Increased Risk of Parvovirus B19 Infection in Young Adult Cancer Patients Receiving Multiple Courses of Chemotherapy

An increased human parvovirus B19 infection rate has been observedin immunocompromised hosts. In this study, we sought to determinethe prevalence of parvovirus B19 infection in adult cancer patientsreceiving multiple courses of systemic chemotherapy. From March1999 through April 2000, 59 men and 68 women, with a medianage of 49 (18 to 79) years, were enrolled in this study. Theyhad received an average of 7.1 (4 to 32) courses of systemicchemotherapy. The median duration from the date of startingchemotherapy to the date of blood sampling was 11 (4 to 88)months. Serum B19 immunoglobulin G (IgG) and IgM levels wereexamined by an enzyme-linked immunosorbent assay, and B19 DNAwas examined by a nested PCR. A group of 400 healthy blood donorsserved as the control group. The overall prevalences of anti-B19IgG in adult cancer patients and healthy blood donors were 61.4and 25.0%, respectively (P < 0.01). Anti-B19 IgM and B19DNA were not detectable in these anti-B19 IgG-seropositive individuals.A further age-stratified comparison revealed that only patientsyounger than 40 years had a significantly higher anti-B19 IgGseropositivity rate than the controls (19 of 39 versus 53 of310; P < 0.001). The increased prevalence of B19 infectionin these 39 adult patients younger than 40 years might be clinicallysignificant, since unexplained anemia, a common sequela of B19infection, was detected in 3 of 20 seronegative patients (15.0%)and in 12 of 19 seropositive patients (63.2%) (P < 0.005).The results of this study suggest that adult patients youngerthan 40 years and receiving multiple courses of systemic chemotherapymay have a significantly increased risk of B19 infection. Prospectivestudies to define the time course and clinical consequence ofB19 infection in this group of patients are needed.

INTRODUCTION

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Introduction

Human parvovirus B19, the only parvovirus known to be pathogenicfor humans, is a small DNA virus with a single-stranded lineargenome which encodes one nonstructural protein, NS-1, and twoviral capsid proteins, VP1 (83 kDa) and VP2 (58 kDa) (23). Thevirus exhibits a remarkable tropism for erythroid progenitorcells (6) and is frequently associated with anemia. ParvovirusB19 infection has also been implicated in a wide range of clinicalmanifestations, the outcome of which depends heavily on thephysiologic status of the individual and the immune responseagainst the virus (29).

In immunologically healthy hosts, B19 may cause a number ofacute, generally self-limiting diseases, notably, fifth diseaseor erythema infectiosum in children, acute polyarthritis inadults, and aplastic crisis in patients with chronic hemolyticanemia such as sickle cell anemia or hereditary spherocytosis(3, 15, 24, 26). In pregnant women, B19 infection may resultin the lysis of nucleated fetal red cells, hydrops fetalis,and subsequent spontaneous abortion and fetal death (16). B19also has been found to be associated with glomerulonephritis,vasculitis, peripheral neuropathies, myocarditis, and fulminanthepatic failure (31). In immunocompromised hosts, B19 infectionmay persist and lead to chronic anemia, red cell aplasia and,less frequently, thrombocytopenia, neutropenia, and pancytopenia(1, 9, 19). In this study, we sought to clarify whether B19infection poses a significant problem in cancer patients receivingmultiple courses of chemotherapy.

MATERIALS AND METHODS

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Materials and Methods

Patients. Serum samples were collected from cancer patients who had previouslyreceived more than four courses of systemic chemotherapy atNational Taiwan University Hospital. Serum samples from 400healthy blood donors, randomly selected from a national bloodbank, served as controls. The medical records of these patientswere carefully reviewed. Pertinent clinicopathologic featureswere correlated with the serologic markers of B19 infection.

Serologic examinations. All the serum samples had been stored at -20°C until testedfor parvovirus B19 immunoglobulin G (IgG) antibody, IgM antibody,and DNA. For testing the prevalence of B19 infection in cancerpatients, a parvovirus B19 IgG and IgM enzyme-linked immunosorbentassay (Biotrin International, Dublin, Ireland) was used accordingto the manufacturer's instructions. This enzyme-linked immunosorbentassay is a µ-capture sandwich enzyme immunoassay, andthe antigen used is the purified parvovirus B19 recombinantVP2 protein. Following a wash step, peroxidase-labeled rabbitanti-human IgG or IgM is added and binds to the human parvovirusB19 IgG or IgM present. The whole complex is then detected bythe addition of a substrate (tetramethylbenzidene), which turnsblue in the presence of peroxidase. The results were expressedas the absorbance at 450 nm and interpreted as positive, equivocal,or negative by using the manufacturer's recommended cutoff values.

Purified DNA equivalent to 2 µl of serum was used fornested PCR as described by Fridell et al. (12). Serum sampleswith and without parvovirus B19 were prepared in the same wayas the test serum samples and used as positive and negativecontrols. In addition, dilution buffer was used as a reagentcontrol. The nested PCR product was a fragment of DNA sequencecoding for VP1 of parvovirus B19. The first-round PCR was performedwith nucleotides (nt) 2955 to 2974 and nt 3364 to 3349, yieldinga product of 410 bp. The second-round PCR was performed withnt 3002 to 3020 and nt 3291 to 3272 as primers, giving a productof 289 bp. The final composition of the reaction mixture was10 mM Tris HCl (pH 9.6), 2 mM MgCl₂, 50 mM NaCl, 0.2 mM eachdeoxynucleoside triphosphate, 0.1 µM each primer, 1 Uof Taq DNA polymerase (Perkin-Elmer Cetus, Foster City, Calif.),purified DNA (or diluted first-round PCR product), and waterin a volume of 25 µl. Twenty-five cycles of both first-and second-round amplifications were performed under the followingconditions: 95°C for 20 s, 55^oC for 30 s, and 72^oC for 30s (Perkin-Elmer Cetus DNA thermal cycler 9600).

Unexplained anemia. Most B19 infection-related symptoms and signs are usually nonspecificand hence left unrecognized; the presence of anemia, on theother hand, can be reliably determined from the medical recordsof patients receiving chemotherapy and was therefore chosenas the surrogate clinical marker of B19 infection. In this study,unexplained anemia was defined as a sudden drop of more than2.5 g of hemoglobin/dl without a readily attributable etiology,such as acute and chronic blood loss, accelerated red bloodcell destruction, iron deficiency, vitamin B₁₂ deficiency, renalinsufficiency, drug-induced marrow suppression, and tumor involvementof the marrow.

Statistical analysis. The relationship between parvovirus B19 infection and clinicopathologicvariables, including sex, age, history of blood transfusion,duration, regimen, and course of chemotherapy, and cancer type,was evaluated by the ${chi}$ ² test, Fisher's exact test, or the t test.A P value of < 0.05 was considered statistically significant.All statistical analyses were performed with the use of SASsoftware (SAS Institute, Cary, N.C.).

RESULTS

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Results

Clinicopathologic features of the patients. From March 1999 through April 2000, a total of 127 cancer patientswere enrolled in this study. Pertinent clinicopathologic featuresof the patients are tabulated in Table 1. There were 59 menand 68 women, with a median age of 49 (18 to 79) years. Theyreceived an average of 7.1 (4 to 32) courses of systemic chemotherapy.The median duration from the date of starting chemotherapy tothe date of blood sampling was 11 (4 to 88) months. The seropositivepatients were significantly older than the seronegative patients;no other significant difference in clinicopathologic featureswas detected.

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TABLE 1. Pertinent clinicopathologic features of all patients

Prevalence of B19 infection. Parvovirus B19 IgG antibodies were detected in 78 (61.4%) ofthe 127 cancer patients and in 100 (25%) of the 400 healthyblood donors. B19 IgM antibodies and B19 DNA were not detectedin any of the patients or donors. A further age-stratified comparisonof B19 infection between cancer patients and healthy blood donorsis shown in Fig. 1. The higher seropositivity rate for B19 IgGwas significantly increased only in cancer patients youngerthan 40 years.

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FIG. 1. Age-stratified comparison of B19 IgG seroprevalence rates between cancer patients (gray bars) and healthy blood donors (white bars). An asterisk indicates a statistically significant difference. P values were calculated by the ${chi}$ ² test or Fisher's exact test.

Association of B19 with unexplained anemia. A further analysis of the clinicopathologic variables in youngcancer patients revealed a significantly higher incidence ofunexplained anemia in seropositive patients (Table 2). Unexplainedanemia was detected in 3 of 20 seronegative and 12 of 19 seropositivepatients (P < 0.005). Eight patients presented with unexplainedanemia 3 to 4 months following chemotherapy. The mean drop inthe hemoglobin level in the 12 seropositive patients was 3.7± 1.2 mg/dl (value is the mean ± the standarddeviation). The mean duration between the date of diagnosisof unexplained anemia and the date of blood sampling in thisstudy was 6.5 ± 2.7 months. It is important that theseropositive young cancer patients had a significantly longerhospital stay than the seronegative patients. Other clinicopathologicfeatures, including the frequency of blood transfusion and thenumber of courses of chemotherapy, were not significantly differentbetween the two groups (Table 2). In patients older than 40years, the history of blood transfusion also was not significantlydifferent between the two groups (P = 0.83).

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TABLE 2. Pertinent clinicopathologic features of patients younger than 40 years

DISCUSSION

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Discussion

This study suggests that adult patients who are younger than40 years and are receiving multiple courses of chemotherapyare at increased risk of parvovirus B19 infection. B19 infectionin this group of patients may cause important sequelae, as exemplifiedin this series by the significantly higher incidence of unexplainedanemia, which is a common complication of B19 infection. Todate, parvovirus B19 has been recognized as an important causeof severe anemia in immunocompromised patients, including organtransplants recipients (1), patients with congenital and acquiredimmunodeficiencies (9, 19), and leukemia patients receivingmaintenance or consolidation chemotherapy (18). This is thefirst report describing an increased prevalence of B19 infectionamong young cancer patients who have received multiple coursesof chemotherapy. Since B19 infection usually presents with nonspecificsymptoms and signs and is easily overlooked, a high degree ofsuspicion and a careful search for clinical evidence of infectionare needed.

In general, an acute B19 infection is diagnosed by detectingB19 IgM antibodies or DNA, and past infection is diagnosed bydetecting B19 IgG antibodies to VP1 and VP2 (2, 8). The meanduration of the IgM response is 4.8 months but can be shorterthan 2 months (22). In contrast, the IgG response persists foryears and, perhaps, for life. The persistence of serum B19 DNAafter infection, as detected by PCR, may last for 2 to 4 months(22). Therefore, the laboratory evidence of B19 infection maygo unnoticed if the infection is not clinically suspected. Wecould not detect B19 IgM and B19 DNA in our B19 IgG-seropositivepatients, indicating that their B19 infection likely occurredat least 2 months before blood sampling. Unexplained anemiafrequently developed 3 to 4 months after the start of chemotherapyin our patients, and the timing appeared to correlate approximatelywith the development of B19 infection in immunocompromised hosts(4, 7, 17, 25, 28, 30). Since the mean duration between theonset of unexplained anemia and the date of blood sampling was6.5 ± 2.7 months in this study, IgM tests and nestedPCR may both yield negative results for sera from patients withB19 infection that began 2 to 6 months earlier. Prospectivestudies to verify this hypothesis are needed.

Serologic studies of the prevalence of antibodies to the B19virus showed that 60% or more of adults 16 to 40 years old inWestern populations are seropositive (3, 10), whereas the antibodyprevalence rates in young adults (15 to 39 years old) in Taiwanrange from 16 to 36% (21). Lower prevalence rates in young adultshave also been observed in Japan and Hong Kong (20, 32). Inour study, the prevalence of anti-B19 IgG in adult cancer patientsyounger than 40 years was significantly higher than that inhealthy blood donors of a similar age. It is well known thatimmune suppression predisposes individuals to unusual manifestationsof B19 infection, such as persistent infection and chronic anemia(1, 9, 19). However, the reasons why only young adults weresusceptible to B19 infection after multiple courses of chemotherapyremain to be defined. We suspect that nosocomial infection mayhave played a role in this scenario. In Taiwan, adult cancerpatients younger than 40 years are more often hospitalized toreceive chemotherapy and hence may have an increased risk ofnosocomial infection with B19 by airborne transmission and person-to-personcontact (2, 5, 27). Our data also indicate that the seropositivegroup of adult cancer patients younger than 40 years had a significantlylonger hospital stay than the seronegative group. Nosocomialspread of B19 infection can occur in the late incubation periodor early in the acute stage of disease and can even occur duringthe chronic phase of infection (11). However, it remains difficultto explain why there was no significant difference in the seropositivityrate between older cancer patients and older healthy blood donors.The already much higher rate of B19 seropositivity in the olderage group may be one of the reasons (2, 10, 21, 32). Anotherpossibility is that older individuals who remain uninfectedby B19 may represent a population subgroup that is inherentlymore resistant to this virus.

Although B19 infection may be transmitted parenterally fromcontaminated blood products, the risk for acquiring B19 fromsingle-unit and single-donor blood transfusions is low (13,14). In this study, we have demonstrated that blood transfusionis not a risk factor for B19 infection in cancer patients undergoingsystemic chemotherapy. This finding is in accordance with thereport by Lim and colleagues, in which the prevalence of anti-B19IgG among patients with Cooley's anemia after multiple transfusionswas only 16.7%, similar to that for the same age group in thegeneral population (20).

In conclusion, the seroprevalence of human parvovirus B19 infectionis increased in adult patients who are younger than 40 yearsand receiving multiple courses of systemic chemotherapy. Theinfection may be associated with significant anemia. A highdegree of clinical suspicion is the only way to make an earlydiagnosis of this easily overlooked infection. Prospective studiesare needed to delineate the course of serologic and clinicalresponses of patients.

ACKNOWLEDGMENTS

This work was supported by the National Health Research Institute,Taipei, Taiwan, and by research grants NTUH 87S2002 from NationalTaiwan University Hospital, Taipei, Taiwan, and NSC 89-2320-B-002-085from the National Science Council, Taipei, Taiwan.

FOOTNOTES

* Corresponding author. Mailing address: Department of Internal Medicine and Department of Oncology, National Taiwan University Hospital, No. 7, Chung-Shan South Rd., Taipei, Taiwan. Phone: 886-2-2312-3456, ext. 7251. Fax: 886-2-2371-1174. E-mail: andrew{at}ha.mc.ntu.edu.tw.

REFERENCES

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