quantifying impact of confounding variables in...

Quantifying Impact of Confounding Variables in Blastocystis/IBS

Association Studies

Ken Boorom Blastocystis Research Foundation

ICOPA 2014 / August 11 1

About BRF / Your Presenter

• Blastocystis Research Foundation est. 2006 in Oregon, USA

• Contact: [email protected] / www.bhomcenter.org

• Supported by researcher volunteer time + public donations

• How do we get highest value from research?

• What research topics are the most relevant?

• Which ideas are most likely to be successful?

• How do we teach researchers to get IRB approval?

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mailto:[email protected]

http://www.bhomcenter.org/

IBS Parameters

• High morbidity, low morality (although associated with 3x suicide rate increase)

• Physicians, researchers, and research groups themselves are often patients (41% of medical professionals in Pakistan)

• QOL scores similar to severe cardiovascular disease

Blastocystis Parameters

• All major subtypes (ST1-4) in symptomatic + asymptomatic patients • ~50% asymptomatic in one study, but depends on ST and patient group • Spontaneous clearance rate 22% in symptomatic (Vanhellemond, 2012) • Metronidazole raises this to 36%, paromycin to 77% • In chronic cases, infection length is indefinite. MZ efficacy=0% • (Probably most important) Only highly prevalent GI protozoal infection

with no reliable eradicative therapy

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Blastocystis and IBS – Brief History • 1986 – Markell et al - Blastocystis patients actually have IBS

• 1997– Hussain, et al - Blastocystis as the cause of IBS (IgG resp)

• 1991 – 1/3 US deployed military return from Gulf War with IBS

• 1999 – Giacommetti, et al - Blastocystis as cause of IBS

• 2005 – Multiple types of Blastocystis – are some non-pathogenic?

• 2007 – Stark, et al– Don’t diagnose IBS in Blastocystis patients

• 2008 – Jones, et al - first US study on Blastocystis

• 2008 – Boorom, et al - First major review, Blastocystis and IBS

• 2010 – IBS/Blastocystis link not significant in Thailand (10% vs 16.7%)

• 2010++ - Yakoob, et al – Blastocystis, IBS, medical students + workers

• 2012 – Jimenez-Gonzalez, et al – Link is significant in Mexico

• 2012 – Olivio-Diaz – First host genetic susceptibility polymorphisms

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2008 – BMC Parasites and Vectors. Most frequently accessed paper (~2008) “IBS is the only functional GI disorder where a protozoal infection has been found In almost half of the diagnosed cases …”

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US CDC

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Patient Statements

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Patient Statements

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Cytokine changes in colonic mucosa associated with Blastocystis spp. subtypes 1 and 3 in diarrhoea-predominant irritable bowel syndrome. Yakoob J, Abbas Z, Usman MW, Sultana A, Islam M, Awan S, Ahmad Z, Hamid S, Jafri W. Parasitology. 2014 Jun;141(7)

In vitro sensitivity of Blastocystis hominis to garlic, ginger, white cumin, and black pepper used in diet. Yakoob J, Abbas Z, Beg MA, Naz S, Awan S, Hamid S, Jafri W. Parasitol Res. 2011 Aug;109(2):379-85.

Blastocystis hominis and Dientamoeba fragilis in patients fulfilling irritable bowel syndrome criteria. Yakoob J, Jafri W, Beg MA, Abbas Z, Naz S, Islam M, Khan R. Parasitol Res. 2010 Aug;

Irritable bowel syndrome: is it associated with genotypes of Blastocystis hominis. Yakoob J, Jafri W, Beg MA, Abbas Z, Naz S, Islam M, Khan R. Parasitol Res. 2010 Apr;106(5):1033-8. doi: 10.1007/s00436-010-1761-x. Epub 2010 Feb 23. Erratum in: Parasitol Res. 2011 Dec;

In vitro susceptibility of Blastocystis hominis isolated from patients with irritable bowel syndrome.

Yakoob J, Jafri W, Jafri N, Islam M, Asim Beg M. Br J Biomed Sci. 2004;61(2):75-7.

Irritable bowel syndrome: in search of an etiology: role of Blastocystis hominis. Yakoob J, Jafri W, Jafri N, Khan R, Islam M, Beg MA, Zaman V. Am J Trop Med Hyg. 2004 Apr;

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http://www.ncbi.nlm.nih.gov/pubmed/24598032















































“Fingerprinting” Infections Mono-infections with Blastocystis spp, Cryptosporidium spp., and Entamoeba histolytica all present this pattern. Giardia intestinalis presents a similar pattern without constipation. Viral/bacterial infections present a different pattern Blastocystis spp., Cryptosporidium spp., and Entamoeba histolytica exhibit similar symptomatic and epidemiological patterns in healthcare-seeking patients in Karachi.

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“Fingerprinting” Infections

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Geographic Patterns Infections (only in Western nations so far)

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Patterns Seen at Clinical Labs (US)

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Goals in IBS-Blastocystis Study

• Identify eradication therapy for long-term cases (Australia, Europe). LTC are highest cost.

• In absence of eradicative therapy: – Avoid expensive testing

– Reduce stress level in patients

– Avoid expensive medical investigations ($1B on GWS)

• Disease Mechanisms: – Reduce symptoms

– Identify factor that produces spontaneous clearance

– IL-8, Nitric Oxide, IgA

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Confounding Variables in Association Studies

• Many diagnostics tests have low sensitivity (~30% for common clinical diagnostics)

• PCR is improvement, but has limited availability. DNA extraction methodologies and laboratory experience are problematic

• About 50% show symptoms. No single symptom (i.e. diarrhea) associated with infection.

• Multiple other factors can produce identical symptoms.

• What does the “p” value mean at the end?

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21 Janoff, 1990

22 Massimo, 2006

What You Can Do With This Model

1. Construct a study to show Blastocystis is strongly associated with IBS using patient selection and appropriate diagnostics.

2. Construct a study to show Blastocystis is not associated with IBS.

3. Make decisions about whether treatment is appropriate in a group, even when a study has found no association

4. Make informed decisions in reviewing papers 5. Apply the methodology to other case control studies 6. Slides focus on Type 1 errors (incorrect rejection of

hypothesis)

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Examples of Acceptable Levels of CV

IBS+ IBS-

BL+ 15 15

BL- 0 70

IBS+ IBS-

BL+ 15 9

BL- 21 55

n=100, 30% exposure, 100% susceptibility, p=4.5E-13

IBS+ IBS-

BL+ 30 0

BL- 0 70


n=100, 30% exposure, 50% susceptibility, 75% sensitivity, 25% other causes, p=0.002404

IBS+ IBS-

BL+ 12 12

BL- 3 73

n=100, 30% exposure, 50% susceptibility, 75% sensitivity p=7.79E-07

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Example of Unacceptable Level of CV

IBS+ IBS-

BL+ 3 7

BL- 0 90

IBS+ IBS-

BL+ 3 4

BL- 24 69


IBS+ IBS-

BL+ 10 0

BL- 0 90

n=100, 10% exposure, 30% susceptibility, p=0.00742

n=100, 10% exposure, 30% susceptibility, 75% sensitivity, 25% other causes, p=0.30. Still have 10% morbidity

IBS+ IBS-

BL+ 2 5

BL- 1 92

n=100, 10% exposure, 30% susceptibility, 75% sensitivity p=0.012294

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Does Increasing n Help?

IBS+ IBS-

BL+ 30 70

BL- 0 900

IBS+ IBS-

BL+ 32 37

BL- 240 691


IBS+ IBS-

BL+ 100 0

BL- 0 900


n=1000, 10% exposure, 30% susceptibility, 75% sensitivity, 25% other causes, p=0.000282

IBS+ IBS-

BL+ 20 49

BL- 10 921

n=1000, 10% exposure, 30% susceptibility, 75% sensitivity p=6.27E-18

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0.1

1

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100

1000

0 200 400 600 800 1000 1200 1400

Erro

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p v

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Study Size in # Partcipants

Predicted Error in p value vs n Susc=10%, Sens=75%, Ocauses=25%

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0

10

20

30

40

50

60

70

80

90

100

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

p v

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Diagnostic Sensitivity

Error in p value vs. Diagnostic Sensitivity

SUSC=50%, n=100, OC=0%

SUSC=10%, n=500, OTR=0%

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As Promised

• To avoid Type 1 errors: – Use larger sample size, higher sensitivity diagnostics, exclude

patients with other causes – Focus on high susceptibility patients (older age, high IL-8

producers, low IL-10 producers)

• To create Type 1 errors – Use lower sensitivity diagnostics – Perform testing on patients with other causes (i.e. unsorted

healthcare seeking patients, patients in endemic areas, patients during norovirus outbreak)

– Focus on younger populations – Prior examples for Giardia intestinalis / Cryptosporidium spp.:

Thai orphanages, US day care, US prisons

• In first example, despite p=0.30, we still had 15% morbidity

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Model Predictions

• How to construct a study to show association – Use high sensitivity diagnostics, exclude participants with other

causes, use susceptible population – Most high quality studies after 2006

• How to construct a study to show no association – Use low sensitivity diagnostics, include participants with other

causes, study children – Thai orphanages – G. intestinalis, Cryptosporidium spp. – US day care – G. intestinalis – US Prisons – G. intestinalis

• Model suggests that severe cases can exist in low susceptibility populations (i.e. p=0.20 does not mean mild pathogen or treatment is inappropriate)

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Backup Slides

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Blastocystis Research Environment

• General agreement at clinical level for treating patients (<3 cases where physician refused treatment 2006-2014)

• Blastocystis as a non-pathogen is a minority opinion in literature (16/102 vs. 86/102 as of 2008, most <1994)

• Over 1000 studies to date

• No organizational body with mission to review literature (i.e. no parallel to the IARC 3 degrees). No generally agreed upon mechanism for defining a pathogen (i.e. LD50). So diversity of opinion will remain.

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Blastocystis surface antigen is stable in chemically preserved stool samples for at least 1 year. Gould R, Boorom K. Parasitol Res. 2013 Jul;112(7):2469-71.

Blastocystis spp., Cryptosporidium spp., and Entamoeba histolytica exhibit similar symptomatic and epidemiological patterns in healthcare-seeking patients in Karachi. Haider SS, Baqai R, Qureshi FM, Boorom K. Parasitol Res. 2012 Sep;111(3):1357-68.

Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD Patients in Ankara, Turkey. Dogruman-Al F, Simsek Z, Boorom K, Ekici E, Sahin M, Tuncer C, Kustimur S, Altinbas A. PLoS One. 2010 Nov 18;5(11):

Molecular characterization of Blastocystis species in Oregon identifies multiple subtypes. Whipps CM, Boorom K, Bermudez LE, Kent ML. Parasitol Res. 2010 Mar;106(4):827-32.

Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt. Souppart L, Moussa H, Cian A, Sanciu G, Poirier P, El Alaoui H, Delbac F, Boorom K, Delhaes L, Dei-Cas E, Viscogliosi E. Parasitol Res. 2010 Jan;106(2):505-11.

Blastocystis subtypes in irritable bowel syndrome and inflammatory bowel disease in Ankara, Turkey. Dogruman-Al F, Kustimur S, Yoshikawa H, Tuncer C, Simsek Z, Tanyuksel M, Araz E,Boorom K. Mem Inst Oswaldo Cruz. 2009 Aug;104(5)

Molecular epidemiology of human Blastocystis isolates in France. Souppart L, Sanciu G, Cian A, Wawrzyniak I, Delbac F, Capron M, Dei-Cas E, Boorom K, Delhaes L, Viscogliosi E. Parasitol Res. 2009 Aug;105(2):413-21.

Emerging infectious diseases are not always obvious. Boorom K. Lancet Infect Dis. 2009 Mar;9(3)

Oh my aching gut: irritable bowel syndrome, Blastocystis, and asymptomatic infection. Boorom KF, Smith H, Nimri L, Viscogliosi E, Spanakos G, Parkar U, Li LH, Zhou XN, Ok UZ, Leelayoova S, Jones MS. Parasit Vectors. 2008 Oct 21;

Association of Blastocystis subtype 3 and 1 with patients from an Oregon community presenting with chronic gastrointestinal illness. Jones MS, Whipps CM, Ganac RD, Hudson NR, Boorom K. Parasitol Res. 2009 Jan;104(2):341-5.

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