Research TeamHutagalung J1, Kusnanto. H 1, Supargiyono2, Hamim S. A3, Novijanti. R4, Triwibowo AG5,

Prihatin MT 5, Bai A6, Bansai I6, Kik Hao. S6

Detection of Asymptomatic, Sub-Microscopic and Spatial Analysis of Malaria Pre-elimination

in Eastern Indonesia.

Center for Tropical Medicine-UGM

Prof. Hari Kusnanto, Dr.PH (Epidemiologist)

Prof. Supargiyono, Ph.D(Parasitologist)

Dr. Hamim Sadewa, Ph.D(Biochemistry)

Dr. Rintis Novianti, Ph.D(Eijkman Institute)

All study respondent

Dr. Ari Winasty, Ph.D(Eijkman Institute)

Brief Outline

1. Introduction

2. Research Purpose

3. Research Methodologies

4. Study Results

5. Conclusions and Recommendations

Introduction: The Prevalence Malaria Diseases In Indonesia

Source: MoH, Republic Indonesia & Eijkman Institute for Molecular, 2014

50 cases

250 cases

Pre-elimination Malaria Criteria (free malaria transmitted by 2025)

WHO CriteriaCurrent Situation Eastern Indonesia

2014-2016

1 case/1.000 Population 15-18 cases/1.000 Population

Strong Surveillance system ACD (active cases detection) & Mass Blood Survey (MBS)

Surveillance ?????Depend funding and political concern

Gold standard: Microscopic & Molecular

Gold standard: Microscopic Only

No local transmission Limited available data

Source: WHO, 2013, Manual for Pre-elimination Malaria. P. 34-54

Research Purpose

To answer, whether Eastern Indonesia should proceed malaria pre-elimination?

Research Methodology

1. Ethic Approval from Fac. Of Medicine UGM No. Ref: KE/FK/85/EC.

2. A survey (systematic random sampling), total 555 healthy people.

3. At 5 districts, Eastern Indonesia by three different Annual Parasite Incidence (API).

4. Data collected and analysis:

– Thick and thin blood smears (Giemsa 3-5%, protocol by MoH and WHO, 2015)

– Molecular analysis

– Multivariate (α= 0,05 & 95% CI)

Molecular Analysis

Assessment Protocol Laboratories

DNA Isolation Promega, Madison, USA. Cat. No. A-1123

Biochemistry Fac. of Medicine-UGM

PCR G6PD gene (Exon 5, 6, 9, 11 and 12)

• Promega, Madison, USA. Cat.No. M-7122.

• Primers: Nguyen et al., 2009

IVRCR&D, Ministry of Health, Salatiga, Central Java

Nested PCR Plasmodium • Promega, Madison, USA. Cat.No. M-7122.

• Primers: Snounou, 1993

Parasitology Fac. Of Medicine-UGM

Sequencing product PCR G6PD

• ABI Prism . 310 Genetic Sequencer.

• MEGA-5, Bio-Edit and Finch TVMacrogen, Korea

PCR-RFLP KAPA HiFi, Boston, USACat. No.

Eijkman Institute for Molecular, Jakarta

STUDY RESULTS

The result Mikroskopis vs Nested PCR (n=555)

0 100 200 300 400 500 600

Mikroskopis, 9 (1.6%)

P. Mal, 181 (32.6%)

P.vivax, 95 (52.5%)

P.Falc, 57 (31.5%)

Mix Infec, 29 (16%)

P. Oval+P. Mal, 0

Total , 555

Profile Schematic Work Flow

Total samples 555

555 Microscopic:a. Positive 9 (1,6%)b. Negative 545 (98,4%)

Exon 56.5% (6/92)Vanua lava 10.883 T>C

555 nPCR:a. Positive 181 (32,6%)b. Mix infection 29 (5,2%) c. Negative 374 (67,4%)

555 G6PD enzyme exam quantitative (<6,97 U/gHb):a. G6PDd 92 (16,6%)b. Non-G6PDd 463 (83,4%)

Exon 6No mutation

Exon 9No mutation

Exon 11No mutation

Exon 12No mutation

a. P. vivax 95 (17,1%)b. P. falciparum 57 (10,3%)c. Mix infection 29 (5,2%)

Ref. Seq= Accession Ref no. X-554481 (Minucci et al., 2012. Elsevier; Blood Cells, Molecules and Diseases)

Five Study Sites (Nusa Tenggara Timur Province)

District Batu Putih (n= 120):

P. vivax= 29 (5,2%)P. falc= 7 (1,3%)G6PDd= 8 (1,4%)

District Oenino (n= 135):

P. vivax= 17 (3,1%)P. falc= 20 (3,6%)G6PDd= 14 (2,5%)

District Oe’ekam (n= 100):

P. vivax= 18 (3,2%)P. falc= 17 (3,1%)G6PDd= 14 (2,5%)

District Panite (n= 100):

P. vivax= 10 (1,8%)P. falc= 11 (2%)G6PDd= 19 (3,4%)

District Oinlasi (n= 100):

P. vivax= 20 (3,6%)P. falc= 2 (0,4%)G6PDd= 37 (6,7%)

Statistical Result Malaria Risk Factors (α=0.05 and 95% CI)

Three Clustering Malaria Cases (Sat-Scan V.9.1.1, Scale 1: 25.000 with 10 km radius)

District Batu Putih

P. vivax= 29 (5,2%)P. falc= 7 (1,3%)G6PDd= 8 (1,4%)

District Oe’ekam:

P. vivax= 18 (3,2%)P. falc= 17 (3,1%)G6PDd= 14 (2,5%)

District Oinlasi:

P. vivax= 20 (3,6%)P. falc= 2 (0,4%)G6PDd= 37 (6,7%)

Figure 1. PCR-RFLP G6PD gene (exon-5) variant Vanua Lava 366 bp, protocol by KAPA HiFi, Cat. No. KK-2101, USA

Figure 2. n-PCR P. Vivax (120 bp), protocol by Promega, Madison, USA.Cat. No. M-7122, USA

Figure 3. n-PCR P. Falciparum (205 bp), protocol by Promega, Madison, USA. Cat. No. M-7122, USA

Result n-PCR and PCR-RFLP Analysis

Sequence Genetic Result G6PD Mutation Variant Analysis (MEGA-6, Bio-Edit and Finch TV)

Ref. Seq= Accession Ref. No X-554481. (Minucci et al., 2012. Elsevier; Blood Cells, Molecules and Diseases )

Sequence G6PD Variant Vanua Lava Heterozygous

a) Age: 48 years/Female heterozygous.

b) nPCR result: Negative malaria.

c) Nucleotide subtitution10.884 C>T ( Vanua Lava) at codon 128 exon-5.

d) Amino Acid substitution Leu>Pro.

e) 1st abortion history

Figure. (A) forwards primer, and (B) reverse primer.

Conclusions and Recommendations

1. Malaria Pre-elimination in eastern Indonesia should be delay (WHO Criteria) Other wise:

▪ Routine treatment (silent transmission)

▪ Outbreak control

▪ Sensitive laboratories support

▪ Surveillance (ACD and MBS)

2. P. Vivax dominant (Relapsing and New Transmitted Concern)

3. G6PDd mutation 16.4% → Be Aware PRIMAQUINE (PQ) Therapy

Acknowledgment goes to;

1. All the Study respondents.

2. Tropical of Medicine Faculty of Medicine (Research Unit), Univ. Gadjah Mada, All mentors & All research team APMEN.

3. APMEN (Asia Pacific Malaria Elimination Network), Menzies, Australia (Full funding).

4. Government and Health Office of District and Health Centre, TTS District and NTT Province.

5. Biochemistry Laboratories Faculty of Medicine, UGM.

6. Parasitology Laboratories, Faculty of Medicine, UGM.

7. Biology Molecular, Faculty of Medicine, UGM.

8. Ministry of Health, IVRCR&D (Biology Molecular Laboratories), Salatiga, Central Java.

9. Eijkman Institute for Molecular, Laboratory RBC & Enzymes Disorder, Jakarta

10. GIS Laboratories, Univ. Gadjah Mada

THANK YOU