Serological and molecular marker analysis in infectious disease identification

Molecular markers are specific short sequences of DNA. They are capable of detecting polymorphism in unique chromosomal locations and can be random too. On the other hand, serological markers are used to quantify the concentrations of an antibody. Moreover, they are potentially the most direct way to understand the dynamics of a population. They are also popularly used in the diagnosis of a disease (Metcalf et al., 2016). The differences between alleles are traced as molecular markers. Here ‘alleles’ are defined as alternative forms of genes arising due to mutations. They help in identifying the desired gene or trait (Gupta et al., 2016). Therefore, serological and molecular marker analysis helps in the identification of different alleles or genes responsible for the identification of specific EIDs.

Importance of serological and molecular marker

The genome sequencing data is used to identify or detect biomarkers. It helps to differentiate non-virulent, virulent, and other human pathogens that are causative agents of many diseases. Genetic markers help in diagnosing the causative agent and this helps in the development of a vaccine. Pathogenic strains possess distinct serotypes or strains that cause different diseases, show selectivity for tissues and host. Pathogens have a relatively small genome. Hence they evolve rapidly and develop resistance against a drug. For example, in 1993 tuberculosis was declared as a global emergency because of its emergence drug-resistant strain (Gupta et al., 2016). In order to treat the problem, it is important to find the root cause. Serological or genetic marker help in confirming the cause. Thus, an important role of serological and molecular marker studies include identifying emerging infections and their sub-categories or mutant strains.

Application of serology in infectious disease diagnosis

Serology deals with the study of serum as well as other bodily fluids (Ryan and Ray, 2014). The term refers to the diagnostic identification of antibodies, which are typically formed in response to an infection, in the serum or given sample. It helps in identifying the viral or pathogen antigens and antibody, diagnose disease and immunity. Serological methods, infections can be classified into four groups (Metcalf et al., 2016):

  • Acute immunizing, antigenically stable pathogens (eg, measles, rubella, and smallpox) for which serology provides a strong signal of lifetime protection and a clear marker of past infection (or vaccination) lie under the first group,
  • Immunizing, but variable pathogens e.g, influenza, invasive bacterial diseases, and dengue,
  • For those, in which infection-induced antibodies are not thought to be protective, e.g., tuberculosis,
  • Infections for which specific antibodies presence do not protect from future infection.
Different types of serological marker analysis techniques

Different types of serological marker analysis techniques

Different types of serological marker analysis techniques

A test’s authenticity determined by the relative degrees of sensitivity, specificity and the time consumed to get a result. Monoclonal-antibody direct fluorescent antibody tests have adequate specificity for a particular virus, but then there is turnaround time and sensitivity exist (Jeong et al., 2017). Immunoglobulins (Ig) is mainly used as a serological marker for diagnostic and immunity check purpose. IgG and IgM are the most common serological markers used for antigen diagnosis.



Serological marker

Technique used


1 HIV/HBV co-infection, May 2006-July 2011 Brazil Hepatitis B surface antigen (HBsAg), hepatitis B “e” antigen (HBeAg). Microparticle Enzyme Immunoassay (Toscano and Corrêa, 2017)
2 HIV/HBV co-infection Uganda, Zimbabwe, Harare HBsAg Enzyme immunoassay (Price et al., 2017)
3 Dengue Fever, 2011-2012 Sri Lanka IgM and IgG ELISA (Senaratne et al., 2016)
4 Zika, 2016 South Korea IgM and IgG ELISA (Jeong et al., 2017)
5 Ebola, 2014 Gulu outbreak, 2001 Mannose-binding lectin and  IgG ELISA (McElroy et al., 2014)

Using molecular marker analysis in diagnosing emerging and reemerging pathogens

A molecular marker, a DNA sequence of known chromosomal location, used to identify an organism and genotyping methods include:

  • Polymerase chain reaction (PCR) amplification followed by strip-based reverse hybridization.
  • PCR followed by Sanger sequencing,
  • Real-time PCR.
  • Reverse transcriptase PCR.

PCR provides a rapid, specific and sensitive method for early detection in,  real-time PCR in addition to standard PCR offers advantages like quantitative measurement, low contamination rate and easy standardization. These methods undoubtedly offer certain advantages, but their limitations are unavoidable as well. Sequencing offers an excellent resolution of genotype, but it can be time-consuming and labour-intensive at the same time, it demands skilled technologists. The accuracy of results cannot be guaranteed either.

Some of the genetic markers used to map Gene sequence and diagnosis as well include:

  • Random amplification of polymorphic DNA
  • Amplified fragment length polymorphism
  • Single nucleotide polymorphism
  • Restriction fragment length polymorphism



Molecular marker

Technique used


1 Dengue, 2016 Pakistan Random Amplified Polymorphic DNA Polymerase chain reaction (PCR) (Ashraf et al., 2016)
2 Malaria Infections, 2015 Myanmar artemisinin-resistance marker K13 (kelch 13 gene) Polymerase chain reaction, nested PCR (Nyunt et al., 2017)
3 Zika, 2016 KDCC Envelope “E” gene real-time reverse transcription-polymerase (Jeong et al., 2017)
4 Dengue Fever, 2011-2012 Sri Lanka Capsid gene and envelope RT-PCR (Senaratne et al., 2016)
5 Dengue, 2007 India Capsid gene Duplex RT-PCR, nested PCR (Neeraja et al., 2013)

Example of serological marker analysis

One recent example of the use of serological marker analysis for HIV and HBV by Toscano and Corrêa, (2017).

  • Pathogen: Human immunodeficiency virus (HIV) and hepatitis B virus (HBV)
  • Disease: HIV and HBV coinfected, São Paulo, Brazil, May 2006-July 2011.
  • The study conducted from June 2011 to July 2012.
  • Serology marker: Hepatitis B surface antigen (HBsAg), hepatitis B “e” antigen (HBeAg).
  • Findings: 2,242 HIV infected patients out of which 105 ( i.e., 4.7%) identified with chronic hepatitis B. All patients received antiretroviral (ARV) therapy during follow-up and follow-up time varied from six months to 20.5 years. 58% of patients with chronic hepatitis B indicated hepatitis B “e” antigen positive. 16 out of 105 of patients with chronic hepatitis B indicated HBsAg, and 8/16 of these patients showed subsequent reactivation or seroreversion of HBsAg. Among HBeAg infected patients, 57% (35/61) presented the clearance of same serologic marker. During clinical follow-up, those initially cleared HBeAg, 28.5% (10/35) of them showed seroreversion or reactivation of this marker. Among HIV co-infected patients undergoing ARV therapy, the evolution of HBV serological markers was frequently observed.

Example of molecular marker analysis

One recent use of molecular marker analysis for dengue by Ashraf et al., (2016)

  • Pathogen: Dengue virus, genetic analysis of Aedes aegypti
  • Disease: Dengue fever, Pakistan.
  • The method used: Random Amplified Polymorphic DNA (RAPD) Markers from Dengue Outbreaks.
  • Advantage: Amplify DNA randomly, and a specific primer not required. Also, it requires only one primer.
  • Findings: The study proposed the existence of genetic diversity in Aedes aegypti population of Lahore is causing the havoc and hence analyzed the genetic variability by RAPD-PCR using 10 oligonucleotide primers. Eighteen populations of mosquitoes were sampled from Faisalabad and Lahore. Polymorphic loci amplified by each primer varied from 22.5% to 51%. The UPGMA (unweight pair-group mean analysis) dendrogram demonstrated two distinct groups of populations. The genetic variation ranged from 0.260 in Faisalabad to 0.294 in Lahore and 0.379 heterozygosity. The overall genetic variation among eighteen populations showed GST (inbreeding indices) = 0.341 and Nm (migration rate)= 1.966. The statistics showed that Aegypti populations had intra-population genetic drift between Faisalabad and Lahore. It was also concluded that Aegypti populations were reportedly genetically more diverse as that of the previous population.

Future trends of serological and molecular markers

Molecular and serological techniques have set a turning point in discovering and characterizing many emerging infectious agents. Challenges are still there for the widespread use of cost-effective, validated, and commercially available molecular tools. Phylogenetic analysis of a study presented that the newly emerged strain of swine influenza contained genes from the 2009 pandemic human H1N1 and swine H3N2 viruses. The report also suggested that a combination of genes from the 2009 pandemic human H1N1 strain and H3N2 Swine Influenza virus endemic in eastern China (Peng et al., 2016). As pathogens evolve continuously and mutate, rapid diagnostic tests needed from single blood, saliva or any sample allow identification of the cause of infection and identify any virus to the species level.


  • Ashraf, H. M. et al. (2016) ‘Genetic Analysis of Aedes aegypti using Random Amplified Polymorphic DNA (RAPD) Markers from Dengue Outbreaks in Pakistan’, Journal of Arthropod-Borne Diseases, 10(4), pp. 546–559.
  • Gupta, V. et al. (2016) ‘Basic and applied aspects of biotechnology’, Basic and Applied Aspects of Biotechnology, pp. 1–520. doi: 10.1007/978-981-10-0875-7.
  • Jeong, Y. E. et al. (2017) ‘Viral and serological kinetics in Zika virus-infected patients in South Korea’, Virology Journal. Virology Journal, 14(1), p. 70. doi: 10.1186/s12985-017-0740-6.
  • McElroy, A. K. et al. (2014) ‘Biomarker correlates of survival in pediatric patients with ebola virus disease’, Emerging Infectious Diseases, 20(10), pp. 1683–1690. doi: 10.3201/eid2010.140430.
  • Metcalf, C. J. E. et al. (2016) ‘Use of serological surveys to generate key insights into the changing global landscape of infectious disease’, The Lancet. Elsevier Ltd, 388(10045), pp. 728–730. doi: 10.1016/S0140-6736(16)30164-7.
  • Neeraja, M. et al. (2013) ‘The clinical, serological and molecular diagnosis of emerging dengue infection at a tertiary care institute in Southern, India’, Journal of Clinical and Diagnostic Research, 7(3), pp. 457–461. doi: 10.7860/JCDR/2013/4786.2798.
  • Nyunt, M. H. et al. (2017) ‘Molecular evidence of drug resistance in asymptomatic malaria infections, Myanmar, 2015’, Emerging Infectious Diseases, 23(3), pp. 517–520. doi: 10.3201/eid2303.161363.
  • Peng, X. et al. (2016) ‘Molecular characterization of a novel reassortant H1N2 influenza virus containing genes from the 2009 pandemic human H1N1 virus in swine from eastern China’, Virus Genes. Springer US, 52(3), pp. 405–410. doi: 10.1007/s11262-016-1303-4.
  • Price, H. et al. (2017) ‘Hepatitis B serological markers and plasma DNA concentrations.’, AIDS (London, England), 31(8), pp. 1109–1117. doi: 10.1097/QAD.0000000000001454.
  • Ryan, K. J. and Ray, C. G. (2014) Sherris medical microbiology, 6th Ed., Sherris medical microbiology. doi: 10.1017/CBO9781107415324.004.
  • Senaratne, T. et al. (2016) ‘Characterization of Dengue Virus Infections in a Sample of Patients Suggests Unique Clinical, Immunological, and Virological Profiles That Impact on the Diagnosis of Dengue and Dengue Hemorrhagic Fever’, Journal of Medical Virology, pp. 1–8. doi: 10.1002/jmv.
  • Toscano, A. L. de C. C. and Corrêa, M. C. M. (2017) ‘Evolution of hepatitis B serological markers in HIV coinfected patients: a case study’, Revista de Saúde Pública, 51, pp. 1–8. doi: 10.1590/s1518-8787.2017051006693.
Avishek Majumder

Avishek Majumder

Research Analyst at Project Guru
Avishek is a Master in Biotechnology and has previously worked with Lifecell International Private Limited. Apart from data analysis and biological research, he loves photography and reading. He loves to play football and basketball in his spare time with an avid interest in adventure and nature. He was also a member of the Scouts in his school and has attended Military training.
Avishek Majumder

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