The diagnostic value of metagenomic next-generation sequencing for identifying Pneumocystis jirovecii infection in non-HIV immunocompromised patients.

Pneumocystis jirovecii pneumonia (PJP) remains an important cause of morbidity and mortality in non-HIV immunocompromised patients especially in transplant recipients. But its diagnosis remains challenging due to the insuffificient performance of conventional methods for diagnosing Pneumocystis jirovecii(P. jirovecii) infection. Therefore, the auxiliary diagnostic function of metagenomics next-generation sequencing (mNGS) in clinical practice is worth of exploring. 34 non-HIV immunocompromised patients who were diagnosed as PJP by clinical manifestations, imaging findings, immune status of the host, and Methenamine silver staining were tested by mNGS from October 2018 to December 2020 in Sichuan Provincial People's Hospital. The clinical performances of mNGS for P. jirovecii infection diagnosis were also evaluated with genome reads abundance and comparing with other traditional diagnostic methods. We diagnosed a total of 34 non-HIV PJP patients by the clinical composite diagnosis. Our data shows that, compared with the clinical microbiological test, the detection rate of mNGS for P. jirovecii in non-HIV infected PJP patients is significantly higher than that of Methenamine silver staining and serum 1-3-β-D-glucan. mNGS can be used as an auxiliary diagnostic tool to help diagnosis. The number of reads mapped to the genome of P. jirovecii and the duration of patients from onset to sampling collection were statistically significant between the two groups (Reads>100 and Reads ≤ 100) (8days vs. 23days, p=0.020). In addition, univariate analysis showed that C-reactive protein (15.8mg/L vs.79.56mg/L, p=0.016), lactate dehydrogenase (696U/l vs. 494U/l, p=0.030) and procalcitonin (0.09ng/ml vs. 0.59ng/ml, p=0.028) was also statistically significant between the two groups. An effective detection rate was achieved in PJP patients using mNGS testing of bronchoalveolar lavage fluid (BALF) or blood. The study also confirmed that the abundance of reads of P. jirovecii is related to the interval between the onset and sample collection. And the inflammation status during simultaneous mNGS detection might determine the abundance of pathogens. Hence, we conclude that the mNGS strategy could benefit disease diagnosis as well as treatment when complicated clinical infections appeared.

Zhao M ，Yue R ，Wu X ，Gao Z ，He M ，Pan L ... -  《Frontiers in Cellular and Infection Microbiology》

Application of metagenomic next-generation sequencing in the diagnosis and treatment guidance of Pneumocystis jirovecii pneumonia in renal transplant recipients.

Pneumocystis jirovecii pneumonia (PJP) is difficult to be diagnosed, so this study explored if PJP could be diagnosed by metagenomic next-generation sequencing (mNGS) and if mNGS could guide the therapy of PJP. mNGS was successfully diagnosed 13 out of 14 PJP recipients with 11 through peripheral blood samples, verified by PCR. Ten non-PJP recipients were enrolled as the control group. Blood tests revealed a high β-D-glucan (BDG) level in all recipients with PJP during the hospitalization. Four (28.6%) of 14 PJP patients were infected with cytomegalovirus simultaneously, while 8 (57.1%) suffered from a combined infection caused by Torque teno virus. Five (35.7%) of 14 cases died of PJP or the subsequent bacteremias/bacterial pneumonia with a longer interval between the onset and diagnosis of/the available therapy against PJP than survival cases. Univariate analysis of characteristics between PJP and non-PJP recipients revealed that BDG assays was higher at the admission in PJP group (P =0.011). This present study supports the value of mNGS detection of blood sample in diagnosing PJP, which could assist clinical decision for therapy against PJ and improve outcome of PJP. The study also highlights the sensitivity of BDG assays. Cytomegalovirus and Torque teno virus infections often occur at the same time of PJP, thus can be alerts of PJP.

Zhang F ，Chen J ，Huang H ，Deng X ，Zhang W ，Zeng M ，Liu R ，Dai L ，Wan Q ... -  《-》

Metagenomic next-generation sequencing promotes diagnosis and treatment of Pneumocystis jirovecii pneumonia in non-HIV infected children: a retrospective study.

Metagenomic next-generation sequencing (mNGS) excels in diagnosis of infection pathogens. We aimed to evaluate the performance of mNGS for the diagnosis of Pneumocystis jirovecii pneumonia (PJP) in non-HIV infected children. Totally 36 PJP children and 61 non-PJP children admitted to the pediatric intensive care unit from March 2018 to December 2021 were retrospectively enrolled. Clinical features of PJP children were summarized. 1,3-β-D glucan (BDG) test and bronchoalveolar lavage fluid (BALF) mNGS were used for evaluation of PJP diagnostic performance. Antimicrobial management modifications for PJP children after the mNGS results were also reviewed. Pneumocystis jirovecii was detected in all PJP children by mNGS (36/36), and the sensitivity of mNGS was 100% (95% confidence interval [CI]: 90.26-100%). The sensitivity of BDG was 57.58% (95% CI: 39.22-74.52%). Of the 26 (72.2%) PJP patients with mixed infection, twenty-four (66.7%) were detected by BALF-mNGS. Thirteen patients (36.1%) had their antimicrobial management adjusted according to the mNGS results. Thirty-six PJP children included 17 (47.2%) primary immunodeficiency and 19 (52.8%) secondary immunodeficiency, of whom 19 (52.8%) survived and 17 (47.2%) died. Compared to survival subgroup, non-survival subgroup had a higher rate of primary immunodeficiency (64.7% vs. 31.6%, P = 0.047), younger age (7 months vs. 39 months, P = 0.011), lower body weight (8.0 kg vs. 12.0 kg, P = 0.022), and lower T lymphocyte counts. The mortality rate of PJP in immunosuppressed children without HIV infection is high and early diagnosis is challenging. BALF-mNGS could help identify PJP and guide clinical management.

Zhang Z ，Liu T ，Ming M ，Shen M ，Zhang Y ，Chen H ，Chen W ，Tao J ，Wang Y ，Liu J ，Zhou J ，Lu G ，Yan G ... -  《BMC Pulmonary Medicine》

Risk factors associated with Pneumocystis jirovecii pneumonia in non-HIV immunocompromised patients and co-pathogens analysis by metagenomic next-generation sequencing.

Pneumocystis jirovecii pneumonia (PJP) is one of the most common opportunistic infections in immunocompromised patients. However, the accurate prediction of the development of PJP in non-HIV immunocompromised patients is still unclear. Non-HIV immunocompromised patients confirmed diagnosis of PJP by the clinical symptoms, chest computed tomography and etiological results of metagenomic next-generation sequencing (mNGS) were enrolled as observation group. Another group of matched non-HIV immunocompromised patients with non-PJP pneumonia were enrolled to control group. The risk factors for the development of PJP and the co-pathogens in the bronchoalveolar lavage fluid (BALF) detected by mNGS were analyzed.  RESULTS: A total of 67 (33 PJP, 34 non-PJP) participants were enrolled from Fujian Provincial Hospital. The ages, males and underlying illnesses were not significantly different between the two groups. Compared to non-PJP patients, PJP patients were more tends to have the symptoms of fever and dyspnea. The LYM and ALB were significantly lower in PJP patients than in non-PJP patients. Conversely, LDH and serum BDG in PJP patients were significantly higher than in non-PJP controls. For immunological indicators, the levels of immunoglobulin A, G, M and complement C3, C4, the numbers of T, B, and NK cells, had no statistical difference between these two groups. Logistic multivariate analysis showed that concomitant use of corticosteroids and immunosuppressant (OR 14.146, P = 0.004) and the lymphocyte counts < 0.7 × 109/L (OR 6.882, P = 0.011) were risk factors for the development of PJP in non-HIV immunocompromised patients. 81.82% (27/33) and 64.71% (22/34) mixed infections were identified by mNGS in the PJP group and non-PJP group separately. CMV, EBV and Candida were the leading co-pathogens in PJP patients. The percentages of CMV and EBV identified by mNGS in PJP group were significantly higher than those in the control group(p < 0.005).  CONCLUSIONS: Clinicians should pay close attention to the development of PJP in non-HIV immunocompromised patients who possess the risk factors of concomitant use of corticosteroids and immunosuppressant and the lymphocyte counts < 0.7 × 109/L. Prophylaxis for PJP cannot rely solely on CD4+ T counts in non-HIV immunocompromised patients. Whether CMV infection increases the risk of PJP remains to be further investigated.

Huang L ，Xu S ，Huang Z ，Chen Y ，Xu N ，Xie B ... -  《-》

Clinical Performance of BAL Metagenomic Next-Generation Sequence and Serum (1,3)-β-D-Glucan for Differential Diagnosis of Pneumocystis jirovecii Pneumonia and Pneumocystis jirovecii Colonisation.

Differentiating Pneumocystis jirovecii infection from colonisation is crucial for appropriate therapy administration. In this study, we evaluated the performance of bronchoalveolar lavage fluid (BAL) metagenomic next-generation sequencing (mNGS) and serum 1,3-β-D-glucan (BDG) tests in differentiating colonisation and infection with P. jirovecii. From January 2018 to March 2021, 47 patients were enrolled in this study at the Hunan Provincial People's Hospital. The final diagnosis was used as a reference, and cases were classified into the P. jirovecii pneumonia (PJP) group or the P. jirovecii colonisation (PJC) group. Clinical data were recorded. The performances of mNGS and BDG were compared. The fungal load significantly differed between patients with PJP and PJC, with median reads of 3,215.79 ± 1,797 vs. 5.61 ± 0.88 in the PJP and PJC groups, respectively (P < 0.0001). BDG also significantly differed between the two groups, with a median titre of 233.60 ± 39.65 pg/ml in the PJP group and 68.48 ± 19.21 pg/ml in the PJC group (P = 0.0006). The area under the curve was 0.973 (95%CI: 0.868-1.007) for mNGS of the BAL and 0.879 (95%CI: 0.769-0.989) for the serum BDG. The optimal threshold value for discriminating P. jirovecii infection from colonisation appeared to be 14 reads (sensitivity, 83.3%; specificity, 95.7%; positive likelihood ratio, 19.2) and BDG = 88.6 pg/ml (sensitivity, 79.2%; specificity, 92.9%; positive likelihood ratio, 18.2). No correlation between mNGS reads and the BDG titre was found in mNGS-positive patients (r2 = 0.0076, P = 0.583). The levels of lactate dehydrogenase and C-reactive protein were significantly higher in the PJP group than in the PJC group. BAL mNGS and serum BDG are useful adjunct tests that can assist with differentiating between colonisation and infection of P. jirovecii.

Liu L ，Yuan M ，Shi Y ，Su X ... -  《-》