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.
We isolated 872 strains of mumps virus from naso-pharyngeal secretions in seven different districts of Japan from January 2000 to July 2001. Among them, 57 strains were geno-typed by nucleotide sequencing in part of the hemagglutinin-neuraminidase (HN) and small hydrophobic (SH) protein regions. Four different genotypes (B, G, K, and L) of mumps virus were co-circulating in Japan and the distribution of genotypes varied in geographically different districts. Two new clusters designated as genotypes K and L had more than 7% nucleotide variation in the SH gene. Among the 57 strains, 11 were classified as B, 35 as G, three as K, and eight as L, which was mainly isolated in Tokyo. We also examined 104 stains isolated in a clinic in Mie prefecture from 1993 to 2003. Genotype B was the indigenous strain and genotype K was introduced in 1994. Genotypes B and K co-circulated in the 1990s and were replaced by genotype G in 2000. There was no significant change in neutralizing test antibody titers against genotypes B, G, K, and L using seven post-vaccination sera with Hoshino strain (genotype B) and these four genotypes had a different antigenicity from genotype A. We should continue to watch on mumps virus molecular epidemiology.
An immunochromatography (IC) kit for human adenovirus (HAdV) was evaluated with 138 patient nasopharyngeal samples. The samples were collected at a sentinel clinic in Japan from January through June 2003. Patients were diagnosed by clinical manifestation of pharyngoconjunctival fever (n ؍ 38) or exudative tonsillitis (n ؍ 100). The IC kit was positive for 84% (116 of 138) of patients diagnosed at bedside. The remaining extract solution of the IC kit test was transferred into maintenance medium and tested via laboratory diagnoses. The IC kit had 95% sensitivity (116 of 122 patients) with HAdV isolation (isolation) as the standard and 91% sensitivity (116 of 128 patients) with PCR as the standard. All of the IC kit-positive samples were isolation and PCR positive. Similarly, all the isolation-positive samples were PCR positive. Twenty-two IC kit-negative samples were evaluated by real-time PCR. Six samples were IC kit negative and isolation positive and contained 3.8 ؋ 10 7 to 2.5 ؋ 10 9 copies of the HAdV genome/ml. Five samples that were only PCR positive contained 3.0 ؋ 10 4 to 3.8 ؋ 10 5 copies of the HAdV genome/ml, but one sample was real-time PCR negative. We conclude that the IC kit is a useful bedside diagnostic tool for HAdV infections because it has 95% sensitivity (compared to isolation), but a negative result does not always rule out HAdV infection.
Clinically apparent mumps reinfection is considered extremely rare, but several cases have been suspected of reinfection in an out-patient clinic. In this study, virological examination, virus isolation, the reverse transcription loop-mediated isothermal amplification (RT-LAMP), and IgG and IgM EIA antibodies, were examined in order to identify mumps reinfection. Patients were divided into three categories; the reinfection group comprised 29 patients with a history of natural infection, the vaccine-failure group consisted of 37 patients with an immunization history, and two patients had histories of both immunization and mumps infection. Another 25 patients were enrolled as a primary infection group. Mumps virus was isolated in 5 (17%) and the genome was detected in 12 (41%) of 29 in the reinfection group. Reinfection was confirmed in 21/28, demonstrating high avidity of IgG EIA. Mumps virus was isolated in 15 (41%) and there was a higher positivity of genome amplification in 25 (68%) of 37 patients in the vaccine-failure group. Among these, 23 were confirmed as secondary vaccine failure by high avidity IgG EIA serology. In the primary infection group, the isolation rate and genome detection rate was higher in 16 (64%) and in 18 (72%) of 25 patients, respectively. There was no significant difference in virus load among the three groups but high mumps virus load was suspected in the IgM EIA-positive group based on the shorter amplification time on RT-LAMP. Mumps virus reinfection was confirmed by RT-LAMP and an IgG avidity test and was not a rare event.
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