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Reporting :

1st IFCC, EFLM, AFCB, FIFIBCML Conference “ Laboratory Medicine: Meeting The Needs of

Mediterranean Nation”

2-4 July, 2018

University of Tor Vergata-Rome

By Dwi Yuniati Daulay

(Prodia Laboratory - Jakarta-Indonesia)

Summary :

1. I got so many experience at the IFCC Congress. Many informations about Mediterranean health care,

and the development of clinical chemistry in that areas (the detail informations are below). Some of

them can be implemented in my work at Laboratory.

2. I was so exciting when someone came in front of my poster, and ask anything about my research.

That was great!

3. That was great opportunity meeting the President of IFCC, Howard Morris. All the young scientist

were so amazing. We gave the facebook and wa number each other, and we made a YS group, so we can

get informations from each country and share so many opportunity.

4. It was new experience for me to feel the different weather, different city and different culture in

Rome. Rome is so amazing!!!

Communicable Diseases in the Mediterranean Area (Ghassan Shannan, Syria)

1. HIV (Northern Syria)

2. Hepatitis : HCV dan HBV prevalensi Southern Mediteranian lbh timggi dari Europian, dan Egypt

yg tertinggi. Control the spread of Hepatitis by stepping up vaccination & treatments)

3. TB (Maroco)

4. Cutaneous Leishmenia

5. Human Brucellosis (Syria)

6. Schistomiosis, parasitic disease (Egypt)

Nothern Mediterran : HIV/AIDS

Southern Mediteran : Infection (poor hygiene)

Pharmacogenomics and Therapy for Hep C Virus Infection (Mohamed Shaaraway, Egypt)

- Viral & Host polymorphism (genotype information) predict response to conventional PEG IFN

Alfa & Ribavirin as well as DAA (Simeprevir & Sofosbuvir)

- Q80K polymorphism testing recommended by the clinical guidelines in all patients before

initiation of simeprevir, PEG and RBV triple regimen. If the Q80K polymorphism is detected in

HCV genotype 1a infection (Olyslo, simeprevir)� need alternative therapy

- 2 SNP host (rs 12979860 dan rs 8099917) are located in IFNL3 gene (previously called IL28B),

associated with SVR in HCV genotype 1 and genotype 4 infection. IFNL3 genotype, one of

strongest predictors of SVR with PEG and RBV therapy as well as IFN-based DAA therapies.

- If an IFN-free regimens becomes more readily available in the future with SVR rates approach

100%, IFNL3 genotype may no longer hold clinical utility.

- Viral polymorphismmay play a bigger role and need to be monitored for the future

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Diet and Life style influences on telomere length as a potential biomarker for various disease (Jelena

Kotur, Serbia)

- Inflammation, oxidative stress/free radical (smoking, diabetes,obesity, hypertention,

hyperglycemia)� telomere shortening� celluler senescence� cardiovascular disease

- Physical activity/exercise� decrease ox stress, inflammation, increase telomerase activity�

telomere protection

- Vit A,D, C,E, Fiber, omega 3, tea and grape seed polyphenols, curcumin� inhibit inflammation

& ox stress

- Refugee status : psychosocial stress� premature telomere shortening, high cortisol level�

direct link between life stress condition and telomere shortening

- Telomere length in cancer : telomerase activity enables cancer cells to be alive in presence of

mutated DNA� Cancer therapy- telomerase inhibition?

- Helthy life style and drugs with anti inflammatory or antioxidative action (physical activity,

strees free life, diet rich in antiox, fibers, meat with low fat, vit D and statin use)� elongate

telomere by increasing the telomerase activity

Diagnostic proteomic markers to detect kidney disease and impaired antiox mechanism. Potential role

for antiox rich Mediterranean diet (Prof Tomris Ozben, Turkey)

- Oxidative stress play a main role in the pathogenesis of uremia, atherosclerosis and type 2

Diabetes

- Measurement of oxidative stress is useful to investigate its role in the initiation and

development of chronic complications and to evaluate preventive actions (antiox therapy and

biocompatible dialysis membrans)

- Increase production of ROS is the major factor leading to oxidative stress during hemodialysis.

Bioincompatibility is an important source of ROS.

The loss of small molecule weight antioxidants via dialysis membranes� HD patients exhibited

altered anti oxidative defences

- Proteins are important targets for free radicals� irreversible modifications on proteins cause

permanent loss of functions/protein degradation

- Protein oxidation products as markers of different disease by proteomic approaches:

comparison of protein patterns between healthy subjects and patients with pathological

condition

- Urinary proteomics : early diagnostic and differentiation of renal and urogenital tract disorders

Methods: 2-DE, Q –TOF-MS/MS, SELDI-TOF-MS, Western Blot

Microalbuminuria is parameter to indicate the presence of nephropathy in diabetic patients, but

it is not a precise indicator to demonstrate DN risk� identification of early biomarkers for DN

risk (urine proteomics analysis)

- Powerful proteomic methodologies could permit to identify promising candidate biomarkers of

kidney dysfunctions and potential nephrotoxicity induced by drugs.

Update on Cardiac Biomarkers (Martina Z, Italy)

- ACS, present and future role of biomarkers:

Myocardial damage : cTn� high sensitivity assay for rule out of MI

Myocardial ischemia : IMA

LV dysfunction : BNP� Diagnosis or exclusion of HF, prognosis and risk stratification

Soluble ST2 and Galectin-3� biomarker of myocardial fibrosis, predictive of hospitalization and

death, additive to natriuretic peptide in their prognostic value.

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Inflammation : CRP

Renal dysfunction : Cystatin C

Vit D and Health Outcomes (Howard Morris, Australia)

- Migration in the Mediterranean region : largely from the lower latitudes with the highest risk to

develop vit D deficiency� increased risk of infectious diseases

- An adequate vit D status enhance activities of the innate and adaptive immune systems

necessary for optimal protection against infectious diseases in dindividuals across all ages

- The beneficial effects of UV exposure to tuberculosis patients� Vit D was a first line treatment

for TB before antibiotics

Vit D deficiency is increased amongst HCV infected pts, and HCV pts with adequate Vit D has less

liver fibrosis and less inflammation

- Serum 25 OH Vit D measurements assess vit D status

- Level for optimal health outcomes range between 24-40 ng/ml (60-100 nmol/L)

- There is no evidence of benefit for serum 25D levels >40 ng/mL (100nmol/L)

Who or What is Sherlock? (Ann Gronowski US)

- SHERLOCK is innovative new diagnostic method to detect nucleic acid

- Analytes in blood : mM (10^-3mol/L)� nM (10^-9 mol/L)� fM (10^-15mol/L)� zM (10^-21

mol/L)

Utility of ultrasensitive methods:

Monitoring (environment, food safety, detection of biological threats)

Clinical diagnostics (early diagnosis of inf disease, testing blood supply, cancer screening)

- PCR the gold standard, however:

Requires expensive equipment with rapid temp cycling

Requires specially trained personnel

Sending to specialized lab delays TAT

In some cases not sensitive enough

- CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

Is a powerful gene editing tool

Part of bacterial immune system to fight against invanding virus

The programmable endonuclease properties of CRISPR has been harnessed to modify genes in

living cells for diagnostic testing

- SHERLOCK (Specific High sensitivity Enzymatic Reporter un Locking) : Nucleic acid detection with

CRISPR-Cas13a/C2c2�diagnostic tools that can be used to detect nucleic acid (RNA or DNA),

high sensitivity, single base specificity, portable platform (no specialized equipment)

- Potential uses of this technology are due to specificity, sensitivity, simplicity, speed & flexibility

- SHERLOCK for lateral flow detection

Proposed uses:

Rapid detection of pneumonia pathogen viral & bacterial in one assay

HIV diagnostics in resource limited area

Liquid biopsy to detect mutations in cell free DNA

Rapid POC detection of pathogen resistance genes

- CRISPER to gene-edit mutation

- SHERLOCK to determine proportion of genes successfully edited

Advancement in POCT molecular testing: the multiplex PCR POCT Devices for Infectious disease

(Alpaslan, Turkey)

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- Multiplex PCR application for tomorrow’s Laboratory

- Ideal POCT device : low complexity and high performance

- Centralized testing: Provider-centered model : Central Lab analyze large numbers of samples at

relatively low cost through automation of analytical processes and consolidation of services

(high complexity and high performance)

- Decentralized Testing: Patient-centered model: Health care is organized around the patient,

greater availability of patients testing primary care and community facilities

- WHO assured criteria for POCT:

Affordable, Sensitive, specific, Rapid, user friendly, Equipment free

- Performance characteristics of MPOCTs:

Panel size (number of disease targets that can be tested in one sample run)

Time to test result, thoughput and usability characteristics

- Potential benefits of MPOCTs:

Improving health care management for the patients

Optimizing antibiotic usage

Limiting the spread of disease

Decreasing health costs

Increasing access to testing in remote or low-resource settings

- Future of MPOC Testing:

Microfluidic devices can provide a fully integrated POC device for sample processing, fluid

handling, and signal generation

Microfluidic technologies reduced assay complexity and enable multiplex analysis and high

thoughput screening

On chip nucleic acid analysis, it miniaturizes and integrates the various assay steps

(lysis/extraction of target cells, purification nucleic acids, amplification and on chip detection of

reaction products)

- Communicability in MPOCT : Linking data to specific geographical locations via global positioning

system (GPS)

- MicroPCR systems: Flow throughmicro PCR doesnot require temperature cycling�much

faster amplification

- Microfluidics-based devices : Microfluidics cartridges (chips), combines solid-phase nucleic acid

extraction, isothermal enzymatic amplification, lyophilized reagent, real-time or endpoint

optical detection

- Microfluidics’lab-on-a-chip’ (LOC) technologies :

Short time (<60 min), reduced reagent consumption, ease of use (minimally trained personel),

minimally lab facility

Minimally-instrumented formats (battery powered or electricity-free diagnostics devices, and

utilization of smartphone (with an operating system to run software application) for detection,

analysis, communication

- BioFire Film Array respiratory panel (BioFire Diagnostics,USA, a bioMerieux Company)> detect

14 respiratory viruses

NGS from research to everyday practice (Maurizio Ferrari, Italia)

- Present & Future Sequencing:

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Sanger (Chain Termination)� Next generation : Reversible Termination( Illumina),

Pyrosequencing (Roche), Ligation (Life Technologies)� Next-next generation : Fluorescence,

Electronic, Atomic

- The Testing Process for NGS :

Indication for testing� Counseling� Sequence analysis (sample preparation, machine

sequencing, alignment (to reference genome), variant call, variant annotation, variant

classification& prioritization, result report)� Communicating results/counseling� Integration

into clinical decision making

NGS diagnostics: shifted towards data analysis (bioionformatics) rather than technical

component

- Clinical utility challenges: NGS data density frequently encountered variants of unknown

significance

Which variants are clinically actionable?

Development of evidence based scientific standard to evaluate utility in different patient

populations for accurate risk estimation

Careful selection of patients for genome sequencing and genetic counseling crucial

- Challenges for use of NGS in clinical practice :

Commom standards for ensuring the reliability of NGS results do not exist

Applying regulatory requirements (ie Clin Lab Improvement Amendments, CLIA) & professional

standards

• Establishment (validation) or verification of performance specifications

• Quality control procedures

• Independent assessment of test performance (Proficiency testing)

• Use of reference materials

- CAP ‘ Lab Standards for Next Generation Sequencing Clinical Test

- Best Practice Guidelines for the use of NGS Applications in Genome Diagnostics: A National

Collaborative Study of Dutch Genome Diagnostic Laboratories

- Diagnostic Spectrum (with increase complexity) : Targeted Gene/Variant Analysis (Sanger)�

Multi-Gene Panels�Whole exome�Whole genome

- Clinical Utility of Human Genomic NGS:

Prenatal : NIPT

Postnatal : Development delay

Adulthood : Disease onset (cancer, heart disease), Risk (Pharmacogenomic, predisposition), Fun

(ancestry, curiosity)

- Application of circulating tumour DNA analysis : cancer detection (screening or earlier diagnosis),

Molecular profiling or prognostication, detection of residual disease, monitoring response

- Body Fuids as a source of tumour-derived molecular information:

Central Nervous system� CSF : CSF-derived circulating tumour DNA better represents the

genomic alterations of brain tumour than plasma

Head & Neck� Saliva : Detection of somatic mutations and HPV in the saliva and plasma of

patients with head and neck squamous cell carcinoma

Respiratory tract� Pleural effusion : EGFR mutation status in tumour derived DNA from pleural

effusion fluid is a practical basis for predicting the response to gefitinib

Urinary tract� urine: A highlysensitive and quantitative test platform for detection of NSCLC

EGFR mutation in urine and plasma

- Genome sequencing in clinical microbiology : genomic variation of the human gut microbiome

Potential mechanisms underpinning the relationship between diet and IBD:

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Mediterranean diet : Fruits and vegetables, whole grains and sea food�Microbiome

diversity�Barrier function (intact permeability)� Immune function (tolerance vs inflammation)

Western diet : Red meat & processed food, refined sugar & saturated fat�Microbiome

dysbiosis� barrier function (impaired permeability)� immune function (loss of tolerance)

- Challenges facing genomic medicine:

Test awareness, Evidentiary framework, clinical implementation, ethical issues

Reimbursement, regulation, physician & patient education

Seminal cell free DNA assessment as a novel prostate cancer biomarker (Giovanni Ponti, Italy)

- Liquid biopsy: Circulating free (CfDNA) and circulating tumour (ctDNA) quantification and their

sequencing, allow the collection of important data regarding cancer diagnosis and

characterization, patient prognosis and management in order to determine therapeutic

strategies and the subsequent follow-up

- Higher blood plasma cell free DNA levels in PCa patiemts with respect to healthy subjects have

been reported as well as significant differences in cfDNA levels and integrity between PCa

patients with BPH patients.

- Liquid biopsy:

• Circulating Nucleic Acids: mainly ctDNA (circulating tumour DNA), miRNA, mRNA, long-non

coding RNA

• Circulating Tumour Cells (CTCs): cancer cells released from primary tumour mass into the

blood stream

• Exosomes: Small membrane-derived vesicles, contain various molecules such as signal

protein,microRNA, mRNA

- The relationship among cfDNA and ctDNA, a percentage of cfDNA in PCa patients was derived

from non cancerous cells because of the induction of apoptosis by pro-apoptotic cytokines

released from prostate cancer cells.

- Circulating tumour DNA can in principle provide the same genetic information as a tissue biopsy

Cell-free circulating DNA may serve as a biomarker for tumour detection and follow up

- Can cfDNA levels can constitute a PCa biomarker for differential diagnosis between PCa and BPH?

• Several non-blood biologic fluids are potential sources for quatification and characterization

of cfDNA in the clinical setting. Among these, seminal fluid is a precious source of nucleic

acids, characterized by higher values of cfDNA with respect to blood

• Seminal fluid, which also contains prostatic secretions, can be adopted as a useful biomarker

for differential diagnosis between PCa and BPH patients

• Seminal fluids of PCa patients were characterized by significantly higher values of cfDNA

whilst BPH patients had low seminal cfDNA concentration levels, similar to those observed

in healthy volunteers.

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