|Year : 2012 | Volume
| Issue : 4 | Page : 407-410
Detection of human parvovirus B19 in cancer patients using ELISA and real-time PCR
Ibn Sina College for Medical Sciences, Gulail, Jeddah, Saudi Arabia
|Date of Submission||08-Feb-2012|
|Date of Acceptance||20-Jun-2012|
|Date of Web Publication||24-Nov-2012|
S A Zaki
Ibn Sina College for Medical Sciences, Gulail, Jeddah
Purpose: Parvovirus B19 (B19) is associated with a wide range of diseases in humans, whose severity depends on the immunological and haematological status of the host. Objective: To determine the incidence of B19 DNA and specific IgM and IgG frequency among patients suffering from different haematological malignancies and to determine the viral load using real-time PCR. Materials and Methods: A total of 70 patients were included in the study, in addition to a control group consisting of 20 apparently healthy volunteers. B19 DNA quantitative analysis was performed using real-time PCR while screening for IgM and IgG anti-B19 antibodies was performed using ELISA. Results: B19 DNA was detected in 26 patients (36.14%) and 3 controls (15%) using real-time PCR. Anti-parvovirus B19 IgM antibodies were detected in 9 patients (12.6%) and 2 controls (10%). Anti-parvovirus B19 IgG antibodies were detected in 32 patients (45.71%) and 5 controls (25%). The difference between the patient and control groups was found to be statistically non-significant in all of the three tests (P < 0.05). The difference in B19 incidence among patients receiving multiple transfusions and non-transfused patients was also found to be statistically non-significant (P < 0.05). Conclusion: We found a high incidence of B19 infection among patients diagnosed with different types of haematological malignancies. We recommend that all cases of haematological disorders should be examined for specific antibodies and tested for the presence of B19 DNA in serum by PCR technique.
Keywords: IgM antibodies, IgG antibodies, Parvovirus B19, real-time PCR
|How to cite this article:|
Zaki S A. Detection of human parvovirus B19 in cancer patients using ELISA and real-time PCR. Indian J Med Microbiol 2012;30:407-10
|How to cite this URL:|
Zaki S A. Detection of human parvovirus B19 in cancer patients using ELISA and real-time PCR. Indian J Med Microbiol [serial online] 2012 [cited 2013 Dec 12];30:407-10. Available from: http://www.ijmm.org/text.asp?2012/30/4/407/103760
| ~ Introduction|| |
Human parvovirus B19 is a small-DNA virus of the family Parvoviridae, which infects red blood cell precursors in the bone marrow. The virus is spread by the respiratory route, primarily during childhood. Infections with this virus are very common and can result in a wide range of clinical manifestations depending on the patient's immunological and haematological status. In immunocompetent individuals B19 infections can be asymptomatic or benign.  Immunodeficient subjects may become chronically infected. It causes a wide range of human diseases, including a febrile rash illness called erythema infectiosum or fifth disease, severe anaemia and transient aplastic crisis.  It may be accompanied, in few cases, by fever, headache, coryza, nausea and diarrhoea. 
The virus also may cause acute or persistent arthropathy and papular, purpuric eruptions on the hands and feet "gloves and socks" syndrome in adults.  In the normal host, the disease is generally mild, with recovery being the rule. Infection of patients with underlying conditions may be more severe. Immunocompromised individuals who are unable to mount an antibody response to the virus can develop persistent infection. Other disorders, rheumatoid arthritis, vasculitis and thrombotic microangiopathy, are linked in human parvovirus B19 infection.  More recently the virus has been associated with hepatitis and myocarditis. Following maternal infection in pregnancy, the virus may be transmitted to the foetus through the placenta, causing hydrops, spontaneous abortion or intrauterine death. 
In the classical natural history, acute B19 infection occurring in immunologically competent individuals is controlled by neutralising antibodies: A transient, high-level viraemia is present for less than one week, and then declines with the appearance of specific immunoglobulin M (IgM) antibodies, and the appearance of lifelong specific IgG antibodies. However, a persistent infection may be observed in immunocompromised patients unable to produce neutralising antibodies and to clear the virus, leading to chronic B19 carriage with or without anaemia.  Persistence of infection in the bone marrow has also been reported in immunocompetent individuals with or without symptoms.  Acute B19 virus infection is thought to confer a protective, lifelong immunity. 
Diagnosis of parvovirus infection is usually possible by the detection of specific antibodies of IgM class in cases of recent infection. The employment of PCR techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility combined with an extremely broad dynamic range.  In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment, and in viruses displaying persistence in defined cell types, distinction between latent and active infection. 
The aim of the present study is to determine the incidence of parvovirus B12 DNA and specific IgM and IgG antibodies in the serum of cancer patients and controls using ELISA and real-time PCR.
| ~ Materials and Methods|| |
Serum samples were collected from 70 adults (n=70) with malignant diseases, including acute and chronic leukaemia, Hodgkin and non-Hodgkin lymphoma and multiple myeloma. During the study, the majority of the patients received multiple blood transfusions and immunosuppressive treatment. Fifty of the 90 patients were males and the median age was 36 years (range 20-65 years). Twenty apparently healthy individuals were used as a control group.
Ten millilitre volumes of whole blood were drawn from patients and collected in red-top tubes. The serum fractions were allowed to clot at room temperature prior to centrifugation. All the serum samples had been stored at -20°C until tested for B19 IgM antibody, IgG antibody and B19 DNA.
A commercial antibody capture EIA based on the use of parvovirus B19 recombinant capsid proteins was used for the presence of anti-B19 IgM and IgG antibodies in serum (parvovirus B19 IgM and IgG Medac Diagnostika, Wedel, Germany). The colorimetric reaction was read using the LP 200 microplate reader (Diagnostic Pasteur, France).
Nucleic acid extraction
For the isolation of DNA, commercially available kits were used. DNA extraction was done using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany) using the manufacturer's instructions.
Detection of viral DNA
For assessment of B19 DNA levels, a parvovirus genotype 1-3-specific TaqMan real-time PCR assay was developed. The assay was performed in a ABI 7700 sequence detection system (Applied Biosystems) in a 50-μL reaction mixture containing 25 μL of TaqMan Universal PCR Master Mix (Applied Biosystems), 5 μL of template DNA, 3 μmol/L of each primer, and 1.5 μmol/L probe for 40 cycles consisting of 15 s at 95°C and 20 s at 60°C. The following primers were used in the amplification: Sense, 5′-ACAAGCCTGGGCAAGTTAGC-3′, and antisense, 5′-GGCCCAGCTTGTAGCTCATT-3′, positioned at B19 genomic nucleotide positions 854-873 and 910-928, respectively (numbers refer to GenBank AY083239). Amplification was performed in a volume of 50 μL in 1× buffer II (Applied Biosystems) and 25 mmol/L MgCl and 10 pmol/L primer at an annealing temperature of 55°C and for 40 cycles. 
Pearson's Chi-Squared test was used for statistical analysis.
| ~ Results|| |
Evidence of B19 infection was found in 26/70 patients (37.14%) by demonstrating viral DNA. Nine patients (12.86%) were positive for specific IgM antibody while 32 patients (45.71%) showed positive IgG antibodies. Regarding the control group, three samples (15%) had positive B19 DNA; two samples (10%) had B19 specific IgM antibodies while five samples were tested positive for parvovirus B19 specific IgG antibodies [Table 1].
|Table 1: Percentage of IgM, IgG and parvovirus B19 DNA detected in serum of cancer patients and controls|
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In the patient group, 41/70 (58.57%) received multiple blood transfusions or blood products before collecting serum samples; 29/70 (41.43%) patients did not receive multiple transfusions. Seropositivity of B19 was 60.98% and 39.02% among patients who received blood transfusion and who were not transfused, respectively [Table 2]. Chi-Square value was 0.24 (P=0.62) indicating that there is no significant difference between transfused and non-transfused patients.
|Table 2: The transfusion status and seropositivity rates among cancer patients|
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| ~ Discussion|| |
Parvovirus B19 infection in humans is distributed worldwide. Seroepidemiologic studies of several countries showed that prevalence of parvovirus B19 infection varied among countries and populations, and increased with age.  The severity of some B19-associated diseases emphasises the importance of specific diagnostic assay systems for B19 infection. In the present study, we have therefore used three different diagnostic tests for detection of B19 DNA, anti-B19 IgM and IgG antibodies. The study was performed by using clinical serum samples from patients suffering from different types of malignancies. Twenty apparently healthy subjects of comparable age and sex were used as a control group.
In the present study, B19 DNA was found in 37.14% of patients. This finding comes in agreement with other studies including Heegaard et al. and Tercan et al. who found evidence of B19 infection in 30% and 29.1% of patients suffering from different haematological malignancies, respectively. These results suggest that B19 infection may be a relatively common finding in patients suffering from haematological malignancies.
Other studies conducted in Brazil and Greece on hospitalised patients showing exanthema showed lower results (14.5% and 17.6%). , A possible explanation is that the patients included in the study were immunocompetent and none of them showed signs of immunosuppression unlike our study group.
In this study, 41% of patients were found to be seropositive for parvovirus B19. This finding is somewhat high because all of the patients involved in this study were in a high-risk population due to their malignancies and immunosuppression. Thirty-two patients were positive for IgG and only nine patients were positive for IgM. Both DNA and IgG indicate the reactivation of latent infection or persistence of infection. The detection of DNA for seronegative patients possibly indicates an early viraemic period or a state of immunosuppression in which patients did not have immune potentials.
A total of nine patients (12.86%) were found to be positive for IgM using ELISA. The discrepancies between DNA and IgM findings indicate that searching for specific IgM may be a cheap and easy diagnostic tool for basic screening but the sensitivity may be very low. Therefore, it is advisable that if IgM turns out to be negative, to continue searching for possible B19 infection by employing more sensitive PCR method as a second line diagnostic assay. Therefore, it is advisable to use the more sensitive PCR method as a second line diagnostic assay in case of IgM-negative cases.
A study that was conducted in Tunisia on children and adolescents suffering from sickle-cell anaemia illustrates the effect of age in the determining the prevalence in B19 virus infection. The study showed a very high incidence of B19 IgG among the study group (56.5%).  This difference in B19 incidence comes in agreement with various epidemiological reports indicating that this infection spreads at a high rate in low age groups to reach 50% of young adolescents by age 15, and continues at a lower rate throughout adult life to reach most of the elderly. Another study that was conducted in Egypt showed an IgG prevalence of 61% among paediatric patients suffering from various malignancies. 
Another clinically important application of quantitative virus tests is the possibility of differentiating between latent infection and reactivation. Persisting viruses may occur after primary infection in healthy immunocompetent individuals, as well as in asymptomatic patients,  and cause universally positive results in qualitative PCR assays. Mere detection of viral pathogens by qualitative PCR may not be relevant to the clinical outcome in these individuals, but consecutive assessment of the virus load seems to play an important role in the diagnosis and prognosis of patients with viral reactivation by providing a basis for timely initiation of appropriate treatment.  Therefore, assessment of viral load by means of real-time PCR is a helpful parameter for clinical decision making.
In this study, 41 out of the 70 patients were multiple transfused subjects (58.57%). There was no significant difference for B19 positivity between patients who received blood transfusion and who did not (P > 0.05). Additionally, there are several studies which have reported B19 IgG positivity among blood donors in different countries. , Although B19 infection is known to be transmitted via transfusion, its incidence may not increase with transfusion; hence studies including larger populations are needed to be performed as our study population was relatively small.
In summary, we have found a high incidence of B19 infection among patients diagnosed with different types of haematological malignancies. Since no accurate clinical or paraclinical features were predictive of a B19 infection, we recommend that all cases of haematological disorders should be examined for specific antibodies and tested for the presence of B19 DNA in serum by means of PCR technique.
| ~ Acknowledgment|| |
The author wishes to thank the staff members of the Clinical Pathology Department, National Cancer Institute, Cairo, Egypt for their help in providing the clinical specimen.
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[Table 1], [Table 2]