Novel Method Identifies Pathogens in Bone Marrow Smears

The rapid identification of four pathogens and the early diagnosis of the four different diseases are of great importance; however some pathogens have a similar size and shape in the Wright‑Giemsa staining of bone marrow smears.

A novel method has been developed to rapidly identify the pathogens Penicillium marneffei, Histoplasma capsulatum, Mucor, and Leishmania donovani in bone marrow smears that can lead to the diseases penicilliosis marneffei, histoplasmosis, mucormycosis, and leishmaniasis, respectively.

Clinical laboratory scientists at Guangxi Medical University (Nanning, China) obtained 55 bone marrow smears (BMS) that were infected. There were 25 invaded by P. marneffei, 10 with H. capsulatum, 3 with Mucor, and 17 infected by L. donovani. The diagnosis of the four diseases was confirmed by pathogen culture. Each BMS was fixed by 95% alcohol for 10 minutes, prior to being washed, dried, stained by 1% periodic acid for 20 minutes and washed and dried once more. Each BMS was then stained by a Schiff reagent for 60 minutes, washed and dried, and subsequently stained by hematoxylin for five minutes, washed and dried. The BMSs were observed using light microscopy. Wright‑Giemsa staining was also performed, following standard protocols.

In the Wright Giemsa staining, the four pathogens were approximately the same size and mainly existed in macrophages. The multiplying P. marneffei had two nuclei, which were on both sides of the fungus, and had light cross walls in the middle. H. capsulatum had a purplish nucleus, which occupied between one third and one half of the spore. The cytoplasm was light blue. Peripheral spores were observed in the form of an empty, bright ring without color, like a capsule.

Generally Mucor were observed to have a long and lightly stained area, which could be confused with the Wright staining of dinuclear P. marneffei. L. donovani exhibited a deep staining kinetoplast near the nucleus. In the Periodic Acid Schiff (PAS) staining, the pathogens of P. marneffei and H. capsulatum were distinct and stained red.

The authors concluded that their staining method was simple and can enable the rapid diagnosis of penicilliosis marneffei, histoplasmosis, mucormycosis and visceral leishmaniasis. Furthermore, the method may have an important role in diag-nosing the four diseases in cases in which it is not possible to conduct pathogen culture and serology tests. The study was published in the May 2015 issue of the journal Experimental and Therapeutic Medicine.

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Guangxi Medical University