A 27-year-old woman was admitted to our hospital (Nov 2, 2018), complaining of two weeks of cough, intermittent low fever and dyspnea during daily activity. Four months prior to the admission, she underwent a renal transplantation because of kidney failure caused by chronic glomerulonephritis. Following successful transplantation, she took immunosuppressive medication, including tacrolimus (2.5 mg twice a day), mycophenolate mofetil (750 mg twice a day) and methylprednisolone (12 mg daily) for four months. During this period, she did not receive influenza immunization or prophylaxis for opportunistic infections including Pneumocystis and cytomegalovirus (CMV) infections as per the Diagnosis and Treatment Guideline of Invasive Fungal Infections in Solid Organ Transplant Recipients in China (2016 edition). Two weeks prior to admission to our hospital, she presented to a local community hospital with complaints of fever, cough and dyspnea during daily activity. Chest X-ray showed bilateral patchy, cloudy opacities in the lower lungs with some adjacent pleural thickening and adhesion (Figure 1A). After empiric treatment with oral moxifloxacin for 7 days, there was no apparent improvement, with fever remaining high (38 C) and dyspnea aggravated.
Upon admission to our hospital, physical examination found a normal body temperature of 36.7 C, blood pressure of 143/93 mm Hg, pulse of 116 beats per minute, and respiration rate of 24 per minute. Moist rales appeared bilaterally, with no lower-extremity edema. Due to hypoxemia (PO2: 63 mmHg, PCO2: 29 mmHg, SO2: 90%), she was given oxygen therapy with mask hydrogen inhalation shortly after admission. High-resolution computed tomography (HRCT) demonstrated diffused stripes and reticular and ground-glass opacity with honeycomb structures, primarily in the lower lungs (Figure 1B and C). Other laboratory tests revealed abnormal values for almost all parameters tested elevated levels of several biomarkers for inflammatory responses and decreased counts of lymphocyte and CD4 cells, as detailed in Table 1. Electrocardiogram was normal while echocardiogram showed left ventricular hypertrophy with decreased diastolic dysfunction. Serological assays showed positive results for IgM antibodies to Parainfluenza virus, but negative results for antibodies to respiratory syncytial virus, influenza virus (types A and B), cytomegalovirus (CMV), Adenovirus, Legionella pneumophila, Mycoplasma pneumonia, Chlamydia pneumoniae, Q-rickettsia, and Cryptococcus neoformans. Serum galactomannan and BDG levels were both normal (Table 1). Diff-Quick staining of sputum smear did not find Pneumocystis organisms. Blood culture was negative. Mycobacterium tuberculosis was negative based on blood test with T-SPOT.TB (Oxford Immunotec Ltd. UK) as well as acid-fast staining of sputum smear. 
Considering the patient was at the high-risk stage to develop opportunistic infections, she was firstly given intravenous antibacterial (meropenem 1g thrice a day) and antiviral (ganciclovir 125g twice a day) treatment on the first day of hospitalization. After 3 days of treatment, she still had fever (38 C) and dyspnea. The antimicrobial treatment was changed to intravenous caspofungin (50mg daily), oral trimethoprim-sulfamethoxazole (TMP-SMX, 320/1600mg quadruple a day), and intravenous methylprednisolone (40mg daily), which resulted in improvement of general conditions and abatement of fever and dyspnea in 3 days. However, chest HRCT showed worsening of the opacity in the lower lungs.
On the 13th day of hospitalization, she underwent endobronchial ultrasound transbronchial lung biopsy combined with bronchoalveolar lavage fluid (BALF) collection under laryngeal mask airway instead of a regular bronchoscopy examination due to respiratory failure. Subsequent microbiological tests of BALF were negative for M. tuberculosis and P. jirovecii by the GeneXpert MTB/RIF assay (Cepheid, Sunnyvale, CA, US) and Diff-Quick staining, respectively. In addition, BALF culture revealed no growth of bacteria or fungi; galactomannan level was in the normal range (OD index <0.5).
The consistently negative results in all etiological tests prompted us to submit 3 mL of BALF for mNGS by the Beijing Genomics Institute (Wuhan, Hubei Province, China) using the MGISeq 2000 platform (MGI Tec, Shenzhen, China). The results of mNGS were received <72h after shipping out the BALF sample. Based on the mNGS report (Table S2), there were 10 bacterial species (5 to 1406 reads per species), 1 fungal species (1,665,693 reads) and 1 viral species (12 reads). None of these species are well-established pathogenic organisms causing respiratory infections in humans except for the fungus P. jirovecii, which is the most dominant species, accounting for 99% of all microbial reads (6.8% of the total filtered reads including human DNA reads). The reads for P. jirovecii allowed assembly of the complete mitogenome of P. jirovecii (35,285 bp, GenBank accession number MT319762), which is 98-99% identical to 13 known P. jirovecii mitogenomes. In addition, these reads allowed assembly of the partial nuclear genome of P. jirovecii, which is ~90% complete compared to the P. jirovecii RU7 reference genome and which is included in a separate manuscript to be published elsewhere. Examination of the mitochondrial and nuclear genome assemblies showed no known nonsynonymous mutations in the dihydropteroate synthase (dhps), dihydrofolate reductase (dhfr) and cytochrome b (cob) genes.
One week after transbronchial lung biopsy (5 days after receiving mNGS report), pathological examination of lung biopsy with hematoxylin-eosin (HE) stain (Figure S1a) reported that alveolar spaces were filled with abundant foamy or granular eosinophilic exudate as well as a few lymphocytes infiltrate; the alveolar septa became thickened due to edema and dilated capillaries. Gomori methenamine-sliver staining of the lung biopsy displayed a plenty of dark brown oval or cup-shaped organisms in alveolar spaces, consistent with the morphology of P. jirovecii (Figure S1b).
After completion of the treatment regimen with caspofungin for 14 days and TMP-SMX for 16 days, her symptoms disappeared, general conditions returned to normal and laboratory findings improved (Table 1). The patient was discharged after 16 days of hospitalization. She continued to take oral TMP-SMX for another two weeks. At her 2-week follow-up visit, she had no symptoms and chest HRCT scan exhibited no abnormal finding except for a faint patchy opacity in the lateral segment (Figure 1D and E). At her 9-month follow up, she appeared healthy with no symptoms and chest HRCT was normal.