Journal of Medical Virology 3826L270 (1992)
The Detection in Urine Specimens of IgG and IgM Antibodies to Hepatitis A and Hepatitis B Core Antigens -
Keith R. Perry, John V. P a r r y , Elise M. Vandervelde, and Philip P. Mortimer PHLS Virus Reference Laboratory, Central Public Health Laboratory, London, United Kingdom The use of urine as a noninvasive specimen for the diagnosis of hepatitis A (HAV) and hepatitis B (HBV) virus infections was investigated. Specimens of urine were collected at the same time as blood or saliva specimens, or singly in cases of previously serologically confirmed recent infection. The specimens were tested for IgG and IgM anti-HAV and anti-HBc by immunoglobulin classspecific capture radioimmunoassays (GACRIA and MACRIA). On the basis of assays on urine specimens it was possible t o distinguish between individuals who were susceptible or immune t o HAV or who had recently been infected with HAV. Using assays on 327 corresponding saliva specimens as reference tests, the observed sensitivity and specificity of tests o n urine specimens by anti-HAV GACRIA were 98.9% and 99.1%, respectively, and by anti-HAV MACRIA were 95.8% and 99.6%, respectively. IgM and IgG anti-HBc were detected readily in the urine of 35 acute or recent cases of hepatitis B but were not found in the urine of seronegative individuals. Of the urine specimens from 52 individuals who were HBsAg carriers or w h o had had long past HBV infections, 49 contained detectable IgG antiHBc. Of urine specimens from 42 HBsAg carriers, 11 contained raised IgM anti-HBc levels. Urine, which is a convenient specimen t o collect, can be used t o study outbreaks of hepatitis A, to ascertain the HAV immune status of individuals, to differentiate hepatitis A from hepatitis B, and to identify individuals w h o have been naturally exposed t o HBV. o 1992 Wiley-Liss, Inc.
KEY WORDS: urinary antibodies, viral hepatitis diagnosis, HAV surveillance, HAV outbreak investigation
INTRODUCTION Class-specific antibody capture assays have made possible the detection of viral antibodies in body fluids with low levels of IgG and IgM. We have previously shown that saliva samples can be used to diagnose re0 1992 WILEY-LISS, INC.
cent or past infection with hepatitis A and B, and to identify susceptible individuals [Parry et al., 1987, 1988,19891. The noninvasive, painless nature of saliva collection permits epidemiological investigations to be carried out that, were they to be based on venepuncture specimens, would be difficult and incomplete. An example is a n investigation of a school outbreak of hepatitis A involving 130 pupils in which collection of venous blood would have been impractical because of reluctance to collect specimens from children [Bull et al., 19891. More recently, urine has been investigated a s a specimen for the detection of viral antibodies, in particular for anti-human immunodeficiency virus (HIV) [Cao et al., 19891.In 1990, Connell et al. described two different forms of a class-specific antibody capture assay suitable for the detection of IgG anti-HIV in urine specimens without preceeding concentration or treatment. These urinary assays could determine accurately the HIV status of 711 subjects including 158 who were seropositive. The assays are effective because urine specimens have low but measurable concentrations of immunoglobulin, derived from plasma either through the kidney or by transudation into the lower renal tract. Connell et al. [1990] also demonstrated that of 690 urine specimens examined for total IgG, 664 had concentrations ranging from 0.1-10 mg/l; 8 had concentrations 10 mg/l. As well a s transudation, local IgG synthesis has been shown in the rabbit, both in pyelonephritis [Lehmann e t al., 19681 and lower urinary tract infection [Hand et al., 19701. There have been few attempts to detect viral antibodies in urine other than for anti-HIV. Neutralising antibodies to poliovirus have been demonstrated in urine following immunisation [Lerner et al., 19621. On occasion antibodies to hepatitis B core (IgG anti-HBc) have been detected by commercial enzyme-linked immunosorbent assay (ELISA) (Abbott Cozyme'"), though after 200-fold concentration of the urine in 3 of 33 se-
Accepted for publication May 15, 1992. Address reprint requests to Mr. Keith R. Perry, PHLS Virus Reference Laboratory, 61 Colindale Avenue, London NW9 5HT, United Kingdom.
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ropositive individuals examined [Cao et al., 19891. We are not aware of any reports describing assays for the detection of specific IgM for any virus in the urine of patients with acute infection. We report the use of antibody capture radioimmunoassays for detection of IgG and IgM specific for antiHAV and anti-HBc in the urine of patients with recent hepatitis, carriers of hepatitis B virus (HBV),and other immune and susceptible individuals. The optimal assay conditions, and the sensitivity and specificity of the assays are described.
MATERIALS AND METHODS Subjects In the course of two outbreaks of hepatitis A, urine and saliva specimens were collected a t the same time from 327 individuals. On the basis of salivary tests by a proven method (see below) 231 subjects were susceptible, 48 were immune, and 48 had evidence of recent hepatitis A virus (HAW infection. In addition, 70 patients with serologically confirmed recent hepatitis A were studied, whose urine specimens were collected from 3 days before to 8 weeks (median 30 days) after the onset of jaundice. Urine specimens were also collected from 35 patients with recent serologically confirmed acute hepatitis B, 10 anti-HBc and anti-HBs seropositive individuals, 42 HBsAg carriers, and 46 anti-HBc seronegative individuals.
The optimal dilution for testing urinary specimens was determined in preliminary experiments for each of the four antibody types. The assays best discriminated between positive and negative samples when the urine specimens were tested undiluted; when urine was tested at dilutions beyond 1/16 the assays became insensitive. For subsequent testing urine specimens were therefore added undiluted to the coated beads. Following incubation for 3 h r a t 37°C the beads were washed and l i l 0 0 HAV tissue culture grown antigen [Parry, 19841 or 112,000 of a “recombinant” HBcAg ( a gift from Wellcome Diagnostics, Dartford, Kent) was added. Incubation with the antigens was, for HAV, 16-18 hr a t room temperature and, for HBcAg, 1 h r at 45°C. The beads were then washed, and purified human IgG with a high titre of anti-HAV or anti-HBc, labelled with “‘1, was added. After incubation for 2% h r and a final wash, bound radioactivity was measured with a y-counter. Test: negative (T:N)values were calculated by dividing the test count by the mean count of four replicates of a negative control serum. In each assay run IgG and IgM anti-HAV or anti-HBc serum controls were included as appropriate, i.e., four replicates of a negative serum and duplicates of positive sera with 100,30,10,3, and 1 arbitrary units of specific antibody I Parry, 1981I.
RESULTS Specificity For the 231 individuals studied who werc susceptible to hepatitis A, the urinary IgG anti-HAV reactivity ranged from 0.3-2.1 times that of the negative control Specimens reaction (T:N) and the median ratio was 1.0: the uriAll saliva samples were collected using the Saliv- nary IgM anti-HAV T:N ratios ranged from 0.3-4.3 and ette device (Sarstedt Ltd., Leicester, U.K.). The de- the median ratio was 1.1. Of the 48 subjects who were vice consists of a 40 mm x 10 mm cylinder of cotton immune to hepatitis A two gave urine samples that wool which is gently chewed by the subject and then were reactive in MACRIA (T:N ratio 4.6 and 5.4): the inserted into a plastic tube with a small hole in its base median ratio was 1.3 (Table I). so that clarified saliva can be extracted by centrifugaTable I1 shows that the 46 anti-HBc seronegative tion. The saliva specimens were then stored a t -30°C individuals did not react in urinary assays for IgG and until tested a s described previously [Parry et al., 19891. IgM anti-HBc. Ten individuals seropositive for IgG Urine specimens were collected in pots, transferred to 6 anti-HBc and anti-HBs had urinary IgM anti-HBc T:N ml bottles with sealers, and posted to the Virus Refer- ratios from 0.7-3.6 and the median ratio was 1.8. Of the ence Laboratory for storage either at 4°C or - 70°C until 42 subjects who were HBsAg carriers, 11 had urinary tested. IgM anti-HBc T:N ratios greater than 3.0 (range for the 11 was 3.7-41.4 median 11.9). That range overlapped Urine Assays with the range of anti-HBc values in urine specimens Urinary IgG and IgM anti-HAV and anti-HBc were from recent cases of acute hepatitis B. Urine specimens from patients with clinical and serodetected by virus-specific IgG and IgM antibody capture radiommunoassay (GACRIA and MACRIA). The logical evidence of recent HAV and HBV infection were same concentrations of anti-y and anti-p for solid phase also tested by GACRIA and MACRIA using the heterolcoating and of antigen and radiolabel and the same ogous antigen and label. Urine specimens from the 23 diluents were used a s described previously for saliva individuals tested who had recently had hepatitis B did tests. Briefly, polystyrene beads (6.5mm diameter code not react by the HAV MACRIA even though 7 were IgG no P201, Northumbria Biologicals) were coated by gen- anti-HAV positive (Table I). Urine specimens from the tle shaking in a dilution of rabbit antibody to human 31 individuals tested who recently had hepatitis A did IgG (y-chain specific, code A424, Dakopatts, Ltd., 11.4 not react for IgG and IgM anti-HBc (Table 11). mg/l) or rabbit antibody to human IgM (p-chain speSensitivity for Urinary Anti-HAV cific, code A426, Dakopatts Ltd., 3.1 mgil) in 0.05 M carbonateibicarbonate buffer, pH 9.6. The beads, after IgG anti-HAV. All individuals whose saliva conwashing, were added to 200 ~1 of untreated urine sam- tained IgG but not IgM anti-HAV were reactive for ples in reaction trays. urinary IgG anti-HAV (Table I). With one exception I\’
Urinary Diagnosis of Viral Hepatitis
267
TABLE I. Reactivities of Urine Specimens for IgM and IgG Anti-HAV Patient cateeorv Recent HAVb Past HAV' Susceptible" Recent HBVd
No. 118 48 23 1 23
IgM anti-HAV Range Median T:Na T:N 2.7-126.0 0.2-5.4 0.34.3 0.7-2.3
28.9 1.3 1.1 1.3
IgG anti-HAV Range Median T:N" T:N 2.7-113.0 4.3-87.8 0.3-2.1 0.9-43.0
21.7 13.5 1.0 1.3
"T:N values are ratios of the test counts divided by the mean count of four negative controls. "One hundred eighteen individuals with recent hepatitis A. Of these, 70 were serologically confirmed convalescent cases and 48 were from hepatitis A outbreaks and identified on the basis of salivary tests. 'Individuals from two hepatitis A outbreaks identified as susceptible or immune to hepatitis A infection based on tests on simultaneously collected saliva specimens. dSerologically confirmed cases of recent hepatitis B.
TABLE 11. Reactivities of Urine Specimens for IgM and IgG Anti-HBc Patient CatePorv Recent HBVb Carrierb Past H B V ~ Seronegative" Recent HAVd
No. 35 42 10 46 31
IgM Anti-HBc Range Median T:N T:N" 3.6-76.1 0.441.4 0.7-3.6 0.8-2 .O 0.6-1.8
32.2 2.1 1.8 1.3 1.1
IgG Anti-HBc Range Median T:N T:N" 8.5-115.0 1.2-91.0 1.6-17.7 0.2-1.3 0.6-1.6
56.3 30.6 6.5 0.9 1.0
"T:N values are ratios of the test counts divided by the mean count of four negative controls. I'Urine specimens were collected from individuals serologically proven to have had previous or recent infection with hepatitis B. 'Urine specimens were collected from individuals serologically proven to have had no evidence of infection-with hepatitis B. dUrine specimens were collected from individuals serologically proven to have had recent hepatitis A infection.
those with evidence of recent HAV infection based on the presence of IgM anti-HAV in saliva had strong IgG anti-HAV reactivity in urine. The exception had a urinary T:N ratio of 2.7 (urinary IgM anti-HAV 27.0) and on the basis of salivary results (IgG anti-HAV 3.2; IgM anti-HAV 54.4) was a n early acute case with still very weak IgG anti-HAV reactions. Two other saliva samples which gave IgM anti-HAV results compatible with acute HAV did not react by the IgG anti-HAV assay, while urine samples collected a t the same time were weakly IgG reactive (Saliva GACRIA T:N: 1.4, 1.8; MACRIA T:N 44.9, 14.5. Urine: GACRIA 4.8,3.9; MACRIA: 55.8,8.1). The distribution of urinary IgG anti-HAV reactivities is shown in Figure l a . IgM anti-HAV. Hepatitis A outbreaks usually include patients with jaundice, with anicteric illness and asymptomatic infection. In order to assess the accuracy of the urinary assay, specimens were examined both from serologically confirmed clinical hepatitis A and from individuals in whom the diagnosis of recent HAV infection was based on IgM anti-HAV in saliva. Of the urine specimens collected within 8 weeks of the onset of jaundice from 70 serologically confirmed cases of HAV infection all but 3 gave strong IgM antiHAV reactions (range 5.9-126.0, median 27.9). The 3 weakly reactive samples, which were collected 40, 47, and 29 days after the onset of jaundice, the T:N ratios were 3.0,2.7, and 4.7, respectively.
Most of the urine specimens collected from individuals with salivary evidence of recent HAV infection had high IgM anti-HAV T:N ratios (median 30.3) although this was not as high a s found in corresponding saliva specimens (median 61.7). Urine specimens from 2 ofthe 48 subjects with salivary evidence of recent HAV infection had IgM anti-HAV T:N ratios less than 5.0 (4.1and 3.9, corresponding salivary ratios values, 5.2 and 17.3). The distribution of urinary IgM anti-HAV reactivities is shown in Figure l b . Comparison between urinary and salivary IgG and IgM anti-HAV reactivities. IgG anti-HAV T:N ratios for paired urine and saliva specimens were plotted against each other for 96 individuals immune to or with recent hepatitis A (ranges: urine, 2.7-87.8; saliva, 1.4-103.0). Sixty-one of the 96 individuals had IgG anti-HAV urinary reactivities which were weaker than for saliva. IgM anti-HAV T:N ratios were similarly plotted for the 48 recently infected individuals from whom paired specimens were available (ranges: urine, 3.9-121.0; saliva, 5.2-131.0). Thirty-seven of 48 urine specimens from cases of recent hepatitis A gave weaker IgM anti-HAV reactions than corresponding saliva specimens. Correlation coefficients (r)were 0.86 for IgG anti-HAV and 0.72 for IgM anti-HAV (Fig. 2).
Sensitivity for U r i n a r y Anti-HBc All 35 patients with recent acute hepatitis B infection (within 8 weeks of onset) had positive urinary IgG
Perry et al.
268 a7 78
0.3
1.5
3
IgG anti-HAV Testnegative (T:N) ratio
50
92 98
b
40
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> ._ U c ._
30
c
0 ij
5
20
z 10
T
0 25
40 >50
IgM anti-HAV Test:negative (T:N) ratio
Fig. 1. The distributirin of (a) IgG anti-HAV and (b) IgM anti-HAV urinary reactivities for 231 susceptible individuals \ El ), 48 with previous HAV infection W, and 118 with recent HAV infection (0).
and IgM anti-HBc reactions. There was no overlap with the reactions of seronegative individuals (Table 11).Of 10 individuals known to be seropositive for anti-HBc and anti-HBs, 8 had detectable IgG anti-HBc in the urine: in the other 2 the urine HBc GACRIA reactions were T:N 1.6 and 1.9. IgG anti-HBc was detected in the
urine specimens of 41 (T:N range 3.6-91.0, median 31.6) of 42 HBsAg carriers.
DISCUSSION In this study the GACRIA cut-off T:N ratio for IgG anti-HAV urine samples was set at 3.0, giving a sensi-
269
Urinary Diagnosis of Viral Hepatitis 140-
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100-
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100
120
140
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for 96 individuals with previFig. 2. IgG anti-HAV reactivities ous or recent HAV infection, and IgM anti-HAV reactivities ( 0 )for 48 individuals with recent HAV infection measured in simultaneously collected urine and saliva specimens.
TABLE 111. Concordance Between Salivary and Urinary IgM and IgG Anti-HAV Results IgM Anti-HAV
Saliva Positive Negative Positive Urine Negative
46 2b
la 278
IgG Anti-HAV Saliva Positive Negative 93 1"
2' 231
"Salivary findings for the discrepancies indicated that there was previous HAV infection. bSalivary findings for the discrepancies indicated that there was recent HAV infection. 'Salivary findings for the discrepancies indicated that there was early acute HAV infection.
tivity of 98.9% and a specificity of 99.1% by reference to results by the salivary GACRIA (Table 111). Excluding the early acute cases, where IgM anti-HAV may be found in the absence of IgG anti-HAV, the sensitivity was 100%.The urinary IgM anti-HAV cut-off was set a s 5.0 and this gave a sensitivity and specificity of 95.8% and 99.6%, respectively, with reference to salivary results. Although a good correlation was found for both IgG and IgM anti-HAV reactivities between saliva and urine specimens collected a t the same time from individuals who were susceptible to, immune to, or recently infected with HAV, urine generally gave weaker reactions than saliva. However, in the majority of cases of recent HAV infection (39/48) urinary IgM anti-HAV T:N ratios were >20.0. Urine specimens which were
collected between 3 days before and 8 weeks after the onset of jaundice from 70 serologically confirmed cases of hepatitis A were all positive for IgG anti-HAV, and 67/70 had IgM anti-HAV reactions that were significant diagnostically. The findings indicate that urine specimens which give IgM anti-HAV T:N ratios between 3.0 and 5.0 should be interpreted with caution. If a specimen gives rise repeatedly to weak IgM anti-HAV reactivity we suggest testing a follow-up urine andlor a saliva or serum specimen. In most instances such reactions arise from individuals who have not been infected recently. IgG and IgM anti-HBc were readily detected in HBV infection in all urine specimens collected between 2 and 8 weeks after onset of jaundice. Urine specimens collected from anti-HBc seronegative subjects did not react. However, some lack of sensitivity was encountered: IgG anti-HBc was not detected in 2 of 10 immune individuals and was not present in 1 of 42 HBsAg carriers. Interpretation of both IgM anti-HAV and IgM antiHBc reactivity can be difficult. Specific IgM appears before specific IgG and often before the onset of jaundice. It can persist in s e r u d p l a s m a a t low levels for at least 6-9 months or, in the case of HBV carriers, indefinitely. Consequently, weak IgM reactivity early in the course of infection is significant, but it can be diagnostically confusing later. IgM anti-HBc was detected in the urine of 11/42 carriers a t levels as high as those found in patients with a recent infection. Smith et al. [1992] reported that in sera from HBV carriers, 14.3% had IgM anti-HBc levels in the range found for acute hepatitis B. A good correlation was also shown between serum IgM anti-HBc levels in carriers and the cytoplasmic expression of HBcAg in liver tissue. Hence, it seems likely that the urinary IgM anti-HBc results are specific even though not associated with primary infection. It may be helpful in these instances to relate IgM to IgG reactivity to assist the interpretation of results [Parry, e t al. 19891. Although the urinary results, particularly for hepatitis A, are closely concordant with those obtained in saliva and serum, there are factors which need further investigation, e.g., urine storage conditions, the possible effect of urinary pH and bacterial contamination, and variability due to changes in fluid intake. Urine specimens were stored in aliquots at room temperature, 4"C, -25"C, and -7O"C, and i t appears from that study (unpublished) that storage at - 25°C is partially detrimental. Positive samples may eventually become negative, especially for virus-specific IgM. Our current practice is to store urine specimens for up to 6 weeks a t 4°C or a t -70°C if longer term storage is required. Because of the apparent lability of immunoglobulins in urine it might, for clinical use, be necessary to test all specimens for total immunoglobulin levels and set a cut-off level below which the specimen would be considered inadequate for testing. However, this precaution is probably unnecessary when these assays are used for epidemiological studies.
Perry et al.
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The prospects for urinary anti-HBc detection are not as clear as for anti-HAV detection. We have identified problems of specificity for IgM anti-HBc and of sensitivity for IgG anti-HBc detection. Although high levels of IgM anti-HBc were detected readily in the urine of nearly all acute cases of HBV, some HBsAg carriers possessed similar levels of urinary IgM anti-HBc. In addition, IgG anti-HBc could not be detected in the urine of some individuals who were immune a s a result of previous infection. It is concluded that urinary antiHBc testing is of less value than urinary anti-HAV testing, although it might still have epidemiological applications. For patients and study subjects who dislike venepuncture and find difficulty in providing a n adequate volume of saliva, urine would be the preferred specimen for diagnosis of hepatitis. In hospitals and general practices, urine specimens are easy to obtain and therefore readily available for clinical and epidemiological studies. In the laboratory, urine specimens have the technical advantage that, unlike blood specimens, preliminary sample preparation or dilution are not needed. I t should not be assumed that because immunoglobulin levels are low, urine specimens are unsatisfactory for antibody based viral diagnosis.
ACKNOWLEDGMENTS We wish t o thank Wellcome Diagnostics for the gift of HBcAg and partial funding, Miss Tracey Carter for typing the manuscript, and Mr. Guy Gabriel for preparing the radiolabels.
REFERENCES Bull AR, Kimmance KJ, Parry JV, Perry KR (1989):Investigation of an outbreak of hepatitis A simplified by salivary antibody testing. Epidemiology and Infection 103:371-376. Cao Y, Hosein B, Borokowsky W, Mirabile M, Baker L, Baldwin DF, Poiesz BJ, Friedman-Kien AE (1989): Antibodies to human immunodeficiency virus type 1 in the urine of HIV-1 seropositive individuals. AIDS Research and Human Retroviruses 5:311-319. Connell JA, Parry JV, Mortimer PP, Duncan RJS, McLean KA, Johnson AM, Hambling MH, Barbara J, Farrington CP (1990): Accurate assays for anti-HIV in urine. Lancet 335:136&1369. Hand WL, Smith JW, Miller TE, Barnett JA, Sandford J P (1970): Immunoglobulin synthesis in lower urinary tract infection. Journal of Laboratory Clinical Medicine 75:19-29. Lehmann JD, Smith J W , Miller TE, Barnett JA, Sandford JP (1968): Local immunoresponse in experimental pyelonephritis. Journal of Clinical Investigation 472541-2550. Lerner AM, Remington JS, Finland M (1962): Neutralizing antibody to polioviruses in normal human urine. Journal of Clinical Investigation 4123055815. Parry J V (1981): Hepatitis A infection: guidance for development of satisfactory assays for laboratory diagnosis. Medical Laboratory Sciences 38:303-311. Parry J V (1984):Diagnosis of hepatitis A infection: comparative specificity of IgM capture assays using antigens derived from tissue cultures and marmoset faeces. Journal of Virological Methods 9:35-44. Parry JV, Farrington CP, Perry KR, Waight P, Mortimer PP, Miller E (1988):Rational programme for screening travellers for antibodies to hepatitis virus. Lancet i:1447-1449. Parry JV, Perry KR, Mortimer P P (1987):Sensitive assays for viral antibodies in saliva; an alternative to tests on serum. Lancet ii:7275. Parry JV, Perry KR, Panday S, Mortimer P P (1989): Diagnosis of hepatitis A and B by testing saliva. Journal of Medical Virology 28:255-260. Smith HM, Lau JYN, Davies SE, Daniels HM, Alexander GJM, Williams R (1992):Significance of serum IgM anti-HBc in chronic hepatitis B virus infection. Journal of Medical Virology 36:16-20.
The Detection in Urine Specimens of IgG and IgM Antibodies to Hepatitis A and Hepatitis B Core Antigens -
Keith R. Perry, John V. P a r r y , Elise M. Vandervelde, and Philip P. Mortimer PHLS Virus Reference Laboratory, Central Public Health Laboratory, London, United Kingdom The use of urine as a noninvasive specimen for the diagnosis of hepatitis A (HAV) and hepatitis B (HBV) virus infections was investigated. Specimens of urine were collected at the same time as blood or saliva specimens, or singly in cases of previously serologically confirmed recent infection. The specimens were tested for IgG and IgM anti-HAV and anti-HBc by immunoglobulin classspecific capture radioimmunoassays (GACRIA and MACRIA). On the basis of assays on urine specimens it was possible t o distinguish between individuals who were susceptible or immune t o HAV or who had recently been infected with HAV. Using assays on 327 corresponding saliva specimens as reference tests, the observed sensitivity and specificity of tests o n urine specimens by anti-HAV GACRIA were 98.9% and 99.1%, respectively, and by anti-HAV MACRIA were 95.8% and 99.6%, respectively. IgM and IgG anti-HBc were detected readily in the urine of 35 acute or recent cases of hepatitis B but were not found in the urine of seronegative individuals. Of the urine specimens from 52 individuals who were HBsAg carriers or w h o had had long past HBV infections, 49 contained detectable IgG antiHBc. Of urine specimens from 42 HBsAg carriers, 11 contained raised IgM anti-HBc levels. Urine, which is a convenient specimen t o collect, can be used t o study outbreaks of hepatitis A, to ascertain the HAV immune status of individuals, to differentiate hepatitis A from hepatitis B, and to identify individuals w h o have been naturally exposed t o HBV. o 1992 Wiley-Liss, Inc.
KEY WORDS: urinary antibodies, viral hepatitis diagnosis, HAV surveillance, HAV outbreak investigation
INTRODUCTION Class-specific antibody capture assays have made possible the detection of viral antibodies in body fluids with low levels of IgG and IgM. We have previously shown that saliva samples can be used to diagnose re0 1992 WILEY-LISS, INC.
cent or past infection with hepatitis A and B, and to identify susceptible individuals [Parry et al., 1987, 1988,19891. The noninvasive, painless nature of saliva collection permits epidemiological investigations to be carried out that, were they to be based on venepuncture specimens, would be difficult and incomplete. An example is a n investigation of a school outbreak of hepatitis A involving 130 pupils in which collection of venous blood would have been impractical because of reluctance to collect specimens from children [Bull et al., 19891. More recently, urine has been investigated a s a specimen for the detection of viral antibodies, in particular for anti-human immunodeficiency virus (HIV) [Cao et al., 19891.In 1990, Connell et al. described two different forms of a class-specific antibody capture assay suitable for the detection of IgG anti-HIV in urine specimens without preceeding concentration or treatment. These urinary assays could determine accurately the HIV status of 711 subjects including 158 who were seropositive. The assays are effective because urine specimens have low but measurable concentrations of immunoglobulin, derived from plasma either through the kidney or by transudation into the lower renal tract. Connell et al. [1990] also demonstrated that of 690 urine specimens examined for total IgG, 664 had concentrations ranging from 0.1-10 mg/l; 8 had concentrations 10 mg/l. As well a s transudation, local IgG synthesis has been shown in the rabbit, both in pyelonephritis [Lehmann e t al., 19681 and lower urinary tract infection [Hand et al., 19701. There have been few attempts to detect viral antibodies in urine other than for anti-HIV. Neutralising antibodies to poliovirus have been demonstrated in urine following immunisation [Lerner et al., 19621. On occasion antibodies to hepatitis B core (IgG anti-HBc) have been detected by commercial enzyme-linked immunosorbent assay (ELISA) (Abbott Cozyme'"), though after 200-fold concentration of the urine in 3 of 33 se-
Accepted for publication May 15, 1992. Address reprint requests to Mr. Keith R. Perry, PHLS Virus Reference Laboratory, 61 Colindale Avenue, London NW9 5HT, United Kingdom.
Perry et al.
266
ropositive individuals examined [Cao et al., 19891. We are not aware of any reports describing assays for the detection of specific IgM for any virus in the urine of patients with acute infection. We report the use of antibody capture radioimmunoassays for detection of IgG and IgM specific for antiHAV and anti-HBc in the urine of patients with recent hepatitis, carriers of hepatitis B virus (HBV),and other immune and susceptible individuals. The optimal assay conditions, and the sensitivity and specificity of the assays are described.
MATERIALS AND METHODS Subjects In the course of two outbreaks of hepatitis A, urine and saliva specimens were collected a t the same time from 327 individuals. On the basis of salivary tests by a proven method (see below) 231 subjects were susceptible, 48 were immune, and 48 had evidence of recent hepatitis A virus (HAW infection. In addition, 70 patients with serologically confirmed recent hepatitis A were studied, whose urine specimens were collected from 3 days before to 8 weeks (median 30 days) after the onset of jaundice. Urine specimens were also collected from 35 patients with recent serologically confirmed acute hepatitis B, 10 anti-HBc and anti-HBs seropositive individuals, 42 HBsAg carriers, and 46 anti-HBc seronegative individuals.
The optimal dilution for testing urinary specimens was determined in preliminary experiments for each of the four antibody types. The assays best discriminated between positive and negative samples when the urine specimens were tested undiluted; when urine was tested at dilutions beyond 1/16 the assays became insensitive. For subsequent testing urine specimens were therefore added undiluted to the coated beads. Following incubation for 3 h r a t 37°C the beads were washed and l i l 0 0 HAV tissue culture grown antigen [Parry, 19841 or 112,000 of a “recombinant” HBcAg ( a gift from Wellcome Diagnostics, Dartford, Kent) was added. Incubation with the antigens was, for HAV, 16-18 hr a t room temperature and, for HBcAg, 1 h r at 45°C. The beads were then washed, and purified human IgG with a high titre of anti-HAV or anti-HBc, labelled with “‘1, was added. After incubation for 2% h r and a final wash, bound radioactivity was measured with a y-counter. Test: negative (T:N)values were calculated by dividing the test count by the mean count of four replicates of a negative control serum. In each assay run IgG and IgM anti-HAV or anti-HBc serum controls were included as appropriate, i.e., four replicates of a negative serum and duplicates of positive sera with 100,30,10,3, and 1 arbitrary units of specific antibody I Parry, 1981I.
RESULTS Specificity For the 231 individuals studied who werc susceptible to hepatitis A, the urinary IgG anti-HAV reactivity ranged from 0.3-2.1 times that of the negative control Specimens reaction (T:N) and the median ratio was 1.0: the uriAll saliva samples were collected using the Saliv- nary IgM anti-HAV T:N ratios ranged from 0.3-4.3 and ette device (Sarstedt Ltd., Leicester, U.K.). The de- the median ratio was 1.1. Of the 48 subjects who were vice consists of a 40 mm x 10 mm cylinder of cotton immune to hepatitis A two gave urine samples that wool which is gently chewed by the subject and then were reactive in MACRIA (T:N ratio 4.6 and 5.4): the inserted into a plastic tube with a small hole in its base median ratio was 1.3 (Table I). so that clarified saliva can be extracted by centrifugaTable I1 shows that the 46 anti-HBc seronegative tion. The saliva specimens were then stored a t -30°C individuals did not react in urinary assays for IgG and until tested a s described previously [Parry et al., 19891. IgM anti-HBc. Ten individuals seropositive for IgG Urine specimens were collected in pots, transferred to 6 anti-HBc and anti-HBs had urinary IgM anti-HBc T:N ml bottles with sealers, and posted to the Virus Refer- ratios from 0.7-3.6 and the median ratio was 1.8. Of the ence Laboratory for storage either at 4°C or - 70°C until 42 subjects who were HBsAg carriers, 11 had urinary tested. IgM anti-HBc T:N ratios greater than 3.0 (range for the 11 was 3.7-41.4 median 11.9). That range overlapped Urine Assays with the range of anti-HBc values in urine specimens Urinary IgG and IgM anti-HAV and anti-HBc were from recent cases of acute hepatitis B. Urine specimens from patients with clinical and serodetected by virus-specific IgG and IgM antibody capture radiommunoassay (GACRIA and MACRIA). The logical evidence of recent HAV and HBV infection were same concentrations of anti-y and anti-p for solid phase also tested by GACRIA and MACRIA using the heterolcoating and of antigen and radiolabel and the same ogous antigen and label. Urine specimens from the 23 diluents were used a s described previously for saliva individuals tested who had recently had hepatitis B did tests. Briefly, polystyrene beads (6.5mm diameter code not react by the HAV MACRIA even though 7 were IgG no P201, Northumbria Biologicals) were coated by gen- anti-HAV positive (Table I). Urine specimens from the tle shaking in a dilution of rabbit antibody to human 31 individuals tested who recently had hepatitis A did IgG (y-chain specific, code A424, Dakopatts, Ltd., 11.4 not react for IgG and IgM anti-HBc (Table 11). mg/l) or rabbit antibody to human IgM (p-chain speSensitivity for Urinary Anti-HAV cific, code A426, Dakopatts Ltd., 3.1 mgil) in 0.05 M carbonateibicarbonate buffer, pH 9.6. The beads, after IgG anti-HAV. All individuals whose saliva conwashing, were added to 200 ~1 of untreated urine sam- tained IgG but not IgM anti-HAV were reactive for ples in reaction trays. urinary IgG anti-HAV (Table I). With one exception I\’
Urinary Diagnosis of Viral Hepatitis
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TABLE I. Reactivities of Urine Specimens for IgM and IgG Anti-HAV Patient cateeorv Recent HAVb Past HAV' Susceptible" Recent HBVd
No. 118 48 23 1 23
IgM anti-HAV Range Median T:Na T:N 2.7-126.0 0.2-5.4 0.34.3 0.7-2.3
28.9 1.3 1.1 1.3
IgG anti-HAV Range Median T:N" T:N 2.7-113.0 4.3-87.8 0.3-2.1 0.9-43.0
21.7 13.5 1.0 1.3
"T:N values are ratios of the test counts divided by the mean count of four negative controls. "One hundred eighteen individuals with recent hepatitis A. Of these, 70 were serologically confirmed convalescent cases and 48 were from hepatitis A outbreaks and identified on the basis of salivary tests. 'Individuals from two hepatitis A outbreaks identified as susceptible or immune to hepatitis A infection based on tests on simultaneously collected saliva specimens. dSerologically confirmed cases of recent hepatitis B.
TABLE 11. Reactivities of Urine Specimens for IgM and IgG Anti-HBc Patient CatePorv Recent HBVb Carrierb Past H B V ~ Seronegative" Recent HAVd
No. 35 42 10 46 31
IgM Anti-HBc Range Median T:N T:N" 3.6-76.1 0.441.4 0.7-3.6 0.8-2 .O 0.6-1.8
32.2 2.1 1.8 1.3 1.1
IgG Anti-HBc Range Median T:N T:N" 8.5-115.0 1.2-91.0 1.6-17.7 0.2-1.3 0.6-1.6
56.3 30.6 6.5 0.9 1.0
"T:N values are ratios of the test counts divided by the mean count of four negative controls. I'Urine specimens were collected from individuals serologically proven to have had previous or recent infection with hepatitis B. 'Urine specimens were collected from individuals serologically proven to have had no evidence of infection-with hepatitis B. dUrine specimens were collected from individuals serologically proven to have had recent hepatitis A infection.
those with evidence of recent HAV infection based on the presence of IgM anti-HAV in saliva had strong IgG anti-HAV reactivity in urine. The exception had a urinary T:N ratio of 2.7 (urinary IgM anti-HAV 27.0) and on the basis of salivary results (IgG anti-HAV 3.2; IgM anti-HAV 54.4) was a n early acute case with still very weak IgG anti-HAV reactions. Two other saliva samples which gave IgM anti-HAV results compatible with acute HAV did not react by the IgG anti-HAV assay, while urine samples collected a t the same time were weakly IgG reactive (Saliva GACRIA T:N: 1.4, 1.8; MACRIA T:N 44.9, 14.5. Urine: GACRIA 4.8,3.9; MACRIA: 55.8,8.1). The distribution of urinary IgG anti-HAV reactivities is shown in Figure l a . IgM anti-HAV. Hepatitis A outbreaks usually include patients with jaundice, with anicteric illness and asymptomatic infection. In order to assess the accuracy of the urinary assay, specimens were examined both from serologically confirmed clinical hepatitis A and from individuals in whom the diagnosis of recent HAV infection was based on IgM anti-HAV in saliva. Of the urine specimens collected within 8 weeks of the onset of jaundice from 70 serologically confirmed cases of HAV infection all but 3 gave strong IgM antiHAV reactions (range 5.9-126.0, median 27.9). The 3 weakly reactive samples, which were collected 40, 47, and 29 days after the onset of jaundice, the T:N ratios were 3.0,2.7, and 4.7, respectively.
Most of the urine specimens collected from individuals with salivary evidence of recent HAV infection had high IgM anti-HAV T:N ratios (median 30.3) although this was not as high a s found in corresponding saliva specimens (median 61.7). Urine specimens from 2 ofthe 48 subjects with salivary evidence of recent HAV infection had IgM anti-HAV T:N ratios less than 5.0 (4.1and 3.9, corresponding salivary ratios values, 5.2 and 17.3). The distribution of urinary IgM anti-HAV reactivities is shown in Figure l b . Comparison between urinary and salivary IgG and IgM anti-HAV reactivities. IgG anti-HAV T:N ratios for paired urine and saliva specimens were plotted against each other for 96 individuals immune to or with recent hepatitis A (ranges: urine, 2.7-87.8; saliva, 1.4-103.0). Sixty-one of the 96 individuals had IgG anti-HAV urinary reactivities which were weaker than for saliva. IgM anti-HAV T:N ratios were similarly plotted for the 48 recently infected individuals from whom paired specimens were available (ranges: urine, 3.9-121.0; saliva, 5.2-131.0). Thirty-seven of 48 urine specimens from cases of recent hepatitis A gave weaker IgM anti-HAV reactions than corresponding saliva specimens. Correlation coefficients (r)were 0.86 for IgG anti-HAV and 0.72 for IgM anti-HAV (Fig. 2).
Sensitivity for U r i n a r y Anti-HBc All 35 patients with recent acute hepatitis B infection (within 8 weeks of onset) had positive urinary IgG
Perry et al.
268 a7 78
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92 98
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20
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0 25
40 >50
IgM anti-HAV Test:negative (T:N) ratio
Fig. 1. The distributirin of (a) IgG anti-HAV and (b) IgM anti-HAV urinary reactivities for 231 susceptible individuals \ El ), 48 with previous HAV infection W, and 118 with recent HAV infection (0).
and IgM anti-HBc reactions. There was no overlap with the reactions of seronegative individuals (Table 11).Of 10 individuals known to be seropositive for anti-HBc and anti-HBs, 8 had detectable IgG anti-HBc in the urine: in the other 2 the urine HBc GACRIA reactions were T:N 1.6 and 1.9. IgG anti-HBc was detected in the
urine specimens of 41 (T:N range 3.6-91.0, median 31.6) of 42 HBsAg carriers.
DISCUSSION In this study the GACRIA cut-off T:N ratio for IgG anti-HAV urine samples was set at 3.0, giving a sensi-
269
Urinary Diagnosis of Viral Hepatitis 140-
0
120-
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100-
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120
140
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for 96 individuals with previFig. 2. IgG anti-HAV reactivities ous or recent HAV infection, and IgM anti-HAV reactivities ( 0 )for 48 individuals with recent HAV infection measured in simultaneously collected urine and saliva specimens.
TABLE 111. Concordance Between Salivary and Urinary IgM and IgG Anti-HAV Results IgM Anti-HAV
Saliva Positive Negative Positive Urine Negative
46 2b
la 278
IgG Anti-HAV Saliva Positive Negative 93 1"
2' 231
"Salivary findings for the discrepancies indicated that there was previous HAV infection. bSalivary findings for the discrepancies indicated that there was recent HAV infection. 'Salivary findings for the discrepancies indicated that there was early acute HAV infection.
tivity of 98.9% and a specificity of 99.1% by reference to results by the salivary GACRIA (Table 111). Excluding the early acute cases, where IgM anti-HAV may be found in the absence of IgG anti-HAV, the sensitivity was 100%.The urinary IgM anti-HAV cut-off was set a s 5.0 and this gave a sensitivity and specificity of 95.8% and 99.6%, respectively, with reference to salivary results. Although a good correlation was found for both IgG and IgM anti-HAV reactivities between saliva and urine specimens collected a t the same time from individuals who were susceptible to, immune to, or recently infected with HAV, urine generally gave weaker reactions than saliva. However, in the majority of cases of recent HAV infection (39/48) urinary IgM anti-HAV T:N ratios were >20.0. Urine specimens which were
collected between 3 days before and 8 weeks after the onset of jaundice from 70 serologically confirmed cases of hepatitis A were all positive for IgG anti-HAV, and 67/70 had IgM anti-HAV reactions that were significant diagnostically. The findings indicate that urine specimens which give IgM anti-HAV T:N ratios between 3.0 and 5.0 should be interpreted with caution. If a specimen gives rise repeatedly to weak IgM anti-HAV reactivity we suggest testing a follow-up urine andlor a saliva or serum specimen. In most instances such reactions arise from individuals who have not been infected recently. IgG and IgM anti-HBc were readily detected in HBV infection in all urine specimens collected between 2 and 8 weeks after onset of jaundice. Urine specimens collected from anti-HBc seronegative subjects did not react. However, some lack of sensitivity was encountered: IgG anti-HBc was not detected in 2 of 10 immune individuals and was not present in 1 of 42 HBsAg carriers. Interpretation of both IgM anti-HAV and IgM antiHBc reactivity can be difficult. Specific IgM appears before specific IgG and often before the onset of jaundice. It can persist in s e r u d p l a s m a a t low levels for at least 6-9 months or, in the case of HBV carriers, indefinitely. Consequently, weak IgM reactivity early in the course of infection is significant, but it can be diagnostically confusing later. IgM anti-HBc was detected in the urine of 11/42 carriers a t levels as high as those found in patients with a recent infection. Smith et al. [1992] reported that in sera from HBV carriers, 14.3% had IgM anti-HBc levels in the range found for acute hepatitis B. A good correlation was also shown between serum IgM anti-HBc levels in carriers and the cytoplasmic expression of HBcAg in liver tissue. Hence, it seems likely that the urinary IgM anti-HBc results are specific even though not associated with primary infection. It may be helpful in these instances to relate IgM to IgG reactivity to assist the interpretation of results [Parry, e t al. 19891. Although the urinary results, particularly for hepatitis A, are closely concordant with those obtained in saliva and serum, there are factors which need further investigation, e.g., urine storage conditions, the possible effect of urinary pH and bacterial contamination, and variability due to changes in fluid intake. Urine specimens were stored in aliquots at room temperature, 4"C, -25"C, and -7O"C, and i t appears from that study (unpublished) that storage at - 25°C is partially detrimental. Positive samples may eventually become negative, especially for virus-specific IgM. Our current practice is to store urine specimens for up to 6 weeks a t 4°C or a t -70°C if longer term storage is required. Because of the apparent lability of immunoglobulins in urine it might, for clinical use, be necessary to test all specimens for total immunoglobulin levels and set a cut-off level below which the specimen would be considered inadequate for testing. However, this precaution is probably unnecessary when these assays are used for epidemiological studies.
Perry et al.
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The prospects for urinary anti-HBc detection are not as clear as for anti-HAV detection. We have identified problems of specificity for IgM anti-HBc and of sensitivity for IgG anti-HBc detection. Although high levels of IgM anti-HBc were detected readily in the urine of nearly all acute cases of HBV, some HBsAg carriers possessed similar levels of urinary IgM anti-HBc. In addition, IgG anti-HBc could not be detected in the urine of some individuals who were immune a s a result of previous infection. It is concluded that urinary antiHBc testing is of less value than urinary anti-HAV testing, although it might still have epidemiological applications. For patients and study subjects who dislike venepuncture and find difficulty in providing a n adequate volume of saliva, urine would be the preferred specimen for diagnosis of hepatitis. In hospitals and general practices, urine specimens are easy to obtain and therefore readily available for clinical and epidemiological studies. In the laboratory, urine specimens have the technical advantage that, unlike blood specimens, preliminary sample preparation or dilution are not needed. I t should not be assumed that because immunoglobulin levels are low, urine specimens are unsatisfactory for antibody based viral diagnosis.
ACKNOWLEDGMENTS We wish t o thank Wellcome Diagnostics for the gift of HBcAg and partial funding, Miss Tracey Carter for typing the manuscript, and Mr. Guy Gabriel for preparing the radiolabels.
REFERENCES Bull AR, Kimmance KJ, Parry JV, Perry KR (1989):Investigation of an outbreak of hepatitis A simplified by salivary antibody testing. Epidemiology and Infection 103:371-376. Cao Y, Hosein B, Borokowsky W, Mirabile M, Baker L, Baldwin DF, Poiesz BJ, Friedman-Kien AE (1989): Antibodies to human immunodeficiency virus type 1 in the urine of HIV-1 seropositive individuals. AIDS Research and Human Retroviruses 5:311-319. Connell JA, Parry JV, Mortimer PP, Duncan RJS, McLean KA, Johnson AM, Hambling MH, Barbara J, Farrington CP (1990): Accurate assays for anti-HIV in urine. Lancet 335:136&1369. Hand WL, Smith JW, Miller TE, Barnett JA, Sandford J P (1970): Immunoglobulin synthesis in lower urinary tract infection. Journal of Laboratory Clinical Medicine 75:19-29. Lehmann JD, Smith J W , Miller TE, Barnett JA, Sandford JP (1968): Local immunoresponse in experimental pyelonephritis. Journal of Clinical Investigation 472541-2550. Lerner AM, Remington JS, Finland M (1962): Neutralizing antibody to polioviruses in normal human urine. Journal of Clinical Investigation 4123055815. Parry J V (1981): Hepatitis A infection: guidance for development of satisfactory assays for laboratory diagnosis. Medical Laboratory Sciences 38:303-311. Parry J V (1984):Diagnosis of hepatitis A infection: comparative specificity of IgM capture assays using antigens derived from tissue cultures and marmoset faeces. Journal of Virological Methods 9:35-44. Parry JV, Farrington CP, Perry KR, Waight P, Mortimer PP, Miller E (1988):Rational programme for screening travellers for antibodies to hepatitis virus. Lancet i:1447-1449. Parry JV, Perry KR, Mortimer P P (1987):Sensitive assays for viral antibodies in saliva; an alternative to tests on serum. Lancet ii:7275. Parry JV, Perry KR, Panday S, Mortimer P P (1989): Diagnosis of hepatitis A and B by testing saliva. Journal of Medical Virology 28:255-260. Smith HM, Lau JYN, Davies SE, Daniels HM, Alexander GJM, Williams R (1992):Significance of serum IgM anti-HBc in chronic hepatitis B virus infection. Journal of Medical Virology 36:16-20.
