Multicentric Evaluation of New Commercial Enzyme Immunoassays for the Detection of Immunoglobulin M and Total Antibodies against Hepatitis A Virus

Abstract

A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively).

TEXT

The etiological agent of hepatitis A is a nonenveloped, positive-stranded RNA virus that is typically transmitted via the fecal-oral route (4).

Hepatitis A virus (HAV) infection is still the most common cause of acute viral hepatitis worldwide and remains a serious health problem not only in the developing world, but also in industrialized countries (6, 10). It can lead to a multiplicity of clinical features, ranging from asymptomatic infection to fulminant fatal disease (16).

Over the last decades, improvement in the living conditions of several world populations has prevented young people from acquiring asymptomatic infection (which provides lifelong protection), leaving them susceptible to hepatitis A infection (2, 4). With regard to adults, certain categories of people, such as men who have sexual intercourse with men, both injection and noninjection drug users, and travelers returning from developing countries, are exposed to a higher risk of infection and therefore are subjected to cyclic outbreaks of hepatitis A (1, 3, 8, 11, 12, 15, 17, 18). It is also worth mentioning that HAV-infected persons with chronic liver disease are likely to develop fulminant hepatitis A (14, 16).

This global scenario of hepatitis A epidemiology calls for an increase in research to be carried out in the coming years and for measures to be taken to control and prevent the spread of this infectious disease. In particular, vaccination against hepatitis A is recommended for all the aforementioned high-risk categories of people and represents an important tool in the prevention of virus spread and epidemic outbreaks (5).

Laboratory diagnosis of hepatitis A is based mainly on the detection of antibodies associated with acute and past infection (IgM and IgG, respectively); complementary tests can be used in some cases for the diagnosis of recent infection (9, 13). In prevaccination programs, however, the detection of total anti-HAV antibodies is also critical for establishing whether individuals have acquired immunity and for identifying those susceptible to HAV infection.

This study aimed to evaluate the performance of two novel anti-HAV enzyme immunoassays (EIA) developed by Bio-Rad Laboratories (the Monolisa anti-HAV IgM EIA and the Monolisa anti-HAV EIA) and to compare their results to those obtained using the FDA-approved ETI-AB-HAV-IgMK Plus and ETI-AB-HAVK Plus assays from DiaSorin S.p.A. The tests were performed and the results interpreted according to the manufacturers' instructions. Relative sensitivity and specificity, as well as agreement between the Bio-Rad and DiaSorin assay results, were calculated, with unresolved equivocal (concordant and discordant borderline) data excluded; 95% binomial confidence intervals (CI) were applied to the results.

The study was conducted on representative serum samples from 1,738 European and U.S. subjects from Parma University Medical School in Italy (n = 586), Paul Brousse Hospital in France (n = 366), and the United States (n = 786).

A retrospective study (to detect total antibodies and IgM) was performed on 235 sera (stored at −20°C) that had been collected from a population with a known hepatitis A status: 84 acutely infected, HAV IgM-positive patients and 151 formerly infected, HAV IgG-positive patients who had recovered.

A prospective study (to detect total antibodies and IgM) was conducted on 1,097 sera from subjects with an unknown hepatitis A status. This population consisted of the following categories: general hospitalized patients (345 Europeans), patients with symptoms of hepatitis (n = 426; 252 Europeans and 174 U.S. individuals), subjects at high risk for hepatitis A (n = 292; 62 Europeans and 230 U.S. individuals), and health care workers (34 Europeans).

A prevalence study (for total antibodies) was performed on 755 subjects (the above-mentioned European groups of 345 general hospitalized patients and 34 health care workers and a further 376 general hospitalized U.S. individuals) whose conditions were not related to hepatitis infections and who were representative of the healthy population.

Total HAV antibody response was also evaluated in 30 subjects who had received one of the three vaccines licensed in the United States (Vaqta; Merck & Co.; Havrix or Twinrix; GlaxoSmithKline).

Samples from individuals with an unknown hepatitis A status were collected after obtaining their written informed consent and upon receiving protocol approval by the ethics committees of the public organizations involved in the study.

The results obtained in the retrospective and prospective studies from the Bio-Rad and DiaSorin (reference) tests for total antibodies and the IgM immunoassays are compared and presented in Table 1. All equivocal (concordant and discordant borderline) results displayed in Table 1 were confirmed by repeating the tests on the same samples in order to avoid any possible technical problem. Only one sample, belonging to the formerly infected/recovered patient category, gave different results: the sample was initially positive for IgM with the Bio-Rad test and borderline with the DiaSorin test, but then it was positive with both assays (thus, it was considered in the statistical analysis).

Table 1.

Anti-HAV total antibodies and IgM in sera from subjects with a known (retrospective study) or an unknown (prospective study) hepatitis A status^a

Study	Specimen category	Total no. of samples	Total antibodies					IgM
			No. of ETI-AB-HAVK Plus samples that were:		No. of Monolisa anti-HAV EIA samples that were:		No. of samples with equivocal results	No. of ETI-AB-HAV IgMK Plus samples that were:		No. of Monolisa anti-HAV IgM EIA samples that were:		No. of samples with equivocal results
			R	NR	R	NR		R	NR	R	NR
Retrospective	Formerly infected/recovered HAV patients	151	150	0	150	0	1	0	149	1	148	2
	Patients with acute hepatitis A	84	84	0	84	0	0	84	0	84	0	0
Total for study type		235	234	0	234	0	1	84	149	85	148	2
Prospective	General hospitalized population	345	237	108	237	108	0	1	343	2	342	1
	Population with symptoms of hepatitis	426	313	108	313	108	5	1	425	6	420	0
	Population at high risk for hepatitis A	292	143	146	145	144	3	0	291	0	291	1
	Health care workers	34	6	28	6	28	0	0	34	0	34	0
Total for study type		1,097	699	390	701	388	8	2	1,093	8	1,087	2
Total for all study types		1,332	933	390	935	388	9	86	1,242	93	1,235	4

^aR, reactive; NR, nonreactive. Equivocal (concordant and discordant borderline) results were confirmed by repeating the tests on the same samples, except for 1 sample in the formerly infected/recovered HAV patient category, which was initially positive for IgM with the Bio-Rad test and borderline with the DiaSorin test and then positive by both assays. Confirmed equivocal results were excluded from statistical analysis.

With regard to total antibodies, 2 discrepant results (i.e., reactive with the Monolisa anti-HAV EIA and nonreactive with ETI-AB-HAVK Plus) were found in the category of patients at high risk for hepatitis A and confirmed upon repetition of the tests. Concerning IgM, 7 discrepant results (i.e., reactive with the Monolisa anti-HAV IgM EIA and nonreactive with ETI-AB-HAV-IgMK Plus) were observed in the formerly infected/recovered, general hospitalized, and high-risk-for-hepatitis A subject categories. The tests were repeated on the same samples, and when possible, complementary assays, such as the anti-HAV IgG avidity and HAV RNA detection tests (9, 13), were also performed in order to ascertain acute infection (Table 2).

Table 2.

Analysis of discrepant IgM samples from subjects with a known and an unknown hepatitis A status^a

Specimen category	Demographics (gender, age [yr])	Monolisa anti-HAV IgM EIA		ETI-AB-HAV-IgMK Plus			Complementary information
		Ratiob	Interpretationc	Sample OD	CODd	Interpretatione	Anti-HAV IgG avidity	HAV RNAf	Total HAV antibodiesg
Formerly infected/recovered population	F, 59	1.13	R	0.06	0.277	NR	NE	NE	R
		1.04	BRD	0.061	0.229	NR
General hospitalized population	F, 76	1.2	R	0.087	0.282	NR	NE	NE	R
		1.01	BRD	0.09	0.278	NR
Symptoms of hepatitis population	M, 42	1.45	R	0.122	0.26	NR	High	Negative	R
		1.71	R	0.136	0.255	NR
	M, 42	1.18	R	0.114	0.262	NR	High	Negative	R
	1.14	R	0.149	0.255	NR
	F, 18	4.08	R	0.037	0.255	NR	High	Negative	R
		2.97	R	0.046	0.257	NR
	M, 36	1.22	R	0.18	0.255	NR	NE	NE	R
		1.29	R	0.144	0.278	NR
	F, 18	3.28	R	0.025	0.28	NR	NE	NE	R
		NE		NE

^aF, female; M, male; R, reactive; NR, nonreactive; BRD, borderline; NE, not evaluated; OD, optical density; COD, cutoff OD.

^bResults are expressed as the ratio of the sample OD to the COD.

^cThe presence or absence of anti-HAV IgM antibodies (Monolisa anti-HAV IgM EIA noncompetitive test) was determined as follows: specimens with ratio values greater than or equal to 1.1 were considered reactive (antibodies present) and those with ratios less than 0.9 as nonreactive (antibodies absent). Specimens with ratios greater than or equal to 0.9 times the COD value and less than 1.1 times the COD value (0.9 < ratio < 1.1) were considered to be equivocal (borderline). For each run, the COD was calculated as the mean of 3 OD values (which must be greater than 0.6 and less than 2.9) of the positive cutoff calibrator divided by 4.

^dFor each run, the COD was determined by adding 0.250 to the mean of the calibrator absorbance values (which must be greater than −0.020 and less than 0.120).

^eThe presence or absence of anti-HAV IgM antibodies (ETI-HA-IgMK Plus noncompetitive test) was determined by comparing the OD value of the patient serum with the COD value. Patient specimens with absorbance values less than the COD value (and not within −20% of the COD) were considered nonreactive and those with values greater than the COD as reactive. Absorbance values ranging between 0 and 20% below the COD were considered equivocal (borderline).

^fThe presence of HAV RNA was evaluated using a one-step reverse transcription (RT)-PCR kit (Qiagen) amplifying a 512-bp fragment that encompasses the VP1/2A junction of the HAV genome. The sensitivity of the RT-PCR assay was 43 IU/ml, as assessed with serial dilutions of the World Health Organization HAV RNA standard.

^gThe presence of total HAV antibodies was confirmed using the DiaSorin ETI-AB-HAVK Plus (competitive format) reference immunoassay. In this case, the COD was calculated for each run as the mean absorbance of three calibration values (which must be greater than the positive-control OD and less than 60% of the negative-control OD). The presence or absence of anti-HAV antibodies was determined by comparing the OD value of the patient serum with the COD value; absorbance values less than the COD were considered reactive, and those with values greater than the COD (and not from 0 to 20% above the COD) nonreactive. Absorbance values ranging between 0 and 20% above the COD were considered equivocal (borderline).

The relative sensitivities of the Monolisa anti-HAV EIA (versus ETI-AB-HAVK Plus) and the Monolisa anti-HAV IgM EIA (versus ETI-HA-IgMK Plus), determined with the samples included in the retrospective and prospective studies, were 100% in both cases (reactive for total antibodies, 933/933 samples [CI, 99.61% to 100%]; reactive for IgM, 87/87 samples [CI, 95.85% to 100%]). The relative specificities were 99.49% (388/390 samples [CI, 98.16% to 99.94%]) for total antibodies and 99.44% (1,235/1,242 samples [CI, 98.84% to 99.77%]) for IgM antibodies. The overall agreement was 99.85% (1,321/1,323 samples [CI, 99.46% to 99.98%]) for total antibodies and 99.47% (1,322/1,329 samples [CI, 98.92% to 99.79%]) for IgM.

HAV antibody prevalence in subjects representative of the healthy population (health care workers and patients hospitalized with conditions unrelated to hepatitis virus infection) was estimated with the Monolisa anti-HAV EIA for Europe (n = 379) and for the United States (n = 376) (Fig. 1). The results obtained from the above-named populations show that prevalence is higher in Europe than in the United States and that, mostly in the former case, it is related to the subjects' age range, with older individuals demonstrating progressively higher prevalences, in accordance with data obtained by other authors (7).

Fig. 1.

HAV antibody prevalence in Europe (n = 379) and in the United States (n = 376) using the Monolisa anti-HAV EIA. The study was performed on subjects representative of the healthy population (health care workers and patients hospitalized with conditions unrelated to hepatitis virus infection). For each age range category, the number of reactive subjects (with total anti-HAV antibodies present) over the total number of subjects is displayed on each histogram.

The HAV total antibody response using the Monolisa anti-HAV EIA and ETI-AB-HAVK Plus was also evaluated in subjects who had received one of the three vaccines licensed in the United States (Vaqta, Havrix, or Twinrix vaccine). It is important to note that only a limited number of vaccinated subjects develop a transient IgM anti-HAV response (13), while immunized people always produce IgG anti-HAV. This is why only total antibodies were assayed in the evaluation of samples from vaccinated populations.

Postvaccination samples from 6 subjects who had received the Vaqta vaccine were assayed, and both tests found all samples to be reactive (data not shown). With regard to the Havrix vaccination, the HAV total antibody response was evaluated in 10 pre- and postvaccination samples (2-dose schedule). Once again, coincident results were found with both tests, showing a correct improvement of the HAV antibody response in postvaccination samples (not shown). For the evaluation of Twinrix (3 injections at 0, 1, and 6 months), 14 subjects were examined. A prevaccination sample was collected the day of the first vaccination dose, and a second sample was obtained before the second dose was injected (1 month after the first one), but the second and third vaccination dose samples were not available. With regard to the Monolisa anti-HAV EIA (Fig. 2), the results show a slight increase in the HAV antibody response after the first injection in the majority of the subjects enrolled in the study (subject 14 was already immunized against HAV before the first vaccination dose); there were 4 cases (subjects 5, 9, 10, and 13) that were found negative after the first dose. The ETI-AB-HAVK Plus assay gave identical results, except for the borderline values of postvaccination samples from subjects 10 and 13 (data not shown).

Fig. 2.

HAV antibody response following Twinrix vaccination. The HAV total antibody response to Twinrix vaccination (GlaxoSmithKline; 3-dose schedule) was evaluated for 14 subjects using the Monolisa anti-HAV EIA. A prevaccination sample (0 Month) was collected the day of the first vaccination dose. A second sample (1 Month) was obtained before the second dose was injected (1 month after the first dose). The second and third vaccination dose samples were not available.

In summary, data derived from this multicentric study indicate that the Monolisa anti-HAV IgM and anti-HAV (total Ig) EIA (Bio-Rad Laboratories) are specific and sensitive assays which may be used effectively in the laboratory diagnosis of acute or past HAV infection and for the identification of HAV-susceptible individuals who should be enrolled in vaccination programs. Their performance is comparable with that of the ETI-AB-HAVK and IgMK Plus tests from DiaSorin S.p.A. The Bio-Rad Monolisa anti-HAV IgM and anti-HAV (total Ig) EIA thus represent valid alternative choices, with the shorter time required to perform the Bio-Rad tests being an additional advantage. The Bio-Rad assays have also been cleared by the FDA.

The few discrepant results (positive with the Bio-Rad versus negative with the DiaSorin tests), mostly related to IgM detection, could be due to a slightly higher sensitivity of the Bio-Rad assays, permitting the detection of small amounts of IgM that in some cases could persist after the acute stage; however, the possibility of false-positive results cannot be ruled out.