpesviruses, John Cunningham virus (JCV), Toxoplasma gondii or C. neoformans. Cranial CT scan was also unremarkable. A transthoracic echocardiogram showed no images suggestive of vegetations or pericardial effusion. Repeated blood, urine, sputum and stool cultures were negative as was a parasitology stool test. Mycobacterial cultures of peripheral blood, bone marrow aspirate, urine, bronchoalveolar lavage and CSF were all negative. A flexible nasolaryngoscopy showed erythematous, vegetative and confluent lesions localised to the posterior wall of the nasopharynx, extending to the oropharynx (figure 4), which were confirmed by biopsy to be Kaposi sarcoma (KS) (figure 3C, D). The same histopathological diagnosis was reached for the penile skin lesions (figure 3B). Upper gastrointestinal endoscopy and colonoscopy showed no visceral involvement of KS and there were no other relevant findings.

Figure 2.

Thoracoabdominopelvic CT demonstrating marked homogeneous hepatosplenomegaly, with the lower border of the liver practically reaching the iliac crest line and the major axis of the spleen measuring 170 mm (coronal and axial planes).

Figure 3.

Histopathology: (A) First bone marrow biopsy (H&E)—HIV-associated dyshaematopoiesis, with no evidence of neoplastic infiltration or microorganisms. (B) Penile skin biopsy (H&E)—vascular proliferation with red blood cell extravasation, compatible with Kaposi sarcoma. (C) Oropharynx lesion biopsy (H&E)—vascular proliferation with haematic extravasation in the corium, positive for CD34 and HHV-8, compatible with Kaposi sarcoma. (D) Oropharynx lesion biopsy—positive immunohistochemical staining for HHV-8. (E) Second bone marrow biopsy (H&E)—histiocyte hyperplasia with haemophagocytosis. (F) Second bone marrow biopsy—positive immunohistochemical staining for CD68, a macrophage marker (HHV-8, human herpesvirus 8).

Figure 4.

Flexible nasolaryngoscopy showing erythematous, vegetative and confluent lesions localised to the posterior wall of the nasopharynx, extending to the oropharynx (biopsy-compatible Kaposi sarcoma).

During the investigation, in spite of several empiric treatment strategies (naproxen challenge test and a trial with antituberculous therapy), fever would not subside, with daily peaks of 38.5–39.5°C and partial response to antipyretics, and several RBC transfusions were required. Interestingly, when antiretroviral therapy (tenofovir+emtricitabine+efavirenz) was initiated, the patient experienced no fever for a few days and a relative improvement in cytopenias, though he soon relapsed (figure 5). With no microbiological isolates or evidence of a lymphoproliferative disease, on reviewing the case, a hypothesis of HLH associated with advanced HIV infection was considered, although lacking histopathological documentation. Another bone marrow specimen was obtained and the case discussed with haematology team, who concurred on starting chemotherapy with dexamethasone and etoposide according to the HLH-94 protocol—oral dexamethasone (10 mg/m²/day for 2 weeks, 5 mg/m²/day for 2 weeks, 2.5 mg/m²/day for 2 weeks, 1.25 mg/m²/day for 1 week, subsequently weaning for 1 week) and intravenous pulses of etoposide (150 mg/m², 2 pulses/week in the first 2 weeks and 1 pulse/week in the remaining 6 weeks). The last bone marrow biopsy did eventually identify focal haemophagocytosis (figure 3E, F). There was no fever almost immediately on initiating treatment (figure 5) and, by the end of the 8-week protocol, the haematological parameters practically normalised.

Figure 5.

Temperature curve during first hospital admission (HAART, highly active antiretroviral therapy; H, isoniazid; R, rifampin; Z, pyrazinamide; E, ethambutol).

However, 2 weeks after chemotherapy completion, the patient presented with fever (39–40°C) and a non-pruritic erythematous micronodular rash extending along his torso. His blood results once again showed pancytopenia (haemoglobin 7.9 g/dL, erythrocytes 2.290×10⁹/L, leucocytes 1 800×10⁶/L, neutrophils 570×10⁶/L and platelets 19 000×10⁶/L) and high ferritin levels (3379 µg/L); microbiological examinations were negative. NK-cell activity was decreased and soluble CD25 levels were high (6130 pg/mL), further confirming an HLH relapse (box 1). Dexamethasone and etoposide were reintroduced as in the induction therapy (for 8 weeks), followed by 14 weeks of continuation therapy—dexamethasone 10 mg/m²/day for 3 days every 2 weeks, alternating with etoposide 150 mg/m² every 2 weeks—with clinical and laboratory improvement.

Outcome and follow-up

On follow-up consultation, 18 months after completing chemotherapy, the patient remains asymptomatic, with no noticeable KS lesions or organomegaly. His haematological parameters and ferritin are within normal range (table 2), with a slowly improving TCD4 cell count and sustained viral suppression.

Discussion

In the HIV setting, HLH has been described at all stages of the infection.⁸ In the majority of cases, AIDS is the underlying disease, in which an opportunistic infection triggers the onset of HLH. However, HIV has itself been attributed as the cause of HLH in several reports, from acute seroconversion⁹ to immune reconstitution,¹⁰ and it is not limited to patients who are profoundly immunosuppressed, but is also seen among patients in a state of a well-controlled or aviraemic HIV infection.¹¹ Although our patient had positive serum PCR (a highly sensitive method) for HSV-1, EBV and HHV-8, there were no clinical features of HSV-1 infection, EBV viraemia was negative and KS involvement was only cutaneous, also, there were no lymphadenopathies and no bone marrow biopsy findings suggestive of multicentric Castleman's disease, an entity known to be associated with HHV-8 infection. Furthermore, the transient resolution of symptoms after the initiation of antiretroviral therapy could support the argument for a pathophysiological role of HIV itself in this patient. HIV infection likely leads to acquired defects in cellular cytotoxicity, which may have similar effects to those of the genetic mutations found in the familial forms.

As most of the features listed as diagnostic criteria for HLH are often present in patients with advanced HIV, it is more likely to be under-recognised in this population. For instance, hypertriglyceridaemia commonly occurs in the context of advanced HIV disease and as a side effect of the highly active antiretroviral therapy (HAART). Cytopenias are also common in different stages of the natural history of HIV infection and secondary to HAART; in this population, it is mandatory to exclude lymphoproliferative disorders and opportunistic infections that may present with febrile pancytopenia; this requires a time-consuming complementary investigation.

Irrespective of HIV status, elevation of serum ferritin may also arise in the context of an acute inflammatory syndrome due to infectious, neoplastic, autoimmune and iron overload diseases, such as haemochromatosis, though a cut-off of >10 000 μg/L greatly increases specificity for HLH.¹²

Histopathological documentation of haemophagocytosis in bone marrow biopsy, the most readily accessible tissue for differential diagnosis of pancytopenia, deserves a further look. Although considered the hallmark of activated histiocytes, it is not a pre-requisite for the clinical diagnosis of HLH, as it is initially absent in a substantial proportion of patients, even during fulminant disease.⁵ This is particularly true in HIV-associated HLH, where haemophagocytosis can be cyclical, thus a given biopsy specimen may yield negative results at first examination and repeat biopsies may be helpful, as shown in our patient. Conversely, it may be found without a clinical diagnosis of HLH. In fact, Sailler et al¹³ documented haemophagocytosis in bone marrow biopsy of around 10% of HIV-infected patients prior to the initiation of HAART, and an autopsy study of 56 patients with AIDS found an approximately 20% rate of haemophagocytosis.¹⁴

In their review of 39 patients with HIV-associated HLH, where 80% had TCD4 counts <200 cells/mm³, Bhatia et al¹⁵ reported a significantly worse recovery rate in patients with HIV (28%) compared to HIV-negative patients (60–70%), with half of deaths occurring within the first month, thus highlighting the fulminant nature of this disease in the HIV setting. Additionally, 11 patients had no opportunistic infection or malignancy, suggesting a possible role of HIV itself as a trigger. A large retrospective study published recently by Fardet et al¹⁶ describes the characteristics of 58 HIV-infected patients with HLH. At the time of diagnosis, the median duration of HIV infection was 4 years, and 57% were receiving HAART; the median CD4 lymphocyte count was 91 cells/mm³ (2–387) and 35% had undetectable viral load. An underlying malignancy or infection was present in 31 and 23 patients, respectively. The prognosis remains poor with a mortality of 31%, which is, however, lower than the 75% mortality rates reported in the pre-HAART era.¹⁷

The immediate aim in the treatment of any patient with HLH is to control the cytokine storm and cellular proliferation that is responsible for the life-threatening symptoms. If a microorganism is identified, specific therapy should be employed, but, usually, hyperinflammation has to be controlled by additional means. In 1994, the HLH Study Group proposed the first international protocol for treatment of HLH (HLH-94), which employed dexamethasone, etoposide (a cytotoxic drug that selectively targets the monocyte line) and, after 7 weeks, cyclosporine A. Subsequent haematopoietic stem cell transplantation is recommended for patients with familial disease or molecular diagnosis, and patients with severe and persistent or reactivated disease. Besides immunosuppressive and cytotoxic therapy, supportive management also involves antimicrobial prophylaxis, but the use of growth factors such as granulocyte colony-stimulating factor (G-CSF) or granulocyte monocyte (GM) CSF is generally not recommended as they can exacerbate HLH. In the revised guidelines, HLH-2004,³ cyclosporine A was moved to the fore in the hope of preventing reactivations of HLH, which were found to be frequent when steroids were tapered. However, these are yet to be widely implemented outside of clinical trials and still fail to address issues such as the use of cyclosporine A in specific populations, such as profoundly immunosuppressed HIV patients.

Learning points.

• The differential diagnosis of febrile pancytopenia in the setting of HIV infection can be challenging, as it includes numerous opportunistic infections and malignancies.

• Haemophagocytic lymphohistiocytosis (HLH) is a rare and potentially life-threatening condition due to cytotoxic immune dysregulation, and is clinically characterised by fever, splenomegaly and cytopenias. High ferritin levels can provide an important diagnostic clue.

• There are several known associated diseases and triggers, including HIV, and HLH has been described at all stages of the infection.

• Histopathological documentation of haemophagocytosis is not a pre-requisite for the diagnosis, as it is initially absent in a substantial proportion of patients, even during fulminant disease.

• A high index of suspicion is critical for early diagnosis, and management with chemotherapy aims to control lymphocyte/macrophage activation and proliferation.