Recent Advance in Acute Lymphoblastic Leukaemia

Volume 14, Issue 2, August 2019  (download full article in pdf)

Editorial note

Dr. Albert Sin
Clinical Assistant Professor

Introduction

Acute lymphoblastic leukaemia (ALL) is an aggressive and highly fatal malignancy resulting from clonal mutations of lymphoid progenitor cells. The incidence of ALL is the most common in childhood and age after 50.¹⁸The prognosis of childhood ALL is good with long-term survival rate approaching 90% treated by intensive chemotherapy.¹²Although the incidence of ALL is less in adolescent, young adult as well as adult, the prognosis of ALL in those people is very poor, with only 30-40% of adult patients able to remit.¹⁸ According to the data from US database which registered all patients with diagnosed ALL from 2000 to 2007, the survival rate was 75% at 17 years old, 45% at 20 years old and 15% at 70 years old.¹⁴An increasing knowledge of disease biology of ALL transformed into insights for development of novel therapies to improve the treatment outcome of ALL.

One of the reasons of adverse prognosis in adolescent and young adult (AYA) as well as adult patients is that they commonly harbored poor-risk genetic aberrations while less patients carried favorable genetic lesion.¹⁴This could explain the sudden drop in survival from 17 years old to 20 years old. 

Ph-like ALL

Ph-like ALL is a newly identified genetic subgroup. The genetic profile of this subgroup of ALL is similar to that of Philadelphia chromosome positive (Ph-positive) ALL but without BCR-ABL1 fusion.¹⁷They have a higher frequency of IKZF1 deletion and mutation in genes of lymphoid transcription factors with poor survival.¹⁹The incidence of Ph-like ALL increases with ages and approaching 27% of cases of adult B-ALL.¹⁴

The nature of genetic aberration is heterogeneous. Despite its complexity, it can be simply classified into five subgroups: 1. CRLF2 rearrangement 2. Rearrangement of ABL-class gene 3. Rearrangement of JAK2 and EPOR 4. Aberrations leading to activation of JAK-STAT or MAPK pathway 5. Other rare kinase alterations.¹⁹The distribution of different types of genetic alterations are different among childhood high risk ALL, young adult and adult (Figure 1). CRLF2 rearrangement is the most common type of genetic alteration in Ph-like ALL. CRLF2 gene is responsible for producing lymphopoietin receptor and regulate the process of lymphopoiesis. Common mechanisms of CRLF2 rearrangement include 1. Translocation of CRLF2 gene into IGH gene 2. Fusion between CRLF2 gene and P2RY8 gene. 3. Point mutation F232C at CRLF2 gene. Nearly 50% of CRLF2 rearranged Ph-like ALL have concomitant JAK mutations.¹⁹

Figure 1

Diagnosis of Ph-like ALL

Genetic profiling is the gold standard for the diagnosis of Ph-like ALL. However, it is difficult to implement in routine diagnostic laboratory. 

Cytogenetics analysis is a standard test for all cases of ALL which allows a global assessment of chromosomal abnormalities. Some of the recurrent genetic abnormalities, for example t(9;22), hyperdiploidy/hyperdiploidy,   rearrangement involving 11q23, etc, can be detected. However, most of the Ph-like ALL genetic alterations are cryptic, e.g. interstitial deletion of CRLF2, ETV6-RUNX1 fusion, etc and thus they cannot be detected by conventional cytogenetics.²

Fluorescent in-situ hybridization (FISH) can be utilized to detect Ph-like ALL genetic abnormalities. Breakapart probes targeting genes most frequently genes including ABL1, ABL2, PDGFRB, JAK2, CRLF2, and P2RY8 are currently available. Although the positive result upon FISH study needs additional fusion probe for confirmation, it provides a readily available and useful diagnostic tool for establishing the diagnosis of Ph-like ALL. However, some of the important Ph-like ALL genetic rearrangement including intrachromosomal inversions (e.g., inv(9) resulting in PAX5-JAK2 fusion), intra-chromosomal deletions (e.g., del(X)(p22p22)/del(Y)(p11p11) resulting in P2RY8-CRLF2 fusion) are undetectable by FISH technique.²Targeted sequencing by NGS platform is an evolving technique for diagnosis.¹⁶

Recently, antibody against CRLF2 is available for flow cytometry study. The expression of CRLF2 as detected by multiparametric flow cytometry is correlated with genetic testing for CRLF2 rearrangement.¹⁵This provides a rapid tool for identifying potential cases of Ph-like ALL before the result of genetic tests is available. 

Most of the genetic alterations of Ph-like ALL are targetable kinase lesions, which could be treated by tailored kinase inhibitor therapy (Table 1).¹⁹This approach of therapy is current undergoing extensive preclinical studies.⁹

Early T-cell precursor ALL (ETP-ALL)

ETP-ALL is recently characterized subtype of T-ALL. It constitutes around 12% of childhood ALL and 7.4% of adult ALL.¹¹Genetic profiling showed ETP cells are similar to that of haemopoietic stem cells and myeloid progenitor cells.⁴This subgroup of ALL is characterized by the unique immunophenotype: cytoplasmic CD3+, surface CD3-, CD1a-, CD2-, CD5 dim (<75% positive), CD7 and positive for one or more stem cell and/or myeloid markers including HLA-DR, CD13, CD33, CD34, or CD117.⁵

While activating mutation of NOTCH1 is a common mutation found in ALL and it account for 50% of cases of childhood ALL, this mutation is less common in ETP-ALL.³ETP-ALL commonly have mutations in FLT3, DNMT3A, IDH1, IDH2, etc.¹¹

ETP-ALL carries a poor prognosis with inferior overall survival when treated with standard chemotherapy regimen comparing with other subtypes of T-ALL.¹¹This subgroup of T-ALL represented a distinct subtype with unique genetic profile and poor prognosis. 

MRD in adult ALL

Minimal residual disease (MRD) describe the very low level of disease burden which cannot be detected by morphology. Measurement of MRD not only pick up a submicroscopic level of disease but also can monitor the disease kinetic during the treatment process of haematological malignancies.¹⁰

The following techniques can be used to detect MRD: 

1. Multiparametric flow cytometry to detect leukaemia-associated immunophenotype (LAIP)
   By using a 4-color or 6-color panel of antibodies, we can identify LAIP in 90% of ALL caes.¹⁰Flow cytometry is a quick method and the result of MRD can be generated in a short period of time for clinical decision. The sensitivity of MRD detection by this method is 0.01%. However, in order to define the positive MRD, we need 10-40 cluster of cells and thus higher number of cells are required for assessment which may be difficult for reassessment samples after intensive chemotherapy.⁷In addition, antigenic shift is commonly occurred in leukaemic cells and normal cells during the therapy. The use of monoclonal antibodies, e.g anti-CD19, anti-CD22 for treatment of ALL will affect the gating strategy used to identify the leukaemic cells.¹⁰


2. Detecting leukaemia-specific fusion transcript by PCR technique
   Quantitative reverse-transcriptase PCR can be employed to detect the amount of leukaemia-specific fusion transcript. The sensitivity is higher compared with flow cytometry (10^-4to 10^-6).⁷The test is relatively easy to be performed in standardized diagnostic laboratory. However, only 30-40% of cases of ALL carry leukaemia-specific fusion transcript and thus limited the eligibility of MRD detection by this method. Moreover, the interpretation is challenging for RNA-based test in those cases will poor RNA quality.


3. Quantitative PCR for immunoglobulin (IG)-T cell receptor (TCR) gene targets
   Quantitative PCR is employed to detect the specific sequence of rearranged IG gene or TCR gene in the sample. The sensitivity of this method is 10^-4to 10^-5and this method can be applied to all cases of ALL. However, this method of MRD detection requires prior characterization of IG or TCR gene rearrangement by sequencing and designs patient-specific primers for each case for subsequent MRD detection. Extensive standardization and experience are needed for the laboratory to set up this test, which limit the use of this method of MRD detection in diagnostic laboratory. Moreover, the clonal evolution in leukaemic blasts during treatment can make the original rearranged sequence to be lost and thus generate a false negative result. Also, the non-specific primer annealing occurs during the process of marrow regenerative may yield false positive result for the test.⁷

Application of MRD in treatment of adult ALL

MRD-guided therapy had been gained extensive experience in childhood ALL.⁸The study group of German Multicenter Study Group for Adult ALL (GMALL) had conducted largest study for the role of MRD in adult Ph-negative ALL. They showed that molecular remission is the only parameters significantly affect the remission duration and survival.⁶Patients with positive MRD after induction therapy achieved better overall survival after receiving haemopoietic stem cell transplant. Early achievement of MRD negativity after induction chemotherapy is associated with good outcome for adult ALL.¹⁰Study showed that MRD level correlates with post-transplant outcome.¹³Another group found that haemopoietic stem cell transplant benefits the patients with positive MRD at week 6.¹These findings may prompt reconsideration of the indications of haemopoietic stem cell transplant for adult patients with ALL, especially those patients achieve MRD negativity after treatment. 

Concluding landmark

The prognosis of acute lymphoblastic leukaemia in young adolescent and adult is poor. The recent discovery of new subtype of acute lymphoblastic leukaemia with characterization of genetic lesions make a breakthrough of understanding of disease biology. Precise disease prognostication can be made. Targeted therapies are being developed for treating those patients. Clinical trials are conducting for evaluating the targeted therapies in those new subtypes of acute lymphoblastic leukaemia. Moreover, the application of MRD monitoring and MRD-adapted therapy in adult ALL can further stratified the patients and select the appropriate candidates of haemopoietic stem cell transplant in order to reduce transplant-related mortality and morbidity. The advances in understanding of molecular mechanism and disease biology of ALL help to improve the risk stratification, rapid development of targeted therapies and hopefully improve the prognosis in young adolescent and adult patients. 

Reference

1. Beldjord K., Chevret S., Asnafi V., Huguet F., Boulland ML., Leguay T., Thomas X., Cayuela JM., Grardel N., Chalandon Y., Boissel N., Schaefer B., Delabesse E., Cavé H., Chevallier P., Buzyn A., Fest T., Reman O., Vernant JP., Lhéritier V., Béné MC., Lafage M., Macintyre E., Ifrah N., Dombret H; Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL). (2014) Oncogenetics and minimal residual disease are independent outcome predictors in adult patients with acute lymphoblastic leukemia. Blood, 2014,12;123(24):3739-49.
2. Bradford J. Siegele., Valentina Nardi. (2018). Laboratory testing in BCR-ABL1-like (Philadelphia-like) B-lymphoblastic leukemia/lymphoma. Am J Hematol, 2018(93):971–977.
3. Chao Gao., Shu-Guang Liu., Rui-Dong Zhang., Wei-Jing Li., Xiao-Xi Zhao., Lei Cui., Min-Yuan Wu., Hu-Yong Zheng and Zhi-Gang Li. (2014) NOTCH1 mutations are associated with favourable long-term prognosis in paediatric T-cell acute lymphoblastic leukaemia: a retrospective study of patients treated on BCH-2003 and CCLG-2008 protocol in China. Br J Haematol, 2014, 166(2):221-8. doi: 10.1111/bjh.12866
4. Elaine Coustan-Smith., Charles G Mullighan., Mihaela Onciu., Frederick G Behm., Susana C Raimondi., Deqing Pei., Cheng Cheng., Xiaoping Su., Jeff rey E Rubnitz., Giuseppe Basso., Andrea Biondi., Ching-Hon Pui., James R Downing., Dario Campana. (2009) Early T-cell precursor leukaemia: a subtype of very high-risk acute lymphoblastic leukaemia.Lancet Oncol, 2009(10):147–56
5. Elizabeth A. Raetz and David T. Teachey. (2016) T-cell acute lymphoblastic leukemia. Am Soc Haemaetolo Educ Program, 2016(1), 580-588
6. Gökbuget N., Kneba M., Raff T., Trautmann H., Bartram CR., Arnold R., Fietkau R., Freund M., Ganser A., Ludwig WD., Maschmeyer G., Rieder H., Schwartz S., Serve H., Thiel E., Brüggemann M., Hoelzer D; German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia. (2012) Adult patients with acute lymphoblastic leukemia and molecular failure display a poor prognosis and are candidates for stem cell transplantation and targeted therapies. Blood, 2012,120(9):1868-76.
7. Jacques J. M. van Dongen., Vincent H. J. van der Velden., Monika Br¨uggemann and Alberto Orfao. (2015) Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies.Blood, 2015;125(26):3996-4009
8. Marianne Ifversen., Dominik Turkiewicz., Hanne V. Marquart., Jacek Winiarski., Jochen Buechner., Karin Mellgren., Johan Arvidson., Jelena Rascon., Lenne-Triin Ko¨rgvee., Hans O. Madsen., Jonas Abrahamsson., Bendik Lund., Olafur G. Jonsson., Carsten Heilmann., Mats Heyman., Kjeld Schmiegelow., Kim Vettenranta. (2019) Low burden of minimal residual disease prior to transplantation in children with very high risk acute lymphoblastic leukaemia: The NOPHO ALL2008 experience. British Journal of Haematology, 2019(184): 982–993
9. Maude SL, Tasian SK, Vincent T, et al. (2012) Targeting JAK1/2 and mTOR in murine xenograft models of Ph-like acute lymphoblastic leukemia. Blood, 2012;120(17):3510-3518.
10. Monika Bru¨ ggemann and Michaela Kotrova. (2017) Minimal residual disease in adult ALL: technical aspects and implications for correct clinical interpretation. Hematology Am Soc Hematol Educ Program.2017(1):13-21. doi: 10.1182/asheducation-2017.1.13.
11. Nitin Jain., Audrey V. Lamb., Susan O’Brien., Farhad Ravandi., Marina Konopleva., Elias Jabbour., Zhuang Zuo., Jeffrey Jorgensen., Pei Lin., Sherry Pierce., Deborah Thomas., Michael Rytting., Gautam Borthakur., Tapan Kadia., Jorge Cortes., Hagop M. Kantarjian., Joseph D. Khoury. (2016) Early T-cell precursor acute lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) in adolescents and adults: a high-risk subtype. Blood, 2016;127(15):1863-1869
12. Paul, S., Kantarjian, H., & Jabbour, E. J. (2016). Adult Acute Lymphoblastic Leukemia. Mayo Clin Proc, 91(11), 1645-1666. doi:10.1016/j.mayocp.2016.09.010
13. Renato Bassan., Monika Brüggemann., Hoihen Radcliffe., Elizabeth Hartfield., Georg Kreuzbauer., Sally Wetten. (2019) A Systematic Literature Review And Meta-Analysis Of Minimal Residual Disease As A Prognostic Indicator In Adult B-Cell Acute Lymphoblastic Leukemia. Haematologica,2019 Mar 19. pii: haematol.2018.201053. doi: 0.3324/haematol.2018.201053.
14. Roberts KG. (2018) Genetics and prognosis of ALL in children vs adults. Hematology Am Soc Hematol Educ Program. 2018(1):137-145. doi: 10.1182/asheducation-2018.1.137.
15. Sergej Konoplev., Xinyan Lu., Marina Konopleva., Nitin Jain., Juan Ouyang., Maitrayee Goswami., Kathryn G. Roberts., Marc Valentine., Charles G. Mullighan., Carlos Bueso-Ramos., Patrick A. Zweidler-McKay., Jeffrey L. Jorgensen and Sa A. Wang. (2017) CRLF2-Positive B-Cell Acute Lymphoblastic Leukemia in Adult Patients: A Single-Institution Experience. Am J Clin Pathol, 2017(147):357-363
16. Stadt UZ., Escherich G., Indenbirken D., Alawi M., Adao M., Horstmann MA. (2016) Rapid Capture Next-Generation Sequencing in Clinical Diagnostics of Kinase Pathway Aberrations in B-Cell Precursor ALL. Pediatr Blood Cancer, 2016;63(7):1283-6. doi: 10.1002/pbc.25975
17. Tasian, S. K., Loh, M. L., & Hunger, S. P. (2017). Philadelphia chromosome-like acute lymphoblastic leukemia. Blood, 130(19), 2064-2072. doi:10.1182/blood-2017-06-743252
18. Terwilliger, T., & Abdul-Hay, M. (2017). Acute lymphoblastic leukemia: a comprehensive review and 2017 update. Blood Cancer J, 7(6), e577. doi:10.1038/bcj.2017.53
19. Thai Hoa Tran and Mignon L. Loh. (2016). Ph-like acute lymphoblastic leukaemia. Haematology Am Soc Haemaetolo Educ Program, 2016(1), 561-566.

Results of College Written Exam in Chemical Pathology & Clinical Microbiology and Infection 2019
 

Candidates who failed in written exam cannot proceed further in Fellowship Assessment in 2019.
Passed candidates can proceed further in Membership or Fellowship Assessment in 2019.

Volume 28, Issue 1 (click here to download the full pdf version)

Message from the President

This is the first issue of our College Newsletter in 2019. In 2018, College was busy with conducting the activities associated with the new training programme in Genetic and Genomic Pathology including the second open forum and First Fellow application. We are now processing numerous applications. An interview will be conducted in October 2019 to finalise the eligibility.

In 2018, the Hong Kong Academy of Medicine was also celebrating her 25th Anniversary with a series of fascinating events including the Congress of Medicine and Gala Dinner with an impressive drummer performance at the opening. Our Fellow, Prof. YUEN Kwok Yung was the Sir David Todd Orator in 2018. He presented his journey to becoming a renowned microbiologist with multiple showcases of Sherlock Holmes-style investigations for our community. In early 2019, the Academy also conducted a visit to Sichuan for our Young Fellows, so as to increase their understanding of the development of specialist care in Mainland China. Dr. MAK Siu Ming and Dr. CHENG Shui Ying, Ivy, represented our College to participate in this visit.

In March 2019, the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPA-QAP) held one of their 30th Anniversary Quality Symposia in the Pao Yue Kong Auditorium at the Hong Kong Academy of Medicine Jockey Club Building. Local and overseas speakers came to give talks and opinions on how quality can be exercised as well as the future of quality in pathology. What an enjoyable experience-sharing opportunity!

In the recent issue of Topical Update, Dr. CHONG Yeow Kuan, Calvin reviewed the technological development of biochemical genetics on three major categories of inherited metabolic diseases. With the introduction of expanded newborn screening using mass spectrometry, there is increasing awareness of these conditions in the community.

Last but not the least, Dr. CHAN Sheung Wai, Gavin will share his concept of modern mortuary development, with a life paradigm shift from just being a place for the dead to a decent environment of bereavement for the family.

I would like to thank all the above Fellows who have contributed so much to the build a positive image of pathologists. Hope you enjoy this issue of Pathologue!

Dr. CHAN Ho Ming
President

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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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.
Passed candidates can proceed further in Membership or Fellowship Assessment in 2018.

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.

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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