Introduction
The recent emergence of the Zika virus (ZIKV) pandemic has underscored the need for a point-of-care (POC) test that can detect viral infections, fostering widespread, accurate and timely diagnostics [1, 2]. Zika infection is often asymptomatic or has comparatively mild symptoms, such as fever and chills that are common to many other infections. However, as is widely known, Zika infection during pregnancy entails substantial risks for the newborn, including severe birth defects [3]. Hence, it is highly desirable to screen women of child-bearing age and their partners for exposure to the ZIKV. Zika episodes have created bottlenecks in conventional laboratory-based diagnostic.
Zika is spread primarily by mosquitos, but sexual and perinatal transmission, as well as transmission via blood transfusions have also been reported. Specific applications for Zika diagnostics thus include determining whether infection has occurred over the course of a pregnancy, whether sexual partners harbour infection, safeguarding the blood supply and tracking the geographic range of Zika infections [4]. In order to inform investment decisions on prevention, control and response, Lee
et al. [5] modelled the economic burden for various scenarios of Zika emergence across six US states, estimating total costs (direct medical, Medicaid, productivity losses) ranging from 0.5 to 2 billion US dollars.
Ideally, a POC technology will enable rapid diagnostics tests that can be performed outside of centralized laboratories such as, for example, in doctors or dentists offices, pharmacies, rural clinics, school infirmaries, border crossings or, ultimately, as an over-the-counter test for home use. Thus, the availability of a mass-produced, inexpensive POC diagnostics device with easy-to-interpret test results, and that could be used in any almost any locale by non-specialists with minimal training, would greatly expand capabilities and options for screening, surveillance, diagnostics, monitoring and therapy.
Current diagnostics technology
According to the World Health Organization (WHO), the pipeline diagnostic ZIKV kits can be categorized into: (i) antibody/antigen-based immunoassay and (ii) nucleic acid-based molecular diagnostics [6]. Immunoassay (antibody detection) for ZIKV infection utilizes envelope proteins and NS1 as targets. The major challenge is that these antibodies cross-react with other highly homologous flaviviruses such as dengue, resulting in non-specific test results [7]. IgM and IgG antibodies, typically emerge, respectively, ~4 and ~10 days after infection, but are usually undetectable until >7–14 days post-infection. Moreover, antibody responses during pregnancy may differ from those in non-pregnant individuals [8], which may adversely impact the effectiveness of immunoassay tests. Moreover, antibody tests may not discriminate between recent and historic exposure. The Food and Drug Administration (FDA) recently authorized for emergency use of the IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (Zika MAC-ELISA) to detect ZIKV [8]. However, this assay requires a lab-format with delays in generating results given current demand. More importantly, these assays are a readout for exposure to ZIKV, whereas active virus infection is not determined. Currently, several companies are developing lateral flow-based rapid diagnostic test for ZIKV antibody detection [9, 10].
Molecular diagnostics-based on reverse-transcription (RT)-PCR is highly specific and sensitive, and considered the gold standard for ZIKV detection. RT-PCR is effective in serum, semen, and saliva within 14 days post-infection, and possibly much longer in urine and semen [11, 12]. Importantly, a recent study has demonstrated that ZIKV is detectable in pregnant women throughout their pregnancy [3]. Indeed, the FDA has authorized the use of the Trioplex rRT-PCR laboratory test to detect ZIKV, dengue virus, and chikungunya virus RNA, under an Emergency Use Authorization (EUA) [13]. Several research groups and companies are developing multiplexed molecular assays to concurrently detect various members of the genus Flavivirus. Most of these RT-PCR kits require, however, instrumentation and are for central laboratory use only.
Instrument-free point-of-care molecular detection of ZIKV
To develop inexpensive molecular detection of ZIKV without complex instrumentation, we utilized reverse-transcription loop-mediated amplification (RT-LAMP) technology [14]. We identified highly conserved regions of the ZIKV genome and designed RT-LAMP primers for the Zika lineage that is prevalent in the Americas. To enable POC molecular diagnostics, we developed a disposable microfluidic cassette (Fig. 1a) that combines viral nucleic acid capture, concentration, isothermal amplification; and detection. Our disposable, microfluidic cassette contains multiple independent amplification reactors, each equipped with a silica-based nucleic acid isolation membrane at its inlet. The advantage of such a design is to decouple the sample volume from the reaction volume, allowing one to use relatively high sample volumes to achieve high sensitivity. Nucleic acids captured by the isolation membrane directly serve as templates in an RT-LAMP amplification process without a need for an elution step, significantly simplifying flow control.
Our microfluidic cassette can be incubated with battery power or operate electricity-free. To eliminate the need for electricity, we used a simple, thermally insulated portable cup (Fig. 1b) heated by an exothermic reaction for chip-based isothermal amplification [14]. One Mg−Fe alloy pouch, which is usually used as a heater of MRE (meal, ready-to-eat), served as the heat source. Tap water was introduced into the drawer, housing the Mg-Fe pouch, through a port in the cup lid to interact with the Mg−Fe alloy to produce heat. To isolate the amplification reactor’s temperature from variable ambient conditions, we used a phase change material (PCM) to regulate the temperature, removing the need for a thermal control circuit. An aluminium heat sink was used to enhance heat transfer from the PCM to the cassette.
We tested the utility of our POC diagnostic system with raw saliva samples spiked with various concentrations of the ZIKV. Our experiments showed that our electricity-free POC diagnostic system could detect ZIKV in saliva with the sensitivity of 5 plaque forming units (p.f.u.) of ZIKV per sample within 40 min (Fig. 1c). Our POC diagnostic system is comparable to that of the benchtop assay without a need for laboratory facilities, expensive equipment and well-trained personnel [14].
Conclusion
Zika molecular diagnostics can be performed at the point of care with a low-cost, portable system based on a microfluidic cassette that integrates nucleic acid isolation and concentration, isothermal amplification, and detection. To achieve electricity-free isothermal amplification, the cassette is combined with a chemically heated cup that generates heat with an exothermic reaction. The platform can be adapted to various sample types and sizes, and multiplex detection. This flexibility is useful in view of the evolving understanding of Zika pathology and Zika biomarker levels and their persistence in different body fluids and tissues. In the future, we plan to expand system capabilities to enable concurrent detection of multiple vector-borne diseases [15]. Our system is very suitable for resource-poor settings, where funds, centralized laboratory facilities and trained personnel are in short supply, as well as for use in remote clinics and at home.
Acknowledgment
The research reported here was supported, in part by the NIH NIDCR R21DE026700, K25AI099160, R01 CA214072, to the University of Pennsylvania.
References
1. Cao-Lormeau VM, Blake A, Mons S, Lastère S, Roche C, et al. Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet 2016; 387(10027): 1531–1539.
2. Tang H, Hammack C, Ogden SC, Wen Z, Qian X, et al. Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell 2016; 18(5): 587–590.
3. Driggers RW, Ho CY, Korhonen EM, Kuivanen S, Jääskeläinen AJ, et al. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities. N Eng J Med 2016; 374: 2142–2151.
4. Kindhauser MK, Allen T, Frank V, Santhana RS, Dye C. Zika: the origin and spread of a mosquito-borne virus. Bull World Health Organ 2016; 94: 675–686C.
5. Lee BY, Alfaro-Murillo JA, Parpia AS, Asti L, Wedlock PT, et al. The potential economic burden of Zika in the continental United States. PLOS Neg Trop Dis 2017; 11(4): e0005531.
6. Current Zika product pipeline. World Health Organization (WHO) 2016. http://www.who.int/csr/research-and-development/zika-rd-pipeline.pdf.
7. Charrel, RN, Leparc-Goffart I, Pas S, de Lamballerie X, Koopmans M, Reusken C. State of knowledge on Zika virus for an adequate laboratory response. Bull World Health 2016; 94: 574–584D.
8. New CDC laboratory test for Zika virus authorized for emergency use by FDA. Centers for Disease Control and Prevention (CDC) 2016. https://www.cdc.gov/media/releases/2016/s0226-laboratory-test-for-zika-virus.html.
9. Zika rapid test. Biocan Diagnostics Inc. http://www.zikatest.com/?page_id=6.
10. Artron Zika test. Artron Laboratories Inc. http://www.artronlab.com/home.html.
11. Gourinat AC, O’Connor O, Calvez E, Goarant C, Dupont-Rouzeyrol M. Detection of Zika virus in urine. Emerg Infect Dis 2015; 21(1): 84.
12. Mansuy JM, Dutertre M, Mengelle C, Fourcade C, Marchou B, et al. Zika virus: high infectious viral load in semen, a new sexually transmitted pathogen. Lancet Infect Dis 2016; 16: 405.
13. Trioplex real-time RT-PCR assay. CDC 2017. https://www.fda.gov/downloads/medicaldevices/safety/emergencysituations/ucm491592.pdf.
14. Song J, Mauk MG, Hackett BA, Cherry S, Bau HH, Liu C. Instrument-free point-of-care molecular detection of Zika virus. Anal Chem 2016; 88: 7289–7294.
15. Song J, Liu C, Mauk MG, Rankin SC, Lok JB, et al. Two-stage isothermal enzymatic amplification for concurrent multiplex molecular detection. Clin Chem 2017; 63(3): 714–722.
The authors
Michael G Mauk PhD, Jinzhao Song PhD, Haim H. Bau PhD, Changchun Liu* PhD
Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
*Corresponding author
E-mail: lchangc@seas.upenn.edu
Point-of-care molecular test for Zika infection
, /in Featured Articles /by 3wmediaThe recent emergence of the Zika virus (ZIKV) pandemic has underscored the need for a point-of-care (POC) test that can detect viral infections, fostering widespread, accurate and timely diagnostics [1, 2]. Zika infection is often asymptomatic or has comparatively mild symptoms, such as fever and chills that are common to many other infections. However, as is widely known, Zika infection during pregnancy entails substantial risks for the newborn, including severe birth defects [3]. Hence, it is highly desirable to screen women of child-bearing age and their partners for exposure to the ZIKV. Zika episodes have created bottlenecks in conventional laboratory-based diagnostic.
Zika is spread primarily by mosquitos, but sexual and perinatal transmission, as well as transmission via blood transfusions have also been reported. Specific applications for Zika diagnostics thus include determining whether infection has occurred over the course of a pregnancy, whether sexual partners harbour infection, safeguarding the blood supply and tracking the geographic range of Zika infections [4]. In order to inform investment decisions on prevention, control and response, Lee et al. [5] modelled the economic burden for various scenarios of Zika emergence across six US states, estimating total costs (direct medical, Medicaid, productivity losses) ranging from 0.5 to 2 billion US dollars.
Ideally, a POC technology will enable rapid diagnostics tests that can be performed outside of centralized laboratories such as, for example, in doctors or dentists offices, pharmacies, rural clinics, school infirmaries, border crossings or, ultimately, as an over-the-counter test for home use. Thus, the availability of a mass-produced, inexpensive POC diagnostics device with easy-to-interpret test results, and that could be used in any almost any locale by non-specialists with minimal training, would greatly expand capabilities and options for screening, surveillance, diagnostics, monitoring and therapy.
Current diagnostics technology
According to the World Health Organization (WHO), the pipeline diagnostic ZIKV kits can be categorized into: (i) antibody/antigen-based immunoassay and (ii) nucleic acid-based molecular diagnostics [6]. Immunoassay (antibody detection) for ZIKV infection utilizes envelope proteins and NS1 as targets. The major challenge is that these antibodies cross-react with other highly homologous flaviviruses such as dengue, resulting in non-specific test results [7]. IgM and IgG antibodies, typically emerge, respectively, ~4 and ~10 days after infection, but are usually undetectable until >7–14 days post-infection. Moreover, antibody responses during pregnancy may differ from those in non-pregnant individuals [8], which may adversely impact the effectiveness of immunoassay tests. Moreover, antibody tests may not discriminate between recent and historic exposure. The Food and Drug Administration (FDA) recently authorized for emergency use of the IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (Zika MAC-ELISA) to detect ZIKV [8]. However, this assay requires a lab-format with delays in generating results given current demand. More importantly, these assays are a readout for exposure to ZIKV, whereas active virus infection is not determined. Currently, several companies are developing lateral flow-based rapid diagnostic test for ZIKV antibody detection [9, 10].
Molecular diagnostics-based on reverse-transcription (RT)-PCR is highly specific and sensitive, and considered the gold standard for ZIKV detection. RT-PCR is effective in serum, semen, and saliva within 14 days post-infection, and possibly much longer in urine and semen [11, 12]. Importantly, a recent study has demonstrated that ZIKV is detectable in pregnant women throughout their pregnancy [3]. Indeed, the FDA has authorized the use of the Trioplex rRT-PCR laboratory test to detect ZIKV, dengue virus, and chikungunya virus RNA, under an Emergency Use Authorization (EUA) [13]. Several research groups and companies are developing multiplexed molecular assays to concurrently detect various members of the genus Flavivirus. Most of these RT-PCR kits require, however, instrumentation and are for central laboratory use only.
Instrument-free point-of-care molecular detection of ZIKV
To develop inexpensive molecular detection of ZIKV without complex instrumentation, we utilized reverse-transcription loop-mediated amplification (RT-LAMP) technology [14]. We identified highly conserved regions of the ZIKV genome and designed RT-LAMP primers for the Zika lineage that is prevalent in the Americas. To enable POC molecular diagnostics, we developed a disposable microfluidic cassette (Fig. 1a) that combines viral nucleic acid capture, concentration, isothermal amplification; and detection. Our disposable, microfluidic cassette contains multiple independent amplification reactors, each equipped with a silica-based nucleic acid isolation membrane at its inlet. The advantage of such a design is to decouple the sample volume from the reaction volume, allowing one to use relatively high sample volumes to achieve high sensitivity. Nucleic acids captured by the isolation membrane directly serve as templates in an RT-LAMP amplification process without a need for an elution step, significantly simplifying flow control.
Our microfluidic cassette can be incubated with battery power or operate electricity-free. To eliminate the need for electricity, we used a simple, thermally insulated portable cup (Fig. 1b) heated by an exothermic reaction for chip-based isothermal amplification [14]. One Mg−Fe alloy pouch, which is usually used as a heater of MRE (meal, ready-to-eat), served as the heat source. Tap water was introduced into the drawer, housing the Mg-Fe pouch, through a port in the cup lid to interact with the Mg−Fe alloy to produce heat. To isolate the amplification reactor’s temperature from variable ambient conditions, we used a phase change material (PCM) to regulate the temperature, removing the need for a thermal control circuit. An aluminium heat sink was used to enhance heat transfer from the PCM to the cassette.
We tested the utility of our POC diagnostic system with raw saliva samples spiked with various concentrations of the ZIKV. Our experiments showed that our electricity-free POC diagnostic system could detect ZIKV in saliva with the sensitivity of 5 plaque forming units (p.f.u.) of ZIKV per sample within 40 min (Fig. 1c). Our POC diagnostic system is comparable to that of the benchtop assay without a need for laboratory facilities, expensive equipment and well-trained personnel [14].
Conclusion
Zika molecular diagnostics can be performed at the point of care with a low-cost, portable system based on a microfluidic cassette that integrates nucleic acid isolation and concentration, isothermal amplification, and detection. To achieve electricity-free isothermal amplification, the cassette is combined with a chemically heated cup that generates heat with an exothermic reaction. The platform can be adapted to various sample types and sizes, and multiplex detection. This flexibility is useful in view of the evolving understanding of Zika pathology and Zika biomarker levels and their persistence in different body fluids and tissues. In the future, we plan to expand system capabilities to enable concurrent detection of multiple vector-borne diseases [15]. Our system is very suitable for resource-poor settings, where funds, centralized laboratory facilities and trained personnel are in short supply, as well as for use in remote clinics and at home.
Acknowledgment
The research reported here was supported, in part by the NIH NIDCR R21DE026700, K25AI099160, R01 CA214072, to the University of Pennsylvania.
References
1. Cao-Lormeau VM, Blake A, Mons S, Lastère S, Roche C, et al. Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet 2016; 387(10027): 1531–1539.
2. Tang H, Hammack C, Ogden SC, Wen Z, Qian X, et al. Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell 2016; 18(5): 587–590.
3. Driggers RW, Ho CY, Korhonen EM, Kuivanen S, Jääskeläinen AJ, et al. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities. N Eng J Med 2016; 374: 2142–2151.
4. Kindhauser MK, Allen T, Frank V, Santhana RS, Dye C. Zika: the origin and spread of a mosquito-borne virus. Bull World Health Organ 2016; 94: 675–686C.
5. Lee BY, Alfaro-Murillo JA, Parpia AS, Asti L, Wedlock PT, et al. The potential economic burden of Zika in the continental United States. PLOS Neg Trop Dis 2017; 11(4): e0005531.
6. Current Zika product pipeline. World Health Organization (WHO) 2016. http://www.who.int/csr/research-and-development/zika-rd-pipeline.pdf.
7. Charrel, RN, Leparc-Goffart I, Pas S, de Lamballerie X, Koopmans M, Reusken C. State of knowledge on Zika virus for an adequate laboratory response. Bull World Health 2016; 94: 574–584D.
8. New CDC laboratory test for Zika virus authorized for emergency use by FDA. Centers for Disease Control and Prevention (CDC) 2016. https://www.cdc.gov/media/releases/2016/s0226-laboratory-test-for-zika-virus.html.
9. Zika rapid test. Biocan Diagnostics Inc. http://www.zikatest.com/?page_id=6.
10. Artron Zika test. Artron Laboratories Inc. http://www.artronlab.com/home.html.
11. Gourinat AC, O’Connor O, Calvez E, Goarant C, Dupont-Rouzeyrol M. Detection of Zika virus in urine. Emerg Infect Dis 2015; 21(1): 84.
12. Mansuy JM, Dutertre M, Mengelle C, Fourcade C, Marchou B, et al. Zika virus: high infectious viral load in semen, a new sexually transmitted pathogen. Lancet Infect Dis 2016; 16: 405.
13. Trioplex real-time RT-PCR assay. CDC 2017. https://www.fda.gov/downloads/medicaldevices/safety/emergencysituations/ucm491592.pdf.
14. Song J, Mauk MG, Hackett BA, Cherry S, Bau HH, Liu C. Instrument-free point-of-care molecular detection of Zika virus. Anal Chem 2016; 88: 7289–7294.
15. Song J, Liu C, Mauk MG, Rankin SC, Lok JB, et al. Two-stage isothermal enzymatic amplification for concurrent multiplex molecular detection. Clin Chem 2017; 63(3): 714–722.
The authors
Michael G Mauk PhD, Jinzhao Song PhD, Haim H. Bau PhD, Changchun Liu* PhD
Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
*Corresponding author
E-mail: lchangc@seas.upenn.edu
Complete laboratory diagnosis of Zika virus infections
, /in Featured Articles /by 3wmediaLaboratory diagnosis of Zika virus (ZIKV) infections is based on two main pillars: direct detection of the viral RNA genome and serological detection of anti-ZIKV antibodies. Direct detection of the virus by reverse transcriptase real-time polymerase chain reaction (RT real-time PCR) is the most important method for diagnosing early acute infections. A new RT real-time PCR system with fully automated data evaluation provides highly standardized and streamlined detection of ZIKV RNA. Serology is useful for acute diagnostics as well as for longer term monitoring and epidemiological studies. An ELISA based on ZIKV NS1 antigen provides exceptionally high specificity with virtually no cross reactivity to other flaviviruses.
by Dr Jacqueline Gosink
Introduction
ZIKV has become firmly established in South and Central America and the Caribbean and is increasingly spreading to other parts of the world. The infection is now classified by the World Health Organization as an enduring public health challenge. Nearly one million people in 48 countries have been infected with ZIKV since the beginning of 2015, according to the Panamerican Health Organization. The actual number of cases is presumably much higher, since many infections are mild and go unreported. The virus is transmitted predominantly by mosquitos of the Aedes genus, which are ubiquitous in many topical and non-tropical regions. Transmission by sexual contact is also increasingly described. ZIKV infections are difficult to distinguish clinically from dengue virus (DENV) and chikungunya virus (CHIKV) infections, which manifest with similar symptoms of fever, exanthema and arthritis and are endemic in much the same geographic regions. There is, however, a growing body of evidence linking ZIKV to birth defects in fetuses and newborns and neurological complications such as Guillain-Barré syndrome in adults. Therefore, accurate diagnosis of ZIKV infections and differentiation between acute and past infections is critical for effective patient care.
ZIKV direct detection
The ZIKV RNA genome can be detected during the viremic phase of infection. The viral RNA is detectable for up to around 5 days after the onset of symptoms in serum and up to 10 days in urine. Molecular diagnostic detection is therefore highly effective for early diagnosis of ZIKV infections and discrimination of ZIKV from clinically similar infections such as DENV or CHIKV.
Novel RT real-time PCR assay
A new assay provides fast detection of ZIKV RNA in serum or urine by reverse transcriptase real-time polymerase chain reaction (RT real-time PCR) with fully automated data analysis. The EURORealTime Zika virus test is based on a one-tube reaction, comprising reverse transcription of the viral RNA into complementary DNA (cDNA) followed by PCR amplification and fluorescence-based real-time detection of defined sections of the ZIKV genome. The reverse transcription, amplification and detection of ZIKV cDNA are carried out by means of ZIKV-specific DNA primers and real-time DNA probes. RNA-based internal and positive controls verify the correct performance, integrity and functionality of the complete procedure. Ready-to-use reagents provide added reliability and convenience.
The evaluation of results is fully automated using the EURORealTime Analysis software and is therefore highly standardized and objective. All results, including those of the controls, are documented and archived. The software also supports simple and error-free test performance by guiding every step of the workflow. The entire detection procedure (excluding RNA extraction) takes less than 90 min.
Specifications and evaluation of the EURORealTime Zika virus test
Highest test sensitivity and specificity is ensured by the meticulous design of the primers and probes. Moreover, cross reactivity with other pathogens that may be present in serum or urine samples and/or are closely related to ZIKV has been excluded experimentally.
In clinical evaluation, 29 serum and 26 urine samples from patients with suspected ZIKV infection were analysed using the EURORealTime Zika virus and another CE/IVD-labelled ZIKV test system. There was a positive agreement of 95.2% and a negative agreement of 97.0% between the results obtained with the two tests (Table 1).
ZIKV serology
Serological detection is effective from soon after symptom onset (4-7 days) to beyond convalescence. Serology serves as a supplement to RT-PCR in acute cases. It is especially useful in cases where viral RNA is no longer detectable, for example if the infection is resolved or has moved into the chronic phase. Serological detection is particularly relevant in prenatal diagnostics, sexual healthcare and epidemiological surveys. Pregnant women with serological evidence of an infection can be offered intense prenatal monitoring, while seronegative women may be spared unnecessary worry. Due to the lengthy presence of ZIKV in semen, men who have resided in or travelled in endemic regions are advised to abstain from unprotected sexual intercourse for six months after returning to prevent sexual transmission, especially when their partner is or could be pregnant. Serological testing can be helpful in these cases for excluding or identifying an infection. As ZIKV continues to move into previously unaffected areas, epidemiological studies using serological methods can help to monitor the spread of the virus and probe its associated complications.
Relevance of immunoglobulin classes
Primary acute ZIKV infections are generally characterized by the occurrence of specific IgM antibodies, with IgG appearing at the same time or shortly afterwards. IgM can remain detectable for several months, while IgG is assumed to persist lifelong. Detection of specific IgM or a rise in the specific IgG titre in a pair of samples taken at least 7 to 10 days apart is evidence of an acute infection.
In secondary flavivirus infections, for example following a previous vaccination or infection with another flavivirus, specific IgM is often found at a low or undetectable titre. Therefore, additional tests like the detection of IgG or plaque reduction neutralization test are recommended.
Specific IgA may also be useful for diagnostics. In secondary flavivirus infections synthesis of IgG is rapidly stimulated. Shortly after infection the IgG titre levels off and is indistinguishable from IgG titres in convalescent infections, making seroconversion difficult to detect. This pattern has been observed in ZIKV patients from regions endemic for other flaviviruses. IgA has recently been proposed as a putative additional marker of acute infection in cases where IgM is not detectable and the IgG titre is already high.
Highly specific NS1-based ZIKV ELISA
Serological diagnosis of ZIKV is challenging due to the high cross-reactivity between flavivirus antibodies. This obstacle has been overcome by the use of recombinant non-structural protein 1 (NS1) from ZIKV as the antigenic substrate in ELISA. Use of this antigen avoids the cross-reactivity typically associated with tests based on whole virus antigens or viral glycoproteins. The NS1-based ELISA provides highly sensitive and specific ZIKV diagnostics, as demonstrated in numerous studies.
Clinical evaluation of IgM/IgG ELISA
The NS1-based Anti-Zika Virus ELISA was used to examine anti-ZIKV antibodies of classes IgG and IgM in various serum panels. In samples from patients with RT-PCR-confirmed infections (n=71), taken 5 days or more after symptom onset, the sensitivity of the test amounted to 100% for IgG/IgM (Table 2) (1). In a panel of blood donors the specificity of the ELISA was 99.8%.
In studies with a total of over 450 patients harbouring other arboviral infections, including DENV, CHIKV, tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and individuals vaccinated against yellow fever virus (YFV) or TBEV, the specificity lay between 96% and 100% (Table 3) (1, 2). In particular, a specificity of 100% was observed in DENV- and CHIKV-infected patients, demonstrating the suitability of the ELISA for discriminating these infections. In a further study (3) the Anti-Zika Virus ELISA showed no cross reactivity (100% specificity) in sera from patients with early convalescent DENV infections or suspected secondary DENV infections.
Usefulness of IgA testing
In a recent study investigating the diagnostic usefulness of IgA antibodies, anti-ZIKV antibodies of class IgA, IgM and IgG were analysed at serial time points in patients with confirmed ZIKV infections (4, 5). In two German travellers, IgM was detected early in infection as expected, followed by IgG seroconversion. IgA antibodies showed an initial increase and subsequent decrease. In two Columbian patients with a presumptive background of past flavivirus infection, IgM was persistently below the cut-off in both NS1-based and full virus-based tests, while IgG was already positive within the first week. Analysis of IgA in these patients demonstrated a titre increase, which peaked above the cut-off in week three and four before dropping below the threshold again (Figure 1). Thus, specific IgA may be useful for the diagnosis of acute infections and discrimination from past infections in IgM-negative patients.
Clinical evaluation of IgA ELISA
The NS-1-based Anti-Zika Virus ELISA was used to analyse anti-ZIKV antibodies of class IgA in Columbian patients (n=31) seven to ten days after positive ZIKV RT-PCR. 29 of the patients were positive for anti-ZIKV IgA, representing a sensitivity of 94%. The specificity of the IgA ELISA amounted to 97% in a control panel of German travellers with confirmed DENV infections and 100% in healthy blood donors and patients with other diseases. With the IgA ELISA, as with the IgM and IgG ELISAs, cross reactivity with antibodies against other flaviviruses, including DENV, TBEV, JEV, WNV and YFV, is almost completely avoided.
Differential diagnostics by IIFT
The indirect immunofluorescence test (IIFT) based on virus-infected cells offers an alternative sensitive screening assay for ZIKV antibodies. Automated microscopy and evaluation of results using the EUROPattern system streamlines the procedure. The ZIKV substrate can be combined with other substrates as a BIOCHIP mosaic, enabling potential cross-reactive antibodies or relevant differential diagnostic parameters to be investigated in parallel. In addition to ZIKV, available substrates include DENV (serotypes 1, 2, 3 and 4) and other flaviviruses (e.g. TBEV, YFV and JEV), as well as other arboviruses (e.g. CHIKV). Endpoint titration of the patient serum provides an indication of the virus causing the infection. As cross reactivity is common in patients with secondary flavivirus infections, BIOCHIP flavivirus mosaics are most useful for patients in non-epidemic countries, for example travellers returning from epidemic regions.
Perspectives
The swift development of sensitive and specific tests for ZIKV antibodies and ZIKV RNA has facilitated the diagnosis and surveillance of this rapidly emerging disease. The EUROIMMUN Anti-Zika Virus ELISA based on NS1 antigen is currently the only commercial serological test whose extremely high specificity has been described in various publications. It is, moreover, the first commercial serological ZIKV test to receive CE Mark (Europe; IgA, IgM and IgG) and ANVISA (Brazil; IgM, IgG, soon also IgA) registrations. The assay is fully automatable, making it ideal for high-throughput application in a routine setting. For direct detection of viral RNA, the new EURORealTime Zika virus test provides software-supported test performance and fully automated result evaluation and documentation, in contrast to many manual ZIKV RT-PCR tests. As ZIKV will likely remain a global health challenge in the foreseeable future, state-of-the-art test systems like these are crucial for monitoring the spread, improving diagnosis and elucidating the mechanisms of this challenging emerging disease.
References
1. Steinhagen et al. Euro Surveill. 2016 15;21(50). pii: 30426.
2. Huzly et al. Euro Surveill 2016;21(16):pii=30203.
3. Granger et al. Poster at the 32nd Clinical Virology Symposium (Florida, USA) 2016
4. Steinhagen et al. Poster at the IMED International Meeting on Emerging Infectious Diseases and Surveillance (Vienna, Austria) 2016
5. Steinhagen et al. Poster at the 1st International Conference on Zika Virus (Washington DC, USA) 2017
The author
Jacqueline Gosink, PhD
EUROIMMUN AG, Seekamp 31,
23560 Luebeck, Germany
www.euroimmun.com
Quality control testing on a random access molecular diagnostics platform running quantitative viral load assays
, /in Featured Articles /by 3wmediaThe DxN VERIS Molecular Diagnostics System* from Beckman Coulter is a real-time PCR analyser for accurate and precise quantitative detection of both RNA and DNA targets. Single sample random access offers workflow flexibility and automation benefits to the laboratory. The design features of the DxN VERIS System and performance characteristics of the VERIS HCV, HIV-1, HBV, and CMV viral load assays enable laboratories to develop Quality Control (QC) programmes tailored to their unique needs. Methods: A QC programme was developed by the Virology lab at the Rennes University Hospital, France. The laboratory evaluated the performance levels of the DxN VERIS System as well as the total number of VERIS HIV-1, HBV, and CMV tests performed over a period of five months. Results: The precision observed over the five-month study period was less than 5.8% CV with standard deviation (SD) within 0.16 log IU/ml. Based on these results the laboratory concluded that performing three levels of QC (negative, low, high) two times per week would provide an acceptable level of system control while significantly reducing QC costs and hands-on time.
Introduction
Consistency in reporting quantitative viral load results is critically important to clinical laboratories, physicians, and the patients they serve. The use of quantitative tests to measure viral load levels in patient samples is especially important for monitoring treatment. With the advent of new quantitative PCR (qPCR) assays for viral load testing, physicians are better able to manage diseases with antiretroviral therapy (ART).
Clinical laboratories are challenged to achieve stringent Quality Control (QC) objectives for viral load testing in an effective and economical manner. The use of external quality controls (EQC) provides laboratories with a means of monitoring variation in the analytical process as well as environmental factors that can affect patient results. In addition, EQC can assist laboratories in identifying when errors are occurring that can impact the utility of viral load assays. For these reasons, manufacturers of qPCR systems may recommend the use of EQC as part of the analytical process for viral load testing.
The DxN VERIS System and VERIS viral load assays are designed to deliver a high standard of clinical performance while providing rapid, convenient, and cost effective QC alternatives to the laboratory.
Quality control for quantitative diagnostic systems – a statistical approach
Statistical QC is defined as a procedure in which stable samples are measured and the observed results compared with limits that describe the variation expected when the measurement method is working properly[2]. Statistical QC is important to ensure the quality of the test results produced by any measurement method. An important concept in statistical QC is the definition of an “analytical run”. With many modern analytical systems, the definition of a run is not always clear. For example, many molecular diagnostics analysers available to laboratories today perform testing in “batch” mode, wherein each run corresponds to a single batch of several tests. While these methods can provide efficiencies in some testing environments (e.g. high volume labs) they can result in delayed results while the laboratory waits to accrue sufficient samples to complete the batch. In addition, batch systems lack the flexibility to adapt to fluctuating testing demand driven by sample volume and clinical needs in the laboratory. New qPCR systems are now available that provide “random access” capability; enabling labs to test individual samples at the precise time that they are most needed. In addition to providing more timely results for physicians and patients, these systems can also increase laboratory work flow efficiency, resulting in less hands-on time. For random access systems, an analytical run can be better understood in terms of the time or number of measurements for which the measurement is stable[2]. Statistical guidance for molecular assays typically suggests that quality control samples should be run at least once during each user-defined analytical run.
The DxN VERIS system
The DxN VERIS System is a fully automated molecular diagnostic system that integrates nucleic acid extraction, reaction setup, real-time PCR amplification and detection, and results interpretation into one system; saving space and time. The system provides single sample random access capability which allows the laboratory to run the right viral load test at the right time for physicians and patients. The DxN VERIS System provides time and workflow advantages compared to batch systems which require the laboratory to accrue a number of patient samples prior to each run.
Designed for quality and accuracy
The DxN VERIS System is engineered to deliver a high level of reliability and process control. The system provides a comprehensive range of individual process checks throughout the analytical process, from sample introduction to result reporting. Listed below are key features of the DxN VERIS System that ensure consistent performance and process control. Collectively, these capabilities may serve to reduce risk of analytical error within run and between runs.
Sample introduction
Nucleic acid extraction
Real-time PCR amplification and detection
An internal process control (PC) is run with each sample to monitor the reaction. The PC may be a plasmid or an inactivated virus that contains a selected or engineered target sequence and is designed to mimic the behaviour of the assay target throughout the extraction, purification, and PCR process.
In addition to these quality features, the DxN VERIS System displays QC results in chart format to provide a graphical view of the data. Depending on the characteristics of the data, the system uses a Levy-Jennings chart or a Shewhart chart. Multiple data sets can be viewed simultaneously in an overlay chart, or in up to four individual charts. On- board QC management software flags when QC is out of range.
QC procedure for VERIS viral load assays
Beckman-Coulter’s QC procedure provides a method of monitoring system performance while minimizing hands-on time and QC cost to the laboratory. Beckman Coulter recommends that Quality Control should be run in each 24-hour period in which test samples are run until variability limits have been established on the DxN VERIS System. Reduced frequency of control testing should be based on data as determined by the individual laboratory. Quality control materials should incorporate the analyte and a negative control.
Each laboratory should establish mean values and acceptable ranges to assure proper performance. Quality control results that do not fall within acceptable ranges may indicate invalid test results. It is recommended that laboratories examine all test results generated after obtaining the last acceptable quality control test point for this analyte.
In some countries or geographic locations, government regulation may define specific requirements that dictate frequency and number of QC data points and specimens used. Each laboratory should establish its own QC protocol based on data as determined by the laboratory in accordance with accrediting organizations and government regulations, as applicable[2].
Customer case study – Rennes University Hospital
Quality Control programmes utilizing the DxN VERIS System have been successfully implemented at customer laboratories across Europe. Described below is an example of a QC protocol developed in the Virology laboratory at Rennes University Hospital, France.
Rennes University Hospital is a 2,000 bed facility serving the Brittany region of France. The Virology lab processes approximately 133,000 analyses per year including 8,000 qPCR tests for HIV-1, HBV, and CMV viral load monitoring. The laboratory adopted the DxN VERIS system in 2016 based on the system’s workflow advantages and assay performance.
The analytical performance of the DxN VERIS System enabled the laboratory to consider the possibility of reducing the frequency of QC testing required to monitor routine patient analyses. In order to determine an appropriate QC frequency for viral load testing, the laboratory evaluated the performance characteristics of the analyser as well as the number of tests performed over a period time. Based on this assessment the laboratory concluded that performing three levels of QC (negative, low, high) two times per week would provide an acceptable level of system control while significantly reducing QC costs and hands-on time. This level of QC testing was appropriate based on the volume of tests performed by the laboratory. For labs that perform a higher volume of tests, QC may need to be performed more frequently in order to provide a sufficient level of QC relative to the number of tests performed. In case of QC out-of range, a procedure has been set-up in Rennes in order to re-test all samples analysed between the two QC measurement times. To validate the twice-weekly QC protocol, the lab evaluated the precision performance of each assay over 5 months. The precision observed over this time frame was less than 5.8% CV with standard deviation (SD) within 0.16 log IU/ml. No values were observed outside of the expected range. These data, summarized below, were determined by Rennes to be sufficient to support the twice-weekly QC protocol.
The Virology laboratory at Rennes University Hospital has been following the twice-weekly QC protocol since April 2016. This has resulted in a reduction in the cost per reportable result and simplified the QC process without impacting the quality of results produced by the lab.
Conclusion
Beckman Coulter’s DxN VERIS System provides a high level of assay performance, ease of use, and workflow efficiency. Effective quality control programs can be developed based on the unique testing requirements of each laboratory, resulting in a high level of system control while reducing hands-on time and QC cost.
* DxN VERIS products are CE-marked IVDs. DxN VERIS product line has not been submitted to U.S. FDA and is not available in the U.S. market. DxN VERIS Molecular Diagnostics System is also known as VERIS MDx Molecular Diagnostics System and VERIS MDx System.
The authors
www.beckmancoulter.comJ. Wyatt, P. Le Roux, V. Thibault1
Beckman Coulter Diagnostics | Brea, CA
1 Rennes University Hospital, France
2 CLSI. Statistical Quality Control for Quantitative Measurement Procedures:
Principles and Definitions; Approved
Guideline-Third Edition. CLSI document C24-A3. Wayne, PA: Clinical and
Laboratory Standards Institute.
DT 100 – The Dual Technology System
, /in Featured Articles /by 3wmediaConsolidate Hemostasis testing on a single platform
, /in Featured Articles /by 3wmediaFree 25OH Vitamin D Elisa
, /in Featured Articles /by 3wmediaMini Collect Capillary Blood Collection System
, /in Featured Articles /by 3wmediaNext Level of Laboratory Automation
, /in Featured Articles /by 3wmediaExperience the power of the Atellica Solution
, /in Featured Articles /by 3wmediaScientific literature review
, /in Featured Articles /by 3wmediaThere are many peer-reviewed papers covering the diagnosis of autoimmune diseases, and it is frequently difficult for healthcare professionals to keep up with the literature. As a special service to our readers, CLI presents a few key abstracts from the clinical and scientific literature selected by our editorial board as being particularly worthy of attention.
Blood biomarkers as outcome measures in inflammatory neurologic diseases
El Ayoubi NK, Khoury SJ. Neurotherapeutics. 2016 Oct 18. [Epub ahead of print]
Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system. Only a few biomarkers are available in MS clinical practice, such as cerebrospinal fluid oligoclonal bands and immunoglobulin index, serum anti-aquaporin 4 antibodies, and serum anti-John Cunningham virus antibodies. Thus, there is a significant unmet need for biomarkers to assess prognosis, response to therapy, or potential treatment complications. Here we describe emerging biomarkers that are in development, focusing on those from peripheral blood. There are several limitations in the process of discovery and validation of a good biomarker, such as the pathophysiological complexity of MS and the technical difficulties in globally standardizing methods for sampling, processing, and conserving biological specimens. In spite of these limitations, ongoing international collaborations allow the exploration of many interesting molecules and markers to validate diagnostic, prognostic, and therapeutic-response biomarkers.
Gene polymorphisms as predictors of response to biological therapies in psoriasis patients
Linares-Pineda TM, Cañadas-Garre M, Sánchez-Pozo A, Calleja-Hernández MÁ. Pharmacol Res. 2016; 113(Pt A): 71–80.
Psoriasis is a chronic inflammatory autoimmune skin disease, characterized by the formation of erythematous scaly plaques on the skin and joints. The therapies for psoriasis are mainly symptomatic and sometimes with poor response. Response among patients is very variable, especially with biological drugs (adalimumab, etarnecept, infliximab and ustekimumab). This variability may be partly explained by the effect of different genetic backgrounds. This has prompted the investigation of many genes, such as FCGR3A, HLA, IL17F, IL23R, PDE3A-SLCO1C1, TNFα and other associated genes, as potential candidates to predict response to the different biological drugs used for the treatment of psoriasis. In this article, we will review the influence of gene polymorphisms investigated to date on response to biological drugs in psoriasis patients.
Biomarker discovery by modeling Behçet’s disease with patient-specific human induced pluripotent stem cells
Son MY, Kim YD, Seol B, Lee MO, Na HJ, Yoo B, Chang JS, Cho YS. Stem Cells Dev. 2016 Oct 12. [Epub ahead of print]
Behçet’s disease (BD) is a chronic inflammatory and multisystemic autoimmune disease of unknown etiology. Due to the lack of a specific test for BD, its diagnosis is very difficult, and therapeutic options are limited. Induced pluripotent stem cell (iPSC) technology, which provides inaccessible disease-relevant cell types, opens a new era for disease treatment. Here, we generated BD iPSCs from patient somatic cells and differentiated them into hematopoietic precursor cells (BD iPSC-HPCs) as BD model cells. Based on comparative transcriptome analysis using our BD model cells, we identified 8 novel BD specific genes, AGTR2, CA9, CD44, CXCL1, HTN3, IL-2, PTGER4 and TSLP, that were differentially expressed in BD patients, compared to healthy controls or patients with other immune diseases. The use of CXCL1 as a BD biomarker was further validated at the protein level using both a BD iPSC-HPC-based assay system and BD patient serum samples. Furthermore, we show that our BD iPSC-HPC-based drug screening system is highly effective for testing CXCL1 BD biomarkers, as determined by monitoring the efficacy of existing anti-inflammatory drugs. Our results shed new light on the usefulness of patient-specific iPSC technology in the development of a benchmarking platform for disease-specific biomarkers, phenotype- or target-driven drug discovery, and patient-tailored therapies.
Overview of laboratory testing and clinical presentations of complement deficiencies and dysregulation
Frazer-Abel A, Sepiashvili L, Mbughuni MM, Willrich MA. Adv Clin Chem. 2016; 77: 1–75.
Historically, complement disorders have been attributed to immunodeficiency associated with severe or frequent infection. More recently, however, complement has been recognized for its role in inflammation, autoimmune disorders, and vision loss. This paradigm shift requires a fundamental change in how complement testing is performed and interpreted. Here, we provide an overview of the complement pathways and summarize recent literature related to hereditary and acquired angioedema, infectious diseases, autoimmunity, and age-related macular degeneration. The impact of complement dysregulation in atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, and C3 glomerulopathies is also described. The advent of therapeutics such as eculizumab and other complement inhibitors has driven the need to more fully understand complement to facilitate diagnosis and monitoring. In this report, we review analytical methods and discuss challenges for the clinical laboratory in measuring this complex biochemical system.