Mumps outbreaks continue to occur worldwide, including in populations with adequate immunization schedules and coverage. In 2016, more than 6000 cases of mumps were reported in the USA, the highest in 10 years . In 2017, more than 5600 cases were reported in the USA, mostly related to an outbreak also involving Canada . The outbreak that started in New York in 2009 and subsequently reached Ontario is one of the biggest reported outbreaks since universal immunization, with more than 3500 reported cases in the USA alone . Phylogenetic analyses confirmed that the strain responsible for the outbreak in Ontario was similar to the one causing the outbreak in New York . Moreover, WGS confirmed that outbreak isolates were > 98.7% similar to sporadic isolates, suggesting that the outbreak strain has been previously circulating. The presumed index case of the New York outbreak was a child who recently returned from the United Kingdom where a large outbreak was happening .
The outbreak in Ontario occurred in a population where 14% of cases were unimmunized, whereas 55 and 28% of cases had previously received 1 and 2 doses, respectively . Therefore, the outbreak occured in a context of suboptimal immunization as well as possible waning of immunity. The relatively low percentage of sequence homology between our strain and the Jeryl Lynn vaccine strain is related to the fact that they belong to genotypes G and A, respectively, which has been suggested as a potential cause of suboptimal vaccine effectiveness [19, 20]. However, additional studies have shown that vaccine-induced antibody effectively neutralizes wild-type virus [19, 21, 22]. Consequently, the pathophysiology of mumps vaccine failure is still unclear.
Our study demonstrated that rRT-PCR was the most sensitive assay for mumps detection. Viral culture was slightly less sensitive, and this difference did not reach statistical significance. However, IgM serology was significantly less sensitive than both rRT-PCR and viral culture. Our observed order of assay sensitivity is consistent with large studies with > 80 laboratory confirmed cases [3, 5, 23, 24]. In addition, a number of studies have shown a better sensitivity of rRT-PCR compared to IgM serology [4, 25,26,27,28,29,30,31,32] and, to a lesser extent, culture [12, 25, 26]. However, inconsistent findings in smaller studies has been observed where IgM yielded similar or better sensitivity than culture [25, 26, 33] or RT-PCR . More than 3500 cases were confirmed in the USA during the 2009-2010 outbreak that reached Ontario . Among the 1648 tested patients in the USA, 831 (50%) were positive by at least one assay. In this large sample, rRT-PCR was the most sensitive assay with 68% positivity (373/550) among cases who had rRT-PCR testing, compared to 64% (283/443) and 35% (550/1563) of cases who underwent cell culture and IgM testing, respectively. The discrepant sensitivity of RT-PCR, culture and serology between the above-mentioned studies could be a result of confounding by the different vaccination status and different time between symptom onset and specimen collection among the studied populations. Indeed, previously immunized patients are less likely to develop an IgM response when subsequently exposed to mumps [2, 6]. Moreover, rRT-PCR and culture are more likely to be positive if specimens were collected in the first 2 days after parotitis onset [2, 6], whereas IgM is more likely to be positive if collected ≥ 3 days after symptom onset . Among the same population, Rota et al. showed that sensitivity of rRT-PCR was higher than serology from day 0 to day 2 after symptom onset, whereas the sensitivity of serology was higher than rRT-PCR from day 3 onwards . The fact that rRT-PCR yield was highest during day one to three after parotitis onset and decreased afterwards was confirmed in two other studies, using rRT-PCR  and RT-PCR , respectively.
Additional testing showed that rRT-PCR and IgM sensitivity varied significantly using different assays [6, 35]. In the Ontario outbreak, negative IgM tests in our confirmed cases could not be explained by early collection of specimens, as we observed a median duration of 4 days from symptom onset to blood collection, which should have been ample time for IgM to develop. The low sensitivity of IgM serology may also be confounded by immunization status. It has been suggested that immunized individuals have modified B cell responses that allow for the rapid generation of IgG antibodies and a blunted, or absent IgM response, when exposed to mumps virus . This phenomenon could also result in a lack of IgG seroconversion being observed following mumps infection in previously immunized patients, due to an IgG boost occurring prior to clinical presentation. Studies performed in the USA during the 2009-2010 outbreak showed that IgM serology was less likely to be positive among immunized (26%) than unimmunized patients (76%, P < 0.05), whereas immunization status had no influence on rRT-PCR and culture results [2, 6]. This low yield of IgM in immunized patients during an outbreak has been reported in other studies [24, 36].
Our study also confirmed that rRT-PCR sensitivity varied with specimen type and was higher for buccal than oropharyngeal swabs or urine. This is consistent with previous studies showing that saliva swabs performed better than pharyngeal swabs, whereas test sensitivity was lowest for urine [4, 23, 37]. Similarly, Hindiyeh et al. showed that RT-PCR sensitivity was higher with pharyngeal or parotid fluid than urine .
The main limitation of the study is the relative lack of clinical data about the studied population, such as symptoms at disease onset. This is related to the fact that clinicians in the community forward specimens to our laboratory for mumps testing, often without providing significant clinical data. As a consequence, it was not possible to know precisely the immunization status of the tested patients which would have allowed comparison of diagnostic methods between immunized and unimmunized patients; however, the immunization status of the same population at this time was reported in another publication, showing that 86% of the population had received at least one dose and more than 55% two doses . Similarly, the time between symptom onset and specimen collection was provided for a minority of patients only. As these data might influence serology and RT-PCR sensitivity, it would have provided important additional information in this setting. As it has been shown that a rRT-PCR targeting the nucleoprotein gene was more sensitive than a rRT-PCR targeting the SH gene , the fact that our assay targeted the F1 and SH genes might also be a potential limitation of the study.
Despite the abundance of previous data showing that RT-PCR, and to a lesser extent viral culture, are the most sensitive detection methods, most patients in our study had serology testing during the acute illness without any rRT-PCR or culture testing. This testing pattern was also observed in the US outbreak and may reflect the current practice in North America. During the same outbreak in the USA, IgM serology was used in 95% (n = 1563) of the 1648 tested patients, whereas rRT-PCR and viral culture were used in only 33% (n = 550) and 27% (n = 443), respectively . This testing practice is in contrast to a 2009 Australian study that showed that RT-PCR was the most frequently used diagnostic method during a mumps outbreak in that country . The current CDC guidelines recommend to perform serology as well as rRT-PCR or culture, but emphasize the lower yield of urine compared to buccal specimens . On the other hand, the Public Health Agency of Canada recommends to perform serology as well as buccal and urine rRT-PCR but no longer recommends culture in the current guidelines . Clinician education is needed to increase awareness of the availability, better sensitivity and test performance of rRT-PCR compared to serology.
Compared to rRT-PCR, IgM serology has lower sensitivity and may be highly influenced by immunization status. As such, rRT-PCR should be considered as the gold standard for mumps detection because of its high sensitivity and its quick turnaround time. However, it is not uncommon that patients with negative rRT-PCR [27, 29, 32] or RT-PCR , have positive IgM. As a consequence, these two assays are somewhat complementary, as IgM yield increases with time following symptom onset, when RT-PCR yield decreases. However, clinicians should keep in mind the poor positive predictive value of IgM when used in a low prevalence setting such as North America, for example in the absence of an outbreak . Due to the impact of time since symptom onset and the relative sensitivity of rRT-PCR and serology, we therefore emphasize the need to continue educating healthcare providers of the importance of submitting both buccal swab and urine specimens for rRT-PCR, in addition to a specimen for IgM/IgG serology when mumps is suspected, in accordance with current guidelines.