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Okafuji Pediatric Clinic, Himeji, Hyougo
Kitasato Institute for Life Sciences, Laboratory of Viral Infection I, Minato-ku
Eiken Chemical Co. Ltd., Kita-ku, Tokyo
Department of Pediatrics, Mie National Hospital, Tsu, Mie, Japan
ABSTRACT
Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis.
INTRODUCTION
Mumps virus is a single-stranded negative-sense RNA virus which belongs to a member of the genus Rubulavirus of the family Paramyxoviridae. It encodes seven main proteins: the nucleocapsid, phospho, membrane, fusion (F), small hydrophobic (SH), hemagglutinin-neuraminidase (HN), and large proteins (1). The F and HN proteins are envelope glycoproteins and the HN protein plays a role in the initial step of viral attachment to the cellular sialic acid receptor and subsequent membrane fusion, thus allowing viral penetration (26). The SH protein is present in infected cells but its function is not clear, although it is not essential for virus infection, transcription, or replication (25).
Mumps virus is still circulating throughout the world but in the United States nationwide acceptance of the measles, mumps, and rubella (MMR) combined vaccine has reduced the number of mumps patients (2, 18). With high vaccine coverage, several authors reported mumps patients suffering from secondary vaccine failure (SVF) (20). Among mumps virus infections, a few mumps patients are hospitalized because of aseptic meningitis. Encephalitis occurs in 1 of 5,000 to 6,000 cases, and deafness is an irreversible complication (19). In Japan, MMR vaccine was introduced in 1989, but it was discontinued in 1993 because of the unexpectedly high incidence of aseptic meningitis after MMR vaccination containing the Urabe strain (9, 23, 27). Since 1993, a monovalent mumps vaccine has been used, but vaccine coverage is now estimated to be <20%. Annual mumps outbreaks occur with different magnitudes every year (5, 8, 16, 24).
The diagnosis of virus infection is typically performed by virus isolation and serological examinations, but these methods are time consuming and not appropriate for clinical settings. Most mumps cases are clinically diagnosed, and acute parotitis is also caused by several virus infections other than mumps virus, bacterial infection, and the obstruction of salivary ducts (1, 19). Mumps virus can be isolated from approximately 60% of salivary swabs obtained within 5 days of illness (1), but Reina et al. (21) reported that a more sensitive shell vial method increased the isolation rate from clinical samples. More-precise laboratory-based surveillance would be required for the control of mumps. In some virus laboratories, molecular-based diagnostic methods are employed using reverse transcription-PCR (RT-PCR) and hybridization. RT-PCR is more sensitive than conventional virus isolation and takes several hours to obtain the results, which requires special equipment and skillful experiences (7). Recently, rapid diagnostic kits have been introduced for many kinds of viruses. Most kits are based on an enzyme-linked or photometric immunoassay for the detection of a specific viral protein(s), but no rapid diagnostic kit is available for the diagnosis of mumps infection.
A sensitive and specific method for DNA amplification method was developed by Notomi et al. (15) and termed loop-mediated isothermal amplification (LAMP). This method employs a DNA polymerase with strand displacement activity and a set of four specifically designed primers that recognize six different sequences on the targeted DNA. The key reaction is the formation of 5' and 3' end loop dumbbell DNA stem-loop structures. The LAMP reaction is characterized by strand displacement DNA synthesis, yielding the original loop DNA structure and new stem-loop DNA products that are twice as long. Through repetitive reactions, the multibranched stem-loop products are amplified. Distinctive features of LAMP include its rapidity, high sensitivity, high specificity, and simplicity, which are required for rapid diagnosis. We developed a new method for the detection of mumps virus genome RNA by LAMP and compared its sensitivity to that of virus isolation and nested RT-PCR.
MATERIALS AND METHODS
Clinical samples. We obtained 75 salivary swab samples from patients clinically diagnosed with mumps infection from January 2003 to July 2003 to compare the sensitivity of LAMP. To investigate the viral load in different pathophysiological conditions of mumps infection, we used 60 salivary swab samples stocked at –70°C from 34 natural mumps infections, 10 samples from patients with cases of acute parotitis after mumps vaccination identified as vaccine adverse events, and 5 samples from patients with SVF confirmed by an avidity test with an enzyme immunoassay kit (Denka Seiken, Tokyo, Japan). Salivary swabs were soaked in minimum essential medium supplemented with appropriate antibiotics, 5% fetal calf serum, and 1% gelatin. They were then transferred to the Kitasato Institute.
Virus isolation. Salivary swabs were centrifuged at 6,000 rpm for 10 min, filtered, and subjected to virus isolation, RT-PCR, and LAMP. The supernatants were inoculated on a monolayer of Vero cell cultures, and we considered virus isolation negative for the samples which did not show a cytopathic effect after two passages (21).
For the determination of virus titer, virus isolates were serially diluted at the ratio of 1:10, and 100 μl of each dilution was placed on a monolayer of Vero cells in 24-well plates in duplicate. The wells were overlaid with 0.5% agar with minimum essential medium, and the plates were kept at 37°C for 7 days in 5% CO2. They were then stained, and the number of plaques was counted. Himeji 89/JPN.00, Takamatsu 1/JPN.00, Sapporo K-4/JPN.00, and Tokyo S-III-10/JPN.01 strains were used as representative strains of genotypes B, G, J, and L, respectively (5).
RNA extraction and RT-PCR. Total RNA was extracted from 200 μl of a salivary swab sample by a magnetic bead-RNA purification kit (Toyobo Co., Ltd., Osaka, Japan), and the RNA pellet was suspended in 25 μl of distilled water. It was subjected to RT-PCR and LAMP. Virus genomic RNA material (5 μl) was converted to cDNA by AMV reverse transcriptase (Life Sciences, Inc.) at 50°C for 1 h with MpF921+ (5'-TCTATAATTCAATTGCCAGA-3'). The first PCR was performed with the primers MpF5+ (5'-ATAGCAGGGAGTTATATGAG-3') and MpL1– (5'-AACCCGTTCTAGACCATCAC-3'). For nested PCR of the HN gene, 548 nucleotides were amplified from genome positions 7,791 to 8,338 with the primers MpHN3+ (5'-GATCCTAGTTACAAATTGCG-3') and MpHN6– (5'-ACCTGCAGTGATAGTCAATCTGGTTAG-3') (5, 7, 24). PCR was performed with 1.25 U of Taq DNA polymerase (TaKaRa BioMedicals, Tokyo, Japan) with the TaKaRa thermal cycler (TaKaRa BioMedicals) and 30 rounds of thermal cycling conditions: denaturation at 93°C for 1 min, reannealing at 58°C for 1 min, and extension at 72°C for 2.5 min. PCR products were confirmed by electrophoresis through a 1.5% agarose gel stained with ethidium bromide (5, 7, 24).
Mumps virus reverse trascription-LAMP (RT-LAMP). LAMP characterized by autocycling strand displacement DNA synthesis was performed with Bst DNA polymerase (New England Biolabs) with high activity on strand displacement and by a specially designed set of primers, as specified in the software program for LAMP primer design (Eiken Chemical Co. Ltd., Tokyo, Japan). The LAMP primers were designed in the HN region similar to the RT-PCR region; the results are shown in Fig. 1. We synthesized six primers from genome position 7931 to 8180 (Fig. 1); two outer primers (F3 and B3), two inner primers, a forward inner primer (FIP), backward inner primer (BIP), and two loop primers (loops F and B). FIP contains a complementary alignment to F1 linked with the F2 sequence, and BIP contains a B1 sequence linked with a complementary sequence to B2. These four primers amplified the target DNA. We synthesized two additional loop primers: primer F, located between F1 and F2, and loop primer B, located between B1 and B2. The addition of two loop primers enhanced the specificity and reactivity (14). For the LAMP reaction, a LAMP mixture was made in 25 μl of reaction mixture containing 40 pmol (each) of FIP and BIP, 5 pmol (each) of F3 and B3, 20 pmol (each) of loop F and loop B, 1.4 mM each deoxynucleoside triphosphate, 0.8 M betaine, 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, 0.1% Tween 20, 0.5 U of AMV reverse transcriptase, 8 U of Bst DNA polymerase, and 5 μl of sample RNA. The reaction mixture was subjected to a real-time turbidimeter LA200 (Teramecs, Tokyo, Japan) (12).
A diagram of LAMP is shown Fig. 2. Genome RNA is first converted to cDNA with the FIP primer (Fig. 2, panel 1), and the F3 primer extends the cDNA synthesis with displacement of RNA and the cDNA double strand (panel 2). The RT process produces two kinds of structures, an RNA and cDNA complex from F3 to the B3 portion and single-strand cDNA primed by the FIP primer (panel 3). This cDNA forms a 5' end loop structure. The BIP primer anneals to the 3' end of cDNA and extends DNA synthesis (panels 3 and 4). The B3 primer attaches itself to the outer portion of B3 and detaches the double-strand DNA (panel 5). Thereafter, double-stranded DNA and the dumbbell loop structure of single-stranded DNA are produced (panel 6). This dumbbell loop structure is the basic structure for further extension of the LAMP reaction, and the FIP primer binds to the 3' end of the single-strand loop region (panel 7). Similar DNA synthesis with displacement activity continues with cycling reactions (panels 8 to 12), and multibranched loop structures are synthesized, as reported by Notomi et al. (15).
As the LAMP reaction progresses, the reaction by-products (pyrophosphate ions) bind to magnesium ions and form a white precipitate of magnesium pyrophosphate. Light (650 nm) emitted by light-emitting diodes passes through PCR tubes containing the LAMP solution and illuminates the photodiode on the opposite side. The turbidity is calculated based upon the ratio between the intensity of light received by the photodiode and the emitted light intensity. Thus, measurement of the turbidity is closely related to the amplification of DNA (12).
Experimental guidelines. The study design was approved by ethical committee of the Kitasato Institute for Life Sciences, and informed consent was obtained from the parents of mumps patients consulting Okafuji Pediatric Clinic and Department of Pediatrics, Mie National Hospital.
RESULTS
Sensitivity of LAMP. The Takamatsu 1/JPN.2000 strain (genotype G) was used for analysis of the sensitivity of LAMP. Viral RNA was extracted from culture fluid containing 6 x 104 PFU/200 μl and serially diluted at a ratio of 1:10. In preliminary examinations, the LAMP reaction was performed at different temperatures of 60, 63, and 65°C, and the reaction at 63°C was the most productive (data not shown). Thereafter, LAMP was carried out at 63°C for 60 min. RT-PCR and LAMP were performed for serial 10-fold dilutions, and the results of LAMP are shown in Fig. 3. Mumps virus genome was detected at a 10–5 dilution by both RT-PCR and LAMP, which contained 0.12 PFU/5 μl of RNA out of 25 μl of RNA extracted from 200 μl of samples. The threshold of LAMP positive for the spectrophotometric measurement was defined as 0.1 (12), and we analyzed the correlation between the time (in seconds) to reach threshold and infectivity (number of PFU). A linear correlation was obtained: y = –0.0056x + 10.03, where y is the number of PFU and x is the number of seconds. Using the equation, we calculated the virus genome quantity related to the infectivity of the samples by real-time LAMP. We examined measles virus, respiratory syncytial virus serotypes A and B, and influenza virus types A and B, but mumps LAMP was negative (data not shown).
We compared the sensitivity of LAMP for different mumps virus genotypes. Recently, four different genotypes (B, G, K, and L) were isolated in Japan (5). Our genotype K was registered as genotype J according to the worldwide nomenclature for mumps virus genotyping (L. Jin, Centre for Infections Health Protection Agency, London, United Kingdom, personal communication). The infectivity was adjusted to 2 x 102 PFU, and genomic RNA was amplified with similar sensitivity (Fig. 4). LAMP products exhibited a typical ladder pattern, and after digestion with Alw44I they became a single band (Fig. 3). The Alw44I site was located at genome positions 8121 to 8126. When the mumps virus genome is correctly amplified, multibranched structures should become a single DNA band after digestion with Alw44I.
Virus isolation and LAMP during the course of mumps infection. In the preliminary examination, we obtained 75 salivary samples from the patients clinically diagnosed with mumps infection who had no past history of mumps vaccination and natural infection. The samples were obtained within 2 days after the onset of parotid swelling, and we compared the sensitivity of three virological examinations. Mumps virus was isolated in 30 samples, and mumps genome was detected in virus isolation positives. Mumps virus was also detected in 18 samples by LAMP and in 17 samples by RT-nested PCR in the remaining 45 samples that were negative by virus isolation. In samples from 27 patients with virus isolation-negative and LAMP-negative results, 18 samples showed no serological response in enzyme immunoassay between acute-phase and convalescent-phase sera. We suppose that the sensitivity of LAMP is similar to that of RT-nested PCR and higher than that of virus isolation.
We obtained a series of salivary swabs from 34 patients with natural mumps infection to investigate the change in the quantity of mumps virus genome during the course of illness. The results of virus isolation and LAMP are shown in Table 1. We designated the day when the parotid swelling was noticed as day 0. Within day 2 of the illness, mumps virus was isolated in 30 of 33 samples, and its genome was amplified by LAMP in 32 samples. From day 3 of the illness onward, mumps virus was isolated in nine samples, and six samples were positive for LAMP. Sixteen samples were negative for virus isolation, but mumps genome was amplified by LAMP in 7 of 16 virus isolation-negative samples.
Virus load for different pathophysiology of mumps infection. We obtained the samples from different categories of mumps infection; 60 samples from 34 patients with natural primary mumps infection, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. RNA was extracted from 200 μl of each sample, and the reaction time was monitored when the spectrophotometric values reached to a threshold of 0.1 of the turbidity. The virus titers (in PFU) are shown in Fig. 5 for different days of illness among different categories of illness. The virus load seemed to decrease after day 4 of the illness during the course of natural infection; in the case of reinfection (SVF), a lower virus load was observed. But scattering virus load was demonstrated in the cases of vaccine-associated parotitis. The virus quantities calculated for each day of illness are shown in Table 2. In natural infection, the mean virus load of 18 LAMP positives was 100.89 ± 0.81 PFU on day 0, 101.54 ± 1.01 PFU on days 1 or 2, and decreased to 100.49 ± 0.77 PFU on day 3 and later (P = 0.0275). The virus load was 100.26 ± 0.52 PFU on day 1 in patients with acute parotitis who had a previous history of mumps vaccination, with a significantly lower load (P = 0.0351). The virus load was 102.00 ± 1.46 PFU on day 0 or 1 in eight patients with vaccine-associated parotitis. Statistical significance was analyzed by Student's t test, and there was no significant difference in viral load between natural infection and vaccine-associated illness.
DISCUSSION
Annual mumps outbreaks still occur with fluctuating magnitude in Japan (5, 8, 16, 24). Acute clinical parotitis is mainly caused by mumps virus infection, but for some patients it is caused by other pathogens such as parainfluenza virus or enterovirus infection, bacterial infection, and Stenson's duct obstruction (1). Mumps is a highly contagious disease among kindergarten or primary school children. Once it is introduced into a closed community, a second or third transmission cycle occurs with typical incubation periods (16, 20). To prevent transmission, an index case should be correctly diagnosed with a reliable virological rapid diagnostic tool, and immunization should be recommended for susceptible individuals. It is difficult to accurately diagnose mumps infection only from clinical observations. The principal of virological examination is virus isolation, but the results are not applicable to clinical practice because of its tardiness. Mumps virus can usually be isolated in 40 to 60% of patients, and virus isolation is time consuming for more than 1 to 2 weeks. Recently, a more sensitive shell vial culture method has been employed (21, 22). In a clinical setting, rapid virological diagnosis is required to prevent further extension of the outbreak. We developed nested RT-PCR in the HN region to detect the genome directly from clinical samples (5, 7, 24), but it takes nearly 8 h with skillful experience to prevent cross-contamination. It was applied mainly for the retrospective laboratory studies, not as a clinical diagnostic tool. Recently, real-time PCR was introduced for the detection of several pathogens and is now being used for RNA viruses in single-tube real-time RT-PCR for the detection of RNA viruses such as respiratory syncytial virus (3, 28). A special apparatus is also required for real-time PCR, but this is beneficial for obtaining virus copies from the samples within a few hours.
LAMP was developed to amplify the target DNA without any temperature shifts, normally required for denaturing, annealing, and extension. LAMP has been used to detect many kinds of virus infection, mainly for DNA virus or bacterial genome infection (4, 6, 10, 11). Recently, RT-LAMP was used to detect West Nile virus (17). We developed RT-LAMP to detect the mumps virus genome and compared the sensitivity of RT-LAMP, nested RT-PCR, and virus isolation. In a preliminary study, the sensitivity of LAMP was the same as that of nested RT-PCR, but LAMP is a simple and timesaving procedure, allowing the results to be obtained within 1 h after extraction of the viral genome. LAMP for the detection of genomes of pathogenic agents is a useful tool for hospital-based rapid diagnosis and will contribute to laboratory-based surveillance studies.
We obtained a linear correlation between the genome quantity and the reaction time when the spectrophotometric values reached to a threshold of 0.1 and calculated the virus load in a sample by monitoring the spectrophotometric value. We found a difference in virus load for different mumps conditions. We detected 101.54 ± 1.01 PFU on days 1 or 2 of natural infection, 100.26 ± 0.52 PFU in patients who had a past history of mumps vaccination (likely due to SVF), and 102.00 ± 1.46 PFU in those who developed vaccine-associated acute parotitis. A lower virus load was noted for those who had been immunized before, and we speculated that the remaining immunity modified virus growth even though SVF occurred. Among recipients with the Hoshino mumps vaccine strain, approximately 2 to 3% developed parotitis 2 to 3 weeks after vaccination (9). Although the symptoms for vaccine-associated parotitis were mild without febrile illness in comparison with natural primary infection, no significant difference in viral load was observed in patients with vaccine-associated acute parotitis or natural primary infection. Vaccine strains are attenuated, and we believe that even though a similar quantity of the genome was detected, the symptoms were mild. As is the nature of live attenuated virus vaccines, mumps vaccine strains have characteristics similar to those of the wild strains. After immunization with mumps vaccine, the vaccine strain of genome was detected 1 to 2 weeks after vaccination among the recipients without any symptoms (13). However, in this study, the number of patients was limited, and we plan to investigate a larger number of the cases to confirm the observation by LAMP.
We reported a simple, sensitive, reliable, rapid diagnostic method for the detection of mumps virus based on genome amplification, results of which can be obtained within 60 min after RNA extraction. No rapid diagnostic tool is available for detecting mumps virus at present, and a mumps LAMP system is a useful, reliable diagnostic tool for hospital- or clinic-based infection control in a clinical setting.
ACKNOWLEDGMENTS
We thank H. Ochiai of Ochiai Pediatric Clinic and M. Watanabe of Suzuka Children's Clinic for their cooperation in this study.
This study was supported in part by a grant from the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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