Improving The Efficiency of Cytomegalovirus (CMV)
The Cytomegalovirus (CMV) is known to be an important pathogen among patients with prolonged confinement to the intensive care unit and longer hospital stays (Vincent, 2010). The virus is recognized to be a common form of the herpes virus, which the Centers for Disease Control described to be present in about 50% to 80% of the people in the United States who are in their 40’s (Mandal, 2014). The infection can actually affect children and adults alike, however the virus has the greatest impact among certain individuals, especially among children and those with a compromised immune system and already had a past history of having contracted the virus that makes them highly susceptible to reactivation (South Australia Health, n. d.). The CMV is also noted for its tremendous impact as a congenital infectious agent in developing countries, and Baumann (2011) indicated that the diagnosis of the infection may involve numerous laboratory methods, which includes the DNA detection from dried spots on a filter paper. The use of filter paper has gained a tremendous importance as a substrate in the surveillance and diagnosis of infectious diseases. This paper will address what the literature studies indicate about the efficiency of this type of diagnosis approach for the CMV and will underpin the various literature evaluations on the efficiency of CMV DNA detection from dried blood spots.
Literature evaluations about the efficiency of cytomegalovirus (CMV) DNA detection from dried blood spots indicate the this type of diagnosis is more effective and less laborious to perform instead of undertaking virus isolation for identifying asymptomatic infected babies and carrying out epidemiological studies (Yamagishi, et. al. 2006). This finding is consistent with the studies conducted by Binda, et. al (2004) citing that the method of detecting the viral DNA in dried blood spots is highly practical when diagnosing congenital CMV infection. The whole blood is more practical sample that one can collect on a filter paper (Barin, 2006), although serum and plasma can also be taken. According to their findings, using only one third of the amount of the dried blood spot can produce 100% sensitive and accurate findings when compared to viral isolation and standard DBS test. It conclusively provides that the process of using DNA detection of the virus using dried blood spot is more economical in terms of saving from the patient materials and testing costs. The use of dried blood spots provided a remarkable means of reducing data collection efforts with impressive performance in terms of its feasibility of combining the biological and contextual measures when conducting a population based research (McDade, Williams, and Snodgrass, 2007). The use of dried blood spots as a bio-marker in detecting the CMV is practical to use in a population where there is a low rate of health services available, lack of facilities in collecting samples, and insufficient laboratory infrastructures (Boerma, Holt, and Black, 2001). It can be noted that the gold standard for congenital CMV diagnosis involves the use of urine and saliva samples that are collected in the first 3 weeks after birth (Lazzarotto, et al., 2008), which is considered not appropriate for the conduct of large scale screening. The dried blood spot assays offer a more practical alternative as the samples can be collected and preserved easily (Shibata, Takano, Hironaka and Hirai, 1994).
The procedure of collecting the blood sample is straightforward, but must be carried out efficiently in order to reduce error. A lancet is used to prick the subject’s finger, which is cleaned with alcohol. The first drop of blood is usually wiped away and the subsequent drops of blood are collected on a filter paper (Turner and Holman, 1978). To dry the blood, it usually takes at least four hours to overnight and are stacked on a resealable plastic bag or container. To avoid contamination, Barbi, et. al. (2000) prescribe the control measures in order to avoid carryover contaminations. This includes proper sterilization of the tools used and the proper handling of the samples. The more efficient means of collecting blood is through the heel or fingers as it requires minimal training (Center for Disease Control and Prevention, 2000).
In order to make an efficient test for the DNA virus, it is essential to collect the blood spots in comparable sizes. In order to avoid the variation in the number of blood samples collected, Adam, et, al. (2000) suggest to marking pre-printed circles on the filter paper in order to standardize the amount of blood to collect. It must be noted that the amount of the degradation of the sample will vary greatly based on the analyte. An assessment must be initiated in terms of the stability of the blood samples to be taken as it can have implications to the handling and storage of the samples. McDade et. al. (2000), for instance, provides that blood samples taken from an Epstein-Barr virus will remain stable at a normal room temperature for eight weeks, but may tend to deteriorate at a room temperature of 37 degrees Celcius within one week. A typical blood drop will contain at least 50 µL of whole blood and the most reasonable amount that one can collect for a single finger prick is 10 to 20 3. 2-mm discs (Williams, Lindau, and McDade 2006). The important of keeping the variance in the amount of blood sample collection as minimal as possible is also cited by Mei, et. al. (2010).
There is less concern with regards to the biosafety issues using the method of CMV DNA detection using filter papers because it involves simpler packaging requirement than liquid blood (Reitmeyer, Ewert, Crawford, Reitmeyer, Mock, 1993). However, precautions must still be observed since the blood dried samples might still contain a culture of the group A streptococci, while Dengue virus may still be present within the first 48 hours on the blood sample (Prado, et. al, 2005). The use of FTA paper offers better protection against pathogenic infections caused by the Avian influence virus within 1 hour after absorption (Abdelwhab, Luschow, Harder and Hafez, 2011). When reporting the dried blood sample evaluation, it is necessary to use the Standards for Reporting of Diagnostic Accuracy guidelines (Bossuyt, 2003), which aims to provide more accurate diagnosis and allowing for the assessment of the potential bias in the internal validity of the study while evaluating its external validity (Stard, 2008).
The sensitivity of using DNA testing for CMV using filter paper was consistent in many literature reports, including the studies performed by Fellous, et. al. (2007) providing that the method offers a reliable tool for the retrospective diagnosis of the CMV congenital infections, and that of de Vries, Claas, Kroes and Vossen (2009), who cited the method as excellent universal neonatal screening for CMV congenital infection. However, the latter provides that there can be a potential difference between the extraction methods in terms of the sensitivity of the result, and recommended to test the dried blood samples in triplicate for optimal sensitivity. In a study conducted by Leruez-Ville (2011), the sensitivity and specificity of the CMV assays of the dried blood spot in diagnosing congenital CMV infection is reportedly high, ranging from 95% to 100% as compared to using a urine sample taken during the first week of life of the child. In order to prevent contamination and to preserve the quality of the blood samples, using the QIAamp DNA micro kit is recommended by Scanga, et. al. (2006), describing its ability to purify the genomic DNA that is extracted. It has also been reported by Gohring, et al. (2010) that the use of the kit can lead to a higher sensitivity result using the whole dried blood spot. It can be assumed based on these findings that the tools used for collecting the dried blood spot and the method of extractions can also affect the outcomes of the test sensitivity when detecting the viral DNA. Another notable use of the dried blood spot test, is using it for neonatal screening for secondary prevention sequelae, as it can serve as a basis for programs on correction and rehabilitation for infected children and prevent further epidemiological infections (Binda, 2004). According to Wang, et al. (2015), the greatest benefit of CMV DNA test is the identification of asymptomatic infants who are likely to develop delayed onset diseases.
The dried blood spot testing appears to be a practical alternative for isolating virus from the urine when diagnosing the CMV from both symptomatic and asymptomatic neonates. The collection of the blood samples on filter papers also suggests that the test works even when using the smallest possible sample (Noorgaard-Pedersen, and Simonen, 1999). This equates to a lot of savings in terms of the cost of study and the screening of the infected babies. In a comparable study of diagnosing congenital CMV infections by isolating the virus from urine and saliva from the DBS testing, Balcarek, et. al. (1993) noted that the former is costly, laborious and requires more extensive epidemiological study that further makes the neonatal screening more complicated. There are also other uses for the DBS test that can help in diagnosing metabolic and genetic disorders, such as congenital hypothyroidism and cystic fibrosis, and neonatal HIV or toxoplasma (Cassol, et al., 1991). The use of the dried blood spot has also been extended in diagnosing CMV specific antibodies that are of maternal origin (Lebech and Petersen, 1992), however, the DBS test is not as sensitive and specific as the viral isolation procedure when detecting the IgM antibody in CMV from the mother and child (Demmler, 1991). Additionally, the use of the Guthrie card or filter paper is viewed to be promising in the early diagnosis of neonatal herpetic disease by means of demonstrating the presence of the herpes simplex virus DNA (Barbi, et al. 1998). It is also useful in ascertaining the etiology of morbid conditions that are diagnosed later and from unknown origin as a neuronal migration defect (Barth, 1987) with the virus already present in the blood at birth or from gestation (Barkovich and Lindan, 1994).
The routine prenatal screening for the CMV infection is not recommended by medical associations (Redehasse, 2013), owing to the danger and risk of terminating the pregnancy and causing harm to a healthy baby since it involves an invasive procedure (Benoist, et al., 2008). Moreover, even in the absence of the CMV DNA screening, there are prenatal screening and diagnosis that may be sufficient in the early detection of hearing loss and developmental disabilities (Gross, et al., 2006). While there are different approaches in diagnosing congenital CMV infections, the method of DBS testing is more helpful in detecting infected neonates who are at risk of the secondary sequelae (Lanari, 2006). Accordingly, many cases of CMV infections are viremic at birth (Boppana, 2005), and the DBS test will offer an opportunity to conduct a straightforward test that can identify neonates who are high risk of long term sequelae caused by the infection (Ross, 2009).
There is currently no international standard on how the DBS testing is undertaken when diagnosing CMV infections, and the methods used in laboratories greatly vary (Kaiser, et al., 2007). Barbi, Binda and Caroppo (2006) cited out critical points that can possibly affect the sensitivity and false positivity of the procedure, namely the method of elution and extraction of the DNA, the amount of blood sample collected, the PRC test and the criteria for positivity. This observation is also consistent with the findings of Smit (2014), providing that there has been an uncritical use of DBS and using inappropriate statistical analysis and the lack of a standardized methodology. The length of the storage for DBS also varies, however Johansson et al (1997) tested DBS samples that are stored at varied periods between 12 and 18 years, which is the longest storage time made for DBS and there appears to be no significant change in terms of the diagnostic sensitivity and specificity that range between 81. 25% and 100%. This is highly suggestive that the CMV DNA collected in dried blood spots is stable enough despite the storage time. Another critical issue involving storage time includes the possibility of cross contamination of adjacent cards carrying the DBS with the virus, but Walters (2008) discounted this possibility after showing that no such cross contamination occurred during his research. The use of DBS is widely used as a diagnostic tool for CMV infection and many researchers advocate its use for an anonymous self test kits for HIV and other viral diseases (Snijdewind, et al., 2012). It is also beneficial to the clinical practice in diagnosing neonates where the current diagnostic procedures can be too demanding for a fragile child (Pandya, Spooner, and Mulla, 2011).
The Research Canvass
Understanding the problem
What is known?
Literature findings were consistent to show that the cytomegalovirus DNA detection using filter paper shows a high rate of sensitivity and accuracy in terms of diagnosing the presence of different infectious diseases caused by the CMV. It is more prominently used in the diagnosis of asymptomatic congenital CMV infection and is considered to be more practical, expeditious and cost effective method of collecting samples for epidemiological studies. The feasibility of DBS for detecting CMV infection in population based researchers is very good and it requires a minimal level of preservation standard and collection of samples. The use of the DBS in detecting CMV infections should be disseminated within the healthcare system because of its practical use and effectiveness as a diagnostic and preventive tool.
What is the gap in the knowledge?
Researches were focused more on determining the sensitivity and accuracy rating of using DBS for detecting CMV infections in comparison to the other diagnostic methods available today. The information regarding the potential contamination of the stored samples is quite limited and what possible errors can occur that can significantly affect the outcomes of the diagnosis remains unclear. Future research aims and objectives should include researches involving the preservation issues that might cause some changes in the quality of the samples, as well as some risks that may be involved during the process of collection to the patient and to the health worker.
The resources used in the analysis include patient samples from the hospitals who are known to be suffering with infectious diseases caused by the CMV, maternal patients with known infectious disease, neonatal patients who are both symptomatic and asymptomatic with CMV. Diagnostic tools include filter paper or Guthrie cards, and the DBS kits that are used for the DNA extraction.
The major barriers in the study include the limitation of the number of patients being evaluated and the lack of basis on the universal screening to perform, since the DBS is not currently the golden standard for CMV infection diagnosis. The researchers were only able to conduct self administering tests, which they pattern and then compare and contrast, with the other researcher’s study outcomes. Some patients (such as mothers with secondary infections) that should have been represented in the study were not included due to constraints of lack of approval from these patients. The majority of the subjects in the study that were infected are neonates born of mothers that have the primary infection.
More data collection is needed in order to expand and dig deeper into the efficiency of the DBS in diagnosing CMV infections and to include its potential use for diagnosing other infectious diseases caused by other viruses.
The research was able to underpin the essential contribution of using the DBS in detecting the CMV infection across health centers, especially in communities that lack laboratory facilities. Being cost effective and practical, the same is easy to implement with the least training required of the health care providers. Using the method as a tool for accurately diagnosing and preventing the secondary sequelae caused by the CMV infection will benefit those patients who are highly susceptible to contracting the infection, including the asymptomatic neonates.
The media is a powerful tool where this valuable information may be disseminated. The research findings may be published in blogs, social media, news, publications, leaflets and journal publishings.
The research has the greatest impact to the public health system, where the diagnostic process used for detecting CMV infections is costly and not practical for large scale screening.
Abdelwhab E. M., Luschow D., Harder T. C., Hafez H. M. 2011. The use of FTA filter papers for diagnosis of avian influenza virus. J Virol Methods 174, pp. 120–122.
Adam, B. W. et, al. 2000. Recoveries of Phenylalanine From Two Sets of Dried-Blood-Spot Reference Materials: Prediction From Hematocrits, Spot Volume, and Paper Matrix.” Clinical Chemistry 46, pp. 126–28.
Balcarek K. B., Warren W., Smith R. J., Lyon M. D., Pass R. F. 1993. Neonatal screening for congenital cytomegalovirus infection by detection of virus in saliva. J Infect Dis167, pp. 1433–1436.
Barbi M, Binda S, Caroppo S. 2006. Diagnosis of congenital CMV infection via dried blood spots. Rev Med Virology 16, pp. 385-392.
Barbi M. 1998. Use of Guthrie cards for the early diagnosis of neonatal herpes simplex virus disease. Pediatr Infect Dis 17, pp. 251–252.
Barbi, M. et. al. 2000. Cytomegalovirus DNA detection in Gruthie cards: a powerful tool for diagnosing congenital infection. Journal of Clinical Virology. 17, pp 159-165.
Barin F., 2006. Human immunodeficiency virus serotyping on dried serum spots as a screening tool for the surveillance of the AIDS epidemic. J Med Virol 78 (Suppl 1), pp. S13–S18.
Barkovich A. J., Lindan C. E. 1994. Congenital cytomegalovirus infection of the brain: imaging analysis and embryologic considerations. Am J Neuroradiol 15, pp. 703–715.
Barth P. G. 1987. Disorders of neuronal migration, Can J Neurol Sci, pp. 1–16.
Baumann, G. 2011. Congenital cytomegalovirus infection: Epidemiology, diagnosis and therapy. Germany: Springer Verlag.
Benoist, G. et al., 2008. The prognostic value of ultrasound abnormalities and biological parameters in blood of fetuses infected with cytomegalovirus. BGOG 115, pp. 823-829.
Binda, S. 2004. Modification of CMV DNA detection from dried blood spots for diagnosing congenital CMV infection. Journal of Clinical Virology 30, pp. 276-279.
Binda, S., et. al 2004. Modification of CMV DNA detection from dried blood spots for diagnosing congenital CMV infection. J. Clin. Virol. 30(3), 276-279.
Boerma, J. T., E. Holt, and R. Black. 2001. “ Measurement of Biomarkers in Surveys in Developing Countries: Opportunities and Problems.” Population and Development Review 27, pp. 303–14.
Boppana S. B., et al. 2005. Congenital cytomegalovirus infection: association between virus burden in infancy and hearing loss. J Pediatr 146, pp. 817–23
Bossuyt P. M, 2003. Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative. BMJ 326, pp. 41–44.
Cassol S., et al. 1991. Use of dried blood spot specimens in the detection of human immunodeficiency virus type 1 bythe polymerase chain reaction. J Clin Microbiol 1 (29), pp. 667–71.
Center forDisease Control and Prevention. 2002. Serologic Assays for Human Immunodeficiency Virus Antibody in Dried-Blood Specimens Collected on Filter Paper. Atlanta, GA: US CDC.
de Vries, J., Claas, E., Kroes, A. and Vossen, A. 2009. Evaluation of DNA extraction methods for dried blood spots in the diagnosis of congenital cytomegalovirus infection. Journal of Clinical Virology 46S, pp. S37-S42.
Demmler G. J. 1991. Summary of a workshop on surveillance for congenital cytomegalovirus disease. Rev Infect Dis 13, pp. 315–29.
Fellous, et. al. 2007. Evaluation of Cytomegalovirus (CMV) DNA Quantification in Dried Blood Spots: Retrospective Study of CMV Congenital Infection. Journal of Clinical Microbiology 45(11), pp. 3804-3806.
Gohring, K. et. al 2010. Influence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards. Journal of Clinical Virology 48, pp. 278-281.
Gross, S. D. et al. 2006. From public health emergency to public health service: The implications of evolving criteria for newborn screening panels. Pediatrics. 117, pp. 923-929.
Johansson P. J. et al. 1997. Retrospective diagnostics of congenital cytomegalovirus infection performed by polymerase chain reaction in blood stored on filter paper. Scand J Infect Dis. 29, pp. 465-468.
Kaiser, et al., 2007. Multicenter proficiency study for detection of Toxoplasma gondii in amnioticfluid by nucleic acid amplification methods. Clin Chim Acta 375, pp. 99-103.
Lanari, M. 2006. Neonatal cyomegalovirus blood load and risk of sequelae in symptomatic and asymptomatic congenitally infected newborns. Pediatrics 117, pp. 76-83.
Lazzarotto T. et al. 2008. New advances in the diagnosis of congenital cytomegalovirus infection. J Clin Virol. 41, pp. 192-197.
Lebech M., Petersen E. 1992. Neonatal screening for congenital toxoplasmosis in Denmark: presentation of the design of a prospective study. Scand J Infect Dis 84, pp. S75–S79.
Leruez-Ville, L. 2011. Prospective Identification of Congenital Cytomegalovirus Infection in Newborns Using Real-time Polymerase Chain Reaction Assays in Dried Blood Spots. Clinical Infectious Disease 52(5), pp. 578-581.
Mandal, A. 2014. What is cytomegalovirus? News Medical Net [online] Available at
McDade, T. W. et. al. 2000. Epstein-Barr Virus Antibodies in Whole Blood Spots: A Minimally-Invasive Method for Assessing an Aspect of Cell-Mediated Immunity.” Psychosomatic Medicine. 62, pp. 560–67.
McDade, T. W., Williams, S., and Snodgrass, J. J. 2007. What a drop can do: Dried blood spots as a minimally invasive method for integrating biomarkers into population based research. Demography. 44(4), pp. 899-925.
Mei, J. V., et. al. 2010. Performance properties of filter paper devices for whole blood collection. Bioanalysis. 2, pp. 1397–1403.
Noorgaard-Pedersen, B., and Simonen, H. 1999. Biological specimens banks in neonatal screening. Acta Paediatr Suppl 432, pp. 106–109.
Pandya, H. C., Spooner, N., Mulla, H., 2011. Dried blood spots, pharmacokinetic studies and better medicines for children. Bioanalysis 3, 779–786.
Prado I, et. al. 2005. PCR detection of dengue virus using dried whole blood spotted on filter paper. J Virol Methods 125, pp. 75–81.
Redehasse, M. J. 2013. Cytomegaloviruses: From molecular pathogenesis to intervention. New York: Horizon Scientific Press.
Reitmeyer J. C., Ewert A, Crawford M. A., Reitmeyer G. R., Mock, L. 1993. Survival of group A streptococci in dried human blood. J Med Microbiol 38, pp. 61–63.
Ross S. A., et al. 2009. Cytomegalovirus blood viral load and hearing loss in young children with congenital infection. Pediatr Infect Dis J 28, pp. 588–592.
Scanga, L. et. al. 2006. Diagnosis of Human Congenital Cytomegalovirus Infection by Amplification of Viral DNA from Dried Blood Spots on Perinatal Cards. Journal of Molecular Diagnostic 8(2), pp. 240-245.
Shibata M, Takano H, Hironaka T, Hirai K. 1994. Detection of human cytomegalovirus DNA in dried newborn blood filter paper. J Virol Methods. 46, pp. 279-285.
Smit, P. W. 2014. Review Article: An overview of the clinical use of filter paper in the diagnosis of tropical diseases. Am. J. Trop. Med. Hyg., 90(2), pp. 195–210.
Snijdewind, I. et al. 2012. Current and future applications of dried blood spots in viral disease management. Antiviral Research 93, pp. 302-309.
South Australia Health. n. d. Cytomegalovirus (CMV) infection – symptoms, treatments and prevention. Government of South Australia. [online] Available at:
Stard. 2008. Startd Statement. Stard [online]. Available at
Turner, R. C., Holman, R. R., 1978. Automatic lancet for capillary blood sampling. Lancet 2, 712.
Vincent, J. L. 2010. Yearbook of intensive careand emergency medicine. Germany: Springer Verlag.
Walter S. 2008. Congenital cytomegalovirus: association between dried blood spot viral load and hearing loss. Arch Dis Child Fetal Neonatal Ed. 93, 280-285.
Wang, L., et al. 2015. Dried blood spots PCR assays to screen congenital cytomegalovirus infection: a meta-analysis. Virology Journal, pp. 2-11.
Williams, S. R., S. Lindau, and T. W. McDade. 2006. “ Evaluation of Dried Blood Spots Collected by Non-medically Trained Interviewers: Number, Size, and Quality of Spots.” Presented at the annual meeting of the Population Association of America, Los Angeles, March 30–April 2.
Yamagishi, Y. et. al. 2006. CMV DNA detection in dried blood spots for diagnosing congenital CMV infection in Japan. J. Med. Virol. 78(7), pp. 923-925.