Background
Zika virus (ZIKV) belongs to the
Flavivirus genus and is related to other viruses that are also transmitted by the bite of mosquitoes, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). The
Flaviviridae family comprises single-strand RNA, membrane-enveloped viruses that frequently use
Aedes aegypti as a vector. Despite ZIKV being discovered over 60 years ago, only since 2014 (in the French Polynesia Islands) and 2015 (Brazil and America) has it been evident that the virus can cause large outbreaks and epidemics that lead to a global public health emergency [1].
ZIKV infection causes a mild severity, undifferentiated febrile syndrome, characterized by rash, arthralgia, myalgia and conjunctivitis, symptoms that are similar to those that appear in DENV fever or chikungunya virus (CHIKV) fever (CHIKV being an unrelated alphavirus transmitted by the same mosquito). The similarities of the symptoms causes confusion between the diseases during clinical evaluation. Also, these three viral illnesses may co-circulate in the same areas, hampering the final diagnosis of patients.
Although the ZIKV morbidity and mortality are considered low, it was demonstrated during the recent outbreaks that infection in pregnant women may be associated with severe birth defects (mostly microcephaly), and with the appearance in infected adults of a severe neurologic disease called Guillain–Barré syndrome (GBS). This neurologic entity increased 2–10-fold the historic cases in Latin America during the 2016 ZIKV epidemic [2]. Epidemiological estimates consider that approximately 75% of ZIKV-infected people do not present signs or symptoms during an outbreak, but they become an efficient transmission focus to mosquitoes and other individuals.
It is well known that mosquito bites are the main transmission route in areas where the insect infestation rates are high; however, it recently has been confirmed that ZIKV is capable of crossing the placental barrier and infecting the fetus. In adult patients, the virus persists in semen and vaginal fluids for two months, producing a viral load sufficient for transmission during sexual intercourse. This finding changes the epidemiological trends, as it is now also possible to detect infected patients in non-tropical countries, challenging the clinical and laboratory diagnosis. However, it is clear that tropical underdeveloped countries will still be the major source of febrile cases and, of course, the congenital malformations and GBS appearance in adults.
ZIKV infection diagnostics
The incubation period of ZIKV disease is not clear but is likely to be a few days, similar to other arboviruses. Symptoms can begin 2 to 7 days after a mosquito bite and last for 3 to 7 additional days. In both early symptomatic or asymptomatic cases, the virus can be detected by reverse transcription (RT)-PCR after purification of plasma or serum RNA. The acute sera can be inoculated in Vero cells or C6/36 mosquito cells to attempt virus isolation, but although this technique is powerful, it is expensive and lacks clinical value. We successfully isolated ZIKV and produced enough inoculum for cell biology and immunologic studies (Fig. 1). As a result of their sensitivity and specificity, ZIKV RNA detection by different nucleic acid tests is used on a routine basis to confirm acute ZIKV cases.
RT-PCR
The real-time RT-PCR protocol designed by the Centers for Disease Control (CDC, USA) during the 2007 Yap Island outbreak is the most used and evaluated, even after the confirmation that a very low viral load occurs during the acute phase and that viremia lasts only a few days in both symptomatic and asymptomatic individuals. This CDC protocol does not amplify RNA from other flaviviruses and alphaviruses [3].
The test was designed as a one-step RT-PCR with fluorogenic probes using serum as the sample and is also used on urine samples, where the virus can be detected until 15 days after symptoms start and when the serum sample has become negative. A comparison between different sample types demonstrated that saliva may be better than serum for confirming ZIKV infection [4]. A very sensitive and specific synthetic biology tool based on isothermal amplification and toehold switch RNA sensors has been reported and is currently under evaluation in field conditions in Colombia, Brazil and Equator [5].
Many other real-time PCR tests have recently been developed, but there are no reports regarding their clinical evaluation. One test with excellent analytical performance is becoming available (Altona Diagnostics), but it has not yet reported clinical assays in ZIKV circulating zones.
Frequently, conventional PCR has been used to follow epidemics and ZIKV circulation in mosquitoes [6], and this reported test was used to confirm the first cases in Brazil. Recently, we used modified primers to perform a double-round one-step RT-PCR to detect DENV, ZIKV and CHIKV in the serum of febrile patients, obtaining samples simultaneously positive for two or even three viruses [7]. This test also detects ZIKV RNA in paired samples of serum, breast milk and urine (Fig. 2).
Serology
The main challenge to serological ZIKV diagnosis is related to its structural proximity to other flaviviruses (DENV, YFV, and WNV) because antibodies against one of them can recognize the other viruses on ELISA platforms, frequently resulting in a false positive diagnostic. For this reason, RNA detection is preferred to confirm the infection during the first week after symptoms appear. However, serological tests are recommended to facilitate the diagnosis of pregnant women living in endemic zones or women or couples wanting pre-conception counselling because ZIKV IgM positivity confirms previous exposure to the virus; in those who are negative, it is recommended to perform periodic tests to prove the absence of virus contact.
Since the first serological diagnostics were performed in Africa, there have been difficulties confirming the infection by antibodies due to cross-reactivity in neutralization tests, hemagglutination inhibition and mouse neutralization used in the 1950s [8]. During the Yap Islands’ 2007 ZIKV outbreak, in addition to molecular confirmation, 14 ZIKV cases were investigated by serology, and it was confirmed that 8/14 individuals who had a previous flavivirus infection (secondary flavivirosis) were positive by DENV IgM ELISA. In addition, ZIKV-confirmed sera had high titres in the plaque reduction neutralization test (PRNT), mainly to DENV (12/14), YFV (3/14) and WNV (6/14) [3].
Currently, the CDC uses IgM antibody capture (MAC)-ELISA in its diagnostic algorithm, in which the ZIKV antigen is obtained from infected mice brains or recombinant proteins (Fig. 3a). This test is being used to confirm recent infections and to counsel women in endemic zones. This ELISA has not yet been tested in endemic zones where other flaviviruses circulate. Recently, an assay based on non-structural protein NS1 from ZIKV adsorbed to ELISA plates has been reported (Euroimmun AG), showing excellent performance to detect both IgM and IgG using samples from endemic zones and samples with confirmed contact with other flaviviruses (Fig. 3b) [9].
Pregnant women: the priority
Considering that ZIKV infection during the first two trimesters of pregnancy can be associated with neurological defects in the fetus, it is important to evaluate the infection risk in three different groups of women: (i) women of childbearing age living in areas with virus circulation; (ii) women travelling frequently to endemic zones; and (iii) women having sexual intercourse with individuals travelling frequently to ZIKV endemic zones (Table 1). Notably, only 25% of infected individuals present signs or symptoms of the disease, but those who are asymptomatic can transmit the virus to mosquitoes and through sexual contact, can develop GBS or can transmit the virus to the fetus during pregnancy. Normally, health authorities do not recommend confirming all cases by PCR or serology, but only those needed to facilitate the surveillance of ZIKV infections or sequelae (Table 1).
CDC testing algorithm
In zones with ZIKV circulation, pregnant women should be assessed for ZIKV exposure (with or without signs or symptoms). If there are fewer than 2 weeks of putative exposition, the recommended test is RT-PCR in both serum and urine samples. If these results are negative, an IgM ZIKV-ELISA should be performed 2–12 weeks later. If the pregnant woman visits the healthcare system 2–12 weeks after having symptoms or the putative exposure, the recommended test is ZIKV IgM ELISA with simultaneous testing of IgM to DENV. If both are positive, it means a recent flavivirus infection. In this case, it is necessary to evaluate antibody titres to each virus using a plaque reduction neutralization assay (PRNT). If the neutralization titres to ZIKV are >10, the diagnosis is a recent ZIKV infection [10].
In both confirmed and presumptive ZIKV infection during pregnancy, serial ultrasounds should be performed every 3–4 weeks to assess fetal anatomy and growth. Amniocentesis to evaluate fetal infection is not recommended. After birth, neonatal serum and urine should be tested by RT-PCR and IgM. If CSF is obtained for other reasons, it can also be tested. The placenta and umbilical cord, as well as tissues from fetal losses, can be processed for PCR and immunohistochemistry.
Conclusion
Emergent ZIKV is here to stay. Virus transmission can occur during the entire year because of the tropical weather and generalized A. aegypti infestation in developing countries. Because of the concurrent arbovirus epidemics and the overlapping endemic regions, the differential diagnosis must always include ZIKV, DENV and CHIKV. The development of new technical approaches to diagnose ZIKV infections and the clinical trials to evaluate them is an imperative need, mainly because of the deep impact on childbearing women in endemic zones.
References
1. Abushouka AI, Negidac A, Ahmed H. An updated review of Zika virus. J Clin Virol 2016; 84: 53–58.
2. Dos Santos T, Rodriguez A, Almiron M, Sanhueza A, Ramon P, et al. Zika virus and the Guillain–Barré syndrome – case series from seven countries. N Engl J Med 2016; 375(16): 1598–1601.
3. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 2008; 14(8): 1232–1239.
4. Musso D, Roche C, Nhan TX, Robin E, Teissier A, Cao-Lormeau VM.Detection of Zika virus in saliva. J Clin Virol 2015; 68: 53–55.
5. Pardee K, Green AA, Takahashi MK, Braff D, Lamber G, et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell 2016; 165: 1255–1266.
6. Faye O, Faye O, Dupressoir A, Weidmann M, Ndiaye M, Alpha Sall A. One-step RT-PCR for detection of Zika virus. J Clin Virol 2008; 43(1): 96–101.
7. Calvo EP, Sánchez-Quete F, Durán S, Sandoval I, Castellanos JE. Easy and inexpensive molecular detection of dengue, chikungunya and zika viruses in febrile patients. Acta Tropica 2016; 163: 32–37.
8. Musso D, Lanteri MC. Thoughts around the Zika virus crisis. Curr Infect Dis Rep 2016; 18(12): 46.
9. Steinhagen K, Probst C, Radzimski C, Schmidt-Chanasit J, Emmerich P, et al. Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016. Euro Surveill 2016; 21(50): pii: 30426.
10. Centers for Disease Control and Prevention. Interim pregnancy guidance: testing and interpretation recommendations for a pregnant woman with possible exposure to Zika virus — United States (including U.S. territories). [https://www.cdc.gov/zika/pdfs/testing_algorithm.pdf]
The authors
Jaime E. Castellanos PhD, Shirly Parra-Álvarez and Eliana P. Calvo* PhD
Grupo de Virología, Universidad El Bosque, Bogotá, Colombia
*Corresponding author
E-mail: calvoeliana@unbosque.edu.co
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, /in Featured Articles /by 3wmediaClinical utility of PIVKA-II in the diagnosis of hepatocellular carcinoma
, /in Featured Articles /by 3wmediaPrimary liver cancer is the seventh most common cancer worldwide and the third most common cause of death from cancer [1]. Seventy-five to eighty-five percent of primary liver cancer cases are associated with HCC. The distinctive features of HCC include relatively large-sized tumours, vascular invasion, intra-hepatic metastasis, low differentiation, common recurrence and poor prognosis [2, 3]. In 70–90% of the cases, development of HCC requires a chronic liver disorder and cirrhosis as a background; these are caused mostly by chronic hepatitis C virus, hepatitis B virus, alcohol abuse, non-alcoholic steatohepatitis and less typically observed in inherited haemochromatosis, autoimmune hepatitis, antitrypsin deficiency, aflatoxin intoxication and also in some cases of oral contraception treatment [4, 5]. In addition, some chronic conditions, such as diabetes mellitus, cholelithiasis, obesity and hormone imbalance, are associated with HCC development [6]. The overall 5-year survival rate is believed to be less than 40%; however, a diagnosis at the early stages, followed by liver resection or transplantation, can improve this rate to 60–70% [7–9].
Taking into account the prevalence and mortality and also poor prognosis of HCC, it is apparent that highly sensitive techniques for diagnosis at the early stages are needed. The main diagnostic tool for HCC screening is radiologic imaging investigations such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI). With the development and introduction of contrast-enhanced ultrasound (CEUS) for analysis of intra-nodular vascularisation pattern, the sensitivity and specificity have been reported to be 90.9% and 100% for progressed HCC and 85.7% and 96.1% for early HCC, respectively [10]. The role of MRI and CT in producing reliable three-dimensional images is very important; however, the relationship between the radiographic and pathological tumour sizes is not yet well established. At this point application of tumour markers as supplementary analysis may provide useful information for making a diagnosis and monitoring of confirmed HCC [11, 12].
Protein induced by vitamin K absence/antagonist-II (PIVKA-II), also known as des-gamma carboxyprothrombin (DCP), is an abnormal form of prothrombin formed as a result of impaired or insufficient post-translational γ-carboxylation that occurs in the presence of vitamin K deficiency and leads to the loss of biological activity of the protein. Following synthesis in the liver, prothrombin, alongside the other hepatic vitamin K-dependent proteins undergoes transformation of specific glutamyl (GLU) residues into γ-carboxyl glutamyl (GLA) residues under the influence of vitamin K-dependent γ-glutamyl carboxylase in the presence of reduced vitamin K concentration (Fig. 1) [13]. Interestingly, carboxylation may not occur at all, which results in the formation of different variants of PIVKA-II with various degree of biological activity [14].
The role of PIVKA-II in HCC pathology is still not well established. It has been shown that PIVKA-II induces the malignant potential of HCC through stimulation of cell proliferation owing to a structural resemblance to hepatocyte growth factor [15–17]. Furthermore, PIVKA-II promotes angiogenesis in HCC resulting in local tissue invasion and metastases via stimulation of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) [13, 18].
Methods and patient samples
The automated chemiluminescent microparticle immunoassay (ARCHITECT PIVKA-II 2P4 CMIA, Abbott) was validated and used for quantitation of PIVKA-II using the Abbott™ Architect iSystem 2000 analyser in the Human Nutristasis Unit at St Thomas’ Hospital, London, UK. Imprecision and recovery evaluations were performed in line with the appropriate standard operating procedures as part of the validation process. The CMIA is based on a two-step sandwich reaction of binding of anti-PIVKA-II antibodies and specific PIVKA-II epitopes with subsequent addition of chemiluminescent labels and registration of the relative light units as a quantitative representation of PIVKA-II concentration in the tested sample [1].
In order to exclude possible interference with anticoagulant therapeutic agents, high PIVKA-II results were tested for warfarin, as it is the most commonly used anticoagulant that interferes with the vitamin K cycle. Samples found to be positive for warfarin were disqualified from further analysis.
Eighty-seven samples from the Gassiott Gastroenterology Clinic (GGC, St. Thomas’ Hospital, London) and the Hepatocellular Carcinoma Clinic in the Institute of Liver Studies (King’s College Hospital, London) were analysed in three groups: high-risk patients with non-HCC pathology of the liver, high-risk patients currently undergoing HCC surveillance, and patients with diagnosed HCC (group A, B and C respectively). Group A (n=29) consisted of randomly selected patients at GGC with viral and non-viral cirrhosis, steatosis, fibrosis, hepatitis and benign lesions. Group B (n=24) represented high-risk patients with changes to the liver suggestive of possible HCC discovered in the course of US/MRI/CT investigations. Finally, group C (n=34) comprised of patients diagnosed with HCC at different stages; the diagnosis was established in the course of histological examination of liver biopsy samples.
All results for PIVKA-II concentrations in patient samples were statistically processed in IBM SPSS Statistics, Version 23. Tests of normality, association between different variables and receiver operating characteristic (ROC) curve were applied for the analysis.
Results and discussion
Using a cut-off of 49.4 mAU/mL, an elevated PIVKA-II concentration was found in just one patient from the negative control group, which represents 3.4% (Table 1). This patient was diagnosed with multiple cysts on the background of hepatitis; therefore, the result may be interpreted as both false positive (elevation of PIVKA-II due to non-malignant pathology) and true positive (in this case the patient would need to undergo more comprehensive screening).
In the positive group, PIVKA-II was elevated in 79.4% of the patients and demonstrated a broad scatter of values (19.06 mAU/mL for the lowest detected concentration and 340 485.5 mAU/mL for the highest detected concentration) owing to various sizes of the tumour masses at different stages of HCC and possibly existence of different PIVKA-II variants depending on the number of GLU residues involved in γ-carboxylation [19]. Normal PIVKA-II results in this group can be explained by the normalisation of PIVKA-II concentration after curative treatment, if performed [16].
Statistical processing of data showed no evidence of dependence of the results on age or gender (P>0.05 for all three groups). Area under the curve (AUC) in ROC analysis for PIVKA-II in the present research was 0.917 (CI 95% 0.847–0.986), which is suggestive of excellent clinical usefulness of PIVKA-II in HCC diagnosis (Fig. 2). AUC for alpha-fetoprotein (AFP) had slightly lower value (0.833 with CI 95% 0.722–0.945), which can still be classified as a fairly useful test (Fig. 3).
In this study the optimal cut-off value for PIVKA-II was identified by means of ROC and is 49.4 mAU/mL with sensitivity of 79.4% and specificity of 96.6%. Analysis of true and false-negative and -positive results revealed, that more than 83% of PIVKA-II results were truly reliable, whereas only 74.6% of AFP results demonstrated true diagnostic value (Table 2).
Unfortunately, sensitivity and specificity of AFP cannot accurately reflect its performance in the present study, as AFP results were available for only 17 patients from group A, which means that the study was possibly deprived of some potentially truly negative results. However, taking into account considerable difference between sensitivity and specificity rates for PIVKA-II and AFP (79.4 vs 96.6% and 70.6 vs 82.4% respectively), allows the conclusion that PIVKA-II displays slightly better clinical utility in HCC diagnosis. Similar results were reported in the previous studies [7, 20–24].
Limitations to the study
The major limitation to this research was the requirement to use anonymised samples, which prevented access to the full clinical history of the patients and impossibility to interpret the results in detail. Another limitation was the number of samples which could be considered to be insufficient to achieve aims of the project with adequate statistical power. A larger number of samples would have given the study more power and allowed a more precise ROC to be constructed and subsequently a more precise cut-off value to be identified.
Conclusion
In the present research PIVKA-II demonstrated high accuracy, sensitivity and specificity in HCC diagnosis. PIVKA-II has several advantages over AFP in terms of clinical utility for HCC diagnosis and prognosis: PIVKA-II is comparatively less frequently elevated in liver pathology [22], is more sensitive to small HCC tumours, correlates with HCC progression significantly better and has shorter half-life than AFP (40–72 hours against 5–7 days), which makes it more suitable for monitoring purposes [14]. Implementation of PIVKA-II as diagnostic test gathers pace in transplantation medicine, as this tumour marker, alongside Milan criteria has been used for recipient selection for living donor liver transplantation [16]. In addition, PIVKA-II concentrations can reflect the responsiveness of the liver to medical treatment (i.e. sorafenib), which cannot be achieved with AFP test. On the other hand, AFP is sensitive to radiological response following transarterial chemoembolisation, whereas PIVKA-II is not [12]. Also, PIVKA-II is affected by potentially interfering pharmacological agents (e.g. warfarin and certain antibiotics), it is dependent on vitamin K metabolism and can give false-positive results in non-HCC conditions which all has to be taken into account while interpreting the results.
Controversy over the best performance of tumour markers traces back to different assays used and various patient groups involved. Fortunately, AFP and PIVKA-II are independent of each other [16, 25]. Therefore, combination of PIVKA-II and AFP alongside AFP-L3, the fucosylated fraction of AFP, is suggested to be the best option for highly accurate laboratory diagnostic of HCC supplementary to imaging techniques. This multi-marker approach has been stated in the guidelines of The Japan Society of Hepatology and successfully used for diagnosis and management of HCC in Japan [26, 27].
Acknowledgement
ARCHITECT PIVKA-II 2P4 CMIA reagents and the graphics used in this article are courtesy of © Abbott Laboratories.
References
1. Kinukawa H, et al. characterization of an anti-PIVKA-II antibody and evaluation of a fully automated chemiluminescent immunoassay for PIVKA-II. Clin Biochem 2015; 48: 1120–1125.
2. Ha TY, et al. Expression pattern analysis of hepatocellular carcinoma tumour markers in viral hepatitis B and C patients undergoing liver transplantation and resection. Transplant Proc 2014; 46: 888–893.
3. Yano Y, et al. Clinical features of hepatitis C virus-related hepatocellular carcinoma and their association with α-fetoprotein and protein induced by vitamin K absence or antagonist-II. Liver Int 2006; 26: 789–795.
4. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132: 2557–2576.
5. Aghemo A, Colombo M. Hepatocellular carcinoma in chronic hepatitis C: from bench to bedside. Semin Immunopathol 2012; 35: 111–120.
6. McMasters K, Vauthey J. Hepatocellular carcinoma: targeted therapy and multidisciplinary care. Springer 2011; Chapters 1–5, 8.
7. Ji J, et al. Diagnostic evaluation of des-gamma-carboxy prothrombin versus α-fetoprotein for hepatitis B virus-related hepatocellular carcinoma in China: a large-scale, multicentre study. PLoS One 2016; 11: e0153227.
8. Huang TS, et al. Diagnostic performance of alpha-fetoprotein, lens culinaris agglutinin-reactive alpha-fetoprotein, des-gamma carboxyprothrombin, and glypican-3 for the detection of hepatocellular carcinoma: a systematic review and meta-analysis protocol. Syst Rev 2013; 2: 37.
9. Song PP, et al. Controversies regarding and perspectives on clinical utility of biomarkers in hepatocellular carcinoma. World J Gastroenterol 2016; 22: 262–274.
10. Giorgio A, et al. Characterization of dysplastic nodules, early hepatocellular carcinoma and progressed hepatocellular carcinoma in cirrhosis with contrast-enhanced ultrasound. Anticancer Res 2011; 31: 3977–3982.
11. Chen H, et al. CT and MRI in target delineation in primary hepatocellular carcinoma. Cancer Radiother 2013; 17: 750–754.
12. Park H, Park JY. Clinical significance of AFP and PIVKA-II responses for monitoring treatment outcomes and predicting prognosis in patients with hepatocellular carcinoma. BioMed Research International 2013; 2013: 310427.
13. Yue P, et al. Des-γ-carboxyl prothrombin induces matrix metalloproteinase activity in hepatocellular carcinoma cells by involving the ERK1/2 MAPK signalling pathway. Eur J Cancer 2011; 47: 1115–1124.
14. Zhang YS, et al. Des-γ-carboxy prothrombin (DCP) as a potential autologous growth factor for the development of hepatocellular carcinoma. Cell Physiol Biochem 2014; 34: 903–915.
15. Suzuki K, et al. Positioning of novel tumor marker NX-PVKA-R in the diagnosis of hepatocellular carcinoma in comparison with PIVKA-II. Dokkyo Journal of Medical Sciences 2013; 40: 163–168
16. Inagaki Y, et al. Clinical and molecular insights into the hepatocellular carcinoma tumour marker des-γ-carboxyprothrombin. Liver Int 2010; 31: 22–35.
17. Jinghe X, et al. Vitamin K and hepatocellular carcinoma: the basic and clinic. World J Clin Cases 2015; 3: 757–764.
18. Fujikawa T, et al. Significance of des-gamma-carboxyprothrombin production in hepatocellular carcinom. Acta Med Okayama 2009; 63: 299–304.
19. Zakhary NI, et al. Impact of PIVKA-II in diagnosis of hepatocellular carcinoma. J Adv Res 2013; 4: 539–546.
20. Mathew S, et al. Biomarkers for virus-induced hepatocellular carcinoma (HCC). Infect Genet Evol 2014; 26: 327–339.
21. Lim TS, et al. Combined use of AFP, PIVKA-II, and AFP-L3 as tumor markers enhances diagnostic accuracy for hepatocellular carcinoma in cirrhotic patients. Scand J Gastroenterol 2015; 51: 344–353.
22. Seo SI, et al. Diagnostic value of PIVKA-II and alpha-fetoprotein in hepatitis B virus-associated hepatocellular carcinoma. World J Gastroenterol 2015; 21: 3928–3935.
23. De J, et al. A systematic review of des-γ-carboxy prothrombin for the diagnosis of primary hepatocellular carcinoma. Medicine 2016; 95: e3448.
24. Ette AI, et al. Utility of serum des-gamma-carboxyprothrombin in the diagnosis of hepatocellular carcinoma among Nigerians, a case–control study. BMC Gastroenterol 2015; 15: 113.
25. Choi JY, et al. Diagnostic value of AFP-L3 and PIVKA-II in hepatocellular carcinoma according to total-AFP. World J Gastroenterol 2013; 19: 339–346.
26. Kudo M. Clinical practice guidelines for hepatocellular carcinoma differ between Japan, United States, and Europe. Liver Cancer 2015; 4: 85–95.
27. Kokudo M, et al. Evidence-based clinical practice guidelines for hepatocellular carcinoma: The Japan Society of Hepatology 2013 update (3rd JSH-HCC Guidelines). Hepatol Res 2015; 45: 123–127.
The authors
Volha Klimovich*1 MSc; Kieran Voong2 MSc; Roy Sherwood3 MSc, DPhil; Dominic J Harrington2 MSc, PhD
1Clinical Biochemistry, Viapath, St Thomas’ Hospital, London, UK
2Human Nutristasis Unit, Viapath, St Thomas’ Hospital, London, UK
3Viapath, King’s College Hospital, London, UK
*Corresponding author
E-mail: klimovichvolha@gmail.com
Improving diagnosis of Zika virus infection: an urgent task for pregnant women
, /in Featured Articles /by 3wmediaZika virus (ZIKV) belongs to the Flavivirus genus and is related to other viruses that are also transmitted by the bite of mosquitoes, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). The Flaviviridae family comprises single-strand RNA, membrane-enveloped viruses that frequently use Aedes aegypti as a vector. Despite ZIKV being discovered over 60 years ago, only since 2014 (in the French Polynesia Islands) and 2015 (Brazil and America) has it been evident that the virus can cause large outbreaks and epidemics that lead to a global public health emergency [1].
ZIKV infection causes a mild severity, undifferentiated febrile syndrome, characterized by rash, arthralgia, myalgia and conjunctivitis, symptoms that are similar to those that appear in DENV fever or chikungunya virus (CHIKV) fever (CHIKV being an unrelated alphavirus transmitted by the same mosquito). The similarities of the symptoms causes confusion between the diseases during clinical evaluation. Also, these three viral illnesses may co-circulate in the same areas, hampering the final diagnosis of patients.
Although the ZIKV morbidity and mortality are considered low, it was demonstrated during the recent outbreaks that infection in pregnant women may be associated with severe birth defects (mostly microcephaly), and with the appearance in infected adults of a severe neurologic disease called Guillain–Barré syndrome (GBS). This neurologic entity increased 2–10-fold the historic cases in Latin America during the 2016 ZIKV epidemic [2]. Epidemiological estimates consider that approximately 75% of ZIKV-infected people do not present signs or symptoms during an outbreak, but they become an efficient transmission focus to mosquitoes and other individuals.
It is well known that mosquito bites are the main transmission route in areas where the insect infestation rates are high; however, it recently has been confirmed that ZIKV is capable of crossing the placental barrier and infecting the fetus. In adult patients, the virus persists in semen and vaginal fluids for two months, producing a viral load sufficient for transmission during sexual intercourse. This finding changes the epidemiological trends, as it is now also possible to detect infected patients in non-tropical countries, challenging the clinical and laboratory diagnosis. However, it is clear that tropical underdeveloped countries will still be the major source of febrile cases and, of course, the congenital malformations and GBS appearance in adults.
ZIKV infection diagnostics
The incubation period of ZIKV disease is not clear but is likely to be a few days, similar to other arboviruses. Symptoms can begin 2 to 7 days after a mosquito bite and last for 3 to 7 additional days. In both early symptomatic or asymptomatic cases, the virus can be detected by reverse transcription (RT)-PCR after purification of plasma or serum RNA. The acute sera can be inoculated in Vero cells or C6/36 mosquito cells to attempt virus isolation, but although this technique is powerful, it is expensive and lacks clinical value. We successfully isolated ZIKV and produced enough inoculum for cell biology and immunologic studies (Fig. 1). As a result of their sensitivity and specificity, ZIKV RNA detection by different nucleic acid tests is used on a routine basis to confirm acute ZIKV cases.
RT-PCR
The real-time RT-PCR protocol designed by the Centers for Disease Control (CDC, USA) during the 2007 Yap Island outbreak is the most used and evaluated, even after the confirmation that a very low viral load occurs during the acute phase and that viremia lasts only a few days in both symptomatic and asymptomatic individuals. This CDC protocol does not amplify RNA from other flaviviruses and alphaviruses [3].
The test was designed as a one-step RT-PCR with fluorogenic probes using serum as the sample and is also used on urine samples, where the virus can be detected until 15 days after symptoms start and when the serum sample has become negative. A comparison between different sample types demonstrated that saliva may be better than serum for confirming ZIKV infection [4]. A very sensitive and specific synthetic biology tool based on isothermal amplification and toehold switch RNA sensors has been reported and is currently under evaluation in field conditions in Colombia, Brazil and Equator [5].
Many other real-time PCR tests have recently been developed, but there are no reports regarding their clinical evaluation. One test with excellent analytical performance is becoming available (Altona Diagnostics), but it has not yet reported clinical assays in ZIKV circulating zones.
Frequently, conventional PCR has been used to follow epidemics and ZIKV circulation in mosquitoes [6], and this reported test was used to confirm the first cases in Brazil. Recently, we used modified primers to perform a double-round one-step RT-PCR to detect DENV, ZIKV and CHIKV in the serum of febrile patients, obtaining samples simultaneously positive for two or even three viruses [7]. This test also detects ZIKV RNA in paired samples of serum, breast milk and urine (Fig. 2).
Serology
The main challenge to serological ZIKV diagnosis is related to its structural proximity to other flaviviruses (DENV, YFV, and WNV) because antibodies against one of them can recognize the other viruses on ELISA platforms, frequently resulting in a false positive diagnostic. For this reason, RNA detection is preferred to confirm the infection during the first week after symptoms appear. However, serological tests are recommended to facilitate the diagnosis of pregnant women living in endemic zones or women or couples wanting pre-conception counselling because ZIKV IgM positivity confirms previous exposure to the virus; in those who are negative, it is recommended to perform periodic tests to prove the absence of virus contact.
Since the first serological diagnostics were performed in Africa, there have been difficulties confirming the infection by antibodies due to cross-reactivity in neutralization tests, hemagglutination inhibition and mouse neutralization used in the 1950s [8]. During the Yap Islands’ 2007 ZIKV outbreak, in addition to molecular confirmation, 14 ZIKV cases were investigated by serology, and it was confirmed that 8/14 individuals who had a previous flavivirus infection (secondary flavivirosis) were positive by DENV IgM ELISA. In addition, ZIKV-confirmed sera had high titres in the plaque reduction neutralization test (PRNT), mainly to DENV (12/14), YFV (3/14) and WNV (6/14) [3].
Currently, the CDC uses IgM antibody capture (MAC)-ELISA in its diagnostic algorithm, in which the ZIKV antigen is obtained from infected mice brains or recombinant proteins (Fig. 3a). This test is being used to confirm recent infections and to counsel women in endemic zones. This ELISA has not yet been tested in endemic zones where other flaviviruses circulate. Recently, an assay based on non-structural protein NS1 from ZIKV adsorbed to ELISA plates has been reported (Euroimmun AG), showing excellent performance to detect both IgM and IgG using samples from endemic zones and samples with confirmed contact with other flaviviruses (Fig. 3b) [9].
Pregnant women: the priority
Considering that ZIKV infection during the first two trimesters of pregnancy can be associated with neurological defects in the fetus, it is important to evaluate the infection risk in three different groups of women: (i) women of childbearing age living in areas with virus circulation; (ii) women travelling frequently to endemic zones; and (iii) women having sexual intercourse with individuals travelling frequently to ZIKV endemic zones (Table 1). Notably, only 25% of infected individuals present signs or symptoms of the disease, but those who are asymptomatic can transmit the virus to mosquitoes and through sexual contact, can develop GBS or can transmit the virus to the fetus during pregnancy. Normally, health authorities do not recommend confirming all cases by PCR or serology, but only those needed to facilitate the surveillance of ZIKV infections or sequelae (Table 1).
CDC testing algorithm
In zones with ZIKV circulation, pregnant women should be assessed for ZIKV exposure (with or without signs or symptoms). If there are fewer than 2 weeks of putative exposition, the recommended test is RT-PCR in both serum and urine samples. If these results are negative, an IgM ZIKV-ELISA should be performed 2–12 weeks later. If the pregnant woman visits the healthcare system 2–12 weeks after having symptoms or the putative exposure, the recommended test is ZIKV IgM ELISA with simultaneous testing of IgM to DENV. If both are positive, it means a recent flavivirus infection. In this case, it is necessary to evaluate antibody titres to each virus using a plaque reduction neutralization assay (PRNT). If the neutralization titres to ZIKV are >10, the diagnosis is a recent ZIKV infection [10].
In both confirmed and presumptive ZIKV infection during pregnancy, serial ultrasounds should be performed every 3–4 weeks to assess fetal anatomy and growth. Amniocentesis to evaluate fetal infection is not recommended. After birth, neonatal serum and urine should be tested by RT-PCR and IgM. If CSF is obtained for other reasons, it can also be tested. The placenta and umbilical cord, as well as tissues from fetal losses, can be processed for PCR and immunohistochemistry.
Conclusion
Emergent ZIKV is here to stay. Virus transmission can occur during the entire year because of the tropical weather and generalized A. aegypti infestation in developing countries. Because of the concurrent arbovirus epidemics and the overlapping endemic regions, the differential diagnosis must always include ZIKV, DENV and CHIKV. The development of new technical approaches to diagnose ZIKV infections and the clinical trials to evaluate them is an imperative need, mainly because of the deep impact on childbearing women in endemic zones.
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3. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 2008; 14(8): 1232–1239.
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The authors
Jaime E. Castellanos PhD, Shirly Parra-Álvarez and Eliana P. Calvo* PhD
Grupo de Virología, Universidad El Bosque, Bogotá, Colombia
*Corresponding author
E-mail: calvoeliana@unbosque.edu.co
Simple, fast and highly sensitive colorimetric detection of Zika virus
, /in Featured Articles /by 3wmediaBackground
An outbreak of Zika virus (ZIKV) in Brazil terrorized the whole world and its explosive spread in the Americas caused the World Health Organization (WHO) to declare it a public health emergency of international concern in February 2016 [1]. This is because ZIKV is a suspected major cause of congenital microcephaly, Guillain-Barré syndrome and other neurologic syndromes [2–4]. ZIKV has a genome consisting of a single-stranded, positive-polarity RNA and belongs to the family Flaviviridae and the genus Flavivirus. Aedes mosquitoes, known as a major ZIKV vector, also transmit dengue and chikungunya viruses across tropical and subtropical regions around the world [5]. Moreover, antigenic similarity between ZIKV and dengue virus gives rise to serological cross-reactivity, precluding antibody-based assays from reliably distinguishing between ZIKV and dengue virus infections [6]. Thus, reliable methods for distinguishing ZIKV from dengue and chikungunya viruses are necessary in practical applications.
WHO target product profiles
In April 2016, the WHO announced Target Product Profiles (TPPs) for a better diagnostic test for ZIKV infection. The TPPs define the desired characteristics of a ZIKV diagnostic test. The proposed TPPs consist of ‘Detection of active infection with ZIKV’ (Table 1a) and ‘Detection of evidence of prior infection’ (Table 1b). Each characteristic in the tables represents essential properties that the newly developed ZIKV diagnostic test should have at least at an acceptable level. To state the obvious, the criteria of specificity for active infection are more stringent [7].
Previous research on ZIKV diagnostics
Due to serological cross-reactivity between ZIKV and other flaviviruses, most of previous studies on ZIKV diagnosis have dealt with molecular diagnostics instead of immunological assays. Faye and colleagues developed and evaluated a one-step reverse transcription (RT)-PCR assay for ZIKV detection. The limit of detection of the assay was found to be 7.7 plaque-forming units (p.f.u.) per reaction in human serum and in the L-15 medium [8]. A quantitative real-time RT-PCR assay for ZIKV was also developed by the same research group. Analytical sensitivity of the assay was estimated at 3.2×102 RNA copies/μL [9]. However, a conventional PCR assay requires a bulky and expensive thermal cycler, prolonged reaction time, and trained technicians; these resources are not available in many low- and middle-income countries. Moreover, the RT-PCR reaction is vulnerable to inhibitors (blood, plasma and urine), thus requiring painstaking and cumbersome RNA extraction steps.
Recent research on ZIKV diagnostics
To overcome such limitations of RT-PCR, a variety of isothermal nucleic acid amplification techniques have recently been developed. Among them, reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid, robust, and highly sensitive isothermal RNA amplification method that uses four to six primers to amplify specific RNA sequences at 60–65°C even in the presence of inhibitors such as blood, plasma, or urine. RT-LAMP is much faster than conventional PCR, and the reaction can even proceed in an oven, water bath or with heating packs [10, 11]. Despite these advantages, the RT-LAMP assays still rely on a conventional bulky amplicon analyser such as a gel electrophoresis apparatus or a fluorescence laser-induced detector for monitoring the LAMP amplicons; this situation precludes the use of RT-LAMP in point-of-care diagnosis.
Our approaches to simple and highly sensitive diagnosis of ZIKV
To eliminate the dependence on a conventional amplicon analyser while retaining the aforementioned advantages of RT-LAMP, we selected the lateral flow assay (LFA) format for RT-LAMP amplicon analysis. The LFA, a driving principle behind pregnancy test strips, is also widely known as a superior diagnostic tool for nucleic acids owing to its high sensitivity, simplicity, selectivity and easy interpretation of results. Moreover, the Bst 3.0 polymerase used in this study for RT-LAMP retains both improved isothermal amplification performance and strong reverse transcription activity, allowing us to avoid addition of exogenous reverse transcriptase and the inhibition of reverse transcription by biological substances. By utilizing the advantages of Bst 3.0 polymerase and combining the RT-LAMP assay with the LFA, we demonstrated simple and highly sensitive detection of ZIKV RNA in human whole blood by merely observing a colorimetric signal within 35 min.
The RT-LAMP reaction and modification of amplicons in our study
As mentioned above, RT-LAMP has excellent tolerance to many inhibitors so that isothermal amplification of ZIKV RNA is possible even when human whole blood is directly used as a sample. We extracted ZIKV RNA and added it into human whole blood to mimic ZIKV-containing blood samples. Then, the spiked human whole blood was serially diluted with blood to set up a concentration range from 106 copies of RNA to a single copy per 2 μL and directly used these dilutions as samples without additional RNA purification steps. To colorimetrically detect the result of the LFA, a special modification is needed: labelling of the amplicon with digoxigenin and biotin. Among our own designed ZIKV-specific primers, two loop primers were tagged with digoxigenin at the 5´end; this approach will allow digoxigenin to label the amplicon when loop primers amplify the ZIKV RNA by the LAMP method. Labelling of the amplicon with biotin is made possible by adding biotin-labelled dUTP (Biotin-dUTP) to the mix of deoxynucleotides (dNTPs) at a certain ratio. When ZIKV RNA is amplified and this reaction consumes dNTPs, Biotin-dUTP will substitute thymine at the adenine sites of the complementary strand, resulting in labelling of the amplicon with biotin.
RT-LAMP was carried out in a 25 μL reaction mixture containing 1× Isothermal Amplification Buffer II [20 mM Tris-HCl, 10 mM (NH4)2SO2, 150 mM KCl, 2 mM MgSO4, and 0.1% Tween 20], additional 2 mM MgSO4, a dNTP mix supplemented with biotin-dUTP (2.2 mM dGTP, dATP, dCTP, 1.375 mM dTTP, and 0.0825 mM biotin-dUTP), a target-specific primer mixture (0.8 μM forward and reverse inner primers, 0.4 μM digoxigenin-labelled loop primers, and 0.2 μM forward and reverse outer primers), 8 U of Bst 3.0 DNA polymerase, and 2 μL of human whole blood spiked with ZIKV RNA ranging from 106 copies to a single copy per 2 μL. The RT-LAMP reaction mixture was incubated for 30 min.
Design and operation of the LFA
Figure 1(a) and 1(b) shows the detailed set-up and operating procedures of the LFA in our study. First, 1 μL of digoxigenin- and biotin-labelled RT-LAMP products was loaded onto the conjugate pad, so that the biotin-labelled RT-LAMP products formed a complex with gold nanoparticles (AuNPs) via streptavidin-biotin interactions. Next, 45 μL of diluent buffer was placed on the buffer loading pad, and then capillary flow transferred AuNPs from the conjugate pad to the test and control line. The AuNP–RT-LAMP complexes were immobilized at the test line by the interaction between digoxigenin and anti-digoxigenin whereas the AuNPs that did not form complexes were captured by biotin. Complexed and uncomplexed AuNPs are indicated by violet bands at the test line and control line, respectively. The colorimetric signal was easily visible with the naked eye within 5 min.
Discussion
Analysis of the limit of detection in human whole blood samples
We evaluated the limit of detection of the LFA to determine whether our method is indeed highly sensitive. Two microliters of human whole blood was directly used as a sample without any purification steps. Figure 1(c) shows the ZIKV RNA detection results for the LFA. The signal intensities on the test line gradually declined as the concentration of ZIKV RNA decreased. Notably, the presence of even a single copy of ZIKV RNA could be detected within 35 min by the LFA. These results imply that our method has a great potential for diagnosis of ZIKV infections.
References
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8. Faye O, Faye O, Dupressoir A, Weidmann M, Ndiaye M, Alpha Sall A. One-step RT-PCR for detection of Zika virus. J Clin Virol 2008; 43: 96–101.
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11. Nyan D-C, Ulitzky LE, Cehan N, Williamson P, Winkelman V, et al. Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay. Clin Infect Dis 2014; 59: 16–23.
The authors
Dohwan Lee MS, Yong Kyoung Yoo PhD, and Jeong Hoon Lee* PhD
Department of Electrical Engineering, Kwangwoon University, Nowon, Seoul 01897, Republic of Korea.
*Corresponding author
E-mail: jhlee@kw.ac.kr