Featured Articles

Simple, fast and highly sensitive colorimetric detection of Zika virus

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

Zika virus (ZIKV) has recently emerged as a global threat, but current diagnostic techniques are cumbersome and time-consuming. We developed a simple, fast and highly sensitive ZIKV molecular detection method. Within 35 min, we could detect even a single copy of ZIKV RNA by merely observing a colorimetric signal with the naked eye.

by Dohwan Lee, Dr Yong Kyoung Yoo and Prof. Jeong Hoon Lee

Background
An outbreak of Zika virus (ZIKV) in Brazil terrorized the whole world and its explosive spread in the Americas caused the World Health Organization (WHO) to declare it a public health emergency of international concern in February 2016 [1]. This is because ZIKV is a suspected major cause of congenital microcephaly, Guillain-Barré syndrome and other neurologic syndromes [2–4]. ZIKV has a genome consisting of a single-stranded, positive-polarity RNA and belongs to the family Flaviviridae and the genus Flavivirus. Aedes mosquitoes, known as a major ZIKV vector, also transmit dengue and chikungunya viruses across tropical and subtropical regions around the world [5]. Moreover, antigenic similarity between ZIKV and dengue virus gives rise to serological cross-reactivity, precluding antibody-based assays from reliably distinguishing between ZIKV and dengue virus infections [6]. Thus, reliable methods for distinguishing ZIKV from dengue and chikungunya viruses are necessary in practical applications.

WHO target product profiles
In April 2016, the WHO announced Target Product Profiles (TPPs) for a better diagnostic test for ZIKV infection. The TPPs define the desired characteristics of a ZIKV diagnostic test. The proposed TPPs consist of ‘Detection of active infection with ZIKV’ (Table 1a) and ‘Detection of evidence of prior infection’ (Table 1b). Each characteristic in the tables represents essential properties that the newly developed ZIKV diagnostic test should have at least at an acceptable level. To state the obvious, the criteria of specificity for active infection are more stringent [7].

Previous research on ZIKV diagnostics
Due to serological cross-reactivity between ZIKV and other flaviviruses, most of previous studies on ZIKV diagnosis have dealt with molecular diagnostics instead of immunological assays. Faye and colleagues developed and evaluated a one-step reverse transcription (RT)-PCR assay for ZIKV detection. The limit of detection of the assay was found to be 7.7 plaque-forming units (p.f.u.) per reaction in human serum and in the L-15 medium [8]. A quantitative real-time RT-PCR assay for ZIKV was also developed by the same research group. Analytical sensitivity of the assay was estimated at 3.2×10² RNA copies/μL [9]. However, a conventional PCR assay requires a bulky and expensive thermal cycler, prolonged reaction time, and trained technicians; these resources are not available in many low- and middle-income countries. Moreover, the RT-PCR reaction is vulnerable to inhibitors (blood, plasma and urine), thus requiring painstaking and cumbersome RNA extraction steps.

Recent research on ZIKV diagnostics
To overcome such limitations of RT-PCR, a variety of isothermal nucleic acid amplification techniques have recently been developed. Among them, reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid, robust, and highly sensitive isothermal RNA amplification method that uses four to six primers to amplify specific RNA sequences at 60–65°C even in the presence of inhibitors such as blood, plasma, or urine. RT-LAMP is much faster than conventional PCR, and the reaction can even proceed in an oven, water bath or with heating packs [10, 11]. Despite these advantages, the RT-LAMP assays still rely on a conventional bulky amplicon analyser such as a gel electrophoresis apparatus or a fluorescence laser-induced detector for monitoring the LAMP amplicons; this situation precludes the use of RT-LAMP in point-of-care diagnosis.

Our approaches to simple and highly sensitive diagnosis of ZIKV
To eliminate the dependence on a conventional amplicon analyser while retaining the aforementioned advantages of RT-LAMP, we selected the lateral flow assay (LFA) format for RT-LAMP amplicon analysis. The LFA, a driving principle behind pregnancy test strips, is also widely known as a superior diagnostic tool for nucleic acids owing to its high sensitivity, simplicity, selectivity and easy interpretation of results. Moreover, the Bst 3.0 polymerase used in this study for RT-LAMP retains both improved isothermal amplification performance and strong reverse transcription activity, allowing us to avoid addition of exogenous reverse transcriptase and the inhibition of reverse transcription by biological substances. By utilizing the advantages of Bst 3.0 polymerase and combining the RT-LAMP assay with the LFA, we demonstrated simple and highly sensitive detection of ZIKV RNA in human whole blood by merely observing a colorimetric signal within 35 min.

The RT-LAMP reaction and modification of amplicons in our study
As mentioned above, RT-LAMP has excellent tolerance to many inhibitors so that isothermal amplification of ZIKV RNA is possible even when human whole blood is directly used as a sample. We extracted ZIKV RNA and added it into human whole blood to mimic ZIKV-containing blood samples. Then, the spiked human whole blood was serially diluted with blood to set up a concentration range from 106 copies of RNA to a single copy per 2 μL and directly used these dilutions as samples without additional RNA purification steps. To colorimetrically detect the result of the LFA, a special modification is needed: labelling of the amplicon with digoxigenin and biotin. Among our own designed ZIKV-specific primers, two loop primers were tagged with digoxigenin at the 5´end; this approach will allow digoxigenin to label the amplicon when loop primers amplify the ZIKV RNA by the LAMP method. Labelling of the amplicon with biotin is made possible by adding biotin-labelled dUTP (Biotin-dUTP) to the mix of deoxynucleotides (dNTPs) at a certain ratio. When ZIKV RNA is amplified and this reaction consumes dNTPs, Biotin-dUTP will substitute thymine at the adenine sites of the complementary strand, resulting in labelling of the amplicon with biotin.

RT-LAMP was carried out in a 25 μL reaction mixture containing 1× Isothermal Amplification Buffer II [20 mM Tris-HCl, 10 mM (NH4)2SO2, 150 mM KCl, 2 mM MgSO4, and 0.1% Tween 20], additional 2 mM MgSO4, a dNTP mix supplemented with biotin-dUTP (2.2 mM dGTP, dATP, dCTP, 1.375 mM dTTP, and 0.0825 mM biotin-dUTP), a target-specific primer mixture (0.8 μM forward and reverse inner primers, 0.4 μM digoxigenin-labelled loop primers, and 0.2 μM forward and reverse outer primers), 8 U of Bst 3.0 DNA polymerase, and 2 μL of human whole blood spiked with ZIKV RNA ranging from 106 copies to a single copy per 2 μL. The RT-LAMP reaction mixture was incubated for 30 min.

Design and operation of the LFA
Figure 1(a) and 1(b) shows the detailed set-up and operating procedures of the LFA in our study. First, 1 μL of digoxigenin- and biotin-labelled RT-LAMP products was loaded onto the conjugate pad, so that the biotin-labelled RT-LAMP products formed a complex with gold nanoparticles (AuNPs) via streptavidin-biotin interactions. Next, 45 μL of diluent buffer was placed on the buffer loading pad, and then capillary flow transferred AuNPs from the conjugate pad to the test and control line. The AuNP–RT-LAMP complexes were immobilized at the test line by the interaction between digoxigenin and anti-digoxigenin whereas the AuNPs that did not form complexes were captured by biotin. Complexed and uncomplexed AuNPs are indicated by violet bands at the test line and control line, respectively. The colorimetric signal was easily visible with the naked eye within 5 min.

Discussion
Analysis of the limit of detection in human whole blood samples
We evaluated the limit of detection of the LFA to determine whether our method is indeed highly sensitive. Two microliters of human whole blood was directly used as a sample without any purification steps. Figure 1(c) shows the ZIKV RNA detection results for the LFA. The signal intensities on the test line gradually declined as the concentration of ZIKV RNA decreased. Notably, the presence of even a single copy of ZIKV RNA could be detected within 35 min by the LFA. These results imply that our method has a great potential for diagnosis of ZIKV infections.

References
1. Lessler J, Chaisson LH, Kucirka LM, Bi Q, Grantz K, Salje H, et al. Assessing the global threat from Zika virus. Science 2016; 353: aaf8160.
2. Schuler-Faccini L, Ribeiro E, Feitosa I, Horovitz D, Cavalcanti D, et al. Possible Association Between Zika Virus Infection and Microcephaly Brazil, 2015. MMWR Morb Mortal Wkly Rep 2016; 65: 59–62.
3. Cao-Lormeau V-M, Blake A, Mons S, Lastère S, Roche C, et al. Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet 2016; 387: 1531–1539.
4. WHO statement on the first meeting of the International Health Regulations (2005) (IHR 2005) Emergency Committee on Zika virus and observed increase in neurological disorders and neonatal malformations. http://www.who.int/mediacentre/news/ statements/2016/1st-emergency-committee-zika/en/ (accessed May 1).
5. Surveillance and Control of Aedes aegypti and Aedes albopictus in the United States. http://www.cdc.gov/chikungunya/resources/ vector-control.html (accessed May 1).
6. Dejnirattisai W, Supasa P, Wongwiwat W, Rouvinski A, Barba-spaeth G, et al. Nat Immunol 2016; doi:10.1038/ni.3515.
7. Target product profiles for better diagnostic tests for Zika virus infection. http://www.who.int/csr/research-and-development/zika-tpp.pdf.
8. Faye O, Faye O, Dupressoir A, Weidmann M, Ndiaye M, Alpha Sall A. One-step RT-PCR for detection of Zika virus. J Clin Virol 2008; 43: 96–101.
9. Faye O, Faye O, Diallo D, Diallo M, Weidmann M, Sall AA. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes. Virol J 2013; 10: 311.
10. Safavieh M, Kanakasabapathy MK, Tarlan F, Ahmed MU, Zourob M, et al. Emerging loop-mediated isothermal amplification-based microchip and microdevice technologies for nucleic acid detection. ACS Biomater Sci Eng 2016; 2: 278–294.
11. Nyan D-C, Ulitzky LE, Cehan N, Williamson P, Winkelman V, et al. Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay. Clin Infect Dis 2014; 59: 16–23.

The authors
Dohwan Lee MS, Yong Kyoung Yoo PhD, and Jeong Hoon Lee* PhD
Department of Electrical Engineering, Kwangwoon University, Nowon, Seoul 01897, Republic of Korea.

*Corresponding author
E-mail: jhlee@kw.ac.kr

Literature Review: NGS

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

Two novel mutations in the PPIB gene cause a rare pedigree of osteogenesis imperfecta type IX

BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic skeletal disorder characterized by increased bone fragility and vulnerability to fractures. PPIB is identified as a candidate gene for OI-IX, here we detect two pathogenic mutations in PPIB and analyze the genotype-phenotype correlation in a Chinese family with OI.

METHODS: Next-generation sequencing (NGS) was used to screen the whole exome of the parents of proband. Screening of variation frequency, evolutionary conservation comparisons, pathogenicity evaluation, and protein structure prediction were conducted to assess the pathogenicity of the novel mutations. Sanger sequencing was used to confirm the candidate variants. RTQ-PCR was used to analyze the PPIB gene expression.

RESULTS: All mutant genes screened out by NGS were excluded except PPIB. Two novel heterozygous PPIB mutations (father, c.25A>G; mother, c.509G>A) were identified in relation to osteogenesis imperfecta type IX. Both mutations were predicted to be pathogenic by bioinformatics analysis and RTQ-PCR analysis revealed downregulated PPIB expression in the two carriers.

CONCLUSION: We report a rare pedigree with an autosomal recessive osteogenesis imperfecta type IX (OI-IX) caused by two novel PPIB mutations identified for the first time in China. The current study expands our knowledge of PPIB mutations and their associated phenotypes, and provides new information on the genetic defects associated with this disease for clinical diagnosis.

Application of next generation sequencing in clinical microbiology and infection prevention

Deurenberg RH, Bathoorn E, Chlebowicz MA, Couto N, Ferdous M et al. J Biotechnol 2017; 243: 16–24

Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements.

A targeted high-throughput next-generation sequencing panel for clinical screening of mutations, gene amplifications, and fusions in solid tumours

Luthra R, Patel KP, Routbort MJ, Broaddus RR, Yau J, Simien C, Chen W, Hatfield DZ, Medeiros LJ, Singh RR. J Mol Diagn 2017; 19(2): 255–264

Clinical next-generation sequencing (NGS) assay choice requires careful consideration of panel size, inclusion of appropriate markers, ability to detect multiple genomic aberration types, compatibility with low quality and quantity of nucleic acids, and work flow feasibility. Herein, in a high-volume clinical molecular diagnostic laboratory, we have validated a targeted high-multiplex PCR-based NGS panel (OncoMine Comprehensive Assay) coupled with high-throughput sequencing using Ion Proton sequencer for routine screening of solid tumours. The panel screens 143 genes using low amounts of formalin-fixed, paraffin-embedded DNA (20 ng) and RNA (10 ng). A large cohort of 121 tumour samples representing 13 tumour types and 6 cancer cell lines was used to assess the capability of the panel to detect 148 single-nucleotide variants, 49 insertions or deletions, 40 copy number aberrations, and a subset of gene fusions. High levels of analytic sensitivity and reproducibility and robust detection sensitivity were observed. Furthermore, we demonstrated the critical utility of sequencing paired normal tissues to improve the accuracy of detecting somatic mutations in a background of germline variants. We also validated use of the Ion Chef automated bead templating and chip loading system, which represents a major work flow improvement. In summary, we present data establishing the OncoMine Comprehensive Assay-Ion Proton platform to be well suited for implementation as a routine clinical NGS test for solid tumours.

Presence of cancer-associated mutations in exhaled breath condensates of healthy individuals by next generation sequencing

Youssef O, Knuuttila A, Piirilä P, Böhling T, Sarhadi V, Knuutila S. Oncotarget 2017; doi: 0.18632/oncotarget.15233 [Epub ahead of print]

Exhaled breath condensate (EBC) is a non-invasive source that can be used for studying different genetic alterations occurring in lung tissue. However, the low yield of DNA available from EBC has hampered the more detailed mutation analysis by conventional methods. We applied the more sensitive amplicon-based next generation sequencing (NGS) to identify cancer related mutations in DNA isolated from EBC. In order to apply any method for the purpose of mutation screening in cancer patients, it is important to clarify the incidence of these mutations in healthy individuals. Therefore, we studied mutations in hotspot regions of 22 cancer genes of 20 healthy, mainly non-smoker individuals, using AmpliSeq colon and lung cancer panel and sequenced on Ion PGM. In 15 individuals, we detected 35 missense mutations in TP53, KRAS, NRAS, SMAD4, MET, CTNNB1, PTEN, BRAF, DDR2, EGFR, PIK3CA, NOTCH1, FBXW7, FGFR3, and ERBB2: these have been earlier reported in different tumor tissues. Additionally, 106 novel mutations not reported previously were also detected. One healthy non-smoker subject had a KRAS G12D mutation in EBC DNA. Our results demonstrate that DNA from EBC of healthy subjects can reveal mutations that could represent very early neoplastic changes or alternatively a normal process of apoptosis eliminating damaged cells with mutations or altered genetic material. Further assessment is needed to determine if NGS analysis of EBC could be a screening method for high risk individuals such as smokers, where it could be applied in the early diagnosis of lung cancer and monitoring treatment efficacy.

Molecular testing for familial hypercholesterolaemia-associated mutations in a UK-based cohort: development of an NGS-based method and comparison with multiplex polymerase chain reaction and oligonucleotide arrays

Reiman A, Pandey S, Lloyd KL, Dyer N, Khan M, Crockard M, Latten MJ, Watson TL, Cree IA, Grammatopoulos DK. Ann Clin Biochem 2016; 53(6): 654–662

BACKGROUND: Detection of disease-associated mutations in patients with familial hypercholesterolaemia is crucial for early interventions to reduce risk of cardiovascular disease. Screening for these mutations represents a methodological challenge since more than 1200 different causal mutations in the low-density lipoprotein receptor has been identified. A number of methodological approaches have been developed for screening by clinical diagnostic laboratories.

METHODS: Using primers targeting, the low-density lipoprotein receptor, apolipoprotein B, and proprotein convertase subtilisin/kexin type 9, we developed a novel Ion Torrent-based targeted re-sequencing method. We validated this in a West Midlands-UK small cohort of 58 patients screened in parallel with other mutation-targeting methods, such as multiplex polymerase chain reaction (Elucigene FH20), oligonucleotide arrays (Randox familial hypercholesterolaemia array) or the Illumina next-generation sequencing platform.

RESULTS: In this small cohort, the next-generation sequencing method achieved excellent analytical performance characteristics and showed 100% and 89% concordance with the Randox array and the Elucigene FH20 assay. Investigation of the discrepant results identified two cases of mutation misclassification of the Elucigene FH20 multiplex polymerase chain reaction assay. A number of novel mutations not previously reported were also identified by the next-generation sequencing method.

CONCLUSIONS: Ion Torrent-based next-generation sequencing can deliver a suitable alternative for the molecular investigation of familial hypercholesterolaemia patients, especially when comprehensive mutation screening for rare or unknown mutations is required.

Analytical validation of the next-generation sequencing assay for a nationwide signal-finding clinical trial: Molecular Analysis for Therapy Choice clinical trial

Lih CJ, Harrington RD, Sims DJ, Harper KN, Bouk CH, et al. J Mol Diagn 2017; 19(2): 313–327

The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap ?4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine.

Targeted next-generation sequencing of FNA-derived DNA in pancreatic cancer

Sibinga Mulder BG, Mieog JS, Handgraaf HJ, Farina Sarasqueta A, Vasen H et al. J Clin Pathol 2017; 70(2): 174–178

To improve the diagnostic value of fine-needle aspiration (FNA)-derived material, we perform targeted next-generation sequencing (NGS) in patients with a suspect lesion of the pancreas. The NGS analysis can lead to a change in the treatment plan or supports inconclusive or uncertain cytology results. We describe the advantages of NGS using one particular patient with a recurrent pancreatic lesion 7 years after resection of a pancreatic ductal adenocarcinoma (PDAC). Our NGS analysis revealed the presence of a presumed second primary cancer in the pancreatic remnant, which led to a change in treatment: resection with curative intend instead of palliation. Additionally, NGS identified an unexpected germline CDKN2A 19-base pair deletion, which predisposed the patient to developing PDAC. Preoperative NGS analysis of FNA-derived DNA can help identify patients at risk for developing PDAC and define future therapeutic options.

Exome sequencing covers >98% of mutations identified on targeted next generation sequencing panels

LaDuca H, Farwell KD, Vuong H, Lu HM, Mu W, Shahmirzadi L, Tang S, Chen J, Bhide S, Chao EC. PLoS One 2017;12(2): e0170843

BACKGROUND: With the expanded availability of next generation sequencing (NGS)-based clinical genetic tests, clinicians seeking to test patients with Mendelian diseases must weigh the superior coverage of targeted gene panels with the greater number of genes included in whole exome sequencing (WES) when considering their first-tier testing approach. Here, we use an in silico analysis to predict the analytic sensitivity of WES using pathogenic variants identified on targeted NGS panels as a reference. METHODS: Corresponding nucleotide positions for 1533 different alterations classified as pathogenic or likely pathogenic identified on targeted NGS multi-gene panel tests in our laboratory were interrogated in data from 100 randomly-selected clinical WES samples to quantify the sequence coverage at each position. Pathogenic variants represented 91 genes implicated in hereditary cancer, X-linked intellectual disability, primary ciliary dyskinesia, Marfan syndrome/aortic aneurysms, cardiomyopathies and arrhythmias.

RESULTS: When assessing coverage among 100 individual WES samples for each pathogenic variant (153,300 individual assessments), 99.7% (n = 152,798) would likely have been detected on WES. All pathogenic variants had at least some coverage on exome sequencing, with a total of 97.3% (n = 1491) detectable across all 100 individuals. For the remaining 42 pathogenic variants, the number of WES samples with adequate coverage ranged from 35 to 99. Factors such as location in GC-rich, repetitive, or homologous regions likely explain why some of these alterations were not detected across all samples. To validate study findings, a similar analysis was performed against coverage data from 60,706 exomes available through the Exome Aggregation Consortium (ExAC). Results from this validation confirmed that 98.6% (91,743,296/93,062,298) of pathogenic variants demonstrated adequate depth for detection.

CONCLUSIONS: Results from this in silico analysis suggest that exome sequencing may achieve a diagnostic yield similar to panel-based testing for Mendelian diseases.

Validation of an NGS mutation detection panel for melanoma

Reiman A, Kikuchi H, Scocchia D, Smith P, Tsang YW, Snead D, Cree IA. BMC Cancer 2017;17(1): 150

BACKGROUND: Knowledge of the genotype of melanoma is important to guide patient management. Identification of mutations in BRAF and c-KIT lead directly to targeted treatment, but it is also helpful to know if there are driver oncogene mutations in NRAS, GNAQ or GNA11 as these patients may benefit from alternative strategies such as immunotherapy.

METHODS: While polymerase chain reaction (PCR) methods are often used to detect BRAF mutations, next generation sequencing (NGS) is able to determine all of the necessary information on several genes at once, with potential advantages in turnaround time. We describe here an Ampliseq hotspot panel for melanoma for use with the IonTorrent Personal Genome Machine (PGM) which covers the mutations currently of most clinical interest.

RESULTS: We have validated this in 151 cases of skin and uveal melanoma from our files, and correlated the data with PCR based assessment of BRAF status. There was excellent agreement, with few discrepancies, though NGS does have greater coverage and picks up some mutations that would be missed by PCR. However, these are often rare and of unknown significance for treatment.

CONCLUSIONS: PCR methods are rapid, less time-consuming and less expensive than NGS, and could be used as triage for patients requiring more extensive diagnostic workup. The NGS panel described here is suitable for clinical use with formalin-fixed paraffin-embedded (FFPE) samples.

Exome sequencing in a family with luminal-type breast cancer underpinned by variation in the

methylation pathway

van der Merwe N, Peeters AV, Pienaar FM, Bezuidenhout J, van Rensburg SJ, Kotze MJ. Int J Mol Sci 2017;18(2): E467

Panel-based next generation sequencing (NGS) is currently preferred over whole exome sequencing (WES) for diagnosis of familial breast cancer, due to interpretation challenges caused by variants of uncertain clinical significance (VUS). There is also no consensus on the selection criteria for WES. In this study, a pathology-supported genetic testing (PSGT) approach was used to select two BRCA1/2 mutation-negative breast cancer patients from the same family for WES. Homozygosity for the MTHFR 677 C>T mutation detected during this PSGT pre-screen step was considered insufficient to cause bilateral breast cancer in the index case and her daughter diagnosed with early-onset breast cancer (<30 years). Extended genetic testing using WES identified the RAD50 R385C missense mutation in both cases. This rare variant with a minor allele frequency (MAF) of <0.001 was classified as a VUS after exclusion in an affected cousin and extended genotyping in 164 unrelated breast cancer patients and 160 controls. Detection of functional polymorphisms (MAF > 5%) in the folate pathway in all three affected family members is consistent with inheritance of the luminal-type breast cancer in the family. PSGT assisted with the decision to pursue extended genetic testing and facilitated clinical interpretation of WES aimed at reduction of recurrence risk.

Proteomics as an alternative diagnostic tool for cervical cancer

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

Cervical cancer is mainly caused by high-risk human papillomavirus (HPV) infection. The Pap test is the gold standard for early cervical cancer diagnosis. However, the lack of Pap test accessibility accounts for the high rates of cervical cancer mostly in developing regions. Here we discuss recent proteomic approaches towards the development of novel diagnostic and prognostic putative biomarkers.

by Georgia Kontostathi, Dr Jerome Zoidakis, Prof. Nicholas P. Anagnou, Prof. Kalliopi I. Pappa and Dr Manousos Makridakis

Background
Cervical cancer is one of the most common gynecological cancers. It shares many common characteristics such as pathways and regulatory networks with vulvar and endometrial cancer [1]. The majority of cervical cancer incidents are attributed to HPV infection by high-risk oncogenic HPV types (mostly HPV16 and HPV18). HPV infects the basal membrane of cervical epithelium leading to upregulated expression of E6 and E7 oncogenes that cause specific histological lesions such as CIN1 [cervical intraepithelial neoplasia or low-grade squamous intraepithelial lesions (LSIL)], CIN2 and CIN3 [or high-grade squamous intraepithelial lesions (HSIL)] [2, 3].

Cervical cancer is the fourth most common cancer in women worldwide, regarding incidence and mortality. It was responsible for 528 000 incidents of malignancy and 266 000 deaths in 2012, of which more than 85% occurred in developing regions [4]. The high number of cervical cancer cases in developing countries is mainly attributed to the limited availability of diagnostic tools such as Pap smear tests or HPV DNA genotyping that enable detection of early-stage lesions [5].

Current diagnostic methods
The Pap test is the most popular diagnostic technique and is based on the nuclear morphology evaluation of cervical epithelial cells. This test enables the detection of possible lesions at an early stage [6]. However, there is a wide range of sensitivity (from 33.8% to 94.0%) [7, 8] that reflects the main limitation of Pap test: the high inter-observer variability.

Other diagnostic options are based on direct probes, such as Southern blot for HPV genomic analysis. This technique has a relatively low sensitivity, is time-consuming and requires large amounts of purified DNA. More sensitive methods include commercially available kits like Digene’s HC2 test (not HPV type specific), which is based on detection of viral RNA by probes. It is used for patients with minor abnormalities detected by Pap test that need further confirmation. Finally, targeted amplification methods such as PCR, are ideal for viral load quantification and genotyping with high sensitivity. However, they are prone to environmental contamination and false-positive results [9]. A recent method [approved by the US Federal Drug Administration (FDA) in 2014] is Roche’s COBAS HPV test for use in primary screening [10]. A major drawback of the above HPV-based tests is the high cost and the requirements for specialized experimental facilities.

Some protein biomarkers have been proposed for early cervical cancer screening. One of them is p16INK4a (cyclin-dependent kinase inhibitor) which is highly expressed at dysplastic epithelium. A combinatorial stain of p16INK4a and the cell proliferation marker Ki-67 has been proposed for increased diagnostic sensitivity [11]. However, lack of a scoring system for immunohistochemistry has hampered the incorporation of the these biomarkers in wide cervical cancer screening. Also, squamous cell carcinoma antigen (SCCA) is a known cancer antigen isolated from tissue, which is usually measured by immunoassays such as ELISA in serum or plasma patients, with limited specificity and sensitivity [12, 13].

Similarly, targets of the E5 HPV protein [e.g. epidermal growth factor receptor, p21 and p27 inhibitors of cyclin-dependent kinase, cyclooxygenase-2 (cox-2), vascular endothelial growth factor, and caveolin-1] have been proposed for early stage cervical cancer detection. Moreover, putative markers that have been suggested are the ProEx C immunocytochemical assay (that targets the expression of topoisomerase II protein and the minichromosome maintenance complex II protein) as well as microRNAs which are regulated by E5, E6, and E7 HPV proteins [14].

Figure 1A summarizes the different methods that have been used for the diagnosis of cervical cancer.

Current proteomic studies
The limitations of the above methods demonstrate the need for the establishment of new reliable diagnostic tests via alternative methods [15]. A novel method that can expose the association of HPV infection and cellular transformation at the molecular level is proteomics [16]. Cervical cancer models (cell cultures or tissue samples) have been studied via several proteomic methods which are either gel-based [two-dimensional gel electrophoresis (2DE)] or gel-free (liquid chromatography (LC)] in combination with mass spectrometry (MS) [17]. Proteomics can be used in order to reveal putative biomarkers for early-stage diagnosis. The role of systems biology that includes the integration of proteomics data with other available ‘OMICS’ datasets, such as genomics and transcriptomics, appears to be vital towards the direction of personalized cervical cancer medicine [18].

In this review, we present some of the most recent and interesting proteomic studies for putative biomarkers in a variety of clinical samples. Diagnostic, prognostic and predictive biomarkers have been assessed in tissue, plasma/serum, cell biopsies/cervical swabs, residual fluid from cell biopsies, cell mucous and cervicovaginal fluid (CVF) by proteomic approaches [19]. Some of the most interesting studies from each category of clinical samples are reported below. The proteomics approach workflow is presented in Figure 1B.

A proteomic study, highlighted keratin 17 as a prognostic biomarker with characteristic gradual increase from normal toward cancer stage in tissue samples. Specifically, stage specific tissue samples [normal squamous mucosa, LSIL, HSIL, and SCC (total number of samples N=22] were analysed by laser capture microdissection in combination with multidimensional liquid chromatography and tandem MS (LC-MS/MS). Proteomics analysis demonstrated differentially expressed and statistically significant proteins and after bioinformatics analysis (gene ontology) keratin 4 and keratin 17 were chosen for validation by immunohistochemistry. The gradual increase from normal towards cancer stage of keratin 17 shown by proteomics, was in total agreement with the immunohistochemistry results. Kaplan–Meier curves of keratin 17 expression and general survival of cervical cancer patients revealed a strong correlation of high keratin 17 expression with poor survival, adding prognostic value to this protein [20]. Another proteomics study focused on the pelvic lymph node metastasis (PLNM) clinical status, which is important in terms of prognosis and treatment of cervical cancer. A two-dimensional difference gel electrophoresis (2D DIGE)/matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS approach was used to compare cervical tissues of patients with PLNM (N=8) to cervical tissues from patients without lymph node metastasis (NPLNM) (N=10). Analysis led to the identification of 41 differentially expressed and statistically significant proteins. Some of them (FABP5, HspB1, and MnSOD) were validated in the PLNM group compared to the NPLNM, by Western blot and immunohistochemistry [21].

The combination of Isobaric tag for relative and absolute quantitation (iTRAQ) and targeted mass spectrometric quantification was used in order to analyse serum from CIN, early- (CES), and late-stage (CLS) cervical cancer patients. A panel of six differentially expressed proteins (alpha-1-acid glycoprotein1, alpha-1-antitrypsin, serotransferrin, haptoglobin, alpha-2-HS-glycoprotein, and vitamin D-binding protein) was validated by MRM (multiple reaction monitoring) in an independent set of 229 serum samples consisting of controls (N=49), CIN-1 to 3 designated as CIN (N=48), CES (N = 49), CLS (N=34), and ovarian cancer (N=49). The above panel discriminated patients with CIN from healthy controls with a sensitivity of 67% and specificity of 88%. Combination of the specific panel with SCCA, a well-studied putative biomarker for cervical cancer, improved discrimination of CIN, CES, and CLS patients from healthy control. Briefly, upon the comparison of the CES versus healthy group, the area under the curve was 0.86 (sensitivity/specificity = 71/90%), when using the six-protein panel and SCCA [12].

An alternative strategy was used by Boylan et al., in order to study the proteome of Pap test clinical samples. The cell-free residual fluid from cell biopsies was collected, proteins were isolated (acetone precipitation) and their concentration upon resolubilization was determined by Bradford assay. Filter aided sample preparation followed by LC-MS/MS analysis yielded 300 protein identifications per sample and 700 unique protein identifications after pooling the samples. Many of the proteins had similar biological functions to the ones identified from CVF. Thus, residual fluid could be an alternative material for the study of proteins related to cervical dysplasia [22].

The cell mucous proteome from 25 HPV-positive and pre invasive cervical disease samples has been investigated by a combination of 2DE MS, gel-based LC-MS/MS and 2DE MS after depletion of highly abundant proteins (e.g. albumin). The above approaches were combined and 107 unique proteins were identified. A bioinformatics study showed that they are related to metabolism, immune response, and transport. Proteins like acute-phase plasma proteins, α-1-antichymotrypsin and α-1-antitrypsin, were found to be both phosphorylated and glycosylated after posttranslational modifications evaluation with appropriate fluorescent dyes [23].

Table 1 highlights the above clinical proteomics studies and some of the most promising putative biomarkers that were identified.

Discussion and conclusions
Cervical cancer is the fourth most common cancer in women. The Pap test is a very efficient diagnostic approach in terms of specificity (77.8-98.8%) but has a variable sensitivity (33.8% to 94.0%) [8). Molecular tests such as HPV DNA detection by PCR and/or hybridization with adequate probes are the reference methods for the detection of HPV. The aforementioned methods are often expensive and unavailable in the developing regions where cervical cancer is very frequent (85% of cervical cancer cases). A promising idea is that proteomics will facilitate the discovery of novel biomarkers that will enable the future cervical cancer screening in developing countries in the form of antibody-based practical diagnostic self-tests (like pregnancy tests). New and advanced proteomic techniques like MRM could validate several biomarkers that will eventually be combined into panels for more accurate testing in the above diagnostic tests. Of course, the future of proteomics studies is not only promising but also challenging. New aspects of research should be taken into consideration. Redox proteomics will be used for the exploration of proteins oxidation status in order to reveal the interaction of oxidative stress and tumour development. In particular, the oxidative status of proteins in HPV-related cervical cancer cells was explored via oxidative isotope-coded affinity tags (OxICAT) and voltage-dependent anion channel 1 (VDAC1) was found to be highly oxidized in HPV-positive cervical cancer cells [24]. The important role of post-translational modifications (PTMs) such as phosphorylation and glycosylation should be thoroughly examined. The combination of systems biology and proteomics offers the possibility to elucidate cervical cancer mechanisms and identify potential biomarkers for early-stage detection.

References
1. Pappa KI, Polyzos A, Jacob-Hirsch J, Amariglio N, Vlachos GD, Loutradis D, Anagnou NP. Profiling of discrete gynecological cancers reveals novel transcriptional modules and common features shared by other cancer types and embryonic stem cells. PloS One 2015; 10(11): e0142229.
2. Doorbar J, Quint W, Banks L, Bravo IG, Stoler M, Broker TR, Stanley MA. The biology and life-cycle of human papillomaviruses. Vaccine 2012; 30(Suppl 5): F55–70.
3. Schiffman MH, Castle P. Epidemiologic studies of a necessary causal risk factor: human papillomavirus infection and cervical neoplasia. J Natl Cancer Inst. 2003; 95(6): E2.
4. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015; 136(5): E359–386.
5. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011; 61(2): 69–90.
6. Wilting SM, Smeets SJ, Snijders PJ, van Wieringen WN, van de Wiel MA, Meijer GA, Ylstra B, Leemans CR, Meijer CJ, et al. Genomic profiling identifies common HPV-associated chromosomal alterations in squamous cell carcinomas of cervix and head and neck. BMC Med Genomics 2009; 2: 32.
7. Nayar R, Wilbur DC. The Pap test and Bethesda 2014. Cancer Cytopathol. 2015; 123(5): 271–281.
8. Wright TC, Jr. HPV DNA testing for cervical cancer screening. FIGO 26th Annual Report on the Results of Treatment in Gynecological Cancer. Int J Gynaecol Obstet. 2006; 95(Suppl 1): S239–246.
9. Hubbard RA. Human papillomavirus testing methods. Arch Pathol Lab Med. 2003; 127(8): 940–945.
10. Hogarth S, Hopkins M, Rotolo D. Technological accretion in diagnostics: HPV testing and cytology in cervical cancer screening. In: Consoli D, Mina A, Nelson RR, Ramlogan R (eds) Medical Innovation: Science, Technology and Practice. Routledge 2015.
11. von Knebel Doeberitz M, Reuschenbach M, Schmidt D, Bergeron C. Biomarkers for cervical cancer screening: the role of p16(INK4a) to highlight transforming HPV infections. Expert Rev Proteomics 2012; 9(2): 149–163.
12. Boichenko AP, Govorukhina N, Klip HG, van der Zee AG, Guzel C, Luider TM, Bischoff R. A panel of regulated proteins in serum from patients with cervical intraepithelial neoplasia and cervical cancer. J Proteome Res. 2014; 13(11): 4995–5007.
13. Kato H, Torigoe T. Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma. Cancer 1977; 40(4): 1621–1628.
14. de Freitas AC, Coimbra EC, Leitao Mda C. Molecular targets of HPV oncoproteins: potential biomarkers for cervical carcinogenesis. Biochim Biophys Acta 2014; 1845(2): 91–103.
15. Wentzensen N, von Knebel Doeberitz M. Biomarkers in cervical cancer screening. Dis Markers 2007; 23(4): 315–330.
16. Lomnytska M, Souchelnytskyi S. Markers of breast and gynecological malignancies: the clinical approach of proteomics-based studies. Proteomics Clin appl. 2007; 1(9): 1090–1101.
17. Di Domenico F, De Marco F, Perluigi M. Proteomics strategies to analyze HPV-transformed cells: relevance to cervical cancer. Expert Rev Proteomics 2013; 10(5): 461–472.
18. Breuer EK, Murph MM. The role of proteomics in the diagnosis and treatment of women’s cancers: current trends in technology and future opportunities. Int J Proteomics 2011; 2011: pii: 373584.
19. Kontostathi G, Zoidakis J, Anagnou NP, Pappa KI, Vlahou A, Makridakis M. Proteomics approaches in cervical cancer: focus on the discovery of biomarkers for diagnosis and drug treatment monitoring. Exp Rev Proteomics 2016; 13(8): 731–745.
20. Escobar-Hoyos LF, Yang J, Zhu J, Cavallo JA, Zhai H, Burke S, Koller A, Chen EI, Shroyer KR. Keratin 17 in premalignant and malignant squamous lesions of the cervix: proteomic discovery and immunohistochemical validation as a diagnostic and prognostic biomarker. Mod Pathol. 2014; 27(4): 621–630.
21. Wang W, Jia HL, Huang JM, Liang YC, Tan H, Geng HZ, Guo LY, Yao SZ. Identification of biomarkers for lymph node metastasis in early-stage cervical cancer by tissue-based proteomics. Br J Cancer 2014; 110(7): 1748–1758.
22. Boylan KL, Afiuni-Zadeh S, Geller MA, Hickey K, Griffin TJ, Pambuccian SE, Skubitz AP. A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics. Clin Proteomics 2014; 11(1): 30.
23. Panicker G, Ye Y, Wang D, Unger ER. Characterization of the human cervical mucous proteome. Clini Proteomics 2010; 6(1–2): 18–28.
24. Zhang C, Ding W, Liu Y, Hu Z, Zhu D, Wang X, Yu L, Wang L, Shen H, et al. Proteomics-based identification of VDAC1 as a tumor promoter in cervical carcinoma. Oncotarget 2016; doi: 10.18632/oncotarget.10562 [Epub ahead of print].

The authors
Georgia Kontostathi^1,2 MSc; Jerome Zoidakis¹ PhD; Nicholas P. Anagnou^2,3 MD, PhD; Kalliopi I. Pappa^3,4 MD, PhD; Manousos Makridakis*¹ PhD
¹Biotechnology Division, Biomedical Research Foundation, Academy of Athens (BRFAA), Athens, Greece
²Laboratory of Biology, University of
Athens School of Medicine, Athens, Greece
³Cell and Gene Therapy Laboratory,
Biomedical Research Foundation,
Academy of Athens (BRFAA), Athens, Greece
⁴First Department of Obstetrics and
Gynecology, University of Athens School of Medicine, Athens, Greece

*Corresponding author
E-mail: mmakrid@bioacademy.gr

BA200 LED Technology

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

Improving obstetric outcome: antenatal thyroid screening

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

Mild hypothyroidism, where plasma levels of thyroid stimulating hormone (TSH) are above the ‘normal’ upper limit but where there is no equivalent change in circulating levels of the thyroid hormones tetraiodothyronine (T4) and triiodothyronine (T3), is common in women of childbearing age; the condition is found in up to 3?% of pregnant women. While normally asymptomatic, in pregnant women mild hypothyroidism has been associated with miscarriage, perinatal death and preterm delivery, the major cause of neonatal death. Several studies have investigated whether treatment with levothyroxine, a synthetic thyroid hormone, would improve the obstetric outcome in women with borderline thyroid function, and results from the most recent study were reported at the Society for Endocrinology (BES) conference in November.
In this study, 645 women out of more than 13?000 tested at the end of the first trimester of pregnancy were found to have sub-clinical hypothyroidism (340) or isolated hypothyroxinemia (305). In the latter condition TSH levels are normal but T4 levels are below the lower reference limit. Five hundred and eighteen women with abnormal thyroid function took part in a randomized trial, with half being prescribed levothyroxine and half acting as control. Rates of stillbirth, neonatal death and delivery before 34 weeks were compared, as well as delivery between 34 and 37 weeks and cesarean sections carried out before 37 weeks. It was found that untreated women with abnormal thyroid function had an increased risk of stillbirth, delivery before 37 weeks and having an early cesarean section when compared with women with normal thyroid function and those treated with the synthetic thyroid hormone. Although the authors emphasize that larger trials are needed to confirm their findings, it seems likely that this cheap and safe drug could have a significant impact on obstetric outcome.
In the more developed countries thyroid autoimmunity is the main cause of hypothyroidism, with iodine deficiency being less frequent. Thyroid autoantibodies, particularly thyroid peroxidase antibodies (TPO), can be measurable even in women with biochemically normal thyroid function, and are a risk factor for miscarriage and preterm delivery. Elevated levels are found in up to 20?% of women, but also in as many as 31?% of sub-fertile women. There is a dearth of robust studies to assess the effect of levothyroxine on pregnancy outcomes in these women but it could be that measuring TPO in both sub-fertile as well as pregnant women, followed by treatment with levothyroxine if indicated, could result in many more healthy, full-term babies.

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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Deurenberg RH, Bathoorn E, Chlebowicz MA, Couto N, Ferdous M et al. J Biotechnol 2017; 243: 16–24

Luthra R, Patel KP, Routbort MJ, Broaddus RR, Yau J, Simien C, Chen W, Hatfield DZ, Medeiros LJ, Singh RR. J Mol Diagn 2017; 19(2): 255–264

Youssef O, Knuuttila A, Piirilä P, Böhling T, Sarhadi V, Knuutila S. Oncotarget 2017; doi: 0.18632/oncotarget.15233 [Epub ahead of print]

Reiman A, Pandey S, Lloyd KL, Dyer N, Khan M, Crockard M, Latten MJ, Watson TL, Cree IA, Grammatopoulos DK. Ann Clin Biochem 2016; 53(6): 654–662

Lih CJ, Harrington RD, Sims DJ, Harper KN, Bouk CH, et al. J Mol Diagn 2017; 19(2): 313–327

Sibinga Mulder BG, Mieog JS, Handgraaf HJ, Farina Sarasqueta A, Vasen H et al. J Clin Pathol 2017; 70(2): 174–178

LaDuca H, Farwell KD, Vuong H, Lu HM, Mu W, Shahmirzadi L, Tang S, Chen J, Bhide S, Chao EC. PLoS One 2017;12(2): e0170843

Reiman A, Kikuchi H, Scocchia D, Smith P, Tsang YW, Snead D, Cree IA. BMC Cancer 2017;17(1): 150

van der Merwe N, Peeters AV, Pienaar FM, Bezuidenhout J, van Rensburg SJ, Kotze MJ. Int J Mol Sci 2017;18(2): E467

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