Recently, the European Journal of Medical Research, a leading international journal in the field of medicine, published online a research paper entitled "Nanopore sequencing of cerebrospinal fluid of three patients with cryptococcal meningitis". The study was done by Prof. Li Wei's team at Henan Provincial People's Hospital.
This study reported three cases of human acute meningitis caused by novel cryptococcal infection, summarized the clinical features of patients with cryptococcal meningitis, and used nanopore sequencing to detect novel cryptococcal sequences from patients' cerebrospinal fluid for the first time, thus demonstrating that nanopore sequencing can assist in the diagnosis of infectious diseases of the central nervous system.
Cryptococcal meningitis is an opportunistic infection caused by the invasion of cryptococci into the central nervous system. The symptoms of the disease are mostly nonspecific and manifest mainly as headache, fever, vomiting, seizures, and focal neurological deficits. The disease progresses rapidly and has a high morbidity and mortality rate. The incidence of cryptococcal meningitis is currently increasing year by year with the increasing number of immunodeficient patients with HIV, cirrhosis and malignancy. Although culture of peripheral blood or cerebrospinal fluid pathogens is the gold standard for diagnosis, the operation is intensive, the testing time is long, and the positive detection rate is low. Testing for cryptococcal antigens is widely used in clinical practice, but can produce false-negative results, thus interfering with the clinician's diagnosis. Therefore, clinicians need a new diagnostic method to assist in diagnosis.
Nanopore sequencing, an emerging molecular diagnostic method with the ability to produce long reads and real-time sequencing, allows for rapid and accurate detection and identification of pathogens without the need for culture and could be used in the future for the detection of cryptococcal meningitis. And for the first time, the team used nanopore sequencing method to test three patients with cryptococcal meningitis.
Of the included patients, one patient had a history of pigeon exposure prior to the onset of symptoms. all three patients developed headache, fever, and neck stiffness after infection. One of the patients presented with seizure symptoms and the other with unconsciousness. Cerebrospinal fluid examination showed elevated intracranial pressure (345-400 mmHg) and elevated white blood cell count (1-343×106/L). Pathogenic findings showed positive cerebrospinal fluid ink staining and cerebrospinal fluid cryptococcal antigen testing in all three patients, while cerebrospinal fluid cultures were negative. Magnetic resonance imaging (MRI) showed enhanced meningeal enhancement in all three patients (Figure 1).
Figures 1.1a and 1b show the enhanced T1WI of case I. 2a and 2b show the enhanced T1WI of case II. 3a and 3b show the enhanced T1WI of case III.
The researchers then performed nanopore sequencing and second-generation sequencing on the cerebrospinal fluid of three patients. Cryptococcal sequences were detected in two patients' cerebrospinal fluid samples using nanopore sequencing at 17,566 reads and 14,366 reads, respectively, and in three patients' cerebrospinal fluid samples using second-generation sequencing at 1,17394 and 97,979 reads, respectively, which matched known novel Cryptococcal sequences in the gene pool. The fragment lengths of the nanopore sequencing reads were much longer than those of the second generation sequencing reads. All three patients were subsequently treated with amphotericin B, flucytosine, and fluconazole for 31 to 46 days, with gradual improvement in symptoms.
This study is the first study in China to apply nanopore sequencing technology to detect pathogens in the cerebrospinal fluid of patients with cryptococcal meningitis. This study demonstrates that nanopore sequencing can assist clinicians in the diagnosis of CNS infections, and provides data to support the future application of nanopore sequencing technology in the clinic.
Jin Ke, MSc, Henan Provincial People's Hospital, is the first author of the paper, and Li Wei, Director of the Department of Neurology, Henan Provincial People's Hospital, is the corresponding author. The study is based on the International Joint Laboratory of Precision Diagnosis and Research and Development of Henan Provincial People's Hospital.