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Improving diagnosis of Zika virus infection: an urgent task for pregnant women

, 26 August 2020/in Featured Articles /by 3wmedia

Mosquito-borne Zika virus (ZIKV) was the cause of the recent large outbreak of Zika disease in America. Despite fever, Zika is a mild disease, although epidemics in recent years have demonstrated an association with the appearance of severe congenital malformations (microcephaly). Owing to ZIKV serology cross-reactivity with other tropical flaviviruses, the final diagnosis relies on nucleic acid amplification. Pregnant women in endemic areas should be investigated to follow infection and sequelae.

By Dr Jaime E. Castellanos, Shirly Parra-Álvarez and Dr Eliana P. Calvo

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

Diagnosis of respiratory tract infections caused by human bocavirus 1

, 26 August 2020/in Featured Articles /by 3wmedia

Human bocavirus 1 (HBoV1) causes respiratory tract infections in infants and children. Diagnosis of acute HBoV1 infections is challenging as viral DNA is frequently detected in asymptomatic controls and as co-finding with other viruses. Recently developed novel HBoV1 mRNA and antigen tests may improve the diagnosis of acute HBoV1 infections.

by Juha M. Koskinen, Dr Andrea Bruning, Dr Petri Susi and Dr Janne O. Koskinen

Background and molecular characteristics of HBoV1
Human bocavirus 1 (HBoV1), a small single-stranded DNA (ssDNA) virus belonging to the Parvoviridae family, was described for the first time in 2005 [1]. Its genome replication is dependent on the formation of double-stranded DNA (dsDNA) intermediates in the nucleus of the host cells. The dsDNA serves as template for transcription of messenger-RNA (mRNA) by the host replication machinery. The mRNA is further translated into viral proteins, such as structural VP2 protein. Structural proteins assemble as empty capsids into which genomic ssDNA is inserted. Thus, during acute infection, the replicating virus produces mRNA transcripts from the viral dsDNA which are translated into viral proteins. Formation of viral proteins and particles are essential for the multiplication and spread of viable viruses.

Epidemiology and clinical outcomes of HBoV1 infections
HBoV1 was originally discovered in hospitalized children with a respiratory tract infection (RTI) [1]. However, HBoV1 can cause RTI illnesses in varying severities. Mainly children at age 6–24 months are affected. By 6 years old almost all children are seropositive for HBoV1. Data on the disease pressure in adults are very scarce but apparently immunity lasts long and acute infections are rare. HBoV1 DNA is detected by PCR in 2–19% of patients with RTI worldwide. The most common symptoms of acute HBoV1 infection are common cold-like complaints, wheezing, bronchiolitis and pneumonia. HBoV1 is associated with asthma exacerbations [2]. Diagnostic positivity rate for HBoV1 has been high in some studies in summer [3]. This would differ from other RTI viruses like influenza and respiratory syncytial virus. However, most cases of HBoV1 DNA detection are reported in winter and spring [2] which may also be linked to the higher frequency of diagnostic testing during the influenza season.

HBoV1 may infect lower airways down to the bronchioles [2]. There has been no difference in HBoV1 prevalence between immunocompetent and immunocompromised patients [2]. It seems that that particularly young children who were born prematurely may be at risk in developing severe RTIs caused by HBoV1 [4, 5].

HBoV1 DNA is often found in stool samples from children. However, detection rates are similar among subjects with or without acute gastroenteritis. Also co-findings with other known gastroenteritis viruses are common. Thus, the detection of HBoV1 from stool is most probably rather a sign of respiratory tract or systemic infection, prolonged viral shedding or persistent infection than acute gastroenteritis [6].

Diagnostic methods and challenges in diagnosis of HBoV1 infections
HBoV1 infection cannot be accurately diagnosed based on clinical symptoms alone. There are four techniques to aid in the diagnosis of HBoV1 infections. These include serology [7], PCR using viral DNA as target [8], reverse transcription (RT) PCR using viral mRNA as target [9], and most recently antigen detection [10]. Also electron microscopy has been used to detect the presence of viral particles [5], although this technique is not suitable for routine diagnostics.

Serology can provide information as to whether the infection is acute or past and it can be used to confirm the findings of other methods. IgM positivity, low IgG avidity, seroconversion or a diagnostic (?4-fold) increase in the IgG level in paired sera are signs of acute HBoV1 infection [2, 7]. A major drawback of serology is that it takes the human body 1–2 weeks to produce the antibody.

A number of commercially available multiplex PCR tests have included the detection of HBoV1 DNA in their test panels and some of the tests may provide results also in stat labs. However, detection of viral DNA from nasal samples may have little clinical significance since HBoV1 DNA is frequently (10–40 %) detected in asymptomatic controls and often found as co-findings (50–70 %) with other respiratory viruses. Prolonged shedding of the virus from infected shells, or long-term presence of virus or viral DNA in the airways may explain the high co-infection rate and prevalence in asymptomatic controls observed in almost every DNA PCR cohort study [11–14]. Currently, the mechanism for persistence is unknown but one possible explanation may be that the virus exists in a latent phase where the transcription of mRNA and protein translation is inhibited by the immune system.

Quantification of viral DNA by Ct-value gives a statistical correlation with severity but is not diagnostic in individual cases owing to, for example, the semi-quantitative nature of sampling. Thus, high viral DNA load and single findings are only indicative of the etiology [3, 8]. Extensive exclusion of the presence of other potential RTI pathogens together with high genome HBoV1 DNA load as single finding, viremia or the presence of the DNA in normally sterile body fluids has shown causality [4, 5]. Instead of extensive exclusion of other RTI viruses with high-cost multiplex PCRs, direct detection of actively replicating HBoV1 viruses by mRNA PCR or an antigen test could be a more straightforward, specific and cost-efficient approach.

mRNA RT-PCR methodology was developed to specifically detect the acute HBoV1 infections before the rise in antibody levels. mRNA RT-PCR is analytically as sensitive as DNA PCR. It provides the same clinical sensitivity but higher diagnostic specificity than DNA PCR. In one HBoV1 case, mRNA was detected up to 10 days from the onset of the symptoms while the DNA was detected at least up to 2 months although the patient was already fully recovered. The time span for positivity based on the mRNA RT-PCR correlated better with acute symptoms than DNA PCR [9].

Serology, mRNA RT-PCR and DNA PCR suffer from being slow, costly and/or labour intensive techniques, and they are only available in highly specialized diagnostic laboratories. Detection of viral antigens (e.g. structural VP2 protein) from nasal samples provides a rapid and specific alternative for testing of acute HBoV1 infections (Fig. 1). Recently the first HBoV1 antigen test, to our knowledge, was introduced into the automated and multianalyte mariPOC respi test (www.arcdia.com). The test provides most of the positive results in 20 minutes and low positives in 2 hours at the point-of-care. The new test has shown similar clinical specificity compared to mRNA RT-PCR test [15]. Antigen testing is feasible only during the acute phase of the infection (active viral replication phase) which seems to be approximately 5 days from the emergence of symptoms [10], as for most of the RTI viruses. The first days are often the most crucial when making clinical decisions and have impact, for example, for the decision on whether to prescribe antibiotics or not. The features of HBoV1 diagnostic methods are compared in Table 1.

Selected diagnostic cases
Case 1
A previously healthy full-term born Finnish girl developed symptoms of rhinorrhea, cough and high fever at 5 months of age. Upper RTI with no lower respiratory tract involvement or signs of otitis was diagnosed. HBoV1 secretion into nasopharyngeal samples was monitored by quantitative mariPOC antigen test up to day 5. Virus peak was at day 3 and viral levels were low at day 5, which coincided with the recovery of symptoms on day 6 [10]. The virus peak sample was estimated to contain 2×1010 viral particles per mL.

Case 2
A prematurely (week 27) born Turkish girl, at 5 months of age, after sepsis, developed high fever, wheezing and was treated for acute bronchiolitis before hospital discharge. The patient was found deceased the same night as the result of respiratory failure caused by pulmonary infection. HBoV was detected as single finding from nasopharyngeal swabs, stools and lung tissues [4].

Case 3
A prematurely (week 25) born Slovene child, at the age of 18 months, with chronic respiratory insufficiency was hospitalized. HBoV1 DNA was detected in tracheal aspirate (2.6×1010 copies/mL), in the nasopharyngeal swab (8.27×106 copies/mL), and in plasma sample (7.42×106 copies/mL). The presence of HBoV1 particles was confirmed by electron microscopy from tracheal aspirate and autologous plasma, which was taken the third day of illness [5].

Conclusions
As demonstrated above, clinical manifestations of HBoV1 range from simple common cold symptoms to fatal respiratory illnesses. Diagnosis of HBoV1 is now significantly more straightforward because of the recent advances in HBoV1 diagnostics. Rapid antigen testing and mRNA RT-PCR provide accurate non-invasive diagnostics for acute HBoV1 infections. mRNA RT-PCR is so far only available in highly specialized diagnostic laboratories while rapid antigen test is applicable at point-of-care. DNA PCR may be most suitable for the detection of viral DNA from body parts, like cerebrospinal fluid during suspected systemic infection. The use of multiple diagnostic methods will provide a more accurate picture about the clinical significance and outcomes of the HBoV1 infections. The method of choice for accurate diagnosis of HBoV1 depends on the elapsed time since the onset of the symptoms, clinical signs and other clinical or research needs. There is no specific medication or vaccine for HBoV1 yet. However, the new diagnostic tests will increase our understanding about the clinical significance of HBoV1 and open new doors for therapy development.

References
1. Allander T, Tammi MT, Eriksson M, Bjerkner A, Tiveljung-Lindell A, Andersson B. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci U S A 2005; 102(43): 12891–12896.
2. Jartti T, Hedman K, Jartti L, Ruuskanen O, Allander T, Söderlund-Venermo M. Human bocavirus-the first 5 years. Rev Med Virol 2012; 22(1): 46–64.
3. Zhou L, Zheng S, Xiao Q, Ren L, Xie X, Luo J, Wang L, Huang A, Liu W, Liu E. Single detection of human bocavirus 1 with a high viral load in severe respiratory tract infections in previously healthy children. BMC Infect Dis 2014; 14(424): 1–8.
4. Ziyade N, Sirin G, Elgörmüs N, Das T. Detection of human bocavirus DNA by multiplex PCR analysis: postmortem case report. Balkan Med J 2015; 32(2): 226–229.
5. Uršic T, Krivec U, Kalan G, Petrovec M. Fatal human bocavirus infection in an 18-month-old child with chronic lung disease of prematurity. Pediatr Infect Dis J 2015; 34(1): 111–112.
6. Paloniemi M. Occurrence and significance of human coronaviruses and human bocaviruses in acute gastroenteritis of childhood. Acta Electronica Universitatis Tamperensis 2016; 1652. (http://urn.fi/URN:ISBN:978-952-03-0079-1)
7. Kantola K, Hedman L, Allander T, Jartti T, Lehtinen P, Ruuskanen O, Hedman K, Söderlund-Venermo M. Serodiagnosis of human bocavirus infection. Clin Infect Dis 2008; 46(4): 540–546.
8. Allander T, Jartti T, Gupta S, Niesters HG, Lehtinen P, Osterback R, Vuorinen T, Waris M, Bjerkner A, Tiveljung-Lindell A, van den Hoogen BG, Hyypiä T, Ruuskanen O. Human bocavirus and acute wheezing in children. Clin Infect Dis 2007; 44(7): 904–910.
9. Christensen A, Døllner H, Skanke LH, Krokstad S, Moe N, Nordbø SA. Detection of spliced mRNA from human bocavirus 1 in clinical samples from children with respiratory tract infections. Emerg Infect Dis 2013; 19(4): 574–580.
10. Bruning AH, Susi P, Toivola H, Christensen A, Söderlund-Venermo M, Hedman K, Aatola H, Zvirbliene A, Koskinen JO. Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test. New Microbes New Infect 2016; 11: 17–19.
11. von Linstow ML1, Høgh M, Høgh B. Clinical and epidemiologic characteristics of human bocavirus in Danish infants: results from a prospective birth cohort study. Pediatr Infect Dis J 2008; 27(10): 897–902.
12. Christensen A, Nordbø SA, Krokstad S, Rognlien AG, Døllner H. Human bocavirus in children: mono-detection, high viral load and viraemia are associated with respiratory tract infection. J Clin Virol 2010; 49(3): 158–162.
13. Martin ET, Fairchok MP, Kuypers J, Magaret A, Zerr DM, Wald A, Englund JA. Frequent and prolonged shedding of bocavirus in young children attending daycare. J Infect Dis. 2010; 201(11): 1625–1632.
14. Rhedin S, Lindstrand A, Rotzén-Östlund M, Tolfvenstam T, Ohrmalm L, Rinder MR, Zweygberg-Wirgart B, Ortqvist A, Henriques-Normark B, Broliden K, Naucler P. Clinical utility of PCR for common viruses in acute respiratory illness. Pediatrics. 2014; 133(3): e538–545.
15. Toivola H, Christensen A, Hedman K, Söderlund-Venermo M, Koskinen JM, Peltola V, Koskinen JO. Advances in the diagnosis of acute human bocavirus infections. 25th European Congress of Clinical Microbiology and Infectious Diseases, Copenhagen, Denmark, 2015. Poster abstract P0329.

The authors
Juha M. Koskinen*^1,2 MSc, Andrea Bruning³ MD, Petri Susi⁴ PhD and Janne O. Koskinen² PhD
Directorate of Laboratory Medicine and Pathology, Royal Hospital, Muscat, Oman
¹Turku Doctoral Programme of Molecular Medicine, Department of Virology, University of Turku, Turku, Finland
²ArcDia International Oy Ltd, Turku, Finland
³Department of Pediatric Infectious Diseases, Emma Children’s Hospital, Academic Medical Center (AMC), Amsterdam, The Netherlands.
⁴Department of Virology, University of Turku, Turku, Finland

*Corresponding author
E-mail: jumako@utu.fi

New serological biomarkers for early detection of uterine cervical lesions and cervical cancer

, 26 August 2020/in Featured Articles /by 3wmedia

There are promising results regarding the use of antibodies for the diagnosis of cervical cancer (CC). This article reviews the antibody response against HPV proteins during the development of the disease as well as their possible use as biomarkers for the progression of cervical lesions and of CC.

by Dr D. A. Salazar-Piña, Berta A. Carrillo-Quiroz and Dr L. Gutierrez-Xicotencatl

Introduction
Cervical cancer (CC) has been a major public health problem among adult women, especially in developing countries. According to the WHO (Word Health Organization) GLOBOCAN project, in 2012 alone, there were more than 440 000 incident cases of CC and over 230 000 deaths due to the disease. Several studies have shown that high-risk (HR) human papillomavirus (HPV) types are important risk factors for the development of CC, and the most common types associated with this disease are HPV-16 and -18 [1].

Initial infection with HPV involves access of viral particles to the basal cell layer of the transform zone of the uterine cervix, which allows viral proliferation through differentiation of the stratified epithelium. During HPV infection, the virus uses cellular mechanisms of the host cell to express essential proteins (E1, E2, E4, E5, E6, E7) for the regulation of the cell cycle and replication of the viral DNA that is then encapsulated in viral particles formed by L1 and L2 capsid proteins [2]. For many years, several groups have studied the different viral proteins to understand the disease, which is caused by persistent infection with HR-HPV.

The HR-HPV types 16 and 18 mainly induce persistent infections without frequent serious complications for the host, and they are highly successful in releasing viral particles transmissible to others. This virus takes the host to a point of balance where the infection does not represent a serious drawback and viral replication is not limited by the host’s immune response because the virus does not have a blood-borne phase or viremia, which allows the HPV infection to persist for a longer period. Under these conditions, it takes a long time for the HPV infection to produce signs of damage to alert the immune system to generate an efficient response to eliminate the infection. Most of these HPV-associated genital lesions are cleared because of a successful cell-mediated immune response, during which cells of the innate immune system (such as keratinocytes, dendritic cells, Langerhans cells, macrophages, natural killer, and natural killer T cells) promote a pro-inflammatory process and eliminate the infection [3].

Diagnosis of cervical cancer
For several decades, actions against this public health problem have been taken in the aspects of prevention, diagnosis and treatment. The use of the Papanicolaou (Pap) smear, which is the primary diagnostic test in most cancer prevention programmes in developed countries, has helped to reduce the global burden of CC, but this has not been the case in developing countries. The main problem is the low and variable sensitivity of the Pap test (50–84%), which makes the identification of premalignant lesions difficult (Table 1) [4, 5]. Among the most frequent complications that the health sector faces is the lack of qualified personnel for sampling, transport, processing and proper evaluation, besides other limitations of the test. More recently, the HPV DNA test has emerged as a good candidate to replace the cytological test. The DNA test has a very high sensitivity for the detection of precancerous lesions (90–100%), but low specificity (47–80%), which makes this test suitable for screening [6]. However, the presence of HPV DNA is not indicative of an active infection; therefore, it has been necessary to develop new diagnostic systems to evaluate progression to CC. Hence, diagnostic systems of greater sensitivity and specificity are needed in order to detect the disease earlier and to prevent the development of CC.

In the newly developed tests for detecting early stages of the disease, the direct detection of HPV E6/E7 proteins, carried out in cell scrapes or cervical tissue samples, has shown low sensitivity as less than 1% of the cells are infected and express the oncogenic proteins. Another test is the surrogate marker p16 that is overexpressed in CC and is an indirect marker of the expression of the HPV E7 oncoprotein [7]. Although these tests have high specificity, unfortunately they still depend on a tissue sample or a cervical lavage, in which the number of HPV-infected cells is reduced (<1%), making it difficult to identify real patients at risk of developing CC [8].
Serological biomarkers for diagnosis of CC
There is, therefore, still a need to look for a test or a combination of tests that are highly sensitive and specific, and less invasive for the detection of early lesions of the uterine cervix that are progressing to CC. Thus, anti-HPV serum antibodies have become a good alternative new biomarker for the detection of CC-associated premalignant lesions. The humoral immune response is a naturally amplified system that allows the detection of low viral antigen concentrations in patients at risk to develop CC associated with persistent HPV infection. The potential of anti-HPV antibodies as biomarkers for CC is because the sequential expression of early HPV proteins in the uterine cervix correlates with the serological data and this might be useful for the identification of previous, current and persistent infections that could be related to the progression of the disease.

The specific antibody response against HPV antigens has been used, first as a method to study the biology of the HPV; later on, it was used to evaluate the efficacy of the new HPV vaccines, and more recently, a possible use as biomarkers of HPV-associated cancers at different anatomical sites has been proposed. Different groups have studied the antibody response against early and late HPV proteins and mixed results have been reported, a variability that could be attributed to the use of different type, purity and source of antigens, as well as the different assays used (Western blot, ELISA, multiplex, slot blot) (Table 2) [9]. However, there are promising studies that combine different viral antigens as new biomarkers for the early detection and progression of HPV-associated CC. For instance, antibodies against HPV16 E6/E7 proteins have predominantly been found in patients with advanced CC (75–80%), and they have been suggested as markers for CC [10]. Antibodies against the VLP L1 protein have shown to be useful to detect new HPV infections in women that initiate sexual life [11], as well as to detect women at risk of developing cervical intraepithelial neoplasia grade 3 (CIN3) and CC [12], or associated with the clearance of the HPV infection [13]. Similarly, anti-E4 antibodies have been associated with premalignant lesions (CIN1–2), and because the E4 protein has been implicated in early stages of viral replication, it has been suggested to be useful as an early marker for CIN1–2 lesions [14].

More recently, a new slot blot system has been used to analyse the presence of antibodies against E4, E7 and L1 proteins, and it was shown that anti-E4 and anti-E7 antibodies were highly associated with women with CC. The clinical performance of the slot blot system for the anti-E4 and/or anti-E7 antibodies was very good to discriminate CC from CIN2–3 with a high sensitivity (93.3%) and moderate specificity (64.1%). These findings suggest that these anti-E4 and anti-E7 antibodies could be used as biomarkers to distinguish pre-neoplastic lesions from CC [15]. Nevertheless, despite the scientific evidence, prospective studies need to be carried out to determine the usefulness of these antibodies as early markers and/or predictors of disease.

Conclusions
The diagnostic systems used for the detection of uterine cervical lesions, such as the Pap test and colposcopy, detect the disease at very late stages. The introduction of HPV DNA detection as a screening test has increased the identification of high-grade lesions, but it has still not been enough to distinguish an active infection that could progress to CC, especially because these premalignant lesions can undergo regression in around 70% of the cases. In order to increase the sensitivity and specificity of detecting women at risk of developing CC, a combination of tests has been suggested. In this scheme, the new serological biomarkers (anti-E4 and anti-E7 antibodies), which have shown to associate with CC and to discriminate it from premalignant lesions, could be used after or in co-testing with the Pap test to discriminate the false negatives of the Pap test from the real CC cases. It is expected that the combination of different tests such as the Pap test, HPV-DNA detection and the serological tests could help to detect over 85% of the CC cases that are missed when only one test is performed. More prospective studies with a larger panel of HPV antigens to evaluate other HPV antibodies need to be carried out to determine the usefulness of these antibodies as biomarkers for the detection and prediction of CC-associated premalignant lesions.

References
1. Faridi R, Zahra A, Khan K, Idrees M. Oncogenic potential of human papillomavirus (HPV) and its relation with cervical cancer. Virol J. 2011; 8: 269.
2. Doorbar J. The papillomavirus life cycle. J Clin Virol. 2005; 32(Suppl 1): S7–15.
3. Stanley MA, Sterling JC. Host responses to infection with human papillomavirus. Curr Probl Dermatol. 2014; 45: 58–74.
4. Hegde D, Shetty H, Shetty PK, Rai S. Diagnostic value of acetic acid comparing with conventional Pap smear in the detection of colposcopic biopsy-proved CIN. J Cancer Res Ther. 2011; 7(4): 454–458.
5. Gutiérrez-Xicotencatl L, De la Fuente-Villarreal D, Astudillo-de la Vega H. Biología molecular en el diagnóstico del cáncer cervicouterino asociado a pailomavirus humano. Gaceta Mexicana de Oncología 2014; 13(Supl 4): 25–32 (in Spanish).
6. Tao K, Yang J, Yang H, Guo ZH, Hu YM, Tan ZY, et al. Comparative study of the Cervista and hybrid capture 2 methods in detecting high-risk human papillomavirus in cervical lesions. Diagn Cytopathol. 2014; 42(3): 213–217.
7. Carozzi F, Gillio-Tos A, Confortini M, Del Mistro A, Sani C, De Marco L, Girlando S, Rosso S, Naldoni C, et al. NTCC working group. Risk of high-grade cervical intraepithelial neoplasia during follow-up in HPV-positive women according to baseline p16-INK4A results: a prospective analysis of a nested substudy of the NTCC randomised controlled trial. Lancet Oncol. 2013; 14(2): 168–176.
8. Gutierrez-Xicotencatl L, Plett-Torres T, Madrid-Gonzalez CL, Madrid-Marina V. Molecular diagnosis of human papillomavirus in the development of cervical cancer. Salud Publica Mex. 2009; 51(Suppl 3): S479–488.
9. Gutierrez-Xicotencatl L, Salazar-Pina DA, Pedroza-Saavedra A, Chihu-Amparan L, Rodriguez-Ocampo AN, Maldonado-Gama M, Esquivel-Guadarrama FR. Humoral immune response against human papillomavirus as source of biomarkers for the prediction and detection of cervical cancer. Viral Immunol. 2016; 29(2): 83–94.
10. Jochmus I, Bouwes Bavinck JN, Gissmann L. Detection of antibodies to the E4 or E7 proteins of human papillomaviruses (HPV) in human sera by western blot analysis: type-specific reaction of anti-HPV 16 antibodies. Mol Cell Probes 1992; 6(4): 319–325.
11. Kjaer SK, van den Brule AJ, Paull G, Svare EI, Sherman ME, Thomsen BL, Suntum M, Bock JE, Poll PA, Meijer CJ. Type specific persistence of high risk human papillomavirus (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: population based prospective follow up study. BMJ 2002; 325(7364): 572.
12. Wang ZH, Kjellberg L, Abdalla H, Wiklund F, Eklund C, Knekt P, Lehtinen M, Kallings I, Lenner P, et al. Type specificity and significance of different isotypes of serum antibodies to Human Papillomavirus capsids. J Inf Dis. 2000; 181(2): 456–462.
13. Bontkes HJ, de Gruijl TD, Walboomers JM, Schiller JT, Dillner J, Helmerhorst TJ, Verheijen RH, Scheper RJ, Meijer CJ. Immune responses against human papillomavirus (HPV) type 16 virus-like particles in a cohort study of women with cervical intraepithelial neoplasia. II. Systemic but not local IgA responses correlate with clearance of HPV-16. J Gen Virol. 1999; 80(Pt 2): 409–417.
14. Vazquez-Corzo S, Trejo-Becerril C, Cruz-Valdez A, Hernandez-Nevarez P, Esquivel-Guadarrama R, Gutierrez-Xicotencatl Mde L. [Association between presence of anti-Ras and anti-VPH16 E4/E7 antibodies and cervical intraepithelial lesions]. Salud Publica Mex. 2003; 45(5): 335–345 (in Spanish).
15. Salazar-Pina DA, Pedroza-Saavedra A, Cruz-Valdez A, Ortiz-Panozo E, Maldonado-Gama M, Chihu-Amparan L, Rodriguez-Ocampo AN, Orozco-Fararoni E, Esquivel-Guadarrama F, Gutierrez-Xicotencatl L. Validation of serological antibody profiles against human papillomavirus type 16 antigens as markers for early detection of cervical cancer. Medicine 2016; 95(6): e2769.
16. Pedroza-Saavedra A, Cruz A, Esquivel F, De La Torre F, Berumen J, Gariglio P, Gutiérrez L. High prevalence of serum antibodies to Ras and type 16 E4 proteins of human papillomavirus in patients with precancerous lesions of the uterine cervix. Arch Virol. 2000; 145(3): 603–623.

The authors
D. Azucena Salazar-Piña¹ PhD, Berta A. Carrillo-Quiroz² MSc, Lourdes
Gutierrez-Xicotencatl*³ PhD
¹School of Nutrition, Autonomous
University of Morelos State (UAEM),
Cuernavaca, Morelos, Mexico
²Center of Information for Decisions in Public Health, National Institute of Public Health, Cuernavaca, Morelos, Mexico
³Center for Research on Infectious Diseases, National Institute of Public Health,
Cuernavaca, Morelos, Mexico

*Corresponding author
E-mail: mlxico@insp.mx

Pediatric reference intervals: tailoring reference intervals to the target population

, 26 August 2020/in Featured Articles /by 3wmedia

The use of suitably matched reference values derived from well-characterized individuals is critical to avoid misdiagnosis. Developing reference intervals in pediatric populations presents unique challenges. Recognizing these issues and bridging international biorepository efforts is essential to improving pediatric healthcare.

by Dr Carmen Gherasim, Sonia L. La’ulu, Sara P.Wyness and Dr Joely A. Straseski

Introduction
Children are unique individuals with dynamic developmental physiology which must be accounted for during clinical evaluation. In addition to a medical history and clinical examination, laboratory investigations constitute an integral part of diagnosis and therapeutic management. Understandably, children have limited ability to describe symptoms and medical providers must rely strongly on clinical and laboratory investigations. The availability of pediatric reference intervals (PRI) is, therefore, essential to avoid missed opportunities for treatment of preventable conditions and adverse consequences due to wrong diagnosis.

Depending on the analyte, PRI can differ substantially from RI in adult populations. Marked differences can even be observed within the pediatric population, with profound changes occurring during timeframes such as the first year of life or puberty. The dynamic process of growth, from the initial adaptation of infants living outside the womb to full sexual maturation associated with adulthood, is accompanied by changes in body composition and metabolic, immune, and hormonal fluctuations [1]. Furthermore, developmental stages in children, particularly during puberty, do not always correlate with age and can be affected by factors including nutritional status, body mass index (BMI), medications, or therapies (i.e. growth hormone therapy) [2]. It is therefore advantageous to develop PRI using clearly defined demographics such as age, sex, and race, along with stratifications accounting for physiological and sexual development to assist in clinical decision making.

From a clinical laboratory perspective, the dynamics of the sampled population along with regulatory and administrative requirements for determining PRI pose unique challenges for establishing high quality RI for pediatric populations [3]. Such challenges include: (1) defining ‘healthy’ in children; (2) obtaining research ethics board approval for sample collections; (3) obtaining informed parental and/or child consent for participation; (4) accommodating small sample volumes; and (5) partitioning of data. Collectively, these can hamper access to a sufficient number of specimens from healthy children, precluding many laboratories from performing PRI studies.

Origination of PRI
While RI studies may be performed by in vitro diagnostic manufacturers for commercially available assays, these often lack pediatric data or have minimal stratification reflecting children’s physiological variability. Although not unique to the pediatric population, recycling of RI determined using obsolete methods/instrumentation or adoption of RI from previously published sources without study traceability are often used to report and interpret laboratory data. Caveats in PRI adoption include the limited number of studies performed in pediatric populations, inclusion of data from hospitalized patients, data analysis techniques used, and lack of detailed information from existing studies including population tested and certain patient demographics. Therefore, careful examination of subjects and methods used in existing studies is critical.

Given the complexity of establishing new PRI, clinical laboratories should carefully consider the option to: (1) transfer, (2) verify or (3) establish PRI based on requirements addressed in guidelines (e.g. Clinical and Laboratory Standards Institute (CLSI) EP28-A3c) [4]. When a laboratory changes analytical methods for measuring an analyte for which they have previously established a RI, that RI may be ‘transferred’ following an acceptable method comparison study. This strategy may prove particularly useful when obtaining pediatric specimens is difficult. Other pre-determined RI must be ‘verified’ before their adoption by examining whether results from a minimum of 20 healthy individuals fall between the proposed limits. Finally, ‘establishing’ new RI requires examining a minimum of 120 samples for each statistically distinct group (partition) selected from qualified, healthy individuals chosen using well-defined inclusion/exclusion criteria (Fig. 1). This is an enormous effort, but particularly onerous in pediatric populations due to limited samples and numerous partitions. Due to this, routine clinical laboratories are often limited to verifying rather than establishing PRI.

Selection of reference populations for PRI
Ideally, reference studies should be conducted using ‘healthy’ volunteers but the identification and recruitment of a healthy representative pediatric population is complex. Health status of recruited children may be assessed by physical examination and health-related questionnaires; however, a definitive delineation of ‘healthy’ is challenging in pediatric populations, as subclinical problems may go unrecognized. Obtaining a sufficient number of pediatric specimens from truly healthy children remains a rate-limiting step of this process.

One strategy that circumvents this challenge is the use of residual blood samples from hospitalized children, deemed healthy due to unrelated or inconsequential medical conditions. This approach eliminates the undesirable blood collection procedure but can be affected by the lack of medical history that can influence test results and overall quality of the PRI established. Additionally, data mining methods exploiting laboratory results from hospitalized children can be used. First introduced by Robert Hoffmann, this approach assumes that the majority of specimens measured in the clinical laboratory represent ‘normal’ values, whereas the most extreme values account for the sickest populations [5].
The effect of common physiological variables such as age and sex on pediatric biomarkers is often evaluated using specific RI partitions. Complex factors such as BMI, Tanner staging, and race may also influence the concentration of select analytes. Despite the rising incidence of pediatric obesity in many developed countries, the effect of BMI on PRIs has been largely overlooked [6]. Challenges in designing studies to address this issue include susceptibility of analytes to BMI variability and delineation between BMI and changes in body composition throughout development. Also, physical and pubertal development in children is not always parallel. Surges in hormone concentrations trigger sexual development and their concentrations vary with the presence of secondary sexual characteristics (described by Tanner stages) rather than age. Finally, race-specific differences can also influence concentrations of pediatric analytes and covariates such as BMI, but their contribution could be underestimated due to their multifactorial etiology.

Statistical methods for PRI analysis
Laboratory data is interpreted in the context of RI, which typically describe the central 95% of results from a population of healthy volunteers. One particular concern with PRI is the availability of sufficient numbers of specimens to allow for statistical significance of each partition [7]. Selection of an appropriate statistical method to compute RI is dependent on the number of specimens in each partition and overall distribution of the data. A small number of pediatric specimens can result in sampling variability which decreases the confidence that a normal result will fall in the established RI. General statistical methods can be employed including parametric (smaller specimen numbers following Gaussian/normal distribution) and nonparametric methods (minimum 120 reference observations per partition). Current CLSI guidelines recommend the use of nonparametric approaches for estimation of RI as they do not make assumptions regarding the distribution of the data [4]. Despite the easy access to data, assumptions regarding data distribution and weak correlation studies often reveal that Hoffmann statistical approaches may be unreliable for establishing new RI in pediatric populations [8]. Increasing the statistical power of PRI determinations and understanding the effect of covariates remains challenging due to the large number of specimens required that can only be reasonably addressed in multicentre RI initiatives.

Current studies emphasizing the need for PRI
The importance of well-defined PRI is highlighted by our recent studies investigating a number of analytes with different concentrations in pediatric populations as compared to adults. A valuable component of our studies is the use of large numbers of well-characterized subjects selected using clearly defined inclusion/exclusion and partition criteria. Whereas some analytes show significant differences between age and sexes (bone markers osteocalcin, procollagen type 1N-terminal propeptide, bone-specific alkaline phosphatase, and C-telopeptide; thyroglobulin and free triiodothyronine), others do not (free thyroxine) [9–11]. PRI for 5α-dihydrotestosterone identified significant differences between sexes and Tanner stages [12]. These differences highlight the need for PRI for individual analytes and the large dataset for each partition in these studies was critical for appropriate data analysis and delineation of proper PRI. As the majority of clinical laboratories verify rather than establish PRI, reported studies should be carefully reviewed for study design, selection of reference individuals, methods used, analytical quality, and appropriate statistical analysis of the data before being considered for PRI adoption.

PRI initiatives
There are a number of initiatives around the world striving to improve the quality and accuracy of PRI (Table 1). Their major advantage is the recruitment of large cohorts of healthy children and adolescents using well-defined selection and partition criteria. Understandably, recruitment strategies vary between initiatives but generally include soliciting members of local communities, organizing clinics at schools and community centres, and/or enrolment prior to undergoing elective, non-invasive, outpatient surgeries. Although each initiative has focused on different biomarkers, most focus on common biochemical markers, blood analytes, vitamins, and/or hormones [13–16]. All studies included partitions for pediatric populations by age and sex and most reference values were analysed using nonparametric statistics to define central 95% PRI. Additional covariates such as Tanner staging, race, or BMI were addressed only by select initiatives including CHILDx, CALIPER and IDEFICS, respectively. A shortcoming of many of the studies was the relatively small representation of other races. Multicentre studies could address this problem, thereby promoting harmonization and diversity amongst PRI. In a recent position statement, the American Association for Clinical Chemistry expressed their support for the foundation of a national repository, enabling a comprehensive evaluation of all PRI covariates and provide and maintain up-to-date PRI databases [17].

Concluding remarks
Reference intervals for pediatric populations weigh heavily in the interpretation of laboratory results and can impact the outcome of clinical decisions. In recent years, we have witnessed an increased awareness of the ‘malpractices’ in PRI including adoption from suboptimal RI studies or from populations that do not mirror the healthy state in children. Recommendations to establish individual PRI that adequately represent the numerous variables in children’s development remain hard to meet by most laboratories, reinforcing a need for a collective effort in establishing PRI. Concentrating national and international efforts can support PRI initiatives and improve pediatric healthcare overall.

References
1. Coffin CM, Hamilton MS, Pysher TJ, Bach P, Ashwood E, Schweiger J, Monahan D, Perry D, Rogers BB, et al. Pediatric laboratory medicine: current challenges and future opportunities. Am J Clin Pathol 2002; 117(5): 683-690.
2. Sikaris KA. Physiology and its importance for reference intervals. Clin Biochem Rev 2014; 35(1): 3-14.
3. Tahmasebi H, Higgins V, Fung AWS, Truong D, White-Al Habeeb NMA, Adeli K. Pediatric reference intervals for biochemical markers: gaps and challenges, recent national initiatives and future perspectives. EJIFCC 2016; 28(1): 43–63.
4. Defining, establishing, and verifying reference intervals in the clinical laboratory. Approved guideline-third edition. CLSI document EP28-A3c. Clinical Laboratory Standards Institute 2008.
5. Hoffmann RG. Statistics in the practice of medicine. JAMA 1963; 185: 864–873.
6. Erhardt E, Foraita R, Pigeot I, Barba G, Veidebaum T, Tornaritis M, Michels N, Eiben G, Ahrens W, et al. Reference values for leptin and adiponectin in children below the age of 10 based on the IDEFICS cohort. Int J Obes (Lond) 2014; 38 Suppl 2: S32–38.
7. Daly CH, Liu X, Grey VL, Hamid JS. A systematic review of statistical methods used in constructing pediatric reference intervals. Clin Biochem 2013; 46(13–14): 1220–1227.
8. Shaw J, Cohen A, Konforte D, Binesh-Marvasti T, Colantonio DA, Adeli K. Validity of establishing pediatric reference intervals based on hospital patient data: a comparison of the modified Hoffmann approach to CALIPER reference intervals obtained in healthy children. Clin Biochem 2014; 47(3): 166–172.
9. Wyness SP, Roberts WL, Straseski JA. Pediatric reference intervals for four serum bone markers using two automated immunoassays. Clin Chim Acta 2013; 415: 169–172.
10. Owen WE, Bunker AM, Straseski JA. Pediatric reference intervals for thyroglobulin using the Beckman Coulter Access 2 immunoassay. Clin Chim Acta 2014; 435: 40–41.
11. La’ulu SL, Rasmussen KJ, Straseski JA. Pediatric reference intervals for free thyroxine and free triiodothyronine by equilibrium dialysis-liquid chromatography-tandem mass spectrometry. J Clin Res Pediatr Endocrinol 2016; 8(1): 26–31.
12. Lin DC, Straseski JA. Tanner stage-stratified pediatric reference intervals for dihydrotestosterone [Abstract]. Clin Chem 2016; 62(10): S188.
13. Colantonio DA, Kyriakopoulou L, Chan MK, Daly CH, Brinc D, Venner AA, Pasic MD, Armbruster D, Adeli K. Closing the gaps in pediatric laboratory reference intervals: a CALIPER database of 40 biochemical markers in a healthy and multiethnic population of children. Clin Chem 2012; 58(5): 854–868.
14. Kohse KP, Thamm M. KiGGS-the German survey on children’s health as data base for reference intervals. Clin Biochem 2011; 44(7): 479.
15. Kant AK, Graubard BI. Race-ethnic, family income, and education differentials in nutritional and lipid biomarkers in US children and adolescents: NHANES 2003-2006. Am J Clin Nutr 2012; 96(3): 601–612.
16. Ridefelt P. Population-based pediatric reference intervals in general clinical chemistry: a Swedish survey. J Med Biochem 2015; 34(1): 64–65.
17. Pediatric lab results: the need for “normal.” AACC Position Statement. AACC 2016; 1–3.

The authors
Carmen Gherasim¹ PhD, Sonia L. La’ulu² BS, Sara P. Wyness² BA, and Joely A. Straseski*^1,2 PhD
¹Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA
²ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA

*Corresponding author
E-mail: joely.a.straseski@aruplab.com

Scientific literature review

, 26 August 2020/in Featured Articles /by 3wmedia

There 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.

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Free 25OH Vitamin D Elisa

, 26 August 2020/in Featured Articles /by 3wmedia

Orion GenRead Campylobacter

, 26 August 2020/in Featured Articles /by 3wmedia

SCIENTIFIC LITERATURE REVIEW: Colorectal cancer

, 26 August 2020/in Featured Articles /by 3wmedia

Highly sensitive stool DNA testing of Fusobacterium nucleatum as a marker for detection of colorectal tumours in a Japanese population

Suehiro Y, Sakai K, Nishioka M, Hashimoto S, Takami T, Higaki S, Shindo Y, Hazama S, Oka M, Nagano H, Sakaida I, Yamasaki T. Ann Clin Biochem. 2016; pii: 0004563216643970. [Epub ahead of print]

BACKGROUND: Accumulating evidence shows an over-abundance of Fusobacterium nucleatum in colorectal tumour tissues. Although stool DNA testing of Fusobacterium nucleatum might be a potential marker for the detection of colorectal tumours, the difficulty in detecting Fusobacterium nucleatum in stool by conventional methods prevented further explorations. Therefore, we developed a droplet digital polymerase chain reaction (PCR) assay for detecting Fusobacterium nucleatum in stool and investigated its clinical utility in the management of colorectal tumours in a Japanese population.
METHODS: Feces were collected from 60 healthy subjects (control group) and from 11 patients with colorectal non-advanced adenomas (non-advanced adenoma group), 19 patients with colorectal advanced adenoma/carcinoma in situ (advanced adenoma/carcinoma in situ (CIS) group) and 158 patients with colorectal cancer of stages I to IV (colorectal cancer group). Absolute copy numbers of Fusobacterium nucleatum were measured by droplet digital PCR.
RESULTS: The median copy number of Fusobacterium nucleatum was 17.5 in the control group, 311 in the non-advanced adenoma group, 122 in the advanced adenoma/CIS group, and 317 in the colorectal cancer group. In comparison with that in the control group, the Fusobacterium nucleatum level was significantly higher in the non-advanced adenoma group, the advanced adenoma/CIS group and the colorectal cancer group.
CONCLUSIONS: This study illustrates the potential of stool DNA testing of Fusobacterium nucleatum by droplet digital PCR to detect individuals with colorectal tumours in a Japanese population.

A genome-wide assessment of variations of primary colorectal cancer maintained in metastases

Cai Z, Han S, Li Z, He L, Zhou J, Huang W, Xu Y. Gene 2016; 595(1): 18–24.

Colorectal cancer (CRC) is a highly heterogeneous disease that is the third leading cause of cancer-related deaths worldwide. This study presents a genome-wide assessment of variations in primary colorectal cancer maintained in metastases, even in distant metastases. The purpose of this study was to determine whether intratumor heterogeneity is related to disease progression and metastasis in CRC. The results showed that 882 single nucleotide polymorphism (SNP) associated genes and 473 copy number variant (CNV) associated genes specific to metastasis were found. In addition, 57 SNPs mapped to miRNAs showed significant differences between primary tumours and metastases. Functional annotation of metastasis-specific genes suggested that adhesion and immune regulation may be essential in the development of tumours. Moreover, the locus rs12881063 in the fourteenth chromosome was found to have a high rate of the G/C type in metastases. The rate of the G/C type in nearby lymph node metastases was 66.7%, while the rate of the G/C type in distance lymph node metastases was 83.3%. These results indicate that rs12881063 may be the basis for clinical diagnosis of CRC metastasis.

High tumour mast cell density is associated with longer survival of colon cancer patients

Mehdawi L, Osman J, Topi G, Sjölander A. Acta Oncol. 2016; 55(12): 1434–1442.

BACKGROUND: Inflammatory cells and inflammatory mediators play an important role in colorectal cancer (CRC). Previous studies have shown that CRC patients with increased expression of cysteinyl leukotriene receptor 1 (CysLTR1) have a poorer prognosis, and Cysltr1-/- mice display fewer intestinal polyps. However, the role of mast cells (MCs) in colon cancer progression remains unclear. The aim of the present study was to explore the relevance of MCs in CRC.
MATERIAL AND METHODS: A tissue microarray from 72 CRC patients was stained with MC anti-tryptase and -chymase antibodies. Mouse colon tissue was stained with MC anti-tryptase antibody. Immunohistochemistry was used to identify MCs in patients and mice.
RESULTS: Patient colon cancer tissue had in comparison with normal colon tissue a reduced number of MCs, predominantly of chymase-positive cells. Further analysis revealed that patients with a relative high MCD in their cancer tissues showed significantly longer overall survival compared to those with a low MCD [hazard ratio (HR) 0.539; 95% confidence interval (CI), 0.302–0.961]. Similar results were observed in subgroups of patients with either no distant metastasis (p = 0.004), or <75 years (p = 0.015) at time of diagnosis. Multivariate Cox analysis showed that MCD independently correlated with reduced risk of death in colon cancer patients (HR 0.380; 95% CI 0.202-0.713). Additionally, a negative correlation was found between cytoplasmic CysLTR1 expression and number of MCs. In agreement, in the CAC mouse model, Cysltr1^-/- mice showed significantly higher MCs in their polyp/tumor areas compared with wild-type mice.
CONCLUSION: A high MCD in cancer tissue correlated with longer patient survival independently from other risk factors for CRC. The concept that MCs have an anti-tumor effect in CRC is further supported by the findings of a negative correlation with CysLTR1 expression in patients and a high MCD in colon polyps/tumors from CysLTR^1-/- mice.

Are hemorrhoids associated with false-positive fecal immunochemical test results?

Kim NH, Park JH, Park DI, Sohn CI, Choi K, Jung YS. Yonsei Med J. 2016; 58(1):150–157.

PURPOSE: False-positive (FP) results of fecal immunochemical tests (FITs) conducted in colorectal cancer (CRC) screening could lead to performing unnecessary colonoscopies. Hemorrhoids are a possible cause of FP FIT results; however, studies on this topic are extremely rare. We investigated whether hemorrhoids are associated with FP FIT results.
MATERIALS AND METHODS: A retrospective study was conducted at a university hospital in Korea from June 2013 to May 2015. Of the 34,547 individuals who underwent FITs, 3946 aged ≥50 years who underwent colonoscopies were analysed. Logistic regression analysis was performed to determine factors associated with FP FIT results.
RESULTS: Among 3946 participants, 704 (17.8%) showed positive FIT results and 1303 (33.0%) had hemorrhoids. Of the 704 participants with positive FIT results, 165 had advanced colorectal neoplasia (ACRN) and 539 had no ACRN (FP results). Of the 1303 participants with hemorrhoids, 291 showed FP results, of whom 81 showed FP results because of hemorrhoids only. Participants with hemorrhoids had a higher rate of FP results than those without hemorrhoids (291/1176, 24.7% vs. 248/2361, 10.5%; p<0.001). Additionally, the participants with hemorrhoids as the only abnormality had a higher rate of FP results than those experiencing no such abnormalities (81/531, 15.3% vs. 38/1173, 3.2%; p<0.001). In multivariate analysis, the presence of hemorrhoids was identified as an independent predictor of FP results (adjusted odds ratio, 2.76; 95% confidence interval, 2.24-3.40; p<0.001).
CONCLUSION: Hemorrhoids are significantly associated with FP FIT results. Their presence seemed to be a non-negligible contributor of FP results in FIT-based CRC screening programmes.

Clinical utility of PIVKA-II in the diagnosis of hepatocellular carcinoma

, 26 August 2020/in Featured Articles /by 3wmedia

The need for cost-effective and reliable early diagnostic tools for hepatocellular carcinoma (HCC) is more than ever of great interest, as incidence rates are on the rise globally and two thirds of the patients are diagnosed with HCC in advanced stages. Alpha-fetoprotein (AFP), the marker that is currently in widespread use for HCC, lacks sensitivity and specificity. Protein induced by vitamin K absence/antagonist-II (PIVKA-II), a novel tumour marker, has been shown to be of good sensitivity and specificity for the diagnosis of HCC.

by Volha Klimovich, Kieran Voong, Prof. Roy Sherwood and Dr Dominic J. Harrington

Introduction
Primary 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

Molecular detection and quantification of human rhinoviruses

, 26 August 2020/in Featured Articles /by 3wmedia

The sequence diversity of the more than 150 currently recognized HRV genotypes poses challenges for the development of robust molecular methods that detect all genotypes with equal efficiency. Real-time reverse-transcription (RT)-PCR was compared to digital RT-PCR for quantification of HRV in clinical specimens when using type-specific and consensus primers and probes.

by Dr Jane Kuypers

Introduction
Human rhinoviruses (HRV) are small, positive-sense RNA viruses within the family Picornaviridae. Over 150 genotypes of this important human pathogen have been recognized within species HRVA, HRVB, and HRVC of the genus Enterovirus (http//:www.picornaviridae.com). HRV infections occur throughout the year and throughout the world. HRV are responsible for a high incidence and wide range of respiratory infections in all populations, including one-half to two-thirds of all common colds and many cases of otitis media and sinusitis in the upper respiratory tract. Lower tract infections include bronchiolitis, pneumonia and exacerbation of disease in children with asthma and cystic fibrosis, and in adults with chronic obstructive pulmonary disease. Cases of serious illness and even death due to HRV have been reported, especially in immunocompromised individuals, the elderly and infants [1, 2].

Laboratory detection of HRV is important for diagnosis and surveillance, especially in high risk populations. HRV are frequently detected as co-infections with other respiratory viruses and in individuals with long-term and asymptomatic shedding [3]. In addition to qualitative detection, accurate quantification of HRV RNA in clinical samples is needed for studies on the association of HRV viral load with viral transmission and with patient symptoms and outcomes. Viral-load studies of other respiratory viruses have shown that a correlation exists between quantity of virus and disease severity. HRV viral-load determinations may also be important for patient management, especially in asymptomatic patients who test positive for HRV at low levels. More importantly, accurate HRV viral-load assessments will be necessary for evaluating the performance of potential HRV antiviral drugs [4].

Detection
HRV were initially detected by growth in cell culture. Approximately 100 serotypes of HRV grown in cell culture were antigenically characterized by their reactions with various antisera. The serotypes were subsequently classified into two groups, A and B, according to their sensitivity towards antivirus agents [5] and are now included in HRV species A (80 genotypes) and B (32 genotypes) based on genetic sequencing. Cell culture is sensitive for detection of many, but not all HRV genotypes; 55 HRV that do not grow in the cell culture lines normally used in the clinical laboratory and have been detected only by molecular methods are classified in HRV species C (http//:www.picornaviridae.com).

The use of molecular methods for the detection of HRV in clinical specimens has provided more accurate information about the disease burden and epidemiology of these ubiquitous viruses. The molecular method most often used to detect HRV is real-time reverse-transcription (RT)-PCR [3]. RT-PCR assays, when accompanied by amplification of serially diluted standards of known RNA copy numbers (RT-qPCR), can be used to quantify the number of viral copies in a sample. By comparing the PCR Ct value (the PCR cycle at which fluorescence reaches a certain threshold) of a clinical specimen to the standard curve, the relative quantity of the analyte can be calculated [6].
Within the HRV genome, the region most frequently targeted for RT-PCR by clinical assays is the 5’ non-coding region (NCR), which exhibits the most sequence homology among the HRV genotypes. However, even in this region, there is a lot of sequence diversity, which makes it challenging to design a single, consensus PCR primer and probe set to amplify all HRV genotypes with equal efficiency. In order to amplify HRV genotypes with diverse sequences in the prime/probe binding regions, consensus PCR primer and probe sets have been designed with degenerate and modified bases or multiple oligonucleotides [7–10]. However, consensus RT-qPCR assays may not give accurate quantitative results for all HRV genotypes due to amplification inefficiency caused by base mismatches between the consensus primers and probe and the viral sequences [11].

Quantitation by RT-qPCR
To determine if a consensus RT-qPCR assay [7] could be used to accurately quantify all genotypes of HRV, including those with sequence differences in the primer and probe binding regions, we compared the efficiency and sensitivity of a consensus RT-qPCR assay to that of genotype-specific RT-qPCR assays [4]. In Figure 1(a), the results of RT-qPCR assays using type-specific primers and probes, which exactly match the target sequences, show standard curves indicating accurate and sensitive quantification of RNA transcripts from six specific HRV genotypes. However, RT-qPCR using a consensus HRV primer and probe set did not give accurate or sensitive quantification for some HRV genotypes, especially types A33 and A88 (Fig. 1b). RNA from HRV genotypes with base mismatches between the consensus primer and probe sequences and the specific viral sequences was inaccurately quantified using the consensus assay, most likely due to poor amplification efficiency.

Quantitation by RT-dPCR
Digital RT-PCR (RT-dPCR), which provides absolute nucleic acid quantification without the need for PCR Ct values and standard curves and is less affected by poor amplification efficiency, may perform better than RT-qPCR for quantification of HRV RNA. In dPCR, an amplification reaction, which contains fluorescent dye to measure amplification, is divided into 12?000 to 200?000 independent partitions, each ideally containing no more than one target molecule. The reaction is amplified to end point and the number of fluorescent (positive) and non-fluorescent (negative) partitions is counted. In specimens with more targets than partitions, Poisson statistics are used to calculate the average number of targets per positive partition and thus, the number of targets in the original sample [12, 13]. Compared to qPCR, dPCR is less susceptible to amplification inefficiency caused by primer/probe sequence mismatches because quantification derives from a PCR reaction that cycles to endpoint rather than from an amplification curve as in qPCR. Accurate quantification by dPCR is also not dependent on a well-calibrated standard [14]. These characteristics make dPCR especially useful for quantifying viral targets with many subtypes and high sequence diversity that leads to mismatches between targets and PCR primer and probe sequences, such as HRV.
To determine if consensus RT-dPCR would perform better than consensus RT-qPCR for quantification of HRV genotypes, we similarly tested RNA transcripts of HRV genotypes, including some with sequence variation in the consensus primer and probe binding region, by RT-dPCR using both type-specific and consensus primers and probes. In Figure 2(a), the results of RT-dPCR assays using type-specific primers and probes show good correlations between the expected number of RNA copies/reaction and the observed number. When amplified by RT-dPCR using the consensus assay (Fig. 1b), in contrast to RT-qPCR, the observed number of RNA copies/reaction was also closely correlated with the expected number for most of the HRV genotypes tested.

In a previous study [4], data from 16 HRV genotypes that represented the consensus primer and probe binding sequences of 128 genotypes indicated that, when using consensus primers and probe, RT-dPCR quantification of HRV RNA was more accurate than that of RT-qPCR for some genotypes. We found that although the consensus RT-qPCR did accurately quantify many HRV genotypes, it did not accurately quantify all genotypes of HRV due to sub-optimal amplification of genotypes with sequences that do not exactly match those of the primers and probe. Consensus RT-dPCR, however, did not overcome all sequence mismatch-induced amplification inefficiency, as evidenced by genotype A88 (Fig. 2b), which has a single mismatch near the middle of the forward primer.

Although RT-dPCR has been shown to be more accurate than RT-qPCR for quantification of HRV and may be applicable to other viruses with high sequence diversity, like HIV and HBV, it has some disadvantages for routine use in a clinical laboratory. RT-dPCR has a more limited dynamic range compared to RT-qPCR (104 for RT-qPCR compared to 108 for RT-qPCR), which would require dilution and retesting of samples with high viral loads. Running an RT-dPCR assay requires more hands-on technician time and has a lower throughput than current RT-qPCR assays. Digital PCR instruments and reagents are also currently more expensive than most qPCR systems.

Conclusion
In conclusion, dPCR was a better alternative to qPCR on RNA templates known to have significant sequence diversity that cannot be avoided during primer and probe design and should be considered the better molecular method for quantification of HRV in respiratory specimens.

References
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The author
Jane Kuypers PhD
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

E-mail: kuypers@uw.edu

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