mNGS-Assisted Diagnosis of Visceral Leishmaniasis Presenting as Hemophagocytic Lymphohistiocytosis: Two Cases Confirmed by rK39

Abstract

Background

Kala-azar, or visceral leishmaniasis (VL), is a parasitic disease caused by Leishmania protozoa. Conventional diagnostic modalities for visceral leishmaniasis—including microscopy, in vitro culture, and serological assays—are constrained by suboptimal sensitivity, invasive sampling, and prolonged turnaround times.

Methods

We report two cases of visceral leishmaniasis-related hemophagocytic lymphohistiocytosis, in which no Leishman-Donovan bodies were detected by conventional assays. Metagenomic next-generation sequencing (mNGS) successfully identified Leishmania pathogens, and the diagnosis was confirmed by the rK39 rapid test.

Results

mNGS successfully identified Leishmania pathogens in both patients. Targeted anti-leishmanial treatment led to rapid clinical improvement in both patients.

Conclusion

This study demonstrates that mNGS can serve as a valuable adjunct for the rapid etiological diagnosis of VL, particularly when conventional tests are negative. Nevertheless, its current use is largely restricted to endemic areas where advanced laboratory infrastructure is available; therefore, mNGS should be regarded as a complementary diagnostic tool rather than a substitute for routine assays. Broader implementation in clinical practice will require further studies on cost‑effectiveness and operational feasibility.

Introduction

Visceral leishmaniasis (VL) is a severe parasitic disease caused by infection with Leishmania parasites, primarily transmitted through the bite of female sandflies, and is clinically known as kala-azar.^1,2 The parasites predominantly reside in the mononuclear phagocyte system, proliferating extensively within macrophages in organs such as the liver, spleen, bone marrow, and lymph nodes, leading to systemic reticuloendothelial hyperplasia and functional impairment.² Currently, endemic foci remain in several provinces in northwestern and southwestern China with Xinjiang being the most heavily affected region, accounting for 846 cases (annual incidence 0.34/100,000) from 2013 to 2023, predominantly in children under 2 years of age (87.12%);³ Kashgar Prefecture reported 978 indigenous cases from 2010 to 2023 with a seasonal peak in October–December.⁴ According to the World Health Organization (WHO), a total of 12,930 new visceral leishmaniasis (VL) cases were reported globally in 2024, with an estimated annual incidence of 50,000–90,000 cases, of which only 25%–45% are notified. Approximately 85% of cases were concentrated in seven countries: Brazil, Ethiopia, India, Kenya, Somalia, South Sudan, and Sudan. In 2024, 449 all‑cause deaths were reported among VL patients, while the cure rate among followed‑up cases was 98%. In 2023, Bangladesh became the first country validated by WHO as having eliminated VL as a public health problem.⁵ The clinical presentation of VL is nonspecific; typical manifestations include prolonged irregular fever, progressive splenomegaly, pancytopenia, and polyclonal hypergammaglobulinemia. The disease course is often protracted, making it easily confusable with various hematologic disorders and infectious diseases.⁶ Without timely diagnosis and effective treatment, the case fatality rate can exceed 95%.

Notably, VL may also serve as a significant trigger for immune hyperactivation, leading to secondary hemophagocytic lymphohistiocytosis (HLH).⁷ HLH is a life-threatening hematologic disorder characterized by excessive immune activation resulting in severe inflammatory responses and multiorgan dysfunction. The etiology of HLH is categorized into primary and secondary forms. Primary HLH is typically caused by genetic mutations, whereas secondary HLH may be triggered by infections, malignancies, autoimmune diseases, or other immune dysregulated states. Common infectious triggers include Epstein-Barr virus, among others. Although VL is a rare trigger of HLH, it remains of considerable importance in endemic regions and among immunocompromised patients. Visceral leishmaniasis-associated hemophagocytic lymphohistiocytosis (VL-HLH) shares strikingly similar clinical features with HLH,^2,8,9 such as fever and pancytopenia, posing significant diagnostic and therapeutic challenges.⁸ Early recognition and timely treatment of VL-HLH are crucial for improving patient outcomes.

Diagnosis of VL remains challenging, especially in HLH presentations. Conventional methods—microscopy, culture, and serology—have major limitations. Microscopy sensitivity is site-dependent (~70% for bone marrow vs >95% for spleen); culture is 60–85% sensitive but requires 2–4 weeks;¹⁰ and serological tests, including rK39, show geographic variability and reduced sensitivity in immunocompromised patients (~40% in HIV co-infection).^11,12 These constraints often delay diagnosis, necessitating faster and more reliable tools.

Herein, we report two cases of VL-HLH in which conventional assays failed to detect Leishmania pathogens, but metagenomic next-generation sequencing (mNGS) successfully identified the causative organisms—with diagnoses subsequently confirmed by rK39 rapid test—and summarize the epidemiological, clinical, diagnostic, and therapeutic lessons learned from these cases alongside previously reported cases in the literature.

Case Patient 1

A 49-year-old female patient was admitted to the People’s Hospital of Xinjiang Uygur Autonomous Region on January 23, 2025, presenting with recurrent high fever for one month as well as chills, fatigue and profuse sweating over the past 10 days. One month prior to admission, the patient developed fever with a maximum temperature of 39.5°C accompanied by chills, fatigue, excessive sweating and cytopenia. She received treatment at a local hospital with unknown specific regimens, yet no clinical improvement was achieved. Her condition deteriorated progressively with aggravated fatigue and nausea, and she was subsequently transferred to our hospital. Significant pancytopenia involving all three hematopoietic lineages was detected, and hemophagocytosis was observed on bone marrow examination. Hemophagocytic lymphohistiocytosis (HLH) was highly suspected, and the patient was transferred to the Department of Hematology for further management.

Admission vital signs: T 36.8 °C, P 78 beats/min, R 20 breaths/min, BP 90/65 mmHg. The patient was alert but lethargic, with no rash or pigmentation on skin, mucous membranes or sclera. Cardiopulmonary auscultation was normal. The liver was non-palpable; the spleen was palpable 3 finger breadths below the costal margin, moderately firm, blunt-edged and non-tender.

Past history: No viral hepatitis or tuberculosis. She had appendectomy and total thyroidectomy, no blood transfusion, hypertension, diabetes or coronary heart disease, and no notable weight change.

Following admission, relevant investigations were performed. Routine blood tests on admission: white blood cell count 1.7×10⁹/L, neutrophil count 1.16×10⁹/L, red blood cell count 3.89×10¹²/L, hemoglobin 103 g/L, platelet count 92×10⁹/L. C-reactive protein 60.55 mg/L, serum amyloid A 40.68 mg/L, procalcitonin 1.25 ng/mL. Coagulation tests: fibrinogen 1.75 g/L, thrombin time 20.6 s. Liver function: total protein 60.5 g/L, albumin 26.9 g/L, globulin 33.6 g/L, albumin/globulin ratio 0.80, alanine aminotransferase 148.0 U/L, aspartate aminotransferase 1075.0 U/L, AST/ALT ratio 7.26, gamma-glutamyl transferase 183.0 U/L, triglycerides 2.72 mmol/L. Serum ferritin: >33,511.2 ng/mL. Tests for hepatitis B and C, syphilis, HIV, influenza A and B, SARS-CoV-2, tuberculosis, and brucellosis were all negative. Non-contrast CT scan revealed: minimal dependent atelectasis in the lower lobes of both lungs; fatty liver; splenomegaly; and a small amount of pelvic fluid. Bone marrow aspiration report indicated: roughly normal granulocyte-to-erythroid ratio; normal megakaryocyte count with predominant granular megakaryocyte proliferation; no hemophagocytes were observed (partially diluted specimen).

Given the presence of pancytopenia, hypertriglyceridemia, hypofibrinogenemia, hyperferritinemia, significantly elevated soluble CD25 (sCD25), splenomegaly, and evidence of hemophagocytosis on bone marrow aspiration, the diagnosis of HLH was confirmed. According to the HLH-2004 chemotherapy protocol, the following treatment was administered: dexamethasone, starting on day 2 after admission, at a dose of 10 mg/m²/day for 2 weeks, followed by 5 mg/m²/day for 4 days; etoposide, starting on day 6 after admission, administered twice weekly at a dose of 5 mg/kg per dose, for a total of 4 doses; and supportive care including granisetron, fluid replacement, urinary alkalinization, recombinant human granulocyte colony-stimulating factor, intravenous immunoglobulin, and blood transfusions.^13,14

The patient’s initial symptoms showed no improvement. We hypothesized the presence of secondary HLH triggered by infection; however, tests for tumor and autoimmune diseases were all negative, and no other pathogenic evidence was identified To investigate the etiology of HLH, peripheral blood was sent for metagenomic next-generation sequencing (mNGS). During epidemiological investigation, the patient had no clear history of outdoor exposure or mosquito bites. During treatment, the peripheral blood mNGS report on January 27 showed: detection of Leishmania genus genomic sequences, with 4,516 reads for Leishmania genus, 104 reads for Leishmania donovani species, and a relative abundance of 45.86%. On the same day, the Xinjiang Center for Disease Control and Prevention performed the rK39 immunochromatographic rapid diagnostic test for visceral leishmaniasis, which yielded a positive result. The hematology laboratory was again requested to re-review the peripheral blood and bone marrow smears, but no Leishmania amastigotes were detected.

Based on clinical presentation, VL-HLH was diagnosed on day 5 after admission, and treatment with sodium stibogluconate at a dose of 0.6 g once daily by intramuscular injection was initiated, with a planned course of 6 to 10 days (Figure 1). One day after initiating antiparasitic therapy, the patient’s body temperature normalized, symptoms gradually resolved, and general condition improved. Follow-up blood tests showed significant recovery of blood cell counts compared with previous values, white blood cell count 2.3×10⁹/L, red blood cell count 4.01×10¹²/L, hemoglobin 110 g/L, platelet count 100×10⁹/L, and liver function returned to nearly normal levels. The patient was discharged after completion of the treatment course (Figure 2). At two weeks after discharge, the patient remained afebrile, and follow-up blood tests revealed normal three-lineage blood cell counts. A telephone follow-up on March 20, 2025, indicated that the patient had experienced no fever or other symptoms since the end of treatment; blood tests showed normal three-lineage blood cell counts, and the spleen had reduced in size compared with previous measurements. Due to local medical conditions and the patient’s financial constraints, no further etiological testing was performed.

Figure 1.

Gantt Chart of Medication Regimen for a Patient with Visceral Leishmaniasis During Hospitalization.

Figure 2.

Dynamic changes of hematological parameters of Case 1 during hospitalization.

Case Patient 2

A 50-year-old male was admitted to the Department of Hematology in our hospital on December 4, 2024, with a chief complaint of fever for two weeks. Two weeks prior to admission, the patient developed fever without an obvious trigger; his body temperature was not measured at that time. The fever was accompanied by cough and fatigue. He initially received symptomatic treatment with antipyretics and anti-inflammatory agents at a local hospital, but the response was poor. The fever persisted, and during the course of illness, pancytopenia developed. He was transferred to a higher-level local hospital for further evaluation, where treatment with anti-infective agents including cephalosporins and levofloxacin, along with fluid replacement, yielded limited efficacy. Bronchoscopy was performed to rule out tuberculosis and brucellosis. Over the course of the illness, progressive decline in blood cell counts was observed, with body temperature ranging between 38.5°C and 40°C. Laboratory findings included ferritin >1,500 ng/mL, hypofibrinogenemia (1.74 g/L), elevated transaminases, and splenomegaly. Hemophagocytic lymphohistiocytosis was considered as a possibility, and the patient was transferred to our hospital for further management. The patient reported a history of black stools for five days prior to admission, which were loose, occurring once to twice daily with small volumes. He had a history of hepatitis B virus carriage for more than 30 years without antiviral treatment, as well as a history of hypertension for over 20 years.

Following admission, comprehensive investigations were performed. Laboratory tests revealed elevated levels of white blood cell count 3.2×10⁹/L, red blood cell count 3.83×10¹²/L, hemoglobin 115.0 g/L, platelet count 83×10⁹/L, C-reactive protein 64.19 mg/L, alanine aminotransferase (155.7 U/L), aspartate aminotransferase (338.51 U/L), total bilirubin (26.25 μmol/L), globulin (45.1 g/L), albumin 29.6 g/L, gamma-glutamyl transferase (473.0 U/L), alkaline phosphatase (529.0 U/L), creatine kinase (80.0 U/L), and lactate dehydrogenase (1448.0 U/L). Hepatitis B virus DNA was detected at 5.70 × 10² IU/mL, immunoglobulin G was 28.9 g/L, and fungal (1,3)-β-D-glucan was >1,000 pg/mL. No other pathogenic evidence was identified; tests for galactomannan and Aspergillus IgG antibodies were negative, and screening for hepatitis C, syphilis, HIV, influenza A and B, SARS-CoV-2, other respiratory pathogens, tuberculosis, and brucellosis were all negative. Peripheral blood and bone marrow smears showed no significant abnormalities. Bone marrow immunohistochemistry (specimen A2436426-1) revealed: E-cadherin (+), CD3 (scattered +), CD42b (+), CD34 (−), CD20 (−), CD15 (+), myeloperoxidase (+), CD117 (−), CD138 (scattered +), kappa (+), and lambda (+). Bone marrow examination from the posterior iliac crest showed a normal granulocyte-to-erythroid ratio with readily visible hemophagocytic histiocytes. Bone marrow karyotyping revealed no clonal numerical or structural abnormalities. Abdominal ultrasound demonstrated splenomegaly (147 mm × 42 mm). Leukemia immunophenotyping revealed no immature cell population with an aberrant immunophenotype. Based on the presence of fever, splenomegaly, pancytopenia, hypofibrinogenemia, hypertriglyceridemia, hyperferritinemia, elevated soluble CD25, and the presence of hemophagocytic histiocytes in the bone marrow, a clinical diagnosis of hemophagocytic lymphohistiocytosis was established. The underlying etiology remained to be clarified.

Given the patient’s history of long-term outdoor work and mosquito exposure, peripheral blood was sent for metagenomic next-generation sequencing (mNGS). The report issued on December 26 revealed the presence of genomic sequences belonging to the genus Leishmania: 9,131 reads were detected for the Leishmania genus, with 5,371 reads specifically identified as Leishmania donovani complex, representing a relative abundance of 58.6%. On the same day, the Xinjiang Center for Disease Control and Prevention reported a positive result for the rK39 immunochromatographic rapid diagnostic test for visceral leishmaniasis. Repeat review of peripheral blood and bone marrow smears still failed to detect Leishmania amastigotes. Treatment was administered according to the HLH-2004 chemotherapy protocol: dexamethasone, starting on day 2 after admission, at a dose of 10 mg/m²/day for 2 weeks, followed by 5 mg/m²/day for 4 days; etoposide, starting on day 6 after admission, administered twice weekly at a dose of 5 mg/kg per dose, for a total of 4 doses; and supportive care including granisetron, fluid replacement, urinary alkalinization, recombinant human granulocyte colony-stimulating factor, intravenous immunoglobulin, and blood transfusions. After the diagnosis of VL-HLH was confirmed, antiparasitic therapy with sodium stibogluconate at a dose of 0.6 g once daily by intramuscular injection was initiated on day 4 after admission, with a planned course of 10 days (Figure 3).

Figure 3.

Gantt Chart of Medication Regimen for a Patient with Visceral Leishmaniasis During 14-Day Hospitalization.

During treatment for HLH, the patient experienced persistent high fever, with a maximum body temperature reaching 41°C. Following initiation of sodium stibogluconate, the fever and associated symptoms gradually resolved three days after starting antiparasitic therapy, and the patient’s general condition improved. By the completion of the 10-day treatment course, body temperature had normalized (Figure 4), follow-up blood tests showed recovery of white blood cell and platelet counts with only mild anemia remaining, white blood cell count 3.6×10⁹/L, red blood cell count 4.38×10¹²/L, hemoglobin 119.0 g/L, platelet count 158×10⁹/L, C-reactive protein 6.4 mg/L, albumin 32.6 g/L, and liver function had returned to nearly normal levels. The patient was discharged after completion of the treatment course. One month after discharge, follow-up blood tests demonstrated normal three-lineage blood cell counts, and the patient reported no fever or other symptoms. A telephone follow-up on March 20, 2025, revealed normal three-lineage blood cell counts; ultrasound showed normal liver and spleen morphology, with no fever or other symptoms reported. Due to local medical conditions and the patient’s financial constraints, etiological follow-up testing was not performed.

Figure 4.

Serial Changes in Body Temperature in a Patient with Visceral Leishmaniasis During Treatment.

The main clinical features of the two patients are summarized and compared in (Table 1).

Table 1.

Comparison of Clinical Features Between Patient 1 and Patient 2

Feature	Patient 1	Patient 2
Age (years)	49	50
Sex	Woman	Man
Fever	Yes	Yes
Pancytopenia	Yes	Yes
Splenomegaly	Yes	Yes
Ferritin elevation	>33511.2ng/mL	>1500ng/mL
HLH	Met(8/8:fever, splenomegaly, cytopenias, hyperferritinemia, Hypertriglyceridemia hemophagocytosis LowNK‑cell activity Elevated soluble)	Met(8/8:fever, splenomegaly, cytopenias, hyperferritinemia, Hypertriglyceridemia hemophagocytosis Elevated soluble)
Bone marrow smear	Negative	Positive
mNGS	Positive	Positive
rK39	Positive	Positive
Treatment	Sodium stibogluconate	Sodium stibogluconate
Outcome	Cured	Cured

Metagenomic Next-Generation Sequencing (mNGS) Detection Procedure

All mNGS tests were performed at the Infection Precision Medicine Laboratory, People’s Hospital of Xinjiang Uygur Autonomous Region. Peripheral blood samples collected from two patients were subjected to pathogen metagenomic next-generation sequencing.

1. Nucleic acid extraction: Cell-free total DNA was extracted from plasma using magnetic bead-based nucleic acid extraction kits in strict accordance with the manufacturer’s standard operating procedures in the DNA preparation room of the laboratory.

2. Library construction: Double-stranded DNA libraries were constructed for all samples, followed by library purification and quality inspection.

3. Sequencing platform and sequencing depth: Sequencing was carried out on the MGISEQ-2000 high-throughput sequencing platform (MGI Tech Co, Ltd). The average sequencing depth of each specimen was 20 million clean reads.

4. Bioinformatic analysis pipeline: Raw sequencing data were filtered to remove low-quality reads and host human genome sequences by Bowtie2 software. The remaining valid microbial reads were aligned to the self-built pathogen reference database of our laboratory.

5. Reference database: The database covered genome sequences of bacteria, fungi, viruses, and parasites including Leishmania donovani.

6. Positive diagnostic criteria for mNGS: Consistent unique specific reads mapped to Leishmania donovani were defined as a positive mNGS result. Combined with clinical manifestations, routine negative pathogen smears and culture results, the mNGS positive finding supported the suspected diagnosis of visceral leishmaniasis.

Discussion

Visceral leishmaniasis (VL) is caused by obligate intracellular protozoan parasites of the genus Leishmania, which primarily infect the mononuclear phagocyte system of the host. The clinical manifestations of this disease are nonspecific. Patients may present with prolonged irregular fever accompanied by chills and hepatosplenomegaly; the fever can persist for several weeks, and the spleen may progressively enlarge to become a massive organ. Other features include progressive anemia, pancytopenia, and lymphadenopathy. The skin of the face, extremities, and abdomen may gradually darken due to rough pigmentation, hence the name “kala-azar,” meaning black sickness.¹⁵

The onset of VL is insidious and lacks characteristic features. In non-endemic regions, it is prone to misdiagnosis or delayed diagnosis because its manifestations overlap with those of common febrile illnesses such as influenza, lymphoma, Epstein-Barr virus infection, and systemic lupus erythematosus.^16–18 Without timely treatment, more than 95% of patients die from complications within one to two years after disease onset.¹⁹ Clinical studies have found that the misdiagnosis and missed diagnosis rate of kala-azar is as high as 84.2%, representing one of the major challenges in the diagnosis of VL in the country.^20,21 In the present study, two patients with VL were treated at our institution. One patient was a resident of a rural area in the Kashi region of Xinjiang, an endemic region, with no clear history of mosquito exposure. The other patient originated from a non-endemic region but had a history of long-term outdoor work with exposure to wild dogs and mosquito bites. Both patients were transferred to the hematology department due to VL complicated by hemophagocytic lymphohistiocytosis (HLH).

Hemophagocytic lymphohistiocytosis (HLH) is a systemic inflammatory syndrome mediated by immune dysregulation triggered by various factors and can be classified as primary or secondary HLH. Early clinical symptoms of HLH may be atypical and easily overlooked; however, once classic manifestations appear, the disease progresses rapidly and carries a high mortality rate. Secondary HLH often develops in the context of infections, rheumatic diseases, or malignancies, leading to abnormal activation and proliferation of lymphoid, monocytic, and macrophage systems, resulting in the secretion of large quantities of inflammatory cytokines and a life‑threatening inflammatory state. Prompt identification and treatment of the underlying etiology can effectively halt disease progression and improve patient outcomes.²²

In the two cases reported here, although conventional diagnostic methods including peripheral blood and bone marrow smears as well as blood and bone marrow cultures failed to detect Leishmania‑Donovan bodies initially, we performed rigorous quality‑control measures to ensure the reliability of these negative results. Specifically, at least three peripheral blood films and multiple bone marrow slides were independently reviewed by two experienced hematologists, and the bone marrow cultures were incubated for up to 4 weeks to exclude slow‑growing organisms. Despite these exhaustive efforts, the results remained negative, which we attribute to the typically low parasitemia and uneven distribution of amastigotes within the bone marrow in immunocompromised hosts—a condition that substantially limits the sensitivity of microscopy (estimated at <50% in such populations).²³ This observation underscores the inherent diagnostic bottleneck of conventional approaches in VL‑associated HLH.

Critically, metagenomic next‑generation sequencing (mNGS) promptly identified Leishmania donovani in both peripheral blood and bone marrow aspirate specimens from both patients, providing robust molecular evidence.²⁴ The findings were subsequently confirmed by serological testing at the Center for Disease Control and Prevention, where the rK39 rapid diagnostic test yielded positive results for specific antibodies. We acknowledge that, at the initial presentation when conventional smears were negative, these two cases could be classified as “clinically suspected” VL. However, the combination of positive mNGS reads (from dual specimens) and confirmatory CDC serology fulfills the etiological criteria for “confirmed cases” according to the national diagnostic guidelines for leishmaniasis (WS 258‑2006).²⁵ Therefore, we emphasize that a negative smear result should never exclude VL, especially in endemic areas or patients with relevant epidemiological exposure, and that early mNGS should be considered to secure a definitive diagnosis.

Regarding the role of rK39 RDT, it serves as a supplementary serological tool rather than a gold‑standard diagnostic method.²⁶ In the context of HLH‑related immunosuppression, delayed seroconversion or false‑negative results are possible; thus, a negative rK39 result cannot rule out VL, whereas a positive result provides valuable confirmatory support. In our diagnostic algorithm, rK39 functioned as a confirmatory adjunct, while mNGS played the decisive role in early etiological identification, offering a critical time window before serological results became available. These cases indicate that mNGS can serve as a useful adjunctive diagnostic method for VL‑associated HLH when conventional diagnostic approaches yield inconclusive results. Further studies are required to verify its clinical utility and cost-effectiveness.

Notably, both patients received specific HLH‑directed immunosuppressive therapy (eg, corticosteroids and/or etoposide) prior to or concurrently with antiparasitic treatment, as is often required for managing the life‑threatening cytokine storm.²⁷ However, despite this immunomodulation, the fever and cytopenias did not resolve until the definitive etiology was established and timely antiparasitic treatment (sodium stibogluconate) was initiated. Following pathogen clearance, all HLH‑related parameters normalized, ultimately leading to clinical cure in both cases. This outcome reinforces the principle that treating the underlying cause remains the cornerstone of managing secondary HLH, even when immunosuppressive agents are used to control acute inflammation; the latter should not replace or delay specific anti‑infective therapy once the pathogen is identified.

Metagenomic next-generation sequencing (mNGS) is an unbiased high-throughput sequencing method that does not rely on clinical culture and can comprehensively detect the microbiome in clinical samples. It is applicable to patients with fever of unknown origin, critical illness, or immunocompromised status, and can be performed on all sample types, including whole blood and cerebrospinal fluid. The technique offers the advantages of comprehensive detection, high sensitivity, and high specificity.^28,29 A systematic review indicated that blood and bone marrow tissue are suitable samples for mNGS, with a detection rate of 100% for Leishmania, whereas the detection rates of bone marrow cytology and rK39 testing were 82% and 80%, respectively.³⁰ A case study from West China Hospital showed that mNGS can serve as a complementary diagnostic tool when conventional testing yields negative results, shortening the time to diagnosis to three to five days. This technique can circumvent false-negative results associated with traditional methods, particularly in immunocompromised or HIV-positive populations.³¹ However, the current application of mNGS is limited by its high cost and the technical constraints of some hospitals. Interpretation of mNGS results requires integration of laboratory findings with clinical evaluation; laboratory personnel must possess robust bioinformatics analysis capabilities to accurately identify microbial sequence reads, while clinicians must interpret results in the context of individual patient characteristics, including symptoms, disease course, underlying conditions, and ancillary testing.

Currently, microscopic detection of Leishmania amastigotes or promastigotes in bone marrow or peripheral blood smears or via culture remains the gold standard for diagnosing VL;³² however, the sensitivity of this method ranges from 53% to 86%, leaving a substantial risk of missed or delayed diagnosis. In the two cases described here, the gold standard method failed to identify the pathogen, whereas mNGS successfully detected the pathogen, and the diagnosis was confirmed by the rK39 test. Timely and targeted antiparasitic therapy led to clinical cure, confirming the diagnosis and preventing missed diagnosis and progression to HLH with its associated poor outcomes. By performing high-throughput sequencing of nucleic acids from clinical samples, mNGS enables rapid, unbiased detection of a wide range of pathogens, including viruses, bacteria, fungi, and parasites, demonstrating significant clinical value for the investigation of infectious diseases. Metagenomic sequencing provides a novel tool for early and accurate diagnosis of VL, offering unique advantages in cases where conventional methods fail or in immunocompromised patients.³³ Future efforts should focus on optimizing technical workflows in clinical practice to promote the widespread application of mNGS for etiological diagnosis in patients with fever of unknown origin.

Funding Statement

This work was supported by the Natural Science Foundation for Distinguished Young Scholars of Xinjiang Uygur Autonomous Region, China (Grant No. 2024D01E21) and the “Tianshan Innovation Team” Project of Xinjiang UygurAutonomous Region, China (Grant No. 2025D14022).

Data Sharing Statement

The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request.

Ethical Considerations

This study was conducted in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of The People’s Hospital of Xinjiang Uygur Autonomous Region (Approval No.: KY2026020501). Written informed consent for publication of this case report, including the use of de-identified clinical data and related information, was obtained from both patients involved. All patient-identifying details have been fully anonymized to protect personal privacy. This report was prepared in compliance with the CARE guidelines, and all relevant ethical procedures were strictly followed.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.