A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy
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JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunosuppressed patients. PML usually has a poor prognosis. Detection and quantification of the JCV genome in cerebrospinal fluid (CSF) is an efficacious tool for the diagnosis and management of PML, for which proper therapeutic interventions are required.
A loop-mediated isothermal amplification (LAMP) assay was applied for the quantitative detection of JCV. The LAMP assay was evaluated for the efficacy in diagnosis of PML in comparison with the TaqMan-based quantitative real-time PCR (qPCR) assay using 153 CSF specimens collected from patients with suspected PML.
The LAMP assay showed no cross-reactivity against other polyomavirus plasmids, viral DNA, and viral RNA, which causes encephalitis, and detected 1 copy of the standard DNA per reaction. Among 50 qPCR-positives, 42 specimens (containing JCV genome ranged from 3.2 × 100 to 3.2 × 106 copies/reaction) showed positive reactions and 8 specimens (containing 0.9 to 19.9 copies/reaction) showed negative in the LAMP assay. Furthermore, 3 of 103 qPCR-negative specimens showed positive reactions in the LAMP assay. The sensitivity, specificity, positive predictive value, and negative predictive values of the LAMP assay were 84% (42/50), 97% (100/103), 93% (42/45), and 93% (100/108), respectively. The kappa statistic was 0.83. The JCV loads determined by the LAMP assay showed a strong positive correlation with those determined by the qPCR assay for 33 specimens with copy numbers of ≥1 copies/reaction (r = 0.89). Additionally, the LAMP assay could monitor the JCV genome copy number in CSF for sequential samples equivalently to qPCR assay.
The newly developed LAMP assay is highly specific against JCV and detect the JCV genome in the sample DNA containing 20 or more copies of JCV genome per reaction with 100% sensitivity (n = 29), which corresponds to ≥3 × 103 copies/mL of CSF. The LAMP assay is useful for the diagnosis and offers valuable information for the evaluation and management of PML in the clinical setting.
KeywordsLAMP PML JC polyomavirus Detection Quantification
Highly active antiretroviral therapy
Human immunodeficiency virus
Herpes simplex virus type 1
Japanese encephalitis virus
Loop-mediated isothermal amplification
Lymphocytic choriomeningitis virus
Magnetic resonance imaging
No template control
Progressive multifocal leukoencephalopathy
Real-time quantitative PCR
Simian virus 40
Time to a positive result
West Nile virus
JC polyomavirus (JCV), a non-enveloped DNA virus, belonging to the Polyomaviridae family, is the causative agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system . The JCV genome is composed of double-stranded, circular, supercoiled DNA that is 5.1 kb in length and encodes six major proteins, including three structural capsid proteins (VP1, VP2, and VP3), the nonstructural agnoprotein, and two regulatory proteins (large T and small t antigens) .
JCV is distributed worldwide and causes ubiquitous infections in humans. Seroprevalence studies on JCV indicate that humans are infected with JCV during childhood, and up to approximately 35–70% of the adult population is positive for the JCV antibody [1, 2, 3]. After primary infection, JCV establishes latent infection in the kidneys, bone marrow, and lymph nodes [2, 3, 4]. It is assumed that the development of PML is a result of the reactivation of JCV from latency in immunosuppressed patients, including hematopoietic stem cell transplant recipients, those with human immunodeficiency virus (HIV) infection, those with hematologic malignancies, and those treated with immunosuppressive therapy [2, 4]. JCV enters the brain and causes lytic infection of the oligodendrocytes, which results in demyelination in such patients . Prompt therapeutic intervention is required, because PML progresses rapidly and the 3 month mortality rate for all cases is 30–50% [2, 5]. Individuals infected with HIV accounted for 85% of all PML patients [6, 7]. Furthermore, PML was found in approximately 4% of all HIV-infected patients before the introduction of highly active antiretroviral therapy (HAART) .
The detection of JCV DNA in cerebrospinal fluid (CSF) is necessary for probable PML, if a patient has suspected clinical features or neurological image findings, and definite PML requires all of clinical, imaging finding and laboratory confirmation and thus the detection of JCV genome in CSF is of great value for making diagnosis [2, 8]. Nested PCR and real-time quantitative PCR assays were developed as highly efficacious methods for the detection of JCV genome [9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20]. Several studies suggested a significant correlation between the JCV load in CSF and clinical outcomes, such as survival time, indicating that quantification of the JCV genome in CSF is of great benefit to predict prognosis [17, 20, 21, 22, 23, 24]. For instance, a high JCV load (> 4.8 × 104 genome copies/mL CSF) is associated with poor prognosis .
Loop-mediated isothermal amplification (LAMP) is a unique nucleic acid amplification method that amplifies the target sequence under isothermal conditions (60–65 °C) . The technique relies on the strand displacement activity of DNA polymerase and a set of four specially designed primers . A set of four primers recognizes six distinct nucleotide sequences on the target genome and an additional primer, named the loop-primer (LF), which improves the efficacy of genome amplification . The LAMP technique has been confirmed for the efficient, specific, and rapid amplification of target genes . In addition to the detection of the target gene, the LAMP technique enables quantification of the target genome by real-time monitoring of turbidity, which is associated with the level of by-product accumulation during the amplification reaction . Therefore, the LAMP assay has been applied as a simple method for the detection of several pathogens associated with viral diseases [29, 30, 31, 32].
Here, we developed a LAMP assay for the detection and quantification of the JCV genome in CSF collected from patients with suspected PML.
Plasmids and viruses
A plasmid containing the whole genome sequence of JCV Mad-1 strain (pJCV1–4- > pJCV: pJCV)  was obtained from the Health Science Research Resources Bank (Osaka, Japan). Plasmids containing the complete genome of other polyomaviruses, including the BK virus (BKV) (pBKV34–2), simian virus 40 (SV40) (pBRSV), and murine polyomavirus (MPyV) A2 strain (pPy-1), were purchased from the American Type Culture Collection (Manassas, VA, USA). Plasmids containing the genome of human immunodeficiency virus (HIV) type 1 subtype B (pNL-432) and subtype C (pINDIE-C1) were kindly provided by Dr. Masashi Tatsumi (National Institute of Infectious Diseases [NIID], Tokyo, Japan). The following viruses were used in this study: herpes simplex virus type 1 (HSV-1) TAS strain, varicella-zoster virus (VZV) V-Oka strain (kindly provided by Dr. Naoki Inoue, NIID), Japanese encephalitis virus (JEV) JaTH160 (GenBank accession no. AB269326), West Nile virus (WNV) NY99–6922 (AB185915), lymphocytic choriomeningitis virus (LCMV) WE strain, measles virus (MeV) Schwarz FF-8 strain (kindly provided by Dr. Katsuhiro Komase, NIID), and rabies virus (RV) HEP-Flury strain. Viral DNA/RNA was extracted from 200 μL of infected culture fluid using the High Pure Viral Nucleic Acid Kit (Roche Molecular Systems, Inc., Pleasanton, CA, USA) and the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) eluted with 50 μL of elution buffer.
The LAMP reaction was conducted with a Loopamp DNA amplification kit (Eiken Chemical Co., Ltd.) according to the manufacturer’s instructions. Briefly, the LAMP reaction mixture (25 μL) contained 40 pmol of FIP and BIP, 5 pmol of F3 and B3, and 20 pmol of LF, 2 × Reaction Mixture (12.5 μL), Bst DNA polymerase (1 μL), and the sample (2–5 μL). The reaction mixture was incubated at 63 °C for 60–120 min in a Loopamp real-time turbidimeter (LA-320C; Teramecs Co., Ltd., Kyoto, Japan), and then incubated at 80 °C for 5 min to terminate the reaction. Distilled water was used as a no template control (NTC). The LAMP assay results were assessed using the LA-320C software package (Teramecs Co., Ltd.). The cutoff turbidity value was fixed at 0.1 to differentiate positive from negative results.
Analysis of the LAMP products was conducted by 2% (w/v) agarose gel electrophoresis to verify specific band pattern and ascertained by restriction enzyme digestion with SspI (Nippon Gene, Toyama, Japan), which is a unique digestion site in the target sequence between F2 and B2 (Fig. 1). Visual fluorescence detection was performed by adding 10 μL of Gel Green™ dye (100 × solution in water; Biotium, Inc., Fremont, CA, USA) to each tube of the LAMP products and then visualized as fluorescent signals under a blue-light transilluminator Dark Reader™ (Clare Chemical Research, Inc., Delores, CO, USA) through an amber screen.
Standard of quantitative LAMP assay
A 207 bp fragment was amplified by PCR from pJCV plasmid using the forward primer 5′-CTT AGT GAT TTT CTC AGG TAG GCC TTA GGT CTG AAA TCT ATT TGC CTT ACA AAT CTG G-3′ and the reverse primer 5′-TGG CAT AAG CAA CCT TGA TTG CCT AAG AGA TTA C-3′ to repair mismatch bases between the LAMP primer target sequence and the plasmid. The amplified fragment was cloned into the pGEM-T® Easy Vector system (Promega Corporation, Madison, WI, USA) using E. coli JM109 cells, and then the sequence of the insert was confirmed. The plasmid was digested with ScaI, and serially fivefold dilutions of purified DNA in EASY Dilution (for real-time PCR) (TakaRa Bio Inc., Shiga, Japan) were used for determining a detection limit and used as standards in the quantitative LAMP assays.
Clinical CSF specimens
A total of 153 CSF specimens collected from 132 patients with suspected PML based on neurological symptoms and/or neuroimaging findings was used. These CSF specimens were sent to the Department of Virology 1, NIID, from the respective hospitals for routine testing for JCV genome by real-time quantitative PCR, as reported previously [13, 35]. Total DNA was extracted from 200 μL of the CSF specimens using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s instructions. The extracted DNA was eluted to a final volume of 60 μL in buffer AE (Qiagen) and stored at − 30 °C until use.
Two μL of each sample DNA was tested twice in independent runs. Samples with at least one positive reaction within the 120 min reaction time of the LAMP assay were regarded as LAMP-positive. For quantification, the viral load was calculated using a standard curve drawn from the serially fivefold dilutions of the standard DNA (1.3 × 102 to 2.0 × 106 copies/reaction) in each run. When a value was calculated as less than 1 copy/reaction, that is, a logarithm of a negative number, the value was omitted. The cutoff value for quantification with the LAMP assay was set to 1.5 × 102 copies/mL (equivalent of 1 copy/reaction). The average of the quantified values was considered as the viral load of the sample.
Real-time quantitative PCR assay
The CSF specimens were tested for the detection and quantification of the JCV using the TaqMan-based quantitative real-time PCR (qPCR) assay as described previously . Briefly, the qPCR primer set was targeted to the large T antigen gene. The qPCR assay was performed using 2 μL of each sample DNA prepared as described above and the cycling condition was 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 1 s.
The agreement between the LAMP and qPCR analyses was evaluated by kappa statistical analysis. Statistical difference in the JCV genome copy numbers in reaction between the LAMP negative and positives was tested by Mann-Whitney U-test. The correlation coefficient between the genome copy numbers in CSF determined by both assays was calculated using Pearson’s correlation coefficient.
The study protocol was approved by the Ethical Committee for Biomedical Science of NIID (approval number 667). All the experiments were conducted in accordance with the ethical standards of the Declaration of Helsinki.
Primer set screening and optimization of temperature for the JCV-LAMP assay
Sequences of the JCV-LAMP primers
FIP (F1c + F2)
BIP (B1c + B2)
Specificity of the JCV-LAMP assay
To evaluate cross-reactivity in the LAMP reaction against other viruses that may cause encephalitis, the viral DNA of HSV-1, VZV, HIV type 1 subtype B and subtype C, viral RNA of JEV, WNV, LCMV, MeV, and RV were subjected to the LAMP reaction at a concentration of not less than 1.0 × 107 genome copies/reaction. As a result, only the sample from the PML patient (ID: P33), which included JCV genome confirmed by qPCR, showed a positive result (Fig. 2d). The other viral DNA (BKV, SV40, MPyV, HSV-1, VZV, and HIV) and RNA (JEV, WNV, LCMV, MeV, and RV) did not show any positive reactions in the JCV-LAMP method.
Detection limit of the JCV-LAMP assay
Efficacy of the JCV-LAMP assay in JCV genome detection with using clinical specimens
Results of JCV detection in clinical specimens with the LAMP assay and qPCR
A JCV-LAMP assay developed in the present study is considered to be an efficacious tool for diagnosis of PML, although it takes a rather longer time than qPCR. The diagnosis of PML should not rely only on the virological tests. The JCV-LAMP assay developed had high sensitivity and specificity based on the highly sensitive qPCR. Three of the 103 qPCR-negative specimens showed a positive reaction in the LAMP assay (Table 2). These three CSF specimens contained JCV genome, and the positive result was confirmed to be evident with the agarose gel electrophoresis analyses as well as the consideration of their clinical backgrounds. Two of these patients were HIV positive, and the other had multiple sclerosis treated with steroid therapy. HIV associated PML cases accounts for approximately 20% of all cases in Japan .
The measurement of the JCV load in CSF is valuable not only for diagnosis but also for clinical management and prediction of the prognosis for PML patients . For instance, the patient #5 (Fig. 6) developed PML following a umbilical cord blood transplant . This patient showed a favorable response to an experimental therapy with mefloquine, which inhibits JCV replication in vitro , although the efficacy of mefloquine in the treatment of PML is not conclusive. In addition, the JCV load in PML patients fluctuates over a rather brief period (Fig. 6); thus, repeated sample collection and testing is required, especially from patients suspected of having PML based on the patients’ clinical backgrounds.
Since PML was reported as an adverse drug event in patients administered with the therapeutic immune-modulatory monoclonal antibodies, such as rituximab, natalizumab, and efalizumab [38, 39], it is suggested that the incidence of PML may increase in the near future. The patients treated with these immune-modulatory monoclonal antibodies should also be monitored closely for PML. The JCV-LAMP assay may become a useful and efficacious tool for monitoring PML for such patients.
The detection limit of the JCV-LAMP assay was higher than that of the qPCR. The detection limit should be lowered to increase the sensitivity of the assay. In this study, 2 μL of extracted DNA from CSF were used as a template. It would be likely that the detection limit can be lowered by using 5 μL-template in a 25 μL reaction mixture. Furthermore, a LAMP reaction is hardly affected by PCR inhibitors. In this study, DNAs extracted from CSF using the extraction kit were used as a template. The usefulness of the JCV-LAMP assay using CSF sample without DNA extraction processing should be evaluated. The target genome in urine sample can be successfully amplified in a LAMP reaction without DNA extraction processing . So, further studies for making the JCV-LAMP assay more usefulness in clinical settings and more sensitive are required.
The LAMP reaction requires no denaturing step or thermal cycling in the reaction process; therefore, LAMP can be performed using relatively simple equipment, such as a heating block or water bath and also be employed by visual examination. Thus, the LAMP assay can be a useful diagnostic tool for the diagnosis of PML in countries with resource-limited setting where MRI or PCR machines are not installed adequately. Although the LAMP primer was designed to detect various kinds of JCV subtypes (Fig. 1), only clinical specimens obtained in Japan were evaluated in this study, where CY and MY are major JCV subtypes . Therefore, the reaction against JCV circulating in the other parts of the world should be tested.
A quantitative JCV-LAMP assay useful not only for the diagnosis of but also for the prognostic prediction of patient with PML was developed.
This study was partly supported by a Grants-in-Aid from the Research Committee of Prion Disease and Slow Virus Infection, Research on Policy Planning and Evaluation for Rare and Intractable Diseases (Grant Number H29-Nanchitou-Nan-Ippan-036) and the research on measures for emerging and reemerging infections (Intractable Infectious Diseases in Organ Transplant Recipients [Grant Number H21-Shinko-Ippan-009]) from the Ministry of Health, Labour and Welfare of Japan and by JSPS KAKENHI (Grant Number 17 K09768). The authors would like to thank Enago (https://www.enago.jp) for the English language review.
This study was partly supported by a Grants-in-Aid from the Research Committee of Prion Disease and Slow Virus Infection, Research on Policy Planning and Evaluation for Rare and Intractable Diseases (Grant Number H29-Nanchitou-Nan-Ippan-036) and the research on measures for emerging and reemerging infections (Intractable Infectious Diseases in Organ Transplant Recipients [Grant Number H21-Shinko-Ippan-009]) from the Ministry of Health, Labour and Welfare of Japan, and by JSPS KAKENHI (Grant Number 17 K09768).
Availability of data and materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
HK designed the primers, optimized the conditions of the LAMP assay, performed the data analysis, and wrote the manuscript. KN collected and prepared the clinical specimens and carried out the qPCR analyses. CKL collected viral samples. LW and II gave technical advice for the LAMP assay. MS, CKL, KN, MTI, and IK revised the manuscript critically. All authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
The study was conducted under the approval from the Ethical Committee for Biomedical Science in the NIID (approval number 667). Written Informed consent from patients or their family members was obtained and the study adhered to the tenets of the Declaration of Helsinki.
Consent for publication
The authors declare that they have no competing interests.
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