A 28-year-old male patient had a dry cough, chest tightness after exercise, and shortness of breath for 1 week. He took oral cephalosporin for 3 days without any efficiency, and his symptoms got worse. On admission, leukoplakia in the mouth, little wet rales at the bottom of both lungs, and multiple swollen lymph nodes were found on clinical examination. His temperature was 37.6 C. His blood test results showed that the white blood cell count was 6.52*109/L, with an elevation in neutrophil ratio of 72.7%, high level of C-reactive protein (CRP) at 166 mg/L and serum amyloid protein A at 130 mg/L, low level of oxygen partial pressure at 87.5 mmHg and albumin at 30.3 g/L. Blood culture, antibody tests of Legionella pneumophila, Streptococcus pneumoniae, and Mycoplasma pneumoniae, and PCR-based detection of SARS-CoV-2 were negative. Cryptococcal capsular antigen, 1-3-beta-D glucan detection, aspergillus galactomannan detection, and interferon-gamma release assays were negative. The viral load of HIV-RNA was 84,100 IU/ml. CD4+ T-cell count was 11 cells/mul. CT of the lung revealed multifocal bilateral ground-glass opacities (Figure 1). According to the clinical diagnosis of PCP, the patient was treated with four pills of sulfamethoxazole-trimethoprim (sulfamethoxazole 1.2 g, trimethoprim 240 mg) three times a day.
After treatment for 2 days (on day 3), the symptoms of the patient were more severe, and his temperature got to 39.9 C. We performed a bronchoscopy on the patient, and it showed bronchial mucosa hyperemia and bronchial inflammatory changes. Nucleated cell counts of bronchoalveolar lavage (BAL) were 20 * 106/L. Cryptococcal antigen, 1-3-beta-D glucan detection, and culture of BAL were negative. Hexamine silver staining of P. jiroveci and PCR-based detection of cytomegalovirus and Mycobacterium tuberculosis were also negative. In addition, next-generation sequencing technologies (NGS) of BAL were carried out, the BAL was sent to a laboratory, and DNA was extracted for whole genomic sequencing, purified, and sonicated to a size of 100-150 bp. The DNA libraries were constructed followed by end repair, joint connection, no bias PCR amplification, and sequenced using the MGlseq-2000 platform after quality control. High-quality sequencing data were generated by removing low-quality reads. After removing human sequences, the remaining sequencing data were aligned to the bacterial, viral, fungal, and protozoan databases.
After treatment for 5 days (on day 6), CT of the lung revealed that it was worse than before (Figure 2). Results of NGS reported 205,658 unique reads of P. jirovecii, with coverage of identified genes 1.70% and 115 unique reads of T. whipplei, with coverage of identified genes 1.00% in BAL. The P. jirovecii-specific SYBR Green quantitative real-time PCR (qPCR) assay targeting a 301 bp fragment using a CFX96 Real-Time System was performed. The forward primer (pH207 5-ACAAATCGGACTAGGATATAGCTGGT-3) and the reverse primer pAZ102-E were used to detect the mtLSUrRNA gene, and the copy number of P. jiroveci was 4,787.36 copies/ml. For specific T. whipplei qPCR, the specimen was tested by using Twhi3F (5-TTGTGTATTTGGTATTAGATGAAACAG-3), Twhi3R (5-CCCTACAATATGAAACAGCCTTTG-3) primer pair, and the specific TaqMan probe Twhi3 (6-FAM-GGGATAGAGCAGGAGGTGTCTGTCTGG-TAMRA). When the specimen was positive in this assay, the result was confirmed by a second qPCR by using Twhi2F (5-TGAGGATGTATCTGTGTATGGGACA-3) andTwhi2R (5-TCCTGTTACAAGCAGTACAAAACAAA-3) primer set, and the Twhi2 probe (6-FAM-GAGAGATGGGGTGCAGGACAGGG-TAMRA). The copy number of T. whipplei was 11,717.3 copies/ml. Considering the progressed condition of the patient, meropenem 1 g q8h and caspofungin 70 mg per day were added for treating the infections of T. whipplei and P. jirovecii. Methylprednisolone 40 mg BID was added due to the severe symptoms. On day 12, there was a clinical improvement, and CT of the lungs showed obviously better than before (Figure 3), and the patient was discharged on day 16.