Chinese Medical Journal 2002;115(12):1892-1894
Clinical blood routine and bone marrow smear manifestations of disseminated penicilliosis marneffei

MO Wuning 莫武宁,  DENG Zhuolin 邓卓霖,  LI Shan 李山

MO Wuning 莫武宁 (Central Laboratory,the First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China)

DENG Zhuolin 邓卓霖 (Department of Pathology, the First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China)

LI Shan 李山 (Central Laboratory,the First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China)

Correspondence to:Mo Wuning,Centrl Laboratory, the First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China (Tel: 0771-5356705. Fax:0771-5356052. E-mail:spymun@163.net)
Keywords
penicilliosis marneffei;bone marrow smear;blood smear
Abstract
Objective To investigate the manifestation of clinical, blood routine and bone marrow smear of disseminated penicilliosis marneffei.
Methods
There were 13 cases of penicilliosis marneffei whose peripheral blood had been drawn for routine tests, as well as blood and bone marrow aspiration for smears. Wright’s-Giemsa stain, Gomori’s methenamine?-silver stain (GMS) and periodic acid Schiff’s reaction (PAS) were performed for light microscopy in consultation with pathologic diagnosis and fungi culture for precise diagnosis.
Results Seven cases of bone marrow and 2 peripheral blood smears were found positive for penicillium marneffei in the test group. The morphology of penicillium marneffei was extremely similar to that of histoplasma capsulatum. However, the observation of sausage cells and central cross wall, which are signs of dividing by fission and not by budding, aided in differential diagnosis.
Conclusion
Bone marrow smear or occasional blood smear examination play an important role in the diagnosis of disseminated penicilliosis marneffei.

Penicilliosis marneffei is a serious fungus infection caused by Penicillium marneffei (P. marneffei), a facultative intracellular pathogen and the only thermally dimorphic Penicillium.[1]P. marneffei usually invades blood vessels and leads to a disseminated and progressive infection[2]and often causes an osteomyelitis and osteolytic lesion, which is why bone marrow is used as a sample for its diagnosis.[3]In order to evaluate the significance of clinical, blood routine and bone marrow smear (BMS) manifestations towards the diagnosis of disseminated penicilliosis marneffei, 13 cases were collected for analysis.

METHODS

There were 13 cases of disseminated penicilliosis marneffei having BMS performed on them available on file (8 males and 5 females). Ages ranged from 4 months to 45 years old. All cases were collected from Guangxi Medical University and confirmed by pathologic examination and fungus culture. Clinical manifestations were based on case records. Blood routines and BMS were examined in every case. The blood smears and BMS were stained with Wright’s-Giemsa, Gomori’s methenamine-silver stain (GMS) and periodic acid Schiff’s reaction (PAS), and were observed by light microscopy. Seven of the 13 cases were found positive for P. marneffei using BMS and 2 cases using blood smears.

RESULTS

Clinical findings
Penicilliosis marneffei was divided into two clinical types: localized and disseminated. The localized type was rarely found in literature, because no characteristic clinical manifestations were suitable for diagnosis of the disease. This was accidentally discovered by autopsy or biopsy, and verified by confirmative pathogens. The most common clinical features observed in patients with disseminated P. marneffei included fever, weight loss and anemia. Many patients had nonproductive coughs. Diarrhea was often seen in infants and young children with intestinal infections caused by swallowed P. marneffei. Through intestinal infection, P. marneffei was able to invade the liver via vena portage, causing liver dyshepatia and icterus. About half of the cases were accompanied by multiform rashes on skin. Molluscum contagiosum-like papules was one characteristic manifestation of late penicilliosis marneffei. Hepatospleno~megaly and lymphadenopathy were observed in many patients, especially children.

Blood routine findings
Documentation of blood routine examination of penicilliosis marneffei were rare. In recorded cases, all patients had reduced hemoglobin, ranging from 30 g/L to 97 g/L, with the most significant reductions in patients with P. marneffei found in BMS. Leukocytosis was often found in patients without P. marneffei in bone marrow smears; the remainder of cases was normal or leucopenial. White blood cell counts ranged from 3.6×10 9 /L to 52.4×10 9 /L. Platelet counts in all patients were normal. Differential blood count was usually normal, but in some cases was neutrocytotic. Eosinophil leukocytes were oormal. Of these 13 cases, 2 were also AIDS patients, and they were the only ones that were P. marneffei positive for blood smears. The pathogens were mostly located in the cytoplasm of monocyte and neutrocyte cells, and a few pathogens were freely scattered extracellularly (Fig. 1). In Wright’s-Giemsa and PAS staining, the morphology of P. marneffei in blood smears was similar to the morphology in BMS.

Bone marrow smear findings
In all cases, bone marrows were normoplastic or hyperplastic. Granulocytic series were normoplastic or hyperplastic; in some cases, left shelf and cytoplasmic vacuolization were observed. Erythrocytic series were usually normoplastic, but hyperplasia had been identified in a few cases. Megakarocytes were usually normal, as were lymphocytes and monocytes. In some cases, plasma cells were slight hyperplastic. Reticulum cells were proliferative, which were noticeable in cases with positive P. marneffei in BMS. The pathogens were mostly located in the cytoplasm of macrophages, but a few pathogens were freely scattered extracellularly (Figs. 2-4) . In Wright’s-Giemsa staining, the yeast forms of P. marneffei were round, ellipsoidal or sausage-like in shape, with one or two purplish-red small nuclei and light blue cytoplasm, but the cell wall could not be seen clearly. In PAS staining, cell walls of P. marneffei were red and distinct. The best stain for P. marneffei was GMS, in which the cell walls were black and distinct. It was notable that some sausage-shaped cells possessing cross-walls could be observed that had a thickness double that of cell walls. Furthermore, the observation of these cells indicated that P. marneffei was divided by fission and not budding, which served as a point of diagnosis and differential diagnosis of the fungus (Figs. 3 and 4).

DISCUSSION

As with most fungi, P. marneffei is a naturally occurring plant saprobe. Human infection is commonly due to congenital or acquired immunodeficiency, such as thymic dysplasia in infants, cancer patients, immunosuppressant therapy and AIDS patients.There are about 300 species in the Penicillium genus; few of them have pathogenicity, suggesting that most of them cannot survive at 37℃ body temperature. P. marneffei is the only dimorphic fungus of the genus Penicillium. The fungus grows similar to the speed of mold at 25℃ on glucose agar medium, with colonies up to 5 mm in size, two days showing classical conidiophores with phialides and conidia. At 37℃, its speed of growth is more similar to yeast on the same medium, also relatively fast. One of P. marneffei’s characteristics is that it invades blood vessels and easily moves with the blood current, leading to a disseminated and progressive infection. It usually invades mononuclear phagocytic systems, including bone marrow, and survives and multiplies intracellularly. Because penicilliosis marneffei is a deeply rooted fungal infection, samples for examination are not easy to obtain, especially for early diagnosis, consequently leading to frequent misdiagnosis. Obtaining samples by superficial lymph node biopsy and bone marrow aspiration is recommended. Fungus cell wall staining with either GMS or PAS yields better results than with Wright’s-Giemsa, such that recognition of size and morphology is possible. Under light microscopy, P. marneffei is identified easily on a well-stained smear. P. marneffei is about (2-3)×(2-8) μm[2] in size. Using Wright’s-Giemsa staining, the morphology of P. marneffei yeast cells is observed as round, ellipsoidal, or sausage-like with light blue cytoplasm, but cell walls cannot be seen clearly.

However, PAS staining results in red and distinct cell walls, while GMS staining obtains distinct and black cell walls that do not fade significantly over time. P. marneffei often clusters inside the cytoplasm of macrophages and enlarges the cells; as the smear is performed, the enlarged macrophages are often pushed forward to the tail or the edge of the smear such that they can easily be found. Some P. marneffei yeast cells become elongated with two blunt ends and are frequently curved like a sausage, measuring about (2-3)×(6-8) μm[2] in size, which is why they are called sausage cells. Occasionally, some of these cells appear to have a clear central septum.[4]The occurrence of sausage cells and central cross walls indicates division by fission, not budding. Another common pathogenic fungus, Histoplasma capsulatum, morphologically is very similar to P. marneffei, but divides by budding. This difference in proliferation method aids in differential diagnosis between the two pathogens.[5]

In our cases, were found to be P. marneffei positive for BMS resulting in the diagnosis of penicilliosis marneffei. This indicates that examination of BMS is an important and faster way (about 1 h) for diagnosis of penicilliosis marneffei, with blood smear helpful only in seriously disseminated cases, such as the involvement of AIDS. When patients do not have skin lesions or superficial lymphadenovarix, fungus culture of secretion and biopsy are difficult, at which point bone marrow puncher should be utilized not only for the smear, but also for culture. Usually the smear offers first information, whereas culture is usually the final step in making a diagnosis. Bone marrow aspiration in multiple locations, as well as overall careful inspection may enhance the detection rate.

REFERENCES

1. Duong TA. Infection due to Penicillium marneffei, an emerging pathogen: review of 155 reported cases. Clin Infect Dis 1996;23:125-130.
2. Deng ZL, Ma Y. Study of vascular wall penetration, an intrinsic feature of Penicillium marneffei. J Clin Exp Pathol 1999;15:30-31.
3. Deng ZL, Ma Y. Disseminated Penicillium marneffei complicated with osteolytic lesions. J Mycol Med 1995;5:44-49.
4. Deng ZL, Li SZ, Ma Y, et al. Penicillium marneffei. Chin J Pathol 1997;16:306-309.
5. Deng ZL, Ma Y. Differential diagnosis between Penicilliosis marneffei and histoplasma capsulatum. Chin J Med Lab 1999;22:313-315.