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Assay Sheet

Test ID

7500 Adenovirus Real-time qPCR

CPT Code

87799

Clinical Utility

Adenovirus is an important cause of morbidity and mortality in the transplant setting, causing pneumonia, hemorrhagic cystitis, hepatitis, encephalitis, pancreatitis, enteritis, and disseminated disease with the mortality rate reaching 60% in some especially high risk situations such as pediatric HSCT. Since proper management is dependent upon early diagnosis, quantitative adenovirus DNA PCR is useful for detecting the virus, tracking the course of infection, and monitoring response to treatment.

Procedure

Extraction of adenovirus DNA from plasma, urine, other biological fluids, or tissues followed by amplification and detection of known adenovirus strains using real-time, quantitative PCR. An internal control is added to ensure the extraction was performed correctly and the PCR reaction was not inhibited. ViraCor’s assay design includes multiple targets to account for viral mutations, which exponentially reduces the chance of false negative results.

Specimen type & Specimen handling

Whole Blood: 4-5 mls collected in EDTA (lavender top) tube. Do not freeze; ship ambient. Testing will be performed on plasma separated from the submitted whole blood specimen. Whole blood specimens are accepted as a matter of convenience for the originating laboratory. 

Plasma: Collect 4-5 mls whole blood in EDTA or ACD tube, centrifuge and transfer 2 mls plasma to sterile, screw top tube. Ship at ambient temperature Monday thru Friday. Specimen must be received within 96 hrs of collection.

Bone Marrow: 2 mls collected in EDTA tube. Ship at ambient temperature Monday thru Friday. Do not centrifuge or freeze. Specimen must be received within 96 hrs of collection.

Bronchial Lavage/Bronchial Wash: 2 mls collected in a sterile, screw top tube. Ship at ambient temperature Monday thru Friday. Specimen must be received within 96 hrs of collection.

Conjunctival/Eye Swab: Sterile swab placed in 2 ml sterile saline, M4, or viral transport media in a sterile, screw top tube.  Do not use calcium alginate swab or wood shafted swab.  Ship at ambient temperature Monday thru Friday.  Specimen must be received within 96 hrs of collection.

CSF: 2 mls, collected in a sterile, screw top tube. Freeze and ship on dry ice Monday thru Friday.  Specimen must be received within 96 hrs of collection.

Fecal: Collect small amount of fecal material (size of pea, or 2 mls liquid stool) and place into screw top tube for shipment. Ship at ambient temperature Monday thru Friday. Specimen must arrive within 96 hrs of collection. Note: Specimen may also be stored at 2 to 8°C or frozen in a non-self-defrosting freezer and shipped with frozen gel packs or dry ice for overnight delivery at Viracor-IBT.

Pleural Fluid: 2 mls collected in a sterile, screw top tube. Ship at ambient temperature Monday thru Friday. Specimen must be received within 96 hrs of collection.

Rectal Swab: Sterile swab placed in 2 ml sterile saline, M4, or viral transport media in a screw top tube. Do not use calcium alginate swab or wood shafted swab.  Ship at ambient temperature Monday thru Friday.  Specimen must be received within 96 hrs of collection. 

Throat Gargle:  2 mls collected in a sterile container then transferred to sterile, screw top tube for shipment. Ship at ambient temperature Monday thru Friday. Specimen must be received within 96 hrs of collection.

Tissue: Place in sterile, screw top container; add small amount of sterile saline to keep moist. Paraffin embedded tissue is acceptable.  Ship at ambient temperature Monday thru Friday.  Fresh tissue must arrive within 96 hrs of collection.

Upper respiratory aspirate (NP aspirate, nasal aspirate, tracheal aspirate, etc.): 2 mls collected in a sterile, screw top tube. Ship at ambient temperature Monday thru Friday. Specimen must be received within 96 hrs of collection.

Upper respiratory swab (NP swab, throat swab): Sterile swab placed in 2 ml sterile saline, M4, or viral transport media in a sterile, screw top tube.  Do not use calcium alginate swab or wood shafted swab.  Ship at ambient temperature Monday thru Friday.  Specimen must be received within 96 hrs of collection.

Sputum: 2 mls collected in a sterile container, then transferred to sterile, screw top tube for shipment. Ship at ambient temperature Monday thru Friday. Specimen must be received within 96 hrs of collection.

Urine: 2 mls sample collected in a sterile urinalysis container then transferred to sterile, screw top tube for shipment.  Ship at ambient temperature Monday thru Friday. Specimen must be received within 96 hrs of collection.

Vitreous Fluid: Small volumes are acceptable. Transfer collected amount to sterile, screw top tube for shipment. Do not ship specimen in syringe.  Ship at ambient temperature Monday thru Friday. Specimen must be received within 96 hrs of collection.

Call ViraCor for authorization prior to sending any specimen type other than those listed above.  

If another specimen type has received authorization for testing the following comment will appear in the final report: "The clinical utility of this result has not yet been demonstrated in the peer reviewed literature and is therefore unknown."

Causes for Rejection

Wood shafted swab, or calcium alginate swab

Call ViraCor at 800-305-5198 if specimen is greater than 96 hrs old

Specimen types other than those listed above that were sent without prior authorization

Specificity

Detects all 52 serotypes in one assay. The primers and probes used in this assay are specific for known adenovirus strains based on similarity search algorithms. Additionally, no cross reactivity was detected when tested against BKV, CMV, EBV, HSV-1, HSV-2, HHV-6 variant A, HHV-6 variant B, HHV-7, HHV-8, JCV, parvovirus B19, SV-40, and VZV.

Adenovirus Assay Range

100 copies/ml to 1 x 10¹⁰ copies/ml

Tissue specimen results will be normalized  to copies/1,000 cells

Fecal specimen results will be in copies

Turnaround Time

Same day, (within 8 to 12 hours of receiving specimen) Monday through Saturday

Shipping

Ship Monday through Friday. Friday shipments must be labeled for Saturday delivery. All specimens must be labeled with patient’s name and collection date. A ViraCor Test Requisition form must accompany each specimen. Multiple tests can be run on one specimen.

Ship specimens FedEx Priority Overnight® to:

Pathogen Overview

ABOUT ADENOVIRUS

Adenoviruses are members of the Adenoviridae family and are nonenveloped, double-stranded DNA viruses comprised of 6 subgroups, A through F, based on common biologic, morphologic, and genetic features. To date, 51 serotypes have been identified. Adenovirus was first isolated in 1953 from adenoid tissue-derived cell cultures, which were observed to spontaneously degenerate over time.

ADENOVIRUS CLINICAL MANIFESTATIONS

Viral infection may occur through direct contact, inhalation of respiratory droplets, or ingestion. After recovery from an active infection, a patient may harbor persistent infections in the tonsils, adenoids, other lymphoid tissues, or intestines. Some adenoviruses establish persistent, asymptomatic infections, and viral shedding may occur for years. Infection most commonly occurs during childhood.

Severe infections are caused primarily by subgroup C adenoviruses. Specific serotypes have been associated with particular diseases. For example, serotypes 40 and 41 are commonly associated with gastroenteritis, while subgroup C serotypes 1, 2, 5, and 6 are commonly associated with respiratory tract illnesses and conjunctivitis in children. In immunocompromised patients, however, multiple localized infections can occur as a manifestation of disseminated infection from a single serotype.

Severe morbidity and mortality due to adenovirus infection affect both hematopoietic stem cell transplant (HSCT) and solid organ transplant recipients. Common illnesses due to adenovirus infection include hemorrhagic cystitis/nephritis, pneumonitis, hepatitis, liver failure, and gastroenteritis, particularly during the acute post-transplant period prior to engraftment. Symptoms of adenovirus infection vary widely, depending on the organ involved. Adenovirus nephritis was associated with acute renal failure in 90% of infected patients. Graft-versus-host disease (GVHD) is also associated with adenovirus infection, and allograft failure may occur in solid organ transplant recipients. Mortality rates of 60% among patients with pneumonia and 50% of patients with hepatitis have been reported.

Retrospective studies of HSCT patients found the incidence of adenovirus infection ranging from 4 to 20%, with similar percentages developing into invasive disease. However, among patients with T cell depleted or mismatched allografts, the infection rate increased to between 20 and 30%, with 30 to 40% of those patients developing invasive disease. This research confirms other reports that found T cell depletion of a graft was associated with a higher incidence of adenovirus infection after transplantation. Polymerase chain reaction (PCR) assays used to detect adenovirus DNA in peripheral blood have demonstrated a strong correlation between viremia and the risk of disseminated adenovirus disease.

Adenovirus infections are particularly associated with significant rates of morbidity and mortality in children who undergo HSCT. In one retrospective study of 328 pediatric HSCT recipients, 37 (11.3%) were infected with adenovirus within 6 months of the transplant procedure. Infection with adenovirus delayed recovery of immunity after HSCT, and infection resulted in 7 deaths (2.1% mortality). Another retrospective study found that over a 4-year period, 42 of 201 HSCT patients (21%) had positive adenovirus cultures post-transplant. The incidence of infection was significantly higher in pediatric patients than in adults. Pediatric patients also had an earlier onset of infection, averaging less than 30 days after transplantation, compared with an average onset of 90 days among adults. Moderate to severe acute GVHD and isolation of adenovirus from 2 or more sites were significant risk factors for developing adenovirus disease.

LABORATORY DIAGNOSIS

Several methods can be used to detect adenoviruses in patients, including virus isolation, serology, and molecular amplification methods, such as PCR. However, not all methods are appropriate for testing transplant recipients. Rates of seroconversion to adenoviruses are high because individuals commonly contract adenoviruses during childhood. Therefore, serology has limited diagnostic value in a clinical setting. Virus isolation through cell culture techniques is a slow process, often taking days to weeks for completion, limiting the clinical usefulness. Furthermore, virus isolation is technically demanding and requires careful sample handling to preserve virus infectivity and, thus, can have low diagnostic sensitivity and yield false negative results. Isolation of viruses may be particularly complicated in immunocompromised patients who have received transfusions of blood products that contain adenovirus antibodies. Additionally, virus isolation does not yield quantitative results, which limits the usefulness of cell culture isolation in monitoring the clinical progress of patients following treatment.

In contrast to these methods, PCR techniques are fast, and extremely sensitive and can identify adenoviruses using many different types of clinical specimens, such as whole blood, urine, cerebrospinal fluid, lung tissue, bone marrow aspirate, throat specimens and fecal specimens, among other fluids and tissues. Quantitative, real-time PCR allows accurate quantification of viral genomic DNA over a range of fewer than 10 copies to as many as 107 copies. These methods are rapid; results can be provided within 24 hours of specimen receipt. This approach provides a substantial advantage over other methods, since patients with viral loads over 10⁶ copies/ ml of adenovirus are considered to be at increased risk for fatal complications. In addition to providing early detection, real-time PCR can also be used to monitor infection progression and response to therapy.

TREATMENT

No specific antiviral therapy has been shown to produce a definite clinical effect against adenovirus infection. The efficacy of ribavirin and cidofovir are under evaluation;  vidarabine has been associated with poor outcomes. If antiviral therapy is used, it should be initiated early in the clinical course and used with restraint to avoid hematologic toxicity and nephrotoxicity associated with these therapies. High-dose intravenous immunoglobulin (IVIG) treatment has been successfully used to treat some patients, and donor leukocyte transfusions for solid organ transplant recipients may be useful as adjunctive treatment with antiviral therapy. Standardized treatment guidelines for transplant recipients have not been developed; treatment decisions should be made on an individual basis. Careful monitoring of viral load during treatment is necessary for optimal outcomes.

CONCLUSION

Although adenovirus can produce severe illness in transplant recipients, sensitive, rapid, quantitative, real-time PCR techniques can quickly and accurately identify the virus. This method allows early diagnosis and differentiation from other infections, giving patients the best chance for recovery. Early diagnosis and intervention provides the best chance of reducing the risk of illness and death among transplant recipients. Real-time PCR may provide the most effective means of detecting adenovirus infection early so that preemptive therapy can be administered.

Selected References

Claas EC, Schilham MW, de Brouwer CS, et al. Internally controlled real-time PCR monitoring of adenovirus DNA load in serum or plasma of transplant recipients. J Clin Microbiol. 2005;43(4):1738-1744.

Echavarria M, Sanchez JL, Kolavic-Gray SA, et al. Rapid detection of adenovirus in throat swab specimens by PCR during respiratory disease outbreaks among military recruits. J Clin Microbiol. 2003;41(2):810-812.

Flomenberg P, Babbitt J, Drobyski WR, et al. Increasing incidence of adenovirus disease in bone marrow transplant recipients. J Infect Dis. 1994;169(4):775-781.

Knipe D, Howley P. Fields Virology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2006. Suparno C, Milligan DW, Moss PA, Mautner V. 2004. Adenovirus infections in stem cell transplant recipients: recent developments in understanding of pathogenesis, diagnosis and management. Leuk Lymphoma. 2004;45(5):873-885.

Van Tol MJ, Kroes AC, Schinkel J, et al. Adenovirus infection in pediatric stem cell transplant recipients: increased risk in young children with a delayed immune recovery. Bone Marrow Transplant. 2005;36(1):39-50.

PAO-01-0707 PCR tests are performed pursuant to a license agreement with Roche Molecular Systems, Inc.

Abstracts & Publications

Carrigan DR. Adenovirus infections in immunocompromised patients. Am J Med. 1997;102(3A):71-74.

Adenovirus infections have been reported in as many as one-fifth of bone marrow transplant (BMT) recipients and patients with acquired immunodeficiency syndrome (AIDS), and in a lesser, though still prominent, proportion of organ transplant recipients. The relative contributions of primary infections versus reactivations from latency in immunocompromised patients remain unclear. Compared with adult BMT recipients, pediatric BMT recipients appear to be infected by adenovirus more frequently and earlier in the post-transplant period. The diagnosis of adenovirus infection is complicated by the existence of >40 viral serotypes, although certain subgroups are more likely to be involved in certain patient populations. Adenoviruses are responsible for a broad range of clinical diseases that may be associated with high mortality, including pneumonia, hepatitis, encephalitis, hemorrhagic cystitis, and gastroenteritis. The clinical and histopathologic features of adenovirus disease may resemble those of cytomegalovirus disease, potentially complicating the diagnosis. Risk factors for clinical adenovirus disease include the number of sites from which the virus is cultured and, in BMT recipients, the presence of moderate to severe acute graft-versus-host disease.

Echavarria M, Forman M, van Tol MJD, Vossen JM, Charache P, et al. Prediction of severe disseminated adenovirus infection by serum PCR. Lancet. 2001;(358):384-385.

Adenoviruses are increasingly recognized as viral pathogens that can cause fatal infections in Immunocompromised patients, particularly recipients of haematopoietic stem-cell grafts. Adenovirus infections are not easily diagnosed and the development of a severe infection cannot be predicted by standard culture techniques. In a pilot study, we investigated the value of adenovirus DNA detection in serum as a marker of disseminated disease in 14 patients with defined patterns of adenovirus infections. The results show that the appearance of adenoviral DNA in serum preceded the development of a severe or fatal adenovirus infection. Because proper management is dependent on early diagnosis and differentiation from other conditions, this test may be a valuable tool in the management of adenovirus infection.

Hale PA, Heslop HE, Krance RA, et al. Adenovirus infection after pediatric bone marrow transplantation. Bone Marrow Transplant. 1999;(23):277-282.

Retrospective analysis of 206 patients undergoing 215 consecutive bone marrow transplants (BMT) at St. Jude's Children's Research Hospital between November 1990 and December 1994 identified 6% (seven male, six female) with adenovirus infection. The affected patients had a median age of 7.9 years (range 3-24 years) at time of transplantation. Although transplants were performed for hematologic malignancies, solid tumors or non-malignant conditions, only patients with hematologic malignancies had adenoviral infections. Adenovirus was first detected at a median of 54 days (range -4 to +333) after BMT. Adenovirus developed in eight of 69 (11.6%) of patients receiving grafts from matched unrelated or mismatched related donors, in four of 52 (7.7%) receiving grafts from HLA-matched siblings, and in one of 93 (1.1%) receiving autografts. The most common manifestation of adenovirus infection was hemorrhagic cystitis, followed by gastroenteritis, pneumonitis and liver failure. The incidence of adenovirus infection in pediatric BMT patients at our institution is similar to that reported in adult patients. Using univariate analysis, use of total body irradiation and type of bone marrow graft were significant risk factors for adenovirus infection. Only use of total body irradiation remained as a factor on multiple logistic regression analysis.

La Rosa AM, Champlin RE, Mirza N, et al. Adenovirus infections in adult recipients of blood and marrow transplants. Clin Infect Dis. 2001;(32):871-876.

Adenoviruses are increasingly recognized pathogens that affect blood and marrow transplant (BMT) recipients. Experience with 2889 adult BMT recipients were reviewed to study the incidence, clinical spectrum, risk factors for dissemination, response to therapy, and outcome of adenovirus infections. Eighty-five patients (3%) were diagnosed by means of culture (n-85) or culture and histopathological examination (n=6). Nine patients had asymptomatic viruria, and 76 had symptomatic infections, which included upper respiratory tract infection (n=20), enteritis (n=18), hemorrhagic cystitis (n=10), pneumonia (n=15), and disseminated disease (n=13). The overall mortality rate was 26%. A higher mortality rate was observed among patients with pneumonia (73%) and disseminated disease (61%). Risk factors for dissemination included receipt of an allogenic transplant, presence of graft-versus-host disease (GVHD), and receipt of concurrent immunosuppressive therapy. Intravenous ribavarin was not associated with an appreciable benefit among 12 patients who received this treatment. In conclusion, adenovirus infections are an important cause of morbidity and mortality in adult BMT recipients, particularly allogeneic transplant recipients with GVHD who are receiving immunosuppressive therapy. The need for an effective, nontoxic antiviral therapy is apparent.

Manna P, Arnoldi S, Vats A, Skopec J, Brewer J, Flomenberg P. Clinical role of quantitative viral load monitoring for adenovirus in immunocompromised patients. Poster presented at: American Society for Blood and Marrow Transplantation; February 16, 2004.

Click here to view poster

Munoz FM, Piedra PA, Demmier GJ. Disseminated adenovirus disease in immunocompromised and immunocompetent children. Clin Infect Dis. 1998;(27):1194.

A retrospective review of adenovirus infections at Texas Children's Hospital during 1990-1996 was performed to evaluate the epidemiology, clinical course, management, and outcome of disseminated adenovirus disease (DAD) in children. DAD with multiorgan involvement occurred in 11 (2.5%) of 440 adenovirus-infected patients. Six (54%) of the 11 were Immunocompromised and 5 (45%) were Immunocompetent. Mortality was 83% among the immunodeficient, 60% in the Immunocompetent, and 73% overall. Two (28%) of the 7 patients receiving Immunoglobulins with or without antivirals and 3 (75%) of the 4 not treated died of DAD. DAD was caused by particular serotypes (3, 5 and 7) and occurred at a younger age in Immunocompetent children. Viremia and prolonged viral excretion were more common in the Immunocompromised. Clinical features and outcome were similar in both groups. Prospective studies addressing the use of new antiviral agents, combination antiviral therapy, and preventive strategies are necessary to determine the optimal therapeutic approach for patients with DAD.