Diagnosis of Parvovirus B19 Infection by Detection of Specific ...

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1996, p. 205–207 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 34, No. 1

Diagnosis of Parvovirus B19 Infection by Detection of Specific Immunoglobulin M Antibody in Saliva RITA C. N. CUBEL,1,2* SOLANGE A. OLIVEIRA,2 DAVID W. G. BROWN,3 BERNARD J. COHEN,3 AND JUSSARA P. NASCIMENTO1 Departamento de Virologia, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro,1 and Departamento de Microbiologia e Parasitologia and Departamento de Doenças Infecciosas e Parasita ´rias, CCM, Universidade Federal Fluminense, Nitero í,2 Rio de Janeiro, Brazil, and Virus Reference Division, Central Public Health Laboratory, London, United Kingdom3 Received 10 August 1995/Returned for modification 7 September 1995/Accepted 13 October 1995

Serum and saliva samples were simultaneously collected from patients with B19 infection. Specimens were collected in a period of 1 to 18 days after the onset of symptoms. Saliva samples were collected with a commercial device, OraSure. The quality of these samples was evaluated by determining the concentration of total immunoglobulin G (IgG) by an enzyme immunoassay. The concentration of IgG in these samples ranged from 4.8 to >250 mg/liter. B19 infection was confirmed for 20 patients by testing sera in a 1:100 dilution by an IgM capture enzyme immunoassay (MACEIA) and an IgM capture hemadherence test (MACHAT). Saliva samples from these IgM-positive patients were tested neat by MACEIA and MACHAT. IgM could be detected in 11 of 20 (55%) samples by MACEIA and in 15 of 18 (83%) samples by MACHAT. Serum and saliva samples from a further 17 patients with rash were also tested. All of these specimens were unreactive by both assays. These results show that saliva may be a convenient alternative to serum for the diagnosis of recent B19 infection. fulfillment of the requirements of a Ph.D. from Instituto de Microbiologia, Universidade Federal do Rio de Janeiro.) Blood and saliva samples were simultaneously collected from patients with suspected B19 infection in 1994 in Nitero í, Rio de Janeiro, Brazil. Saliva was collected with a commercial collection device, OraSure (Epitope, Beaverton, Oreg.), according to the manufacturer’s instructions. First, a small absorbent pad was placed between the patient’s cheek and gum and was rubbed back and forth several times. Then the pad was kept in place for 2 min. After this the vial containing the preservative was opened, the pad was inserted in the bottom of the vial, and the pad was broken by hand, snapping it against the side of the vial. Finally, the cap was replaced and the samples were kept refrigerated before being transported to the laboratory, where they were centrifuged. The fluids recovered from the absorbent pads were stored at 2208C until analyzed. In order to investigate the quality of the saliva samples collected, we determined the IgG concentrations in the samples by using an EIA as described by Azevedo Neto et al. (4) but with minor modifications. Microtiter plates (Immulon II; Dynatech, Chantilly, Va.) were coated with 75 ml of anti-human IgG (Dako, Glostrup, Denmark) diluted 1:1,000 in 0.05 M carbonate buffer, pH 9.6. After incubation for 18 h at 48C, the plates were washed three times with phosphate-buffered saline (PBS), pH 7.2, plus 0.05% Tween 20 (PBST). Fifty-microliter portions of saliva samples diluted 1:50 and 1:500 in PBST were added, and the plates were incubated for 2 h at 378C. After the plates were washed with PBST, 75 ml of peroxidase-conjugated goat anti-human IgG (Sigma) diluted 1:20,000 in PBST was added, and the wells were incubated for 30 min at 378C. Finally, the plates were washed five times with PBST, 100 ml of tetramethylbenzidine substrate solution was added to each well, and the mixtures were incubated for 15 min at room temperature. The reaction was stopped with 100 ml of 2 M H2SO4, and the optical densities at 450 nm were measured with a Titertek Multiscan Plus spectrophotometer. The IgG

Parvovirus B19, the only known human pathogenic parvovirus, is associated with a wide range of clinical symptoms. B19 is the etiologic agent of erythema infectiosum, an innocuous exanthem in children (3) which can cause acute arthritis in adults, particularly women (14). It also causes transient aplastic crisis in patients with chronic hemolytic anemia (12) and persistent anemia in immunocompromised patients (7). B19 has also been implicated as a cause of hydrops fetalis and fetal loss during pregnancy (5). The diagnosis of B19 infection largely depends on detection of antibody in serum. At present, the virus cannot be cultured in conventional cell lines for diagnostic purposes, and in most clinical presentations, e.g., erythema infectiosum and arthralgia, the virus cannot be detected after the onset of symptoms. Recent infections are mostly diagnosed by detection of specific immunoglobulin M (IgM) antibodies in patients’ sera. The techniques most used are radioimmunoassay and enzyme immunoassay (EIA), with virus antigen prepared directly from the plasma of viremic blood donors (1, 2). Recently, recombinant B19 capsids produced in insect cells have also been used as antigens for serological assays (15). The use of saliva as a noninvasive alternative to serum for detecting virus-specific antibodies was first described by Parry et al. (10). Subsequently, detection of virus-specific IgM in saliva by antibody capture radioimmunoassays was reported for patients with recent hepatitis A or B, measles, mumps, and rubella (11, 13). The present study was carried out to investigate whether saliva could be used for the diagnosis of recent parvovirus B19 infection. (This research was conducted by R. C. N. Cubel in partial

* Corresponding author. Mailing address: Departamento de Virologia, Instituto Oswaldo Cruz, FIOCRUZ, Avenida Brasil 4365, 21045900, Rio de Janeiro, RJ, Brazil. Phone: 00 55 21 598 4360. Fax: 00 55 21 270 6397. 205

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TABLE 1. Comparison of detection of B19-specific IgM by MACEIA and MACHAT in saliva samples collected from 20 B19-infected patientsa

TABLE 3. Duration of disease and detection of anti-B19 IgM in saliva from patients with or without recent B19 infection Detection of salivary anti-B19 IgM

MACEIA result MACHAT result

Positive Negative Noneb Total a b

Positive

Negative

Total

7 2 2 11

8 1 0 9

15 3 2 20

B19 diagnosis was confirmed by detection of IgM in patients’ sera. Samples were not tested.

concentrations in the saliva samples were estimated by comparing the optical density values of the saliva samples with the optical density values of an IgG control serum (5 mg/liter; Binding Site, Birmingham, United Kingdom) diluted to concentrations of 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, and 0.039 mg/liter. This allowed the salivary IgG concentrations to be expressed in milligrams per liter. In order to diagnose recent B19 infection, serum samples were tested for the presence of anti-B19 IgM antibodies by using an antibody capture EIA (MACEIA) and an antibody capture hemadherence test (MACHAT) as described before (6). B19 infection was confirmed for 20 patients. Specific IgM in patients’ sera diluted 1:100 was detected by both assays. The age range of individuals with recent B19 infection was from 2 to 36 years. For 15 of the patients, a rubella-like exanthem was reported; for 5 patients, the exanthem had the classic ‘‘slapped cheek’’ appearance. For 4 of the 20 patients, the main symptoms were exanthem and arthralgia, and for 1 patient, only arthralgia was reported. For control purposes, paired serum and saliva samples from 17 patients with rash but no evidence of recent B19 infection (anti-B19 IgM negative) were examined. The ages of the patients ranged from 4 months to 55 years. We tested saliva samples from the 20 patients with acute B19 infection for anti-B19 IgM. Undiluted saliva was tested by MACEIA and MACHAT. Anti-B19 IgM was detected in 11 of 20 samples by MACEIA and in 15 of 18 by MACHAT (Table 1). Two of the samples found to be IgM positive by MACEIA were not available for testing by MACHAT. The concentrations of total IgG in these specimens ranged from 4.8 to 120 mg/liter (mean, 51.04 mg/liter). No relationship between IgG concentration and positive results by IgM tests could be observed (Table 2). Saliva samples were collected from 1 to 18

TABLE 2. Comparison of total-IgG concentrations in saliva and detection of salivary anti-B19 IgM in patients with and without recent B19 infection Detection of salivary anti-B19 IgM Salivary IgG concn (mg/liter)a

#15 .15 to ,100 $100 a

Patients with recent B19 infectionb MACEIA

MACHAT

Patients with no evidence of recent B19 infectionc

2/4 7/14 2/2

4/4 10/12 1/2

3/3 10/10 4/4

Measured by enzyme-linked immunosorbent assay. Serum anti-B19 IgM positive. Each result is the number of positives per the number of samples tested. c Number of negatives per the number of samples tested. MACEIA and MACHAT results were identical. b

Duration of disease (days)

01–05 06–10 11–15 16–18 Not available

Patients with recent B19 infectiona MACEIA

MACHAT

Patients with no evidence of recent B19 infectionb

7/12 1/3 0/2 2/2 1/1

8/10 3/3 2/2 1/2 1/1

15/15 2/2 0/0 0/0 0/0

a Serum anti-B19 IgM positive. Each result is the number of positives per the number of samples tested. b Number of negatives per number of samples tested. MACEIA and MACHAT results were identical.

days after the onset of symptoms, and 55% of them were positive by MACEIA and 83% were positive by MACHAT (Table 3). Saliva samples collected from the 17 patients with rash not related to B19 infection were tested for anti-B19 IgM by MACEIA and MACHAT. All of them proved to be negative for anti-B19 IgM by both assays (Table 1). The total-IgG concentrations in saliva samples from these negative patients ranged from 7.8 to .250 mg/liter (mean, 70.8 mg/liter) (Table 2), the samples were collected from 1 to 6 days after the onset of the rash (Table 3). We have demonstrated that anti-B19 IgM can be detected in saliva from 55 to 83% of patients with acute B19 infection and that anti-B19 IgM is not present in the saliva of individuals without recent parvovirus B19 infection. For detection of anti-B19 IgM in saliva, MACHAT was more sensitive than MACEIA, which is in agreement with the results we obtained previously using MACHAT to detect anti-B19 IgM in sera (6). Up till now detection of IgM in saliva samples for virus diagnosis has mostly been performed by radioimmunoassay (11, 13). Probably we were able to detect anti-B19 IgM in saliva by a less sensitive EIA because we used a commercial device, OraSure, to collect saliva. The device contains substances that attract crevicular fluid. It is known that immunoglobulin concentrations in crevicular fluid are much higher than in salivary gland secretions, so it is important that crevicular fluid is well represented in any salivary samples collected for diagnosis (8). To determine the quality of specimen collection, which is an important factor in the detection of virus-specific antibodies in saliva, the concentration of total IgG in each saliva sample was measured. Samples containing IgG concentrations lower than 15 mg/liter are not considered adequate for diagnosis by antibody detection (8), but we were able to detect anti-B19 IgM in this kind of sample by both assays, probably because we were searching for IgM antibodies. The use of the total-IgG concentration for assessing the quality of samples has been questioned by others (4), even though levels around 0.5 mg/liter are acceptable for human immunodeficiency virus tests (9). For diagnosis of recent measles, mumps, and rubella infections by detection of virus-specific IgM in saliva, Perry et al. found that the optimal time for collection of samples was from 1 to 5 weeks after onset of illness (13). As 60% of our saliva samples were collected in the first 5 days after the onset of the rash, we may have missed some positive results. More specimens should be investigated to determine the optimal time of saliva collection for diagnosis of B19 infection.

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NOTES

The results from this study indicate that saliva specimens may be a convenient alternative to serum for diagnosis of recent B19 infection, especially during outbreaks, since saliva can be obtained from children much more readily than blood samples.

7.

8.

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