FINAL DIAGNOSES
The RNA viral PCR test came back the 3 days after collection, positive for Influenza A. The patient's sputum culture was positive for methicillin resistant Staphylococcus aureus (MRSA). Admission nasal swab was also MRSA positive. The bone marrow biopsy was hypocellular for age with myeloid immaturity, likely secondary to growth factor therapy, scattered hemophagocytic histiocytes and atypical megakaryocytes with abnormal nuclear lobation. Classical cytogenetic studies performed on the bone marrow demonstrated a normal male karyotype. Flow cytometry studies and immunohistochemical stains performed on the bone marrow did not demonstrate an aberrant myeloid or lymphoid population of cells. Immunohistochemical stains for CMV and parvovirus were negative. In situ hybridization to EBV encoded RNA (EBER) was also negative. Molecular studies were not performed.
The patient went into acute respiratory and renal failure and expired four days after admission. An autopsy was performed which confirmed viral and secondary bacterial bronchopneumonia as the cause of death. There was a severe ulcerative tracheobronchitis. The lungs showed severe, diffuse bilateral acute necrotizing and hemorrhagic pneumonia of all lobes. There was acute lung injury with diffuse alveolar damage. The right upper lung also showed an early abscess formation. Bilateral serosanguineous pleural effusions with a mild fibrinous pleuritis were also noted. There was also a mild lymphocytic myocarditis and rhabdomyolysis, with a creatine phosphokinase up to 8796 U/L, 3 days after admission.
At our institution, real-time PCR test is performed for the detection of influenza A and B. For this patient, testing for Influenza B was negative. The Influenza A subtype was not performed, as the threat of H1N1 was not yet realized at the time of testing (months before outbreak). Real-time PCR is used to determine the presence ribonucleic acids of influenza A or B in a patient's specimen. Virus may be detected by PCR prior to diagnosis by immunological methods. PCR provides the most sensitive results, which are reported as positive, negative or equivocal. Currently at our institution, Influenza A subtype can now be determined on those samples that test positive for Influenza A. Influenza A is now subtyped as either swine origin 2009 influenza A (H1N1), seasonal H1 influenza A, or seasonal H3 influenza A (1). This patient had a nasal/nose specimen sent for testing, which is an acceptable specimen, but the laboratory's preferred specimens for increased sensitivity include a nasopharyngeal swab collected in viral transport medium such as M4 transport Medium (3 ml). With the nasopharyngeal specimen, a thin, flexible metal or plastic swab is inserted through the nostril deeply into the nasopharynx. The swab is rotated or held in place for 10 seconds to ensure the collection of an adequate specimen and to reduce the possibility of false negative results. The lab does note to clinicians that the specimen should be kept at "refrigerator" temperature (not frozen) during transit. For other specimens, including sputum, the sample is collected in a sterile, leak proof container. All specimens' stability is up to 48 hours at room temperature, 7 days at 4oC, and 30 days if frozen (1).
Other methods of testing include culture from nasopharyngeal secretions. Expectorated sputum is also an acceptable specimen, if bronchial columnar cells, alveolar macrophages, or neutrophils are present. Shell vial culture is preferred over culturing with traditional cell monolayers and red cell hemadsorption, as the incubation time is shorter with shell vial culture (2). The direct fluorescent antigen testing for influenza antigen is also a useful test with quick turn-around-time if nasopharyngeal aspirates specimens are used. The sensitivity of methods is highly dependent on specimen quality, as nasopharyngeal aspirates yield the highest amount of viral-infected columnar epithelial cells compared to nasopharyngeal or throat secretions (2).
Mortality from seasonal influenza virus is typically associated with children less than 1 year of age, adults older than 65 years, pregnant woman (especially in the 3rd trimester), and individuals of any age with comorbid illnesses (5). However, the H1N1 2009 influenza infection has been described in a younger population, with a reported mean age of 23.4 years with 54% male patients (6). In a Canadian study, the median age of patients with H1N1 infection was 23 years; these patients also had the highest risk of severe outcome, including death in those over 20 years, and/or having an underlying medical condition (7). Furthermore, while our patient was neither immunosuppressed nor had significant comorbid illness, he was obese (BMI = 33), which may be an independent risk factor in H1N1 influenza morbidity and mortality. Therefore, it is recommended that these individuals should be followed closely (8). However, our patient presented before the H1N1 influenza pandemic was recognized, and therefore, the specimen was not subtyped. Given the patient's age and clinical presentation, there remains a strong suspicion that this may have been an early, unrecognized case of H1N1 influenza infection.
The bone marrow findings were of interest in this case. Hemophagocytic histiocytes in association with viral illness have been described in relation to the hemophagocytic syndrome (HPS). Viral-associated hemophagocytic syndrome (VAHS) is a disorder of the mononuclear phagocytic system with a non-malignant proliferation of histiocytes in various organs (bone marrow, lymph nodes, spleen, liver) that engulf red blood cells and platelets (3). Typically Epstein-Barr virus, cytomegalovirus, herpes simplex virus, adenovirus, parvovirus B19, HIV, respiratory syncytial virus, and hepatitis A, B, and C viruses have been described in VAHS. A few case reports of Influenza A as the causative virus have been reported in immunocompromised patients (4). One case report has described a fatal case of VAHS in a previously immunocompetent child (3). Another case report of VAHS caused by Influenza A (H3N2) has been described in a 40-year old Japanese woman, who had been previous treated with chemotherapy/radiation 2 years prior for a lingual carcinoma. She presented with severe respiratory failure, myocarditis, and acute renal failure but was successfully treated with aggressive immunosuppressive medications, including pulsed methylprednisolone, gamma-globulin, and granulocyte colony-stimulating factor (9). Our case-patient fulfilled some of the criteria of VAHS (see bold table 1); however, all diagnostic criteria must be met in order to diagnose VAHS (Table 1). In addition to treating the underlying virus in VAHS, immunosuppressive medications are also given (dexamethasone, etoposide, cyclosporine A) in order to control the abnormal, overactive immune response. The proposed pathogenesis is that the virus infects macrophages and activates T-lymphocytes to elicit a marked increase in the levels interleukins and other cytokines and thus leads to a profound pro-inflammatory state (3).
It is also important to note that while viral infections are associated with hemophagocytic syndrome, they can also produce certain bone marrow findings unassociated with VAHS. Hepatitis infection can present with bone marrow granulomas and a hypocellular marrow with increased lymphocytes and/or plasma cells, along with an aplastic anemia (10). With advanced anti-retroviral therapies, there are less bone marrow evaluations performed for Human Immunodeficiency Virus (HIV) and Acquired Immunodeficiency Syndrome (AIDS). Typical bone marrow findings in these entities can include a hypercellular marrow with multilineage dysplasia, including dyserythropoiesis, dysgranulopoiesis, with a left-shifted maturation, and abnormal megakaryocytes. The virus can also induce reticulin fibrosis. Less commonly there is marrow hypocellularity with red cell aplasia, lymphoid aggregates and increased histiocytes (10). Epstein Barr Virus (EBV) bone marrow infection can demonstrate nonspecific reactive changes, not limited to lymphocytosis (mainly of T-cell origin) with plasma cells and scattered immunoblasts that may be concerning for Hodgkin and Reed-Sternberg cells. Rarely there are increased histiocytes and small non-necrotizing granulomas (10). An immunohistochemical stain for the latent-membrane antigen and/or in situ hybridization to EBV encoded RNA (EBER) is helpful for identifying infected cells. Cytomegalovirus (CMV) is another virus that can be identified with immunohistochemical staining, if the classic cytomegalic inclusions, both intranuclear and intracytoplasmic are not apparent with routine staining. Marrow findings are similar to those seen in EBV-infection, including reactive lymphocytosis (with a predominance of T-cells), small granulomas, and decreased megakaryocytes (10). Parvovirus B19 infection is another viral infection with bone marrow findings significant for decreased erythroid progenitor cells, secondary to viral proteins inducing cell cycle arrest and cell death. Megakaryocytes and myeloid cells are also variably affected, though less common. Classic giant proerythroblasts (lantern cells) with a prominent eosinophilic nuclear inclusion helps confirm but are not diagnostic of the virus; however, immunohistochemical stains and/or in situ hybridization studies can help identify infected cells. It is also important to correlate with serum PCR and serology studies with all above aforementioned viral infections.
In conclusion, despite therapy, this young male patient died of an acute respiratory illness caused by multifactorial infectious processes with associated reactive and myelosuppressive effects on the marrow. Given the patient's clinical and pathologic findings, it remains a likely possibility that H1N1 influenza subtype contributed to this patient's demise.
Contributed by Jennifer Picarsic, MD and Lydia Contis, MD
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