Abstract
Introduction
CMV is a member of the herpes virus family. It is a 235-kb double-stranded linear DNA virus and is spread via saliva, urine, and most other bodily fluids of infected individuals. This ease of transmission results in particularly high disease prevalence in certain parts of the world. Seroprevalence differs greatly from country to country and can range from as little as 50% up to 90%, depending on many factors including ethnicity, socio-economic status, and lack of understanding of disease prevention (1). It is well documented that infection in immunocompetent individuals is generally asymptomatic or some mild symptoms may present, however the consequences of infection become more serious in immunocompromised individuals or pregnant woman. Primary infection gives rise to lifelong latency and reactivation from latency can occur.
Primary HCMV infection in pregnant women carries a risk of intrauterine transmission with 30%–40% of all primary infections resulting in vertical transmission, and 10%–20% of congenitally infected babies will be symptomatic at birth compared to secondary infection that results in a much lesser likelihood of symptomatic infection of 0.2% to 2%. It has been documented that women who become infected in the first trimester of pregnancy pose a greater risk of delivering a newborn with congenital defects than a woman who becomes infected in the later stages of pregnancy (2,3). Furthermore, reinfection with a different strain of the virus cannot be ruled out and may lead to intrauterine transmission and symptomatic congenital infection (3).
Although the impact of cCMV is well documented, there is no current government coordinated screening program in place in Europe and many other parts of the world. However, some countries (Israel, France, Belgium, Spain, Italy, Germany, Austria, Portugal, and The Netherlands) have begun to screen pregnant woman routinely without government recommendations or guidelines (4). No such screening currently exists in Ireland.
CMV DNA detection in urine and saliva of neonates remain the gold standard for laboratory confirmation of cCMV infection. However, since these samples are not available retrospectively and since early postnatal infection with CMV is common, DBS, which are routinely taken in the first week of life, are often the only sample available for retrospective diagnosis of cCMV infection. Recent publications have presented results that alluded to the sensitivity and stability of DBS in relation to detection of CMV DNA. These studies were recently reviewed in Snijdewind et al. (5) who showed that, although the specificity for detection of CMV DNA in DBS is close to 100% in all studies, the sensitivity ranges from 15%–100%. This discrepancy in sensitivity could be explained by several factors, including different DNA extraction methods (6), use of single, two, or nested primer sets (7,8), real-time amplification techniques, and the proportion of symptomatic and asymptomatic cases in the sample cohort investigated (9).
The largest study to date that included samples from 7 US medical centres concluded that PCR analysis of DBS has low sensitivity for correctly identifying infants with cCMV and therefore are not suitable for screening newborns for cCMV infection as they miss approximately two-thirds of the infections (8). The study population consisted of 20,425 DBS samples; the first 11,407 samples were tested using a single-primer DBS PCR and the second set of 9018 samples were tested using a two-primer DBS PCR. The results showed 99.9% specificity for both single and two-primer PCR; however the sensitivity was very low, 28.3% and 34.4% respectively. This study collected both a saliva sample and DBS sample from each infant. The saliva rapid culture method showed sensitivities for the same study groups of 98% and 100%, respectively. This study concluded that mass screening of newborns using saliva samples may be more effective than DBS, until advances are made to improve the sensitivity of DBS PCR assays (8).
Other factors compounding detection of CMV DNA in DBS include the possibility that congenitally-infected infants may have become infected early in the pregnancy and so may not have detectable CMV DNA in their blood at birth (8). With regards to detection of CMV DNA in DBS stored for prolonged periods of time, discrepant results have been reported. Soetens and co-workers (6) reported detection of CMV DNA in DBS up to 10.8 years later. However, another report showed degradation of CMV DNA during 2 years of storage at room temperature (10).
A key study reported that the combined use of RT PCR and IgM-specific antibodies in DBS significantly improved the sensitivity and specificity for diagnosis of acute measles infection (11) and allowed a highly accurate diagnosis of measles to be made with a positive predictive value of 99%. Interestingly, the authors showed that the sensitivity and specificity of measles-specific IgM alone on DBS was 95% and 96%, respectively. With regards to congenital infections, Tan et al. (12) showed that retrospective Toxoplasma-specific IgM testing of neonatal DBS could be useful to distinguish between congenital and postnatal acquired toxoplasmosis in children presenting with symptoms later in childhood. Since recent findings by Fabbri and colleagues (13) showed that a combined measurement of CMV IgM and CMV DNA significantly improved the diagnosis of cCMV in fetal blood with sensitivity and specificity of 86% and 89%, respectively (G. Gerna, personal communication), and this approaches levels similar to that of saliva samples in Boppana's studies (8,14,15), it is feasible that measurement of both CMV IgM and CMV DNA in DBS may improve the restrospective diagnosis of cCMV.
Preliminary data on 72 DBS received for retrospective cCMV diagnosis from children who were asymptomatic at birth but who have subsequently shown clinical symptoms of cCMV infection such as sensorineural hearing loss, developmental delay, and microcephaly is shown in Table 1. CMV DNA was detected in 15 DBS (21%) and CMV IgM detected in 36 eluted DBS (50%). The DBS groups were divided as follows: 7 (10%) were CMV IgM+/PCR+, 27 (37.5%) were CMV IgM-/PCR-, 30 (41.5%) were CMV IgM+/PCR-, and 8 (11%) were CMV IgM-/PCR+. CMV IgM was detected in 7 of the 15 DNA+ DBS. Since there are no published reports on the measurement of CMV IgM on DBS, we examined the time elapsed between the date of birth and the date the DBS was analyzed in our laboratory. The overall time span for the 15 DNA+ DBS was from 4 days to 17 years. Of the 7 DBS with the longest time elapsed (between 1.5 and 17 years), only 1 had detectable CMV IgM. Of the remaining 8 DBS with least time elapsed (4 days–1.5 years), 6 had detectable CMV IgM. This could indicate that the assay has a higher sensitivity for CMV IgM detection on DBS less than 1.5 years old. If this is the case, the statistical probability for CMV IgM/PCR+ in DBS increases from 47% (7/15) to 75% (6/8) (p<0.05). Future large studies are required to determine the kinetics of CMV IgM stability in DBS.
Literature reports on the stability of IgM in DBS are limited. The repeated reconstitution and measles-specific IgM testing of 201 samples confirmed that IgM is fairly stable during storage of filter paper blood samples at 4°C (16). The authors showed that despite the 16 months between the first and second measurement, the results of the two assays were almost identical. Other studies reporting on measurement of anti-dengue IgM in DBS showed stability for at least 166 days (17); however, further timepoints were not examined. A recent kinetic study showed that Toxoplasma-specific IgM in DBS remained stable up to 400 days if stored at 4°C or lower (12). Several other studies have reported the detection of IgM virus-specific antibodies in DBS in individuals with hepatitis A (18,19), measles (20,21), rubella (22,23), toxoplasma (24), and dengue viral infections (17,25 –28).
In summary, these findings indicate that the detection of CMV IgM in DBS is feasible. However, future large studies are needed to show that CMV IgM detection may be a useful additional diagnostic tool. Finally, The WHO Measles and Rubella Laboratory Network supports and have validated the use of dried blood spots as an alternative to serum in the measles/rubella program since antibody is stable in this form (29).
Footnotes
Acknowledgments
The authors would like to thank Professor Gerna for kindly sharing unpublished data on congenital cytomegalovirus infection studies.
Author Disclosure Statement
No competing financial interests exist.