Next-generation Sequencing with cerebrospinal fluid for the diagnosis of progressive multifocal leukoencephalopathy in HIV-infected patients

Background To investigate the clinical manifestations, imaging findings, characteristics of cerebrospinal fluid, treatment and prognosis in patients with AIDS-related progressive white matter lesions diagnosed by second-generation sequencing with cerebrospinal fluid. Methods Patients with clinically suspected PML were diagnosed by second-generation sequencing with cerebrospinal fluid, and the clinical data of these patients were retrospectively reviewed. Results Ten patients with AIDS-related PML were diagnosed, including eight males (80%). The average age was 38.7±8.2 years, and the median CD4+ T cell count was 46×106/L. The median HIV RNA was 5.99 × 104 copies/ml. Main clinical manifestations included: dyskinesia in 4 patients (40%) and dizziness in 3(30%), cognitive decline in 2(20%), and speech disorder in 1(10%). Three patients (30%) developed convulsions throughout the course of the disease. Image findings involved lesions cerebellum in 3 cases (30%), and lesions above the cerebellum in 7 cases (70%). Mild increase of total protein was observed in the cerebrospinal fluid of 4 cases (40%), while white blood cell count, sugar and chloride were normal in all of the cases. The copies of JCV sequence in the cerebrospinal fluid ranged from 3-12531 reads (median=67 reads). All the patients received antiviral therapy including integrase inhibitors. Among the 10 patients, 2 died in hospital and 8 survived, 2 of them have survived for more than 1 year. Symptoms were improved in 5 patients and unchanged in 3 patients. Conclusions PML is common in patients with HIV /AIDS. It is reliable to diagnose the disease through clinical manifestations, routine detection of cerebrospinal fluid, imaging and cerebrospinal fluid NGS sequencing. Strong and effective antiretroviral treatment may improve the prognosis of patients.


Background
Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating disease of the central nervous system (CNS) caused by reactivation of the JC polyomavirus (JCV) which occurs almost exclusively in immunocompromised patients. In the pre-antiretroviral therapy (ART) era, 3-7% of human immunodeficiency virus (HIV)-infected patients developed PML [1][2][3]. With the widespread use of ART, the incidence and mortality of PML have declined significantly [4][5][6][7][8]. PML typically manifested as altered mental status, motor deficits, ataxia, and visual symptoms and is progressive and usually fatal. Currently, no specific treatment is available, but restoration of immune function with ART may lead to disease remission or improved survival.
More importantly, diagnosis of PML in HIV-infected patient is still challenging due to the complicated clinical characteristics in late stage of diseases, various imaging manifestations and the sensitivity of JCV DNA detection in cerebrospinal fluid (CSF) [9,10].
Next-generation sequencing technology has been successfully used in detecting pathogens in CSF [11][12][13]. Here, we reported a series of cases diagnosis of PML through detecting the JC virus in CSF using NGS in clinically suspected patients.

Methods
HIV-infected patients was suspected of PML by clinical manifestations, typical cranial imaging features and examination of CSF which rule out common CNS infections (e.g., cryptococcal meningitis, tuberculosis meningitis and toxoplasmic encephalitis). CSF of suspected patients was sent for NGS.

CD4 + T cell count and HIV viral load
CD4 T cell count and HIV viral load were quantified using flow cytometry with FACSCalibur (BD Company, USA) and COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (Roche, Switzerland) at the clinical laboratory of SPHCC, respectively.

DNA extraction and next -generation sequencing
Sterile cerebrospinal fluid (CSF) of patients was retained for high-throughput sequencing.
The glass bead grinding process was carried out firstly. A glass bead with a diameter of 0.5mm was added to the broken tube, and then 0.6ml of the sample was added in. The sample was vibrated at 2800-3200 rounds/minute for 30 minutes. Then 300μl was used for nucleic acid extraction. The genome extraction kit (DP316) of Tiangen microsample was operated according to the instructions. Five hundred nanogram of extracted DNA was interrupted, repaired, jointed, amplified, repositored and sequenced according to the standard process. A CSF sample produced 20M sequence data. Low-quality and short sequence (less than 35bp) was removed from the sequence data, so as to obtain highquality sequence. The sequence was then compared with the human reference genome (H19), with the software of Burrows-Wheeler Alignment. After removing the human sequence, the sequence was compared with 4 databases of bacteria (2473 species), fungi (199 species), viruses (4061 species) and parasites (135 species), and the numbers of sequences matched to any a certain pathogen were obtained. The possible pathogens were judged according to the number of sequences and other clinical judgement.

Treatment and follow-up
Patients with positive JCV in CSF were diagnosis of PML. ART were continued during hospitalization or initiated immediately after exclusion of the previous-mentioned common CNS infection. Patients were followed-up on the outpatient clinic or by phone call. Patients with aggravation of clinical symptoms and CD4 count increase following ART were diagnosis of immune reconstruction syndrome (IRIS) after excluded other causes.

Statistical methods
Descriptive analysis to describe demographic data. The baseline characteristics of patients were described by median values with continuous variables. The classified variables were counted and proportioned. All the data were processed using STATA 13 software (StataCorp, Texas, USA).

Basic information of patients:
From Oct 2017 to Jan 2019, a total of 10 cases were diagnosed of PML, including eight males (80%) and two females (20%). These patients were at their middle age (38.7±8.2 years) with advanced immunodeficiency (CD4 T cell count 46 (2-241) cells/ml). HIV RNA was detectable in the majority patients (80%). The value of HIV RNA was ranged from less than 40 to 2.81 *10 5 copies/ml, with a median of 5.99 *10 4 copies/ml.
HIV infection was not diagnosed until onset of PML symptoms in four patients. Two patients never received ART although HIV infection has been diagnosed for 6 and 8 years, respectively. Two patients who had resistance to current ART regimens developed symptoms after ART for 6 months and 12 years, with HIV viral load of 1.3 *10 5 copies/ml and 2.3 *10 4 copies/ml, respectively. One patient developed symptoms after 1 month of ART. Two cases were complicated with pulmonary non-tuberculosis Mycobacterium infection, and no other co-infection was found in all the patients.

Symptom, imaging and CSF examination
The onset of the disease is occult, gradually aggravated. The time from symptoms onset to admission was 0.5-3 months. Dyskinesia (40%), convulsions (30%), dizziness (30%), cognitive impairment (20%) and speech impairment (10%) were the most common symptom. Symptoms in case 9 were related to immune reconstruction syndrome after antiviral therapy. (See Table 1) Lesions were found in various parts of the brain in MRI examination. The lesions were located under the tentorium cerebellum in 3 cases (30%) and above the tentorium cerebellum in the rest (70%) (see Figure 1) . For CSF test, levels of white blood cell, glucose and chloride were normal while slight increase of protein was observed in four patients (40%).

Treatment
All patients received ART. Four of them started antiretroviral treatment immediately, 4 had been already treated for 1-3 months, 2 adjusted regimen according to drug resistance reports. Either raltegravir or dolutegravir was included in the regimen for every patient.
Three patients received quadruple regimen to strengthen antiviral therapy. Supportive treatment were also given to alleviate symptoms.

Therapeutic Response
Two patients died at 2 and 5 months after onset of symptoms respectively. The remaining 8 cases survived and were followed-up for 2-18 months. Two patients had survived for more than one year. Symptoms in five of the patients were relieved, but did not improve in the other three patients.

Discussion
The diagnosis of PML can be challenging in HIV-infected patients as variable CNS Written informed consent were obtained from all the study patients.
-Consent for publication Written informed consent to publish this information was obtained from study participants.
-Availability of data and material Not applicable.

-Competing interests
There is no competing interests to be declared. Tables Table 1 Basic characteristic of patients with PML Figure 1 Lesions locations in the brains of all the cases