INTESTINAL MICROSPORIDIOSIS IN TUNISIA: GROUPS AT RISK, PREVALENCES & SPECIES IDENTIFICATION

CHABCHOUB N, ABDELMALEK R, AISSA S, KANOUN F, TIOUIRI H, BOURATBINE A & AOUN K

LR 11-IPT-06 Medical Parasitology, Biotechnologies & Biomolecules Institut Pasteur de Tunis-TUNISIA

Intestinal Microsporidiosis (Desportes, 1985) Emerging opportunistic infection (HIV infected +++) Microsporidia pathogens: Protozoa / Fungi

1st descriptions in Tunisia in 1997: HIV patients, Congenital immunodefisciency & Immunocompetent child

Objective of the study:

Estimation of the prevalence of intestinal microsporidia and identification of the species involved in Tunisia

Institut Pasteur de Tunis

Studied Subjects

177 Immunocompromised patients:

123 VIH infected & 54 Haematological malignancies (HIV -) 142 Healthy controls (children)

One Stool Sample

Techniques
Direct Exam
(Ritchie concentration)

Trichrome/Weber Staining

PCR V1/PMP2

Universal PCR V1/PMP2 (Fedroko et al, 1995)

Gene of the small sub unit rRNA

279 pb

250 pb

250 pb E. bieneusi, 270 pb Enc. intestinalis, 268 pb Enc. cuniculi, 279 pb Enc. hellem

Sequencing

Species Specific PCRs

E. bieneusi: V1/EB450 (Zhu et al, 1993)

Enc. intestinalis: V1/IS500 (Coyle et al, 1993)

375 pb 353 pb

Results

Institut Pasteur de Tunis

Prevalence in HIV patients Trichrome VIH (+) (123) 8 (6.5%) PCR 18 (14.6%) p 0,02

Prvalence in patients with Haematologic malignancies Trichrome PCR p 0,028

VIH (-) (54)

5 (9.3%)

All Healthy Controls revealed negative

Species identification: 23 positive samples

Suggestive Size of amplified fragment (V1/PMP2): 22/23 (95.7%)
Sequencing: E. bieneusi L16868, E. intestinalis SIU09929, E. hellem

L19070, E. cuniculi L17072 (BLAST http:www.ncbi.nlm.nih.gov) 19/23 (82.6%)

Specific PCR: E. bieneusi, Enc. Intestinalis 11/19 (57.9%)

Correlation in all cases

Species
E. bieneusi (n= 8) Enc. intestinalis (n = 10) Enc. hellem (n = 1)

Proportion
42.1% 52.6% 5.3%

Patient

PCRs

PCR (V1/PMP2)
R42 R44 R106 R56 R14 R110 R119 Sce1 I14 R60 R92 I44 R68 R82 R86 R104 R117 I11 R15 250 pb 250 pb 250 pb 250 pb 250 pb 250 pb 250 pb 250 pb 270 pb 270 pb 270 pb 270 pb 270 pb 270 pb 270 pb 270 pb 270 pb 279 pb 270 pb 250 pb 270 pb 270 pb 270 pb

Sequencing
E. bieneusi E. bieneusi E. bieneusi E. bieneusi E. bieneusi E. bieneusi E. bieneusi E. bieneusi Enc. intestinalis Enc. intestinalis Enc. intestinalis Enc. intestinalis Enc. intestinalis Enc. intestinalis Enc. intestinalis Enc. intestinalis Enc. intestinalis Enc. hellem Enc. intestinalis ND ND ND ND

V1/EB450 E. bieneusi

V1/IS500 Enc. intestinalis

-

+ + + + + +
-

+ + + +
-

+
-

R29 R30 I22

A8

240 pb ND

ND

Clinical & Biological parameters

Stool aspect of HIV infected Stool Patients Total

+ 7 (18.4%) 11 (12.9%) 18

31 74 105

Diarrheal Molded Total

38 85 123

=> No statistical association

The infection was significantly associated with low T Lymphocyte CD4 cell counts (p=0,008)

Haematological malignancies cases

Patient
I11 I14 I22

Age
51 55 ND

Stools
Diarrheal Diarrheal Diarrheal

Disease
Myeloma Myeloma Myeloma

I44
A8

35
ND

Diarrheal
Molded

Myeloma
BM aplasia

Genotyping of E. bieneusi (6 isolates): PCR-RFLP & Sequencing

CONCLUSION
High

prevalence of Microsporidia immunocompromised patients in Tunisia

infection

in

PCR much more sensitive than Microscopy First microsporidia species identification in Tunisia: E. bieneusi & Enc. intestinalis; but also Enc. hellem Co-transmission of Genotype B (Anthroponotic) et D (Zoonotic) of E. bieneusi

Institut Pasteur de Tunis

THANK YOU

JFMPM Rabat 23-26 Oct 2013

Institut Pasteur de Tunis