Results of College Supplementary Autopsy Exam 2018 (Council Meeting 19 March 2019)

Biochemical genetics in the expanded newborn screening era

Volume 14, Issue 1 January 2019  (download full article in pdf)

Editorial note

In this topical update, Dr. Calvin Chong reviews and updates on the technological development of biochemical genetics for the three major classes of metabolic disorders, namely aminoacidopathies, organic acidurias and fatty acid oxidation defects. These conditions have increasing local awareness, in particular with the introduction of universal expanded newborn screening. With a rising clinical demand for confirmatory tests in biochemical genetics, the ways to achieve better analytical quality and capacity were discussed. We welcome any feedback or suggestions. Please direct them to Dr. Sammy Chen (email: chenpls@ha.org.hk) of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Dr. Calvin Yeow-Kuan Chong
Department of Pathology, Princess Margaret Hospital

Introduction

Biochemical genetics refers to the diagnosis of genetic disorders with biochemical markers. Sir Archibald Garrod first described the biochemical features of alkaptonuria in 1902¹, and is often named as the founding father of biochemical genetics. Throughout the past century, the practice of biochemical genetics has evolved from spot chemical tests towards the use of chromatography and mass spectrometry^2,3. The recent implementation of the pilot study of expanded newborn screening means, on one hand, disorders are diagnosed earlier, and patients with such conditions would fare better, and on the other hand, this represents an increase in the use of confirmatory tests in biochemical genetics which is most acutely felt at the major chemical pathology laboratories.

The screening panel of the government-initiated pilot study included 8 aminoacidopathies, 7 organic acidurias, 6 fatty acid oxidation defects, and 3 other disorders⁴. Apart from the three disorders which required separate measurements, all three other major classes of disorders (viz. the aminoacidopathies, organic acidurias, and fatty acid oxidation defects) are screened by the use of tandem mass spectrometric measurement of succinylacetone, amino acids and acylcarnitines, all done within a period of around two minutes.

Table 1 lists the confirmatory markers and tests for the screened conditions ^5,6: as can be seen, plasma amino acids, urine organic acids, and plasma acylcarnitines represents the main bulk work as confirmatory tests for these conditions. The present article aims therefore to explore the contemporary techniques available for these three major confirmatory tests in biochemical genetics and attempts to identify the approaches a laboratory may seek in order to cater for the increased demands.

Table 1. Confirmatory markers and tests for conditions covered in the government-initiated pilot study of expanded newborn screening. Specialized tests not discussed in the present article are italicized.

Amino acids

Organic acids

GC-MS based urine organic acid analysis

For GC-MS analysis of urine organic acids, a creatinine-corrected amount of urine is subject to liquid-liquid extraction with ethyl acetate after acidification using hydrochloric acid, the organic extract is then dried and derivatized by N,O-bis-tri(methylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS)²². The resultant product is injected to the GC-MS operating in scan mode. For reliable detection of ketones, oximation with hydroxylamine hydrochloride can be performed prior to acidic extraction²³.

An alternative way of performing urine organic acid analysis is to bypass the extraction step. As no acidic extraction step is done, a limited panel of amino acids, purine, pyrimidines, and mono- and di-saccharides can be detected in the same analytical run. This method has been in-use in the author’s laboratory in the past 2 decades^24,25, and allows the detection of a much wider range of metabolites in urine. With no extraction, the chromatograms are extremely complex due to co-elution of analytes and therefore this approach requires expertise in post-analytical data processing to be viable as a routine method.

The interpretation of GC-MS data for urine organic acid analysis is commonly based on examination of the chromatogram (Figure 1), followed by a combination of peak integration in the total ion chromatogram followed by library searching and examination of extracted ion chromatogram at particular retention times for analytes which gives lower responses (Figure 2). The raw analyte response is then compared to locally-established age-specific reference intervals which allow clinical interpretation²³.

Figure 1. Total ion chromatogram of a urine sample from a patient with malonic acidemia circulated in the ERNDIM qualitative organic acid program in 2016. The two abnormal peaks are indicated by asterisks: the first peak represents malonic acid (di-TMS derivative) and the second peak represents methylmalonic acid (di-TMS derivative).

Figure 2. Extracted ion chromatogram showing the quantifier ions (m/z 375, for aconitic acid in red; and m/z 254, for orotic acid, in black) and qualifier ions (m/z 285, for aconitic acid in green; and m/z 357, for orotic acid, in blue). They are co-eluting in many GC-MS based urine organic acid assays.

LC-MS based urine organic acid analysis

In the recent years, liquid chromatography-mass spectrometry-based methods have been published for analysis of urine organic acids. The benefit of liquid chromatography-mass spectrometry is clear: there is no need for the cumbersome derivatization step, and heat-labile analytes can be detected^26,27. The major drawbacks of LC-MS based methods are the poorer separation of analytes and higher susceptibility towards ion suppression, as electrospray ionization is much less robust against matrix effects compared to electron ionization as used in GC-MS based methods²⁸. The difficulty of developing an in-house LC-MS based method for urine organic acids lies in the procurement of a practically endless list of organic acid standards to establish the retention time and multiple reaction monitoring ratios. At the time of writing, there is at least one commercial kit that has been made available (Zivak Organic Acids LC-MS/MS analysis kit), but this commercial kit did not utilize isotopic internal standards even for critical analytes (such as hexanoylglycine and orotic acid) and one may wish to validate extensively the robustness of such assays against matrix effects.

Choice of method and the future

Out of the three assays discussed in the present article, urine organic acid analysis represents the most difficult of the three to establish in a laboratory. There is first the difficulty in establishing age-specific reference intervals, and then difficulty in acquiring a large number of chemical standards. A good starting point would be obtaining the bi-level quality controls for urine organic acid and special assays for urine from the ERNDIM network which consist of a number of critical analytes. As for choice of internal standards, while traditional choices such as tropic acid and pentadecanoic acid are often employed, deuterated internal standards covering critical analytes are much more available nowadays (e.g. methylmalonic acid-d3 and orotic acid-1,3-15N2) and their addition may improve quantitation of these critical analytes, the former being commonly quantified and the latter being prone to analytical errors²⁹.

For laboratories with existing GC-MS based urine organic acid assay, the question is probably whether to adopt LC-MS based solution. The belief that organic aciduria always results in sky-high level of abnormal metabolites in urine is flawed: the low-excretor phenotype of glutaric aciduria type I can serve as the classical example³⁰. The difficulty may be mitigated somewhat if acylcarnitines and acylglycines are measured by the LC-MS based assays as glutarylcarnitine has been shown to be informative even for low-excretor GA I patients³¹. Overall, it remains to be seen whether LC-MS based organic acid analysis could stand alone replacing, rather than complementing, GC-MS based assays.

Free carnitine and acylcarnitines

Figure 3. Cumulative precursor scan mass spectrum for a blood-spot sample distributed in the ERNDIM Qualitative Acylcarnitine Program in 2014. In this specimen, elevated signals at m/z ratio 260 (C6-carnitine), 288 (C8-carnitine), and 314 (C10:1-carnitine) can be seen. The pattern would be compatible with MCAD deficiency. (Mass spectra courtesy of Mr CK Lai, Chemical Pathology Laboratory, PMH)

In the analysis of carnitine and acylcarnitines, butyl-ester derivatization enhances the formation of positively-charged ion by reacting with carboxylic groups, and causes mass separation of dicarboxylic-carnitines and hydroxy-acylcarnitines (e.g. C4DC-carnitine and C5OH-carnitine), which are isobaric when underivatized³⁶. Derivatization is typically performed at highly acidic conditions (e.g. 3N hydrochloric acid at 65°C for 15 minutes)³⁵. On the other hand, chromatographic separation allows the separate determination of individual isomeric constituents (e.g. C4DC-carnitines include succinylcarnitine and methylmalonylcarnitine; and C5OH-carnitines include 3-hydroxyisovalerylcarnitine and 2-methyl-3-hydroxybutyrylcarnitine) (Figure 4). With meticulous chromatographic separation, most biologically relevant isomeric species could be separately quantified³⁷.

Choice of method and the future

The question for the laboratory is, first, whether to employ the derivatization procedure. The advantage of derivatization is the mass separation of hydroxylacylcarnitines and carnitine derivatives of dicarboxylic acids; the problems associated with derivatization are the partial hydrolysis of acylcarnitines because of the high temperature and strongly acidic condition employed³³. The other considerations are whether to employ chromatography and if so, how extensive should it be: it would then be a delicate balance between through-put, diagnostic specificity, and analyser-time that is available. With the improvement of separation capability of liquid chromatographs and sensitivity of modern mass spectrometers, it is suggested that a short UHPLC program combined with both scheduled MRM and precursor ion scan function would be a good compromise.

Figure 4. SRM chromatogram of m/z 262>85 showing chromatographic separation of different species of isobaric (C4DC/C5OH) acylcarnitines (viz. succinylcarnitine at 4.68 minutes, two diastereomeric peaks of methylmalonylcarnitine at 5.2 and 5.35 minutes, and 3-hydroxyisovalerylcarnitine at 5.82 minutes) could be individually identified and quantified with liquid chromatography-tandem mass spectrometry without derivatization. (a) sample with normal amounts of succinylcarnitine; (b) sample with abnormal amounts of methylmalonylcarnitines (Chromatograms courtesy of Mr CK Lai, Chemical Pathology Laboratory, PMH)

Conclusions

Reference

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2. Millington DS, Kodo N, Norwood DL, Roe CR. Tandem mass spectrometry: a new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism. Journal of inherited metabolic disease. 1990;13(3):321–4.

3. Schulze A, Lindner M, Kohlmüller D, Olgemöller K, Mayatepek E, Hoffmann GF. Expanded newborn screening for inborn errors of metabolism by electrospray ionization-tandem mass spectrometry: results, outcome, and implications. Pediatrics. 2003;111(6):1399–406.

4. Mak, CM. Newborn Screening: Past, Present and the Future. Topical Update, The Hong Kong College of Pathologists. 2016;11(2):1–10.

5. Ozben T. Expanded newborn screening and confirmatory follow-up testing for inborn errors of metabolism detected by tandem mass spectrometry. Clinical Chemistry and Laboratory Medicine. 2012;51(1):157–176.

6. Saudubray J-M, Berghe G van den, Walter JH. Inborn Metabolic Diseases. Springer-Verlag Berlin Heidelberg; 2012.

7. Kaspar H, Dettmer K, Gronwald W, Oefner PJ. Advances in amino acid analysis. Anal Bioanal Chem. 2009 Jan;393(2):445–52.

8. Duran M. Amino Acids. In: Blau N, Duran M, Gibson KM, editors. Laboratory Guide to the Methods in Biochemical Genetics [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008 [cited 2018 Nov 24]. p. 53–89. Available here.

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10. Cunico RL, Schlabach T. Comparison of ninhydrin and o-phthalaldehyde post-column detection techniques for high-performance liquid chromatography of free amino acids. Journal of Chromatography A. 1983;266:461–70.

11. Ferreira CR, Cusmano-Ozog K. Spurious Elevation of Multiple Urine Amino Acids by Ion-Exchange Chromatography in Patients with Prolidase Deficiency. JIMD Rep. 2016 Apr 12;31:45–9.

12. Fürst P, Pollack L, Graser TA, Godel H, Stehle P. Appraisal of four pre-column derivatization methods for the high-performance liquid chromatographic determination of free amino acids in biological materials. Journal of Chromatography A. 1990;499:557–69.

13. Schuster R. Determination of amino acids in biological, pharmaceutical, plant and food samples by automated precolumn derivatization and high-performance liquid chromatography. Journal of Chromatography B: Biomedical Sciences and Applications. 1988;431:271–84.

14. Terrlink T, Van Leeuwen PA, Houdijk A. Plasma amino acids determined by liquid chromatography within 17 minutes. Clinical chemistry. 1994;40(2):245–9.

15. Narayan SB, Ditewig-Meyers G, Graham KS, Scott R, Bennett MJ. Measurement of plasma amino acids by Ultraperformance® Liquid Chromatography. Clinical Chemistry and Laboratory Medicine. 2011;49(7):1177–1185.

16. van Wandelen C, Cohen SA. Using quaternary high-performance liquid chromatography eluent systems for separating 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate-derivatized amino acid mixtures. Journal of Chromatography A. 1997;763(1–2):11–22.

17. Armenta JM, Cortes DF, Pisciotta JM, Shuman JL, Blakeslee K, Rasoloson D, et al. A sensitive and rapid method for amino acid quantitation in malaria biological samples using AccQ•Tag UPLC-ESI-MS/MS with multiple reaction monitoring. Anal Chem. 2010 Jan 15;82(2):548–58.

18. Gray N, Zia R, King A, Patel VC, Wendon J, McPhail MJW, et al. High-Speed Quantitative UPLC-MS Analysis of Multiple Amines in Human Plasma and Serum via Precolumn Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate: Application to Acetaminophen-Induced Liver Failure. Anal Chem. 2017 Feb 21;89(4):2478–87.

19. Sharer JD, Biase ID, Matern D, Young S, Bennett MJ, Tolun AA. Laboratory analysis of amino acids, 2018 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG). Genetics in Medicine. 2018 Oct 19;1.

20. Walker V, Mills GA. N-(pyrrole-2-carboxyl) glycine a diagnostic marker of hyperprolinaemia type II: mass spectra of trimethylsilyl derivatives. Clin Chim Acta. 2009 Jul;405(1–2):153–4.

21. Christou C, Gika HG, Raikos N, Theodoridis G. GC-MS analysis of organic acids in human urine in clinical settings: a study of derivatization and other analytical parameters. Journal of Chromatography B. 2014;964:195–201.

22. Rinaldo P. Organic Acids. In: Blau N, Duran M, Gibson KM, editors. Laboratory Guide to the Methods in Biochemical Genetics [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008 [cited 2018 Nov 30]. p. 137–69. Available here.

23. Gallagher RC, Pollard L, Scott AI, Huguenin S, Goodman S, Sun Q, et al. Laboratory analysis of organic acids, 2018 update: a technical standard of the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2018;20(7):683–91.

24. Lee H-CH, Lai C-K, Yau K-CE, Siu T-S, Mak CM, Yuen Y-P, et al. Non-invasive urinary screening for aromatic l-amino acid decarboxylase deficiency in high-prevalence areas: A pilot study. Clinica Chimica Acta. 2012 Jan 18;413(1):126–30.

25. Au KM, Lai CK, Yuen YP. Diagnosis of dihydropyrimidine dehydrogenase deficiency in a neonate with thymine-uraciluria. Hong Kong Medical Journal. 2003;9(2):130–3.

26. Körver-Keularts IMLW, Wang P, Waterval HWAH, Kluijtmans LAJ, Wevers RA, Langhans C-D, et al. Fast and accurate quantitative organic acid analysis with LC-QTOF/MS facilitates screening of patients for inborn errors of metabolism. J Inherit Metab Dis. 2018 May 1;41(3):415–24.

27. Yassine MM, Dabek-Zlotorzynska E. Application of ultrahigh-performance liquid chromatography–quadrupole time-of-flight mass spectrometry for the characterization of organic aerosol: Searching for naphthenic acids. Journal of Chromatography A. 2017 Aug 25;1512:22–33.

28. Taylor PJ. Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography–electrospray–tandem mass spectrometry. Clinical biochemistry. 2005;38(4):328–34.

29. Scott C, Langhans C-D, Roux C. Qualitative Organic Acid. In: ERNDIM Workshop. Manchester, UK; 2017.

30. Kölker S, Christensen E, Leonard JV, Greenberg CR, Boneh A, Burlina AB, et al. Diagnosis and management of glutaric aciduria type I–revised recommendations. Journal of inherited metabolic disease. 2011;34(3):677.

31. Tortorelli S, Hahn SH, Cowan TM, Brewster TG, Rinaldo P, Matern D. The urinary excretion of glutarylcarnitine is an informative tool in the biochemical diagnosis of glutaric acidemia type I. Molecular Genetics and Metabolism. 2005 Feb 1;84(2):137–43.

32. Dhillon KS, Bhandal AS, Aznar CP, Lorey FW, Neogi P. Improved tandem mass spectrometry (MS/MS) derivatized method for the detection of tyrosinemia type I, amino acids and acylcarnitine disorders using a single extraction process. Clinica Chimica Acta. 2011;412(11–12):873–9.

33. Ho CS, Cheng BSS, Lam CWK. Rapid Liquid Chromatography–Electrospray Tandem Mass Spectrometry Method for Serum Free and Total Carnitine. Clinical Chemistry. 2003 Jul 1;49(7):1189–91.

34. Matern D. Acylcarnitines, Including In Vitro Loading Tests. In: Blau N, Duran M, Gibson KM, editors. Laboratory Guide to the Methods in Biochemical Genetics [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008 [cited 2018 Nov 30]. p. 171–206. Available here.

35. Rinaldo P, Cowan TM, Matern D. Acylcarnitine profile analysis. Genetics in Medicine. 2008 Feb;10(2):151–6.

36. Vreken P, Van Lint AEM, Bootsma AH, Overmars H, Wanders RJA, Van Gennip AH. Quantitative plasma acylcarnitine analysis using electrospray tandem mass spectrometry for the diagnosis of organic acidaemias and fatty acid oxidation defects. Journal of inherited metabolic disease. 1999;22(3):302–6.

37. Giesbertz P, Ecker J, Haag A, Spanier B, Daniel H. An LC-MS/MS method to quantify acylcarnitine species including isomeric and odd-numbered forms in plasma and tissues. Journal of lipid research. 2015;jlr. D061721.

38. Letters and Papers, Foreign and Domestic, Henry VIII, Volume 13 Part 1, January-July 1538 [Internet]. 1892. Available here.

Application for First Fellow in Genetic and Genomic Pathology is now opened and please download the application form here.

Results of College Exam 2018 (Council Meeting 27 September 2018)

Fellowship Assessment:

Membership Examination:

Results of College Written Exam in Chemical Pathology, Clinical Microbiology & Infection, and Haematology 2018
 

Candidates who failed in written exam cannot proceed further in Fellowship Assessment in 2018.
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Antimicrobial resistance – a global health crisis

Volume 13, Issue 2 July 2018  (download full article in pdf)

Editorial note

With increasing prominence of the threat of antimicrobial resistance both internationally and locally, awareness and knowledge on the problem and prospects are essential, in order for rational application of control measures and monitoring of their effectiveness. In this issue of the Topical Update, Dr. Dominic Tsang and Dr. Christopher Lai present an updated overview of this important subject. We welcome any feedback or suggestion. Please direct them to Dr. Janice Lo (e-mail: janicelo@dh.gov.hk), Education Committee, The Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists. 

Dr. Dominic TSANG and Dr. Christopher LAI
Consultant Microbiologist and Associate Consultant, Department of Pathology, Queen Elizabeth Hospital, Hong Kong 

End of modern medicine as we know it

The World Health Organization (WHO) described in 2001 antimicrobial resistance (AMR) as a global problem and an impending crisis. The apocalyptic term “Post-antibiotic era” was mentioned.¹ The situation did not improve since. In fact, it deteriorated. In the United Kingdom (UK) government-commissioned “Review of Antimicrobial Resistance” by Lord Jim O’Neill, it was estimated that 700,000 people died from AMR infections in 2016 in the UK alone, and that drug-resistant infections could cause 10 million human deaths annually by 2050, costing the world up to $100 trillion.² The Chief Medical Officer for England, Dame Professor Sally Davies, has predicted that unless tackled now, AMR could lead to the end of modern medicine as we know it. It could lead to routine operations and even childbirth becoming increasingly dangerous without the required antibiotics. In the UK, over 25,000 deaths a year are attributed to drug resistant infections.³ European Commission estimated the costs associated with AMR infection at €1.5 billion annually.⁴

The driving force behind emergence and dissemination of AMR is directly related to the use of antibiotics, i.e. the antibiotic selection pressure.⁵ It is recognized that non-human indiscriminate use of antibiotics in agriculture and animal husbandry to promote growth in animals and the consequential persistence of antibiotics in soil and aquatic environment select for AMR that could be disseminated widely.⁶ Human overuse nonetheless needs to be controlled by dedicated efforts on strengthening regulations on over-the- counter purchase of prescription only antibiotics, enhancing training in antibiotic prescriptions, monitoring compliance with antibiotic guideline and antibiotic stewardship programme (ASP).⁷

Concerted effort against AMR

Antibiotics on one hand is an agent required for life saving. But on the other, its usage and the resultant selective pressure have been recognized as the main drivers for AMR. Therefore, its use in human and non-human settings should be balanced against the risk of driving AMR. As such, antibiotic usage data has been linked to AMR surveillance data in the AMR containment strategy.^8,9

There are already in place a few surveillance systems on AMR, covering healthy animals, diseased animals, food and humans in countries such as Canada, Denmark, Finland, Germany, Italy, Japan, USA, the Netherlands, Norway, France, and Sweden.¹⁰

The UK and China will establish the Global AMR Research Innovation Fund and encourage further investment from other governments and the private sector, helping to address AMR. The new fund will invite bids from industry, academia and other bodies. It aims to create international partnerships to build a global response and support new research to reduce the spread of antibiotic resistance.

The World Health Assembly in May 2015 endorsed a WHO global action plan to tackle antimicrobial resistance to ensure continuity of successful treatment and prevention of infectious diseases with effective and safe medicines.¹¹ The plan sets out five strategic objectives including: to improve awareness and understanding of antimicrobial resistance; to strengthen knowledge through surveillance and research; to reduce the incidence of infection; to optimize the use of antimicrobial agents; and to develop the economic case for sustainable investment that takes account of the needs of all countries, and increase investment in new medicines, diagnostic tools, vaccines and other interventions. Under each of the objectives, specific actions were listed out for member states as well as international partners to implement.

Hong Kong Strategy against Multidrug resistant organisms (MDROs)

The Hong Kong Strategy and Action Plan on Antimicrobial Resistance 2017-2022¹² was released in July 2017 with the goals to develop a territory-wide network on surveillance on AMR and antimicrobial use, promote appropriate therapeutic use of antimicrobials in human and animals and to promote research on diagnostic and related interventions. Six key areas were targeted in the plan, including strengthening knowledge, optimizing antimicrobial use, reducing infections, improving awareness, promoting research and fostering partnerships among stakeholders.

In terms of AMR surveillance, the overall resistance profiles of MDROs have all along been closely monitored in public hospitals under the Hospital Authority in Hong Kong¹³ Among the concerned MDROs, Gram positive organisms include methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE). Gram negative organisms include extended spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) and the WHO top priority organisms of carbapenem-resistant Enterobacteriaceae (CRE), carbapenem- resistant Acinetobacter baumannii (CRAB), and carbapenem-resistant Pseudomonas aeruginosa (CRPA).¹⁴

Trends in antibiotic resistant organisms

MRSA: In Hong Kong, MRSA bloodstream infection was made a key performance indicator (KPI) to gauge the performance of infection control practices in all public hospitals.¹⁵ The corresponding MRSA bacteraemia rate was 0.18- 0.19 per 1,000 acute bed days when the monitoring began in 2008 and declined gradually to 0.144 per 1,000 acute bed days in 2017. At the same time, MRSA constituted 43.1% among S. aureus isolated from local HA hospitals.

VRE: The total number of VRE cases detected remains below 50 a month since 2015, after an upsurge and successful control in the Queen Elizabeth Hospital in 2013 when the peak number of cases detected in a month reached 300.¹⁶

CRAB/multi-drug resistant Acinetobacter baumannii (MDRA) is commonly associated with patients with prolonged hospital stay and who required ventilator-assisted ventilation. These patients are also usually put on multiple antibiotics for the treatment of underlying infections.¹⁷ Substantial environmental contamination with the resistant bacteria is frequent as a result of nursing care procedures, leading to explosive outbreaks which are difficult to abort unless attention is given to patient segregation and effective disinfection of instruments and environmental surfaces.^18,19

ESBL-E began to emerge in late 1980s and peaked in UK in 2006 with 12% E. coli isolated from bacteraemic cases being ESBL positive. ESBL-E has been common locally, at 22% in 2017 compared to 24.3% since 2012. They are especially important as they commonly caused community-acquired as well as hospital-acquired infections. In fact, a local report showed 62% of imported chicken were found to be contaminated with ESBL-E.²⁰ Community carriage rates for ESBL-E are high in East Mediterranean areas and South East Asia, from 30-60%.^21,22 For treatment of ESBL-E infections, imipenem has been universally effective since its launch in 1987, and the same applies to meropenem, another carbapenem antibiotic which was launched in 1996.

CPE are members of the Enterobacteriaceae including E. coli and Klebsiella species which possess the gene on plasmids coding for the enzymes such as KPC, NDM and OXA-48 under the Ambler Classification on molecular class A, B or D respectively which enable them to inactivate carbapenem antibiotics and also other beta- lactams. CPE are usually multiply antibiotic resistant, therefore rendering limited options in treatment of their infections.²³ The emergence and subsequent spread of CPE followed human movement closely as exemplified by the clustering of KPC-CPE in New York, North-West England, Israel and the spreading of NDM-CPE to Sweden by a returning patient from New Delhi, India.^24-26 In Hong Kong, CPE cases were being increasingly detected, from 19 cases in 2011 to 473 cases in 2017. Active bacterial screening (ABS) of patients based on a set of consensus criteria in public hospitals including admission screening of all patients who have stayed in overseas hospitals in the past 12 months, suffering from antibiotic associated diarrhoea, staying in the same cubicle of a known CPE case, etc., helped to pick up an increasing number of CPE. Fortunately, most of the cases (90%) were asymptomatic carriers and did not require specific treatment. Of note, the number of confirmed CPE isolates in England in 2016 was more than 2,500 with KPC, NDM and OXA-48 predominant. Effective treatment options for CPE are few. The activities of ceftazidime and aztreonam with and without avibactam were tested against a large, contemporary, international collection of carbapenemase-producing Gram negative bacilli (CP-GNB) with diverse resistance mechanisms. Aztreonam-avibactam was active against all isolates except two NDM producers with elevated MICs of 8/4 and 16/4 mg/litre; ceftazidime- avibactam was active against all KPC-, IMI-, SME-, and most OXA-48 group-producing isolates (93%) but not metallo-β-lactamase producers. Among the older and contemporary antimicrobials, the most active were colistin, tigecycline, and fosfomycin, with overall susceptibilities of 88%, 79%, and 78%, respectively.²⁷ Among local CPE isolates, the resistance rate of colistin is around 5.8%.

The “Find and Confine” Control strategy of AMR in HA hospitals

“Find”: This is the single most important control measures aiming to uncover the carriage state of any asymptomatic carrier, especially of VRE and CPE, who could shed the MDRO in the excreta. This is commonly done by ABS in the form of admission screening based on high risk factors such as hospitalization in recent months, a history of exposure to a known case, prolonged hospital stay (e.g. for 14 days or more), development of antibiotic associated diarrhoea etc. The yield of ABS at present is not high, ranging from 0.6% to 1.4% (average 1.0%) based on 2017 CPE ABS data in one of the hospitals, but ABS has undoubtedly provided a substantial impact in mitigating otherwise uninterrupted dissemination of VRE and CPE in the hospital setting. The availability of commercial agar media with the function to differentiate CPEs after overnight incubation has greatly facilitated ABS. Coupled with other rapid enzyme detection and PCR confirmation of CPE, which are also available commercially, the time to detection of CPE carriage in patients has been shortened to one to two days.

“Confine”: It is an important part of standard precautions (SP) plus contact precautions (CP) in the care of a known MDRO case. A room with en- suite is the preferred placement especially for those who suffer from diarrhoea and therefore at a higher risk of transmission of MDROs. This is widely practiced in developed countries. When en-suite rooms are not available, segregation of MDRO carriers by “cohorting” patients carrying the same MDRO is a pragmatic alternative.

Hand hygiene (HH): Healthcare workers (HCWs) strictly observing proper HH, e.g. WHO 5 moments, is undoubtedly the most important and effective measure in aborting the dissemination of MDROs in healthcare setting. The use of alcohol handrub in place of hand washing in situations where soiling is minimal has greatly improved HH compliance. However, HH is an action governed by human behavior which unfortunately also suffers from all factors that affect our behavior, such as physical fatigue, forgetfulness, motivation, persistence, peer influence, etc. For the same reason, the reliability of HH compliance monitoring by direct observation is also limited by the well-known “Hawthorn effect” in that, as humans, we tend to perform when we are aware of being observed.²⁸ Further, in periods of high bed occupancy like influenza seasons, it is very hard to expect HCWs to fully comply with HH and as a consequence, these periods are more prone to cross transmission and outbreaks of MDROs. Apart from HCWs, patients and visitors are equally important in observing HH in order to avoid cross transmission. Similar moments of HH are being promoted for patients in local hospitals. In the long run, the development of a positive and motivated infection control culture and HH habit would not only maintain the cross transmission of MDROs at a low level but also mitigate the risk of other cross infections.

Environmental hygiene: The inanimate environment plays an important role inperpetuating the dissemination of MDROs in the hospital settings, especially VRE and MDRA.^29,30 High-touch (frequently touched) surfaces such as patients’ privacy curtain, bed rails, door knobs, nursing trolleys, drip stands are often contaminated with the MDROs during outbreaks.³¹ The conventional cleansing and disinfection by using diluted sodium hypochlorite solution, although effective, is labour intensive. Also, to ensure the cleaning procedure is meticulously performed, it is commonly monitored by the use of a surrogate marker (e.g. UV-fluorescent marker) to ensure satisfactory performance.³² To circumvent such drawback, there are now plenty of effective new products on environmental disinfection, ranging from self- disinfecting surface coating sprays, hydrogen peroxide vapor, UV-C device, 2-in-1 disinfectant wipes to antimicrobial privacy curtains that could achieve effective decontamination with less labour. In a multi-centre study, 42.7% of standard hospital curtains were contaminated with MRSA and 42.3% with CRAB.³³ The use of disposable antibacterial privacy curtains, e.g. nanoparticle silver or quaternary ammonium impregnated curtains, have been shown to prolong the time to contamination and reduce the bio-burden even after extended usage in acute care setting.³⁴

Reducing overuse of antibiotics: It is imperative to maintain the use of antibiotic at the minimum essential level in order to prevent the emergence of resistance.34 Education and training at an early stage of the medical curriculum is critical in establishing the concept and skills in prudent antibiotic use. Guideline such as the local IMPACT guideline on antibiotic use is indispensable in providing the guidance on the right indications, choice, dose, route, and duration of antibiotic use. In addition, antibiotic stewardship program (ASP) has been widely practiced and proven to be effective in ensuring appropriate use of antibiotics.³⁵ Locally, a multi- disciplinary team of clinical microbiologists, physicians, pharmacists and infection control nurses has been put in place in all HA hospitals to provide concurrent feedback on the use of targeted “big-gun” antibiotics. The percentage of appropriate use stays at above 80% in general and feedbacks on antibiotic use are welcomed in most of the cases.

Another very important aspect in the control of AMR is diagnostic support in infection.³⁶ The rapid isolation and identification of an aetiological agent lends strong support in the continuation of antibiotic treatment, or its discontinuation in the absence of any evidence of infection. This is made possible with the introduction of molecular platforms such as 16S ribosomal RNA PCR and metagenomic studies.³⁷

Surrogate biomarker of infection, in particular procalcitonin (PCT) which exhibits greater specificity than other proinflammatory markers such as C reactive protein (CRP) helps in identifying patients with sepsis and can be used for diagnosing infections, especially ventilator- associated pneumonia (VAP).^38,39 The short half- life (25-30 hours in plasma) of PCT and its absence in healthy state make it the preferred biomarker for bacterial infections. PCT levels

The outlook of the challenges from AMR

The outcome in our battle against MDROs depends on how successful we are in preserving the efficacy of our existing antibiotics for treatment of infections and the result of our search for new antibiotics. Both require resources, efforts and dedication. While preserving existing effective antibiotics demands the aforementioned One Health approach, research breakthroughs arguably provide us the only hope in winning the battle against MDROs and to ensure effective treatment of infections for the continual practice of modern medicine.

Not long ago, researchers have identified from a soil sample a new cell wall inhibitor, teixobactin, from a previously unknown Gram-negative bacterium that lives in soil but which cannot be cultured in the laboratory using standard technique.⁴⁰ The researchers used the “Ichip”, an isolation chamber, in which a soil sample is diluted with agar and a single bacterial cell in a chamber is then placed in soil where the bacteria could access to nutrients and growth factors. The teixobactin identified has excellent activity against Gram-positive pathogens including MRSA, Clostridium difficile, Bacillus anthracis and Mycobacterium tuberculosis. Another recent breakthrough was reported⁴¹ on accessing hidden natural products (NP) made by bacteria not by culturing but by sequencing, bioinformatics analysis and heterologous expression of biosynthetic gene clusters captured on DNA extracted from environmental samples. Such technique has led to the discovery of malacidins, a distinctive class of antibiotics which are active against MRSA infections without selection for resistance under the laboratory conditions. These discoveries are definitely good news given the great potential for more to be discovered by using these innovative technologies. Of course, there are still tests to be done before they could become clinically useful, but at least these discoveries through our human creative and innovative minds are holding promise in the battle against AMR. Meanwhile, we must not be distracted away from the momentum in tackling the rapidly deteriorating AMR situation.

Reference

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21. Barreto Miranda I, Ignatius R, Pfuller R, Friedrich-Janicke B, Steiner F, Paland M, Dieckmann S, Schaufler K, Wieler LH, Guenther S, Mockenhaupt FP. High carriage rate of ESBL- producing Enterobacteriaceae at presentation and follow-up among travellers with gastrointestinal complaints returning from India and Southeast Asia. J Travel Med [Internet]. 2016 [cited 2016 Feb];23(2):tav024. In: Ovid MEDLINE(R) [Internet].

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24. Woodford N, Tierno PM Jr, Young K, Tysall L, Palepou MF, Ward E, Painter RE, Suber DF, Shungu D, Silver LL, Inglima K, Kornblum J, Livermore DM. Outbreak of Klebsiella pneumoniae producing a new carbapenem- hydrolyzing class A beta-lactamase, KPC-3, in a New York Medical Center. Antimicrob Agents Chemother [Internet]. 2004 [cited 2004 Dec];48(12):4793-9. In: Ovid MEDLINE(R) [Internet].

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26. Khong WX, Xia E, Marimuthu K, Xu W, Teo YY, Tan EL, Neo S, Krishnan PU, Ang BS, Lye DC, Chow AL, Ong RT, Ng OT. Local transmission and global dissemination of New Delhi Metallo-Beta-Lactamase (NDM): a whole genome analysis. BMC Genomics [Internet]. 2016 [cited 2016 06 13];17452. In: Ovid MEDLINE(R) [Internet].

27. Vasoo S, Cunningham SA, Cole NC, Kohner PC, Menon SR, Krause KM, Harris KA, De PP, Koh TH, Patel R. In Vitro Activities of Ceftazidime-Avibactam, Aztreonam-Avibactam, and a Panel of Older and Contemporary Antimicrobial Agents against Carbapenemase-Producing Gram-Negative Bacilli. Antimicrob Agents Chemother [Internet]. 2015 [cited 2015 Dec];59(12):7842-6. In: Ovid MEDLINE(R) [Internet].

28. Srigley JA, Furness CD, Baker GR, Gardam M. Quantification of the Hawthorne effect in hand hygiene compliance monitoring using an electronic monitoring system: a retrospective cohort study. BMJ Qual Saf [Internet]. 2014 [cited 2014 Dec];23(12):974-80. In: Ovid MEDLINE(R) [Internet].

29. Chemaly RF, Simmons S, Dale C Jr, Ghantoji SS, Rodriguez M, Gubb J, Stachowiak J, Stibich M. The role of the healthcare environment in the spread of multidrug-resistant organisms: update on current best practices for containment. Ther. adv. infect. dis. [Internet]. 2014 [cited 2014 Jun];2(3-4):79-90. In: Ovid MEDLINE(R) In- Process & Other Non-Indexed Citations [Internet].

30. Tan TY, Tan JS, Tay H, Chua GH, Ng LS, Syahidah N. Multidrug-resistant organisms in a routine ward environment: differential propensity for environmental dissemination and implications for infection control. J Med Microbiol [Internet]. 2013 [cited 2013 May];62(Pt 5):766-72. In: Ovid MEDLINE(R) [Internet].

31. Dancer SJ. Controlling hospital-acquired infection: focus on the role of the environment and new technologies for decontamination. Clin Microbiol Rev [Internet]. 2014 [cited 2014 Oct];27(4):665-90. In: Ovid MEDLINE(R) [Internet].

32. Ling ML, Apisarnthanarak A, Thu le TA, Villanueva V, Pandjaitan C, Yusof MY. APSIC Guidelines for environmental cleaning and decontamination. Antimicrob. resist. infect. control [Internet]. 2015 [cited 2015];458. In: Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations [Internet].

33. Shek K, Patidar R, Kohja Z, Liu S, Gawaziuk JP, Gawthrop M, Kumar A, Logsetty S. Rate of contamination of hospital privacy curtains on a burns and plastic surgery ward: a cross-sectional study. J Hosp Infect [Internet]. 2017 [cited 2017 May];96(1):54-58. In: Ovid MEDLINE(R) [Internet].

34. Canton R, Morosini MI. Emergence and spread of antibiotic resistance following exposure to antibiotics. FEMS Microbiol Rev [Internet]. 2011 [cited 2011 Sep];35(5):977-91. In: Ovid MEDLINE(R) [Internet].

35. Plachouras D, Hopkins S. Antimicrobial stewardship: we know it works; time to make sure it is in place everywhere. Cochrane Database Syst Rev [Internet]. 2017 [cited 2017 02 09];2ED000119. In: Ovid MEDLINE(R) Revisions [Internet].

36. Center for Infectious Disease Research and Policy (CIDRAP). ASP Policy Update: Rapid Diagnostic Testing in Antimicrobial Stewardship [Internet]. Minneapolis: University of Minnesota; 2017 Oct [cited 2018 May].

37. Bogaert D, van Belkum A. Antibiotic treatment and stewardship in the era of microbiota-oriented diagnostics. Eur J Clin Microbiol Infect Dis [Internet]. 2018 [cited 2018 05];37(5):795-798. In: Ovid MEDLINE(R) In- Process & Other Non-Indexed Citations [Internet].

38. Stolz D, Smyrnios N, Eggimann P, Pargger H, Thakkar N, Siegemund M, Marsch S, Azzola A, Rakic J, Mueller B, Tamm M. Procalcitonin for reduced antibiotic exposure in ventilator- associated pneumonia: a randomised study. Eur Respir J [Internet]. 2009 [cited 2009 Dec];34(6):1364-75. In: Ovid MEDLINE(R) [Internet].

39. Schuetz P, Wirz Y, Sager R, Christ-Crain M, Stolz D, Tamm M, Bouadma L, Luyt CE, Wolff M, Chastre J, Tubach F, Kristoffersen KB, Burkhardt O, Welte T, Schroeder S, Nobre V, Wei L, Bucher HC, Bhatnagar N, Annane D, Reinhart K, Branche A, Damas P, Nijsten M, de Lange DW, Deliberato RO, Lima SS, Maravic-Stojkovic V, Verduri A, Cao B, Shehabi Y, Beishuizen A, Jensen JS, Corti C, Van Oers JA, Falsey AR, de Jong E, Oliveira CF, Beghe B, Briel M, Mueller B. Procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections. Cochrane Database Syst Rev [Internet]. 2017 [cited 2017 10 12];10CD007498. In: Ovid MEDLINE(R) In- Process & Other Non-Indexed Citations [Internet].

40. Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Schaberle TF, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K. A new antibiotic kills pathogens without detectable resistance. Nature [Internet]. 2015 [cited 2015 Jan 22];517(7535):455-9. In: Ovid MEDLINE(R) [Internet].

41. Hover BM, Kim SH, Katz M, Charlop-Powers Z, Owen JG, Ternei MA, Maniko J, Estrela AB, Molina H, Park S, Perlin DS, Brady SF. Culture- independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant Gram-positive pathogens. Nat. microbiol. [Internet]. 2018 [cited 2018 Apr];3(4):415-422. In: Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations [Internet].

Volume 27, Issue 2 (click here to download the full pdf version)

Message from the President

Time flies! The Year of 2018 is coming to an enjoyable end as we are continuously receiving good news from our seniors and young Fellows.

I would like to congratulate Prof. KY Yuen, Prof. Dennis Lo and Prof. Malik Peiris for their recent awards and honours received locally and internationally. Between them, they have added significant new dimensions to the reach of their science and the way we view and understand their disciplines. In addition, two of our Fellows, Dr. SM Mak and Dr. HW Ip were nominated as Distinguished Young Fellows and received their certificates from Prof. CS Lau, President of the Hong Kong Academy of Medicine in September. Dr. SM Mak, as the representative of the Young Fellows’ Chapter of our College, will give a detailed report on its activities including the Medical Indemnity Forum, and Cultivating Professionalism and Research in Medical Education.

The Training and Examinations Committee would like to announce that a total of 20 candidates passed our Membership Examination and Fellowship Assessment this year. Congratulations to the successful candidates on their achievements! In addition, the second Open Forum on the Fellowship in Genetic and Genomic Pathology was successfully conducted on 26th October 2018. More importantly, the Genetic and Genomic Pathology First Fellows Application is now open for submission till the end of March 2019.

As College President, I wish to report to you that I attended the Malaysia-Singapore-Hong Kong Tripartite Congress of Medicine in Kuala Lumpur in August as a joint function of the three Academies of Medicine as well as the Meeting of the International Liaison of Pathology Presidents (ILPP) 2018 in London in October to discuss the common interests and issues faced by the rest of the pathology world. This year, the ILPP has determined to use social media to gain public interest. Therefore, we have used our facebook homepage to publicize the successful celebration of the International Pathology Day at Queen Mary Hospital on 18th November 2018, where the College organized a series of live demonstrations and simple experiments to attract the attention of 180 secondary school students nominated by the Principals of various schools.

Being one of the 15 constituent colleges of the Academy of Medicine, our College has nominated Fellows to be interviewed by the Radio Television Hong Kong “Healthpedia”《精靈一點》to talk about their interesting encounters for the celebration of the 25th Anniversary of the Academy. The interviews can be reviewed at this link.

Finally, Dr. Dominic Tsang and Dr. Christopher Lai will share with us a Topical Update on Antimicrobial Resistance which is a worldwide issue to be tackled not only by pathologists but also by clinicians, nurses and other related healthcare workers. I would like to thank all the above Fellows who have contributed so much to the building of a positive image of pathologists. Hope you enjoy this issue of Pathologue!

Dr. CHAN Ho Ming
President
November 2018

Volume 27, Issue 1 (click here to download the full pdf version)

Message from the President

The Year of 2018 would be a year of celebration as our College has had a very good start!

After months of deliberation and negotiation in the Genetics and Genomics Taskforce, the Training and Examinations Committee as well as the College Council, various pathology specialties including Anatomical Pathology, Chemical Pathology, Haematology and Immunology have finally come to a consensus on the draft training programme for Genetic and Genomic Pathology. On 17 January 2018, the College held an Open Forum to introduce the background and the draft training programme to all Fellows and to receive comments and suggestions from them. It turned out to be a success! With the blessings from our Fellows, the Genetic and Genomic Pathology training programme was approved at one of the Hong Kong Academy of Medicine’s Council Meetings on 12 April 2018. It would be a 2-year post- fellowship training programme with knowledge-based, cross-college and specialty-based components. This new Genetic and Genomic Pathology programme will be implemented within 24 months of the date of the Academy’s approval. To accommodate the changes, the College has also updated all the fellowship training programmes, which again were endorsed at the Academy’s Council Meeting on 15 March 2018.

The College has recently established a Young Fellows’ Chapter to encourage our younger generations to increase their participation in College activities. During their participation, various generations of Fellows can sit down together and exchange their views and opinions. In addition, one of the Young Fellows, Dr. Elaine Au, would like to share a Topical Update on immune-mediated demyelinating disease.

To celebrate the ILPP(International Liaison of Pathology Presidents) International Pathology Day 2017, and to promote the image of Pathologists to the public, the College organised a workshop at the Prince of Wales Hospital to receive over 100 senior secondary school students to enjoy some interactive laboratory experiments and demonstrations.

Lastly, Dr. NG Wai Fu would like to share his hobby that can make him young and fit!

Dr. CHAN Ho Ming
June 2018

Results of College Supplementary Exam 2017 (Council Meeting 25 January 2018)

An overview of NMO Spectrum Disorder and the diagnostic utility of anti-NMO antibodies

Volume 13, Issue 1 December 2017  (download full article in pdf)

Editorial note

NMOSD is an immune mediated demyelinating disease. Though its clinical presentation may overlap with multiple sclerosis, distinguishing these two entities is important in view of differences in treatment. Anti-NMO antibodies play a crucial role in the workup and diagnosis of NMOSD. In this review, Dr Elaine Au provided an overview of the NMOSD condition and the use of anti-NMO antibody assays. We welcome any feedback or suggestions. Please direct them to Dr Elaine Au (email: ayl436@ha.org.hk) of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists. 

Dr Au Yuen Ling Elaine
Associate Consultant, Division of Clinical Immunology, Department of Pathology, Queen Mary Hospital 

NMO is an idiopathic immune mediated demyelinating disease that predominantly affects optic nerves and spinal cord. The prevalence range from 0.3 to 4.4 per 100000 people, with Asian and African-American more affected than Caucasian, where multiple sclerosis is more common in the white population (1-5). The condition has been named as Devic’s disease in the past (6), which described a monophasic disorder presenting with simultaneous bilateral optic neuritis and transverse myelitis. With the availability of specific serological marker, antibodies that targeted the water channel aquaporin-4 (AQP4), the clinical and neuroimaging spectrum of NMO disease is broadened. Instead of being a monophasic disorder, NMO antibodies positive patients with recurrent attacks are not uncommonly found. Moreover, the clinical presentations are more variable than the traditional Devic disease. There is no pathognomonic clinical feature of spectrum disorder (NMOSD), though certain clinical presentations are particularly suggestive of the disorder. These include simultaneously bilateral optic neuritis, complete spinal cord syndrome and area postrema clinical syndrome.

Multiple sclerosis is an important differential diagnosis of this condition in view of the overlapping clinical features of these two conditions. In NMOSD, optic neuritis tends to be more severe, more often with simultaneous bilateral involvement or sequential in rapid succession, compare to multiple sclerosis. Complete spinal cord syndrome, with longitudinally extensive transverse myelitis in MRI, is more suggestive of NMOSD than multiple sclerosis. Differentiating NMOSD from other demyelinating disease, i.e. multiple sclerosis is important since the treatment is different. Indeed, some multiple sclerosis therapies may aggravate NMO disorders (7-10).

Diagnostic Criteria

In 2006, the serological marker was first incorporated into the revised NMO diagnostic criteria. In 2007, NMOSD was introduced to include seropositive patients who do not follow the classical monophasic bilateral optic neuritis and transverse myelitis. Lately, the International Panel for NMO Diagnosis (IPND) has further revised the diagnostic criteria (11). For anti-NMO antibodies positive cases, presenting at least one core clinical characteristic of the disease is required for diagnosis. Core clinical characteristics include optic neuritis, acute myelitis, area postrema syndrome (episode of otherwise unexplained hiccups or nausea and vomiting), acute brainstem syndrome, narcolepsy or acute diencephalic syndrome with typical Magnetic Resonance Imaging (MRI) lesions and symptomatic cerebral syndrome with typical MRI lesions. On the other hand, more stringent clinical criteria, with additional neuroimaging findings, are required in seronegative patients to fulfill the diagnostic criteria (See appendix). 

For the workup of the disease, MRI, cerebral spinal fluid (CSF) exam and serological test for anti-NMO antibodies are important. In some patients, CSF pleocytosis, usually in the form of monocytosis and lymphocytosis, are present. Increased CSF protein levels are noted in 46-75% of cases (12, 13). Nevertheless, presence of CSF oligoclonal bands is uncommon in NMOSD, and at most transient, in contrast to the presence of persistent CSF oligoclonal bands in the case of multiple sclerosis. Finally, visual evoked potentials, somatosensory evoked potentials and brainstem acoustic evoked potentials examination may also be helpful in the workup.

Clinical course and prognosis

In NMOSD syndromes affecting regions other than optic nerve and spinal cord, not uncommonly patients will relapse with more classical involvement in subsequent attacks. Majority of NMOSD patients suffer from recurrent attacks (80- 90%), less frequently monophasic attack (10-20%) (14), while gradual progressive course with neurological deterioration is very rare (15). Relapses usually occur in clusters, but unpredictable intervals. In the Mayo Clinic series, the second relapse occurred within 1 year in 60% of cases, and within 3 years in 90% of cases (16). Repeated NMO attacks not uncommonly lead to accumulation of neurological impairment. NMO is associated with more adverse outcome than MS in general(14).

NMOSD has been shown to be frequently associated with other autoimmune disorders, including lupus, Sjogren’s syndrome, etc (17-19). On contrary, NMOSD is not common in rheumatic disease patients.

Anti-NMO antibodies

Among the different investigations, NMO antibodies test is central to the workup. Anti-NMO antibodies are pathogenic. The third extracellular loop of AQP4 is the major epitope for the anti- NMO antibodies. Biopsy and autopsy tissue obtained from seropositive patients demonstrate loss of AQP4 immunoreactivity. Perivascular complement activation in actively demyelinating lesions is also happened. In the central nervous system (CNS), AQP4 is expressed at the foot processes of astrocytes, near the basement membranes, in the optic nerve, in a subpopulation of ependymal cells, in hypothalamic nuclei and in the subfornical organ (20, 21). Truncated astrocyte processes or cell loss were found in demyelinating lesions (11). In rat models, passive transfer of the antibodies leaded to the development of disease (22, 23). These pathological findings distinguish NMOSD from multiple sclerosis.

The Anti-NMO antibodies can be detected by indirect immunofluorescence staining on tissue slide using mouse cerebellum tissue section, cell- based assays, radioimmunoprecipitation assays and enzyme-linked immunosorbent assays (ELISA). Overall, cell-based assay is preferred in view of better assay sensitivity and specificity compared to other methods. Ideally, confirmatory testing with one or more techniques is suggested (11), especially in cases with atypical presentation or borderline results are obtained.

In tissue based indirect immunofluorescence test, NMO antibodies positive case is characterized by the binding to structures adjacent to microvasculature, the Virchow-Robin spaces (VRS) and pia mater (Fig.1).

Fig. 1. Mouse cerebellum tissue section stained with anti-NMO antibodies.

This assay allows the detection of any coexisting anti-neuronal antibodies, which may be important as differential diagnosis and workup. However, the method is observer dependent and subjective. The interpretation of antibody staining may easily be affected by non-specific background staining on the tissue. In some rare occasions, antibodies other than anti-NMO may mimic the staining pattern and lead to false positive results. Moreover, indirect tissue based immunofluorescence test has relative low sensitivity (63-64%) (24-27).

Cell-based assay utilize cell lines such as human embryonic kidney 293 (HEK293) cells or Chinese hamster ovary (CHO) cells that have been transfected with AQP4 gene expression vector, so that expressing much higher level of antigen comparing to normal tissues. Cell lines from different units may use different ratio of the two isoform of AQP4: M1 and M23 in order to obtain optimal antigen presentation. Cell-based assay can be assessed by indirect immunofluorescence staining or flow cytometry. For indirect immunofluorescence cell- based assay, slide with fixed AQP-4 gene transfected cells and non- transfected cells growing on different biochips are placed side by side for comparison. Therefore, false positivity is minimized with the inclusion of control non-transfected cells. A higher expression of antigen in the transfected cells also enhance the assay sensitivity compared to tissue-based indirect immunofluorescence testing. (Fig.2)

Fig.2. HEK 293 cells transfected with AQP-4 gene expression vector and stained positive with anti- NMO antibodies.

Overall, cell-based assay is the recommended assay in view of good sensitivity and specificity (mean sensitivity 76.7% in a pooled analysis; 0.1% false positivity in a multiple sclerosis cohort) (24- 27). Commercial kits for indirect immunofluorescence cell-based assay are available, which facilitate the assay setup in service laboratories. Nevertheless, indirect immunofluorescence method is semi-quantitative and observer dependent.

Protein-based assays, like ELISA and radioimmunoprecipitation assays, in general, have lower sensitivity compared to cell based assays. In addition, ELISA, in particular at low titer, may yield nonspecific results. However, these assays provide quantitative results which may potentially be used for serial monitoring (24).

Though NMO antibody testing in serum is well- established, the diagnostic role of testing the antibody in CSF is controversial. Most of the cases reported in literature are diagnosed by serum test, though there have been rare cases reported that were CSF positive but serum test negative (28, 29). When studying paired CSF and serum samples with antibody indices calculated, intrathecal production of the NMO antibody is rare (24). NMO antibodies can be present in patients few years before and after the disease presentations. Lately, there is increasing evidence that the antibody titre may reflect disease activity. Elevated antibody levels at relapse and decrease in titre after immunosuppressant treatment has been reported in literature (30-34). Therefore, serial monitoring may possibly facilitate management and medication adjustment. However, there is no general threshold value for clinical relapse and the absolute level varies with individual patients. Rising level may not predict relapse in all cases. In addition, some methodologies, like indirect immunofluorescence test, only provide semi-quantitative results, and inter-run reproducibility is another issue. Other factors including the frequency of test necessary to achieve meaningful disease status monitoring and the cost involved are also important consideration. Therefore, it remains to be determined whether the marker should be serially monitored for treatment response and disease activity monitoring.

Treatment

The treatment for classical Devic’s disease presentation and relapsing NMOSD presentation is no different. High dose steroid is commonly employed as first- line of treatment in acute presentation. Plasma exchange may be considered in treatment refractory cases. Immunomodulatory treatment with interferon β, which is a treatment option in multiple sclerosis, may exacerbate NMOSD disorders. Therefore, differentiating between these two conditions is important. Options of steroid sparing immunosuppressants to consider in NMOSD include azathioprine, methotrexate, mycophenolate mofetil, rituximab, etc (14).

Conclusion

NMOSD is a rare but increasingly recognized condition, which present as an inflammatory and demyelinating autoimmune disorder affecting the central nervous system. With the availability of a serological marker, anti-NMO antibody, the diagnosis and differentiating from related conditions is facilitated. Timely diagnosis and treatment is important for the management of these patients.

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Appendix

NMOSD diagnostic criteria for adult patients

Diagnostic criteria for NMOSD with NMO-IgG

1. At least 1 core clinical characteristic

2. Positive test for NMO-IgG using best available detection method (cell-based assay strongly recommended)

3. Exclusion of alternative diagnoses

Diagnostic criteria for NMOSD without NMO-IgG or NMOSD with unknown NMO-IgG status

1. At least 2 core clinical characteristics occurring as a result of one or more clinical attacks and meeting all of the following requirements:

   - At least 1 core clinical characteristic must be optic neuritis , acute myelitis with LETM, or area postrema syndrome

   - Dissemination in space (2 or more different core clinical characteristics)

   - Fulfillment of additional MRI requirements, as applicable

2. Negative tests for NMO-IgG using best available detection method, or testing unavailable

3. Exclusion of alternative diagnoses

Core clinical characteristics

1. Optic neuritis

2. Acute myelitis

3. Area postrema syndrome: episode of otherwise unexplained hiccups or nausea and vomiting

4. Acute brainstem syndrome

5. Symptomatic narcolepsy or acute diencephalic clinical syndrome with NMOSD-typical diencephalic MRI lesions

6. Symptomatic cerebral syndrome with NMOSD-typical brain lesions

Additional MRI requirements for NMOSD without NMO-IgG and NMOSD with unknown NMO-Ig status

1. Acute optic neuritis: require brain MRI showing (a) normal findings or only nonspecific white matter lesions, OR (b) optic nerve MRI with T2-hyperintense lesion or T1-weighted gadolinium-enhancing lesion extending over >1/2 optic nerve length or involving optic chiasm

2. Acute myelitis: requires associated intramedullary MRI lesion extending over >=3 contiguous segments (LETM) OR >=3 contiguous segments of focal spinal cord atrophy in patients with history compatible with acute myelitis

3. Area postrema syndrome: requires associated dorsal medulla/ area postrema lesions 4. Acute brainstem syndrome: requires associated periependymal brainstem lesions

Neurology 2015;85:177-189

Clinical features and laboratory findings atypical for NMOSD, that need to consider alternative diagnoses

1. Progressive overall clinical course (neurologic deterioration unrelated to attacks: Consider MS)

2. Atypical time to attack nadir: less than 4 hours (consider cord ischemia/ infarction); continual worsening for more than 4 weeks from attack onset (consider sarcoidosis or neoplasm)

3. Partial transverse myelitis, especially when not associated with LETM MRI lesion (consider MS)

4. Presence of CSF oligoclonal bands (oligoclonal bands occur in < 20% of cases of NMO vs > 80% of MS

Comorbidities associated with neurologic syndromes that mimic NMOSD

1. Sarcoidosis, established or suggestive clinical, radiologic or laboratory findings thereof (e.g. mediastinal adenopathy, fever and night sweats, elevated serum angiotensin converting enzyme or interleukin-2 receptor level)

2. Cancer, established or with suggestive clinical, radiologic or laboratory findings thereof; consider lymphoma or paraneoplastic disease ( e.g. collapsing response mediator protein-5 associated optic neuropathy and myelopathy or anti-Ma- associated diencephalic syndrome)

3. Chronic infection, established or with suggestive clinical radiologic, or laboratory findings thereof ( e.g. HIV, syphilis)

Neurology 2015;85:177-189

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