Update on hepatitis B and C virus diagnosis

Table 1 Clinical significance of hepatitis B virus serological markers

Marker	Clinical significance
HBsAg	First marker to appear in course of infection
	Appears one to 3 wk before the onset of symptoms
	The permanence of this marker for more than 24 wk indicates chronicity
Anti-HBc IgM	Marker of recent infection marker
	Appears with the onset of symptoms and persists up to 32 wk after infection
Anti-HBc IgG	This marker did not indicate immunity and it is not elicited by vaccination
	This presence indicates prior contact with the virus
HBeAg	It appears before the onset of symptoms and indicates viral replication independent of disease phase (acute or chronic)
	This presence indicates high infectivity
Anti-HBe	It appears after the disappearance of HBeAg
	Their presence suggests reduction or absence of viral replication, except when infection is due to HBV strains with pre-core mutant (not producing the protein “e”)
Anti-HBs	It appears one to 3 mo after HBV vaccination or after recovery of HBV acute infection and indicates immunity to HBV infection

HBV: Hepatitis B virus; HBeAg: Hepatitis Be Antigen; HBsAg: Hepatitis B surface antigen; Anti-HBe: Antibodies against HBeAg; Anti-HBs: Antibodies against HBsAg.

Table 2 Advantages and disadvantages characteristics of hepatitis B virus serological assays

Technique	Advantages	Disadvantage	Commercial assays	Ref.
RIA	High sensitivity	High cost	IRMA kit (North Institute of Biological Technology, Beijing, China)	[38,39,44]
		Risk to operator
EIA	Automation	Time consuming, need sophisticated equipment and trained technicians, continuous supply of electricity, not suitable for field settings	ETI-AB-AUK-3 (DiaSorin)	[41,42,47,48]
	High reproducible results		Enzygnost anti-HBs (Dade Behring)
	Low cost		Monolisa anti-HBs (Bio-Rad Laboratories)
MEIA	High sensitivity	Sophisticated equipment, trained technicians, continuous supply of electricity	Abbott Axsym AUSAB assay	[47]
	Faster than other immunological methods
ECLIA	High sensitivity	High cost, sophisticated equipment, trained technicians, continuous supply of electricity	Modular E170 analyzer, Roche Diagnostics	[43,44,46]
	Results available in few minutes
	Automation
CLIA	High sensitivity and specificity Automation	High cost, sophisticated equipment, trained technicians, continuous supply of electricity	Advia Centaur anti-HBs	[47]
			Vitros anti-HBs on the Vitros ECI Immunodiagnostic system (Ortho Clinical Diagnostics)
			Roche Elecsys anti-HBs on the Modular System (Roche Diagnostics)
			Liaison anti-HBs on the Liaison system (DiaSorin) Abbott Architect anti-HBs assay on the Architect i2000 system (Abbott)
CMIA	High sensitivity and specificity Automation	High cost, sophisticated equipment, trained technicians, continuous supply of electricity	Architect i2000 analyzer (Abbott Diagnostics)	[44]
Rapid point-of-care tests	Simplicity, do not need sophisticated equipaments, minimum training to execute the assay, storage and performance at room temperature, results can be read in up to 10 min	Poor performance in seroconversion panels and among individuals infected by several HBV mutants	Determine™ HBsAg (Abbott Laboratories)	[49-56]
			Virucheck® HBsAg (Orchid Biomedical Systems)
			Cypress HBsAg (Cypress Diagnostics)
			Hexagon® HBsAg (Human)
			Cortez Rapidtest (Cortez Diagnostics)
			VIKIA HBsAg (Biomériuex)
			Quick Profile™ (Lumiquick)
			DRW-HBsAg v2.0 (Diagnostics for the Real WorldTM)
			AMRAD ICT
			Binax
			Advanced quality™ one step test (Intec Products)

HBV: Hepatitis B virus; RIA: Radioimmunoassay; EIA: Enzyme immunoassay; MEIA: Micro-particle enzyme immunoassay; ECLIA: Electrochemiluminescence immunoassay; CLIA: Chemiluminescence immunoassay; CMIA: Chemiluminescent microparticle immunoassay; HBsAg: Epatitis B surface antigen; Anti-HBs: Antibodies against HBsAg.

Table 3 Performance characteristics of commercial methods for hepatitis B virus and hepatitis C virus detection and quantification

Assay (manufacture)	Method	Measurable range (IU/mL)	Limit of detection IU/mL (using WHO HBV standard)	Conversion factor (IU/mL to copies/mL)
HBV
Cobas ampliPrep/Cobas TaqMan HBV test v2.0 (Roche Diagnostics, California, United States)	Semi-automated qPCR	20-1.7 × 107	20	5.82
Cobas TaqMan HBV test (for use with high pure system)	Semi-automated real time PCR	29-1.1 × 107	6	5.82
Abbott real time HBV (Abbott Diagnostic, Chicago, United States)	Automated real time PCR	10-1 × 109	10	3.41
Versant HBV 3.0 assay (Siemens Healthcare, United States)	Branched DNA	2000-1 × 108	2000	5.6
HCV qualitative
Cobas Amplicor HCV v2.0 (Roche)	Semiautomated RT-PCR	50	50	-
Versant HCV RNA qualitative assay (TMA)	Transcription-mediated amplification	< 9.6	5-9.6	-
Quantitative
Versant HCV RNA 3.0 (Siemens)	Branched DNA	7.7 × 106	615	5.2
Cobas Amplicor Monitor HCV v2.0 (Roche)	Semiautomated RT-PCR	5 × 105	600	2.7
Real time HCV (Abbott)	Real time PCR	1 × 107	10	3.8
Cobas AmpliPrep/Cobas TaqMan (Roche)	Automated real time PCR	6.9 × 107	43	3
High Pure/Cobas TaqMan (Roche)	Semiautomated real time PCR	3.9 × 108	25	3

WHO: World Health Organization; HBV: Hepatitis B virus; HCV: Hepatitis C virus; PCR: Polymerase chain reaction; RT-PCR: Reverse transcriptase-PCR; qPCR: Quantitative PCR.

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.

Table 5 Main Characteristics of serological assays for hepatitis C virus diagnosis

Serological assays		Antigen (region of the genome)	Assay/manufacturer	Sensitivity	Specificity	Ref.
EIA	1st generation EIA	c100-3 (NS3-NS4)	HCV-Ac-EIE Salck	70%-80%	50%-70%	[118,119]
	2nd generation EIA	c100-3 (NS3-NS4), c33-c (NS3), c22-3 (core)	ORTHO HCV ELISA test system	92%-95%		[118,119]
	3rd generation EIA	c100-3 (NS3-NS4), c33-c (NS3), c22-3 (core), NS5	ORTHO HCV 3.0 ELISA (Ortho)/ETI-AB-HCVK Sorin	95%-99%	99%	[57,118]
	4th generation EIA		Monolisa™ HCV AgAb ULTRA (BioRad)/HCV Murex AgAb (Abbott)	100%	99.5%	[117,133,144]
MEIA		HCr43 (Fusion core e NS3), c200 (NS3 - NS4), c100-3 (NS3-NS4), NS5	AxSYM® HCV 3.0 (Abbott)	100%	99.8%	[117,118]
ECLIA		Core, NS3 and NS4 proteins	Elecsys anti-HCV assay (Roche)	100%	99.7%	[117,118]
CLIA		[c22-3 (core), c200 (NS3 - NS4) and NS5]	ARCHITECT i4000 anti-HCV assay (Abbott); VITROS Eci anti-HCV assay (Ortho)	99.5%	98.2%	[145]
CMIA		HCr43 (core - NS3), c100-3 (NS3-NS4)	ARCHITECT® anti-HCV (Abbott)	99.1%	99.6%	[146]
RIBA	RIBA-1	5-1-1 (NS4) e c100-3 (NS3-NS4)		NP	NP	[118]
	RIBA-2	5-1-1 (NS4), c100-3 (NS3-NS4), c33-c (NS3), c22-3 (core)	Chiron RIBA-2.0 RIBA-2	NP	NP	[148]
	RIBA-3	c100-3 (NS3-NS4), c33-c (NS3), c22-3 (core), NS5	Chiron RIBA HCV 3.0 SIA	NP	NP	[118]
Biosensor technology		Core protein (p22 fusion protein), NS3, NS4 and NS5	mBio Diagnostics® company	NP	NP	[57,137]

NP: Not presented; EIA: Enzyme immunoassay; MEIA: Micro-particle enzyme immunoassay; ECLIA: Electrochemiluminescence immunoassay; CLIA: Chemiluminescence immunoassay; CMIA: Chemiluminescent microparticle immunoassay; RIBA: Recombinant immunoblot assays; HCV: Hepatitis C virus; ELISA: Enzyme-linked immunosorbent assay.