Mahmood et al (2021): Parvovirus infection in SLE patients © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24534

Parvovirus infection in systemic lupus erythematous patients in Najaf governorate, Iraq

Thikra Abdullah Mahmood1*, ThanaaShamsulddin Abbas2, SaifJabbar Yasir3,MunaSachit Hashim4

1Department of Community, Faculty of Medicine, University of Kufa, Iraq 2, 3Department of Microbiology, College of Medicine, University of Kufa, Iraq 4 Department of Pathology, college of veterinary medicine, University of Baghdad/Iraq Corresponding author Thikra Abdullah Mahmood [email protected]

Abstract Background: The emergence of symptoms is consistent with systemic autoimmune diseases in relation to many viral infections, especially (PVB19).This study was conducted to investigate the IgM and IgG antibodies, in addition to the DNA of PVB19 in systemic lupus erythematosus (SLE) patients, and its relationship to disease activity, depending on the evolution of the age and immune status of the host. Aim of the study: Determination of PVB19 infection by different biomarkers in SLE patients in different age groups and genders. Methods: Serum samples were collected from 62 patients with SLE and 62 normal controls, in order to test for the PVB19 infection by detection of anti PVB19 IgM and IgG, and viral DNA, using ELISA and PCR, respectively. Results: The ELISA tests for IgM antibodies showed 21(33.9%) seropositive in 62 patients, and for IgG29 (46.8%) seropositive have been found. Out of 62 samples, 18 samples showed positive results for viral DNA. In the control group, 4(6.5%) IgM, 8(12.9%) IgG and 1(1.6%) positive viral DNA,have been found. These data were significantly different in SLE patients, compared with the healthy controls (P< 0.05). Conclusions: Viral infections, particularlyPVB19,are one of the most important infections associated with autoimmune diseases. We could not find any significant correlation between the emergence of PVB19 and SLE in affected patients, in terms of age groups and genders.

Keywords: Parvovirus B19, IgM, IgG, PCR, SLE

DOI: http://doi.org/10.36295/ASRO.2021.24534 Page: 424-430

Volume/Issue: Volume: 24 Issue: 05

Introduction Parvoviruses are single-stranded, non-segmented, linear DNA with a mean genome size of 56 Kbp. (1).According to the of , they are classified as group II viruses. Hence the name, from Latin Parvus meaning small with diameter ranged from 23to28 nm, soparvoviruses are among the smallest viruses (2). The B19 also known as primate erythroparvovirus 1 causes many diseases in humans, especially the , which is a member of the major red blood viruses’ species in the genus of the red virus. Red precursors affect blood cells and were the first small virus that appears to cause human disease (3).The primary spread of the virus is by respiratory droplets, and blood-borne transmission, which has been reported in some cases (4).

About 50%, has the risk of a secondary attack on exposed family members, and class contacts were about the other half (5,6).In some autoimmune diseases, such as systemic lupus erythematosus (SLE),the host immune attacks healthy tissue incorrectly in many parts and organs (7). Symptoms may vary from mild to severe among peoples (8). Common symptoms include fever, swollen joints, swollen lymph nodes, painful chest pain, mouth ulcers, tiredness, hair loss and most commonly a red rash on the face(9). Regularly there are remission periods during which there are few symptoms, termed flares (10). Recent research has focused on the role of the parvovirus in SLE patients in relation to different ages and genders.

Materials and Methods In this study, 62 serum samples, including 39 females and 23 males, were collected from SLE patients. The age groups of patients were from 15 to 55 years old. Blood samples were collected from patients during the period between March 2019and December 2019.Samples were taken from the Al-Saader Medical City Hospital in Najaf, Al-Najaf province, Iraq. The recent study also included 62healthy individuals, as a control group. Enzyme-linked immunoassay(ELISA) was used for clinical diagnosis of SLE, detecting antiviral IgM and IgG antibodies. Patients

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Mahmood et al (2021): Parvovirus infection in SLE patients © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24534 were excluded if they were pregnant, had breast-feeding, underwent splenectomy, smoked or were taking medication.

Study design: A cross-sectional study was performed wherein the sample size was calculated, using the equation of the study type. Serological test: 1. Human Anti-nuclear antibodies (ANA) Screen ELISA test kit, Diagnostic Automation, Cortez Diagnostics Inc. California, USA. 2. Parvoscan‐B19TMIgM/ Euro‐Diagnostica, Ideon, Sweden, for detection of anti-B19 IgM antibody. 3. Parvoscreen‐B19TMIgG, Euro‐Diagnostica, for detection of anti-B19IgGantibody.

Detection of parvovirus B19 nucleic acid by nested PCR: Amplification was performed, using ready-to-use PCR master mix beads (Bioron, Ludwigshafen, Germany).Two pairs of primers were designed to cover a well-conserved region in viral DNA, and amplified in 2 steps. The primer pairs (from The Midland Reagent Co., Germany) were as follows: 5’-AAAGCTTTGTAGATTATGAG-3’/5’-GGTTCTGCATGACTGCTATGG-3’(11). The amplification was performed, using a DNA thermal cycler (Cetus; Perkin Elmer, Emeryville, California, USA) as follows: Twenty-five cycles of amplification were achieved with the same parameters as those in the first round. Negative controls containing all components, except the DNA extract, were involved in each cycle to exclude non-specific amplification. Amplified viral PCR products were visualized on the agarose gel, using gel electrophoresis. Products with 322 bp were documented as being positive for the DNA of PVB19 (11).

Data analysis: Statistical analysis was carried out, using SPSS software (version 22.0).A chi-squared (χ2) test was performed to define the parvovirus infection in SLE, p value ˂0.05 was considered significant. Consent: All official approvals were obtained from the hospital administration and a consent form was signed by the participating patients after informing them of the purpose of the research. The study was approved by the Medical Ethics Committee of the Medical Faculty.

Results IgM seropositivity in SLE according to the age groups Among 62 patients, 21 patients were seropositive for IgM antibody (33.9%) and 41 patients were seronegative (66.1%). SLE patients were found to be most commonly aged between 40 and 55 years old (60.7%) for both males and females, as shown in Table 1.

Table 1: The IgM seropositivity in SLE patients according to the age groups

Age groups Cases ParvoIgM+ ParvoIgM (Years) No. (%) No. (%) No. (%) P value 15-25 13(100) 3(23.0) 10(77.0) 25-40 21(100) 7(33.4) 14 (66.6) 0.593 40-55 28(100) 11(39.3) 17(60.7) Total 62(100) 21(33.9) 41(66.1)

IgGseropositivity in SLE according to the age groups Table 2 shows the IgG seropositivity according to the age group. About 29 samples were seropositive for IgG(46.8%), while 33 samples were seronegative (53.2%), there isno significant difference between age groups, P ˃0.785.

Table 2: The IgG seropositivity in SLE patients according to the age groups

Age groups Cases Parvo IgG+  ParvoIgG P value (Years) No.(%) No. (%) No. (%) 15-25 13(100) 5(38.5) 8(61.5)

25-40 21(100) 10(47.6) 11(52.4) 0.785 40-55 28(100) 14(50.0) 14(50.0) Total 62(100) 29(46.8) 33(53.2)

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Mahmood et al (2021): Parvovirus infection in SLE patients © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24534

Distribution of IgM and IgG seropositivity of blood samples of SLE patients by gender. The distribution of IgM and IgG seropositivity based on the gender was shown in Table 3.There isno significant difference in IgM and IgG seropositivity of blood samples of SLE patients according to the gender, p value>0.05 (0.12 for male and 0.56 for female).

Table 3:The distribution of IgM and IgG seropositive blood samples in SLE patients by gender

Seropositivity IgM serology Parvo IgG serology Parvo

P value Positive Negative Positive Negative Gender No. (%) No. (%) No. (%) No. (%) Male 7(11.3%) 16(25.8%) 2(6.9%) 21(33.9%) 0.56 Female 14(22.6%) 25(40.3%) 27(93.1%) 12(19.4%) 0.12 Total 21 41 29 33

The distribution of parvo DNA based onIgM positivity in SLE patients is shown in Table 4. No significant differences were observed between different age groups and variations of Parvo DNA and IgM positivity in SLEs patient(p value ≥ 0.05).

Table 4: The distribution of Parvo DNA and IgM positivity in SLE patients according to the age groups

Parvo DNA Parvo IgM Age groups Cases Positive Negative Positive Negative (Years) No.(%) No. (%) No. (%) No. (%) No. (%) 15-25 13(100 3(23.1) 10(76.9) 3(23.1) 10(77.0) 25-40 21(100) 5(23.0) 16(76.2) 7(33.4) 14 (66.6) 40-55 28(100) 10(35.7) 18(64.3) 11(39.3) 17(60.7) Total 62(100) 18(29) 44(71) 21(33.9) 41(66.1) P Value 0.574 0.593

The distribution of parvo DNA based on IgG positivity in SLE patients, is shown in Table 5. No significant differences were observed between the different age groups and variations of Parvo DNA and IgGpositivity in SLEs patient (p value ≥ 0.05). In Table 5, no significant difference (p value = 0.536) in parvo DNA and parvo IgG (p value = 0.785) was found in SLE cases, according to the age groups per year.

Table 5: The distribution of parvo DNA and IgG positivity in SLE patients based on the age groups

DNA ParvoIgG Age groups Cases Positive Negative Positive Negative (Years) No.(%) No. (%) No. (%) No. (%) No. (%) 15-25 13 (100) 0(0.0) 13(100) 5(38.5) 8(61.5) 25-40 21(100) 2(9.5) 19(90.5) 10(47.6) 11(52.4) 40-55 28(100) 2(7.1) 26(92.9) 14(50.0) 14 (50.0) Total 62(100) 4(6.5) 58(93.5) 29(46.8) 33(53.2) P value 0.536 0.785

Control groups: Parvo IgM was used to detect any recent parvovirus infection in the control group,in different age groups. A serological assay was required to determine if the patients were seropositive or seronegative for the parvo IgM. The sera of 62 SLE patients were collected. The coefficients of variation for parvo IgM were 6.5% for positive parvo IgM and 93.5% for negative parvo IgM. No significant differences were observed between different age groups and variations of parvo IgM (p value > 0.05), as shown in Table 6.

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Mahmood et al (2021): Parvovirus infection in SLE patients © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24534

Table 6: The IgM positivity in healthy individuals according to the age groups

Age groups Healthy ParvoIgM ParvoIgM P value (Years) No.(%) Positive Negative No. (%) No. (%) 15-25 11(100) 0(0.0) 11(100.0) 25-40 22(100) 1(4.5) 21(95.5) 0.862 40-55 29(100) 3(10.3) 26(89.7) Total 62(100) 4(6.5) 58(93.5)

Parvo IgG was used to detect any recent and previous parvovirus infection in the control group for different age groups. A serological assay was performed to determine if the patients were positive or negative for the parvo IgG. The sera of 62 SLE patients were collected. The coefficients of variation for parvo IgG were 12.9% for positive parvoIgG and 70.1% for negative parvo IgG. No significant differences were observed between different age groups and variations for parvo IgG (p value > 0.05), as shown in Table 7.

Table 7: The parvoIgG positivity in healthy individuals according to the age groups

Parvo IgG Parvo IgG Age groups Health P value Positive Negative (Years) No. (%) No. (%) No. (%) 15-25 11(100) 0(0.0) 11(100)

25-40 22(100) 3(13.6) 19(86.4) 0.635 40-55 29(100) 5(1.2) 24(82.8) Total 62(100) 8(12.9) 54(70.1)

Parvo DNA positive and negativewere used to assess if the patients were positive or negative, compared to healthy individuals. Parvo DNA was quantified in 62 patients and grouped by age.The age groups 1525, 2540 and 4055 years old were determined to have 0.0%,0.0% and3.4% parvo DNA positive. No significant differences in the parvo DNA were detected between different age groups (p ≥ 0.05),as indicated in Table 8.

Table 8: The parvo DNA positivity in healthy individuals according to the age groups

Parvo Parvo DNA Age groups Healthy DNA P value (Years) No. (%) Positive Negative No. (%) No. (%) 1525 11(100) 0 (0.0) 11 (100) 2540 22(100) 0 (0.0) 22 (100) 0.719 4055 29(100) 1 (3.4) 28 (96.6) total 62(100) 1(1.6) 61(98.4)

Discussion The PVB19 infection and its association with SLE are an important concern. There are striking resemblances between clinical features of SLE symptoms and B19 infection;however, it is difficult to clinically distinguish this viral infection from SLE. B19 virus infection occurrence in adults has been recorded in some patients with systemic autoimmune diseases (12, 13, 14, and 15). PVB19 may mimic or increase SLE,and usually isconnected with a transient clinical autoimmune complaint(13, 17, and 22).The development of SLE may be associated with B19 infection,as well as other chronic diseases (12, 16).

In this study, viral DNA was detected in the serum of 18(24%) patients with SLE and confirmed by PCR. Parvoviral DNA has not been allocated in the serum of other autoimmune diseases, including Sjogren's syndrome (SS), rheumatoid arthritis (RA) and primary biliary cirrhosis (PBC)(23, 24). However, the presence of B19 DNA in patients with SLE may not be a cause. Alternatively, it may reflect overlapping B19 infection in patients with SLE

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Mahmood et al (2021): Parvovirus infection in SLE patients © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24534 due to a lack and absence of anti-B19antibodies. The prevalence of anti-B19 IgM and IgG antibodies in sera of SLE patients with B19 DNA, was significantly lower than in patients without the B19 DNA (P <0.05).

Kurtzmanet al. (19, 20) explained that antibody against the protein capsid of PVB19 plays a key role in human HIV infection reduction. It has also been reported that B19 antibodies’ production in an immunosuppressive host within a few days leads to the cleansing of viremia, while the virus persists in immunocompromised patients (29, 20, and 21). The prevalence of antibodies against the virus may modify the course of the viral infection. Parvovirus persistence in patients with SLE may be due to a lack of antibodies against the virus because of the immunocompromised host or due to immunosuppressive management. Only in patients with EI or SLE, the B19 DNA was also detected, and their clinical significance was studied as well. Hypocalcemia and Raynaud's phenomenon (RP) were significantly more common in patients with a viral infection, compared to cases without the B19 DNA(22, 23).

B19 infection may worsen the SLE clinical course. However, there was no clear correlation between the presence of B19 DNA with other clinical characteristics, such as rashes, proteinuria and arthritis in SLE patients. Another study reveals that cytomegalovirus (CMV) infection isa significant risk factor for RP (22). Viral infection may affect the course of SLE, leading to specific clinical types of complications. These preliminary conclusions necessitate confirmation to clarify the importance of the presence of B19 DNA in SLE. Various studies have sought a possible link between the SLE pathogenesis and infection with certain viruses. Several exogenous viruses have been associated with SLE, where Epstein-Barr virus (EBV) contains the greatest evidence that it is a causative candidate. Other viruses involved in SLE are endogenous human , human papillomavirus (HPV), HIV, CMV, human dengue virus and human herpesviruses (HHV), such as HHV-6, HHV-7, HHV-8(14).

Overall, serological and molecular investigations demonstrated that antibodies and PCR products of these viruses were higher in SLE patients, compared to the control group. In some cases, along with enhanced viral loads, these phenomena have been linked to an outbreak of disease activity. In general, these tests indicate that SLE has occurred or worsened as a direct result of a particular virus, or indirectly through internal viral factors present in humans. Currently, there is no consensus about the virus being most relevant to the causative agents of SLE, due to difficult agreement through publication bias and the absence of unpublished negative data (30).

Similarities in clinical characteristics led to speculation about the relationship between B19 and SLE infection. The relationship between B19 and lupus can be visualized in three ways: First, some SLE patients with B19 infection produce a common antibody, in addition to creating B19 polymorphic antibodies (18). Secondly, clinical and analytical features similar to those found in SLE may appear in B19 patients, for example, "cheek rash", acute arthritis and/or cell deficiency with constitutional symptoms, including elevated fever and antibody production, DNA and antibodies against phospholipid. However, the accuracy of these symptoms within one to two weeks provides the correct diagnosis immediately. Thirdly, SLE can be developed after infection with B19. Additionally, there have been few reports of B19 infection in conjunction with known SLE exacerbation [18,19]; this association may reflect a shift from B cell activation to the production of antibodies in eligible individuals, and the secretion of aqueous antibodies to ssDNA in 3070% of patients, leading to viral ssDNA breakdown in blood and other body fluids (10).

Although there are similarities in clinical manifestations between B19 and SLE, there are obvious differences such as the scarcity of long fever or lack of characteristic of renal involvement or seizures in B19 infection (24).This study is an attempt to find out the relationship between B19 and SLE infection in a reasonable number of patients based on serum viral DNA detection via synaptic PCR. This technique has the advantage of being highly sensitive and suitable for clinical use. Our results do not show any positive ratios, compared to viral DNA in SLE patients and healthy controls. In this study, the prevalence of B19DNA was 13.5% in healthy controls, compared with 40% of SLE patients.

Our results for SLE patients were in agreement with those previous studies and case reports. Pingtonet al.(20)showed no increase in the prevalence of B19 infection in SLE patients,concerning the general population. Unlike our results, Hsu and Tsay(17) detected B19 DNA in 24% of patients with SLE, but not in any of those with other systemic rheumatic diseases; this discrepancy may be related to the difference in many factors, including sample size (72 cases compared to 30 cases in our study). In the study of Trapani et al. [12],cases of SLE caused by B19 have also been reported. Cobb et al. (16), suggested that this virus may trigger mechanisms that initiate an autoimmune response in the form of SLE. Some authors state that the B19 virus causes indications that mimic only lupus, such as arthralgia, vasculitis and fever episodes (26).Some Arabic studies have been published concerning SLE patients, describing SLE or SLE associated with human immunodeficiency virus (HIV) and B19. In the study of Aichi et al. (27), HPV was detected in 30% of the study population in Egypt, compared to any of the

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Mahmood et al (2021): Parvovirus infection in SLE patients © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24534 normal controls (P <0.05). A prospective Tunisian epidemiological study was conducted to illustrate the potential role of this infectious agent, in the etiology of SLE. The results supported a direct correlation between B19 infection and the cause of SLE(29, 30).

Many of the observations in this study are noteworthy. First, somewhat unexpectedly, there were positive natural controls for B19 DNA. As Cossart et al. suggest, the presence of viral DNA in healthy blood donors (28) can last for a long period despite the absence of clinical symptoms.

Conclusion Viral infections, especially PVB19 is one of the most important pathogenic agents in autoimmune diseases, and may play a critical role in the initiation of pathogenesis in these cases. We could not find a significant correlation between the appearance of PVB19 and SLE, in different age groups and genders. Future directions Multicenter prospective studies are needed to assess the role of many infectious agents, including B19 on the pathogenesis of SLE.On the other hand, SLE patients should be monitored continuously and the association of genetic and social variables should be further investigated.

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