A 22-year-old man presented with a 70-day history of bilateral lower extremities weakness, followed by fever, productive cough and jaundice. On March 20th 2017, the patient suddenly developed lower limb weakness without any other notable symptoms. After the patient’s admission to the local hospital, brain computed tomography (CT) scan found no abnormalities while the contrast magnetic resonance imaging (MRI) detected multiple lesions in the brain and abnormal signals in spinal cord on T2 (Fig. 1a,b). Chest CT scan revealed multiple nodules in both lungs and a large lesion in lower left lung combined with cavity formation and gas-fluid levels inside (Fig. 1c). Abdomen CT scan showed hepatosplenomegaly and multiple retroperitoneal lymph node enlargement. Laboratory tests reported a leucocyte count of 12.7 × 103 cells/μL, a alanine aminotransferase activity (ALT) of 116 U/L, a total bilirubin concentration (TBil) of 37 μmol/L (direct bilirubin (DBil): 31.8 μmol/L), an serum albumin level of 25 g/L and moderate anemia. HIV serology test, HBV/HCV serology test, serum T-SPOT.TB test, blood culture and cryptococcal latex agglutination test (CLAT) were all negative. Lumbar puncture revealed a decreased glucose concentration, an increased protein concentration and a normal leucocyte count of the cerebrospinal fluid (CSF). The results of bacterial and fungal cultures, CLAT, indian ink staining and acid-fast staining of CSF were all negative. The local hospital suspected disseminated tuberculosis infection and empirical anti-tuberculosis treatment with isoniazid, rifampin, ethambutol and pyrazinamide was prescribed.
After the treatment, no remission of the previous symptoms was observed and the patient developed new discomforts including fever (Tmax 39 °C), productive cough and jaundice in the early April. Repeated blood, sputum and CSF cultures all came back negative. During the next two months, the patient continuously to have intermittent low grade fever, weakness of lower extremities and jaundice and by the end of May, rashes started to appear over his face and back. The local medical facility then transferred the patient to our hospital on June 7th, 2017.
A careful history was taken and the patient reported no significant past medical history and denied any history of addiction, drug abuse, and exposure to toxic matter. During examination, physician identified multiple papules on the patient’s face and backside, which were 0.5 cm to 2 cm in diameter with crusted center (Fig. 2). His skin and conjunctiva were yellow and both legs’ muscle strength were rated grade 0. He had slightly increased muscle tone and positive Babinski signs in both lower extremities. Laboratory testing revealed a C-reactive protein concentration of 29 mg/L, a ferritin concentration over 2000 ng/ml, an ALT of 36 U/L, an aspartate transaminase activity (AST) of 119 U/L, and a Tbil of 302.6. HIV RNA test were negative, and the lymphocyte subsets were within normal range. Blood gas analysis suggested a type II respiratory failure and other laboratory results were all insignificant. Contrast MRI scans revealed multiple lesions in the brain with ring enhancement, and abnormal enhanced lesions in spinal cord, pia mater spinals and vertebrae similar to previous imaging results. Contrasted CT scan further discovered suspicious lesions in liver, spleen, left adrenal gland, prostate, intra-abdominal lymph nodes and positron emission tomography/computed tomography scan found abnormal uptakes in these regions as well. After obtaining written consents from the patient, bone marrow biopsy and skin biopsy of the facial lesions were conducted on June 8th followed by lumbar puncture and fibro bronchoscope in later days. All samples were sent for culture and NGS (See Additional file 1 for NGS method) and during the following days, only supportive treatments were given.
On July 11th, NGS reported detecting 18,987 of 28,648,375 T.marneffei nucleotide sequences (Cover rate: 0.066%) in bone marrow and 144,780 of 28,648,375 T.marneffei nucleotide sequences (Cover rate: 0.51%) in DNA extracted from the skin lesions (Fig. 3a). Considering the notably high sequencing reads compared to the negative control (in which no T.marneffei nucleotide sequence was detected) and the patient’s clinical manifestation, physicians reached the diagnosis of T.marneffei infection and intravenous anti-fungi therapy consisting of 25 mg amphotericin B deoxycholate and 250 mg itraconazole per day was initiated. On July 14th, culture of the skin lesion tissue reported T.marneffei (Fig. 2) and 7 days later, repeated blood and bone marrow culture for bacteria, fungi and tuberculosis all came back negative. On July 19th, NGS reported detecting 25,906 of 28,648,375 T.marneffei nucleotide sequences (0.09%) in the BALF sample and 17,877 of 28,648,375 T.marneffei nucleotide sequences (0.062%) in the CSF sample. The cultures of both CSF and BALF for fungi and bacteria came back negative (Both Myco/F lytic system from BD company and Sabouraud’s agar medium were used in fungal culture).
We then conducted sequencing of the isolated strain from the skin lesion culture, and a 97% coverage of T.marneffei was identified (Fig. 3b). Close relationship between our strain and other T.marneffei strains was revealed in phylogenetic analysis (Fig. 3c). Confirmatory nested PCR [4] was done in patient’s blood, CSF, BALF and bone marrow samples and all PCR results were positive.
In consideration of all the facts, the physicians diagnosed the patient with disseminated T.marneffei infection, which invaded lung, bone marrow, central nervous system and skin. The anti-fungi therapy consisting of amphotericin B deoxycholate and itraconazole was continuously prescribed and the patient’s fever resolved three days after the initiation of the treatment, while other clinical symptoms such as cough, jaundice, skin lesions and lower limb weakness resolved within a month of the therapy (Fig. 4). Until this article was submitted, the total amount of amphotericin B deoxycholate reached 1170 mg, and the patient was discharged from the hospital with itraconazole oral solution.