Although many studies investigated the prevalence and manifestations of HPV-B19 infection in patients with sickle cell anemia (SCA), thalassemia, and hereditary spherocytosis (HS) separately, there is limited information about the extent to which HPV-B19 infection leads to severe complications and chronic infection.

HPV-B19 is the smallest single-stranded, linear DNA virus that causes human diseases. Exposure to respiratory secretions is the most common means of transmission of this virus. Infection is associated with a variety of manifestations ranging from asymptomatic to severe complications that depending on the host's hematological and immunological status. No prospective comparative studies investigated the risks associated with this pathogenic virus and predicted the disease course and morbidity among hereditary hemolytic anemia (HHA) patients classified by their underlying diseases.

Nobody has studied the underlying causes of the different and unusual presentations in acute symptomatic HPV-B19 infection in hereditary hemolytic anemias patients. So that, this first head-to-head study to compare thalassemia, sickle cell anemia (SCA), and spherocytosis (HS) patients presented with acute symptomatic HPV-B19 infection.

Studying the relationship between viremia, humoral immune response, and the underlying disease in patients presenting with acute symptomatic HPV-B19 infection should be useful for clarifying the undiagnosed pathophysiological mechanisms and other accessory factors to improve the diagnosis and management.

Moreover, early diagnosis of risk factors of unusual presentations of acute symptomatic HPV-B19 infection will be a useful tool for better treatment strategy, follow up and prognosis as well as prevent future complications

Patients admitted from haematology clinics and emergency departments of Sohag and Assiut University Hospitals for HHA-related complications or manifestations suggesting HHA were screened continuously between February 2018 to February 2020 for enrolment in this study and monthly followed for at least one-year and 3 years post HPV-B19 infection.

Design of the study

1. Patients

First 2 years, the candidate patients who will have evidence of HHA and/or will present with manifestations suggesting HHA and agree to comply with follow-up instructions will be screened for enrollment in this study and additional will be followed for at least one-year post-HPV-B19 infection. Patient data will be included demographics, relevant comorbidities, need for hospital admission, need for blood or plasma transfusion, renal replacement therapy or plasma exchange, hospital length of stay, completion of hospitalization (hospital discharge and death), cardiac events, neurological and respiratory events, type of treatments administered, and inflammatory laboratory parameters.

Diagnosis of HHA in the study patients

Laboratory investigations

1. Routine Biochemical Investigations Liver and renal function tests (LFTs), bilirubin, blood sugar, lipid, and indicated hormonal profiles will be performed on Cobas c 311& modular P auto analyzer (Roche Diagnostics, Mannheim, Germany), lactate dehydrogenase (LDH)

   : analysis will be done with Dimension Rx Max-Siemens & urine analysis, and albumin creatinine ratio will be performed for all patients.

2. Hematological analysis Complete blood count: peripheral blood samples will be withdrawn for diagnostic laboratory investigations and routine follow-up. 2- ml blood will be collected on potassium- ethylene diamine tetra-acetic acid (EDTA) anticoagulant coated tube for CBC using Cell-Dyn 3700, automated cell counter (Abbott diagnostic, Dallas, USA).

   Hereditary Hemolytic Anemia Diagnosis HHA will be suspected by clinical/family history of patient, examination, and laboratory tests including full complete blood count (CBC), calculation of red blood cells (RBCs) indices, corrected reticulocyte count, peripheral blood smear (PBS), iron study, total and direct bilirubin (TB, DB), increased serum LDH, decreased serum haptoglobin, and negative direct antiglobulin (Coomb's) test.

   Confirmation of HHA and identification of subtypes will be done by a series of specialized investigations as following:

   A. Diagnosis of hemoglobinopathy

   The protocol of Hb electrophoresis, sickle solubility testing, and studying Hb fractions with high-performance liquid chromatography (HPLC) will be used for the diagnosis of hemoglobinopathies, such as SCA and thalassemia as previously described.

   B. Diagnosis of RBC Membranopathy and Enzymopathy Hereditary red cell membrane disorders, such as HS and elliptocytosis will be identified by blood smear examination, red cell osmotic fragility test (OFT) with and without 24 hours incubation, acidified glycerol lysis test (AGLT), and RBC labeling with eosin-5'-maleimide (EMA) dye and subsequent measurement of RBC mean fluorescence intensity (MFI) by flowcytometry as previously described.

   Patients considered having HS will base on clinical and laboratory signs of chronic hemolysis, presence of spherocytes at PBS, plus two positive tests (OFT, AGLT, and EMA binding test) or at least one test with a family history of HS, and exclusion of other causes of secondary spherocytosis.

   All patients will be evaluated for RBC enzymes' activity, such as glucose-6-phosphate dehydrogenase (G6PD) to detect the coexistence of RBC enzymopathies and membranopathy.

3. Detection of HPV-B19 and HHA-Patients' Comparator Groups During the study period, any HHA-patient will have manifestations suggesting HPV-B19 infection (fever/ muscle pains /rash/ arthropathy/ lymphadenopathy/ rapid drop in Hb level and reticulocytopenia) and/or contact with a suspected case of erythema infectiosum will be investigated for anti-parvovirus B19 IgM and IgG immunoglobulin status. Those HPV-B19 seronegative will be retested 2 to 3 weeks later. HPV-B19 immunoglobulins (IgM and IgG) status will be determined by chemiluminescent immunoassays (CLIA) technology from (Diasorin, Saluggia, Italy). Additionally, serological tests for hepatitis, HIV, CMV, and EBV viruses will be done to exclude other recent viral infections. Blood cultures and sensitivity will be performed to exclude septicemia. The presence of signs, symptoms, HPV-B19 specific IgM, and absence of IgM of other viruses will be considered as proof of recent symptomatic HPV-B19 infection. Patients will be divided based on previous criteria into two groups.

Group I= HHA patients with acute symptomatic HPV-B19 infection, Group II= HHA patients without acute symptomatic HPV-B19 infection. Then group I patients will be subdivided according to underlying disease into three subgroups: Group Ia: HPV-B19(+)β-thalassemia, Group Ib: HPV-B19(+)HS, and Group Ic: HPV-B19(+)SCA.

The different presenting features of HPV-B19 infection, such as a number of cytopenias, acute kidney injury (AKI), hepatitis, and level of hyperbilirubinemia in group I will be compared. AKI will be diagnosed and classified according to the last guidelines. Extreme hyperbilirubinemia will be considered when total bilirubin >25 mg/dL. The clinical presentation at baseline was recorded.

7.4. Other Investigations The diagnosis of HPV-B19 extra-hematological manifestations, associated autoimmune disorders, cellular and humoral immune deficiencies will be determined using several methods such as detection of several autoantibodies, flowcytometry, serum immunoglobulins level.

Histopathological studies of BM, spleen, and liver biopsies will be done after patients' acceptance in restricted conditions and according to guidelines for an indication of biopsy as:

• BM aspiration and biopsy will be done for patients with persistent severe BMF non responded to treatment.
• Splenic biopsy from patients who will be submitted for elective splenectomy according to recommendations regarding splenectomy in HHA(13).
• Liver biopsy will be done for patients with persistently abnormal liver function with serological test positive to autoimmune or sclerosing cholangitis to differentiate the underlying disease for proper management.

Radiologic assessment by echocardiography, X-ray chest and long bones, and abdominal ultrasound will be routinely performed for screening all patients. The HHA patients with extreme hyperbilirubinemia or obstructive jaundice will be screened by magnetic resonance cholangiopancreatography (MRCP).

Group I patients will be monthly followed for at least one-year post HPV-B19 infection. Group II patients will be diligently followed for the time of writing this manuscript. During this period, complications, and laboratory profiles including BM aspiration if the patient had severe cytopenia, will be checked. All patients will be scanned by echocardiography and examined by ultrasound to view the gallbladder, kidneys, spleen, and liver size (cm). The events of interest will be recorded, such as bone marrow failure (BMF), spleen sequestration, chest pain, vaso-occlusive crisis, cholelithiasis, cholecystitis, and choledocholithiasis and transfusion frequency

Inclusion Criteria:

• Confirmed hereditary hemolytic anemias patients (age above 12 years) presented with signs and symptoms of hereditary hemolytic anemia (HHA) and admitted or treated in emergency departments or Hematology Units at Internal Medicine Departments of various university hospitals will be screened for enrollment in this study.

Exclusion Criteria:

• • Patients will be diagnosed with non-HHA as ( autoimmune hemolytic anemia, microangiopathic hemolytic anemia (MAHA), Wilson disease, paroxysmal nocturnal hemoglobinuria (PNH).

  • HHA-patients will refuse to consent to this study.
  • Serologic evidence of recent virus infection other than human parvovirus B19 (HPV-B19); hepatitis A (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis E virus (HEV,) cytomegalovirus (CMV), Epstein-Barr virus (EBV), or positive test for HIV.
  • HHA patients with uncontrolled diabetes mellitus (DM).
  • HHA patients with a history of nephrotic syndrome or chronic kidney disease(CKD).
  • HHA patients with a history of treatment by immunosuppressive drugs.
  • HHA patients with clinical and laboratory evidence of relevant toxicity related to iron chelation.
  • HHA patients with severe systemic diseases (such as cardiovascular, renal, and hepatic disease) or surgical/medical conditions that might interfere with follow-up instructions.
  • HHA patients with a life expectancy of less than 1 year.
  • HHA patients with psychiatric disorders or a history of drug abuse,
  • Pregnant women will be also excluded.
  • Patients are not a candidate for investigation (Refusal

HHA Patients will be classified according to of presence or absence of acute symptomatic HPV-B19 infection