Table 4.

Main characteristics of hepatitis B virus and hepatitis C molecular techniques

HBV and HCV molecular diagnosis	Application	Method	Advantages	Disadvantages	Ref.
HBV DNA qualitative methods	Diagnose infection	PCR	Low cost; high sensitivity	It only determines the presence or absence of HBV DNA	[81,82,85]
	HBV occult cases identification
	Screening on blood donors
HBV DNA quantitative methods	Evaluate the prognosis and risk of progression	bDNA	Wide dynamic range	Low sensitivity to detect low HBV DNA levels	[81,82]
	Define the beginning of antiviral treatment
	Monitor antiviral treatment
		Hybrid capture	More sensitive than bDNA; less labor-intensive	Low sensitivity to detect low HBV DNA levels; individual probes are required	[83]
		Real time PCR	Capacity of detecting low viral loads; broad dynamic range; do not carry over contamination; can be fully automated	High cost	[85,91]
HBV DNA genotyping methods	Determination of HBV genotype	RFLP	Easily done; low cost; simple, rapid and suitable for large number of samples	Low sensitivity for typing samples with low HBV DNA levels; poor accurate to determine some genotypes	[85,104]
		Genotype specific PCR assays	Automated systems; high sensitivity; easy to perform; suitable for detecting mixed genotype infections	High cost	[107]
		Sequence analysis	Identification of patients infected with recombinant genotypes	Technically demanded; time consuming	[107]
HBV DNA aminoacid substitution identification	Identify antiviral resistance to treatment	Direct DNA sequencing	Accurate	Technically demanded; time consuming; necessity of cloning for identification of mixed population	[107,110,111]
		Commercial methods	Sequencing of mixed population, relative quantification of individual mutations with extremely high coverage	Differences between the statistical and biological/clinical relevance of HBV mutation maximal sequence read length and PCR amplification bias	[114]
HCV RNA qualitative methods	To confirm chronic hepatitis C in patients with positive HCV antibodies	RT-PCR	High sensitivity	It only determines the presence or absence of HCV RNA	[121,168]
	To identify virological response during, at the end or after antiviral therapy		Equal sensitivity for all genotypes
	To screen blood donations for evidence of infection with HCV
		Transcription-mediated amplification	High sensitivity; amplifies viral RNA; more sensitivity for detection of genotype 1	It only determines the presence or absence of HCV RNA	[121,168]
HCV RNA quantitative methods	To guide treatment decisions; To evaluate the prognosis; To monitor the antiviral efficacy of treatment	bDNA	Wide range of detection of HCV independent of HCV genotype (615 IU/mL to 8 million IU/mL)	Low sensitivity to detect samples presenting low HCV RNA levels	[172]
		qRT-PCR	Capacity of detecting low viral loads; broad dynamic range; not carry over contamination; can be fully automated	High cost	[170-173]
HCV RNA genotyping methods	HCV genotyping is mandatory for double antiviral treatment (interferon and ribavirin), since patients infected with genotypes 1 or 4 are treated for longer times than patients infected by genotypes 2 and 3	RFLP	Easily done; low cost; simple, rapid and suitable for large number of samples	Low sensitivity for typing samples with low HCV RNA levels; Poor accurate to determine some genotypes	[186,187]
		Probes	Easily done; low cost; useful to detect HCV genotypes and subtypes based on region 5’UTR and core and has a low limit of detection	Identify only subtypes 1a and 1b; discrepant results among subtypes when compared to sequence analysis of NS5B region	[180-184]
		qPCR	Can be fully automated avoiding contamination; determines the viral genotype and subtypes 1a, 1b, 2a, 2b, 3, 4, 5 and 6	High cost	[186,187]
		Direct sequencing	Gold standard; identification of patients infected with recombinant genotypes	Technically demanded; time consuming	[120,182]
HCV RNA aminoacid substitution identification	Identify antiviral resistance to treatment	Direct Sequencing	Identification of antiviral resistance in majority population	Technically demanded; time consuming; necessity of cloning for identification of quasispecies	[188,189,193]
		Deep Sequencing	Identification on resistant variants predominate in the HCV population; powerful tool for obtaining more profound insight into the dynamics of variants in the HCV quasispecies	Need for in-depth knowledge to analyze the results	[112,113,195,196]

PCR: Polymerase chain reaction; HCV: Hepatitis C virus; HBV: Hepatitis B virus; RFLP: Restriction fragment-length polymorphism; RT-PCR: Reverse transcriptase-PCR; qPCR: Quantitative PCR; RFLP: Restriction fragment-length polymorphism; bDNA: Branched DNA technology.