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INTESTINAL MICROSPORIDIOSIS IN TUNISIA : G ROUPS AT RISK , P REVALENCE S & S PECIES IDENTIFICATION
CHABCHOUB N, ABDELMALEK R, A ISSA S, K ANOUN F, T IOUIRI H,BOURATBINE A & AOUN K
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Institut Pasteur de Tunis
Intestinal Microsporidiosis (Desportes, 1985)Emerging opportunistic infection (HIV infected +++)
Microsporidia pathogens: Protozoa / Fungi
1st descriptions in Tunisia in 1997: HIV patients, Congenitalimmunodefisciency & Immunocompetent child
Objective of the study:
Estimation of the prevalence of intestinal microsporidiaand identification of the species involved in Tunisia
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177 Immunocompromised patients:123 VIH infected & 54 Haematological malignancies (HIV -)
142 Healthy controls (children)
One Stool Sample
Studied Subjects
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279 pb
250 pb
250 pb E. bieneusi , 270 pb Enc. intestinalis , 268 pb Enc. cuniculi , 279 pb Enc. hellem
Universal PCR V1/PMP2 (Fedroko et al , 1995)Gene of the small sub unit rRNA
Sequencing
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E. bieneusi : V1/EB450(Zhu et al, 1993)
Enc. intestinalis : V1/IS500(Coyle et al, 1993)
353 pb375 pb
Species Specific PCRs
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Results
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Trichrome PCR pVIH (+)
(123)8 (6.5%) 18 (14.6%)
Prevalence in HIV patients
0,02
Prvalence in patients with Haematologic malignancies
Trichrome PCR p
VIH (-)(54)
0 5 (9.3%) 0,028
All Healthy Controls revealed negative
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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 identification: 23 positive samples
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Species Proportion
E. bieneusi (n= 8) 42.1%
Enc. intestinalis (n = 10) 52.6%
Enc. hellem (n = 1) 5.3%
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PatientPCR (V1/PMP2) Sequencing
PCRsV1/EB450 V1/IS500
E. bieneusi Enc. intestinalis
R42 250 pb E. bieneusi -
R44 250 pb E. bieneusi -
R106 250 pb E. bieneusi -
R56 250 pb E. bieneusi -
R14 250 pb E. bieneusi -
R110 250 pb E. bieneusi -
R119 250 pb E. bieneusi - -
Sce1 250 pb E. bieneusi -
I14 270 pb Enc. intestinalis -
R60 270 pb Enc. intestinalis -
R92 270 pb Enc. intestinalis -
I44 270 pb Enc. intestinalis -
R68 270 pb Enc. intestinalis - -
R82 270 pb Enc. intestinalis - -
R86 270 pb Enc. intestinalis - -
R104 270 pb Enc. intestinalis - -
R117 270 pb Enc. intestinalis - -
I11 279 pb Enc. hellem - -
R15 270 pb 250 pb
Enc. intestinalis ND
--
+
-
R29 270 pb ND - -
R30 270 pb ND - -
I22 270 pb ND - -
A8 240 pb ND ND - -
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Stool aspect of HIV infected
Stool Patients Total
Diarrheal 7 (18.4%) 31 38
Molded 11 (12.9%) 74 85
Total 18 105 123
No statistical association
The infection was significantly associated with low T Lymphocyte CD4cell counts ( p=0,008 )
Clinical & Biological parameters
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Patient Age Stools Disease
I11 51 Diarrheal Myeloma
I14 55 Diarrheal Myeloma
I22 ND Diarrheal Myeloma
I44 35 Diarrheal Myeloma
A8 ND Molded BM aplasia
Haematological malignancies cases
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Genotyping of E. bieneusi ( 6 isolates): PCR-RFLP & Sequencing
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C ONCLUSION
High prevalence of Microsporidia infection inimmunocompromised patients in Tunisia
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
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16/16Institut Pasteur de Tunis
JFMPMRabat 23-26 Oct 2013