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Jingwen Lyu,1,2,* Qianyun Deng,2,* Rongqing Li,3 Benshun Tian,1,2 Yunhu Zhao,2 Xuejiao Hu,2 Maohua Zhou,2 Bing Gu2 1Xuzhou Key Laboratory of Laboratory Diagnostics, School of Medical Technology, Xuzhou Medical University, Xuzhou, 221004, People’s Republic of China; 2Laboratory Medicine, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, 510000, People’s Republic of China; 3Jiangsu Province Key Laboratory of Immunity and Metabolism, Xuzhou Medical University, Xuzhou, 221004, People’s Republic of China*These authors contributed equally to this workCorrespondence: Maohua Zhou; Bing Gu, Laboratory Medicine, Guangdong Provincial People’s Hospital, 106 Zhongshan 2nd Road, Yuexiu District, Guangzhou, Guangdong, 510000, People’s Republic of China, Tel +86-20-83827812-62462, Email zhoumaohua@gdph.org.cn; gb20031129@163.comBackground: Pneumonia produced by coinfection with Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) in infants and young children without timely diagnosis and treatment is often fatal due to the limitations of traditional tests. More accurate and rapid diagnostic methods for multiple infections are urgently needed.Case Presentation: Here, we report a case of a 2-month-old boy with pneumonia caused by Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) without HIV infection. Chest computed tomography (CT) showed massive exudative consolidation in both lungs. Microscopic examination of stained sputum and smear specimens and bacterial and fungal culture tests were all negative, and CMV nucleic acid and antibody tests were positive. After a period of antiviral and anti-infective therapy, pulmonary inflammation was not relieved. Subsequently, sputum and venous blood samples were analysed by metagenomic next-generation sequencing (mNGS), and the sequences of PJ and CMV were acquired. The patient was finally diagnosed with pneumonia caused by PJ and CMV coinfection. Anti-fungal combined with anti-viral therapy was given immediately. mNGS re-examination of bronchoalveolar lavage fluid (BALF) also revealed the same primary pathogen. Therapy was stopped due to the request of the patient’s guardian. Hence, the child was discharged from the hospital and eventually died.Conclusion: This case emphasizes the combined use of mNGS and traditional tests in the clinical diagnosis of mixed lung infections in infants without HIV infection. mNGS is a new adjunctive diagnostic method that can rapidly discriminate multiple causes of pneumonia.Keywords: metagenomic next-generation sequencing, Pneumocystis jirovecii, cytomegalovirus, pneumonitis, HIV-negative