The “WHO” and “HOW” of HBV AND HCV testing: A glimpse of the latest WHO guidelines 2017

“WHO” TO TEST FOR CHRONIC HBV INFECTION?


Testing Approach and Population Recommendations
General Population Testing In settings with a = 2% or =5% HBsAg seroprevalence in the general population, it is recommended that all adults have routine access to and be offered HBsAg serological testing with linkage to prevention, care and treatment services General population testing approaches should make use of existing community/ or health facility based testing opportunities or programmes such as at antenatal clinics, HIV or TB clinics.
Routine Testing in Pregnant Women In settings with a = 2% or =5% HBsAg seroprevalence in the general population, it is recommended c that HBsAg serological testing is routinely offered to all pregnant women in antenatal clinics, with linkage to prevention, care and treatment services. Couples and partners in antenatal care settings should be offered HBV testing services.
Focused Testing in Most Affected Populations In all settings (and regardless of whether delivered through facility/ or community- based testing), it is recommended that HBsAg serological testing and linkage to care and treatment services be offered to the following individuals:

  • • Adults and adolescents from populations most affected by HBV infection (i.e. who are either part of a population with high HBV seroprevalence or who have a history of exposure and/or high-risk behaviours for HBV infection)
  • • Adults, adolescents and children with a clinical suspicion of chronic viral hepatitis
  • • Sexual partners, children and other family members, and close household contacts of those with f HBV infection
  • • Health-care workers: in all settings, it is recommended that HBsAg serological testing be offered and hepatitis B vaccination is given to all health-care workers who have not been vaccinated g previously ( adapted from existing guidance on hepatitis B vaccination)
Blood donors (WHO guidance on screening donated blood for transfusion transmissible infections) In all settings, screening of blood donors should be mandatory with linkage to care, counselling and treatment for those who test positive.

Abbreviations: HBsAg hepatitis B surface antigen, PWID people who inject drugs, MSM men who have sex with men a The GRADE system (Grading of Recommendations, Assessment, Development and Evaluation) was used to categorize the strength of recommendations as strong or conditional (based on consideration of the quality of evidence, balance of benefits and harms, acceptability, resource use and programmatic feasibility) and the quality of evidence as high, moderate, low or very low

A threshold of ≥2% or ≥5% seroprevalence was based on several published thresholds of intermediate or high seroprevalence. The threshold used will depend on other country considerations and epidemiological context

Many countries have chosen to adopt routine testing in all pregnant women, regardless of seroprevalence in the general population, and particularly where seroprevalence ≥2%. g A full vaccination schedule including birth dose should be completed in all infants, in accordance with the WHO position paper on hepatitis B vaccines 2009 d Includes those who are either part of a population with higher seroprevalence (e.g. some mobile/migrant populations from high/intermediate endemic countries, and certain indigenous populations) or who have a history of exposure or high-risk behaviours for HBV infection (e.g. PWID, people in prisons and other closed settings, MSM and sex workers, HIV-infected persons, partners, family members and children of HBV-infected persons)

Features that may indicate underlying chronic HBV infection include clinical evidence of existing liver disease, such as cirrhosis or hepatocellular carcinoma (HCC), or where there is unexplained liver disease, including abnormal liver function tests or liver ultrasound

In all settings, it is recommended that HBsAg serological testing with hepatitis B vaccination of those who are HBsAg negative and not previously vaccinated be offered to all children with parents or siblings diagnosed with HBV infection or with clinical suspicion of hepatitis, through community- or facility-based testing

WHO position paper. Hepatitis B vaccines. Weekly Epidemiological Record. 2009;4 (84):405–20

Screening donated blood for transfusion transmissible infections. Geneva: World Health Organization; 2010

“HOW” TO TEST FOR CHRONIC HBV INFECTION AND MONITOR TREATMENT RESPONSE?


Topic Recommendations
Which Serological Assays to Use? For the diagnosis of chronic HBV infection in adults, adolescents and children (>12 months of b c d age ), a serological assay that meets minimum quality, safety and performance standards (with regard to both analytical and clinical sensitivity and specificity) is recommended to detect hepatitis B surface antigen (HBsAg).

  • • In settings where existing laboratory testing is already available and accessible, laboratory based immunoassays are recommended as the preferred assay format
Serological Testing Strategies
  • • In settings or populations with an HBsAg seroprevalence of ≥0.4%, a single serological assay for detection of HBsAg is recommended, prior to further evaluation for HBV DNA and staging of liver disease
  • • In settings or populations with a low HBsAg seroprevalence of
Detection of HBV DNA: Assessment for treatment (WHO HBV 2015 guidelines )
  • • Directly following a positive HBsAg serological test, the use of quantitative or qualitative nucleic acid testing (NAT) for detection of HBV DNA is recommended as the preferred strategy and to guide who to treat or not treat
Monitoring for HBV Treatment Response and Disease Progression (WHO HBV 2015 Guidelines ) It is recommended that the following be monitored at least annually:

  • • ALT levels (and AST for APRI), HBsAg, HBeAg, & HBV DNA levels (where HBV DNA testing is available)
  • • Non-invasive tests (APRI score or transient elastography) to assess for the presence of cirrhosis in those without cirrhosis at baseline
  • • If on treatment, adherence should be monitored regularly and at each visit More frequent monitoring is recommended:
  • • In persons on treatment or following treatment discontinuation: more frequent on- treatment monitoring (at least every 3 months for the first year) is indicated
  • • In persons with more advanced disease (compensated or decompensated cirrhosisj)
  • • During the first year of treatment to assess treatment response and adherence
  • • Where treatment adherence is a concern;
  • • In HIV-coinfected persons
  • • In persons after discontinuation of treatment
  • • In persons who do not yet meet the criteria for antiviral therapy: i.e. persons who have intermittently abnormal ALT levels or HBV DNA levels that fluctuate between 2000 IU/mL h and 20,000 IU/mL (where HBV DNA testing is available) and in HIV coinfected persons

Abbreviations: ALT alanine aminotransferase, AST aspartate aminotransferase, APRI aspartate-to-platelet ratio index, HBeAg HBV e antigen, HBsAg HBV surface antigen, NAT nucleic acid test, RDT rapid diagnostic test

The GRADE system (Grading of Recommendations, Assessment, Development and Evaluation) was used to categorize the strength of recommendations as strong or conditional (based on consideration of the quality of evidence, balance of benefits and harms, acceptability, resource use and programmatic feasibility) and the quality of evidence as high, moderate, low or very low

 A full vaccination schedule including birth dose should be completed in all infants in accordance with the WHO position paper on Hepatitis B vaccines, 2009. Testing of exposed infants is problematic within the first six months of life as HBsAg and hepatitis B DNA may be inconsistently detectable in infected infants. Exposed infants should be tested for HBsAg between 6 and 12 months of age to screen for evidence of hepatitis B infection. In all age groups, acute HBV infection can be confirmed by the presence of HBsAg and IgM anti-HBc. CHB is diagnosed if there is persistence of HBsAg for six months or more

Laboratory-based immunoassays include enzyme immunoassay (EIA), chemoluminescence immunoassay (CLIA), and electrochemoluminescence assay (ECL)

Assays should meet minimum acceptance criteria of either WHO prequalification of in vitro diagnostics (IVDs) or a stringent regulatory review for IVDs. All IVDs should be used in accordance with manufacturers’ instructions for use and where possible at testing sites enrolled in a national or international external quality assessment scheme e Based on results of predictive modelling of positive predictive values according to different thresholds of seroprevalence in populations to be tested, and assay diagnostic performance

A repeat HBsAg assay after 6 months is also a common approach used to confirm chronicity of HBV infection g For further details, see Chapter 5: Who to treat and who not to treat. Guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection: World Health Organization; 2015

In persons on treatment, monitor for HBsAg loss (although this occurs rarely), and for seroreversion to HBsAg positivity after discontinuation of treatment

Monitoring of HBeAg/anti-HBe mainly applies to those who are initially HBeAg positive. However, those who have already achieved HBeAg seroconversion and are HBeAg negative and anti-HBe positive may serorevert

Decompensated cirrhosis is defined by the development of portal hypertension (ascites, variceal haemorrhage and hepatic encephalopathy), coagulopathy, or liver insufficiency (jaundice). Other clinical features of advanced liver disease/cirrhosis may include: hepatomegaly, splenomegaly, pruritus, fatigue, arthralgia, palmar erythema and oedema

“WHO” TO TEST FOR CHRONIC HCV INFECTION?


Testing Approach and Population Recommendations
Focused Testing in Most Affected Populations In all settings (and regardless of whether delivered through facility/ or community- based testing), b it is recommended that serological testing for HCV antibody (anti- HCV) be offered with linkage to prevention, care and treatment services to the following individuals:

  • • Adults and adolescents from populations most affected by HCV infection (i.e. who are either part of a population with high HCV seroprevalence or who have a history of exposure and/or high-risk behaviours for HCV infection)
  • • Adults, adolescents and children with a clinical suspicion of chronic viral hepatitis
    Note: Periodic re-testing using HCV NAT should be considered for those with an ongoing risk of acquisition or reinfection
General population Testing
  • • In settings with a ≥ 2% or ≥5% HCV antibody seroprevalence in the general population, it is recommended that all adults have access to and be offered HCV serological testing with linkage to prevention, care and treatment services
  • • General population testing approaches should make use of existing community/ or facility-based testing opportunities or programmes such as HIV or TB clinics, drug treatment services and f antenatal clinics
Birth Cohort Testing
  • • This approach may be applied to specific identified birth cohorts of older persons at higher risk of g infection and morbidity within populations that have an overall lower general prevalence

Abbreviations: NAT nucleic acid test, anti-HCV HCV antibody, PWID people who inject drugs, MSM men who have sex with men

The GRADE system (Grading of Recommendations, Assessment, Development and Evaluation) was used to categorize the strength of recommendations as strong or conditional (based on consideration of the quality of evidence, balance of benefits and harms, acceptability, resource use and programmatic feasibility) and the quality of evidence as high, moderate, low or very low

This may include fourth-generation combined antibody/antigen assays c Includes those who are either part of a population with higher seroprevalence (e.g. some mobile/migrant populations from high/intermediate endemic countries, and certain indigenous populations) or who have a history of exposure or high-risk behaviours for HCV infection (e.g. PWID, people in prisons and other closed settings, MSM and sex workers, and HIV-infected persons, children of mothers with chronic HCV infection especially if HIV-coinfected)

Features that may indicate underlying chronic HCV infection include clinical evidence of existing liver disease, such as cirrhosis or hepatocellular carcinoma (HCC), or where there is unexplained liver disease, including abnormal liver function tests or liver ultrasound

A threshold of ≥2% or ≥5% seroprevalence was based on several published thresholds of intermediate and high seroprevalence. The threshold used will depend on other country considerations and epidemiological context Routine testing of pregnant women for HCV infection is currently not recommended

Because of historical exposure to unscreened or inadequately screened blood products and/or poor injection safety

“HOW” TO TEST FOR CHRONIC HCV INFECTION AND MONITOR TREATMENT RESPONSE?


Testing Approach and Population Recommendations
Which serological Assays to Use?
  • • To test for serological evidence of past or present infection in adults, adolescents and children b c (>18 months of age ), an HCV serological assay (antibody or antibody/ antigen) that meets d minimum safety, quality and performance standards (with regard to both analytical and clinical sensitivity and specificity) is recommended
  • • In settings where there is limited access to laboratory infrastructure and testing, and/or in populations where access to rapid testing would facilitate linkage to care and treatment, RDTs are recommended
Serological Testing Strategies
  • • In adults and children older than 18 months, a single serological assay for initial detection of serological evidence of past or present infection is recommended prior to supplementary nucleic acid testing (NAT) for evidence of viremic infection
Detection of Viremic Infection
  • • Directly following a reactive HCV antibody serological test result, the use of quantitative or qualitative NAT for detection of HCV RNA is recommended as the preferred strategy to diagnose viremic infection
Assessment of HCV Treatment Response
  • • Nucleic acid testing for qualitative or quantitative detection of HCV RNA should be used as a test of cure at 12 or 24 weeks (i.e. sustained virological response (SVR12 or SVR24)) after completion of antiviral treatment

Abbreviations: DBS dried blood spot, IVD in vitro diagnostics, NAT nucleic acid test, RDT rapid diagnostic test

The GRADE system (Grading of Recommendations, Assessment, Development and Evaluation) was used to categorize the strength of recommendations as strong or conditional (based on consideration of the quality of evidence, balance of benefits and harms, acceptability, resource use and programmatic feasibility) and the quality of evidence as high, moderate, low or very low

HCV infection can be confirmed in children under 18 months only by virological assays to detect HCV RNA, because transplacental maternal antibodies remain in the child’s bloodstream up until 18 months of age, making test results from serology assays ambiguous  

Laboratory-based immunoassays include enzyme immunoassay (EIA), chemoluminescence immunoassay (CLIA), and electrochemoluminescence assay (ECL) Assays should meet minimum acceptance criteria of either WHO prequalification of IVDs or a stringent regulatory review for IVDs. All IVDs should be used in accordance with manufacturers’ instructions, and where possible at testing sites enrolled in a national or international external quality assessment scheme

PROCALCITONIN AS A BIOMARKER FOR SERIOUS BACTERIAL INFECTIONS AND SEPSIS

Procalcitonin for the diagnosis, prognosis and monitoring of sepsis

Procalcitonin has been used in Europe for many years and was also approved for use in the United States by the US Food and Drug Administration (FDA) as a diagnostic aid for sepsis in 2005. It also gained an FDA indication in 2016 for serial use to assess sepsis progression and 28-day mortality risk. Several different procalcitonin assays exist, but all demonstrate good concordance at clinically relevant cutoffs.

Serum procalcitonin levels rise rapidly in response to systemic inflammatory insults, with peak levels that correlate with the intensity of the stimulus. Procalcitonin has a short half-life (25–30 hours), and levels decline rapidly with resolution of inflammation. These properties make it potentially useful in helping decide whether to start antibiotics and when to stop antibiotics in a clinically improving patient.

procalcitonin levels are significantly higher in culture-positive sepsis versus culture negative-sepsis, and thus diagnostic performance may be better in the former group of patients. Furthermore, in critically ill patients with microbiologically documented infection, procalcitonin levels differ by site of infection, with the highest levels in those with positive blood cultures and lowest with pulmonary cultures.

The use of procalcitionin in therapy decision making

Because of the lack of a reliable reference standard for identifying bacterial pneumonia and sepsis, particularly in culture-negative patients, the utility of procalcitonin cannot be determined through observational studies alone.

However, numerous randomized intervention studies have examined whether procalcitonin-based algorithms can help clinicians decide whether to start or stop antibiotic therapy, without adversely impacting patients through delayed antibiotic therapy or inadequate treatment courses.

These studies have primarily demonstrated efficacy in 2 clinical scenarios: adult patients with suspected respiratory infections, and critically ill adults with any type of suspected infection. Improving antibiotic use in these 2 clinical scenarios carries the potential for significant benefit given that respiratory infections are the most common indication for antibiotics in hospitalized patients and antibiotic use is most prevalent in ICUs.

Procalcitonin to guide antibiotic therapy for respiratory infections

The largest study to date examining procalcitonin for respiratory infections, the Procalcitonin Guided Antibiotic Therapy and Hospitalisation in Patients With Lower Respiratory Tract Infections (ProHOSP) concluded that procalcitonin levels can be safely used in patients with lower respiratory tract infections like pneumonia; to guide treatment decisions regarding initiation and discontinuation of therapy.

A subsequent Cochrane review concluded that procalcitonin guidance for respiratory patients in a variety of settings (including the emergency department, ICU, and primary care settings) resulted in a significant reduction in total antibiotic exposure (median 4 days vs 8 days), with no difference in mortality or rates of treatment failure.

How to interpret and use procalcitonin levels for guiding antibiotic therapy in stable and critically ill patients with infections?

Non-critically ill patients with suspected or proven respiratory infection

Procalcitonin can be safely used at a cutoff of <0.25 µg/L to withhold antibiotics in stable, low-risk patients with suspected respiratory infections. If antibiotics are given, procalcitonin can help inform early discontinuation of antibiotics based on serial measurements, even in those with documented bacterial infections.

Critically ill patients with suspected infection/sepsis

Clinicians are understandably reluctant to withhold antibiotics when sepsis is first suspected in unstable patients, regardless of procalcitonin levels, but serial procalcitonin measurements can be used at a cutoff of <0.5 µg/L or >80% decrease in peak level to help guide early antibiotic discontinuation once patients stabilize. Using procalcitonin in this fashion is safe, even in those with documented infections, and the largest trial to date (SAPS) suggests that it may even reduce mortality.

Precautions and Conclusions
  • Clinicians must remember that false positives and negatives can occur with procalcitonin testing and that sepsis is a complex and heterogeneous syndrome
  • In most of the procalcitonin trials, overruling of the algorithm was allowed and in fact common in the ICU-based studies, underscoring the fact that procalcitonin should not replace clinical judgment
  • Clinicians should also be aware of the patients typically excluded from trials, particularly severely immunocompromised patients. However, when used properly as a clinical decision aid, the evidence is robust that procalcitonin can be a powerful antibiotic stewardship tool

DIRECT VERSUS CALCULATED (LDL)

LDL TESTING: DIRECT MEASUREMENTIS SUPERIOR TO CALCULATED!
Why is a Low Density Lipoprotein (LDL) test necessary?
  • Every fifth Indian dies due to a heart attack and 80% Indians have a poor lipid profile (Times of India, ICMR study – 2014)
  • Elevated LDL levels are a strong independent risk factor for heart disease (Arterioscler Thromb Vasc Biol. 2000 Mar;20(3):830-5.)
  • Increased LDL levels are also an independent risk factor for Type 2 diabetes (Adv Lab Med Int. 2012; 2(1): 9 -18). With over 50 million Type 2 diabetics, India is now the diabetes capital of the world (Times of India, 2016)
  • High LDL levels significantly contribute towards increased obesity, hypertension and the metabolic syndrome. (Position Paper of The Obesity Society and the American Society of Hypertension – 2012)
  • As Indians, we have a genotype-phenotype that’s more vulnerable to high LDL levels, CVD and metabolic syndrome (Dr. V. Mohan, Indian J Med Res 125, March 2007, pp 217-230
THE IMPORTANCE OF ACCURATE LDL MEASUREMENTS

As LDL levels majorly contribute towards CVD, Hypertension, Type 2 diabetes, Obesity and the Metabolic Syndrome – Ensuring that we get the accurate and precise measurements of LDL is of paramount importance. An error in the value of LDL could misguide treatment planning and outcomes

HOW IS LDL MEASURED IN A LABORATORY?

Indirect or calculated LDL is derived by the Friedwald formula

Total Cholesterol = HDL + LDL + VLDL

VLDL = Triglyceride / 5

LDL = Total cholesterol – (HDL + Triglyceride / 5)

Friedewald formula

The Direct LDL measurement uses a homogenous assay based on a beta quantification kit that directly measures the LDL component from a serum sample; without needing to rely on TC, TG or HDL levels.

WHY IS THE CALCULATED LDL AN INFERIOR METHOD OF MEASUREMENT?

The drawbacks of using the Friedwald Formula for determining levels of LDL cholesterol are:

  • Calculated LDL is not a directly measured value. It’s an indirect estimation derived via a formula
  • It requires multiple assays and multiple steps each adding a potential source of error
  • It is inaccurate in assessing LDL levels among individuals with elevated triglyceride levels (>400 mg/dL). In addition, it has been reported that the formula becomes increasingly inaccurate at borderline triglyceride levels (200-400 mg/dL). Any lapses in TC, TG and HDL measurements can confound the calculated LDL values.(Indian J EndocrinolMetab. 2014 Jul-Aug; 18(4): 502–504
WHAT MAKES DIRECT LDL MEASUREMENT, A SUPERIOR AND PREFERRED METHOD?
  • It is a direct measurement of LDL by a homogenous assay. This excludes the possibility of any error occurring due to indirect and derived estimations
  • Direct LDL is measured on an automated platform with 2- level controls at a defined interval. The process is standardized
  • It provides an accurate estimation of LDL results regardless of the TG levels
  • As the direct measurement does not rely on other parameters like TC, TG and HDL – it is not influenced or confounded by any errors in the reporting of these other lipid values. It ensures accurate risk stratification of CVD and guides correct treatment planning

Ref: Indian J EndocrinolMetab. 2014 Jul-Aug; 18(4): 502–504.

Pharmacotherapy. 2004 Feb;24(2):167-72.

Pak J Med Sci. 2016 Jul-Aug; 32(4): 955–960

INTERNATIONAL GUIDELINES RECOMMEND DIRECT LDL

Recommend using only direct LDL methods, especially when the LDL is below 70 mg/dL and TG is higher than the range of 15 to 200 mg/dL

ATHEROSCLEROTIC CARDIOVASCULAR DISEASE (ASCVD) PRIMARY PREVENTION GUIDELINE

LDL values can come from either a fasting lipoprotein panel or a direct LDL test. The lipoprotein panel indirectly calculates LDL cholesterol by using the Friedewald equation (LDL = TC-HDL-TG/). In general, either test can be used, but be aware that the Friedewald equation tends to underestimate LDL when LDL is low (-70) or triglycerides are high (> 150-200). In these cases, the true LDL value can be 10-20 points higher. So, in such cases, consider using a direct LDL test or ensuring that the patient is well below target.

The NCEP ATP III report recognizes the benefit of a truly accurate direct LDL-C that could be used in the non-fasting state.

Third Report of the National Cholestrol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II).

INDIAN STUDY SPANNING APPROX. 15,000 PATIENTS SUPPORTS THE USE OF DIRECT LDL IN TG > 200 AND LDL 130 mg/dL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC413 8905/ Original Article

LDL-cholesterol: Friedwald calculated versus direct measurement-study from a large Indian laboratory database

Subramanian Kannan, Shriraam Mahadevan , Bharath 2 3 4 Ramji , Muthukumaran Jayapaul , V. Kumaravel Consultant, Endocrinology Diabetes and Bariatric Medicine, 1 Narayan Health City, Banglore, Karnataka, Endocrine and 2 Speciality Clinic, Chennai, Assistant Professor Endocrinology, Chettinad Medical College, Chennai, 3Consultant Endocrinologist, Arka Cenre for Hormonal 4 Health, Chennai, Department of Clinical Endocrinolgy, Alpha Hospital and Research Centre, Anuppanady, Madurai, Tamil Nadu, India Background: Validity of Friedwald Formula (FF) in patients with serum triglycerides (TGs)

Results: LDL by FF correlateed with directly measured LDL with correlation coefficient of 0.89 with the best correlation seen in TG levels 100-150. Higher level of TG (>200) underestimates the LDL calculated by FF particularly at LDL values 130 mg/dl. Conclusion: We suggest repeating the LDL by direct assay techniques particularly in patients with TG 200 and when LDL 130. This helps in correctly stratifying the coronary artery disease’ (CADs’) risk and goals of treatment

IN-HOUSE STUDY AT SUBURBAN DIAGNOSTICS

We performed an in-house retrospective analysis of around 1 lakh samples that were tested at our labs for lipid profile. We performed the LDL test for these samples by both the direct and the calculated methods. The objective was to understand the correlation of results between the two methods. Summary of Results:

Triglycerides Category Number of Bookings Mean of LDL Cholesterol Direct Mean of LDL Cholesterol Calculated
Less than 200 88962 114 105
200-300 12683 134 114
300-400 2750 133 107
400 or more 1628 118 77

Across all ranges of TG, the LDL value was under-reported by the calculated or indirect estimation. At the level of TG >200, the calculated LDL method seemed inefficient in reporting the actual LDL and gave consistently lower values. These findings seem consistent with the findings of other larger studies that have shown an underestimation of 20 mg/dL with calculated LDL as compared to direct LDL (Lindsey et al. Pharmacotherapy. 2004;24(2))

KEY TAKE HOME MESSAGES

As per recommendations of international guidelines, for TG levels more than 200 and for LDL levels less than 70 mg/dL, the calculated or indirect method of LDL estimation is not accurate as it provides an underestimated value.

Guidelines recommend opting for the direct LDL measurement in such cases. Direct LDL is an automated assay with controls and provides an accurate estimation of LDL values regardless of TG levels

The FDA Warns that Biotin May Interfere with Lab Tests: FDA Safety Communication

Whom does this concern?
  • People taking or considering taking biotin, vitamin B7, supplements
  • Physicians of all specialities and other health care providers who order lab tests
  • Lab personnel
  • Lab test developers
Product:

Many lab tests use biotin technology due to its ability to bond with specific proteins which can be measured to detect certain health conditions. For example, biotin is used in hormone tests and tests for markers of cardiac health like troponin. Biotin, also known as vitamin B7, is a water-soluble vitamin often found in multi-vitamins, prenatal vitamins, and dietary supplements marketed for hair, skin, and nail growth.

Purpose:

The FDA is alerting the public, health care providers, lab personnel, and lab test developers that biotin can significantly interfere with certain lab tests and cause incorrect test results which may go undetected.

Summary of Problem and Scope:

Biotin in blood or other samples taken from patients who are ingesting high levels of biotin in dietary supplements can cause clinically significant incorrect lab test results. The FDA has seen an increase in the number of reported adverse events, including one death, related to biotin interference with lab tests. Biotin in patient samples can cause falsely high or falsely low results, depending on the test. Incorrect test results may lead to inappropriate patient management or misdiagnosis. For example, a falsely low result for troponin, a clinically important biomarker to aid in the diagnosis of heart attacks, may lead to a missed diagnosis and potentially serious clinical implications. The FDA has received a report that one patient taking high levels of biotin died following falsely low troponin test results when a troponin test known to have biotin interference was used.

The FDA is aware of people taking high levels of biotin that would interfere with lab tests. Many dietary supplements promoted for hair, skin, and nail benefits contain biotin levels up to 650 times the recommended daily intake of biotin. Physicians may also be recommending high levels of biotin for patients with certain conditions such as multiple sclerosis (MS). Biotin levels higher than the recommended daily allowance may cause interference with lab tests. Patients and physicians may be unaware of biotin interference in laboratory assays. Even physicians who are aware of this interference are likely unaware as to whether, and how much biotin, patients are taking. Since patients are unaware of biotin interference, patients may not report taking biotin supplements to their physicians, and may even be unware they are taking biotin (e.g., when taking products generally labeled for their benefits to hair and nails).

Recommendations:
For Consumers:
  • Talk to your doctor if you are currently taking biotin or are considering adding biotin, or a supplement containing biotin, to your diet
  • Know that biotin is found in multivitamins, including prenatal multivitamins, biotin supplements, and supplements for hair, skin, and nail growth in levels that may interfere with laboratory tests
  • Be aware that some supplements, particularly those labeled for hair, skin, and nail benefits, may have high levels of biotin, which may not be clear from the name of the supplement
  • If you have had a lab test done and are concerned about the results, talk to your health care provider about the possibility of biotin interference.
For Health Care Providers:
  • Talk to your patients about any biotin supplements they may be taking, including supplements marketed for hair, skin, and nail growth
  • Be aware that many lab tests, including but not limited to cardiovascular diagnostic tests and hormone tests, that use biotin technology are potentially affected, and incorrect test results may be generated if there is biotin in the patient’s specimen
  • Communicate to the lab conducting the testing if your patient is taking biotin
  • If a lab test result doesn’t match the clinical presentation of your patient, consider biotin interference as a possible source of error
  • Know that biotin is found in multivitamins, including prenatal multivitamins, biotin supplements, and dietary supplements for hair, skin, and nail growth in levels that may interfere with lab tests
  • Report to the lab test manufacturer and the FDA if you become aware of a patient experiencing an adverse event following potentially incorrect laboratory test results due to biotin interference
For Lab Personnel:
  • If you use assays with biotin technology, be aware that it is difficult to identify samples that contain biotin; therefore, it is important to communicate with health care providers and patients to prevent incorrect test results
  • If you are collecting samples in the lab, ask whether the patient is taking biotin
  • Educate health care providers about biotin interference with certain lab tests used in your lab
  • Consider that the daily recommended allowance for biotin is 0.03 mg and these biotin levels do not typically cause significant interference. However, supplements containing high biotin levels including those marketed for hair, skin, and nail benefits, may contain up to 20 mg of biotin, and physicians may recommend up to 300 mg per day for conditions such as multiple sclerosis. Biotin levels higher than the recommended daily allowance may cause significant interference with affected lab tests
  • Be aware that specimens collected from patients taking high levels of biotin may contain more than 100 ng/mL biotin. Concentrations of biotin up to 1200 ng/mL may be present in specimens collected from patients taking up to 300 mg per day
  • Currently available data is insufficient to support recommendations for safe testing using affected tests in patients taking high levels of biotin, including about the length of time for biotin clearance from the blood
  • Communicate with the lab test manufacturer if you have questions about biotin interference
For Lab Test Manufacturers and Developers:
  • If your assay uses biotin technology, contact the FDA to discuss biotin interference
  • Investigate interference from biotin (up to at least 1200 ng/mL biotin) in your assays that use biotin technology. Determine the lowest concentration of biotin that may cause clinically significant interference with your test(s)
  • Communicate with your customers if they may be unaware that your test uses biotin technology and how it may be affected
  • Contact the FDA if you have any questions about biotin technology and interference
FDA Actions:

The FDA is working with stakeholders to better understand biotin interference with laboratory tests, and to develop additional future recommendations for safe testing in patients who have taken high levels of biotin when using laboratory tests that use biotin technology. The FDA is monitoring reports of adverse events associated with biotin interference with laboratory tests and will update the public if significant new information becomes available.

Adapted from: https://www.fda.gov/MedicalDevices/Safety/AlertsandNotices/ucm586505.htm

VARIATIONS IN TSH: A CASE STUDY

Patient Details: 29 year-old female, from Mumbai.

Medical History: No major systemic disorder, healthy patient.

Clinical presentation: The patient presented to her family physician to get basic lab tests done as she wanted to conceive shortly and evaluate her fitness.

Clinical Action: The doctor ordered basic blood tests. In particular, the doctor was keen to assess thyroid function prior to conception. Thyroid assessment was also done as it is a key parameter for women who want to conceive.

Tests done: CBC, LFT, Blood sugar, HbA1c, AMH, urinalysis and lipid profile. Thyroid tests included free T3/T4 and TSH. She gave the sample for TSH testing at one of our centres in the morning.

Results: We performed TSH testing on the CLIA platform. The patient’s result was 7.6 microIU/mL and reported as elevated as per the reference range of the CLIA platform. No other tests showed any derangements.

Clinical Problem: As the patient did not have any symptoms, the clinician was not sure whether the TSH result was correct. The same patients, 2 days later, gave another sample for TSH testing at another lab. The sample was given in the evening. The other lab performed the test by the CMIA method. As per that platform, the patient was reported to have a normal TSH level with a value of 4.5 microIU/mL.

Clinical Question: The clinician and the patient were confused and did not know which report to consider true and correct as there was a 40% difference in the TSH values between the 2 reports.

Points to Note: The patient gave samples for TSH testing in the two labs at different times of the day (morning and evening). Besides, the second sample was given two days later. Both labs used a different method to test TSH levels. Diurnal variations can cause large fluctuations in TSH levels. So also, different platforms that labs use for testing TSH can cause wide variations in the result.

Let’s delve deeper to precisely understand the reasons for TSH variations and why is it that a large number of hypothyroid patients may actually have elevated TSH and not show any apparent features

UNDERSTANDING TSH VARIATION AND THE TREATMENT ALGORITHM FOR SUBCLINICAL HYPOTHYROIDISM
CAUTION – VARIATIONS IN THYROID STIMULATING HORMONE CAN MISLEAD DIAGNOSIS!!

In the context of the clinical and laboratory diagnosis of hypothyroidism, the frequent variations that occur in the levels of TSH need to be factored in.

  • Biological rhythm, weather, medications, diet, alcohol, other associated disorders and stress are among the many extraneous factors that can significantly alter TSH levels
  • The diagnosis of hypo and hyperthyroidism cannot solely depend upon TSH levels. Testing also needs to be performed for T3, T4 and other metabolic parameters for a conclusive diagnosis
  • The lab profile of the patient needs to be accurately correlated with clinical features and with history – in order to arrive at a conclusive diagnosis

Ref: Biological Variation: From Principles to Practice-Callum G Fraser (AACC Press)

Let’s take a closer look at commonly known causes that bring about changes in TSH levels

Causes of variation:
  • TSH levels exhibit a diurnal variation with the peak occurring during the night and the levels deplete to approximate 50% of the peak value, between 10 am to 4 pm in the day. (TSH measurement, Review paper, Medscape at http://www.medscape.com/viewarticle/452667_4 )
  • TSH reference interval widens to 0.1–20 µIU/mL; in central hypothyroidism. (Central hypothyroidism refers to thyroid hormone deficiency due to a disorder of the pituitary, hypothalamus, or hypothalamic-pituitary portal circulation)(Indian J Clin Biochem. 2014 Apr; 29(2): 189–195)
  • Very slight changes in the concentrations of free thyroid hormones bring about much greater opposite changes in the TSH levels (Surks MI, Chopra IJ, Mariash CN, et al. American Thyroid Association Guidelines for the Use of Laboratory Tests in Thyroid Disorders. JAMA 1990;263:1529-1532)
  • Physiologically TSH rises in colder months and drops in warmer months (http://www.thyroidinfo.com/articles/tsh-fluctuating.htm)
  • High fibre diet reduces the ability to absorb thyroid medications, hence variation in TSH results (http://www.apiindia.org/pdf/medicine_update_2005/ch apter_63.pdf)
  • Variation/erratic time of taking medications can affect TSH levels Co-ingestion of calcium supplements with thyroid medication causes TSH to rise (http://www.apiindia.org/pdf/medicine_update_2005/ch apter_63.pdf)
  • Intense surge in estrogen during early pregnancy can increase TSH (http://www.apiindia.org/pdf/medicine_update_2005/ch apter_63.pdf)
  • TSH levels decline in the first trimester when serum HCG levels are high and rise after 10-12 weeks of gestation (MolsheHod et al. Textbook of Diabetes and Pregnancy, Third Ed)
  • Serum TSH may be slightly higher in obesity and may be reduced after weight loss (Indian J EndocrinolMetab. 2016 Jul-Aug; 20(4): 554–557)
Inter-Instrument Variation:

At Suburban Diagnostics, we report TSH values by the Chemiluminescence Immunoassay (CLIA). The other commonly used methodology is Chemiluminescent Microparticle Immunoassay (CMIA).

What are the reference ranges of the CLIA and CMIA platforms used for measuring TSH?

CLIA reference range for TSH (0.27–4.20 µIU/mL) is actually on the narrower side than the range of the CMIA assay (0.35–4.94 µIU/mL) Indian J Clin Biochem 2014 Apr; 29(2): 189–195 (Note: The lab uses its own derived ranges)

Why do the CLIA and CMIA platforms have different reference ranges?

The CLIA platform used at Suburban diagnosticsis based on a 95% central interval limit which means that while determining the biological reference range for this assay, 95% central values of the general population were taken into consideration – excluding 2.5% values at the bottom and 2.5% values at the top of the range, from the general population.

This is the central interval limit recommended by the regulating authority – Clinical Laboratory Standards Institute (CLSI)

On the other hand, the CMIA platform is based on a 99% central interval which means that while determining the biological reference range for this assay, 99% central values of the general population were taken into consideration – excluding 0.5% values at the bottom and 0.5% values at the top of the range, from the general population.

Ref: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory, 3rd Edition

Why is it beneficial to test TSH values on the CLIA platform as done by Suburban Diagnostics?
  • The CLIA platform has a narrower reference range as compared to the CMIA platform (95% central interval of CLIA versus 99% of CMIA)
  • As a result, a certain proportion of patients which are shown to have abnormal values by the CLIA method at Suburban, could be reported as normal by the CMIA method (due to difference in reference intervals)
  • This subgroup of subclinical hypothyroid patients (laboratory evidence of hypothyroidism with no apparent symptoms) which actually has deranged TSH, gets reported as normal on the CMIA method and goes undetected
  • Consequently, no follow-up is done for these patients. Over time, these patients could worsen and move from subclinical hypothyroidism to overt disease
  • Had the same patients been tested on the CLIA platform as done at Suburban Diagnostics, these patients would be reported to have deranged TSH and would have received follow-up and monitoring
  • Treatment is necessary only when elevations above 10 mU/L are sustained over a 3-month period.(Harrison, Textbook of Medicine)
HOW TO INTERPRET TSH VALUES FOR DIAGNOSING AND MANAGING SUBCLINICAL HYPOTHYROIDISM?

TREATMENT ALGORITHM FOR SUBCLINICAL HYPOTHYROIDISM

Ref: Harrison – Textbook of Medicine As per recommendations, treatment need not be initiated if TSH levels are below 10 mU/L

Any elevations above this level can be considered for treatment. However, lab-based evidence of such elevations above 10 mU/L, sustained for at least a period of 3 months; should be available before treatment is begun.

DENGUE INFECTION

DENGUE IS CLASSIFIED INTO PRIMARY AND SECONDARY INFECTIONS
  • When a person gets infected with Dengue for the first time with a certain serotype of the virus – the infection is labelled as primary dengue
  • When the same individual is again infected by the dengue virus, but by a serotype other than the one which caused the initial infection – this is called secondary dengue
  • There are four virus serotypes which are designated as DEN-1, DEN-2, DEN-3 and DEN-4.
  • At present DEN1 and DEN2 serotypes are widespread in India.
CLINICAL AND PATHOLOGICAL DIFFERENCES BETWEEN PRIMARY AND SECONDARY DENGUE
Clinical Differences
  • Primary infection is milder in intensity and presents generally with coryza, fever, rash, body ache, joint ache and headache, chills and malaise
  • Secondary infection presents a much more serious clinical course comprising Grade 3 dengue hemorrhagic fever and complications like dengue shock syndrome
Serological Differences
  • Serologically, primary infection is characterized by rise in dengue-specific IgM by 4–5 days of fever onset, followed by rise of IgG antibody with 7–10 days.
  • IgM antibodies for dengue may remain elevated for 2 to 3 months after the illness.
  • The diagnosis of primary dengue in the initial 5 days, therefore, requires utilization of polymerase chain reaction techniques (RT-PCR) or antigen detection, especially the NS1 antigen
  • Secondary infection is recognized by an early rise in the IgG antibody titer and a lower titer of IgM rise.
  • Again, early phases of secondary dengue also show PCR and NS1 positivity

Ref: Mandavdhare HS, Sharma V. Differentiating primary and secondary dengue infections: Why and how? Med J DY PatilUniv 2016;9:594-5

IgG antibody IgM antibody NS1 antigen Polymerase chain reaction
Early primary infection +++ +++
Late primary infection -/+ ++
Early primary infection ++ -/+ ++ +++
Late primary infection ++ +

* – Likely to be negative, + Likely to positive, +/- Varibale, ++ very likely to be positive, +++ Extremely likely to be positive

Ref: Center for Disease Control and Prevention. Laboratory Guidance and Diagnostic

Testing. Available from: http://www.cdc.gov/dengue/clinicallab/laboratory.html

Cross Reactivity in Serology needs to be factored in prior to a diagnosis
  • Serological cross-reactivity across the flavivirus group is common (i.e. between dengue 1, 2, 3 & 4, Japanese encephalitis, Murray Valley encephalitis, St. Louis encephalitis, West Nile, and yellow fever viruses). These diseases must be excluded before confirmation of diagnosis.
  • Heterophilic antibodies are a well-recognised cause of interference in immunoassays . These antibodies to animal IgG may cross-react with reagent antibodies and generate a false positive signal. This must be excluded before confirmation of diagnosis.
  • Cross reactivity is also known due to interference with antibodies against Epstein-Barr virus, Malarial parasite, Influenza A and B, Anti-nuclear antibody, Rheumatoid factor Hepatitis A, Leptospira, Salmonella typhi, Scrub typhus and West Nile virus

Ref: WHO Report: Evaluation of commercially available anti-dengue virus IgM tests.

Diagnostic evaluation series No.3

HEALTH AUTHORITIES STRONGLY DISCOURAGE RAPID DENGUE TESTS

The use of rapid tests for dengue has been discouraged by the Health Ministry of India as the rapid diagnostic kits are generating up to 50% of false ‘positives’ for dengue (Times of India report, Sep 17, 2015)

PCR OFFFERS A CLEAR ADVANTAGE OVER OTHER MODALITIES IN DIAGNOSING DENGUE
Infection Sensitivity
RT – PCR NS1 Clinical Benefit
Primary 95% 90% ↑5%
Secondary 95% 85% ↑10%
Additional 5-10% patients accurately detected positive by PCR

Ref: Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control:

New Edition- WHO 2009.ECDC training Workshop on laboratory diagnosis of dengue

virus infections Berlin, 23-27 January 2012. CENTER FOR DISEASE CONTROL

WHICH INFORMATION DOES THE LABORATORY NEED FOR FURTHER INTERPRETATION OF THE RESULTS?

The availability of the following data points pertaining to the Clinical and personal history of the patient could prove instrumental in corroborating the lab test results with the clinical presentation

Mandatory:
  • Date of onset of illness ( not fever alone )
  • Date of sample collection
  • Travel history (country, dates…)
Recommended:
  • Other diagnosis
  • Previous flavivirus infections or vaccinations
  • Nationality
  • History of previous travel to endemic areas
  • Previous fever episodes after travel
  • Concise clinical findings
DENGUE IS OFTEN SUBCLINICAL!

Quite a large proportion of patients who test positive for Dengue infection, do not show any symptoms.

In case of elevated IgG and IgM with no symptoms (seropositive but asymptomatic) – it could be the convalescent phase of the disease as antibodies can be found for upto 3 months after the illness

This is in fact the “Unapparent” or “Subclinical” form of Dengue which forms a large portion of the total disease burden

The WHO’s regional office for South East Asia confirms the following

There are many people who are infected with the virus and do not suffer from any signs or symptoms of the disease. For every patient with symptoms and signs, there may be four or five persons with either no symptoms or very mild symptoms.

Ref: http://www.searo.who.int/srilanka/areas/dengue/faqs-on-dengue.pdf?ua=1

https://www.cdc.gov/dengue/clinicallab/laboratory.html



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