Development of a Droplet Digital Polymerase Chain Reaction for Sensitive Detection of Pneumocystis jirovecii in Respiratory Tract Specimens

Background:Pneumocystis jirovecii is a human-specific opportunistic fungus that causes Pneumocystis pneumonia (PCP), a life-threatening opportunistic lung infection that affects immunocompromised patients. P. jirovecii colonization may be linked to the transmission of the infection. The detection of P. jirovecii in immunocompromised patients is thus especially important. The low fungal load and the presence of PCR inhibitors limit the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of P. jirovecii in specimens. Droplet digital PCR (ddPCR), however, presents a methodology that allows higher sensitivity and accuracy. Here, we developed a ddPCR method for detecting P. jirovecii DNA in respiratory specimens, and evaluated its sensitivity against qPCR.Materials and Methods: One bronchoalveolar fluid (BALF) sample each was collected from 82 patients with potential PCP to test the presence of P. jirovecii DNA using both ddPCR and qPCR, and samples with inconsistent results between the two methods were further tested by metagenomic next generation sequencing (mNGS). In addition, 37 sputum samples from 16 patients diagnosed with PCP, as well as continuous respiratory tract specimens from nine patients with PCP and treated with sulfonamides, were also collected for P. jirovecii DNA testing using both ddPCR and qPCR.Results: ddPCR and qPCR gave the same results for 95.12% (78/82) of the BALF samples. The remaining four specimens tested positive using ddPCR but negative using qPCR, and they were found to be positive by mNGS. Detection results of 78.37% (29/37) sputum samples were consistent between ddPCR and qPCR, while the other eight samples tested positive using ddPCR but negative using qPCR. The P. jirovecii load of patients with PCP decreased to undetectable levels after treatment according to qPCR, but P. jirovecii was still detectable using ddPCR.Conclusions: ddPCR was more sensitive than qPCR, especially at detecting low-pathogen-load P. jirovecii. Thus, ddPCR represents a useful, viable, and reliable alternative to qPCR in P. jirovecii testing in patients with immunodeficiency.

Pulmonary Nocardia infection in child with idiopathic pulmonary hemosiderosis: case report and literature review

BackgroundIdiopathic pulmonary hemosiderosis (IPH) encompasses a rare and agnogenic group of diffuse alveolar capillary hemorrhagic diseases. Corticosteroid treatment is the globally preferred therapeutic strategy for IPH. However, its long-term administration can cause immunodeficiency. Nocardia infection often occurs in immunocompromised patients and primarily involves the pleura and lungs. Herein, we describe a case of pediatric pulmonary Nocardia infection complicated by IPH.Case presentationA 7-year-old girl presented with chief complaints of pale complexion persisting for 1 year and a cough for 20 days. Abundant hemosiderin-laden macrophages were detected in the gastric juice during the last hospitalization. Uninterrupted small doses of corticosteroids (1–2 mg/kg/day) were administered to the patient to treat the IPH. After nearly two months of corticosteroids therapy, the children began to cough. Next-generation sequencing of the bronchoalveolar lavage fluid (BALF) sample revealed the presence of Nocardia abscessus (N. abscessus) DNA and confirmed IPH again. Linezolid was administered to treat the N. abscessus infection. She recovered well and was discharged after 18 days of hospitalization. After 1 month of follow-up, her pulmonary lesions exhibited gradual resorption, the iron deficiency anemia had resolved, and the IPH appeared to be well-controlled.ConclusionsThis pediatric case of N. abscessus infection complicated by IPH, including the nonspecific clinical manifestations, time-consuming diagnosis, and timely adjusted treatment, provided considerable clinical experience and enlightenment.