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Neglected Tropical Diseases (NTDs): Importance of Diagnosis

Nilanjan Lodh, Ph.D., M.Sc. Assistant Professor, Department of Clinical Laboratory Science,

Marquette University, Milwaukee, Wisconsin, USA

Thursday, August 2nd, 2018

Presentation outline Introduction to neglected tropical diseases (NTDs):

current situation.

Our research approach.

Perspective about three NTDs: diagnostic is the focus.

Future endeavor.

INTRODUCTION/BACKGROUND

Expected number of cases in 2010 as extrapolated from the Global Burden of Disease Study 2010.

Hotez et al., 2014, PLoS NTD

Neglected Tropical Diseases (NTDs)

Center for Disease Control and Prevention (CDC)

Global overlap of seven of the common NTDs Post Millennium Development Goals (MDGs)

2012 London Declaration on NTDs.

2013 World Health Assembly resolution on the

NTDs (WHA66.12).

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Hotez et al., 2016, PLoS NTD

Timeline for elimination (top) and eradication (bottom) of targeted NTDs by preventive chemotherapy.

Drugs

Diagnostics

Vaccines

Vector control agents

Inadequacies

RESEARCH APPROACH

Diseases under investigation

Schistosomiasis.

Strongyloidiasis.

Chagas disease.

Sample collection to infection detection

Workflow

PCR Gel Electrophoresis

LAMP Gel Electrophoresis

WORKFLOW1

WORKFLOW2

SCHISTOSOMIASIS (Bilharzia)

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Background Schistosomiasis has been called the deadliest of the Neglected Tropical Diseases

(NTDs) by the US Centers for Disease Control and Prevention (CDC).

It is a blood parasite disease that undergoes asexual reproduction in freshwater snails (intermediate host) and sexual reproduction in humans (primary host).

More than 200 million people are currently infected worldwide and at least 700 million people are at risk.

Higher burden of infection is carried by school aged children.

Two most prevalent species in sub-Saharan Africa:

Schistosoma mansoniS. haematobium.

Why is Schistosome diagnosis important? Kato-Katz (KK - Parasitological test): Fecal smear on slide to look for parasite egg.

Low sensitivity and High specificity

Circulating Cathodic Antigen test (CCA - Antigen capture test): CCA captures parasite antigen from urine on reagent strip.

Low sensitivity and Low specificity

Hematuria (Parasitological sign): Presence of blood in urine. Low sensitivity, Low specificity, possible False Positive

PCR test (Molecular test): Detects parasite DNA in urine or parasite egg DNA in fecal matter.

High sensitivity and High specificity

Schistosome species repeat fragments

S. haematobium repeat fragment

S. mansoni repeat fragment

Diagnosis of Schistosoma mansoni from filtered urine by PCR and comparison with other diagnostic methods. [Lodh et al. 2013, American Journal of Tropical medicine and Hygiene]

Diagnosis of multi schistosome parasite (S. mansoni and S. haematobium) infection from single urine specimen by PCR. [Lodh et al. 2014, PLoS ONE]

Diagnosis of multi schistosome parasite (S. mansoni and S. haematobium) infection from single urine specimen by LAMP. [Lodh et al. 2017, Acta Tropica]

Diagnosis of multi schistosome parasite (S. mansoni and S. haematobium) infection from single urine specimen after MDA. [Hessler et al. 2017, PLoSONE]

Focus on symptomatic and asymptomatic infection.

Research Overview

Identification of multi Schistosome parasites by PCR

Ghana

Lodh et al. 2014, PLoS ONE

PCR detected species-specific DNA for both Schistosome parasites from a single urine specimen.

Primers were specific for each parasite species and did not cross amplify confirmed by the result of sequencing.

Species-specific DNA detection by PCR was the strongest indicator of positive cases with sensitivity of ~100% in comparison to KK (76%) and haematuria (30%).

Study outcome 1

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PCR detected 11 such individuals infected by both parasites who showed false negative results by both KK and hematuria indicated presence of asymptomatic infection.

Study outcome 2

Schistosome identification by loop-mediated isothermal

amplification (LAMP) in the field

Lodh et al. 2017, Acta Tropica

Ghana

LAMP procedure

Eiken Chemical Co., Ltd.

Single temperature.

Uses Bst polymerase and four primers to amplify six distinct regions of target DNA.

Duration: 1.5 – 2 hours.

Visual confirmation by SYBR Green.

S. mansoni: LAMP performed better than PCR, whereas not LAMP_PURE.

S. haematobium: LAMP, PCR and LAMP_PURE all performed equally well.

Study outcome 1 S. mansoni: test positives by LAMP_QiaAmp were highly unlikely to be the same with PCR and

LAMP_PURE.

S. haematobium: test positives by LAMP_QiaAmp are highly likely to be same with PCR and LAMP_PURE.

Study outcome 2

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Schistosome identification by sensitive and specific molecular

approach after mass drug administration (MDA)

ZambiaHessler et al. 2017, PLoS ONE

PCR detected three times more positive S. mansoni infection compared to KK. Similar findings were also evident for S. haematobium by PCR compared to hematuria and

urine filtration.

Study outcome 1

Parasite DNA detection effectively identified positive infection irrespective of sex and age.

Study outcome 2

STRONGYLOIDIASIS (Threadworm)

Background Causative agent: Strongyloides stercoralis.

Worldwide distribution except for Antarctica.

30-100 million people are infected.

Diagnosis is difficult: Parasitological tests are insensitive and time consuming. Serological tests are difficult to standardize and replicate on a larger

scale. Real time PCR tests based on stool require future validation.

S. stercoralis specific repeat DNA detection from filtered urine samples.

Lodh et al., 2016, Acta Tropica, 163: 9 - 13

S. stercoralis diagnostic stage

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Study objectiveDemonstrate the feasibility of using a urine-based diagnostic test for S. stercoralis to detect species-specific repeat DNA

fragment from urine residues captured on filter paper

S. stercoralis repeat fragmentLodh et al., 2016, Acta Tropica

Study outcome 1

34 individuals were negative in both stool and urine based analyses. 8 individuals were positive for stool but were negative by urine based

PCR. 56 individuals were positive by PCR, whereas only 27 individuals were

positive by stool analysis. Overall increase of prevalence by 48.2% using PCR.

N: 125 samplesStool analysis

+ 35 (28%) - 90 (72%)

Urine PCR+ 83 (66.4%) 27 (21.6%) 56 (44.8%)

- 42 (27.1%) 8 (6.4%) 34 (27.2%)

Study outcome 2

CHAGAS DISEASE (American

trypanosomiasis)

Background Causative agent: Trypanosoma cruzi.

Distribution is in North, South, and Central America with the highest prevalence in Central America.

At least 8 million people in the Americas are infected.

Diagnosis is difficult: Blood smears: low sensitivity, high specificity Xenodiagnosis: low sensitivity, high specificity Haemoculture: low sensitivity, high specificity Serology: high sensitivity, low specificity

T. cruzi specific repeat DNA detection from filtered urine samples.

Price and Lodh, 2018, Parasite & Vectors, Under review

T. cruzi diagnostic stage

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Study objectiveWe intended to demonstrate the effectiveness of detecting species-specific repetitive satellite DNA fragment via PCR

from the urine sediment captured on filter paper after filtration to evaluate the possibility of detection of T. cruzi

over the classical methods of detection.

T. Cruzi repeat satellite fragment

Study outcome 1 Satellite DNA was detected from 70pg/ul concentration to 175fg/ul

concentration, which is less than one copy of the target DNA fragment. The TcB primer set consistently performed well.

Study outcome 2 One clinical sample came out as positive for T. cruzi by TcA-F/ TcA-R

and TcC-F/TcC-R sets of primers.

TAKE HOME MESSAGES

Overall summary Post nephritic species-specific repeat DNA detection by PCR was highly

successful for schistosomes, Strongyloides and Chagas from urine sediment collected on the filter paper after filtration.

Successful amplification of S. mansoni and S. haematobium repeat DNA by LAMP from single urine sample.

These were achieved for different parasites in different countries, such as Ghana, Zambia, Argentina.

Molecular xenomonitoring via isothermal assays, such as LAMP will help achieve the goals of the London Declaration of 2020.

As we change the way we live we also change the

ways diseases are spread.

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Acknowledgement Johns Hopkins University, Baltimore, MD, USA

Dr. Clive Shiff (Postdoctoral Advisor)

Dr. Jean M. Naples

Keio University, Tokyo, Japan

Dr. Kei Mikita

Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana

Dr. Kwabena M. Bosompem (Director), Dr. Nicaise Ndam

University of Zambia, Zambia

Dr. James Mwansa, Dr. Chummy Sikasunge

University of Salta, Argentina

Dr. Alejandro KrolewieckiFunding Sources:

Acknowledgement

Nilanjan (Nil) Lodh, Ph.D. M.Sc.Assistant Professor

Marquette UniversityCollege of Health Sciences

Department of Clinical Laboratory Science561 North 16th street, Schroeder Complex 274

Milwaukee, WI 53233Email: nilanjan.lodh@marquette.edu

Phone(Office): 414-288-3404