The early diagnosis of common colds caused by coronavirus is a crucial step in preventing the recurrence of a global outbreak. The goals of this article are to discuss a prokaryotic-expressed recombinant nucleocapsid protein used in the development of a sensitive diagnostic assay for the diagnosis of human coronavirus infection.
by Dr Ming-Hon Hou
An overview of coronavirus
Human coronavirus (HCoV) was identified in the 1960s and has generally been associated with symptoms of the common cold. Although HCoV infections are generally mild, more severe upper and lower respiratory tract infections, such as bronchiolitis and pneumonia, which are particularly severe in infants, elderly individuals, and immunocompromised patients, have been documented. There have also been reports of clusters of HCoV infections that cause pneumonia in adults. In addition, a previous study reported that the neurotropism and neuroinvasion of HCoV are associated with multiple sclerosis.
In recent years, several emerging human coronaviruses have been discovered, and between 2003 and 2004, the SARS-CoV outbreak caused a worldwide epidemic that had a significant economic impact in countries where the disease outbreak occurred. Phylogenetic analyses have shown that SARS-CoV contains sequences that are closely related to sequences found in the betacoronaviruses. In 2004, another alphacoronavirus, HCoV-NL63, which was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis, was reported in the Netherlands. In 2005, a novel betacoronavirus, HKU1 was found in patients with respiratory tract infections. Recently, a novel SARS-like coronavirus was found in patients with respiratory tract infections in the Middle East.
The RNA genomes of coronaviruses include genes encoding the structural proteins S (spike), M (matrix), E (envelope), and N (nucleocapsid). Additionally, some coronaviruses encode a third glycoprotein, HE (hemagglutinin-esterase), which is present in most of the betacoronaviruses. A helical nucleocapsid exists in the centre of the viral particle. The primary function of CoV N protein (NP) is to recognize a stretch of RNA that serves as a packaging signal, leading to the formation of the ribonucleoprotein (RNP) complex or to a long helical nucleocapsid structure during viral assembly. The formation of the RNP is important for maintaining the RNA in an ordered conformation suitable for replication and transcription of the viral genome. The CoV NP was shown to be involved in the regulation of cellular processes, such as gene transcription, actin reorganization, host cell cycle progression, and apoptosis.
Coronaviruses cause colds of mild to moderate severity and are transmitted by aerosols of respiratory secretions, the fecal–oral route, and mechanical transmission. The most common symptoms of coronavirus infection are nasal catarrh and a sore throat, and the illness typically lasts approximately 6 to 8 days. The early diagnosis of common colds caused by a coronavirus is an important step in preventing the recurrence of a global outbreak. Previously, rapid viral diagnosis has also been critical in the control of epidemics and the management of SARS patients. HCoVs are difficult to detect, and the current diagnosis of coronaviral infection is based on reverse transcription polymerase chain reaction with real-time PCR and antibody detection.
Previous studies have shown that NPs are the immunodominant domain in hosts infected with several coronaviruses. Additionally, it has been shown that NPs can accumulate intracellularly before being packaged into mature viruses and are the most abundant viral protein. NP is involved in the pathological reaction to human coronavirus and is a key antigen for the development of a sensitive diagnostic assay. These characteristics make NP a suitable candidate for the early diagnosis of coronavirus infection.
Nucleocapsid protein for coronavirus serodiagnosis
NP is involved in the pathological reaction to CoV infection and has been used in the development of a sensitive diagnostic assay. Previous studies reported that NP can be detected in the serum samples of SARS patients as early as 1 day after disease onset. Prokaryotic-expressed NPs have successfully been used as antigens for the detection of antibodies specific to many viruses, including SARS-CoV and several animal coronaviruses, and were produced for establishing an antigen-capture ELISA (or Western blot assay) for the diagnosis of HCoV infection
These methods are highly sensitive and specific. For example, Shi et al. [10] used recombinant SARS-CoV NP to establish an antigen-capture ELISA for SARS diagnosis. Anti-NP antibodies could be detected in approximately 90% of SARS patients 11 to 61 days after illness. No false positives were observed in non-SARS patients or health care workers.
An immunofluorescence assay is the gold standard for the detection of SARS. However, it requires efficient SARS-CoV replication in vitro to use whole virus or infected cell lysates as antigens. There are several reasons for selecting a recombinant protein rather than whole virus for this assay. The prokaryotic expression system is high yield, inexpensive, highly efficient, does not require viral cultures, and is non-toxic. Despite these advantages, viral proteins expressed in prokaryotic cells lack post-translational modifications that are present in proteins expressed in baculovirus expression systems.
Using recombinant nucleocapsid protein as an antigen for coronavirus infection diagnosis: one recent case study
HCoV is distributed worldwide. Recently, we produced soluble recombinant human coronavirus OC43 (HCoV-OC43) NP to analyse the antigenicity of the betacoronavirus HCoV-OC43 NP. To express soluble HCoV-OC43 NP as a set of fusion proteins in E. coli, the NP gene was cloned into the pET-28a expression vector. His-tagged NP was purified from the soluble fraction using Ni-NTA column chromatography [Figure 1]. The yield from 1 L of bacterial culture was as large as 10 mg of pure NP after extraction and column chromatography. A recombinant protein-based Western blot assay was used to screen human serum from young adults, middle-aged and elderly patients with respiratory infection symptoms and cord blood units.
Western blotting is generally accepted as the most effective method for unequivocally locating linear or continuous immunodominant epitopes. Pohl-Kooppe et al. [8] also reported that Western blotting is a more sensitive test system than an immunofluorescence assay for the analysis of sera from pediatric groups. Our results showed that approximately 80–90% of serum samples from young adults and middle-aged and elderly patients with respiratory infections reacted strongly to the HCoV-OC43 NP, indicating prior exposure to this disease. In addition, the serum samples tested in this study were 81% seropositive for HCo-229E NP [Fig. 2].
This finding is consistent with previous epidemiological surveys that concluded that seroprevalence increases rapidly during childhood, attaining a seroprevalence rate of up to 90% in adults. Additionally, antibodies against HCoV-OC43 NP were detected in over 90% of cord blood samples tested. Maternally acquired antibodies may help to protect a newborn baby from HCoV-OC43 infection, although this protection appears to wane by 4 to 5 months of age. HCoV is responsible for approximately 30% of all common colds, and it is expected that 80–90% of serum samples from healthy donors and patients have antibodies to HCoV-OC43.
CoV NPs contain multiple immunodominant epitopes and antigenic sites. To compare the immunoreactivity of the three structural regions of HCoV-OC43 NP, three truncated recombinant fragments [aa 1–173 (the N-terminal domain), aa 174–300 (the central region), and aa 301–448 (the C-terminal domain)] were produced in E. coli; these regions were chosen based on PONDR (predictor of naturally disordered regions) predictions. The reactivity of human serum against these fragments was determined through Western blotting. The human serum reacted strongly with the central region and the C-terminal domain of the NP, whereas the N-terminal domain demonstrated low reactivity with the antibody. The findings of the current study are consistent with those of Chen et al. [2], who found that the antigenicity of the C-terminus of SARS-CoV NP was higher than that of the N-terminus.
The polyclonal antibody against coronavirus NP could be used to develop a rapid, easy and specific diagnostic tool for the detection of HCoV infections through immunofluorescence or ELISA-based tests. Many studies have reported that NP polyclonal antibody does not cross-react with other human CoV NPs, including those of SARS-CoV and HCoV-229E, despite the presence of highly conserved motifs in these coronavirus NPs. Previous studies also showed that the anti-SARS CoV NP and anti-HCoV-229E NP polyclonal antibodies did not cross-react with other human CoV NPs.
In our recent studies, using purified recombinant NP as an antigen, a polyclonal antibody was generated from rabbit serum with specificity for HCoV-OC43 NP; this antibody reacted specifically with HCoV-OC43 NP and did not cross-react with other human CoV NPs (including those of SARS-CoV and 229E) through Western blotting.
Conclusion
A novel SARS-like coronavirus was found in patients with respiratory tract infections in the Middle East. Thus, new and convenient diagnostic methods for CoV infection are urgently needed. The prokaryotic expression of recombinant HCoV NP is suitable for high-sensitivity, highly specific antibody production and can be used for the epidemiological screening of HCoV infection in the future.
References
1. Che XY, Qiu LW, Liao ZY, Wang YD, Wen K, Pan YX, Hao W, Mei YB, Cheng VC, Yuen KY. Antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229E and OC43. J Infect Dis 2005; 191: 2033–7.
2. Chen Z, Pei D, Jiang L, Song Y, Wang J, Wang H, Zhou D, Zhai J, Du Z, Li B, Qiu M, Han Y, Guo Z, Yang R. Antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein. Clinical Chem 2004; 50: 988–95.
3. He Q, Chong KH, Chng HH, Leung B, Ling AE, Wei T, Chan SW, Ooi EE, Kwang J. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin Diagn Lab Immunol 2004; 114: 417–22.
4. Huang CY, Hsu YL, Chiang WL, Hou MH. Elucidation of the stability and functional regions of the human coronavirus OC43 nucleocapsid protein. Protein Sci 2009; 18: 2209–18.
5. Huang LR, Chiu CM, Yeh SH, Huang WH, Hsueh PR, Yang WZ, Yang JY, Su IJ, Chang SC, Chen PJ. Evaluation of antibody responses against SARS coronaviral nucleocapsid or spike proteins by immunoblotting or ELISA. Journal Med Virol 2004; 73: 338–46.
6. Liang FY, Lin LC, Ying TH, Yao CW, Tang TK, Chen YW, Hou MH. Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protein by Western blot: Implications for the diagnosis of coronavirus infection. J Virol Methods 2013; 187: 413–20.
7. Mourez T, Vabret A, Han Y, Dina J, Legrand L, Corbet S, Freymuth F. Baculovirus expression of HCoV-OC43 nucleocapsid protein and development of a Western blot assay for detection of human antibodies against HCoV-OC43. J Virol Methods 2007; 139: 175–80.
8. Pohl-Koppe A, Raabe T, Siddell SG, ter Meulen V. Detection of human coronavirus 229E-specific antibodies using recombinant fusion proteins. J Virol Methods 1995; 55: 175–83.
9. Shao X, Guo X, Esper F, Weibel C, Kahn JS. Seroepidemiology of group I human coronaviruses in children. J Clin Virol 2007; 40: 207–13.
10. Shi Y, Yi Y, Li P, Kuang T, Li L, Dong M, Ma Q, Cao C. Diagnosis of severe acute respiratory syndrome (SARS) by detection of SARS coronavirus nucleocapsid antibodies in an antigen-capturing enzyme-linked immunosorbent assay. J Clin Microbiol 2003; 41; 5781–2.
11. Timani KA, Ye L, Zhu Y, Wu Z, Gong Z. Cloning, sequencing, expression, and purification of SARS-associated coronavirus nucleocapsid protein for serodiagnosis of SARS. J Clin Virol 2004; 30: 309–12.
The author
Ming-Hon Hou PhD
1 Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
2 College of Life Science, National Chung Hsing University, Taichung, Taiwan
3 Institute of Genomics and Bioinformatics, National Chung Hsing University,
Taichung, Taiwan
E-mail: mhho@dragon.nchu.edu.tw
Genetic diagnostics in pediatric hearing loss
, /in Featured Articles /by 3wmediaHearing impairment in newborn children is one of the most frequent forms of sensorineural disorders, affecting 1 in 1000 infants. In half of the cases the hearing loss has a genetic basis, and over 70 genes have been identified so far, making hearing loss genetically exceptionally heterogeneous. Early detection in newborns, in combination with a genetic diagnosis is critical for the selection of a proper intervention and the development of speech, language and communication skills.
by Dr Isabelle Schrauwen
Hearing impairment in infants can be due to environmental influences such as cytomegalovirus infection, but in industrialized countries, however, most cases of early-onset hearing impairment have a genetic basis. Genetic hearing loss is non-syndromic in 70% of cases, whereas other symptoms (apart from hearing loss) are noticeable in 30% of cases (syndromic hearing loss). Autosomal recessive non-syndromic hearing loss (ARNSHL) is most common (80%) and is typically prelingual in onset, and autosomal dominant non-syndromic hearing loss (ADNSHL), X-linked and mitochondrial hearing loss are less frequent (20 and <1% respectively). To date, over 70 genes have been found to be implicated in non-syndromic hearing loss (NSHL), of which 40 are autosomal recessive. The most frequent causes of ARNSHL in most populations are mutations in GJB2, with a frequency ranging from 10 to 50% of all ARNSHL cases.
The implementation of newborn hearing screening in many countries has lead to an early detection of hearing loss and deafness in infants. This, together with improved genetic diagnostics and neuroimaging, has lead to a better understanding and better intervention of hearing loss overall [1].
The importance of a genetic diagnosis in pediatric hearing impairment
Clinical tests are not always sufficient for an accurate diagnosis and genetic diagnostics can provide answers that clinical tests cannot. Identification of the genetic cause can help predict the progression of the hearing loss and also direct the choice of the most appropriate treatment or method of communication. In addition, some apparent forms of non-syndromic hearing loss can be diagnosed to be syndromic as they give other symptoms at a later age (such as goitre in Pendred syndrome or retinitis pigmentosa in Usher syndrome). For Usher syndrome, preventative measures can be taken including sunlight protection and vitamin therapy to minimize the rate of progression of retinitis pigmentosa [2]. Furthermore, autosomal recessive mutations in GJB2 often cause a stable form of hearing loss and patients usually have good prospects with a cochlear implant. Knowing the gene responsible can also be very important to the parents, reducing their feelings of guilt and predicting the likelihood of subsequent children having hearing loss.
In addition, more extensive screening will also be very useful in providing a more accurate picture of the prevalence of different types of deafness affecting people across the world. Finally, advances in molecular and cellular therapies for hearing loss are also gene-specific [3], and identification of the genetic cause is key.
Gene-specific sequencing
Until recently, routine molecular diagnostics for hearing impairment consisted of the gene-specific sequencing of certain deafness genes, mainly with Sanger sequencing. GJB2 testing is offered most frequently in routine diagnostics, as it is responsible for a large number of ARNSHL cases. When there is evidence of progression of the hearing loss, or the presence of a goitre, an enlarged vestibular aqueduct (EVA), or Mondini dysplasia, SLC26A4 will be analysed, and when a specific phenotype is seen, other genes might also be analysed (OTOF, TECTA, COCH, WFS1, or a mitochondrial mutation). The selection criteria are typically: (1) high frequency cause of deafness (i.e. GJB2); (2) association with another recognizable feature (i.e. SLC26A4 and EVA); or (3) a recognizable
audioprofile (i.e. WFS1) [4].
Syndromic forms of deafness usually only have one or a few candidate genes responsible for each syndrome. However, for non-syndromic deafness, it is very difficult, and often impossible, to determine candidate genes because of the large number of causative genes leading to a relatively indistinguishable phenotype. GJB2 sequencing will identify 10–50% of ARNSHL cases, but the remaining cases of hearing loss display a high degree of genetic heterogeneity and unless a specific audioprofile is present it is hard to diagnose these with a gene-specific test. Traditionally, with gene-specific tests, it has therefore been difficult to establish a genetic diagnosis due to extreme genetic heterogeneity and a lack of phenotypic variability.
Microarrays
The analysis of multiple mutations in several genes in parallel was made possible by the development of single nucleotide extension microarrays [5]. These microarrays detect a specific mutation by hybridizing primers to patient DNA, followed by a single base extension. This technology therefore only detects known mutations, and a panel of 198 mutations in 8 genes [GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5 and the mitochondrial genes encoding 12S rRNA and tRNA-Ser(UCN)] underlying sensorineural (mostly non-syndromic) hearing loss has been developed [5]. Although new mutations cannot be picked up, this technique can provide some additional diagnostic value in GJB2 negative cases.
An Affymetrix resequencing microarray capable of resequencing 13 genes mutated in NSHL was also developed (GJB2, GJB6, CDH23, KCNE1, KCNQ1, MYO7A, OTOF, PDS, MYO6, SLC26A5, TMIE, TMPRSS3, USH1C) [6], but the number of genes here is also limited and specific kinds of mutations such as insertion/deletion (indel) mutations cannot be detected accurately.
Custom gene enrichment with next-generation sequencing
The need for new and better diagnostic methods for extremely heterogeneous diseases has been filled by the availability of next-generation sequencing, which has made it possible to sequence a large number of genes at the same time. This has lead to an immense growth of custom hearing-loss gene panels. Several labs have adopted this approach in-house already [7–9], and several labs offer this test for ARNSHL, ADNSHL, some cases of syndromic hearing loss, or all of the above.
The most commonly available systems for massive parallel sequencing are: Illumina, 454, or SOLiD. The Illumina platform is the most widely used platform to date and relies on cyclic reversible termination technology. Before massive parallel sequencing, DNA will be enriched for a custom selection of hearing-loss genes. In a diagnostic setting, sensitivity and specificity are important, and different enrichment methods perform differently in these criteria. Capture enrichment methods have been used more often and are easy to use, but PCR-based methods seem to have a better performance. A portion of targeted bases in repetitive regions cannot be captured, whereas PCR is able to enrich 100% of the desired target area. This is crucial to the sensitivity of detecting variants.
Although PCR-based techniques are usually more labour-intensive, microdroplet PCR methods have improved this greatly [9]. By using barcoding, custom hearing-loss panels are now offered for a competitive price in several labs across the world, and depending on the genes included in the panel, will offer a genetic diagnosis in the majority of cases.
Exome sequencing
Exome sequencing is also emerging as a diagnostic tool for many diseases and has decreased in price significantly in recent years. Exome sequencing targets every coding exon in the genome for enrichment prior to next-generation sequencing. Though current exome kits provide insufficient target enrichment in a diagnostic setting for deafness [9], as the regions of interest might not been completely covered and coverage depth may not be high enough for a diagnostic setting. Exome sequencing has therefore a decreased sensitivity to detect mutations in known genes compared to the custom panels available, but does allow the identification of new genes. In addition, given the amount of data that arises from exome sequencing, identification of the causative mutation among the list of variants will be more challenging. Although over 70 genes have already been discovered, there are still many more to be found, and the identification of new genes will greatly improve our understanding of deafness. Since its introduction, exome sequencing has lead to a fast rise in the identification of hearing-loss-related genes.
Future techniques and conclusions
Other technologies, such as Ion torrent, Pacific Biosystems, and specifically the emerging Oxford Nanopore technique, might offer very cost-effective sequencing methods for the future of molecular diagnostics in many diseases. Furthermore, genome sequencing might be shown useful in the diagnosis of hearing loss if the price of sequencing keeps dropping.
In conclusion, a genetic test ideally has to be sensitive, specific, accurate and low in cost. Gene-specific analysis of GJB2 will detect a 10–40% of ARNSHL cases, and custom gene panels with next-generation sequencing will provide a diagnosis in the majority of genetic hearing-loss cases. It is anticipated that within the coming years genetic testing will be routinely implemented in pediatric hearing loss, leading to better intervention and choice of treatment.
References
1. Paludetti G, et al. Infant hearing loss: from diagnosis to therapy Official Report of XXI Conference of Italian Society of Pediatric Otorhinolaryngology. Acta Otorhinolaryngol Ital 2012; 32: 347–70.
2. Hamel C. Retinitis pigmentosa. Orphanet J Rare Dis 2006; 1: 40.
3. Hildebrand MS, et al. Advances in molecular and cellular therapies for hearing loss. Mol Ther 2008; 16: 224–36.
4. Hilgert N, et al. Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? Mutation Res 2009; 681: 189–96.
5. Gardner P, et al. Simultaneous multigene mutation detection in patients with sensorineural hearing loss through a novel diagnostic microarray: a new approach for newborn screening follow-up. Pediatrics 2006; 118: 985–94.
6. Kothiyal P, et al. High-throughput detection of mutations responsible for childhood hearing loss using resequencing microarrays. BMC Biotechnol 2010; 10: 10.
7. Shearer AE, et al. Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing. Proc Natl Acad Sci U S A 2010; 107: 21104–9.
8. Brownstein Z, et al. Targeted genomic capture and massively parallel sequencing to identify genes for hereditary hearing loss in Middle Eastern families. Genome Biol 2011; 12: R89.
9. Schrauwen I, et al. (2013) A sensitive and specific diagnostic test for hearing loss using a microdroplet PCR-based approach and next generation sequencing. Am J Med Genet A 2013; 161A: 145–52.
The author
Isabelle Schrauwen PhD 1,2
1 Department of Medical Genetics, University of Antwerp, Antwerp, Belgium
2 The Translational Genomics Research Institute (TGen), Phoenix, AZ, USA
E-mail: isabelle.schrauwen@ua.ac.be
Molecular techniques used in the diagnosis of cutaneous lymphoma
, /in Featured Articles /by 3wmediaCutaneous lymphomas are a heterogenic group of conditions often difficult to diagnose. The diagnosis requires careful correlation between clinical presentation pathology and molecular analysis. Molecular analysis includes inmunophenotyping, clonality assays and rarely chromosomal analysis. The importance of molecular analysis hinges on two main reasons: firstly to confirm the diagnosis and secondly to further characterize the nature of the lymphoma. In addition, molecular analysis may provide some further insight on the origin of the malignancy, for example if it is primarily cutaneous or if the skin is a secondary site of involvement.
by Dr Belén Rubio González and Dr Joan Guitart
Recently, cancer has been defined by unlimited growth of cells derived from a single mutated cell or a clonality expansion. The detection of a monoclonal population may help to distinguish a lymphoma from a reactive process. However, on the one hand, clonality by itself does not imply malignancy and, on the other hand, a negative clonality result does not rule out a malignant condition. During this process, genes encoding the antigen receptor immunoglobulin (Ig) for B cells and the T-cell receptor (TCR) for T cells are rearranged as commonly seen in primed lymphocytes, resulting in a wide diversity of unique antigen receptors providing high antigenic specificity.
The clonal nature of several skin conditions may help us recognize pre-malignant stages or the concept of cutaneous lymphoid dyscrasias (CLD) which has been recently introduced and includes parapsoriasis, pigmented purpuric dermatosis, idiopathic follicular mucinosis, pityriasis lichenoides, syringolymphoid hyperplasia with alopecia, and idiopathic generalized erythroderma (pre-Sézary). Although almost all of these conditions never progress to a frank malignancy, they have the potential risk of converting into cutaneous T-cell lymphoma (CTCL). The recognition of a T-cell clone may identify these dermatoses, which have been difficult to categorize in the past.
Clonality methods
T-cell clonality studies are based on the detection of specific T-cell receptor gene rearrangements (TCR-GR) by Southern blot analysis (SBA) or polymerase chain reaction (PCR). We should expect that the tumour cells contain identical TCR-GRs, reflecting a monoclonal T-cell population.
SBA used to be the gold standard for detection of T-cell clonality, but the procedure is laborious and lengthy. Furthermore, fresh or frozen tissue and radioactive probes are required. If this method is used, the clonal population must represent at least 5% of the total DNA extracted, which includes cells other than T-cells decreasing the sensitivity of the test. For the reasons above, SBA has been gradually replaced by PCR techniques.
The overall sensitivity of PCR-based methods for detection of T cell clonality ranges between 70 and 90%, with specificity range depending on the sample population. The test amplifies extracted DNA using primers directed against the TCR beta, gamma and delta chains. The gamma chain gene is most commonly used because of the lower complexity of the gene. Adding probes to the beta TCR gene allows for a higher sensitivity and specificity of the clonal analysis.
In the case of B-cells, PCR uses primers for four conserved reliable targeted regions for immunoglobulin heavy-chain. In our experience the sensitivity of PCR for the immunoglobulin heavy chain is lower than for T-cell clonality. The detection of light chain restriction by immunophenotypic test (often referred as monotypical immunoglobulin expression) is also consistent with a clonal B-cell population. This can be accomplished with immunohistochemistry at the protein level or in situ hybridization at the RNA level. Monoclonality can also be demonstrated with flow cytometry targeting kappa and lambda light chain expression at the B-cell membrane. An international consensus on B- and T-cell clonality assays was established with the BIOMED-1 proposal.
In most of the conventional PCR methods monoclonality is defined by the presence of a band after high-resolution capillary gel electrophoresis of the PCR product. Using temperature- or chemical-gradient gel electrophoresis can enhance separation of DNA products. After that, fluorescent fragment analysis using consensus primers for the TCR gene and the fluorescence input is analysed by capillaroscopy. Furthermore, clonal definition should be confirmed using multiple PCR probes labelled with different fluorochromes.
The detection of a dominant T-cell clone, defined as the same PCR product at different sites (two skin biopsies, skin and blood, skin and lymph node, etc.) implies dissemination of a prevailing T-cell clone, and has been associated with a higher incidence of tumour progression. Clonal heterogeneity has been reported in patients with early stage or indolent mycosis fungoides (MF) and in CLD conditions without a malignant process.
The value of the detection of circulating clonal T-cells in peripheral blood has been debated. That is much more common in patients with erythrodermic MF (42%) compared to other lower stages (12.5%). It may also help in distinguishing a dominant CTCL clone from innocent cytotoxic T-cell clones, which are often detected in the blood of elderly patients.
In the context of palpable lymphadenopathy, detection of the same clone in the lymph node and the skin CTCL lesions may indicate a poor prognosis, similar to the identification of lymphoma by histology.
T-cell clonality and significance
TCR clonality should be tested for in skin and blood samples at the time of diagnosis when a cutaneous lymphoma is suspected. The detection of a dominant clone in both sites is important to confirm the diagnosis and for prognostic guidance. T-cell clonality is particularly helpful in the early stage of an MF which does not include sufficient clinical or microscopic evidence for the diagnosis. TCR gamma clonality was positive in 53% of the patch stage and in 100% of plaque or tumour stage in different series. An increased rate of clonality was observed in connection with more advanced cutaneous disease and higher histopathological diagnostic score.
False-positive monoclonal and oligoclonal bands may be identified in inflammatory dermatosis, where the T-cell infiltrate is sparse. Amplification of TCR-GRs from a few T-cells may result in a false-positive clone or ‘pseudomonoclonality’. A pseudoclone is infrequently associated with a malignant T-cell process. Repeating the analysis using the same DNA template or fresh DNA extraction may solve the problem because in reactive conditions, the predominant PCR product typically varies in repeated analysis of the same sample. In contrast, in lymphomas, dominant TCR clones are reproducible and should be routinely verified to confirm monoclonality.
A correlation between TCR clonality by PCR methods and response to treatment has been suggested in several studies. The absence of a detectable clone in CTCL was associated with a higher rate of complete remission, but was not necessarily associated with improved survival.
Also immunophenotypic and immunogenotypic assays have been used to monitor the response of CTCL to therapy. The concept of minimal residual disease is defined as the persistence of the tumour T-cell clone in tissue or blood despite clinical complete remission status. Minimal residual disease as detected by deep sequencing methods may help identify patients at risk of relapse but the real prognosis is still uncertain. In the future, the presence or absence of the dominant or persistent clone may guide our therapeutic approach, aiming for more durable remissions while minimizing the adverse effects of therapy.
Other methods used in the olecular diagnosis of cutaneous lymphomas
Flow cytometry analysis
Blood flow cytometry analysis (FCA) is routinely performed in erythrodermic patients to rule out Sézary syndrome (SS). This method is based on the abnormal expression of various surface markers of malignant T-cells compared with normal T-cells. Other helpful findings are the demonstration of overwhelming dominance of specific T-cell subsets (clusters of differentiation CD4 vs CD8) and the loss of one or more pan-T-cell antigens (i.e. CD2, CD3, CD5, and CD7). A high CD4 : CD8 ratio of more than 10 : 1 and loss of CD7 and CD26 are the most reliable findings in SS. However, low CD7 expression has lower specificity because some inflammatory diseases also show the same deletion. The addition of CD26 to standard T-cell panels enhances the sensitivity of FCA in the diagnosis of SS.
Moreover, flow cytometry is able to detect a clonal population by using antibodies against different subsets of T-lymphocytes based on the expression of V beta family antibodies. This is used mainly as a research tool because the extensive panel of antibodies is expensive, incomplete and does not include the entire spectrum of V beta families.
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) involves annealing of fluorescently labelled nucleic acid probes with complementary DNA or RNA sequences and the subsequent detection of these probes within fixed cells. FISH is used to detect major chromosomal gains or losses, as well as specific translocations, and requires a target specific probe. Although FISH is not routinely used in the diagnosis of cutaneous lymphomas, recent publications have shown its potential for future applications in various areas.
Genomic analysis by microarray assays or comparative genomic hybridization
Comparative genomic hybridization (CGH) allows the identification of chromosomal imbalances but it is not able to identify specific genes involved due to its measurement resolution. The microarray-based CGH is more precise, and chromosomal imbalances can be quantified and defined appositionally. A high frequency of gains in chromosomes 1, 7, 8, and 17 and losses of chromosomes 5, 9, and 13 was demonstrated using array-based CGH for identification of genomic differences between SS and MF.
Conclusion
Molecular diagnosis, in combination with a meaningful correlation with histological results and clinical presentations can provide an important tool in the evaluation of cutaneous lymphoid infiltrate. While PCR-based clonality techniques need to be interpreted with caution, modern capillaroscopy methods offer clone-specific data that allow us to improve the accuracy for diagnosis, prognosis and staging implication.
References
1. Deonizio JM, Guitart J. Semin Cutan Med Surg 2012; 31: 234–240.
2. Groenen PJ, Langerak AW, et al. J Hematop 2008; 1: 97–109.
3. Guitart J, Magro C. Arch Dermatol 2007; 143: 921–932.
4. Rübben A, Kempf W, et al. Exp Dermatol 2004; 13: 472–483.
5. Kulow BF, Cualing H, Steele P, et al. J Cutan Med Surg 2002; 6: 519–528.
6. Nihal M, Mikkola D, et al. Hum Pathol 2003; 34: 617–622.
7. Meyerson HJ. G Ital Dermatol Venereol 2008; 143:21–41.
8. Van Dongen JJ, Lamgerak AW, et al. Leukemia 2003; 17: 2257–2317.
9. Sandberg Y, Heule F, et al. Haematologica 2003; 88: 659–670.
10. Guitart J, Camisa C, et al. J Am Acad Dermatol 2003; 48: 775–779.
The authors
Belén Rubio González* MD and Joan Guitart MD
Northwestern Medical Hospital, Chicago, IL, USA
*Corresponding author
E-mail: rubiogonzalezbelen@gmail.com
DNA microarrays for SNP profiling in thrombosis and hemochromatosis
, /in Featured Articles /by 3wmediaSpecialized diagnostic DNA microarrays provide fast and reliable determination of factor V and factor II gene mutations associated with thrombosis or HFE gene defects linked to hereditary hemochromatosis. The simple microarray procedure includes fully automated data analysis and can be performed on whole blood samples, circumventing the need for preanalytical DNA isolation. Patient genotyping aids diagnosis in symptomatic individuals and risk assessment in healthy individuals, thus facilitating decision making in therapy and prevention.
by Dr Jacqueline Gosink
Laboratory analysis of genetic determinants is gaining momentum as ever increasing numbers of disease-associated alleles are discovered. With cutting edge diagnostic microarray technology, newly identified DNA parameters can progress rapidly from the research laboratory to routine diagnostics. Microarray platforms such as the EUROArray provide quick and easy determination of DNA mutations, enriching diagnosis and risk evaluation in a range of genetically linked diseases.
This article focuses on DNA microarray systems for genetic analysis in two common hereditary hematological disorders. The first detects single nucleotide polymorphisms (SNPs) in the factor V (FV) and/or factor II (FII) genes that lead to thrombosis and embolism. The second identifies up to four SNPs in the HFE (high iron) gene that contribute to hereditary hemochromatosis.
Thrombosis and embolism
Deep and superficial venous thrombosis and thromboembolism of the brain, lung and coronary vessels are among the most frequent causes of death, especially in western industrialized countries. These conditions result from a combination of genetic susceptibility and exogenous factors such as old age, immobility, smoking, diabetes mellitus, pregnancy, oral contraceptives or hormone replacement therapy. Notably, more than half of all cases can be attributed to genetic factors, particularly if the disease occurs before the age of 45 without any obvious external factors or at an atypical location.
The most important and most frequent genetic risk factors are the FV Leiden 1691G>A mutation (APC resistance) and the FII 20210G>A mutation in the prothrombin gene. These DNA mutations result in amino acid substitutions which disrupt the blood coagulation functions of FV and FII.
Factor V and II mutations
In healthy individuals activated FV is normally prevented from triggering coagulation by proteolytic cleavage catalysed by activated protein C (APC) and its cofactor protein S. Persons with the FV Leiden mutation exhibit an altered form of FV resulting from an exchange of the amino acid at position 506 from arginine to glutamine. The modified structure of FV makes it resistant to inactivation by APC (APC resistance), which leads to hypercoagulability and an increased risk of thrombosis. More than 95% of cases of APC resistance are caused by the autosomal, dominant FV Leiden mutation. In Europe around 3-7% of the population is a heterozygous carrier. In these individuals the thrombosis risk is 3-8 times higher than in non-affected persons, and if oral contraceptives are taken up to 30 times higher. The homozygous FV Leiden mutation occurs in around 0.2% of the European population and is associated with a 50-100-fold increased risk of thrombosis.
The 20210G>A mutation in the FII gene leads to a raised plasma concentration of the coagulation factor prothrombin via an as yet unidentified mechanism. The resulting thrombosis can be venous or arterial. The heterozygous genotype is present in 1-3% of the population in Europe and is associated with a 3-fold higher risk of deep venous thrombosis. If oral contraceptives are taken, the risk of venous thrombosis is increased 16-fold and of brain venous thrombosis up to 150-fold.
The factor V and factor II gene defects have an additive effect, and thrombophilia patients who exhibit the FII 20210G>A mutation often also have the FV Leiden mutation. In these patients the risk of venous thrombosis is elevated by a factor of 20.
Genetic analysis of FV and FII mutations is of outstanding importance in individuals with a high thrombosis risk based on their personal or family history, as well as in patients with unexplained recurrent miscarriages, biochemically proven resistance to APC or proven protein C or protein S deficiency. Genetic risk determination should also be undertaken before prescribing oral contraceptives or hormone replacement therapy to women with a familial tendency to thrombosis, especially young smokers.
Hereditary hemochromatosis
Hereditary hemochromatosis is the most frequent autosomal, recessive inherited metabolic disorder and is characterized by increased resorption of iron in the upper small intestine. The augmented iron uptake leads to an increase in the total iron content in the body from around 2–6 g (normal value) to up to 80 g. Since the human body cannot excrete the excess iron, it is deposited in various organs such as the liver, pancreas, spleen, thyroid gland, pituitary gland, heart and joints. In untreated patients irreversible damage occurs, resulting in an increased risk of cardiomyopathy, arthropathy, diabetes mellitus, liver cirrhosis and liver and pancreas carcinoma. Most cases of hereditary hemochromatosis are caused by defects in the HFE gene, which lead to functional flaws in the encoded iron regulatory protein.
HFE mutations
There are four SNPs in the HFE gene that are associated with hereditary hemochromatosis. The two most frequent, representing 90% of cases, result in the amino acid substitutions C282Y or H63D which cause a loss or reduction of the physiological function of the Hfe protein. The penetrance of the mutations is dependent on age and gender. Thus, the disease does not necessarily manifest itself in all carriers of these mutations. The strongest disease association is observed in patients with a homozygous C282Y mutation, whereby the penetrance is much lower in young women than in men due to menstruation. While 80% of men under 40 with this gene defect develop hemochromatosis, less than 40% of women do so. The penetrance increases to 95% of men and 80% of women for the population group of over 40 year olds. The two further SNPs in the HFE gene that are associated with hereditary hemochromatosis are S65C, which results in an amino acid substitution in the Hfe protein, and E168X, which causes early termination of protein synthesis, whereby both of these mutations are rare.
Around 10% of the population in northern Europe is heterozygous for one of the disease-associated mutations in the HFE gene and 0.3–0.5% is homozygous. New studies show that 90–100% of hemochromatosis patients exhibit homozygous gene defects. However, even a mutation in one HFE allele is sufficient to cause at least minor abnormalities in iron metabolism. The early identification of HFE gene defects enables suitable preventative measures to be implemented, for example a reduction in the consumption of high-iron-containing foods.
Simple microarray analysis
DNA mutations associated with thrombosis and hemochromatosis can be reliably determined using DNA microarray systems such as EUROArray [1, 2]. This microarray system provides fast and efficient SNP detection with fully automated data analysis, and can easily be used by persons unfamiliar with molecular biology. A special feature of the thrombosis and hemochromatosis microarray procedures is the use of pretreated whole blood as sample material, which eliminates the need for a preanalytical DNA isolation step. The hands-on processing time for the direct procedure is thus reduced to as little as 1.5 minutes per sample.
In the microarray procedure [Figure 1], the sections of DNA containing the disease-associated alleles are amplified by multiplex polymerase chain reaction (PCR) using highly specific primers. During this process the PCR products are labelled with a fluorescent dye. The PCR mixture is then incubated with a microarray slide containing immobilized DNA probes [Figure 2]. The PCR products hybridize with their complimentary probes and are subsequently detected via the emission of fluorescence signals. The evaluation of the microarrays [Figure 3] proceeds quickly and objectively using the special microarray scanner and EUROArrayScan software. The software interprets the results, produces patient genotype reports, and archives all data and patient information [Figure 4]. It can be integrated seamlessly into existing laboratory software.
Reliable biochip technology
EUROArrays are based on proven biochip technology which has been adapted for DNA analysis. Each biochip is composed of DNA spots of wild type and mutant alleles and contains in addition integrated control sequences to verify correct performance of the test. The microarray slides are incubated using the established TITERPLANE technique, which provides standardized, parallel incubation of multiple samples. Up to five samples can be analysed per slide. The reproducibility and convenience of the analysis is further enhanced by ready-to-use PCR reagents and meticulously designed amplification primers and hybridisation probes. The entire procedure from sample arrival to report release is IVD validated and CE labelled.
In clinical evaluation using molecular genetically precharacterized samples, each microarray demonstrated a sensitivity of 100% and a specificity of 100% [Table 1]. Homozygous and heterozygous genotypes were reliably discriminated for every position.
Comprehensive microarray range
The thrombosis diagnostic microarray system is available in different constellations for separate or parallel analysis of the FV Leiden and FII 20210G>A mutations, while the hemochromatosis microarray system is available in two versions encompassing either just the two most frequent mutations C282Y and H63D or, for a more extensive analysis, the four disease-associated mutations C282Y, H63D, S65C and E168X.
In addition to the determination of FV/FII and HFE mutations, EUROArray technology can also be used to analyse further genetic risk factors such as HLA-DQ2/ DQ8 in celiac disease, HLA-Cw6 in psoriasis or HLA-B27 in ankylosing spondylitis. New parameters soon to be added to the platform include HLA-DR Shared Epitope in the diagnosis of rheumatoid arthritis and human papilloma virus detection and subtyping.
Summary
The current pace of genetic discoveries combined with advances in microarray technology is resulting in a plethora of novel DNA tests for the routine diagnostic laboratory. New DNA microarrays for rapid identification of thrombosis-associated mutations in the factor V/factor II genes and hemochromatosis-linked mutations in the HFE gene have greatly enhanced diagnosis and risk evaluation in susceptible individuals. Early awareness of a genetic predisposition enables individuals to adopt appropriate lifestyle or medical interventions to reduce the impact or even prevent development of these debilitating diseases.
References
1. Voss J. et al. to be presented at IFCC EuroMedLab, Milano, Italy (2013).
2. Axel K. et al. to be presented at IFCC EuroMedLab, Milano, Italy (2013).
The author
Jacqueline Gosink PhD
Euroimmun AG
Luebeck, Germany
RND efflux pumps in P. aeruginosa: an underestimated resistance mechanism
, /in Featured Articles /by 3wmediaAn adequate initial antibiotic therapy is a key determinant of therapeutic success in Pseudomonas aeruginosa – infected patients. Antibiotic efflux is an underestimated resistance mechanism because it may occur in strains categorized as susceptible. It is rarely or not at all diagnosed in routine laboratories and often masked by high-level resistance mechanisms.
by Dr Laetitia Avrain, Dr Pascal Mertens and Professor Françoise Van Bambeke
P. aeruginosa: state of the art of antibiotic susceptibility
P. aeruginosa is a Gram-negative bacterium recognized as a major cause of infections in hospitalized patients or in patients with impaired defences as observed in burn wounds or cystic fibrosis. In spite of improved hygiene measures, the risk of infection by P. aeruginosa in ICU remains high (infection incidence > 30/100 patients hospitalized in ICU). P. aeruginosa infections are associated with mortality rates as high as 30 % to 50 % in bacteremia [1] and up to 70% in patients with nosocomial pneumonia [2].
Yet, empirical selection of antibiotics is made difficult by the continuously evolving resistance of P. aeruginosa to antibiotics, notably due to the emergence of Multi Drug Resistance (MDR) phenotype (R ≥ 3 antibiotic classes). The MDR status of the strain as well as an initial inappropriate treatment negatively influence patient outcome [3].
Acquired high level resistance is due to the acquisition of genes coding for aminoglycoside-modifying enzymes or beta-lactamases, or to mutations in fluoroquinolone targets. Intrinsic antibiotic resistance is due to low outer membrane permeability mediated either by under production of the oprD porin, or by the expression of multidrug resistance efflux pumps. The genome of P. aeruginosa codes for numerous efflux pumps, among which MexAB-OprM and MexXY-oprM are of first clinical importance due to their large prevalence in clinical strains and their ability to expel several classes of chemically-unrelated antibiotics.
RND efflux pumps in P. aeruginosa
The main efflux pumps in P. aeruginosa belong to the Resistance-Nodulation-Division (RND) superfamily, which uses proton motive force as energy source. They constitute a tri-partite system, composed of an integral cytoplasmic membrane drug-proton transporter, an outer membrane channel and a periplasmic fusion protein linking the two other proteins. This assembly allows expelling the substrate from the inner membrane directly to the extracellular medium [Fig. 1, reproduced from [4]].
Ten efflux systems have been characterized in P. aeruginosa, among which MexAB-OprM and MeXY-OprM are constitutively expressed at a basal level in wild-type strains (expression of MeXY-OprM being however much lower than that of MexAB-OprM). Both systems are inducible when exposed to antibiotic substrates. The other systems (MexCD-OprJ, MexEF-OprN, MexJK, MexGHI-OpmD, MexVW, MexPQ-OpmE, MexMN, and TriABC are not expressed in wild type strains but may contribute to antibiotic or biocide resistance when expressed in resistant strains [5].
Antipseudomonal antibiotics released by P. aeruginosa multidrug efflux systems
RND efflux systems release multiple antimicrobials components including first-line antipseudomonal antibiotics such as β-lactams and β-lactamase inhibitors, fluoroquinolones, aminoglycosides [Table 1]. More specifically MexAB-OprM transports β-lactams fluoroquinolones, macrolides, tetracyclines, trimethoprim, sulfamides and chloramphenicol; MexXY-OprM, aminoglycosides, fluoroquinolones, macrolides, and tetracyclines; MexCD-oprJ, some β-lactams, fluoroquinolones, macrolides, tetracyclines, trimethoprim and chloramphenicol, and MexEF-OprN, fluoroquinolones, trimethoprim and chloramphenicol. The latter is also involved in resistance to meropenem and doripenem, but this may rather result from the fact that the OrpD porin is downregulated in strains expressing this efflux system.
Colistin, the last resort drug for MDR P. aeruginosa, is not substrate for these efflux pumps. Thus, efflux is responsible for multidrug resistance, a single pump being able to transport several classes of drugs while at the same time some redundancy exists among transporters, fluoroquinolones for example being universal substrates for the main efflux systems. Moreover, the subsequent reduction in antibiotic concentration inside the bacteria may help selecting high level resistance mechanisms, in particular target mutations [6].
Over-expression of efflux pumps: impact on antimicrobial susceptibility
A study published in 2010 examined the impact of antibiotic treatment on the susceptibility of P. aeruginosa, by collecting successive isolates from ICU patients at the time of diagnosis of infection and during treatment [7]. Globally, mean minimum inhibitory concentration (MIC) values increased after exposure to antibiotics, with statistically significant effects being observed for amikacin, ciprofloxacin, cefepime, meropenem and piperacillin/tazobactam, bringing mean MICs to values higher than the EUCAST susceptibility breakpoints. Three quarters of the isolates showed a moderate elevation of the MIC (≤16X initial MIC), suggesting the involvement of low to moderate levels resistance mechanisms as those affecting membrane permeability [Fig 2, reproduced from [7]].
The analysis of the expression of efflux pumps in this collection revealed that a high proportion of the strains (34 %) did overexpress MexAB-OprM and MexXY-OprM in the initial isolate, but that this proportion further increased during the antibiotic treatment, with about two third of the strain overexpressing at least one of these efflux systems [Fig.3, adapted from [8]].
Diagnosis of efflux in clinical laboratory
Because efflux in P. aeruginosa almost always co-operates with other mechanisms of resistance, differential diagnosis by phenotypic antimicrobial analysis is complex, high levels resistance mechanisms masking the effect of the expression of efflux systems on MICs. Moreover, efflux pumps can be overexpressed during treatment, which may explain therapeutic failures with antibiotics that are considered active based on the original determination of the susceptibility profile.
Resistance by efflux can be detected using Efflux Pumps Inhibitors (EPI), which revert MICs to those strains that do not express efflux systems. Among them MC-207,110 (phenylalanine arginyl beta-naphthylamide) is a broad spectrum inhibitor that has been widely used in vitro to investigate the impact of efflux on susceptibility to antibiotics of P. aeruginosa. Inhibitors specific of a given transporter are also under investigation. Yet, in MDR strains with additional resistance mechanisms, EPI do not allow restoring antibiotic activity, which may lead to false-negative results [9].
In this context, molecular methods appear as the only way to evidence the expression of efflux pumps in clinical isolates. Immunoblotting methods were developed first but were rapidly replaced by Reverse Transcriptase quantitative PCR (RT-qPCR) due to its higher specificity and rapidity. RT-qPCRs were thus developed to detect and quantify the expression of the genes coding for the different proteins of a given RND pump. This method remains applicable whatever the other resistance mechanisms present in the clinical strain and can thus be applied in clinical laboratories. Typically, a 2-fold increase in the expression of mexA and mexB genes causes a decrease in antibiotic susceptibility, while overexpression of mexX needs to be higher (≥ 5-fold) to increase MIC values. This low level of overexpression implies that all the steps for RT-qPCR should be carefully standardized [10]. The commercial mex Q-TesT kit includes two housekeeping genes to standardize the RT-qPCR and facilitates the interpretation of mexA and mexX genes expression of clinical Pseudomonas aeruginosa strains in comparison to wild type strain PAO1.
Conclusion
Resistance by efflux has now well been characterized in specialized laboratories but is still rarely or not at all diagnosed in routine laboratories. The complexity of resistance in P. aeruginosa with MDR phenotypes and the lack of diagnostic tools are probably the main reasons why this mechanism is neglected. Because this resistance mechanism can also contribute to therapeutic failures, accurate diagnosis is of prime importance for selecting adequate therapy.
References
1. Aliaga L, Mediavilla JD, et al. A clinical index predicting mortality with Pseudomonas aeruginosa bacteraemia. J Med Microbiol 2002; 51(7): 615-619.
2. Alp E, Guven M, et al. Incidence, risk factors and mortality of nosocomial pneumonia in intensive care units: a prospective study. Ann Clin Microbiol Antimicrob 2004; 3: 17.
3. Hirsch EB, Tam VH. Impact of multidrug-resistant Pseudomonas aeruginosa infection on patient outcomes. Expert Rev Pharmacoecon Outcomes Res 2010; 10(4): 441-451.
4. Mesaros N, Van Bambeke F, et al. L’efflux actif des antibiotiques et la résistance bactérienne: état de la question et implications. La lettre de l’infectiologue 2005; (4): 117-126.
5. Lister PD, Wolter DJ, et al. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev 2009; 22(4): 582-610.
6. Zhanel GG, Hoban DJ, et al. Role of efflux mechanisms on fluoroquinolone resistance in Streptococcus pneumoniae and Pseudomonas aeruginosa. Int J Antimicrob Agents 2004; 24(6): 529-535.
7. Riou M, Carbonnelle S, et al. In vivo development of antimicrobial resistance in Pseudomonas aeruginosa strains isolated from the lower respiratory tract of Intensive Care Unit patients with nosocomial pneumonia and receiving antipseudomonal therapy. Int J Antimicrob Agents 2010; 36(6): 513-522.
8. Riou M, Avrain L, et al. Influence of antibiotic treatments on gene expression of RND efflux pumps in successive isolates of Pseudomonas aeruginosa collected from patients with nosocomial pneumonia hospitalized in Intensive Care Units from Belgian Teaching Hospitals. ECCMID, 10-13 April 2010, Vienna, Austria. P780.
9. Van Bambeke F, Pages JM, et al. Inhibitors of bacterial efflux pumps as adjuvants in antibiotic treatments and diagnostic tools for detection of resistance by efflux. Recent Pat Antiinfect Drug Discov 2006; 1(2): 157-175.
10. Avrain L, Hocquet D, et al. Pre-Real-Time PCR steps standardization for appropriate interpretation of mexA and mexX gene expression by mex Q-Test in P. aeruginosa. ECCMID, 10-13 April 2010, Vienna, Austria. P590.
The authors
Laetitia Avrain PhD1*, Pascal Mertens PhD1 and Françoise Van Bambeke, Professor, Maître de Recherche FNRS, PhD2
1 Coris BioConcept, Gembloux, Belgium
2 Molecular and cellular pharmacology,
Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium
*Corresponding author
E-mail: laetitia.avrain@corisbio.com
Prokaryotic-expressed recombinant nucleocapsid protein for the detection of coronavirus infection
, /in Featured Articles /by 3wmediaThe early diagnosis of common colds caused by coronavirus is a crucial step in preventing the recurrence of a global outbreak. The goals of this article are to discuss a prokaryotic-expressed recombinant nucleocapsid protein used in the development of a sensitive diagnostic assay for the diagnosis of human coronavirus infection.
by Dr Ming-Hon Hou
An overview of coronavirus
Human coronavirus (HCoV) was identified in the 1960s and has generally been associated with symptoms of the common cold. Although HCoV infections are generally mild, more severe upper and lower respiratory tract infections, such as bronchiolitis and pneumonia, which are particularly severe in infants, elderly individuals, and immunocompromised patients, have been documented. There have also been reports of clusters of HCoV infections that cause pneumonia in adults. In addition, a previous study reported that the neurotropism and neuroinvasion of HCoV are associated with multiple sclerosis.
In recent years, several emerging human coronaviruses have been discovered, and between 2003 and 2004, the SARS-CoV outbreak caused a worldwide epidemic that had a significant economic impact in countries where the disease outbreak occurred. Phylogenetic analyses have shown that SARS-CoV contains sequences that are closely related to sequences found in the betacoronaviruses. In 2004, another alphacoronavirus, HCoV-NL63, which was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis, was reported in the Netherlands. In 2005, a novel betacoronavirus, HKU1 was found in patients with respiratory tract infections. Recently, a novel SARS-like coronavirus was found in patients with respiratory tract infections in the Middle East.
The RNA genomes of coronaviruses include genes encoding the structural proteins S (spike), M (matrix), E (envelope), and N (nucleocapsid). Additionally, some coronaviruses encode a third glycoprotein, HE (hemagglutinin-esterase), which is present in most of the betacoronaviruses. A helical nucleocapsid exists in the centre of the viral particle. The primary function of CoV N protein (NP) is to recognize a stretch of RNA that serves as a packaging signal, leading to the formation of the ribonucleoprotein (RNP) complex or to a long helical nucleocapsid structure during viral assembly. The formation of the RNP is important for maintaining the RNA in an ordered conformation suitable for replication and transcription of the viral genome. The CoV NP was shown to be involved in the regulation of cellular processes, such as gene transcription, actin reorganization, host cell cycle progression, and apoptosis.
Coronaviruses cause colds of mild to moderate severity and are transmitted by aerosols of respiratory secretions, the fecal–oral route, and mechanical transmission. The most common symptoms of coronavirus infection are nasal catarrh and a sore throat, and the illness typically lasts approximately 6 to 8 days. The early diagnosis of common colds caused by a coronavirus is an important step in preventing the recurrence of a global outbreak. Previously, rapid viral diagnosis has also been critical in the control of epidemics and the management of SARS patients. HCoVs are difficult to detect, and the current diagnosis of coronaviral infection is based on reverse transcription polymerase chain reaction with real-time PCR and antibody detection.
Previous studies have shown that NPs are the immunodominant domain in hosts infected with several coronaviruses. Additionally, it has been shown that NPs can accumulate intracellularly before being packaged into mature viruses and are the most abundant viral protein. NP is involved in the pathological reaction to human coronavirus and is a key antigen for the development of a sensitive diagnostic assay. These characteristics make NP a suitable candidate for the early diagnosis of coronavirus infection.
Nucleocapsid protein for coronavirus serodiagnosis
NP is involved in the pathological reaction to CoV infection and has been used in the development of a sensitive diagnostic assay. Previous studies reported that NP can be detected in the serum samples of SARS patients as early as 1 day after disease onset. Prokaryotic-expressed NPs have successfully been used as antigens for the detection of antibodies specific to many viruses, including SARS-CoV and several animal coronaviruses, and were produced for establishing an antigen-capture ELISA (or Western blot assay) for the diagnosis of HCoV infection
These methods are highly sensitive and specific. For example, Shi et al. [10] used recombinant SARS-CoV NP to establish an antigen-capture ELISA for SARS diagnosis. Anti-NP antibodies could be detected in approximately 90% of SARS patients 11 to 61 days after illness. No false positives were observed in non-SARS patients or health care workers.
An immunofluorescence assay is the gold standard for the detection of SARS. However, it requires efficient SARS-CoV replication in vitro to use whole virus or infected cell lysates as antigens. There are several reasons for selecting a recombinant protein rather than whole virus for this assay. The prokaryotic expression system is high yield, inexpensive, highly efficient, does not require viral cultures, and is non-toxic. Despite these advantages, viral proteins expressed in prokaryotic cells lack post-translational modifications that are present in proteins expressed in baculovirus expression systems.
Using recombinant nucleocapsid protein as an antigen for coronavirus infection diagnosis: one recent case study
HCoV is distributed worldwide. Recently, we produced soluble recombinant human coronavirus OC43 (HCoV-OC43) NP to analyse the antigenicity of the betacoronavirus HCoV-OC43 NP. To express soluble HCoV-OC43 NP as a set of fusion proteins in E. coli, the NP gene was cloned into the pET-28a expression vector. His-tagged NP was purified from the soluble fraction using Ni-NTA column chromatography [Figure 1]. The yield from 1 L of bacterial culture was as large as 10 mg of pure NP after extraction and column chromatography. A recombinant protein-based Western blot assay was used to screen human serum from young adults, middle-aged and elderly patients with respiratory infection symptoms and cord blood units.
Western blotting is generally accepted as the most effective method for unequivocally locating linear or continuous immunodominant epitopes. Pohl-Kooppe et al. [8] also reported that Western blotting is a more sensitive test system than an immunofluorescence assay for the analysis of sera from pediatric groups. Our results showed that approximately 80–90% of serum samples from young adults and middle-aged and elderly patients with respiratory infections reacted strongly to the HCoV-OC43 NP, indicating prior exposure to this disease. In addition, the serum samples tested in this study were 81% seropositive for HCo-229E NP [Fig. 2].
This finding is consistent with previous epidemiological surveys that concluded that seroprevalence increases rapidly during childhood, attaining a seroprevalence rate of up to 90% in adults. Additionally, antibodies against HCoV-OC43 NP were detected in over 90% of cord blood samples tested. Maternally acquired antibodies may help to protect a newborn baby from HCoV-OC43 infection, although this protection appears to wane by 4 to 5 months of age. HCoV is responsible for approximately 30% of all common colds, and it is expected that 80–90% of serum samples from healthy donors and patients have antibodies to HCoV-OC43.
CoV NPs contain multiple immunodominant epitopes and antigenic sites. To compare the immunoreactivity of the three structural regions of HCoV-OC43 NP, three truncated recombinant fragments [aa 1–173 (the N-terminal domain), aa 174–300 (the central region), and aa 301–448 (the C-terminal domain)] were produced in E. coli; these regions were chosen based on PONDR (predictor of naturally disordered regions) predictions. The reactivity of human serum against these fragments was determined through Western blotting. The human serum reacted strongly with the central region and the C-terminal domain of the NP, whereas the N-terminal domain demonstrated low reactivity with the antibody. The findings of the current study are consistent with those of Chen et al. [2], who found that the antigenicity of the C-terminus of SARS-CoV NP was higher than that of the N-terminus.
The polyclonal antibody against coronavirus NP could be used to develop a rapid, easy and specific diagnostic tool for the detection of HCoV infections through immunofluorescence or ELISA-based tests. Many studies have reported that NP polyclonal antibody does not cross-react with other human CoV NPs, including those of SARS-CoV and HCoV-229E, despite the presence of highly conserved motifs in these coronavirus NPs. Previous studies also showed that the anti-SARS CoV NP and anti-HCoV-229E NP polyclonal antibodies did not cross-react with other human CoV NPs.
In our recent studies, using purified recombinant NP as an antigen, a polyclonal antibody was generated from rabbit serum with specificity for HCoV-OC43 NP; this antibody reacted specifically with HCoV-OC43 NP and did not cross-react with other human CoV NPs (including those of SARS-CoV and 229E) through Western blotting.
Conclusion
A novel SARS-like coronavirus was found in patients with respiratory tract infections in the Middle East. Thus, new and convenient diagnostic methods for CoV infection are urgently needed. The prokaryotic expression of recombinant HCoV NP is suitable for high-sensitivity, highly specific antibody production and can be used for the epidemiological screening of HCoV infection in the future.
References
1. Che XY, Qiu LW, Liao ZY, Wang YD, Wen K, Pan YX, Hao W, Mei YB, Cheng VC, Yuen KY. Antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229E and OC43. J Infect Dis 2005; 191: 2033–7.
2. Chen Z, Pei D, Jiang L, Song Y, Wang J, Wang H, Zhou D, Zhai J, Du Z, Li B, Qiu M, Han Y, Guo Z, Yang R. Antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein. Clinical Chem 2004; 50: 988–95.
3. He Q, Chong KH, Chng HH, Leung B, Ling AE, Wei T, Chan SW, Ooi EE, Kwang J. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin Diagn Lab Immunol 2004; 114: 417–22.
4. Huang CY, Hsu YL, Chiang WL, Hou MH. Elucidation of the stability and functional regions of the human coronavirus OC43 nucleocapsid protein. Protein Sci 2009; 18: 2209–18.
5. Huang LR, Chiu CM, Yeh SH, Huang WH, Hsueh PR, Yang WZ, Yang JY, Su IJ, Chang SC, Chen PJ. Evaluation of antibody responses against SARS coronaviral nucleocapsid or spike proteins by immunoblotting or ELISA. Journal Med Virol 2004; 73: 338–46.
6. Liang FY, Lin LC, Ying TH, Yao CW, Tang TK, Chen YW, Hou MH. Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protein by Western blot: Implications for the diagnosis of coronavirus infection. J Virol Methods 2013; 187: 413–20.
7. Mourez T, Vabret A, Han Y, Dina J, Legrand L, Corbet S, Freymuth F. Baculovirus expression of HCoV-OC43 nucleocapsid protein and development of a Western blot assay for detection of human antibodies against HCoV-OC43. J Virol Methods 2007; 139: 175–80.
8. Pohl-Koppe A, Raabe T, Siddell SG, ter Meulen V. Detection of human coronavirus 229E-specific antibodies using recombinant fusion proteins. J Virol Methods 1995; 55: 175–83.
9. Shao X, Guo X, Esper F, Weibel C, Kahn JS. Seroepidemiology of group I human coronaviruses in children. J Clin Virol 2007; 40: 207–13.
10. Shi Y, Yi Y, Li P, Kuang T, Li L, Dong M, Ma Q, Cao C. Diagnosis of severe acute respiratory syndrome (SARS) by detection of SARS coronavirus nucleocapsid antibodies in an antigen-capturing enzyme-linked immunosorbent assay. J Clin Microbiol 2003; 41; 5781–2.
11. Timani KA, Ye L, Zhu Y, Wu Z, Gong Z. Cloning, sequencing, expression, and purification of SARS-associated coronavirus nucleocapsid protein for serodiagnosis of SARS. J Clin Virol 2004; 30: 309–12.
The author
Ming-Hon Hou PhD
1 Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
2 College of Life Science, National Chung Hsing University, Taichung, Taiwan
3 Institute of Genomics and Bioinformatics, National Chung Hsing University,
Taichung, Taiwan
E-mail: mhho@dragon.nchu.edu.tw
Functional evaluation of chemistry analyser
, /in Featured Articles /by 3wmediaThe aim of this study was to assess the practicability and evaluate the analytical characteristics of the Mindray BS-2000M, a new automatic chemistry analyser, manufactured by Shenzhen Mindray Bio-Medical Electronics Co., Ltd (Mindray Shenzhen, China).
The evaluation involved 21 clinical chemistry parameters measured using indirect potentiometry, spectrophotometric and immunoturbidimetric assays.
Spectrophotometric assays
Alanine aminotransferase (ALT, according to IFCC with-out pyridoxal phosphate), aspartate aminotransferase (AST, according to IFCC method), gamma-glutamyltransferasa (GGT, according to Szasz), total bilirubin (TBIL, Diazotized sulfanilic acid method), calcium (Ca, Arsenazo III method), creatine kinase (CK, IFCC method), creatinine (Crea, modified Jaffe method), glucose (Glu, Hexokinase method), high density lipoprotein-cholesterol (HDL-C, direct method), magnesium (Mg, Xylidyl blue method), phosphorus (P, Phosphomolybdate method), total cholesterol (TC, Cholesterol oxidase – Peroxidase method), triglycerides (TG, Glycerokinase Peroxidase – Peroxidase method), total protein (TP, Biuret method), uric acid (UA, Uricase-Peroxidase method), iron (Fe, Colorimetric assay-Ferrozine), α-amylase [α-AMY, substrate: 4, 6- ethylidene-(G7)-1, 4-nitrophenyl-(G1) –α, D-maltoheptaoside (EPS-G7), enzymatic colorimetric assay according to IFCC method] and urea (Urea, Urease-glutamate dehydrogenase).
Immunoturbidimetric assays
Apolipoprotein A1 (ApoA1).
Indirect potentiometry assays (ISE)
Sodium (Na), potassium (k) and chloride (Cl).
Analytical evaluation
Among all the available parameters to verify the Mindray BS-2000M analytical performance, those which are more frequently requested in routine clinical practice were selected (e.g., glucose, creatinine, total protein).
An imprecision study was carried out according to the CLSI EP5-A2 guideline [1]. The within run imprecision was evaluated using two control materials. Final results were expressed as coefficient of variation (CV%). We checked that these CV satisfied the allowable maximum imprecision based on biological variability [2]. These data were taken from the listing of biological variation by Ricos et al [3], recently updated in 2012.
The inaccuracy study was done according to the CLSI EP9-A2 guideline [4], measuring at least 40 patient samples for the two analysers (Mindray BS-2000M and ADVIA 2400 Siemens Healthcare Diagnostics, USA) for 5 days.
In the inaccuracy study the mean bias and 95% confidence interval (CI) was calculated with the Bland-Altman plot analysis, and the linear regression was assessed using Passing-Bablok regression method [5-6].
The results of the imprecision study [Table 1] showed that for all the parameters imprecision was lower than the maximal allowable applying to biological variability based criteria, with the exception of sodium (0.7%) and chloride (0,8%) in control 1 and total proteins (1,7%) in control 2. Nevertheless, these three parameters fulfill the commonly used “State of the art” criterion. According to this criterion, the maximal allowable imprecision for physiological concentrations must be less than the 0.20 fractile of the between-run imprecision (CV) of the laboratories in a external quality assessment scheme (7). The CV% limit for these three parameters following this approach would be 0.9% for Na, 1.6% for Cl and 1.7% for TP.
In the comparison study, a close correlation was observed for all parameters studied (r range: 0.92 – 1.00) [Table 2]. It is noted that there were no significant differences for 11 of the 21 parameters studied. For the other parameters statistically significant differences were found but, except for creatinine, those differences were not considered to have a clinical significance. The constant and proportional differences may be due to different standardization of both procedures. Traceable calibration materials should be used related to the reference method and also switchable materials that reveal the degree of deviation of multiple methods with respect to the true value should be used [8].
References
1. Clinical and Laboratory Standards Institute. Evaluation of precision performance of quantitative measurement methods; approved guideline -second edition. CLSI document EP5-A2. Wayne, PA:CLSI, 2004.
2. Fraser CG, et al. Proposed quality specifications for the imprecision and inaccuracy of analytical systems for clinical chemistry. Eur J Clin Chem Clin Biochem 1992; 30: 311.
3. Ricos C, et al. Desirable quality specifications for total error, imprecision, and bias, derived from biological variation. http://www.Westgard.com/biodatabase1.htm. Accessed on February 15, 2013.
4. Clinical and Laboratory Standards Institute. Method comparison and bias estimation using patient samples; approved guideline – second edition. CLSI document EP9-A2. Wayne, PA: CLSI, 2002.
5. Bland JM, et al. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; 1: 307.
6. Passing H, et al. A new biometrical procedure for testing the equality of measurements from two different analytical methods. Application of linear regression procedures for method comparison studies in clinical chemistry, Part I. J Clin Chem Clin Biochem 1983; 21: 709.
7. Sebastian-Gámbaro, et al. An improvement on the criterion of the state of the art to estimate the maximal allowable imprecision. Eur J Clin Chem Biochem. 1996; 34: 445.
8. Documento de consenso. Sociedad Española de Bioquímica Clínica (SEQC) y Sociedad Española de Nefrología (SEN). Recomendaciones sobre la utilización de ecuaciones para la estimación del filtrado glomerular en adultos. Química Clínica 2006; 25: 423.
The author
Dr. Jose Luis Bedini,
Hospital Clinic I Provincial De Barcelona,
Barcelona,
Spain
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