Zika virus (ZIKV) has recently become a global threat owing to the link between infection, Guillain–Barré syndrome and serious neurological defects in unborn fetus and infants. There are major challenges associated with the detection methods that are currently available for the virus, and there is no point-of-care test to accurately and quickly detect ZIKV. Herein, we describe the advantages and disadvantages of the methods that are used presently, and provide an insight into developing technologies that will yield improved detection in the future.

by Devon Pawley, Dr Emre Dikici, Dr Sapna Deo and Prof. Sylvia Daunert


Background
Infectious diseases are a serious public health concern and are the leading cause of death in low income countries [1]. The World Health Organization (WHO) declared the potential impact of the Zika virus (ZIKV) a global public health emergency in 2016, and considers the virus an ongoing threat [2]. Of particular concern is its association with Guillain–Barré syndrome and the link between ZIKV infection of pregnant women and microcephaly, neurological impairment and distress in their offspring [3, 4].

The ZIKV belongs to the genus Flavivirus, and is most commonly transmitted via different species of mosquitoes of the Aedes genus frequently found in tropical environments [5, 6]. The virus has also been shown to be transmitted from mother to fetus, as well as during sexual intercourse between individuals through bodily fluids [7]. The virus is closely related to other flaviviruses, such as the dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), which often complicates correct diagnosis of ZIKV [8]. Although the virus was discovered in Uganda in 1947, the potential for the virus to infect mammals was not described until 1971 [9, 10]. Interestingly, the first clinical reports of perinatal transmission and association with Guillain–Barré syndrome due to ZIKV occurred in 2013 in French Polynesia following a major change in the virus epidemiology [11–14]. This outbreak was complicated by concurrent outbreaks of patients of DENV and chikungunya virus (CHIKV) transmitted by the same Aedes mosquito vector [15]. Since then, other reports from Brazil have chronicled a rapidly spreading epidemic that, once more, co-exists with transmission of DENV and CHIKV, and is characterized by fever, conjunctivitis, and a maculopapular rash [16]. More ominously, there are reports of microcephaly and ocular damage in aborted fetuses and infants born to mothers infected with ZIKV. In these cases, evidence of ZIKV infection came from the recovery of the virus from amniotic fluid, placental, and brain tissue. Additionally, it is known that the virus can persist in body fluids such as urine, saliva, and semen beyond the short time (<7 days) that it is present in blood, which becomes an important consideration when developing methods of ZIKV detection [17, 18].

Developing rapid diagnostics is central to prevent and control ZIKV spread, while also providing women with the necessary information to make informed decisions regarding pregnancy. It is particularly important to distinguish ZIKV infection from that of the structurally related DENV in areas where DENV is endemic and ZIKV is increasing in prevalence. Regions with the highest incidence of ZIKV infection also tend to be resource-limited. There is, therefore, an urgent and unmet need for rapid, simple, on-site, and cost-effective diagnostics that can specifically identify ZIKV and ZIKV-specific antibody (Ab) responses in body fluids.

Current ZIKV detection methods, although rapid (<30 min), are not cost effective and require specialized equipment and trained personnel. These methods are not ideal in resource-limited settings where the virus is frequently found. Additionally, these methods are regularly used concurrently for detection of ZIKV in more than one bodily fluid, most commonly urine and serum, to accurately identify the presence of the virus. Because 20–25% of infected individuals do not demonstrate symptoms, the short window of time in which ZIKV is actively present in the body is often missed [7]. Thus, tests for previous exposure to ZIKV are also performed in conjunction with tests for active infection. It is important to note that test development, validation, and optimization have proven difficult thus far due to the low amount of samples available.

Current ZIKV tests and their limitations
RNA nucleic acid tests (NATs)
The presence of active ZIKV can usually be detected early in the infection in bodily fluids using RNA NATs, such as the Trioplex real-time polymerase chain reaction (RT-PCR) Assay, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), reverse-transcription isothermal recombinase polymerase amplification (RPA) and reverse-transcription strand-invasion based amplification (RT-SIBA) assay [19, 20]. The Trioplex RT-PCR is currently the test used by the Centers for Disease Control and Prevention (CDC) for evaluating symptomatic pregnant women in conjunction with IgM serology. Briefly, the viral RNA is first converted to cDNA via reverse transcription. If the sample contains the desired DNA sequence, a specially designed probe will bind to the target area and is detected via fluorescence. RNA nucleic acid testing is highly sensitive and can identify extremely low concentrations of viral RNA, 1.93×104 genome copy equivalents per millilitre of serum according to the CDC, present during the first 10 days of ZIKV infection (21). However, NATs require expensive machinery, technical expertise, and are associated with high costs. Additionally, because viral RNA degrades rapidly in the body, NATs cannot detect prior exposure to ZIKV. Under updated recommendations of the CDC, negative NATs should be repeated with new sample extractions because of the low levels of virus present during infection.

Plaque-reduction neutralization test (PRNT)
PRNTs involve an intensely laborious process that is performed by the CDC or at a laboratory designated by CDC to detect neutralizing antibodies of a virus. If a sample has a negative ZIKV NAT and a non-negative or inconclusive serology result, a PRNT is required. PRNTs take several days to deliver a result as the process involves mixing the sample with live virus, growing this treated sample in a dish over a monolayer of host cells, and leaving the plate to incubate until plaques grow. Plaques grow when the sample added contains neutralizing antibodies, indicating previous exposure to the virus. Besides the inherent downfall of the time it takes from sample collection to plaque identification, PRNTs require specific equipment, trained personnel and do not provide information on active
ZIKV infection.

Serologic test for ZIKV
The first antibodies produced in response to initial exposure to ZIKV, IgMs, are manifested towards the end of the first week of infection. These antibodies, as well as neutralizing antibodies, can be detected via the Zika IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA). A plate is coated with the anti-IgM capture, the patient’s sample is added and detection is achieved by consequential addition of an enzyme-conjugated anti-viral antibody. The enzyme interacts with a chromogenic substrate producing a colorimetric change, which can then be detected using a spectrophotometer. Important limitations to address include (1) length of assay time (2.5 days to complete); (2) detection of previous exposure to ZIKV only rather than active infection; (3) occurrence of false-negative and false-positive results. False-negatives occur when the samples were collected before IgMs have been generated, usually 4 days post-onset of symptoms or when the samples were collected after IgMs levels have fallen below detectable levels, approximately 12 weeks post-onset of symptoms. Equally, false-positives occur due to cross-reactivity with structurally similar antigens, most commonly other flaviviruses, such as DENV. Follow-up testing is necessary to rule out a false-positive result.

Active infection ELISA
Active ZIKV can be detected using a sandwich-format ELISA. Specific anti-ZIKV antibodies sandwich the virus, if it is present in the sample, and can be detected via an enzyme-conjugated secondary antibody in the same manner as the MAC-ELISA. Until recently, developing an accurate active infection ELISA proved difficult owing to the lack of specific antibodies towards ZIKV, which caused high instances of cross-reactivity with other structurally similar flaviviruses.

The previously described methods are conducted under an ‘Emergency Use Authorization’ issued by the FDA except for the active infection ELISA. In collaboration with Dr David Watkins and Dr Esper Kallas, our lab is working on developing a highly specific active infection ELISA using monoclonal antibodies isolated from ZIKV-infected patients in Sao Paulo, Brazil, that bind only to ZIKV and no other flaviviruses. Currently, our assay is under optimization to detect levels of ZIKV in urine and serum samples.

The advantages and limitations of the methods of ZIKV detection discussed above are summarized in Table 1.

Ongoing and future developments: point-of-care testing for active infection for ZIKV
Recently, paper-based detection methods have gained considerable interest because of the low cost, portability, stability at various storage conditions, and ease of use associated with their handling. These testing platforms do not require external equipment, allowing them to be carried out in remote and resource-limited areas, such as those where ZIKV flourishes. Thus, there is an emphasis on the translation of common assay principles to more portable and affordable platforms.

Lateral flow assays employ ELISA principles, and, as such, antibodies that are selective towards the desired antigen are immobilized onto a membrane. Briefly, the primary and secondary antibodies are dispensed onto the membrane via inkjet technologies and function as the test and control lines, respectively. The top portion of the membrane is laminated with an adsorbent pad to facilitate capillary action. A separate set of selective primary antibodies are conjugated to detection molecules such as gold nanoparticles, latex particles or coloured cellulose nanobeads and are immobilized onto the conjugate pad. The sample is added to the sample pad and then migrates, via capillary action, through the membrane to the conjugate pad. If the sample contains the antigen, the dried primary Ab conjugated to the coloured particles will be remobilized and the antigen will bind to these conjugated primary antibodies. The formed complexes will flow through the reaction matrix, which is usually a porous matrix such as nitrocellulose. The labelled antigen will then be captured by the immobilized primary antibodies forming a coloured band (Fig. 1). The control line will bind the coloured labelled primary antibodies regardless of the presence of antigen. This verifies that the test is working properly and the labelled conjugate can flow and bind to its respective antibody pair. When the antigen is present, the antibody/bead complex will bind to the antigen, and this Ab/antigen complex is captured by the antibody that is immobilized as the test line. One line at the control region indicates a functional but negative test and two lines indicates a functional positive test. Using our highly specific anti-ZIKV antibodies, we have additionally developed a sandwich-format lateral flow assay for the detection of ZIKV in urine that is currently under optimization.

DNA/RNA detection methods on paper are also of particular interest because of the high selectivity of hybridization. In 2015, the Whitesides group described a novel “paper machine” device that uses LAMP to detect a signal using a hand-held UV source and camera phone [22]. The paper-based device costs $1.83, an extreme improvement when compared with traditional nucleic acid testing. The only drawback of this device is that it requires incubation steps at 65 °C throughout the assay to dry the reagents present on the paper strip, which sometimes can be challenging in a point-of-care situation. Furthering the research on paper-based methods of viral RNA detection, our group described a different paper-based platform that has only one step involving incubation in a boiling water bath [23]. We have continued our pursuit to develop a point-of-care paper-based viral detection system and have constructed another test that utilizes RPA and requires incubation at much lower temperature, namely at 37 °C.

The threat of ZIKV creating serious health issues has not lessened and continues to afflict women who are pregnant and wish to become pregnant. Without proper methods of detection, the virus is difficult to characterize, document, and study. While many of the progressive paper-based platforms described herein are promising, none are currently FDA approved and on the market for use for the detection of ZIKV. It is, therefore, imperative that researchers continue to investigate and design innovative detection methods that can detect ZIKV in an easy, accurate, and affordable manner.

References
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4. Štrafela P, Vizjak A, Mraz J, Mlakar J, Pižem J, Tul N, Županc TA, Popović M. Zika virus-associated micrencephaly: a thorough description of neuropathologic findings in the fetal central nervous system. Arch Pathol Lab Med 2017; 141(1): 73–81.
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6. Haddow AJ, Williams MC, Woodall JP, Simpson DI, Goma LK. Twelve isolations of Zika virus from Aedes (Stegomyia) africanus (Theobald) taken in and above a Uganda forest. Bull World Health Organ 1964; 31: 57–69.
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15. Roth A, Mercier A, Lepers C, Hoy D, Duituturaga S, Benyon E, Guillaumot L, Souares Y. Concurrent outbreaks of dengue, chikungunya and Zika virus infections – an unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012-2014. Euro Surveill 2014; 19(41): pii: 20929.
16. Cardoso CW, Paploski IA, Kikuti M, Rodrigues MS, Silva MM, Campos GS, Sardi SI, Kitron U, Reis MG, Ribeiro GS. Outbreak of exanthematous illness associated with Zika, chikungunya, and dengue viruses, Salvador, Brazil. Emerg Infect Dis 2015; 21(12): 2274–2276.
17. Gourinat AC, O’Connor O, Calvez E, Goarant C, Dupont-Rouzeyrol M. Detection of Zika virus in urine. Emerg Infect Dis 2015; 21(1): 84–86.
18. 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.
19. Eboigbodin KE, Brummer M, Ojalehto T, Hoser M. Rapid molecular diagnostic test for Zika virus with low demands on sample preparation and instrumentation. Diagn Microbiol Infect Dis 2016; 86(4): 369–371.
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22. Connelly JT, Rolland JP, Whitesides GM. “Paper machine” for molecular diagnostics. Anal Chem 2015; 87(15): 7595–7601.
23. Zhang DH, Broyles D, Hunt EA, Dikici E, Daunert S, Deo SK. A paper-based platform for detection of viral RNA. Analyst 2017; 142(5): 815–823.

The authors
Devon Pawley, Emre Dikici PhD, Sapna Deo PhD, Sylvia Daunert PhD
Department of Biochemistry and Molecular Biology,
Miller School of Medicine, University of Miami,
Miami, FL 33136, USA

*Corresponding author
E-mail: sdaunert@med.miami.edu

Vitamin D status is currently assessed by measurements of total 25-hydroxyvitamin D [25(OH)D]. However, over 99% of circulating 25(OH)D is bound to protein, vitamin D binding protein in particular. The free hormone hypothesis stipulates that only the free form crosses the cell membrane to exert biologic action. Measurement of free 25(OH)D is now available.

by Professor Daniel D Bikle

Introduction
Circulating levels of 25-hydroxyvitamin D [25(OH)D] are the most commonly used marker for the assessment of vitamin D nutritional status. This is because its concentration in blood is higher than all other vitamin D metabolites, making it easier to measure, and because its conversion from vitamin D is substrate dependent with minimal regulation. However, 25(OH)D is not the most biologically active metabolite of vitamin D. Instead 25(OH)D must be further metabolized to 1,25 dihydroxyvitamin D [1,25(OH)2D] for vitamin D to achieve its full biologic potential. 1,25(OH)2D is the ligand for a nuclear transcription factor, the vitamin D receptor (VDR), that mediates the genomic and at least some of the nongenomic actions of vitamin D within the cell. Nearly all, if not all, cells express the VDR at some stage in their development or activation. As the appreciation that vitamin D and its metabolites affect numerous physiologic processes and not just bone and mineral metabolism, and that these physiologic processes may have different requirements for these vitamin D metabolites, interest in determining optimal levels of the vitamin D metabolites to effect these different biologic processes has grown. Complicating this determination is the fact that all the vitamin D metabolites circulate in blood tightly bound to proteins, of which the vitamin D binding protein (DBP) plays the major role. For most cells, these binding proteins limit the flux of the vitamin D metabolites from blood into the cell where they exert their biologic activity. This raises the issue of what should we measure to determine vitamin D status: the total levels of these metabolites or their free levels?

The free hormone hypothesis: why measure free 25(OH)D
The free hormone hypothesis postulates that only the non-bound fraction (the free fraction) of hormones that otherwise circulate in blood bound to their carrier proteins is able to enter cells and exert their biologic effects. This hypothesis applies to steroid hormones, thyroid hormone and vitamin D. For the vitamin D metabolites this hypothesis needs to be qualified in that some tissues, kidney and parathyroid glands in particular, express a transport system, the megalin/cubilin complex, that enables 25(OH)D bound to DBP to be transported into these cells. However, for cells lacking this complex the free fraction is felt to be the fraction capable of entering these cells. In serum samples from normal individuals, ~85% of circulating vitamin D metabolites are bound to DBP, whereas albumin with its substantially lower binding affinity binds only ~15% of these metabolites despite its 10-fold higher concentration than DBP. Approximately 0.4% of total 1,25(OH)2D and 0.02–0.03% of total 25(OH)D is free in serum from normal non-pregnant individuals. The fraction of ‘bioavailable’ vitamin D metabolites is composed of the fraction of the free vitamin D and the fraction bound to albumin, thus measuring around 15% in normal individuals. At this point there is little evidence that the albumin fraction is truly bioavailable. A simple strategy might be to estimate the free concentration based on measurements of DBP and total 25(OH)D with known binding constants of DBP for 25(OH)D. This has in fact been done, but as subsequent research has documented, this relationship is affected by numerous clinical conditions and the different DBP variants with different affinities for 25(OH)D.

DBP
DBP is a 51–58 kDa multifunctional serum glycoprotein synthesized primarily in the liver. Initially, isoelectric focusing migration patterns identified phenotypic variants termed Group-Specific Component (Gc), the most common of which are Gc1f, Gcs and Gc2. Two common missense point mutations (SNPs) in exon 11 of the DBP gene, rs7041 (G/T single-nucleotide variation) and rs4588 (an A/C single-nucleotide variation), result in the three most common isoforms with amino acid changes at positions 416 and 420: Gc1f (Asp416, Thr420), Gc1s (Glu 416, Thr420), and Gc2 (Asp416, Lys420). Gc2 is the least abundant and Gc1f the most abundant. The distribution of the Gc alleles varies by race. Black and Asian populations are more likely to carry the Gc1f form, whereas the Gc2 form is rare, whereas Whites more frequently express the Gc1s and the Gc2 alleles. Although affinities of these DBP variants for 25(OH)D appear to vary, the rank order remains controversial, and their contribution of total 25(OH)D levels and the relationship between free and total 25(OH)D is modest in comparison to differences influenced by clinical condition. In the absence of disease or pregnancy, DBP levels are relatively constant over time in adults. That said, various substances in the blood such as polyunsaturated fatty acids may alter the affinity of DBP for the vitamin D metabolites, as can various clinical conditions. Liver disease leads to reduced levels of DBP, as do protein-losing nephropathies and acute illness (DBP is an acute phase reactant), whereas DBP levels are elevated during the latter stages of pregnancy. Moreover, various clinical conditions appear to shift the relationship between free and total 25(OH)D seemingly independent of DBP levels or DBP haplotypes. Thus, the measurement of total 25(OH)D may not provide the best assessment of vitamin D status. Calculation of free 25(OH)D from DBP and total 25(OH)D measurements using affinity constants obtained by measurements in normal sera may be inaccurate, at least in some clinical situations. Therefore, direct measurement of free 25(OH)D would appear to offer information about vitamin D nutritional status that at least complements that of total 25(OH)D.

The free 25(OH)D assay
The original free 25(OH)D assay employed centrifugal ultrafiltration. This was a labour- and reagent-intensive assay suitable only for a dedicated research laboratory. However, it sufficed to determine free 25(OH)D levels in a number of patient groups including cirrhotics and pregnant women, providing proof of concept that the free 25(OH)D measurement would add to the assessment of vitamin D nutritional status. This assay has subsequently been superseded by a much simpler method capable of high throughput.

A two-step ELISA that directly measures free 25(OH)D levels was recently developed by Future Diagnostics Solutions using monoclonal antibodies from DIAsource Immunoassays. In the first incubation step, an anti-25(OH)D monoclonal antibody immobilized on a microtitre plate binds the free 25(OH)D in the serum sample. The serum is removed and biotinylated 25(OH)D in a known amount is added to react with the unoccupied binding sites on the monoclonal antibody attached to the plate. The non-bound biotinylated 25(OH)D is then removed followed by the addition of streptavidin peroxidase conjugate and the substrate 3,3ʹ,5,5ʹ-Tetramethylbenzidine (TMB). The bound streptavidin peroxidase can be quantified by measuring the absorbance at 450 nm generated in the reaction. The intensity is inversely proportional to the level of free 25(OH)D. The limit of detection is 2.8 pg/mL. The antibody in the current assay does not recognize 25(OH)D₂ as well as 25(OH)D₃ (77% of the 25(OH)D₃ value), and so it underestimates the free 25(OH)D₂. However, under most situations where the predominant vitamin D metabolite is 25(OH)D₃ this issue is not a major concern. The data for both normal subjects and those with different DBP levels (cirrhotics, pregnant women) compare quite well to those obtained from similar populations using the centrifugal ultrafiltration assay.

Clinical implications
In a study currently under review for publication we compiled data from over 1600 individuals in whom free 25(OH)D had been measured by this ELISA. The samples included sera from both normal subjects and those with a variety of clinical conditions and a variety of DBP alleles. In the nearly 1000 normal and community dwelling outpatient subjects the normal range for free 25(OH)D was established at 4.3±1.9 pg/mL with a mean total 25(OH)D of 21.9±9.9 ng/mL, providing a percent free 25(OH)D of 0.02%. These results are essentially identical to those reported by the author using centrifugal ultrafiltration 30 years ago. As expected, clinical conditions affecting DBP values made a big difference. Liver disease resulted in lower DBP levels and higher percentage free 25(OH)D resulting in the population of cirrhotics studied having among the highest free 25(OH)D despite the lowest total 25(OH)D. Nursing home patients also had unexpectedly high free 25(OH)D, higher than that of the cirrhotics, with only modest reductions in DBP levels. Pregnancy (third trimester), however, resulted in increased DBP levels and the lowest free 25(OH)D levels, although the free fraction was not lower than that of the normal subjects. Overall, these results indicate that the free fraction is altered by the clinical situation not only in terms of altered DBP levels but in the relationship between total and free 25(OH)D for any given DBP level. Therefore, it is recommended that the free 25(OH)D level needs to be measured directly if the free level is thought to have particular relevance to the clinical situation that cannot be captured by measuring total 25(OH)D.

At this point it is not yet clear whether the determination of free 25(OH)D is a better marker of vitamin D nutritional status and biologic action than the determination of total 25(OH)D. Using a convenient marker such as parathyroid hormone (PTH), much as we use thyroid-stimulating hormone (TSH) as a marker of thyroid status, is problematic. First of all PTH levels are controlled by calcium as well as
vitamin D. Second, regulation of PTH secretion is mediated primarily by the 1,25(OH)₂D produced within the gland itself (much as TSH secretion is controlled by triiodothyronine (T3) produced within the pituitary). Third, the parathyroid gland has the megalin/cubilin transport system to enable 25(OH)D bound to DBP to enter the cells, obviating any advantage free 25(OH)D might have in cell uptake. However, several studies have demonstrated a stronger correlation between free 25(OH)D and bone markers than that observed with total 25(OH)D. But at this point, determining the role that free 25(OH)D measurements play in the assessment of vitamin D nutrition and action requires further investigation.

Bibliography
1. Bikle DD. Vitamin D Assays. Front Horm Res 2018; 50: 14–30.
2. Malstroem S, Rejmark L, et al. Current assays to determine free 25-hydroxyvitamin D in serum. J AOAC Internl 2017; 100: 1323–1327.
3. Bikle D, Bouillon R, et al. Vitamin D metabolites in captivity? Should we measure free or total 25(OH)D to assess vitamin D status? J Steroid Biochem Mol Biol 2017; 173: 1054–1116.
4. Bikle DD, Malmstroem S, Schwartz J. Current controversies: are free vitamin metabolite levels a more accurate assessment of vitamin D status than total levels? Endo Clinics NA 2017; 46: 901–918.
5. Lai JC, Bikle DD, et al. Total 25(OH) vitamin D, free 25(OH) vitamin D, and markers of bone turnover in cirrhotics with and without synthetic dysfunction.  Liver Int 2015; 35: 2294–2300.
6. Schwartz JB,  Lai J, et al. A comparison of direct and calculated free 25-OH vitamin D levels in clinical populations. J Clin Endocrinol Metab 2014; 99: 1631–1637.

The author
Daniel D Bikle MD, PhD
VA Medical Center and University of
California San Francisco, San Francisco,
CA 94158, USA

E-mail: Daniel.bikle@ucsf.edu

Mitochondrial DNA mutations (mtDNA) have been described that are associated with leukemia. To identify somatic mutations it is necessary to have a control tissue from the same individual for comparison. In this review we describe a new next-generation sequencing approach to identify leukemia-associated mtDNA mutations by using remission samples as control.

by Dr Ilaria Stefania Pagani

Introduction
The identification of acquired somatic mutations in leukemic samples is of considerable importance for diagnosis and prognostication. In order to identify somatic mutations it is necessary to have a control tissue from the same individual for comparison. Non-hematopoietic tissues, such as mesenchymal stromal cells (MSCs) or hair follicles are preferred, but not always available. When patients with leukemia achieve remission, the remission peripheral blood (PB) may be a suitable and easily available control tissue. This article will provide recommendations for the identification of tumour-associated mtDNA somatic mutations, highlighting advantages and disadvantages of the method.

mtDNA characteristics
Human mitochondrial (mt) DNA is a 16 569 bp double-stranded, circular DNA molecule that encodes 13 polypeptides of the oxidative phosphorylation system (OXPHOS), 22 transfer RNAs and 2 ribosomal RNAs. Several important differences between the mt genome and the nuclear genome complicate the study of mtDNA mutations. Ninety-three percent of the sequence consists of coding DNA, introns are absent, the only non-coding region is at the level of the D-loop containing the promoters of the genes and it is maternally inherited. Each cell has a variable number of mitochondria (typically several hundred) and each mitochondrion contains a variable number of genomes (typically 2–10). Consequently, mtDNA mutations do not follow the pattern of a diploid genome: rather, a cell may have a single mt genotype (homoplasmy) or multiple mt genotypes (heteroplasmy). Heteroplasmy may be at any frequency, could vary between cells and many variants will be below the limit of detection of Sanger sequencing, and therefore technically difficult to validate [1]. To date, more than 400 mtDNA mutations have been associated with human diseases, most of them being heteroplasmic. Therefore, an accurate determination of the level of heteroplasmy is important for disease association studies [2].

mtDNA mutations and cancer
MtDNA mutations may potentially contribute to a cell to becoming cancerous, leading to invasion and metastasis [3]. Heteroplasmic somatic mtDNA mutations have been reported in hematological neoplasms, including myelodysplastic syndromes, chronic lymphocytic leukemia, chronic myeloid leukemia (CML), acute myeloid leukemia, and acute lymphoblastic leukemia (ALL) [1]. Many cancer types, including leukemia, have a tendency to be highly glycolytic, increasing the production of the reactive oxygen species (ROS), that lead to genomic instability. The mtDNA genome is susceptible to ROS-induced mutations owing to the high oxidative stress in the mitochondrion and limited DNA-repair mechanisms [3]. The identification of acquired somatic mutations in leukemic samples is of considerable importance for diagnosis and prognostication. In a study in acute myeloid leukemia, for example, patients with mutated NADH dehydrogenase subunit 4 (ND4) showed greater overall survival than patients with wild-type ND4 [4].

mtDNA somatic mutations: the problem of control tissue
MtDNA acquires somatic mutations at a rate 10-fold higher than nuclear DNA, so mtDNA single nucleotide variants (SNVs) accumulate with age, and may be tissue-specific [5]. This means that there is no absolutely reliable source of ‘germline’ mtDNA, especially in older individuals [1]. Somatic mutations must be distinguished from non-pathogenic germline variants by comparison with a control tissue sample. Non-hematopoietic tissues, such as buccal cells, hair follicles or MSCs  are  preferred, but not always available. PB cells from a post-treatment remission sample may be used as alternative. This method is widely used for nuclear mutations, but less commonly for mt mutations [1]. Blood samples are readily accessible from leukemia patients who achieve morphological remission after treatment. Therefore, a method for the detection of leukemia-associated mtDNA mutations based on comparison with a remission sample may be useful.
A new approach to identify mtDNA somatic mutations at diagnosis by using remission samples as control tissue
Pagani IS and colleagues developed a next-generation sequencing (NGS) approach for the identification of leukemia-associated mtDNA mutations using samples from CML patients at diagnosis and in remission following treatment with tyrosine kinase inhibitors (TKIs) [1]. This approach could also be applied to both hematopoietic and  non-hematopoietic cancers, such as epithelial tumours, in which a tumour biopsy specimen can be compared with the normal mucosa.

Twenty-six chronic phase CML patients enrolled in the Australasian Leukaemia and Lymphoma Group CML9 trial (TIDEL-II; ID: ACTRN12607000325404) [6] took part in the study [6]. PB samples from leucocytes at diagnosis before commencing TKI treatment, and remission after 12 months of therapy were compared. Hair follicles (n=4), bone marrow MSCs (n=18), or both (n=4) were used as non-hematopoietic control samples. The comparison of a diagnostic sample with a non-hematopoietic control tissue is the standard method to identify somatic mutations in leukemia [1]. The concordance between this classic method and the diagnosis versus remission approach has been investigated.

NGS assay for the mt genome
The workflow chart is represented in Figure 1. Briefly the genomic DNA (comprising a mixture of nuclear and mtDNA) was extracted by a phenol/chloroform method from PB leukocytes and non-hematopoietic tissues. The mtDNA was amplified by long-range PCR, generating two or three overlapping fragments covering the entire mt genome. The PCR amplicons were then pooled at equimolar concentrations and sequencing libraries were prepared using the Nextera XT kit (Illumina). Indexed libraries were multiplexed and run on an Illumina MiSeq instrument using the 600 cycle MiSeq Reagent kit (v3) generating 300-bp paired-end reads [1].
Somatic mutation calling from high-throughput sequencing datasets and validation
The majority of the variant-calling methods in use are based on low-coverage human re-sequencing data and diploid calls with discrete frequencies of interest (0%, 50% or 100%) [7, 8]; however, these assumptions do not apply to mtDNA. The LoFreq software (loFreq-star version 2.11, genome Institute of Singapore; http://csb5.github.io/lofreq/) was chosen because it was developed for viral and bacterial genomes as well as diploid data, and because of its ability to automate comparison with a matched control tissue for the detection of somatic mutations [8]. The revised Cambridge Reference Sequence (rCRS) for the human mt genome (NC_012920) was used as reference sequence to identify SNVs. Tumour tissue (test) and control were then compared to identify somatic mutations specific only for the tumour tissue.  Variants in common between the test and the control sample were considered to represent germline polymorphisms or mutations and were filtered out by the software. A binomial test was applied to the remaining variants to determine whether an apparent difference between samples could be due to inadequate read coverage in the control. Variants passing the binomial test were retained in the final list of putative somatic mutations (Fig. 2a) [8]. The identified mutations should be considered putative and, in common with most other NGS strategies for the discovery of novel mutations, any specific mutation of clinical interest would need to be confirmed using an independent method, as Sanger sequencing (limit of detection 20%), Sequenom MassArray, digital array (Fluidigm) or another NGS platform.

NGS: error rate, false positives and threshold
Before the application of NGS technologies, no evidence of heteroplasmy was detected, probably because of the lower sensitivity of earlier techniques [9]. NGS technologies enable the inquiry of mt heteroplasmy at the genome-wide scale with much higher resolution because many independent reads are generated for each position [2]. However, the higher error rate associated with the more sensitive NGS methodology must be taken into consideration to avoid false detection of heteroplasmy. Short-read sequencing technologies (like in Illumina systems) have a high intrinsic error rate (approximately 1 in 102–103 bases) when applied at the very high depth required to detect and measure low-level heteroplasmy. Thus, appropriate criteria for avoiding false positives due to sequencing errors are required. The most obvious way to distinguish between sequencing errors and heteroplasmy is to invoke a threshold. Two duplicate sequencing run, of which one was ultra-deep (validation run), were compared to determine sensitivity (proportion of true positives that are correctly identified as such) and specificity (proportion of true negatives that are correctly identified as such). An empirical threshold of 2% was therefore applied to distinguish true variants from sequencing errors. Variants with a variant allele fraction (VAF, the variant allele’s read depth divided by total read depth at each nucleotide position) between 2 and 98% where then considered as heteroplasmic, and variants with a VAF >2% were called homoplasmic [1]. This threshold could be refined by an iterative process in which a different threshold is identified for each nucleotide position [10], as some variation in error rate was observed. The incorporation of molecular barcodes in the initial long-range PCR would also  reduce the risk of false-positive mutations due to PCR artefact [1].

Remission samples as control tissue in the identification of the mtDNA somatic mutations at diagnosis
In the four patients who had both MSC and hair follicle DNA available as control tissue, the same mutations at diagnosis have been identified, therefore the results using the non-hematopoietic tissues as control were combined. Remission samples were then used as control tissue to determine mtDNA somatic mutations at diagnosis, and the concordance between this method and the conventional diagnosis versus the MSC/hair follicle approach was examined. Seventy-three somatic mutations (81%) were identified in common, 11 mutations (12%) were identified only in comparison with the non-hematopoietic control, and six (6.7%) only by comparison with remission samples (Fig. 2b) [1]. Divergent results occurred as the result of differences in read quality or depth at a specific nucleotide not reaching statistical significance in the algorithm. False-negative results could be encountered using remission samples as the control tissue, because of low-level heteroplasmic mutations in the control sample that would lead to the same mutation at diagnosis being removed through filtering.

Concluding remarks
Remission samples can be used as control tissues to detect candidate mtDNA somatic mutations in leukemic samples when non-hematopoietic tissues are not available. The presence of mutations at low VAF in the remission samples in common with the diagnosis tissue, could be filtered out by the LoFreq software leading to false-negative results. Therefore visual inspection of the unfiltered variants is recommended.

References
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2. Li M, Schonberg A, Schaefer M, Schroeder R, Nasidze I, Stoneking M. Detecting heteroplasmy from high-throughput sequencing of complete human mitochondrial DNA genomes. Am J Hum Genet 2010; 87(2): 237–249.
3. van Gisbergen MW, Voets AM, Starmans MH, de Coo IF, Yadak R, Hoffmann RF, Boutros PC, Smeets HJ, Dubois L, Lambin P. How do changes in the mtDNA and mitochondrial dysfunction influence cancer and cancer therapy? Challenges, opportunities and models. Mutat Res Rev Mutat Res 2015; 764: 16–30.
4. Damm F, Bunke T, Thol F, Markus B, Wagner K, Gohring G, Schlegelberger B, Heil G, Reuter CW, et al. Prognostic implications and molecular associations of NADH dehydrogenase subunit 4 (ND4) mutations in acute myeloid leukemia. Leukemia 2012; 26(2): 289–295.
5. Gattermann N. Mitochondrial DNA mutations in the hematopoietic system. Leukemia 2004; 18(1): 18–22.
6. Yeung DT, Osborn MP, White DL, Branford S, Braley J, Herschtal A, Kornhauser M, Issa S, Hiwase DK, et al. TIDEL-II: first-line use of imatinib in CML with early switch to nilotinib for failure to achieve time-dependent molecular targets. Blood 2015; 125(6): 915–923.
7. Meldrum C, Doyle MA, Tothill RW. Next-generation sequencing for cancer diagnostics: a practical perspective. Clin Biochem Rev 2011; 32(4): 177–195.
8.  Wilm A, Aw PP, Bertrand D, Yeo GH, Ong SH, Wong CH, Chiea CK, Rosemary P, Martin LH, Niranjan N. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res 2012; 40(22): 11189–11201.
9. Chatterjee A, Dasgupta S, Sidransky D. Mitochondrial subversion in cancer. Cancer Prev Res 2011; 4(5): 638–654.
10. Kerpedjiev P, Frellsen J, Lindgreen S, Krogh A. Adaptable probabilistic mapping of short reads using position specific scoring matrices. BMC Bioinformatics 2014; 15: 100.

The author
Ilaria Stefania Pagani^1,2 PhD
¹Cancer Theme, South Australian Health & Medical Research Institute, Adelaide, Australia
²School of Medicine, Faculty of Health Sciences, University of Adelaide, Adelaide, Australia

*Corresponding author
E-mail: Ilaria.pagani@sahmri.com

The award recognizes the ‘care, dedication and commitment’ of ordinary people in the battle against HIV/AIDS.

A 28-year-old African community youth worker, Evah Namakula, has won the first, global CARES HIV/AIDS award, designed to recognize ordinary people who have shown ‘care, dedication and commitment’ in their communities as part of the fight against the disease. Ms Namakula was also part of the 2018 award launch at the International Aids Society meeting in Amsterdam (July 23 – 27).
In its first year, the CARES award focused on the dedication of ordinary people in Africa, one of the areas in the world most affected by HIV/AIDS. The award has two categories of winners – an individual, Ms Namakula, and an organization, the Hillcrest AIDS Centre Trust (HACT). This is a South African charity that cares for some of the poorest and most disadvantaged people in Africa. 
Each year the winning organization will receive a grant of 5500 USD provided by Beckman Coulter Life Sciences through the Beckman Coulter Foundation.  The grant is made in the name of the individual winner, but their work cannot be linked.  
An independent judging panel described as ‘remarkable’ Ms Namakula’s achievements in her local Ugandan community to dispel the stigma of HIV/AIDs. She is also global youth ambassador for Reach Out Integrity (ROI) Africa, where she helps to promote health and sexual responsibility to young people. Evah has recently founded her own charity, IGNITE, to carry her work forward.
Ms Namakula is part of the Young African Leaders Initiative (YALI) set up by President Barak Obama to empower leadership skills in African youth. As a YALI volunteer, she has been working as a leadership mentor in local communities and schools, helping to develop public speaking skills.
Inspired as a child by the determination of her mother and siblings, Evah said: “l had already become a campaigner, but it was while l was working in my local hospital laboratory that I realized how I could use my medical knowledge to reduce the myth young people in my community had about HIV/AIDs.”
“Evah is an inspirational young woman and will be a hard act to follow,” said Samuel Boova, Beckman Coulter’s Director Alliance Development, High Burden HIV Markets. “She is exactly the kind of youth leader that President Obama wanted to encourage to develop the Africa of the future and we are honoured not only to have her as our first winner, but to have her support in launching the global initiative.
“The award gives a platform to the work and stories of those we see as the unsung heroes of individual communities. These are people who have shown individual dedication, commitment and courage or who have made a difference in the battle against HIV/AIDS. 
“However, it is not just the final winner we want to publically recognize. We hope the award will encourage communities to learn about and honour the work of every nominee, so that more people will come forward to help and support those living with HIV/AIDS.”
Potential candidates for the CARES award can be nurses, healthcare workers, national coordinators, lab scientists and even clinicians. It could include lay people who are active in community outreach work or a social worker providing AIDS counselling.
CARES supports the UNAIDS 90-90-90 target to ensure that by the year 2020, 90% of people living with HIV will know their status, 90% of those with diagnosed HIV infection will receive sustained antiretroviral therapy, and 90% of all people receiving antiretroviral therapy will have viral suppression.
It focuses on encouraging innovative solutions for the monitoring of HIV and AIDS treatment. It was inspired by the work of Professor Debbie Glencross, a leading South African laboratory pathologist, who found an inexpensive way to measure a patient’s CD4 count, a special type of white blood cell that can indicate how compromised a person’s immune system might be. Prof Glencross is Director and Principle Pathologist in the Flow Cytometry unit of the Department of Hematology at the Charlotte Maxeke Johannesburg Academic Hospital. 
Monitoring a patient’s immune system by counting the CD4 cells has to be carried out by laser technology in a special blood analyser, the flow cytometer. However, in many parts of rural Africa, the equipment and infrastructure simply hasn’t been available to test patients, get their blood samples to a laboratory, and then report the results. As Prof Glencross explained: “We are working to empower smaller community laboratories so that they can extend the availability of the test to meet demand while still meeting the requirements of the National Health Laboratory Service. This will enable best clinical and laboratory practice while reducing the time it takes to deliver the result.”
When counting CD4 cells, large global hospital labs first differentiate between the types of white blood cells, count them and then work out the number of CD4 cells in each millionth of a liter of blood. While accurate, this method can be laborious. In contrast, rather than going through the time-consuming and costly process of isolating individual antibodies, Glencross’s ingenious approach uses a mathematical equation. She realized that using the white cell count as a stable reference point would eliminate the need for additional quality control steps, while still maintaining standards.

http://info.beckmancoulter.com/CARES-Award-Intro