Evaluation of sera with a low signal to cut-off ratio using two chemiluminescent assays for detecting Hepatitis C Virus, and their correlation with the detection of Viral RNA (2023)

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Tables (2)

Table 1. Results of samples with S/CO ≥1.00 and ≤3.00 with DiaSorin and their correlation with ARCHITECT and real-time PCR.

Table 2. Results of samples with S/CO 1–3 by ARCHITECT and their correlation with DiaSorin and PCR.

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Abstract

Objective

All commercial assays used to measure the presence of Hepatitis C virus (HCV) antibodies set cut-off points to categorise the results, but the problem of false positive results in screening hepatitis C sera is well known.

The aim of this study was to evaluate the results obtained by two chemiluminescent assays in selected sera, and compare these results with the detection of viral RNA in the specimens studied.

Material and methods

Two hundred reactive sera (positive) were selected, although with a low signal to cut-off ratio (S/CO), were selected, using two chemiluminescent assays and were then subjected to genome amplification.

Results and discussion

Viral RNA could be only be detected in 8 (4%) of the selected specimens. Taking these results into account, we believe that the design of the current chemiluminescent assays do not provide sufficient specificity when they are used as the only tests for the diagnosis of hepatitis C.

Keywords:

Chemiluminescence

Hepatitis C

S/CO ratio

(Video) SARS CoV 2 Quality Control for Molecular and Serology assays

Resumen

Objetivo

Los ensayos comerciales utilizados para demostrar la presencia de anticuerpos anti-VHC establecen, cada uno de ellos, puntos de corte determinados que categorizan los resultados en función de los mismos, aunque el problema de los resultados falsos positivos en el cribado de sueros de hepatitis C sea bien conocido.

El objetivo de este trabajo ha sido valorar los resultados obtenidos por 2 ensayos quimioluminiscentes en una serie de sueros seleccionados, contrastando estos resultados con la detección de ARN viral en las muestras estudiadas.

Material y métodos

Se seleccionaron 200 sueros considerados como reactivos (positivos), aunque con un bajo índice S/CO, utilizando 2 ensayos de quimioluminiscencia y posteriormente fueron sometidos a amplificación genómica.

Resultados y discusión

Solamente en 8 (4%) de las muestras seleccionadas pudo detectarse ARN viral. A la vista de estos resultados, consideramos que el diseño de los ensayos de quimioluminiscencia empleados no ofrecen una especificidad suficiente utilizados como pruebas únicas para el diagnóstico de la hepatitis C.

Palabras clave:

Quimioluminiscencia

Hepatitis C

Índice S/CO

Full Text

Introduction

Hepatitis C virus (HCV) infection is a significant global public health problem. It is estimated that more than 175 million people in the world may be infected by this agent, generating between 3 and 4 million new cases per year.1 In western countries, the prevalence of infection is below 3%, and is around 2% in Spain. However, in other geographical areas such as North Africa, the prevalence reaches 15%, with the main risk factor being the use of contaminated material for the administration of injectables. In these risk groups the prevalence can reach 70%.2

Although the acute form of the infection is asymptomatic in most cases, up to 70–80% of those infected remain as chronic carriers of the virus and approximately 20% of them, in the absence of treatment, have a considerable risk of developing liver cirrhosis. In addition, after 20 years of evolution, about 5% end in a hepatocarcinoma. Currently, in our environment, HCV is the main cause of liver transplantation.3

The first link in the microbiological diagnosis of HCV infection consists of the detection of antibodies against the virus, complemented later with the confirmation by Recombinant ImmunoBlot Assay (RIBA) or the quantification of the viral RNA that allows, besides confirming the infection, for differentiating stages in its evolution.4

Positive anti-HCV results, but weakly reactive ones (low values of the S/CO index), regardless of the system used, have a low positive predictive value of current HCV infection and correspond more frequently to false reactivities or to infections already resolved in the past.5

The objective of this study was to learn the correlation between the weakly reactive results of 2 latest-generation screening tests and how many of these sera with low reactivity correspond to current infection or are past infections that have been resolved or are false positives.

Material and methods

100 samples were collected whose anti-HCV S/CO index was between ≥1.00 and ≤3.00, using a LIAISON® XL MUREX HCVAb chemiluminescent immunoassay (CLIA) (DiaSorin) that uses the NS4 and NS3 biotinylated core viral antigens and another 100 samples with the same S/CO index according to the CMIA ARCHITECT Anti-HCV® (Abbott) assay that uses the HCr43 and c100-3 viral antigens. Both tests were conducted and interpreted following the instructions of the manufacturers.

The results obtained with both systems were categorised as “reactive” or “non-reactive”, according to the manufacturer's instructions: the samples with signal-to-cut-off ratio below 1.00 were classified as non-reactive for anti-HCV antibodies, and the samples with signal-to-limit ratio equal to or greater than 1.00 were classified as reactive for anti-HCV antibodies.

Subsequently, a quantification of HCV RNA was carried out in all samples by real-time PCR using the COBAS® Ampliprep/COBAS® Taqman® HCV Quantitative v2.0 (ROCHE) test whose lower limit of detection is 15IU/ml.

Results

Of the 100 samples initially evaluated with LIAISON with an anti-HCV S/CO ratio between ≥1.00 and ≤3.00, when tested with ARCHITECT, only 29 were positive, showing the results that appear in Table 1.

Table 1.

Results of samples with S/CO ≥1.00 and ≤3.00 with DiaSorin and their correlation with ARCHITECT and real-time PCR.

S/CO ratioResults with LIAISON XLLIAISON XL results with PCR (+)Results with ARCHITECTARCHITECT results with PCR (+)
<1.00065 (65%)0
1.0–3.0100629 (29%)1
>3.0006 (6%)5

The results of the 100 samples tested by the Abbott reagent, and then by the DiaSorin assay, appear in Table 2: only 40 showed a positive result with the second assay.

Table 2.

Results of samples with S/CO 1–3 by ARCHITECT and their correlation with DiaSorin and PCR.

S/CO ratioResults with ARCHITECTARCHITECT results with PCR (+)Results with LIAISON XLLIAISON XL results with PCR (+)
<1.00056 (56%)0
1.0–3.0100240 (40%)2
>3.0004 (4%)0

Viral RNA was only detected in 8 (4%) of all of the selected samples.

Discussion

Although the CDC recommends that in order for a patient to present diagnostic evidence of hepatitis C, a confirmation with a second test equal or more specific than the one used – such as RIBA or a positive nucleic acid amplification of the virus – is necessary to avoid false positive diagnoses,6 especially in populations with low prevalence of the disease, in reality, numerous clinical laboratories simply offer a single positive result when establishing a presumptive diagnosis of hepatitis C, based on a single serological test.7 In most of these situations, the serological test used is usually a chemiluminescence test (CLIA or CMIA) designed to be used as screening in a low-risk population, such as health examinations or job evaluations, where, due to its high sensitivity, very low S/CO ratios can be detected, but, given their lower specificity, they are not sufficient to establish the clinical diagnosis of the disease.

Although with these techniques the presence of a high S/CO, clearly above the limit, presents a high possibility of defining the corresponding serum as carrier of anti-HCV antibodies, the same does not occur with those that have a low S/CO ratio, close to the limit of reactivity, which for this equipment would be between ≥1 and ≤3, and which, even serving as alarms, do not guarantee in any way the individual diagnosis and need to be confirmed preferably with the detection of circulating virus.

The problem of false positives in hepatitis C screening is also relatively well known and may be due to the increase in gammaglobulins, autoimmune diseases, liver diseases or other viral or parasitic infections.8

In our study, when the 200 sera considered as reactive (positive) by some of the assays were subjected to the detection of viral RNA by genomic amplification – considered as a highly reliable diagnostic test – viral RNA was only detected in 8 (4%) of them, while on the other hand, in 10 sera, in which some of the tests had high S/CO ratios of between 3.1 and 6.0, 5 (50%) harboured circulating viruses.

Therefore, the samples that were negative in either of the 2 assays presented a negative PCR, which suggests the irrelevance of performing confirmatory tests in this group, which accounted for more than 60% of the samples. On the other hand, we consider that the design of the chemiluminescence assays employed does not offer sufficient specificity when used as a single test for the individual diagnosis of hepatitis C, requiring, when they are positive, the endorsement of a second confirmatory test.

Although it is known that the design of the chemiluminescence assays, including the 2 tested, are intended primarily for use as screening tests and, therefore, establish low cut points trying to enhance their sensitivity at the expense of limiting their specificity, their use in numerous clinical laboratories with diagnostic criteria, sometimes as a single determination, makes it advisable for manufacturers to consider the values in which the S/CO ratios are low as included in a “grey area” of categorisation, which absolutely require confirmation with another test of greater specificity.

Conflicts of interest

The authors declare that they have no conflicts of interest.

References

[1]

World Health Organization; 2014. Available from: http://www.who.int [accessed March 2016].

[2]

A. Delgado-Iribarren García-Campero, J.M. Echevarria Mayo, P. León Rega.

Procedimientos de Microbiología clínica: diagnóstico de las hepatitis víricas.

SEIMC, (2004),

[3]

P. Sharma, A. Lok.

Viral hepatitis and liver transplantation.

Semin Liver Dis, 26 (2006), pp. 285-297

[4]

P. Vermeersch, M. van Ranst, K. Lagrou.

Validation of a strategy for HCV antibody testing with two enzyme immunoassays in a routine clinical laboratory.

J Clin Virol, 42 (2008), pp. 394-398

http://dx.doi.org/10.1016/j.jcv.2008.02.015 | Medline

[5]

M. Moretti, B. Pieretti, A. Masucci, D. Sisti, M. Rocchi, E. Delprete.

Role of signal-to-cutoff ratios in hepatitis C virus antibody detection.

Clin Vaccine Immunol, 19 (2012), pp. 1329-1331

[6]

Centers for Disease Control and Prevention (CDC).

Testing for HCV infection: an update of guidance for clinicians and laboratorians.

MMWR Morb Mortal Wkly Rep, 62 (2013), pp. 362-365

[7]

M.J. Alter, W.L. Kuhnert, L. Finelli.

Guidelines for laboratory testing and results reporting of antibody to hepatitis C virus.

MMWR Recomm Rep, 52 (2003), pp. 1-16

[8]

A. Berger, H. Rabenau, R. Allwinn, H.W. Doerr.

Evaluation of the new ARCHITECT anti-HCV screening test under routine laboratory conditions.

J Clin Virol, 43 (2008), pp. 158-161

http://dx.doi.org/10.1016/j.jcv.2008.05.009 | Medline

Please cite this article as: López-Fabal MF, Pérez-Rivilla A, Gómez-Garcés JL. Valoración de sueros con bajo índice S/CO utilizando 2 sistemas de quimioluminiscencia para la detección de anticuerpos frente al virus de la hepatitis C y su correlación con la detección de ARN viral. Enferm Infecc Microbiol Clin. 2018;36:222–224.

Copyright © 2017. Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica

FAQs

What is HCV signal-to-cutoff ratio? ›

The S/Co ratio of anti-HCV reactive samples ranged from 0.9-11.1 [mean = 5.1; SD ± 2.9] whereas S/Co ratio of anti HCV and NAT reactive samples (concordant positives) ranged from 4.1-11.1 [mean 7.3]. In our analysis we found that S/CO ratio of 4 showed positive predictive value (PPV) and sensitivity of 100%.

What is a signal to cut off ratio? ›

Signal to cut-off ratio, calculated by dividing the OD value of the sample being tested by the OD value of the. assay cut-off for that run. Screening tests. Serologic immunoassays for detection of anti-HCV (e.g., Abbott HCV EIA 2.0, ORTHO HCV Version 3.0 ELISA, and VITROS Anti-HCV) Screening-test--negative.

What does HCV signal-to-cutoff mean? ›

Reactive hepatitis C virus (HCV) antibody screening results with signal-to-cutoff (S/Co) ratios of below 8.0 are not predictive of the true HCV antibody status. Additional testing is recommended to confirm anti-HCV status.

What is HCV S CO ratio? ›

Samples with an S/CO ratio of 3.8 or above have a >95% probability of predicting true positive anti-HCV and are indicative of past or present infection. Supplemental testing by qualitative or quantitative PCR on these patients allows assessment of viral activity.

What is low HCV? ›

Low viral load: This is a count below 800,000 IU/mL. Your odds that treatment will make all or most of your HCV go away are better than with a high viral load.

What is cut off ratio How does it affect the air standard efficiency? ›

In diesel engines the fuel cut off ratio depends on the output and therefore unlike the otto cycle the efficiency of the diesel cycles depends on the output. When the cut-off ratio of diesel cycle increases, the air standard efficiency of the cycle is decreased when compression ratio is kept constant.

Is 11.0 positive for hep C? ›

Hepatitis C Antibody (HCV Ab)

Positive results with an index of > 11.0 are accepted by CDC as having anti-HCV status with no need of confirmation.

Can you test positive for hep C and not have it? ›

If you receive a positive hepatitis C antibody test it does not necessarily mean you are currently infected. Up to 20% of people clear the virus from their bodies naturally. This is called 'spontaneous clearance'. Although they have cleared the virus, tests will still show the presence of hepatitis C antibodies.

What is a positive HCV result? ›

What does a reactive HCV antibody test result mean? A reactive or positive antibody test means you have been infected with the hepatitis C virus at some point in time. Once people have been infected, they will always have antibodies in their blood.

What is a good viral load for hep C? ›

A viral load of less than 800,000 IU/mL (international units per milliliter) is considered low. Successful treatment is more likely with a low viral load.

How long is HCV undetectable? ›

After exposure to the hepatitis C virus, it can take 8–11 weeks for an HCV antibody test to be positive. For most people who are infected, the anti-HCV blood test will become positive by 6 months after exposure.

What does non reactive anti HCV mean? ›

What does a non-reactive HCV antibody test result mean? ► A non-reactive or negative antibody test means that you are not currently infected with the hepatitis C virus. ► However, if you think you might have been exposed to hepatitis C in the last 6 months, you will need to be tested again.

What does 0.3 mean on a hep C test? ›

Patients were grouped into three categories according to the OD obtained: < 0.3 (negative test); 0.3-0.6 (intermediate positivity); > 0.6 (high positivity).

What lab values indicate hepatitis? ›

A positive HBeAg indicates high levels of virus in the blood and a person is considered infectious. A negative HBeAg indicates very low to no virus in the blood and a person is usually considered less infectious; sometimes this can indicate a person has a mutant hepatitis B virus (see below).

Why would my doctor order a Hep C antibody test? ›

A doctor may order hepatitis C testing for screening, diagnosis, and to guide and monitor treatment. Acute hepatitis C: This occurs in the first six months after you are exposed to the virus. Early in the illness, acute hepatitis C is mild and may cause no symptoms.

Which hepatitis is not curable? ›

A vaccine can prevent hepatitis B, but there's no cure if you have the condition. If you're infected, taking certain precautions can help prevent spreading the virus to others.

Can you have hep C without liver damage? ›

How the disease progresses varies significantly from person to person. After many years some people will have minimal liver damage with no scarring while others can progress to cirrhosis (extensive scarring of the liver) within less than ten years.

How long can you live without hep C treatment? ›

A 2000-2011 study of the lifespan impact of chronic hepatitis C in New York City (NYC) found that people with hepatitis C died at an average age of 60 years, while those without hepatitis C infection lived to an average age of 78 years.

How do you calculate cutoff ratio for dual cycle? ›

Answer and Explanation:
  1. Compression ratio, r=(V1V2)=12.
  2. Cutoff ratio, rc=(V4V3)=1.4.
  3. Pressure ratio for constant volume heat addition process, (P3P2)=1.25.

What is cut off ratio in dual cycle? ›

9-54 An ideal dual cycle has a compression ratio of 14 and cutoff ratio of 1.2. The thermal efficiency, amount of heat added, and the maximum gas pressure and temperature are to be determined.

How does cut off ratio affect the thermal efficiency of a diesel cycle? ›

The cutoff ratio is the ratio of the volumes prior and following the combustion. The Diesel cycle thermal efficiency decreases with the increase of the cutoff ratio.

What is the normal range of hepatitis? ›

For hepatitis B surface antibody (anti-HBs), a level less than 5 mIU is considered negative, while a level more than 12 mIU is considered protective. Any value between 5 and 12 mIU is indeterminate and should be repeated.

What level of liver enzymes indicate hepatitis? ›

A transaminitis greater than 1000 is suggestive of acute viral hepatitis, ischemic injury, medication/toxin induced injury (most commonly acetaminophen) or autoimmune hepatitis. More rare causes of transaminitis greater than 1000 include Wilson's disease and acute biliary obstruction.

What levels are high in autoimmune hepatitis? ›

Patients typically have an elevated AST, ALT, and gamma globulins typical of AIH and also elevated alkaline phosphatase and IgM characteristic of PBC.

Can you test negative for hepatitis and still have it? ›

A single positive PCR test indicates infection with HCV. A single negative test does not prove that a person is not infected. Virus may be present in the blood and just not found by PCR. Also, a person infected in the past who has recovered may have a negative test.

When is hep C test conclusive? ›

Even at 15 weeks, only about 80% of HCV-infected persons will have positive HCV Ab [MMWR rr5005a1]. Therefore, the6-month (24-week) HCV antibody test is considered to be conclusive in excluding HCV acquisition: ≥97% will be positive at 6 months post exposure [MMWR rr5005a1].

Can hepatitis be undetectable? ›

Inactive Carrier State: Most people transition into the inactive hepatitis B phase, which is characterized by undetectable or low viral loads (less than 2,000 IU/mL) and improvement in liver inflammation and fibrosis. Adults in this phase will also typically have normal ALT levels and minimal inflammation.

What can cause a Hep C false positive? ›

Causes of a false-positive hepatitis C test

You may receive a false-positive result if your antibodies are triggered by another infection. People who've recovered from hepatitis C on their own may also get a false-positive anti-HCV test result. In rare cases, lab error leads to a false positive.

Can your body fight off Hep C on its own? ›

Can hepatitis C go away on its own? Yes. From 15% to 20% of people with hep C clear it from their bodies without treatment. It's more likely to happen in women and people who have symptoms.

What is the acceptable viral load? ›

An undetectable viral load will be under 40 to 75 copies in a blood sample. This means there are relatively few copies of HIV in the blood. When this happens, it is no longer possible to pass the virus on to another person during sex, according to the CDC .

What is the normal number of viral load? ›

Without treatment, the range of viral loads seen is anything from undetectable to >10 million copies/mL. However, most patients fall within the range of approximately 10,000 to 200,000 copies/mL, if you do random viral loads on them.

What is a good number for a viral load? ›

A lower HIV viral load is below 10,000 copies per milliliter of blood. The virus probably isn't actively reproducing as fast and damage to the immune system may be slowed, but this is not optimal. An undetectable HIV viral load is generally considered to be less than 20 copies per milliliter of blood.

Can hep C come back after you get rid of it? ›

It's possible, but rare, for hepatitis C infection to reappear after apparently successful treatment. Relapses usually occur in the first few months after blood testing to confirm that the virus is no longer detectable. Sometimes, however, a relapse becomes evident much later.

What is a normal HCV viral load? ›

Understanding the viral load range

Fewer than 15 IU/mL: The virus is detected, but the amount can't be measured exactly. You may need to return later for another test to see if the measurement changes. Fewer than 800,000 IU/mL: A low viral load is detected. More than 800,000 IU/mL: A high viral load is detected.

What is a high viral load for HCV? ›

For each patient, the result can be described as either a "high" viral load, which is usually >800,000 IU/L, or a "low" viral load, which is usually <800,000 IU/L. It's not uncommon to have a viral load in the millions. Today's hepatitis C treatments are very effective with both high and low viral loads.

What is a positive HCV antibody result? ›

What does a reactive HCV antibody test result mean? A reactive or positive antibody test means you have been infected with the hepatitis C virus at some point in time. Once people have been infected, they will always have antibodies in their blood.

What is the reference range for hep C? ›

Reference Range:

A result of <15 IU/mL (<1.18. log IU/mL) indicates that HCV RNA is detected, but the HCV RNA level present cannot be quantified accurately below this lower limit of quantification of this assay. When clinically indicated, follow-up testing with this assay is recommended in 1 to 2 months.

Can chronic HCV be cured? ›

Today, chronic HCV is usually curable with oral medications taken every day for two to six months. Still, about half of people with HCV don't know they're infected, mainly because they have no symptoms, which can take decades to appear.

When do you treat Hep C viral load? ›

Antiviral therapy for chronic hepatitis C should be determined on a case-by-case basis. However, treatment is widely recommended for patients with elevated serum alanine aminotransferase (ALT) levels who meet the following criteria : Age older than 18 years. Positive HCV antibody and serum HCV RNA test results.

When HCV test is conclusive? ›

After 6 months , most people will have developed enough antibodies for an HCV test to detect. In rare cases, however, antibodies can take up to 9 months to develop. If a person has a test during this window period, a hepatitis C antibody test may return a negative result.

How accurate is HCV Antibody Test? ›

According to a 2016 review , third-generation anti-HCV tests have an average specificity of 97.5% to 99.7%. The sensitivity of these tests varies from 61.0% to 81.8%. These findings indicate that anti-HCV tests detect true negatives (specificity) more accurately than true positives (sensitivity).

Can HCV antibodies disappear? ›

Even when a person has cleared HCV or been cured by treatment, HCV antibodies remain in a person's blood for years.

How high are ALT levels in hepatitis? ›

ALT levels often rise to several thousand units per liter in patients with acute viral hepatitis. The highest ALT levels—often more than 10,000 U per L—are usually found in patients with acute toxic injury subsequent to, for example, acetaminophen overdose or acute ischemic insult to the liver.

What liver levels indicate hepatitis? ›

The AST is typically in the 100 to 200 IU/L range, even in severe disease, and the ALT level may be normal, even in severe cases. The AST level is higher than the ALT level, and the ratio is greater than 2:1 in 70% of patients. A ratio greater than 3 is strongly indicative of alcoholic hepatitis.

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