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Quantitative Real-Time PCR for Detection of Adenovirus in Immunosuppressed Patients

(Reference ― 2013.03.008)

Notice of Assessment

April 2014

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1 GENERAL INFORMATION 1.1 Requestor

CHU Sainte-Justine

1.2 Application for Review Submitted to MSSS January 21, 2013

1.3 Application Received by INESSS November 1, 2013

1.4 Notice Issued February 28, 2014

Note: This notice is based on the scientific and commercial information submitted by the requestor and on a complementary review of the literature according to the data available at the time that this test was assessed by INESSS.

2 TECHNOLOGY, COMPANY, AND LICENCE(S) 2.1 Name of the Technology

Quantitative real-time polymerase chain reaction (PCR) for detection of adenovirus (AdV) in blood, respiratory, urine, fecal matter, and cerebrospinal fluid (CSF) samples.

2.2 Brief Description of the Technology, and Clinical and Technical Specifications The objective of the technique is to quantify virus particle copy numbers by measuring the number of AdV genome copies present in blood or other specific biological specimens. DNA is measured by quantitative PCR using an integrated standard curve generated from commercial quantified controls. This assay uses hydrolysis probes (TaqMan® MGB), and is carried out in triplicate on each clinical sample in 96-well plates with an ABI 7500 machine. The assay targets a unique conserved sequence of the Hexon gene and can detect 51 serotypes from 7 species (A to G) of AdV. The real-time PCR used by the requestor is based on one described by Heim et al. [2003]. The protocol was adapted in terms of the reaction mixture (master mix) and by the addition of an internal control (spike). Heim’s reaction mixture (FastStart Hybridization kit from Roche Diagnostics) was replaced with a kit from QIAgen with better performance. The requestor’s protocol is similar to the one used in the Viracor laboratory (Lee’s Summit, Montana, US). Real-time PCR analysis can identify and quantify 6 subgroups (A to F) and 51 serotypes of AdV.

2.3 Company or Developer In-house PCR protocol, developed and validated internally.

2.4 Licence(s) Not applicable.

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2.5 Patent, If Any Not applicable

2.6 Approval Status (Health Canada, FDA) Not applicable. In-house protocol.

2.7 Weighted Value 67.0

3 CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES 3.1 Targeted Patient Group

Immunosuppressed patients who have undergone allogeneic hematopoietic cell transplantation or solid organ transplantation, or who have a hematological cancer, solid cancer, or a primary or secondary immune deficiency, to detect and monitor disseminated AdV infections. Most target patients are children, but the test can also be performed on some adults. According to the requestor, it can also be used to investigate encephalitis and myocarditis.

3.2 Targeted Disease(s) Patients who undergo allogeneic hematopoietic cell transplantation or solid organ transplantation are at risk for a variety of bacterial, viral, and parasitic infections, depending on their degree of immunosuppression. The specific pathogens that cause these infections can vary depending on whether they occur before the transplant, immediately after the transplant (3 weeks to 3 months), or later (more than 3 months) [Anaissie, 2010]. Adenoviruses are included in pathogens responsible for infections in the immediate post-transplantation period, either as a primary infection or reactivation of a previous infection. Reactivation generally occurs within 30 days of transplantation in children, and more than 90 days after transplantation in adults. More than 80% of patients who undergo autologous and allogeneic transplantation experience a reactivation of an adenovirus infection, but this causes serious disease in less than 2% of cases. Adenoviral infection presents as pneumonitis, nephritis, diarrhea, and hemorrhagic colitis or cystitis. Infection can become generalized and cause multiple organ failure [Anaissie, 2010]. According to information provided by the requestor, there is a high incidence of adenovirus infection in children who have undergone allogeneic hematopoietic stem cell transplantation (between 20% and 26%) or solid organ transplantation. Between 1% and 7% of these infections can develop into a disseminated syndrome and result in death in 8% to 26% of cases. A recent review of the literature indicates an incidence of 8% to 50% in children who have undergone stem cell transplantation and a mortality rate of 3.2% to 6%. The incidence is lower in adults (from 2% to 5%), with a mortality rate of 1% or less [Ganzenmueller and Heim, 2012].

3.3 Number of Patients Targeted

The requestor estimates the number of quantitative PCR assays to detect adenovirus in blood at approximately 1,000 per year in Quebec. The majority of patients come from the adenovirus detection program for children who have undergone allogeneic hematopoietic cell transplantation

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3.4 Medical Specialties and Other Professions Involved Microbiology, pediatrics, hematology-oncology, molecular diagnostics.

3.5 Testing Procedure The test can be performed on whole blood, throat, urine, bronchoalveolar lavage (BAL), and cerebrospinal fluid (CSF) samples, and other normally sterile fluids. Collection methods depend on the type of sample.

4 TECHNOLOGY BACKGROUND 4.1 Nature of the Diagnostic Technology

The technology is unique; no tests performed in Quebec can identify adenoviruses in blood. The technology would mean that tests would no longer have to be sent outside Quebec. There are two tests for adenovirus in the Index:

41202 Adenovirus (rapid detection in a clinical specimen) (immunofluorescence). Micro-Viro. Supraregional assay; WV: 29.0. This is a qualitative test with low sensitivity that cannot be used on blood samples.

41203 Adenovirus (NAAT of a clinical specimen). Micro-Molecular. Supraregional assay; WV: 40.0. This qualitative conventional PCR is inadequate for monitoring of immunosuppressed patients.

4.2 Brief Description of the Current Technological Context The test is currently sent outside Quebec, either to Ontario (The Hospital for Sick Children, Toronto) or the United States (Viracor laboratory, Lee’s Summit, Montana) in exceptional cases. Data gathered by MSSS indicate that 80 PCR assays to detect adenovirus were performed in these centres between April 1, 2012 and March 31, 2013, at an average cost per test of almost C$200.

4.3 Brief Description of the Advantages Cited for the New Technology Performing the assay in Quebec would reduce costs and delays caused by sending the test outside the province. The results, which would be available in less than four working days, would make it possible to begin treatment more quickly and provide better monitoring, thereby reducing infection-related mortality.

4.4 Cost of Technology and Options Not assessed.

5 EVIDENCE 5.1 Clinical Relevance

Other Tests Replaced

No test is replaced, but conducting the test in Quebec would replace sending tests outside the province.

5.1.1 Diagnostic or Prognostic Value

Several retrospective studies examined the prognostic value of viral load for development of generalized infection and death. Some of these studies are presented in Table 1. A recent review of the literature on techniques and application of AdV viral load diagnostics by quantitative PCR indicated that blood viral loads > 104 copies/mL are predictive for disseminated AdV disease, although the threshold value is not yet well established. Determination of viral load is becoming an accepted method for monitoring AdV infection in immunosuppressed

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patients following allogeneic stem cell transplantation, permitting early diagnosis, prompt treatment initiation before the symptom onset, and monitoring of antiviral therapy. Studies are required to establish the viral load threshold [Ganzenmueller and Heim, 2012]. Without an absolute value correlating viral load and prognosis, viral kinetics should be monitored during certain critical periods rather than acting on an absolute value. The studies found examined the relationship between viral load and clinical results, but they do not provide results on the advantages of the test in terms of mortality or morbidity reduction, survival benefits, and quality of life. Additionally, no adverse effects were reported with respect to the test or testing.

5.1.2 Availability of Treatment and Treatment Modification Based on Test Results

Various antiviral medications have been suggested to treat AdV infections in immunosuppressed patients. These medications include cidofovir, ganciclovir, and ribavirin. Other treatments have also been proposed. Lindemans et al. [2010] published a treatment guide for adenoviral infections in immunosuppressed patients. They propose a treatment algorithm with cidofovir, either as prevention (or pre-emptive treatment) or secondary treatment. The algorithm reflects the patient’s risk of infection and viral load detected by quantitative PCR [Lindemans et al., 2010]. More recently, Ip and Qasim [2013] published a review of clinical management of children following allogeneic hematopoietic stem cell transplantation. They also suggest an algorithm a definition for a high-risk group at a threshold of 103 copies/mL, and different monitoring and treatment modalities based on risk and viral load [Ip and Qasim, 2013]. Cidofovir is not in the Quebec formulary. It is available through Health Canada’s Special Access Programme (SAP).

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Table 1: Viral load detected by quantitative PCR and relationship to clinical results

STUDY NUMBER OF

PATIENTS

SAMPLE VIRAL LOAD (copies/mL) CLINICAL OUTCOME DETECTION TIME

THRESHOLD RESULT

Ganzenmueller et al., 2011

38 A and C

Peripheral blood

1.0 × 104 > 1.0 × 104 Between 4.0 × 104 and 5.2 × 106

Between 1.6 × 104 and 5.2 × 107

Between 2.0 × 107 and 1.8 × 109

27/38 9/27 survivors

11/27 deaths unrelated to AdV 7/27 deaths AdV disease

Lion et al., 2010 153 C

Peripheral blood Fecal

matter

n.a. 51 cases + fecal matter 29: ≤ 1 × 106 copies/g

22: > 1 × 106 copies/g

87 cases –

1 case intestinal infection

5/29 deaths, none related to AdV

14/22 viremia 8/22 disseminated AdV disease 8/8 deaths associated with AdV

AdV disease or viremia: 0

Engelmann et al., 2009

21 C*

n.a. < 104

1 × 104 Between 9.9 × 103 and 4.2 × 108

8 cases + 6/8 asymptomatic 1 case of diarrhea

1 case of hepatitis and sepsis

2 weeks post-transplantation Pre-transplantation and 4

weeks post-transplantation

Erard et al., 2007 62 668 Plasma

n.a. Between 0 and 1.05 × 109 Between 0 and 1.74 × 108 Between 0 and 4.03 × 101

Gr 1: confirmed AdV disease (24)

Gr 2: AdV infection (19) Gr 3: not infected (1/19 patient

had 40.3 copies/mL)

Time between detection and diagnosis: 15 days (between 2

and 48 days) (Gr 1)

Claas et al., 2005 48 C

> 106 to 107

12/48 had + PCR 6/12 had disseminated

infection (at least 2 consecutive + PCR).

3/6 deaths; 2 associated with AdV

Risk of severe complications

Leruez-Ville et al., 2004

44 A and C

127 Plasma Other

n.a. Between 1.0 × 103 and 1.0 × 108 8/44 (38 plasma samples) + 8 disseminated AdV disease

3/8 deaths associated with AdV

Between 19 and 300 days post-transplantation

Abbreviations: A = adults; AdV = adenovirus; C = children; Gr = group; n.a. = not available *Hepatic transplantation.

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5.1.3 Therapeutic Value

Viral load detected by the test is used in treatment algorithms for prevention or secondary treatment.

5.2 Clinical Validity Several methods to detect adenovirus have been developed for diagnostic applications: viral culture, detection through immunospecific approaches and electron microscopy. However, these analytical approaches are inadequate for detecting adenovirus in the blood of immunosuppressed patients because of poor sensitivity, the time required for the technique, and difficulties intrinsic to blood samples. A recent review of the literature indicates that PCR-based assays are preferred for quantitative, rapid, sensitive, and specific detection of AdV in all types of specimens for immunosuppressed patients following allogeneic hematopoietic stem cell transplantation. PCR screening is required for high-risk patients [Matthes-Martin et al., 2013].

TERM PRESENCE ABSENCE NOT

APPLICABLE

Sensitivity x

Specificity x

Positive predictive value (PPV)

Negative predictive value (NPV)

Likelihood ratio (LR)

ROC curve

Accuracy

Table 2: Diagnostic performance

STUDY NUMBER OF

PATIENTS

OBJECTIVE VIRAL LOAD (copies/mL) Se Sp

Ganzenmueller et al., 2011

38 A and C

Identification of patients with fatal

outcome

Threshold: 1.3 × 107 in

peripheral blood 100 95

Erard et al., 2007 62 A and C

Detection of adenoviremia*

All Sustained and

increasing† ≥ 10

3 copies/mL

87.5 83.3 87.5

86.8 89.4 89.4

Abbreviations: A = adults; C = children; Se = sensitivity; Sp = specificity. * Proven cases with positive AdV culture. † At least two consecutive plasma samples confirmed positive for adenoviral DNA.

5.3 Analytical (or Technical) Validity The requestor has been performing routine quantitative PCR for AdV since December 2012. The protocol has undergone internal validation by comparison with other protocols in use in the United Kingdom, the commercially available R-Gene kit [Jeulin et al., 2010] and in-house cell cultures. The requestor also indicates that the method has undergone three series of external validation tests by CAP since it was implemented in 2012.

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TERM PRESENCE ABSENCE NOT APPLICABLE

Repeatability x

Reproducibility x

Analytical sensitivity x

Analytical specificity x

Matrix effect

Concordance x

Correlation between test and comparator x

Other depending on the type of test

5.3.1 Precision/Concordance

Comparison between an in-house method as described by Heim et al. [2003] and the Adenovirus R-Gene kit (Argene Inc, NT): 141/150 concordant results [Jeulin et al., 2010]. Comparison between an in-house PCR method and viral culture: 92.1% concordant results (139/151). Twelve discordant results: 8 respiratory specimens and 4 fecal matter specimens gave a negative culture result and a positive PCR result [Damen et al., 2008]. Comparison between the requestor’s protocol (modified Heim method) and viral culture (requestor’s data): 93.3 % concordant results (121/127); 6 discordant results: 4 negative by virology and undetermined by their method and 2 positive by virology and negative by PCR.

5.3.2 Repeatability and Reproducibility

Intra-assay variability: 2.7% for 1.5 × 105 copies/run and 1.1% for 1.5 × 104 copies/run.

5.3.3 Analytical Sensitivity Limit of Detection (LOD): 8 copies in the sample, with 50% positivity (6/12 runs), for 400 copies/mL. With the internal control, 16 copies were detected, with 42% positivity (5/12 runs), for a detection limit of 103 copies/mL [Damen et al., 2008]. Between 50 and 250 copies/mL [Claas et al., 2005]. Lower Limit of Detection (LLOD): viral load ≥ 1E + 3 particles/mL or /g. On cellular material: 10 particles per 1E + 6 cells [Ebner et al., 2005]. The results of the validation study for the method by Heim et al. [2003] showed very good analytical sensitivity as follows:

1.5 × 101 copies reliably detected;

1.5 × 100 copies occasionally detected;

higher dilutions: negative;

negative controls: negative result.

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5.3.4 Analytical Specificity Of 61 control samples containing different viruses, 4 (3 EBV1 and 1 BK virus) were positive for AdV [Damen et al., 2008].

No signal was obtained when CMV,2 EBV, herpesvirus, parvovirus, hepatitis B virus, hepatitis C virus, HIV,3 and BK virus (polyomavirus) were used as targets [Claas et al., 2005]. Marginal cross-reactivity between detection of subgroups ACF and BDE. Complete concordance of more than 200 specimens tested in parallel by two-reaction RQ-PCR and species-specific five-reaction RQ-PCR, and no false positives [Ebner et al., 2005].

5.3.5 Linear Regression

Linear dynamic range: between 5 × 103 and 5 × 108, regression coefficient: 0.991 (internal control: 104 copies; 12 [10-fold] serial dilutions) [Damen et al., 2008]. In the study by Heim et al. [2003], the linear range of quantitation was between 1.5 × 101 and 1.5 × 108 copies. The slope of –3.50 ± 0.075 indicates more than 93% efficiency of the amplification reaction [Heim et al., 2003].

5.4 Recommendations From Other Organizations Clinical guidelines for diagnosis and treatment of adenovirus infection in immunosuppressed patients, developed by the 4th European Conference on Infections in Leukemia [ECIL-4], indicate that quantitative PCR is the recommended technique for monitoring high-risk patients [Matthes-Martin et al., 2012]. Guideline recommendations for monitoring AdV infection are graded. Grades are indicated in parentheses and an explanation of each is presented in the Appendix. Patients undergoing allogeneic stem cell transplantation:

Routine monitoring of viral load is not recommended for low-risk patients, such as those receiving a transplant from an HLA-identical sibling (B II);

Quantitative PCR monitoring of viral load in peripheral blood is recommended on a weekly basis for patients with at least one risk factor (A II for children; B III for adults);

The monitoring period must be adjusted to the risk period based on the degree of immune reconstitution (B III children; C III adults).

Autologous stem cell transplantation:

Routine monitoring is not recommended (C II);

Quantitative PCR must be performed when AdV infection or disease is clinically suspected (C II). ECIL-4 also indicates that data from recent years show that quantitative PCR is more sensitive and predictive of adenoviral disease than culture or direct immunofluorescence.

1 EBV: Epstein-Barr virus. 2 CMV: cytomegalovirus. 3 HIV: human immunodeficiency virus.

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In clinical practice guidelines for preventing infectious complications in patients undergoing hematopoietic cell transplantation, published in 2009, the Infectious Diseases Society of America (IDSA) states that, for patients at very high risk of adenovirus infection,4 weekly monitoring for active adenovirus infection by PCR for either the first six months after transplantation or the duration of severe immunosuppression/lymphopenia could be considered. IDSA qualified the recommendation as optional (CII). Additionally, it recommended quantitative PCR testing for monitoring progression of infection and response to treatment (BII or Should generally be offered) [Tomblyn et al., 2009].

6 OUTCOMES OF INTRODUCING THE TEST 6.1 Impact on Material and Human Resources

Not analyzed.

6.2 Economic Consequences of Introducing Test Into Quebec's Health Care and Social Services System Not analyzed.

6.3 Main Organizational, Ethical and Other (Social, Legal, Political) Issues

Not analyzed.

7 IN BRIEF 7.1 Clinical Relevance

This is a useful test for detecting AdV in immunosuppressed patients. AdV infection is fatal for a large percentage of these patients. The test can provide early detection, allowing treatment to be provided for patients.

7.2 Clinical Validity Few of the studies found provide information on the test’s clinical validity in terms of sensitivity and specificity. When these data are available, they indicate good test performance. There is a relationship between viral load and clinical results, although there is no clearly established viral load threshold.

7.3 Analytical Validity Analytical validity has been thoroughly studied; the results from various studies and the validation conducted by the requestor indicate good performance.

7.4 Recommendations From Other Organizations At least two guidelines found recommend quantitative PCR monitoring of viral load in peripheral blood using different approaches based on the patient’s clinical condition.

4 Patients at very high risk of adenovirus infection: refractory graft versus host disease (GvHD), umbilical cord blood transplantation, haploidentical transplantation, stem cell graft T-cell depletion of > 2-3 log10, use of anti-T cell antibodies (e.g., antithymocite globulin, alemtuzumab).

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8 INESSS NOTICE IN BRIEF

Quantitative Real-Time PCR for Detection of Adenovirus in Immunosuppressed Patients

Status of the Diagnostic Technology

Established

Innovative

Experimental (for research purposes only)

Replacement for technology: , which becomes obsolete

INESSS Recommendation

Include test in the Index

Do not include test in the Index

Reassess test

Additional Recommendation

Draw connection with listing of drugs, if companion test

Produce an optimal use manual for children and adults

Support collection of clinical results data

NOTE Suggestion for the Index ―

Discipline: Microbiology

Subsection: Molecular diagnostics.

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REFERENCES

Anaissie E. Overview of infections following hematopoietic cell transplantation. Waltham, MA: Wolters Kluwer Health; 2010. Available at: http://www.uptodate.com.

Claas ECJ, Schilham MW, de Brouwer CS, Hubacek P, Echavarria M, Lankester AC, 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):1738-44.

Damen M, Minnaar R, Glasius P, van der Ham A, Koen G, Wertheim P, et al. Real-time PCR with an internal control for detection of all known human adenovirus serotypes. J Clin Microbiol 2008;46(12):3997-4003.

Ebner K, Suda M, Watzinger F, Lion T. Molecular detection and quantitative analysis of the entire spectrum of human adenoviruses by a two-reaction real-time PCR assay. J. Clin Microbiol 2005;43(7):3049-53.

Engelmann G, Heim A, Greil J, Schmitt CP, Flechtenmacher C, Daum E, et al. Adenovirus infection and treatment with cidofovir in children after liver transplantation. Pediatr Transplant 2009;13(4):421-8.

Erard V, Huang ML, Ferrenberg J, Nguy L, Stevens-Ayers TL, Hackman RC, Corey L, Boeckh M. Quantitative real-time polymerase chain reaction for detection of adenovirus after T cell-replete hematopoietic cell transplantation: Viral load as a marker for invasive disease. Clin Infect Dis 2007;45(8):958-65.

Ganzenmueller T and Heim A. Adenoviral load diagnostics by quantitative polymerase chain reaction: Techniques and application. Rev Med Virol 2012;22(3):194-208.

Ganzenmueller T, Buchholz S, Harste G, Dammann E, Trenschel R, Heim A. High lethality of human adenovirus disease in adult allogeneic stem cell transplant recipients with high adenoviral blood load. J Clin Virol 2011;52(1):55-9.

Heim A, Ebnet C, Harste G, Pring-Akerblom P. Rapid and quantitative detection of human adenovirus DNA by real-time PCR. J Med Virol 2003;70(2):228-39.

Ip W and Qasim W. Management of adenovirus in children after allogeneic hematopoietic stem cell transplantation. Adv Hematol 2013;2013:Article ID 176418.

Jeulin H, Salmon A, Bordigoni P, Venard V. Comparison of in-house real-time quantitative PCR to the Adenovirus R-Gene kit for determination of adenovirus load in clinical samples. J Clin Microbiol 2010;48(9):3132-37.

Leruez-Ville M, Minard V, Lacaille F, Buzyn A, Abachin E, Blanche S, et al. Real-time blood plasma polymerase chain reaction for management of disseminated adenovirus infection. Clin Infect Dis 2004;38(1):45-52.

Lindemans CA, Leen AM, Boelens JJ. How I treat adenovirus in hematopoietic stem cell transplant recipients. Blood 2010;116(25):5476-85.

Lion T, Kosulin K, Landlinger C, Rauch M, Preuner S, Jugovic D, et al. Monitoring of adenovirus load in stool by real-time PCR permits early detection of impending invasive infection in patients after allogeneic stem cell transplantation. Leukemia 2010;24(4):706-14.

Matthes-Martin S, Boztug H,Lion T. Diagnosis and treatment of adenovirus infection in immunocompromised patients. Expert Rev Anti Infect Ther 2013;11(10):1017-28.

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Matthes-Martin S, Feuchtinger T, Shaw PJ, Engelhard D, Hirsch HH, Cordonnier C; Fourth European Conference on Infections in Leukemia. European guidelines for diagnosis and treatment of adenovirus infection in leukemia and stem cell transplantation: Summary of ECIL-4 (2011). Transpl Infect Dis 2012;14(6):555-63

Tomblyn M, Chiller T, Einsele H, Gress R, Sepkowitz K, Storek J, et al. Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: A global perspective. Biol Blood Marrow Transplant 2009;15(10):1143-238.

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APPENDIX

Grading Scale for Evidence and Recommendations Used by ECIL-4 [Matthes-Martin et al., 2012]. Quality of evidence I Evidence from ≥1 properly randomized, controlled trial II Evidence from ≥1 well-designed clinical trial, without randomization; from cohort or case-controlled analytic studies (preferably from >1 center); from multiple time-series; or from dramatic results from uncontrolled experiments III Evidence from opinions of respected authorities, based on clinical experience, descriptive studies, or reports of expert committees Strength of recommendation A. Good evidence to support a recommendation for use B. Moderate evidence to support a recommendation for use C. Poor evidence to support a recommendation