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Molecular diagnosis of intestinal helminths

Jaco J. Verweij

Laboratory for Medical Microbiology and Immunology

St. Elisabeth Hospital, Tilburg

j.verweij@elisabeth.nl

LUMC

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Microscopic diagnosis

1681 1910 2010

Mul+plex  real-­‐+me  PCR  

10-1 10-2 10-3 10-4 10-5 10-6

Cycle threshold-value (Ct)

•  Validation –  Specificity ~ 100%

–  Design of primers and probes –  Control samples (n=150)

–  Sensitivity PCR >>> Microscopy

DNA  polymerase  

•  Automation, high-throughput

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Examples of (multiplex) real-time PCRs

Protozoa Helminths

Entamoeba histolytica Ancylostoma duodenale

Entamoeba dispar Necator americanus

Giardia lamblia Oesophagostomum bifurcum

Cryptosporidium Strongyloides stercoralis

Cyclospora cayetanensis Ascaris lumbricoides

Cystoisospora belli Schistosoma mansoni

Dientamoeba fragilis Schistosoma haematobium

Blastocystis Schistosoma genus

Enterocytozoon bieneusi Opistochis felineus

Encephalitozoon Clonorchis sinensis

Taenia solium

Taenia saginata

Hymenolepis nana

Trichuris trichiura

Bacteria & viruses

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N. americanus PCR quantitative result vs egg count

10 100 1000 10000 1000000.1

1

10

100

1000

10000

100000

EPG

Arbi

trary

cop

y nu

mbe

rs

N  =  148  CC  =  -­‐0.76  p  <  0.001  

Verweij  et  al,  AJTMH,  2007  

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PLoS Negl Trop Dis. 2016 Jan 28;10(1):e0004380. Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial. Stacey Llewellyn, Tawin Inpankaew, Susana Vaz Nery, Darren J. Gray, Jaco J. Verweij, Archie C. A. Clements, Santina J. Gomes, Rebecca Traub, James S. McCarthy.

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0%

20%

40%

60%

80%

100%

baseline 1 year 2 year

Albendazole

0%

20%

40%

60%

80%

100%

baseline 1 year 2 year

Placebo

0

Effect  of  albendazole  interven+on  on  N.  americanus  

557   562   497   530   559   484  

Plos  One,  2013  

0%

20%

40%

60%

80%

100%

baseline 1 year 2 year

Placebo

Effect  of  albendazole  interven+on  on    A.  lumbricoides  

0%

20%

40%

60%

80%

100%

baseline 1 year 2 year

Albendazole

557   562   497   530   559   484  

Plos  One,  2013  

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DNA load after treatment

Mejia et al, AMJTMH 2013

Rapid clearance of Giardia lamblia DNA from the gut after successful treatment

0,020

0,025

0,030

0,035

0,040

0,045

0,050

0,055

0,060

pre post 0 post 7 neg

van den Bijllaardt et al, 2014, Clin. Microb. Infect.

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Th1 and Th2 cytokines to Ascaris antigen decrease with increasing N. americanus load

Pearson correlation IL-2, n=405; R2= 0.215 ; P<0.000 IFN-g, n=407; R2= 0.15 ; P=0.002 IL-5, n=406; R2= 0.168 ; P=0.001

correla+on  with  immunological  markers  

                       

 Microscopy  nega+ve  

 

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Hookworm  species-­‐specific  prevalence  in  Malawi  

0%

10%

20%

30%

40%

50%

A. duodenale N. americanus

Plos  NTD,  2012  

N=830  pre-­‐school  children  cases  and  controls  

Association between anemia and A. duodenale DNA load

A.  duodenale  

0%

10%

20%

30%

40%

50%

cases Hospital controls

Community controls

N.  americanus  

0%

10%

20%

30%

40%

50%

cases Hospital Controls

Community Controls

252   291   287   252   291   287  

Plos  NTD,  2012  

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Associa+on  between  anemia  and  A.  duodenale  DNA  load  

Plos  NTD,  2012  

In this rural Ghanaian population current N. americanus infections are not associated with altered immune function, while infection with P. falciparum is associated with pro-inflammatory innate immune responses.

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DNA isolation

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Algorithm parasitological diagnostics •  Standard (Gastroenteritis)

–  PCR protozoa: E. histolytica, Cryptosporidium, Giardia

•  Standard (Ova & Cysts) –  PCR protozoa: E. histolytica, Cryptosporidium, Giardia –  PCR helminths: Ancylostoma, Necator, Ascaris, Trichuris, Strongyloides, Schistosoma

•  Adoption, refugee, eosinophilia, hepatic impairment, (sub) tropic, immunosuppression, visual inspection

–  Additional microscopy

Helminth PCR Elisabeth ziekenhuis 2014-2016

Positive/Negative Ct Ancylostoma 0 Necator americanus 0 Ascaris lumbricoides 0 Trichuris trichiura 2 28.2; 36.3 Strongyloides stercoralis 2 24.7; 34.6 Schistosoma 2 19.7; 21.5 Negative 2031 Inhibition 3

Microscopy (N=248) Taenia 1 Enterobius 2

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Medical  Research  Unit  Albert  Schweitzer  Hospital  in  Lambaréné,  Gabon    

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Implemented multiplex PCRs on the Corbett Rotorgene real-time PCR

•  A. duodenale; N. americanus; A. lumbricoides with PhHV (internal control) PCR.

•  Schistosoma genus PCR with PhHV.

•  Malaria PCR –  P. falciparum; P. vivax with

PhHV PCR. –  P. malariae; P. ovale with

PhHV PCR.

Training  and  Technology  Transfer  

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Molecular diagnostics of intestinal helminths

•  One  method  fits  all!  Integrated  approach!  

•  Short  +me  in  the  field,  transport  to  central  laboratory  

•  Observer  independent,  highly  sensi+ve  and  specific  detec+on  

•  Quan+fica+on  correlates  with  intensity  of  infec+on  and  disease  

•  Monitoring  the  effect  of  interven+on  

•  Correla+on  with  immunological  markers  

•  EPG  standard  for  Mdx  of  intes+nal  helminths  (like  WHO  standard  Mdx  malaria).  

•  Pooling  samples?  

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Thanks for listening

Questions?