Multiple primer pairs for polymerase chain reaction (PCR) amplification of human parvovirus B19 DNA.

Human parvovirus B19 is the etiologic agent of erythema infectiosum and transient aplastic crisis in patients with hemolytic anemias and has been associated with fetal death, arthritis, and chronic anemia. Acute B19 infection is best diagnosed by detection of IgM antibodies, whereas the diagnosis of chronic infection often requires the sensitivity of PCR to demonstrate presence of virus over time. To improve our ability to detect B19 DNA by polymerase chain reaction (PCR), we evaluated 19 primers combined into 16 different primer pairs for their ability to detect temporally and geographically diverse B19 isolates. All 16 pairs reacted with all isolates tested but with different sensitivity. Sequence analysis showed few nucleotide changes compared with published sequences. These changes did not explain observed differences in sensitivity between primer pairs. The most sensitive primer pairs detected 350 to 3500 DNA copies after 35 cycles. A second amplification cycle with nested primers improved the sensitivity 100-fold. These 16 primer pairs provide the diagnostic virologist with multiple options for B19 PCR assays.

Characterization and seroepidemiology of a type 5 astrovirus associated with an outbreak of gastroenteritis in Marin County, California.

The Marin County strain of type 5 astrovirus was associated with two separate outbreaks of nonbacterial gastroenteritis in California in 1978. A safety-tested, bacterium-free filtrate prepared from a stool specimen of an individual who was ill during the original outbreak was given orally to 19 adult volunteers. One volunteer developed a gastrointestinal illness, and nine had serologic responses. Several diarrheal stool specimens from the ill volunteer contained a large number of 27-nm particles. By using immune electron microscopy with acute- and convalescent-phase sera from the original outbreak, these 27-nm particles were shown to be identical to the viral inoculum. The Marin County virus, purified from the stool of the ill volunteer, was shown by immunoprecipitation and polyacrylamide gel electrophoresis to contain a single structural protein with a molecular weight of 30,000. The buoyant density of the virion was 1.39 g/cm3 in cesium chloride. By electron microscopy, approximately 5% of the particles had the characteristic stellate configuration of astrovirus, and serologic studies by immunofluorescence technique confirmed previous classification of the Marin County virus as a type 5 astrovirus. Radioimmunoassay and biotin-avidin immunoassay were used to detect antibody to the Marin County virus in paired acute- and convalescent-phase sera from 32 outbreaks of nonbacterial gastroenteritis, but none of these outbreaks could be attributed to this virus. Prevalence of antibody to this strain of astrovirus was approximately 13% in children 6 months to 3 years of age and increased to 41% in older children and young adults.

Detection of human parvovirus B19 in early spontaneous abortuses using serology, histology, electron microscopy, in situ hybridization, and the polymerase chain reaction.

OBJECTIVE: To determine whether there is an association between parvovirus B19 infection and early spontaneous abortion at less than 20 weeks' gestation. METHODS: Eighty samples of early spontaneous abortions were analyzed. Each sample was examined histologically for the presence of viral inclusions, and selected cases were analyzed for parvovirus using electron microscopy and in situ hybridization. Polymerase chain reaction DNA amplification for the virus was done in each case. Maternal sera were analyzed for immunoglobulin (Ig) M and IgG parvovirus antibodies and compared with temporally matched controls. RESULTS: Five cases in the study group had evidence of seroconversion for parvovirus, compared with two controls. Products of conception from two of these five cases were positive for virus by polymerase chain reaction amplification, and only one of these two had a characteristic inclusion of parvovirus histologically. Conversely, five chorionic vesicles from mothers who had not seroconverted had histologic changes suggesting parvovirus infection, but all of these cases were negative for parvovirus using in situ hybridization, polymerase chain reaction, and electron microscopy. CONCLUSIONS: Parvovirus B19 DNA was found in two of 80 early spontaneous abortuses. Although viral DNA was detected in two cases, there was no clear evidence that the infections caused fetal death. Neither case showed erythroblastosis with large numbers of inclusions, as is seen in hydropic fetuses with parvovirus infection. In addition, in five cases in which parvovirus infection was not documented serologically or by the polymerase chain reaction, there was erythroid nuclear clearing suggestive of parvovirus B19 inclusions. This indicates that histologic evaluation for parvoviral inclusions is not always reliable in early spontaneous abortuses.

Chronic parvovirus infection in a presumably immunologically healthy woman.

Infection due to parvovirus B19 is common and usually resolves over several weeks. Prolonged infection has been reported primarily in immunodeficient hosts. The present report describes a chronic infection in an apparently immunologically healthy woman. The illness was characterized by recurrent episodes of paresthesia without anemia. Laboratory studies demonstrated persistence of parvovirus-specific DNA for nearly 4 years.

Prenatal diagnosis of intrauterine infection with parvovirus B19 by the polymerase chain reaction technique.

Human parvovirus B19 is a recently recognized cause of fetal hydrops and death. Efforts to characterize the natural history of fetal infection with this virus have been hampered by the lack of sensitive and specific tests for diagnosis in utero. Using the highly sensitive polymerase chain reaction (PCR) assay, we determined the fetal infection status in 56 pregnancies by testing amniotic fluid, fetal serum, and maternal serum for B19 DNA and antibodies. Factors associated with a high risk of B19 infection were fetal disease, exposure to persons with erythema infectiosum, or signs or symptoms of acute B19 infection. Fifteen women (27%) were B19 IgM-positive, a status suggesting recent infection; the positivity of all of the corresponding fetal specimens for B19 DNA in the PCR was indicative of fetal infection. In four of these cases, serial ultrasonographic examinations documented spontaneous resolution of fetal hydrops. Twenty-four women (43%) were IgG-positive and IgM-negative; this pattern suggested prior infection. The PCR gave positive results, consistent with recent maternal infection, in four of these cases. Seventeen women (30%) were IgG-negative and IgM-negative, a pattern suggesting no prior infection; the PCR results in four cases were indicative of a possible early maternal infection or a possible atypical immune response. The PCR is a sensitive and rapid method for the diagnosis of intrauterine infection with human parvovirus B19 and promises to facilitate studies of the natural history and treatment of this infection.

Lupus-like presentation of human parvovirus B19 infection.

The diagnosis of systemic lupus erythematosus (SLE) was a leading initial consideration in 2 patients with rash, arthritis and hypocomplementemia. One patient also had leukopenia and thrombocytopenia. Spontaneous regression occurred. In both patients antinuclear antibodies were negative. Serologic studies indicated recent human parvovirus B19 infection. We propose adding human parvovirus B19 infection to the list of conditions that may masquerade as SLE.

Human parvovirus B19 specific IgG, IgA, and IgM antibodies and DNA in serum specimens from persons with erythema infectiosum.

To determine the diagnostic use of different markers of acute parvovirus B19 infection, serum specimens obtained from 128 persons with erythema infectiosum were tested for specific immunoglobulin G (IgG), IgA, and IgM antibodies by capture enzyme immunoassay (EIA) using Chinese hamster ovary (CHO) cell-expressed B19 antigen, and tested for circulating B19 DNA by polymerase chain reaction (PCR). A significant rise in specific IgG and IgA antibodies was detected in 87% and 77%, respectively, of persons from whom acute- and convalescent-phase serum specimens were available. Specific IgA antibodies were detected in single serum specimens from 90% of cases and were present in 22 (18%) of 120 persons from a control group without a history of recent exposure to B19. Specific IgM antibodies were detected in 97% of cases and one person (1%) from the control group. B19 DNA was detected in 94% of cases and was absent in 20 persons from the control group positive for both IgG and IgA antibodies. Serum specimens obtained between 4 and 6 months after onset of illness from six additional persons were also tested. All had specific IgG antibodies, four (67%) had IgA, five (83%) had IgM, and none had detectable B19 DNA. Our data indicate that 1) specific IgA antibodies are too persistent to be a useful indicator of recent B19 infection; 2) specific IgM antibodies are the most sensitive indicator of acute B19 infection in immunologically normal persons but can persist up to 6 months; and 3) B19 DNA can often be detected up to 2 months after onset of illness even in immunologically normal hosts and might be a useful adjunct test for diagnosis of acute B19 infection.

Occupational risk factors for infection with parvovirus B19 among pregnant women.

To identify exposures associated with parvovirus B19 infection during pregnancy, two groups of pregnant women were studied during an outbreak of erythema infectiosum (EI). Of 796 pregnant women from Connecticut who were tested serologically because of perceived exposure to B19, 53% (419/796) had serologic evidence of previous B19 infection, and 6% (23/376) of the rest had evidence of recent infection. Of 121 pregnant women who had not requested testing but who lived in a community where a large outbreak of EI had occurred among schoolchildren, 36% (43/121) had serologic evidence of previous infection, and only 3% (2/78) of the rest had had a recent infection. In the exposed group, 479 women returned a supplemental exposure questionnaire. The highest infection rates among susceptible women were for schoolteachers (16%, 10/64), followed by day care workers (9%, 2/22) and homemakers (9%, 4/46). Women working outside the home but not in school or day care settings had the lowest risk (4%, 3/80). This study suggests that there is risk for B19 infection in selected occupational settings and in households.

Management and outcomes of pregnancies complicated by human B19 parvovirus infection: a prospective study.

During a large statewide outbreak of fifth disease in Connecticut in 1988, 39 pregnant women were identified who had serologic evidence of recent human B19 parvovirus infection. The patients were followed up prospectively with targeted fetal ultrasonographic examinations to detect signs of fetal hydrops. Of these 39 pregnant women, 37 had healthy infants and two patients had miscarriages. None of the fetuses developed hydrops. We propose that pregnant women exposed to B19 parvovirus be tested for evidence of IgG and IgM B19-specific antibodies and that targeted fetal ultrasonography be considered when IgM antibodies are found. Percutaneous umbilical blood sampling and intrauterine transfusion can be considered in cases of B19 parvovirus-associated hydrops and anemia. The overall fetal loss rate in this prospective follow-up group was 5%.

Risk of infection following exposures to human parvovirus B19.

In response to concern about the effect of human parvovirus B19 infection of the fetus, we have developed estimates of the risk of adults becoming infected following B19 exposures at home, in schools or day-care centers, and in hospitals. These estimates can then be used with other data to estimate the risk to the fetus of a B19 exposure during pregnancy. The risk to the fetus equals the rate of maternal susceptibility to infection times the rate of maternal infection following the specific type of exposure times the rate of fetal death following maternal infection. Data from studies of outbreaks of B19 associated erythema infectiosum and aplastic crisis suggest that the risk of infection among susceptible adults following household exposure to a B19 infected person is approximately 50% and following school exposures during outbreaks of erythema infectiosum is 20% to 30%. All susceptible school staff members, not just teachers, appear to be at risk for infection during outbreaks. Additional study is needed to determine the risk of infection following exposure to B19 infected patients in the hospital. Based on these and other data we can estimate that pregnant women whose serologic status is unknown have less than 2.5% chance of suffering fetal loss after household exposure and less than 1.5% chance after school exposure.

Occupational risk of human parvovirus B19 infection for school and day-care personnel during an outbreak of erythema infectiosum.

Human parvovirus B19, the cause of erythema infectiosum, has recently been associated with adverse fetal outcomes. During a large outbreak of erythema infectiosum in Connecticut, a survey was conducted on 571 (90%) of 634 school and day-care personnel to determine the risk of acquiring B19 infection. Serologic evidence of B19 infection was determined by using an enzyme-linked immunosorbent assay. Of the school and day-care personnel, 58% had evidence of previous B19 infection. The minimal rate of B19 infection in susceptible personnel during the outbreak was 19%. The risk was increased for teachers and day-care providers who had contact with younger children and with greater numbers of ill children. These results suggest that B19 infection is an occupational risk for school and day-care personnel.

Serum immunoglobulin A response to Norwalk virus infection.

We describe the serum immunoglobulin A (IgA) antibody response to Norwalk virus infection in human volunteers and compare it with previously described IgM and total antibody responses. Whereas specific IgA and IgM peak within 2 weeks after onset of symptoms, titers of total blocking antibody continue to rise, implying mediation by IgG antibody.

Development and evaluation of an IgM capture enzyme immunoassay for diagnosis of recent Norwalk virus infection.

A capture enzyme immunoassay specific for Norwalk IgM class antibody was developed. The assay was moderately sensitive, identifying 33/53 (62%) of patients with naturally acquired Norwalk virus infection and 17/18 (94%) of experimentally infected volunteers. The assay was also specific for IgM class antibody and acute Norwalk virus infection and results were generally reproducible. A specific IgM response correlated with seroconversion by total antibody blocking assay and occurred independently of clinical symptoms. Among 81 symptomatic cases composing seven Norwalk outbreaks, specific IgM was absent from acute phase sera collected less than or equal to three days post onset, and was uncommon in sera collected within one week and after five weeks, with an optimal collection time at about two to three weeks. The Norwalk IgM capture immunoassay may be used to augment paired sera assays in the identification of Norwalk-associated outbreaks of acute gastroenteritis.

An outbreak of acute infectious nonbacterial gastroenteritis in a high school in Maryland.

An outbreak of acute infectious nonbacterial gastroenteritis (AING) occurred in a high school in Maryland in 1984. Thirty-six percent of students surveyed met the case definition of gastroenteritis, as did 24 percent of school employees. Eating lunch in the cafeteria on January 30 was significantly associated with illness. After controlling for other food items consumed during the January 30 lunch, only the sandwiches were significantly associated with illness, but the source of the contamination was not identified. Four of 17 serum pairs from sick students and none of the 8 serum pairs from exposed controls (a nonsignificant difference) showed at least a 4-fold rise in antibody titre to Norwalk virus between acute- and convalescent-phase specimens. This outbreak of AING is believed to be the first to implicate epidemiologically sandwiches as vehicles of transmission. The outbreak highlights the need for investigators to look for a viral etiology in gastroenteritis outbreaks.

Severe anemia caused by human parvovirus in a leukemia patient on maintenance chemotherapy.

A 6-year-old boy on maintenance chemotherapy for acute lymphocytic leukemia developed severe hypoplastic anemia during chemotherapy previously well tolerated. The hypoplastic episode persisted for approximately 30 days. Human parvovirus (B19), the etiologic agent of aplastic crisis in persons with underlying hemolytic syndromes, was detected in the patient's serum 25-30 days after onset of hemoglobin decrease, and B19 IgM seroconversion occurred 1 week later. The patient's hypoplastic anemia was presumably caused by prolonged B19 infection resulting from a blunted immune response. An immune response to the B19 infection and resolution of the illness were temporally associated with brief cessation of chemotherapy.

Risk factors for secondary transmission in households after a common-source outbreak of Norwalk gastroenteritis.

In November 1984, a foodborne outbreak of Norwalk gastroenteritis occurred in a K-12 public school in northern Vermont. The outbreak offered an opportunity to systematically study in detail secondary transmission rates in households. Eating salad at Tuesday's school-sponsored Thanksgiving Banquet was associated with illness among students and staff members (p less than 0.025). Seven of 11 serum pairs from ill persons showed a fourfold or greater rise in antibody titer to Norwalk virus compared with one of nine controls (p = 0.028). The study of secondary household transmission revealed that households with persons with primary illness were 5.5 times more likely to experience secondary illness than households with well school children or adults. As the number of individuals with primary illness in the household increased, the secondary illness rates increased. Pre-school children were twice as likely as adults to develop secondary illness.

Norwalk virus antigen and antibody response in an adult volunteer study.

To better define the optimum timing of specimen collection and identify alternate ways to diagnose Norwalk virus outbreaks, we looked at the timing of the antibody response and virus excretion in a human volunteer study. The Norwalk virus antibody titers and antigen in stool specimens were examined by biotin-avidin immunoassay. Our data suggest that in epidemic situations, convalescent-phase sera could be collected as soon as 13 days after the onset of illness and acute-phase sera could be collected as late as 5 days after onset. Our data also suggest that if sufficient serum samples are collected, convalescent-phase case and control serum samples can be used to identify Norwalk virus outbreaks. Antigen detection was much less sensitive than seroconversion for detecting infection.

Detection of antibody to group B adult diarrhea rotaviruses in humans.

Group B rotaviruses have been responsible for annual epidemics of severe diarrhea affecting both adults and children in China. We developed a specific and sensitive enzyme-linked immunosorbent blocking assay to detect antibody to group B rotaviruses that will be useful to assess the role of group B rotavirus infections as a cause of human gastroenteritis. We tested 219 human sera and 18 immunoglobulin pools collected from eight countries for antibodies to both group A and group B rotaviruses. Overall, a low proportion (10 of 237 or 4.2%) of sera contained antibody to group B rotaviruses. Antibody to group B rotavirus was detected in only 1 of 155 serum samples from healthy or hospitalized individuals in the United States, including patients with the chronic inflammatory bowel diseases Crohn's disease and ulcerative colitis. No antibody was detected in 15 serum samples from Australia and from an outbreak of gastroenteritis on a cruise ship or in nine immunoglobulin pools from Japan and the United Kingdom. Antibody to group B rotaviruses was detected in 8 convalescent-(but not acute-)phase serum samples from Chinese patients with group B gastroenteritis, in five immunoglobulin pools from China, in 1 of 6 serum samples from Chinese students in the United States, and in 1 each of 10 serum samples from Kenya, 20 from Thailand, and 15 from Canada. In contrast, most of these samples (226 of 237 or 95.4%) had antibody to group A rotaviruses. These results indicate that human infection with group B rotavirus has not been widespread in areas outside China. Seroconversion observed between the acute-and convalescent-phase serum samples from China also suggests that infections with this virus are primary infections. Continued surveillance for this new group of rotaviruses should determine whether the many susceptible people become infected of whether other factors influence the severe pathogenicity of human infections with these viruses in China.