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Background Information

The herpes simplex virus (HSV) is a common sexually transmitted disease virus that infects the skin, lips, oral cavity, eyes, genital tract, and central nervous system. HSV disease can range from the usual mild illness, indistinguishable in most patients, to sporadic, severe, and life-threatening disease in infants.

Infections with HSV-1 and HSV-2 can differ somewhat in their clinical manifestations and severity. HSV-1 is more frequently associated with orolabial infection (cold sores), whereas HSV-2 primarily is more frequently associated with urogenital infections in adults. Although there are differences in the frequency of infections at particular sites, in reality, both viral subtypes can cause either of these diseases.¹

HSV also can cause viral central nervous system (CNS) disease. For example, HSV-1 is the most common cause of sporadic encephalitis that, if left untreated, has a high mortality rate. HSV-2 is a cause of meningitis, which occurs most frequently among young adults. Both HSV subtypes are also a cause of neonatal disease acquired during exposure of the infant to HSV-infected secretions during birth.

Early and rapid diagnosis of HSV meningitis/meningoencephalitis is important to direct therapy and minimize the suffering in affected patients. It also helps to reduce empiric antibacterial therapy and the likelihood of side effects from unnecessary therapy. In the past, diagnosis relied on brain biopsy. Fortunately, new molecular diagnostic tests, such as rapid-cycle PCR on cerebrospinal fluid, have virtually replaced the need for brain biopsy.²

Clinical Indications

Laboratory testing with PCR is routinely used as an aid in the diagnosis of HSV infections for patients with meningitis and meningoencephalitis. Despite its prevalence, HSV remains an underdiagnosed disease, and early diagnosis and detection results in improved patient care.

This test should not be used to screen patients not exhibiting signs or symptoms supportive of an HSV infection.

Limitations

Although HSV PCR represents a significant advance in the diagnosis of HSV CNS disease, a negative result does not eliminate the possibility of HSV infection.

In some patients, HSV DNA may only be present in cerebrospinal fluid for three to four weeks after the initial presentation of symptoms, and DNA levels may be undetectable over a period of time. Although false-positive results may occur, they are rare. Repeat testing should be considered for patients with a low likelihood of HSV infection if a false-positive reaction is suspected.

Methodology

A polymerase chain reaction (PCR) test on spinal fluid can detect the genetic material (DNA) of the HSV virus. This assay also differentiates the HSV-1 and HSV-2 subtypes.

Nucleic acid amplification assays, such as PCR, are the best methods for testing spinal fluid for the presence of HSV when HSV meningitis or meningoencephalitis is suspected.

The PCR test detects viral DNA in specimens and is quicker, safer, and more sensitive and specific than earlier methods. Cleveland Clinic Laboratories uses rapid-cycle PCR, which is considered the gold standard for detection of HSV in CSF specimens.

References

1. Genital Herpes: Centers for Disease Control and Prevention. Retrieved 7/18/06. Available from http://www.cdc.gov/std/healthcomm/fact_sheets.htm.

2. Herpes simplex virus infections of the central nervous system. Kimberlin DW. Semin Pediatr Infect Dis. 2003. Apr;14(2):83-9.

Order a Bone Marrow Biopsy Consultation Kit

Chromosomal Single Nucleotide Polymorphism (SNP) Microarray for Hematology on Bone Marrow

Technical Brief

Chromosomal Single Nucleotide Polymorphism (SNP) Microarray for Hematology on Bone Marrow

Test Name

Bone Marrow Cancer Chromosome Microarray + SNP (BMHSNP)

CPT Code

81406

Methodology

Genomic microarray

Turnaround Time

14 days

Specimen Requirements

Type:
Bone marrow

Volume:
4 mL

Specimen Container:
Lavender BD Hemogard™ K₂EDTA Tube

Transport Temperature:
Ambient

Collect the specimen Monday–Friday only.

Type:
Bone marrow

Volume:
4 mL

Specimen Container:
Green BD Hemogard™ Sodium Heparin Tube

Transport Temperature:
Ambient

Collect the specimen Monday–Friday only.

Specimen Collection & Transport

Pathology Consultation Kits

For hassle-free specimen shipping, our laboratories can provide clients with Bone Marrow Biopsy Pathology Consultation Kits.

Each kit includes the following:

– (1) White CCL box (6.5 x 6 x 2.75 inches)
– (2) Slide holders
– (2) 10% neutral buffered formalin
– (2) Lavender K₂EDTA tubes
– (2) Green Sodium Heparin tubes
– (1) Foam insert
– (1) Specimen bag
– (1) Absorbent sheet
– Shipping Label and Bag

Stability

Ambient:
48 hours

Refrigerated:
Unacceptable

Frozen:
Unacceptable

Additional Information

Background

Background Information

Chromosomal microarrays are applied for the detection of genomic and allelic imbalances in hematological malignancies and solid tumors. Chromosomal microarray (CMA) and single-nucleotide polymorphism array (SNP-A) provide a combination of karyotyping and FISH with whole-genome scan and a targeted approach at a higher resolution.

Cleveland Clinic Laboratories uses a cancer-specific microarray designed by the Cancer Cytogenomics Microarray Consortium (CCMC) that targets genomic regions associated with cancer. This array can be used for diagnostic testing of hematological malignancies. Common indications include, but are not limited to, acute and chronic leukemia, myelodysplastic syndrome (MDS), and myeloproliferative disease.

This array has the ability to detect genome-wide copy number variants (CNV) with simultaneous detection of loss of heterozygosity (LOH), also known as copy neutral LOH (cnLOH). The array contains approximately 20,000 cancer-associated 60-mer CGH probes covering more than 500 cancer-related genes (1 probe per 0.5-1kb) in addition to 60,000 single nucleotide polymorphism (SNP) probes and backbone probes evenly distributed across the genome. This analysis can interrogate the whole genome for recurrent and novel clinically-relevant copy number variation (CNV) at an increased resolution over FISH or chromosome analysis.

Chromosomal microarray aids in diagnosis, prognosis, and therapeutic management by identifying gains, losses, or LOH in hematological disorders. It is also helpful in monitoring disease progression. Chromosome analysis or karyotyping for detection of genetic abnormalities is hindered by suboptimal cell growth or chromosome morphology, and clonal or subtle unbalanced chromosomal abnormalities may be missed. FISH provides an improved rate of detection of clonal abnormalities when compared to karyotyping, but only for the targeted region.

Chromosome or FISH testing cannot detect copy-neutral events that are associated with hematological disorders, which are often due to mutations and subsequent selection of mutant tumor-suppressor genes or oncogenes. However, SNP microarray detects submicroscopic chromosomal variants involving gains or losses in chromosomes across the genome in addition to LOH.

Clinical Indications

This array can be used for diagnostic testing of hematological malignancies for myeloid and lymphoid disease. Common indications include, but are not limited to, acute and chronic leukemia, myelodysplastic syndrome (MDS), and myeloproliferative disorders. The assay can assess prognosis and monitor disease progression and response to therapy (loss/gain of genomic DNA; loss of heterozygosity (LOH)).

Methodology

Designed by the Cancer Cytogenomics Microarray Consortium (CCMC), the Agilent SurePrint G3 Cancer CGH + SNP 4x180K Microarray Kit is a cancer-specific microarray designed to target genomic regions associated with cancer. This array has the ability to detect genome-wide copy number variants (CNV) while simultaneously detecting LOH.

The array contains 20,000 cancer-associated 60-mer CGH probes covering more than 500 cancer-related genes (1 probe per 0.5-1Kb) in addition to 60,000 single nucleotide polymorphism (SNP) probes and backbone probes evenly distributed across the genome.

Interpretation

A written summary and interpretation of the microarray findings are provided in the Test Overview:

1. Normal.

2. Pathogenic.

3. Unclear clinical significance, likely pathogenic.

4. Unclear clinical significance, not otherwise specified.

5. Unclear clinical significance, likely benign.

Limitations

Microarray analysis cannot detect balanced rearrangements like translocations, inversions, and insertions that may be important for diagnosis and prognosis of hematological disorders.

In addition:
• Low-level mosaicism may not be detected.
• May not be appropriate for individuals with expected lower levels of malignant cells.
• Not recommended for minimal residual disease

The assay does not detect:
• Balanced chromosomal rearrangements.
• Base pair mutations and small deletions/duplications.
• Positional information for chromosome rearrangements.
• Low-level mosaicism (small clones).

Therefore, microarray findings should be interpreted together with other concurrent test results, such as flow cytometry, morphology, FISH, chromosome analysis, and other studies
as appropriate.

The chromosomal SNP Array for hematology may or may not detect low-level mosaicism; therefore, this test is not recommended for detecting minimal residual disease.

Lipoprotein (a) in Serum

Technical Brief

Lipoprotein (a) in Serum

Test Name

Lipoprotein (a) (LPA)

CPT Codes

83695

Clinical Information

Evaluation of coronary artery disease risk associated with elevations of the atherogenic lipoprotein (a).

Methodology

Nephelometry (NEPH)

Turnaround Time

1 – 2 days

Specimen Requirements

Type:
Serum

Volume:
1 mL

Minimum Volume:
0.5 mL

Specimen Container:
Gold BD Hemogard™ Serum Separation Tubes (SST)™

Transport Temperature:
Refrigerated

Allow specimen to clot completely at room temperature. Separate serum from cells within 2 hours of collection.

Stability

Ambient:
8 hours after separation from cells

Refrigerated:
14 days after separation from cells

Frozen:
30 months after separation from cells

Patient Preparation

Patients should fast for at least 12 hours before blood is drawn.

Reference Range

0-40 mg/dL

Additional Information

Background

Background Information

Lipoprotein (a) [Lp(a)] is a spherical lipid particle that is genetically determined and remains at relatively constant levels over an individual’s lifetime. It contains two crosslinked proteins as part of its structure: apolipoprotein(a) covalently bound to apolipoprotein B-100. Lp(a) is important as a serum marker for coronary artery disease independent of diet and lipid levels. Elevated Lp(a) levels are associated with increased risk and severity of atherosclerosis, coronary heart disease, and stroke.

Similar to LDL-cholesterol, Lp(a) is synthesized in the liver. Although Lp(a) shows some homology to LDL-cholesterol in structure, Lp(a) differs from LDL in molecular weight, electrophoretic mobility, and protein/lipid ratio. Physiologic circulating levels of Lp(a) do not appear to be regulated by the same mechanisms of LDL-cholesterol. Likewise, cholesterol feeding does not appear to increase levels of Lp(a) in plasma, although it does increase levels of LDL cholesterol. Most pharmacologic agents that have an effect on lowering LDL-cholesterol levels have little effect on levels of Lp(a), thus also indicating regulation under different metabolic control.

The causes of high Lp(a) are kidney disease and certain family (genetic) lipid disorders.

Clinical Indications

Patients with a family history of elevated Lp(a) and/or a family history of premature cardiovascular disease that is not explained by high LDL or low HDL.

Also used for heart disease patients with a normal lipid profile and mildly elevated cholesterol and/or low-density lipoprotein cholesterol (LDL-C), as it is believed that an elevated Lp(a) may worsen other heart and vascular disease processes.

An elevated Lp(a) may suggest the need for more aggressive treatment of LDL and other, more treatable risk factors down to acceptable levels.

Limitations

For the most accurate results, wait at least two months after a heart attack, surgery, stroke, infection, injury, or pregnancy to check blood level.

In general, lipids should not be measured right after excessive alcohol intake, with severely uncontrolled diabetes, or during rapid weight loss.

Methodology

Lipoprotein (a) in serum is quantitatively measured on the IMMAGE 800 Immunochemistry system by rate nephelometry. Antibody to human Lp(a) is brought into contact with Lp(a) in a sample.

The IMMAGE 800 Test measures the rate of increase in light scattered from particles suspended in solution as a result of complexes formed during an antigen-antibody reaction.

The increase in light scatter resulting from the antigen-antibody reaction is converted to a peak rate signal, which is a function of the sample Lp(a) concentration. Following calibration, the peak rate signal for a particular assay is automatically converted to concentration units by the analyzer.

References

1. IMMAGE 800 Immunochemistry System Operations Manual, Instructions #A11403, March 2004, Beckman Coulter Instruments, Inc., Fullerton, CA 92834-3100.

2. Beckman Coulter IMMAGE 800 Immunochemistry System, Chemistry Information Manual, Beckman Coulter Instructions #962248, March 2000, Beckman Coulter Instruments, Inc., Fullerton, CA 92834-3100.

3. Tietz, NW. Specimen Collection and Processing: Sources of Biological Variation. Textbook of Clinical Chemistry. WB Saunders, Philadelphia, PA. 1986;478-518.

4. National Committee for Clinical Laboratory Standards. Procedures for the Handling and Processing of Blood Specimens, Approved Guideline. NCCLS publication H18-A, Villanova, PA.1990.

5. Schreiner, JP, Heiss G, Tyroler HA, Morrisett JD, Davis CD, Smith R. Race and Gender Differences in the Association of Lp(a) with Carotid Artery Wall Thickness: The Atherosclerosis Risk in Communities (ARIC) Study. Arterioscler Thromb Vasc Biol. 1996;16:471-478.

6. National Committee for Clinical Laboratory Standards, How to Define, Determine, and Utilize Reference Intervals in the Clinical Laboratory: Proposed Guideline. NCCLS publication C28-P, Villanova, PA. 1990.

7. Tietz, NW. Clinical Guide to Laboratory Tests. 2nd ed, WB Saunders, Philadelphia, PA. 1990.

8. Henry JB, ed. Clinical Diagnosis and Management by Laboratory Methods. 17th edition. 1984.

9. Statland, Bernard E. Clinical Decision Levels for Lab Tests. Medical Economic Book, Oradel, New Jersey.1983.

10. Tietz, NW, ed. Fundamentals of Clinical Chemistry. 3rd Edition, WB Saunders, Philadelphia, PA.1987.

11. National Committee for Clinical Laboratory Standards. Method Comparison and Bias Estimation Using Patient Samples: Tentative Guideline. NCCLS publication EP9-T, Villanova, PA. 1993.

12. National Committee for Clinical Laboratory Standards. Precision Performance of Clinical Chemistry Devices: Tentative Guideline, 2nd Edition. NCCLS publication EP5-T2, Villanova, PA. 1992.

13. Wild SH, Fortmann SP, Marcovina Sm. A Prospective Case-Control Study of Lipoprotein(a) Levels and Apo(a) Size and Risk of Coronary Heart Disease in Stanford Five-City Project Participants. Arterioscler Thromb Vasc Biol. 1997;17:239-245.

Testing for Heparin-Induced Thrombocytopenia

Technical Brief:

Testing for Heparin-Induced Thrombocytopenia

Test Name

Anti-Platelet Factor 4 (PLATF4)

All positive specimens (OD>= 0.400) will have confirmatory platelet aggregation testing (PLHEPP or PLLMWP) ordered and charged.

CPT Codes

86022 (x2)

Methodology

Enzyme-Linked Immunosorbent Assay (ELISA)

Turnaround Time

1 day

Specimen Requirements

Volume:
2 mL

Minimum Volume:
1 mL

Specimen Type:
Plasma

Collection Container:
Light Blue Sodium Citrate Coagulation Tube

Transport Temperature:
Froze

3.2% sodium citrate is the preferred anticoagulant recommended by CLSI.

Stability

Ambient:
4 hours

Refrigerated:
Unacceptable

Frozen:
2 months

Additional Information

Background

Background Information

Heparin-induced thrombocytopenia (HIT) is a clinically significant immune-mediated disorder, characterized by antibodies forming immune complexes with the chemokine platelet factor 4 (PF4) bound to unfractionated heparin (UFH), leading to paradoxical thrombosis.¹ The IgG immune complexes engage Fc-gamma receptor IIa (FcγRIIa) expressed on platelets and possibly leukocytes,² initiating a signal transduction cascade resulting in cellular activation. Activated platelets may potentiate thrombin generation, form thrombi, and induce a prothrombotic state involving both the venous and arterial systems.³ In the process, platelets are consumed, which leads to the observed thrombocytopenia. It is critical that HIT is recognized early so that patients can be alternatively anticoagulated and avoid further exposure to heparin.

Based on the findings of a consensus report,⁴ HIT is regarded as a clinicopathologic syndrome, requiring both clinical features and laboratory detection of the pathologic antibodies. Clinically, HIT needs to be differentiated from other potential causes of thrombocytopenia, particularly the well-characterized non-immune heparin-associated thrombocytopenia (HAT).

A useful clinical scoring system, referred to as the “4Ts,” is helpful in determining whether a patient fits into the “high probability,” “intermediate probability,” or “low probability” category.⁵

The criteria below should be assessed to determine the pretest probability of HIT:

4Ts Scoring System for Assessing Clinical Risk of HIT

Score each category, then add all points to determine maximum score. (Maximum possible score = 8 points)

Thrombocytopenia

0 points = <30% fall or nadir <10×109/L

1 point = 30-50% fall or nadir 10-19×109/L

2 points = >50% fall and nadir 20-100×109/L

Timing of Platelet Count Fall

0 points = <4 days without recent exposure

1 point = Consistent with day 5-10 (but not clear), or >10 days, or ≤1 day with heparin 30-100 days prior

2 points = Day 5-10 or ≤1 day if recent heparin (within 30 days)

Thrombosis

0 points = None

1 point = Progressive, recurrent, or silent thromboses

2 points = Proven thrombosis, skin necrosis, or acute systemic reaction with heparin bolus

Other Causes for Thrombocytopenia

0 points = Definite

1 point = Possible

2 points = None evident

Pretest Probability

<3 points = Low

4-5 points = Intermediate

6-8 points = High

Clinical Indications

Suspicion for HIT in patients exposed to UFH or low molecular-weight heparin (LMWH).

Interpretation

Testing for HIT encompasses both immunologic (Anti-PF4 IgG ELISA) & functional assays (heparin-induced platelet aggression [HIPA] and serotonin release [SRA]).

Positive Anti-PF4 IgG ELISA Result

• When the anti-PF4 IgG antibody ELISA and platelet functional assay (heparin-induced platelet aggregation testing [HIPA] or serotonin release assay [SRA]) are both positive, the diagnosis of HIT is supported by laboratory findings.

• If the anti-PF4 IgG antibody ELISA is positive or equivalent, but the platelet functional assays (see Methodology) are both negative, the diagnosis of HIT is less likely. However, clinical judgement is important since the confirmatory tests are not as sensitive for detecting HIT as the ELISA. Reassessing the pretest probability is suggested. If clinical suspicion remains high, retesting may be justified in case the antibody titer was too low to induce immune complex-mediated platelet activation in the initial work-up.

• If the anti-PF4 IgG antibody ELISA is positive, but the HIPA is negative, ordering the SRA is suggested if clinical suspicion remains high. The SRA has a higher sensitivity than the HIPA.

Negative Anti-PF4 IgG ELISA Result

• If the anti-PF4 IgG antibody ELISA is negative, HIT is very unlikely and further testing is unnecessary. In rare situations where clinical necessity dictates confirmatory testing despite a negative anti-PF4 result, the SRA is the recommended confirmatory assay since it has a higher sensitivity than the HIPA.

Limitations

A positive reaction obtained by the anti-PF4 IgG antibody screening ELISA does not confirm the diagnosis of HIT; a functional assay must be performed to confirm HIT.

The presence of immune complexes or other immunoglobulin aggregates in the patient sample may cause nonspecific binding in the ELISA and produce false positives.

The platelet functional assays are not as sensitive as the anti-PF4 IgG antibody ELISA, but have higher specificity and, therefore, should be used only as confirmatory tests.

Non-heparin dependent antibodies, such as anti-HLA, can cause platelet activation independent of heparin/PF4 immune complexes, rendering the functional assays indeterminate.

Methodology

Laboratory testing for HIT is a two-stage process. Testing begins with a high-sensitivity ELISA screen for anti-PF4 IgG antibodies.

No Antibodies Detected:

If the O.D. < 0.4, a diagnosis of HIT is unlikely (negative predictive value 97-99%), and no further testing is necessary.

Heparin Dependent Anti-PF4 IgG Antibodies are Present:

If the O.D. > 0.4 and % inhibition with soluble heparin is > 50%, the assay is positive. The ordering clinician is immediately notified of the result.

The HIPA platelet functional assay (with higher specificity) is then performed to confirm the diagnosis of HIT. Recommendations to order the SRA will be made.

Heparin-Dependent Anti-PF4 IgG Antibodies Equivocal:

If the O.D. > 0.4 and % inhibition with soluble heparin is < 50%, the assay is equivocal.

Platelet functional assays (with higher specificity) are performed to further evaluate.

The anti-PF4 IgG assay (GTI Diagnostics, Waukesha, WI) is performed by a solid-phase ELISA method. The antibody in the patient’s specimen will bind to microwells coated with PF4 complexed with polyvinyl sulfonate. After serial addition of alkaline phosphatase labeled anti-human IgG and the substrate p-nitrophenyl phosphate, the optical density (O.D.) of developed color is measured in a spectrophotometer. The anti-PF4 IgG assay is performed with the patient’s specimen with and without additional soluble heparin (final heparin concentration of 100 U/mL) as well as positive and negative controls.

If the O.D. is less than 0.4, it is considered a negative result.

If the anti-PF4 IgG result is positive with the O.D. greater than 0.4, % inhibition will be calculated as follows:

Patient specimen with heparin – Negative control
[1- (___________________________ )] x 100 = % Inhibition
Patient specimen without heparin – Negative control

The inhibition of a positive reaction by 50% or more (by calculation) in the presence of excess heparin is considered confirmatory for the presence of specific antibodies that react with PF4: heparin.

Heparin-induced platelet aggregation testing (HIPA) is performed by mixing donor platelet-rich plasma (providing the platelets) with patient plasma (providing the antibodies). Low-dose UFH (0.1-0.5 U/ml) is added. If antibodies are present, immune complexes form. The antibody/PF4/heparin complexes activate the platelet FcγRIIa and the platelets aggregate (> 30% aggregation indicates a positive confirmatory result). As an additional control for specificity, high-dose UFH (100 U/ml) is also tested. The excess heparin prevents the formation of immune complexes and should not induce platelet aggregation. If aggregation is seen with the high-dose heparin, the test is considered indeterminate due to non-specific cross reactivity. This test has a higher specificity for pathologic HIT than the screening assay, but the sensitivity is only 50-80%.

The serotonin release assay (SRA) (sent out) and other washed platelet assays offer more sensitive confirmatory testing (80-90%). These tests are performed in a similar manner to the HIPA, relying on donor platelets and patient plasma or serum. With the SRA, a > 20% release of serotonin in the presence of low-dose UHF is considered positive. If there is > 20% serotonin release in the presence of high-dose UFH, the test is considered indeterminate. If the screening test is positive with a high pretest probability, and the HIPA is negative, performing the SRA may be helpful.

In patients suspected of developing a HIT-like syndrome while on low molecular weight heparin (LMWH), confirmatory testing is performed with LMWH instead of UFH.

References

1. Kelton JG, Warkentin TE. Heparin-induced thrombocytopenia: a historical perspective. Blood. 2008 Oct 1;112(7):2607-16.

2. Xiao Z, Visentin GP, Dayananda KM, Neelamegham S. Immune complexes formed following the binding of antiplatelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion. Blood. 2008 Aug 15;112(4):1091-100.

3. Walenga JM, Jeske WP, Messmore HL. Mechanisms of venous and arterial thrombosis in heparin-induced thrombocytopenia. J Thromb Thrombolysis. 2000 Nov;10 Suppl 1:13-20.

4. Warkentin TE, Chong BH, Greinacher A. Heparin-induced thrombocytopenia: towards consensus. Thromb Haemost. 1998;79:1-7.

5. Warkentin TE, Heddle NM. Laboratory Diagnosis of Immune Heparin-Induced Thrombocytopenia. Current Hematology Reports. 2003;2:148-157.

Stool Culture for Yersinia

Technical Brief

Stool Culture for Yersinia

Test Name

Yersinia Culture (YERCUL)

CPT Codes

87046

Methodology

Culture

Turnaround Time

5 days

Specimen Requirements

Specimen Type:
Stool

Volume:
5 mL

Collection Container:
Para-Pak™ C&S (Culture & Sensitivity) Stool Transport System

Transport Temperature:
Refrigerated

Stability

Ambient:
2 hours

Refrigerated:
Preserved – 24 hours
Unpreserved – 2 hours

Frozen:
Unacceptable

Additional Information

Background

Background Information

Bacterial, parasitic, and viral agents can cause infectious gastroenteritis. Approximately 48 million people become ill and 128,000 require hospitalization from foodborne diseases in the United States each year.¹ Enterohemorrhagic Escherichia coli (EHEC) is one of the top five causes of foodborne illness requiring hospitalization in the United States.¹ Most clinical laboratories that perform enteric cultures on stool samples routinely include media and methods to rule out Campylobacter spp., Salmonella spp., Shigella spp., and EHEC.

Shigella, which accounts for less disease prevalence in the U.S. (~14,000 cases/year), is highly infectious and its detection in all stool samples is important to prevent further disease from spreading person to person. Yersinia enterocolitica can also cause foodborne disease, but at a much lower incidence; consequently, most laboratories do not routinely look for this pathogen. The incidence of Y. enterocolitica, as reported by CDC Food Net activities, is about one culture-confirmed case per 100,000 population per year. This compares to the > 1 million cases of Salmonella and > 800,000 cases of Campylobacter reported annually to CDC in the U.S.² Blood transfusion reactions have occurred from blood products contaminated with Y. enterocolitica, however, gastroenteritis is not usually a part of the resultant bacteremia.

Y. enterocolitica can cause gastroenteritis in an individual who has consumed contaminated food or water. Y. enterocolitica has been isolated from raw meats, such as beef, lamb, pork and chicken, but can also be found in cooked, pre-packaged deli meats. Consumption of raw or improperly cooked pork is the main source of gastroenteritis in humans, with the specific association with improperly prepared and handled pork chitterlings.^3,4,5,6 Drinking unpasteurized milk, untreated water, or coming into contact with infected animals also can be the source of infection.

The severity of the disease is related to the specific serotype of Y. enterocolitica as well as load of organism consumed. The range of diseases associated with Y. enterocolitica is self-limited gastroenteritis to terminal ileitis, to mesenteric lymphadenitis that is often mistaken as appendicitis. Occasionally, skin rash and joint pains can accompany Yersinia gastroenteritis.^1,3

Children are more commonly diagnosed with Y. enterocolitica than adults. Patients with gastroenteritis associated with Yersinia are more likely to present with fever, diarrhea, and abdominal pain that can last for seven days. A carrier state of the organism can ensue for up to several months. Septicemia can result if organisms migrate out of the gastrointestinal tract via the lymphatics and find their way to lymph nodes. Persons at the highest risk for systemic disease are the elderly and immunocompromised populations. In particular, persons with underlying metabolic diseases that are associated with iron overload (hemochromatosis), cancer, liver disease, and steroid therapy are at the highest risk of more serious Y. enterocolitica disease. A case of ileal perforation post gastroenteritis has been reported with a review of other surgical complications of Yersinia gastroenteritis.⁶

Clinical Indications

A request for Yersinia culture from stool should be made if a patient is suspected of having gastroenteritis associated with Yersinia, for example, when there has been exposure to undercooked pork chitterlings, or when routine stool cultures are negative for Salmonella and Campylobacter. In addition, patients presenting with diarrhea and associated symptoms of appendicitis and/or septicemia should be considered for a Yersinia culture request.

This test should be done as an adjunct to the routine culture and not in place of it.

Children, the elderly, and immunocompromised patients are the more likely patients at risk for Yersinia gastroenteritis.

Interpretation

Yersinia enterocolitica can grow on most routine laboratory media, including Maconkey’s agar, but grows more slowly than other members of the normal GI flora Enterobacteriaceae. Cefsulodin-irgasan-novobiocin (CIN) agar will be planted in addition to a Maconkey’s agar when a request is made for isolation of Y. enterocolitica.

Most results should be available within 48-72 hours after collection and processing of the stool samples.

Growth and identification, specifically of Y. enterocolitica, will be considered a positive result; no growth as a negative result.

Serotyping of the Y. enterocolitica is not performed routinely in clinical laboratories and is not widely available in reference laboratories.

Most cases of Yersinia gastroenteritis are self-limited and do not require treatment; however, if there is concomitant systemic disease and/or if the patient is immunocompromised, susceptibility testing can be performed.

Limitations

Overgrowth with normal flora GI bacteria and/or other GI pathogens may limit the growth of Y. enterocolitica.

Culture of stool samples after beginning treatment may limit detection.

Methodology

Y. enterocolitica is a gram-negative bacterium that can be isolated in culture from stool specimens.

Stool should be submitted to the laboratory within two hours of collection or transported in Cary Blair transport media and refrigerated if there will be delays. The order should be placed for a Yersinia culture when the stool is submitted.

Cultures are performed seven days per week.

References

1. http://www.cdc.gov/foodborneburden/2011-foodborneestimates.html. CDC website.

2. Long C, Jones TF, Vugia DJ, Scheftel J, Strockbine N, Ryan P, Shiferaw B, Tauxe RV, Gould LH. Yersinia pseudotuberculosis and Y. enterocolitica infections, FoodNet, 1996-2007. Emerg Infect Dis. 2010;16:566-7.

3. Wanger, A. Chapter 44. Yersinia. In Murray PR et al. (eds). Manual of Clinical Microbiology 9th ed. ASM Press: Washington, 2007.

4. Fosse J, Seegers H, Magras C. Prevalence and risk factors for bacterial foodborne zoonotic hazards in slaughter pigs: a review. Zoonoses Public Health. 2009;56:429-54.

5. Centers for Disease Control and Prevention. Yersinia enterocolitica gastroenteritis among infants exposed to chitterlings—Chicago, Illinois, 2002. MMWR Morb Mortal Wkly Rep. 2003;52:956-8.

6. De Berardis B, Torresini G, Brucchi M, Marinelli S, Mattucci S, Schietroma M, Vecchio L, Carlei F. Yersinia enterocolitica intestinal infection with ileum perforation: report of a clinical observation. Ata Biomed. 2004;75:77-81.

OSOM® Trichomonas Rapid Test

Technical Brief

OSOM^® Trichomonas Rapid Test

Test Name

Trichomonas Prep (TRICHO)

CPT Codes

87808

Methodology

Immunochromatography

Turnaround Time

1 day

Specimen Requirements

Type:
Swab, gential

Collection Device:
BD CultureSwab™ Liquid Amies Double Swab

Transport Temperature:
Ambient

Swabs in gel or other transport medium, dry swabs, and swabs with wooden shaft will be rejected.

Stability

Ambient:
Less than 24 hours

Refrigerated:
36 hours

Frozen:
36 hours

Additional Information

Background

Background Information

Trichomonas is a common cause of vaginitis and the most common nonviral sexually transmitted disease worldwide.¹ Studies show that T. vaginalis is an important cause of premature rupture of membranes, premature delivery, pelvic inflammatory disease, urethritis, and chronic prostatitis.

Diagnosis of Trichomonas infection proves challenging in that the traditional wet mount method relies on detecting motility of the parasite, which is often lost during delays in transport or refrigeration. Wet mount preparations are less than optimal when the transport time exceeds six hours.² Optimal transport time is one hour or less. Alternative methods for diagnosis of Trichomonas infection include EIA methods, in-office physician-performed microscopy, culture, and molecular methods. Molecular diagnostic methods have cost-to-patient charges that are significantly higher than other methods. Wet mount microscopy has a reported sensitivity of 58% versus culture.³

The OSOM® Trichomonas Rapid Test is an FDA-approved, CLIA-waived test that offers a simple, inexpensive option with improved sensitivity for diagnosis of Trichomonas infection as compared to wet prep. The test is intended for qualitative detection of Trichomonas vaginalis antigen from vaginal swabs or from the saline solution prepared when making wet mounts from vaginal swabs.

Clinical Indications

The OSOM^® Trichomonas Rapid Test is intended for the qualitative detection of Trichomonas vaginalis antigen from genital swabs. The test is intended for use in patients with symptoms of vaginosis or suspected exposure to the organism.

Interpretation

If Trichomonas is present in the sample, it will form a complex with the primary anti-Trichomonas antibody conjugated to blue-colored particles. The complex will then be bound by a second anti-Trichomonas antibody coated on the nitrocellulose membrane. The appearance of a visible blue test line along with the red control line (internal control) indicates a positive result.

Limitations

The OSOM^® Trichomonas Rapid test has not been approved for urine samples; the test has only been validated for qualitative detection of T. vaginalis antigen from vaginal swabs. Molecular methodology is recommended for urine specimens.

A negative result may be obtained if the specimen is inadequate or if the antigen concentration is below the sensitivity of the test.

Samples contaminated with preparations containing iodine or by the immediate prior use of vaginal lubricants are not recommended.

The test does not differentiate between viable and non-viable organisms, nor does it differentiate between acute infection and carrier states.

Staphylococcus aureus in specimens at concentrations higher than 1 X 108 cfu/ml may interfere with the test results in negative samples. These concentrations are higher than would be expected to be present in normal patient samples.

The OSOM^® Trichomonas Rapid Test is reported to detect as little as 2500 organisms/ml per manufacturer’s package insert.

Methodology

The OSOM^® Trichomonas Rapid Test is an immunochromatographic test that utilizes capillary flow dipstick technology. The test requires solubilization of the Trichomonas proteins from the swab by mixing the sample in the sample buffer. A sample test strip is then added allowing the subsequent migration of the sample along the membrane surface of the dipstick.

References

1. Campbell L, Woods V, et al. Evaluation of the OSOM Trichomonas Rapid Test versus Wet Preparation Examination for Detection of Trichomonas vaginalis Vaginitis in Specimens from Women with a Low Prevalence of Infection. Journal of Clinical Microbiology. 2008; 3467-3469.

2. Huppert JS, Batteiger BE, et al. Use of an Immunochromatographic Assay for rapid Detection of Trichomonas vaginalis in Vaginal Specimens. Journal of Clinical Microbiology. 2005; 684-687.

3. Kingston MA, Bansal D, Carlin EM. Shelf life of Trichomonas vaginalis, International Journal of STD & AIDS. 2003;14:28-29.

4. OSOM® Trichomonas Rapid Test product information and package insert, Sekisui Diagnostics, Framingham, MA, 2011.

Mixing Study, Incubated Activated Partial Thromboplastin Time (APTT)

Technical Brief:

Mixing Study, Incubated Activated Partial Thromboplastin Time (APTT)

Test Name

PTT Incubated Mixing Study (PTTIM)

CPT Codes

85730
85610
85732
85670
85520
85390

Methodology

Clot Detection

Turnaround Time

1 – 3 days

Specimen Requirements

Volume:
4 mL

Minimum Volume:
1.5 mL

Specimen Type:
Plasma

Collection Container:
Light Blue Sodium Citrate Coagulation Tube

Transport Temperature:
Frozen

3.2% sodium citrate is the preferred anticoagulant recommended by CLSI.

Specimen Collection & Handling

The presence of heparin, fondaparinux, dabigatran, or a direct thrombin inhibitor in the specimen may interfere with test results.

Discontinue coumadin therapy for 14 days prior to collection.

Discontinue direct thrombin inhibitors and heparin 2 days prior to collection.

Stability

Ambient:
7 days

Citrated plasma for ADAMSTS13 Antibody remains stable at room temperature and refrigerated for 7 days, but no tests for ADAMSTS13 Activity or Inhibitor can be performed on such specimens.

Reference Range

See Interpretation

PT Screen:
8.4-13.0 seconds

APTT Screen:
< 33.2 seconds

APTT Immediate Mix:
33.2 seconds

APTT Incubated Mix:
< 36.0 seconds

Thrombin Time:
< 18.6 seconds

Heparin Assay:
< 0.1 U/mL

Additional Information

Background

Background Information

The activated partial thromboplastin time (APTT) is one of the most commonly used tests to investigate bleeding patients, monitor anticoagulant therapy, and screen patients before surgery. The APTT measures the integrity of the intrinsic and common pathways of the coagulation cascade. The prothrombin time (PT), another common screening test, measures the integrity of the extrinsic and common coagulation pathway.

The APTT is measured as the number of seconds for the patient’s plasma to form a fibrin clot after the addition of an intrinsic pathway activator, phospholipid, and calcium. A prolonged APTT can be caused by a coagulation factor deficiency or the presence of an inhibitor. The mixing study, incubated APTT, is used to investigate the cause of a prolonged APTT result. The mixing study is performed by measuring the APTT in the patient’s plasma, then mixing an equal volume of the patient’s plasma and normal pooled plasma (NPP), and repeating the APTT tests immediately and after one-hour incubation.

The components of the panel include PT screen, APTT screen, APTT Immediate Mix, and APTT Incubated Mix, as well as a thrombin time and heparin anti-Xa assay if needed.

The principle of the mixing study can be summarized as:

1. If the prolonged APTT screen is due to a factor deficiency, mixing with an equal volume of NPP (which has approximately 100% of all coagulation factors) will replace the patient’s deficient factor. This result in an APTT immediate mix is shortened or corrected into the reference range.

2. If the prolonged APTT screen is due to the presence of an inhibitor, mixing with an equal volume of NPP will not shorten or correct the prolongation of APTT in repeated tests. The reason is the inhibitor in the patient’s plasma is present in excess and binds to coagulation factors or protein/phospholipid complexes in both the patient’s plasma and NPP.

Correction of the APTT in the mixing study suggests a coagulation factor deficiency in either the intrinsic pathway (factors VIII, IX, XI and XII, high-molecular-weight kininogen [HMWK] or prekallikrein [PK]), or in the common pathway (also prolonged PT) such as factor II, V, and X. Deficiency of factors VIII, IX, and XI will present with bleeding; however, deficiency of factor XII or prekallikrein will not increase bleeding risk, but may increase thrombotic risk. Further testing, such as clotting factor assays, is necessary to diagnose a specific factor deficiency. See Figure 1 for the diagnostic algorithm used in the laboratory.

There are three different types of inhibitors:

1. Inhibitors directly against specific factors, such as factor VIII or factor V inhibitors.

2. Anticoagulants such as heparins, fondaparinux, dabigatran, and other direct thrombin inhibitors.

3. Non-specific inhibitors, such as lupus anticoagulants.

Some inhibitors will demonstrate a delayed-type inhibitor pattern, with time and/or temperature dependence. In cases with a delayed-type inhibitor, the APTT Immediate Mix will correct to within the reference range; however, the APTT Incubated Mix will be prolonged.

Although rare, the presence of a factor inhibitor, such as a factor VIII inhibitor, will increase the risk of life-threatening bleeding. The presence of a factor inhibitor can be confirmed by a Bethesda assay for that factor. The presence of heparins, fondaparinux, dabigatran, or other direct thrombin inhibitors can cause prolongation of both the APTT Immediate Mix and APTT Incubated Mix. Careful clinical and medication history, and additional thrombin time with heparin assay (anti-Xa inhibition assay) can exclude the presence of anticoagulants.

The presence of lupus anticoagulants, which are antibodies against protein-phospholipid complexes, will increase the risk of thromboembolism. The presence of low-level, nonspecific inhibitors in the patient’s plasma may demonstrate a prolonged APTT Incubated Mix similar to a delayed-type inhibitor. If the clinical history suggests a lupus anticoagulant, further testing, including phospholipid based screening tests, phospholipid dependency assays, and exclusion of the presence of inhibitors, in addition to mixing study, incubated APTT, is necessary (refer to Figure 1 for lupus anticoagulant).

The adequate performance of the mixing test and accurate interpretation is important because the presence of a specific factor inhibitor, non-specific inhibitors, such as lupus anticoagulant, anticoagulants, or factor deficiency, have different clinical manifestations and require different clinical management.

Clinical Indications

The mixing test, incubated APTT, is indicated when the cause of a prolonged APTT result needs to be investigated.

Interpretation

APTT Screen:
Prolonged

APTT, Immediate Mix:
Normal

APTT, Incubated Mix:
Normal

If the APTT Screen is prolonged with a normal APTT Immediate Mix and APTT Incubated mix, this indicates a factor deficiency in the intrinsic or final common pathway.

If the PT is normal, this suggests an intrinsic pathway deficiency (VIII, IX, XI, XII, PK, HMWK).

If the PT is prolonged, this suggests a common pathway deficiency (fibrinogen, II, V, X).

APTT Screen:
Prolonged

APTT, Immediate Mix:
Normal

APTT, Incubated Mix:
Abnormal

If the APTT Screen is prolonged, with a normal APTT Immediate Mix, but an abnormal APTT Incubated Mix, this indicates the presence of a delayed inhibitor such as specific factor inhibitors, most commonly factor VIII inhibitor, and small numbers of lupus anticoagulant.

APTT Screen:
Prolonged

APTT, Immediate Mix:
Abnormal

APTT, Incubated Mix:
Abnormal

If the APTT Screen is prolonged, with an abnormal APTT Immediate Mix and abnormal APTT Incubated Mix, this favors a non-specific inhibitor, such as a lupus anticoagulant, and anticoagulants such as heparin, fondaparinux, dabigatran, or other direct thrombin inhibitors.

Methodology

The PT Screen is performed using Innovin® (Dade Behring, Inc.) reagent and STAR Evolution® Analyzer (Diagnostica Stago, Inc.). The PT Screen is included to localize abnormalities to common, intrinsic, and extrinsic pathways.

The APTT Screen is performed using the PTT-Automate reagent and STAR Evolution® analyzer (both Diagnostica Stago, Inc).

The mixing studies are performed by mixing the patient’s plasma with an equal volume of the NPP (Cryocheck; Precision Biologic, Inc).

For the APTT Immediate Mix, the APTT is performed immediately after mixing the plasmas. For the APTT Incubated Mix, the APTT is performed after one-hour incubation at 37ºC. The NPP serves as a negative control; two levels of positive control are performed; lupus positive plasma (Precision Biologic, Inc) and weak lupus positive plasma (Precision Biologic, Inc).

The thrombin time (Diagnostica Stago, Inc) will be measured in specimens with prolonged APTT. If the TT is prolonged, a heparin assay (anti-Xa inhibition assay; Rotachrom Heparin kit, Diagnostica Stago, Inc.) by a chromogenic assay will be performed to distinguish a heparin effect from a direct thrombin inhibitor.

Legionella pneumophila by Real-Time PCR

Technical Brief:

Legionella pneumophila by Real-Time Polymerase Chain Reaction (PCR)

Test Name

Legionella pneumophila PCR (LEGPCR)

CPT Codes

87541

Methodology

Polymerase Chain Reaction (PCR)

Turnaround Time

3 days

Specimen Requirements

Specimen Type:
Bronchoalveolar lavage (BAL)

Volume:
3 mL

Minimum Volume:
2 mL

Collection Container:
Sterile specimen container

Transport Temperature:
Ambient

Alternative Specimen

Specimen Type:
Sputum, induced
Aspirate, tracheal

Volume:
1 mL

Collection Container:
Sterile specimen container

Transport Temperature:
Ambient

If aliquoting is necessary, sterile tubes must be used.

Stability

Ambient:
24 hours

Refrigerated:
7 days

Frozen:
30 days

Reference Range

No Legionella pneumophila detected

Additional Information

Background

Background Information

Legionella pneumophila is the most common pathogenic species of the 42-recognized Legionella species and is associated with significant mortality in elderly patients and those with severe underlying disease. Diagnostic delay also may result in increased mortality. Target sequences within the genes that encode the 5S and 16S ribosomal subunits, as well as the MIP (macrophage infectivity potentiator) gene, in conjunction with real-time PCR, are useful for the detection of the Legionella genus and, specifically, the species L. pneumophila.

The MIP gene, which encodes a 24-kDa protein virulence factor that facilitates the entry of legionellae into amoebae and macrophages, has sufficient sequence variability between the Legionella species to allow for the specific detection of L. pneumophila by real-time PCR. This assay has 100% specificity and sensitivity.

Clinical Information

Multiple laboratory methods should be employed to ensure the diagnosis of Legionnaire’s disease (LD), a bacterial pneumonia caused by L. pneumophila (90% of cases) or other Legionella species. Molecular methods are more sensitive than culture for the diagnosis of LD.

The PCR assay performed at Cleveland Clinic will not detect disease caused by Legionella species other than L. pneumophila.

Culture for Legionella species from respiratory sites is a sensitive (~80-90%) method for diagnosing severe, untreated disease, but insensitive (~20%) for the diagnosis of mild disease. Specimens from non-respiratory sites should not be submitted for Legionella culture unless there is a high index of clinical suspicion to support the request. Urine antigen assays for L. pneumophila serogroup 1 will be positive in ~90-95% of patients with severe disease due to the Pontiac monoclonal subtype of serogroup 1, but positive in only 50% of these patients with mild disease. The urine antigen assay is unreliable for the diagnosis of severe LD caused by L. pneumophila other than serogroup 1 or a different Legionella species (detects less than 5-40% of cases).

Interpretation

PCR results are reported qualitatively as positive or negative for Legionella pneumophila.

Limitations

This assay does not contain an internal amplification control; instead, the false-negative rate, which to date has been 0%, is monitored according to the College of American Pathologists’ guidelines.

This assay only detects L. pneumophila, as Legionella species other than L. pneumophila infrequently cause legionellosis. The implementation of this test was intended to replace the less-sensitive direct immunofluorescence assay, and it is intended to be used in conjunction with culture.

Methodology

The LightCycler FastStart DNA Master Hybridization Probe Kit (Roche Diagnostics, Indianapolis, Ind.) is used in conjunction with species-specific Legionella pneumophila primers and probes. The PCR is performed on the LightCycler system (Roche).

Qualitative positive and negative results are determined based on analysis of the amplification curves and post-amplification melt curve.

Hepatitis C Virus Genotyping

Technical Brief

Hepatitis C Virus Genotyping

Test Name

Hepatitis C Genotype (HEPGEN)

CPT Codes

87902

Methodology

Reverse Transcription/Polymerase Chain Reaction (RT/PCR)

Turnaround Time

5 – 7 days

Specimen Requirements

Type:
Plasma

Volume:
3 mL

Minimum Volume:
1 mL

Tube/Container:
White BD Hemogard™ K₂EDTA Plasma Preparation Tube

Transport Temperature:
Refrigerated

Separate plasma from whole blood by centrifugation within 6 hours of collection.

Do not pour-off.

Alternative Specimen

Type:
Plasma

Volume:
3 mL

Minimum Volume:
1 mL

Tube/Container:
Lavender BD Hemogard™ K₂EDTA Tube

Transport Temperature:
Refrigerated

Separate plasma from whole blood by centrifugation within 6 hours of collection. Transfer plasma to a sterile, polypropylene screw-cap tube.

Alternative Specimen

Type:
Serum

Volume:
3 mL

Minimum Volume:
1 mL

Tube/Container:
Gold BD Hemogard™ Serum Separation Tubes (SST)™

Transport Temperature:
Refrigerated

Centrifuge within 6 hours of collection.

Do not pour-off.

Stability:

Plasma

Ambient:
3 days

Refrigerated:
3 days

Frozen:
60 days

Stability:

Whole blood

Ambient:
15-30°C for up to 6 hours prior to centrifugation

Refrigerated:
2-8°C for up to 6 hours prior to centrifugation

Frozen:
Unacceptable

Reference Ranges

Capable of discriminating among HCV subtypes 1a, 1b, 2a-2c, 3a-3c, 4a-4h, 5a, and 6a

Additional Information

Background

Background Information

Chronic infection with the hepatitis C virus (HCV) remains one of the world’s most important clinical and public health problems. It has been estimated that approximately 3% of the world’s population is infected with HCV, which represents nearly 170 million people worldwide.^1,4 In the United States, up to 3.9 million people (1.8% of the population) are currently living with HCV, of which as many as 2.7 million suffer from chronic infection.^2,3 In the Western world, chronic damage from hepatitis C is the primary cause for end-stage liver disease that requires liver transplantation.

Clinical Significance

The 1989 discovery of the hepatitis C virus was a major development. Previously, it was clear that a major cause of acute hepatitis after a blood transfusion was related to neither hepatitis A nor to hepatitis B. This resulted in the early name for this disease: non-A, non-B hepatitis. It is now known that HCV is the cause for most of the non-A, non-B hepatitis cases.

HCV is a single-stranded RNA virus that has approximately 9400 nucleotides that demonstrate significant genetic variation. HCV replicates in the liver and is detectable in serum during acute and chronic infection. The RNA polymerase lacks the proofreading functions of DNA polymerase and introduces random nucleotide errors. This results in a relatively high rate of spontaneous nucleotide substitutions. As a consequence, HCV is a highly-heterogeneous virus with at least six known major genotypes and more than 80 subtypes identified worldwide.⁵ Genotypes are classified based on differences in the amino acid sequence of specific proteins.

Recent understanding of the natural history of chronic hepatitis C has greatly expanded, and more effective therapeutic strategies have been developed. Several studies have shown a strong correlation between HCV genotype and a patient’s response to various treatments. Determining genotypes is necessary because some hepatitis C viruses with certain genetic variations are harder to treat successfully and usually require a different treatment approach; others are much easier to treat and respond well to shorter treatment schedules.

For many years, the standard of care for patients with chronic hepatitis C infection was peginterferon (PegIFN) alfa and ribavirin (RBV) taken for 24 to 48 weeks, depending on the genotype; however, less than 50% of patients responded to this therapy. In May 2011, the FDA approved the use of two new protease inhibitors – Incevik (telaprevir) and Victrelis (boceprevir) – for the treatment of hepatitis genotype 1, the most common form of hepatitis in North America. The drugs, used in conjunction with the standard interferon and ribavirin therapies, represent the first major therapeutic advance in the treatment of hepatitis C in more than a decade. The new protease inhibitors block the replication of an enzyme that is crucial for the hepatitis C virus to reproduce. In patients with hepatitis genotype 1, the use of the new protease inhibitor combined with the traditional antiviral treatment eliminated the virus in 70 to 80% of all cases. Patients with the other types of hepatitis C continue to be treated with peginterferon and ribavirin, which is successful in 80% or more in those with genotypes 2 and 3 infections.^6-8

Clinical Indications

Pre-treatment analysis of hepatitis C genotype is used to determine the duration of therapy and predict therapeutic response.

Interpretation

The predominant HCV genotypes in the United States are 1a, 1b, 2a, 2b, and 3a; the other subtypes (4, 5, 6) are more prominent in other parts of the world.⁹

Limitations

This assay is based on the sequence variability of the 5’UTR, allowing differentiation between HCV genotypes 1 to 6. Subtyping may occasionally be limited.

Methodology

The gold standard for genotyping is determining the nucleotide sequence of an HCV isolate. Currently, this method is not practical for the clinical diagnostic laboratory, and a less labor-intensive technique, such as Line Probe Assay (LiPA), is employed.

The LiPA method uses genotypic-specific oligonucleotide probes immobilized onto membrane strips. The end products obtained from RT-PCR of the 5’UTR region of the clinical isolate are then hybridized onto the membrane containing the immobilized-oligonucleotides. A purple-brown line develops where sequence homology occurs between the biotinylated PCR products and the probe. Hybridization of 5’UTR amplification products with genotype-specific probes is capable of discriminating among HCV subtypes 1a, 1b, 2a-2c, 3a-3c, 4a-4h, 5a, and 6a.¹⁰

References

1. Organization WH. Hepatitis C surveillance and control. Available at: http://www.who.int/=csr/disease/hepatitis/whocdscsrlyo2003/en/index4.html#incidence.

2. Armstrong GL, Wasley A, Simard EP, McQuillan GM, Kuhnert WL, Alter MJ. The prevalence of hepatitis C virus infection in the United States, 1999 through 2002. Ann Intern Med. 2006;144:705-14.

3. Centers for Disease Control. www.cdc.gov/hepatitis/PDFs/disease_burden.pdf.

4. WHO Hepatitis C fact sheet. 2011.

5. Davis GL. Hepatitis C Virus genotypes and quasispecies. Am J Med. 1999;107(6B):21S-25S.

6. Poordad, F, et al. (March 2011). “Boceprevir for Untreated Chronic HCV Genotype 1 Infection”. N Engl J Med. 364(13):1195 206. doi:10.1056/NEJMoa1010494. PMID 21449783.

7. Bacon, B, et al. (March 2011). “Boceprevir for Previously Treated Chronic HCV Genotype 1 Infection”. N Engl J Med. 364(13):1207-17. doi:10.1056/NEJMoa1009482. PMC 3153125. PMID 21449784.

8. McHutchison JG, Manns MP, Muir AJ, et al. (April 2010). “Telaprevir for previously treated chronic HCV infection”. N Engl J Med. 362 (14): 1292–303. doi:10.1056/NEJM oa0908014. PMID 20375406.

9. Mahaney K, Tdeschi, V, Maertens G, et al. Genotypic analysis of hepatitis C virus in American patients. Hepatology; 1994; 20:1405-1411.

10. Stuyver L, Wyseur A, Van Amhem W et al. Second generation line probe assay for hepatitis C virus genotyping. J Clin Microbiol. 1996; 24:2259-2266.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine, Endocervical, Vaginal, and Urethral Specimens

Technical Brief

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine, Endocervical, Vaginal, and Urethral Specimens

Test Name

GC/Chlamydia Amplification, Genital, Rectal and Oral Specimens (GCCT)

GC Amplification, Genital, Rectal and Oral Specimens (GC)

Chlamydia Amplification, Genital, Rectal and Oral Specimens (CT)

CPT Codes

GCCT

87491
87591

87591

87491

Methodology

Target amplification nucleic acid probe, qualitative

Turnaround Time

1 – 4 days

Specimen Requirements

Type:
Swab

Source:
Vaginal, urethral, endocervical, rectal, throat

Specimen Container:
Aptima^® Unisex Swab Specimen Collection Kit

Transport Temperature:
Ambient

Alternative Specimen

Type:
Cervical

Specimen Container:
ThinPrep^® Pap Test

Transport Temperature:
Ambient

Cytyc PreservCyt Solution (ThinPrep) is not recommended unless performed in conjunction with a ThinPrep^® PAP test.

Prior to cytology testing, and within 30 days of collection, transfer a 1 mL aliquot into an Aptima^® Specimen Transfer Tube. Note: the specimen must have been stored at 2 – 30°C.

Stability:

Aptima^® Swab

Ambient:
60 days

Refrigerated:
60 days

Frozen:
1 year

Stability:

ThinPrep^® Solution in Aptima^® Transport Media

Ambient:
14 days

Refrigerated:
30 days

Frozen:
1 year

Stability:

ThinPrep^® Solution, unprocessed

Ambient:
30 days

Refrigerated:
30 days

Frozen:
Unacceptable

Additional Information

Background

Background Information

Sexually transmitted diseases (STDs) continue to be a major cause of deteriorating reproductive health throughout the world. Chlamydia trachomatis and Neisseria gonorrhoeae remain as two of the most common causes of STDs in the United States.¹ C. trachomatis infections have comprised the largest proportion of all STDs reported to the CDC since 1994, with a reported 1,244,180 cases in 2009 for a rate of 409.2/100,000 population. This was a 2.8% increase in rate from that reported in 2008.^1,2 The increase in reported chlamydial infections during the last 20 years reflects the expansion of chlamydia screening activities and the use of increasingly sensitive assays for the detection of C. trachomatis. The CDC recommends annual chlamydia screening of all sexually active women younger than 25 years of age.³

In 2009, there were 301,174 cases of N. gonorrhoeae infections reported for a rate of 99.1/100,000 population. This rate was a 10.5% decrease since 2008. The national gonorrhea rate declined by 74% between 1975 and 1997 following the implementation of a national gonorrhea control program in the mid-1970s. However, since 1997, these rates have reached a plateau and are not continuing to decline.^1,2 Infections due to both C. trachomatis and N. gonorrhoeae are a major cause of pelvic inflammatory disease (PID) in the U.S. and both have been shown to facilitate the transmission of HIV as well.

Rapid and sensitive methods for the laboratory diagnosis of these two agents have been developed, making it reasonable to test for both simultaneously when the diagnosis of an STD is being considered.² The estimate of mixed infections with both agents can be as high as 40%, making it important to consider ordering both agents when sending material off to the laboratory for testing. Nucleic acid amplification tests (NAAT) are recommended for detection of reproductive tract infections caused by C. trachomatis and N. gonorrhoeae infections in men and women with and without symptoms. NAAT should be used for diagnosing both C. trachomatis and N. gonorrhoeae in women with cervicitis; testing can be performed on vaginal, cervical, or urine samples. In men with urethritis, NAAT testing of urine or urethral swabs is recommended.³

Clinical Indications

Both C. trachomatis and N. gonorrhoeae cause urethritis in the male and cervicitis in the female. A significant number of cases, however, remain asymptomatic in both males and females. In addition, both can cause epididymitis and rectal infections in the male, and PID in the female.

Neonates, who contract chlamydial infection during birth, can develop inclusion conjunctivitis and/or pneumoniae; pregnant women can infect their newborns, causing ophthalmia neonatorum; gonorrheal infections can produce joint infections, pharyngitis, and disseminated disease.

Cleveland Clinic Laboratories offers a target amplification nucleic acid probe (APTIMA, Gen-Probe, Inc, San Diego, CA) for the laboratory diagnosis of C. trachomatis and N. gonorrhoeae from urethral and urine specimens from males suspected of these infections, and from cervical, vaginal, and urine samples from females. Numerous articles have been published demonstrating the excellent performance of NAAT testing for the diagnosis of both of these STD agents.^4-8

Methodology

The laboratory diagnosis of Neisseria gonorrhoeae can include culture of urethral or cervical specimens, gram stain of the urethral secretions in symptomatic males, detection by specific nucleic acid gene probes, and amplification of N. gonorrhoeae nucleic acids. Amplification of N. gonorrhoeae nucleic acids has been shown to be a very sensitive and specific method of detection.^4,5 The sensitivity is equivalent to culture, but it is not fraught by the problem of organism fragility that can easily occur with delays in specimen transport.

Although culture or the use of a nucleic acids probe can be employed for the detection of C. trachomatis, nucleic acid amplification is the most sensitive method, with studies indicating that it may be up to 40% more sensitive than culture. The same assay that detects Chlamydia trachomatis nucleic acids is also used by Cleveland Clinic Laboratories to detect Neisseria gonorrhoeae nucleic acids, thus providing a convenient approach to dual detection.

Collection & Transport

Specimen Collection & Transport

Acceptable specimens include urethral, endocervical, and vaginal swabs, as well as urine. A vaginal swab is optimal for screening asymptomatic females, while a first-catch urine is optimal for screening asymptomatic men.

Specimen collection/transport using Aptima^® devices is preferred.

Urethral, Endocervical Specimens

The Aptima^® Unisex Swab Specimen Collection Kit for urethral or endocervical specimens contains a white cleaning swab to be used for removing excess mucus. The blue swab must be used for collection of specimens that are submitted for testing.

For urethral specimens:
- Patients should not urinate within 1 hour prior to specimen collection.
- Insert the blue shaft swab 2 to 4 cm into the urethra.
- Gently rotate swab clockwise for 2 to 3 seconds and withdraw carefully.

For endocervical specimens:
- Remove excess mucus using cleaning swab and then insert blue shaft swab into the endocervical canal.
- Rotate swab for 10-30 seconds in the endocervical canal to ensure adequate sampling and withdraw carefully (avoid contact with vaginal mucosa).

Place swab into the transport tube and carefully break at the scoreline. Use care to avoid splashing contents. Discard top portion of swab shaft and recap transport tube tightly.

Maintain the specimen at 2ºC to 30ºC.

Vaginal Specimens

The Aptima^® Multitest Swab Transport Media Kit is optimal for testing asymptomatic women.

For vaginal specimens:
- Hold the swab with forefinger and thumb covering the scoreline (do not hold the shaft below the scoreline).
- Carefully insert the swab about 2 inches into the vagina and gently rotate the swab for 10-30 seconds.
- Make sure the swab touches the walls of the vagina so that moisture is absorbed by the swab, then withdraw the swab without touching the skin.
- While holding the swab in the same hand, unscrew the cap from the tube, being careful not to spill contents of the tube.

Immediately place the swab in the transport tube and carefully break swab shaft at score line against side of the tube. Use care to avoid splashing contents. Discard top portion of swab shaft and recap transport tube tightly.

Maintain the specimen at 2ºC to 30ºC.

Urine Specimens

The Aptima^® Urine Specimen Collection Kit is used for the collection and transport of male or female urine specimens for chlamydia and/or gonorrhea testing.

For urine specimens:
- Patients should not urinate within one hour of collection.
- Collect the first catch urine (approximately 20-30 ml of initial urine stream; collecting larger volumes of urine will reduce test sensitivity).
- Within 24 hours of collection, transfer 2 mL of urine into the Aptima^® urine transport tube using the disposable pipette provided in the collection kit. The correct volume of urine has been added when the fluid level is between the black lines on the transport tube label.

Maintain the specimen at 2ºC to 30ºC.

ThinPrep^® Pap Test Specimens

Alternatively, if a ThinPrep^® vial is being used for a Liquid Cytology PAP Test, the same sample can be submitted for detection of C. trachomatis and N. gonorrhoeae as well.⁹

The assay can only be performed on ThinPrep^® vials if 1 mL of Cytyc PreservCyt Solution is transferred to an Aptima^® Specimen Transfer Tube before the specimen is processed in Cytology for a PAP test.

Maintain the specimen at 2ºC to 30ºC.

Interpretation

Amplification is performed Monday through Friday. Internal controls are run with each specimen in order to detect any inhibitors in the sample.

Results will be reported as “positive for C. trachomatis and/or N. gonorrhoeae by amplification” when the relative light unit (RLU) result is above our positive cut-off value.

Within a narrow range of RLU results, as determined by the assay manufacturer, an “equivocal result” will be reported with a request that a repeat specimen be submitted.

If the internal control indicates inhibition, and the result is negative for C. trachomatis and/or N. gonorrhoeae, the report will be: “Inhibition detected; N. gonorrhoeae, and/or C. trachomatis, if present, would not be detectable. Please send an additional specimen.”

All results for N. gonorrhoeae and/or C. trachomatis that are lower than the laboratory’s derived positive cut-off, but within the instrument derived positive results, will be confirmed with a repeat amplification assay before reports are released. This is done to avoid any problems with false-positive results that might occur with low positive results.¹⁰

Limitations

There is currently no FDA clearance for use of amplification assays on specimens outside of the genitourinary tract. Culture is recommended for testing specimens from the throat, eye, or rectal area. However, a laboratory can validate the use of NAAT for rectal and pharyngeal specimens. In addition, for specimens obtained from infants and children, or if the information from the laboratory is to be used for legal purposes, culture is the preferred method.⁷

Since NAAT is more sensitive, it may be run in conjunction with culture for purposes of treatment decision-making. Although “test-of-cure” samples are not recommended from patients in whom the diagnosis has previously been made within the last 4-6 weeks, if required, a culture is the preferred request.

If a culture is needed for any of these purposes, the collection swab for Neisseria gonorrhoeae needs to be placed into a culturette and NOT the Aptima^® transport tube, and a specific request for culture should be made.

References

1. Centers for Disease Control and Prevention. Sexually Transmitted Disease Surveillance, 2009. Atlanta, GA: U.S. Department of Health and Human Services; November 2010. Printed copies and the on-line version of this report can be obtained at the following website: http://www.cdc.gov/std/pubs.

2. http://www.cdc.gov/std/stats09/chlamydia.htm

3. Centers for Disease Control and Prevention. Sexually transmitted Diseases treatment Guidelines. MMWR. 2010;59(RR#2):1-110.

4. Chernesky M, Martin DH, Hook EW et al. Ability of Aptima CT and Aptima GC assays to detect Chlamydia trachomatis and Neisseria gonorrhoeae in male urine and urethral swabs. J Clin Microbiol. 2005;43:127-31.

5. Gaydos CA, Quinn TC, Willis D, Weissfeld A, Hook EW, Martin DH, Ferrero DV, Schachter J. Performance of the APTIMA Combo 2 assay for the multiplex detection of Chlamydia trachomatis and Neisseria gonorrhoeae in female urine and endocervical swab specimens. J Clin Microbiol. 2003;41:304-309.

6. Fang J, Husman C, Dasilva L et al. Evaluation of self collected vaginal swab, first void urine, and endocervical swabs for the detection of C. trachomatis and N. gonorrhoeae in adolescent females. J Pediatr Adolesc Gynecol. 2008;21:355-60.

7. Blake DR, Maldeis N, Barnes MR et al. Cost-effectiveness of screening strategies for C. trachomatis using cervical swabs, urine, and self-obtained vaginal swabs in a sexually transmitted disease clinic setting. Sex Transm Dis. 2008;35:649-55.

8. Schachter J, Chernesky MA, Willis DE, et al. Vaginal swabs are the specimens of choice when screening for C. trachomatis and N. gonorrhoeae: results from a multicenter evaluation of the Aptima assays for both infections. Sex Transm Dis. 2005;32:725-8.

9. Chernesky M, Jang D, Smieja M et al. Validation of the Aptima Combo 2 assay for detection of C. trachomatis and N. gonorrhoeae in Sure-Path liquid-based pap test samples taken with different collection devices. Sex Transm Dis. 2009;36:581-2.

10. Farrell, DJ. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR. J Clin Microbiology. 1999;37:386-90.