Microsporidiosis in South Africa: PCR detection in stool samples of HIV-positive and HIV-negative individuals and school children in Vhembe district, Limpopo Province

Abstract

SummaryMicrosporidia were initially recognized as pathogens of insects and fish but have recently emerged as an important group of human pathogens, especially in immune-compromised individuals, such as those with HIV infection. In this study, we used a PCR-RFLP assay confirmed by quantitative real-time PCR and trichrome staining to determine the prevalence of microsporidian infections among hospital patients and school children in Vhembe region. Enterocytozoon bieneusi was the only microsporidian species detected in these stool samples. It was found in 33 (12.9%) of 255 samples from the hospitals and in 3 (4.5%) of 67 samples from primary school children and was significantly associated (P = 0.039) with diarrhea in HIV-positive patients (21.6%) compared to HIV-negative individuals (9%). However, microsporidian infections were not associated with intestinal inflammation as indicated by the lactoferrin test. These results suggest that microsporidia might be a cause of secretory diarrhea in HIV-positive patients. To our knowledge, this is the first report of E. bieneusi in the Vhembe region of South Africa. Further investigations are needed in order to clarify the pathogenesis of E. bieneusi in HIV-positive patients. Authors' contributions: AS, CLO, ST and RLG conceived the study; AS, ST, LMW and RLG designed the study; RLG provided supplies for the laboratory studies that were conducted in his laboratory; AS ran all the laboratory analysis; AS, CLO, LMW and RLG participated in the data analysis; AS and RLG drafted the paper; CLO, ST, LMW and RLG critically revised the manuscript for important intellectual content. All authors revised and approved the final version of the manuscript. AS and RLG are guarantors of the paper. Conflict of interest: None declared.Ethical approval: The study was approved by the research and ethical committee of the University of Venda and the Department of Health and Welfare and the Department of Education in Polokwane, Limpopo Province, South Africa, before the initiation of the study. The molecular analysis was conducted according to the University of Virginia ethics guidelines on non-identified samples.

Microsporidiosis and Malnutrition in Children with Persistent Diarrhea, Uganda

“…However, E. bieneusi is the more common of 2 species known to cause intestinal microsporidiosis (13,14). In previous studies at Mulago Hospital, 16.8% of children with acute diarrhea and 16.8% of children without diarrhea had microsporidiosis (7).…”

Evaluation of pulmonary microsporidiosis in iatrogenically immunosuppressed patients

“…Small-subunit ribosomal DNA (SSU-rDNA) regions at 250-279 bp were amplified with the primers PMP1 (5'-CACCAGGTTGATTCTGCCTGAC-3') and PMP2 (5'-CCT CTCCGGAACCAAACCCTG-3') specifically designed for Enterocytozoon bieneusi, Encephalitozoon intestinalis, E. cuniculi and Encephalitozoon hellem (14,18,(20)(21). For each reaction, 2.5 μL 10x reaction buffer, 2.5 μL MgCl 2 (25 mM stock solution), 2.5 μL dNTP (2 mM stock solution), 1 μL Hot start Taq DNA polymerase (1 U/μL stock solution), 0.75 μL primer (10 μM stock solution) and 5 μL DNA sample were added and the final volume was completed to 25 μL with distilled water.…”

“…For each reaction, 2.5 μL 10x reaction buffer, 2.5 μL MgCl 2 (25 mM stock solution), 2.5 μL dNTP (2 mM stock solution), 1 μL Hot start Taq DNA polymerase (1 U/μL stock solution), 0.75 μL primer (10 μM stock solution) and 5 μL DNA sample were added and the final volume was completed to 25 μL with distilled water. Amplification was performed under the following conditions: after an initial denaturation at 94°C for 10 minutes, 35 cycles at 94°C for 30 seconds, at 60°C for 30 seconds, at 72°C for 30 seconds and final extension at 72°C for 10 minutes (20,21). In order to enhance sensitivity, a 5-μL specimen taken from the first amplification product was amplified again with the same primers under the same conditions as in the first reaction (20,21).…”

“…Amplification was performed under the following conditions: after an initial denaturation at 94°C for 10 minutes, 35 cycles at 94°C for 30 seconds, at 60°C for 30 seconds, at 72°C for 30 seconds and final extension at 72°C for 10 minutes (20,21). In order to enhance sensitivity, a 5-μL specimen taken from the first amplification product was amplified again with the same primers under the same conditions as in the first reaction (20,21). To evaluate amplification of target DNA, 10 μL-amplification products were analysed by electrophoresis on 1.5% agarose gel under 100 volt for one hour.…”

AIDS Diarrhea and Antiretroviral Drug Concentrations: A Matched-Pair Cohort Study in Port au Prince, Haiti

“…Aliquots of unpreserved stool were frozen for batch testing by polymerase chain reaction for Cryptosporidium sp., enteroaggregative Escherichia coli , Campylobacter jejeuni , Clostridium difficile , and Entero cytozoon bineusi . [21][22][23][24][25][26] Quantitative lactoferrin enzyme-linked immunosorbent assay (IBD-Scan; Techlabs, Blacksburg, VA) was also performed on frozen stool samples. Antimicrobial susceptibility testing was performed by disk diffusion.…”