Nanopore targeted sequencing-based diagnosis of central nervous system infections in HIV-infected patients.

BACKGROUND: Early and accurate etiological diagnosis is very important for improving the prognosis of central nervous system (CNS) infections in human immunodeficiency virus (HIV)-infected patients. The goal is not easily achieved by conventional microbiological tests. We developed a nanopore targeted sequencing (NTS) platform and evaluated the diagnostic performance for CNS infections in HIV-infected patients, with special focus on cryptococcal meningitis (CM). We compared the CM diagnostic performance of NTS with conventional methods and cryptococcal polymerase chain reaction (PCR). METHODS: This study included 57 hospitalized HIV-infected patients with suspected CNS infections from September 2018 to March 2022. The diagnosis established during hospitalization includes 27 cases of CM, 13 CNS tuberculosis, 5 toxoplasma encephalitis, 2 cytomegalovirus (CMV) encephalitis and 1 Varicella-zoster virus (VZV) encephalitis. The 2 cases of CMV encephalitis also have co-existing CM. Target-specific PCR amplification was used to enrich pathogen sequences before nanopore sequencing. NTS was performed on stored cerebrospinal fluid (CSF) samples and the results were compared with the diagnosis during hospitalization. RESULTS: 53 (93.0%) of the patients were male. The median CD4 cell count was 25.0 (IQR: 14.0-63.0) cells/uL. The sensitivities of CSF culture, India ink staining, cryptococcal PCR and NTS for CM were 70.4% (95%CI: 51.5 - 84.1%), 76.0% (95%CI: 56.6 - 88.5%), 77.8% (59.2 - 89.4%) and 85.2% (95%CI: 67.5 - 94.1%), respectively. All those methods had 100% specificity for CM. Our NTS platform could identify Cryptococcus at species level. Moreover, NTS was also able to identify all the 5 cases of toxoplasma encephalitis, 2 cases of CMV encephalitis and 1 VZV encephalitis. However, only 1 of 13 CNS tuberculosis cases was diagnosed by NTS, and so did Xpert MTB/RIF assay. CONCLUSIONS: NTS has a good diagnostic performance for CM in HIV-infected patients and may have the ability of simultaneously detecting other pathogens, including mixed infections. With continuing improving of the NTS platform, it may be a promising alterative microbiological test for assisting with the diagnosis of CNS infections.

Coinfection of Cedecea lapagei and Aspergillus sydowii detected in bronchoalveolar lavage fluid of a patient with pulmonary infection using metagenomic next-generation sequencing: A case report.

Background: Cedecea lapagei (C. lapagei), as a potential human pathogen, has been reported in limited cases of human infections in medical literature. However, the increasing frequency of isolating Cedecea lapagei from clinical specimens underscores its growing clinical significance that should not be underestimated. Aspergillus sydowii (A. sydowii), commonly isolated from various environments, serves as a pathogen of human cryptic aspergillosis. Clinical pathological changes caused by A. sydowii are not obvious, posing a significant challenge in clinical diagnosis. Consequently, metagenomic next-generation sequencing (mNGS) are required for precise differentiation and identification of pathogens. Case description: Here we present a case demonstrating successful treatment outcome in a patient with pulmonary infection caused by coinfection of C. lapagei and A. sydowii, as identified through metagenomic next-generation sequencing. The patient, a 50-year-old male, presented with worsening cough, sputum production, and hemoptysis. Metagenomic next-generation sequencing (mNGS) analysis of the bronchoalveolar lavage fluid (BALF) revealed the presence of both C. lapagei and A. sydowii. Subsequently, C. lapagei was also detected by culture in the same BALF sample, however while clinical fungal cultures and (1-3)-ß-D glucan testing yielded negative results. Based on these findings along with imaging features and clinical symptoms of the patient, the final diagnosis was determined to be a co-infection of C. lapagei and A. sydowii. Conclusion: The clinical manifestations of human infections caused by C. lapagei are not specific; patients with cryptic aspergillosis may have been previously overlooked due to improper specimen selection or negative routine tests. Therefore, precise identification of pathogens is crucial. This case report highlights the value of mNGS in detecting C. lapagei and A. sydowii in BALF, enabling timely diagnosis with coinfections.

Osteomyelitis of the femur caused by Metamycoplasma orale in an immunocompromised patient using metagenomic next-generation sequencing: A case report.

Background: Metamycoplasma orale (M.orale), a symbiotic bacterium observed in the human oral cavity, is generally regarded as non-pathogenic to humans. Although infrequent, symptomatic infections caused by M.orale may occur in individuals with compromised humoral immunity. Accurate identification and early diagnosis of M.orale still present significant challenges due the limitations associated with conventional detection methods. Although metagenomic next-generation sequencing (mNGS) is currently widely utilized in clinical practices and exhibits a remarkable specificity and sensitivity for detecting various pathogens, its application in the diagnosis of M.orale-induced osteomyelitis remains largely unexplored. Case description: In this report, we present a case study of osteonecrosis caused by M.orale in a 20-year-old female patient with nephrotic syndrome and other comorbidities. She was administered long-term hormone therapy and immunosuppressants, leading to her admission to the hospital due to recurrent fever, hip abscess and left thigh pain. Imaging examination revealed bilateral mid-femoral lesions, with the extensive nature of the left femoral lesion suggesting a potential secondary infection. Although no pathogen was detected in pus culture, mNGS analysis identified M.orale in the sample. Following treatment with doxycycline and levofloxacin, the patient's symotoms improved and she was discharged with favorable outcomes. Conclusion: mNGS enables rapid identification of etiology in patients with osteomyelitis caused by the rare pathogen M.orale. This case accentuate the strength of mNGS for early detection and targeted clinical treatment of infectious diseases caused by uncommon pathogens.