Metagenomic next-generation sequencing assists in the diagnosis of visceral leishmaniasis in non-endemic areas of China

Abstract

Introduction: Leishmaniasis, a protozoan disease caused by infection by Leishmania, is a critical issue in Asia, South America, East Africa, and North Africa. With 12 million cases globally, leishmaniasis is one of the most serious neglected tropical diseases worldwide. Direct identification of infected tissues is currently the primary method of diagnosis; however, the low sensitivity and inconvenience of microscopic examination in detecting amastigotes, parasitic manifestations of Leishmania, leads to the possibility of misdiagnosis, delayed diagnosis, and underdiagnosis.

Methods: With the development of metagenomic nextgeneration sequencing (mNGS) technology for pathogen identification, it is possible to detect specific nucleic acid sequences characteristic of Leishmania parasites, which opens new avenues for the more accurate diagnosis of leishmaniasis. In this study, we report two cases of leishmaniasis from Henan Province, China, in which Leishmania parasites were identified using mNGS technology, massively expediting diagnosis and treatment.

Results: Our report demonstrates that the mNGS method is applicable to peripheral blood samples (PB), which are far more readily available in clinical settings, in addition to bone marrow aspirate samples (BM), which are traditionally used for diagnosis of visceral leishmaniasis.

Conclusion: Our report validates the efficacy of mNGS technology as a rapid and accurate method of diagnosis for leishmaniasis.

Keywords: clinical diagnosis; endemic area; leishmaniasis; metagenomic next-generation sequencing assists; therapy.

Figure 1

Transverse section from chest CT scan of patient 1 showing pleural effusion, hyperdense regions surrounding the lungs, as indicated by the arrows. Pleural effusion is characteristic of pulmonary infection.

Figure 2

The genome coverage map of Leishmania infantum as detected by mNGS in case 1. In the first mNGS test, genome coverage of Leishmania infantum in the PB sample was 0.04% (A) and 0.02% in BALF (B). In the second mNGS test, the genome coverage of Leishmania infantum in the PB sample was 0.16% (C) and 0.02% in sputum (D).

Figure 3

Bone marrow micrographs from patient 2. Active bone marrow hyperplasia, reticular cells, hemophagocytosis and Leishmania amastigotes, as indicated by the arrows, can be seen. (A) Macrophages contain numerous Leishman-Donovan bodies. (B) Leishman-Donovan body infect more circulating or fixed macrophages. (C) The macrophage dies, Leishman-Donovan body are released. (D) Macrophages engulf a large number of lydosomes. Cells visualized with H&E stain, 100X magnification. Scale bar represents 5 µm.

Figure 4

The genome coverage map of Leishmania infantum as detected by mNGS in case 2. The genome coverage of Leishmania infantum in the PB sample was 2.79% (A) and 4.01% in BM (B).