JC Polyoma Virus DNA, Quantitative, Real-Time PCR, CSF

JC Polyoma Virus DNA, Quantitative, Real-Time PCR, CSF is used for the quantification of JC polyomavirus DNA. The assay is based on real-time PCR amplification and detection of JCV genomic DNA. The quantifiable reportable range is 50 to 50,000,000 IU/mL (1.70 to 7.70 Log IU/mL) and the LOD of the assay is 25 IU/mL based on a 95% detection rate and 16 IU/mL based on probit analysis.

The LOD and LOQ are critical elements for interpreting the performance of real-time PCR assays. LOD refers to the lowest concentration of JCV DNA in cerebrospinal fluid (CSF) that the assay can reliably detect with 95% confidence. It indicates the minimum amount of viral DNA that can be detected, even if it cannot be precisely quantified.

LOQ is the lowest concentration of JCV DNA that the assay can accurately quantify in a specimen with an acceptable level of precision and accuracy. This quantity is reported in international units per milliliter (IU/mL). The LOQ is often higher than the LOD. A result may be reported as detected but less than the LOQ (ie, for JCV, it would be reported as Detected <50 IU/mL).

A “Not Detected” result should not be considered equivalent to absence of infection with JCV, particularly when clinical suspicion is high. A “Not Detected” result can also suggest that the genomic DNA present in the sample is below the level of detection. In early-stage progressive multifocal leukoencephalopathy (PML), the JCV viral load in CSF may be extremely low, resulting in a false-negative PCR result.^1,2

The current JC Polyoma Virus DNA, Quantitative, Real-Time PCR, CSF assay (test code 16442) has similar analytical performance as the discontinued Ultra-Sensitive JC Virus Quantitative PCR (discontinued test code 18939). The quantifiable reportable range for the current assay is 50 to 50,000,000 IU/mL (1.70 to 7.70 Log IU/mL), compared to the reportable range of the discontinued Ultra-Sensitive assay of 50 to 1,000,000 IU/mL (1.70 to 6.00 Log IU/mL). The limit of detection (LOD) of both assays is 25 IU/mL based on a 95% detection rate and 15 to 16 IU/mL based on probit analysis.

A significant advantage of the current test offering is the reduction of irreplaceable cerebrospinal fluid (CSF) volume requirements (0.3 mL) compared to the ultrasensitive version (0.5 mL) with equivalent performance. As technology for nucleic acid extraction and PCR amplification has improved over the years, so has the performance of the assay, allowing for a more streamlined test offering. 

No, comparing viral load results across laboratories is not recommended. Significant variability in the following elements may result in discrepancies between laboratories:

• Calibration material may not be standardized to measure the viral load.
• Testing platforms and analytical performance may not be equivalent (including target of amplification; target recovery; amplification efficiency, stability, and analytical performance; Limit of Detection [LOD] and Limit of quantification [LOQ]; and even units of measure across different laboratories and assays).
• Units of measurement and analytical measurement range may not be equivalent.

No, cp/mL is not equivalent to IU/mL. An IU is a carefully developed unit of measurement based on World Health Organization (WHO) international standards. WHO standards allow for assays to use the same calibration material, thus using the same reference to measure a viral load. The 1st WHO International Standard for JC polyomavirus (JCV) is intended for the standardization of nucleic acid amplification test (NAAT)-based assays for JCV. It should be used primarily for the calibration of secondary and/or in-house working standards. The WHO material has been evaluated in a worldwide collaborative study involving 23 laboratories using a range of JCV NAAT-based tests and was subsequently established by the WHO Expert Committee on Biological Standardization (ECBS).³ A conversion factor for cp/mL to IU/mL is not possible unless a specific assay using cp/mL is empirically compared to an assay calibrated using WHO standards in IU/mL. 

A "Not Detected" JC polyomavirus (JCV) PCR result, particularly if clinical suspicion remains high, should be considered in the context of the clinical picture. The presence of small PML lesions with a negative PCR result may be a consequence of a viral load under the limit of detection (LOD) and may require additional testing. All test results must be evaluated within the context of the patient's individual immunological status, treatment history, and overall clinical course. In such cases, clinicians may consider the following^1,2:

• Repeat testing using another collection of cerebrospinal fluid (CSF)
• Consider conducting a brain biopsy if clinical and radiological suspicion remains strong 

In natalizumab-treated multiple sclerosis (MS) patients, routine MRI surveillance facilitates the diagnosis of PML at a very early, asymptomatic, or oligosymptomatic stage. In CSF, patients with MS on natalizumab can have viral loads below the detection limit.^4,5 Retrospective reviews show that 10% to 16% of later-confirmed natalizumab-related PML cases have undetected JCV DNA in their CSF at initial presentation. ⁴ Therefore, PML diagnosis is based on a combination of clinical, immunological, and radiological factors.^1,2.

The clinical performance of test code 16442 has not been evaluated. This test was developed and its analytical performance characteristics have been determined by Quest Diagnostics. It has not been cleared or approved by the US Food and Drug Administration (FDA). This assay has been validated pursuant to the CLIA regulations and is used for clinical purposes.