Clinical Indications

Findings suggestive of community-acquired pneumonia (CAP) include productive cough, fever, pleuritic chest pain, hypoxemia, and an imaging study demonstrating a pulmonary infiltrate.

Microbiologic tests to determine the specific cause of community-acquired pneumonia are often negative; however, if the etiology of CAP is determined, empiric therapy can be optimized to decrease morbidity and mortality. De-escalation of therapy can reduce pressure on the emergence of drug resistance and avoid toxic drug effects.

Sputum and blood cultures (prior to initiation of antimicrobial therapy) are recommended for the evaluation of patients with suspected CAP. Recovery of an organism confirms the diagnosis and allows in vitro susceptibility testing to be performed.

Gram stain of sputum is helpful to guide therapy and for correlation with culture results.

Antigen testing is useful when empiric antimicrobial therapy prevents culture confirmation of pneumococcal disease. Urine antigen testing for S. pneumoniae and Legionella pneumophila are recommended for patients with severe CAP.^[1]

The S. pneumoniae antigen test has a specificity of >90% and a sensitivity of 50-80% for the diagnosis of pneumococcal pneumonia in adults.^[1] However, urinary antigen tests are not recommended for the diagnosis of pneumococcal pneumonia in children because false positive tests are common and attributed to nasopharyngeal colonization with S. pneumoniae.^{[2, 3]}

Methodology

Cleveland Clinic Laboratories uses a qualitative immunochromatographic membrane assay (BinaxNOW^® Streptococcus pneumoniae) to detect pneumococcal soluble antigen in human urine. The S. pneumoniae antigen test provides a rapid, simple method for the presumptive diagnosis of pneumococcal pneumonia.

A urine specimen should be collected prior to antimicrobial therapy in a clean, leak-proof container. The specimen may be stored at room temperature if assayed within 24 hours of collection.

Urine is stable for two weeks if refrigerated or frozen. Boric acid may be used as a preservative; however, other preservatives are unacceptable.

Interpretation & Limitations of the Assay

A positive result is presumptive evidence of pneumococcal pneumonia. Correlation of test results with clinical findings is required.

A negative result does not exclude infection by S. pneumoniae since the antigen present in the sample may be below the detection limit of the test.

This test has not been evaluated on patients taking antibiotics for more than one day, or on patients who recently completed a course of antibiotic therapy.^[4]

Cross-reactivity with closely-related bacteria in the Streptococcus mitis group may occur.

Streptococcus pneumoniae vaccine may cause false positive results within two days following vaccination, and is not recommended within five days following pneumococcal vaccination.

This test has only been validated for urine samples.

Antigen testing is not recommended for the diagnosis of pneumococcal pneumonia in children.^{[2, 3]}

References

1. Mandell LA, Wunderink RG, Anzueto A, et al. Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis. 2007 Mar 1; 44 (Suppl 2):S27-72.

2. Dowell SF, Garman RL, Liu G, et al. Evaluation of Binax NOW, an assay for the detection of pneumococcal antigen in urine samples, performed among pediatric patients. Clin Infect Dis. 2001;32:824-825.

3. Bradley JS, Byington CL, Shah SS, et al. The management of community-acquired pneumonia in infants and children older than 3 months of age: clinical practice guidelines by the Pediatric Infectious Diseases Society and the Infectious Diseases Society of America. Clin Infect Dis. 2011;53(t):e25-e76.

4. BinaxNOW® Streptococcus pneumoniae, Package Insert, Alere, Ref. IN710050, Rev. 3, 5/7/10.

Targeted Gene Regions

Genes interrogated, including relevant transcripts and exons, are listed in alphabetical order.

Clinical Indications

This test is intended for the diagnosis of benign or malignant mesenchymal tumors (sarcomas and their benign mimics) as well as other solid tumors.

Interpretation

The results of this test are to be interpreted in the context of the histological, immunohistochemical, and clinical features of the neoplasm.

Methodology

This test relies on Anchored Multiplex PCR (AMP™) technology to generate scalable, target-enriched libraries for NGS from formalin-fixed, paraffin-embedded tissue sections.

In AMP, unidirectional gene-specific primers (GSPs) are used to enrich libraries for known and unknown mutations. Adapters that contain both molecular barcodes and sample indices enable quantitative multiplex data analysis, read de-duplication, and accurate variant calling. Libraries are sequenced on the Illumina MiSeq instrument, which employs “sequencing by synthesis;” a fluorescence, image-based, reversible-terminator technology to sequence targeted regions of the 59 genes included in the panel.

Sequencing data are analyzed for fusion variant detection using Archer® Analysis bioinformatics tools. Specimen quality control is monitored and recorded by in-house developed software (scripts). Raw sequencing data are de-multiplexed based on a unique index sequence using the Illumina bcl2fastq program. The fastq.gz files are de-duplicated according to the unique molecular barcode present and aligned to the human reference genome hg19. Part of the fusion calling and annotation is performed utilizing the Archer® Quiver™ Fusion Database.

Limitations

This test does not detect missense mutations, insertions, deletions, or copy number changes, and does not distinguish between variants that are inherited versus acquired.

References

1. Archer Dx, FusionPlex Anchored MultiPlex PCR (AMP) technology http://archerdx.com/fusionplex/ [Accessed: July 2018]

2. MiSeq System user Guide, Publication Number 15027617 Rev.0. Illumina, San Diego, CA. 9/2014.

3. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®), Soft Tissue Sarcoma, version 1.2019

4. Taylor BS, Barretina J, Maki RG, Antonescu CR, Singer S, Ladanyi M. Advances in sarcoma genomics and new therapeutic targets. Nat. Rev. Cancer. Jul 14 11(8), 541-57 (2011).

Targeted Gene Regions

Genes interrogated, including relevant transcripts and exons, are listed in alphabetical order.

Clinical Indications

This assay is intended for patients with known or suspected hematologic neoplasms including myelodysplastic syndromes, myeloproliferative neoplasms, acute myeloid leukemia, acute lymphoblastic leukemia, and selected mature lymphoid leukemias.

Interpretation

All variants are classified using Association for Molecular Pathology guidelines for interpretation of somatic variants in cancer.^[8]

Detailed interpretations are provided for each variant, and an overall interpretation of the entire mutational profile summarizes the case findings.

Reported variants include those of strong or potential clinical significance as well as variants of unclear clinical significance.

Known benign polymorphisms are not reported.

Methodology

Nucleic acid extracted from the specimen is subjected to nested multiplex PCR-based target enrichment.

Coding and non-coding regions of targeted genes are amplified and sequenced on an Illumina instrument (San Diego, CA) with paired-end, 150×2 cycle reads.

A customized bioinformatic analytical pipeline is used to map reads to the reference human genome (Genomic Build GRCh37/hg19).

Limitations

This test does not detect structural variants or copy number changes and does not distinguish between variants that are inherited versus acquired.

During internal validation, this test delivered an average of >500X coverage and >98% of targeted regions showed over 100X coverage. The test demonstrated 95.2% sensitivity and 99.9% specificity in identifying single nucleotide variants, small insertions and deletions (indels) (≤10bp) of >5% variant allele fraction (VAF). For the identification of large indels (>10bp) at >5% VAF the test demonstrated 87.5% sensitivity and 99.9% specificity.

Due to the limitations of next-generation sequencing technology, some large insertions may not be detected.

References

1. Papaemmanuil E, Gerstung M, Malcovati L, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013 Nov 21; 122(22):3616-27.

2. Bejar R, Stevenson K, Abdel-Wahab O, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med. 2011 Jun 30;364(26):2496-506.

3. Döhner H, Weisdorf DJ, Bloomfield CD. Acute Myeloid Leukemia. N Engl J Med. 2015 Sep 17;373(12):1136-52.

4. Nazha A, Zarzour A, Al-Issa K, et al. The complexity of interpreting genomic data in patients with acute myeloid leukemia. Blood Cancer J. 2016 Dec 16;6(12):e510.

5. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016 May 19;127(20):2391-405.

6. “NCCN Guidelines for Treatment of Cancer by Site,” National Comprehensive Cancer Network: https://www.nccn.org/professionals/physician_gls/default.aspx#site.

7. McClure RF, Ewalt MD, Crow J, et al. Clinical Significance of DNA Variants in Chronic Myeloid Neoplasms: A Report of the Association for Molecular Pathology. J Mol Diagn. 2018 Nov;20(6):717-737. DOI: 10.1016/j.jmoldx.2018.07.002. Epub 2018 Aug 20.

8. Li MM, Datto M, Duncavage EJ, et al. Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn. 2017 Jan;19(1):4-23.