Prevalence of cryptosporidium parvum, giardia intestinalis and molecular characterization of rotavirus assoceated with diarrhea in children below five years old in Gaborone, Botswana

Abstract

Background: Diarrhea remains a major cause of mortality and morbidity in children under the age of five years across the globe. In Botswana, the control of diarrheal diseases aims at reducing the burden caused by diarrhea as well as improving the quality of lives of children. Despite all these efforts, gastroenteritis is still one of the major causes of death and illnesses in young children in Botswana. To ensure the acceleration towards the reduction of diarrhea in children, recent information on pathogens causing the disease should be documented. The aim of this study was to determine the occurrence of Cryptosporidium parvum, Giardia intestinalis and molecular characteristics of rotaviruses in Botswana. Methods: A case study was carried out on 200 stool specimens from symptomatic pediatric patients and 100 asymptomatic children under the age of five years from selected hospitals and clinics in Gaborone. The Ziehl Neelsen staining technique was used for detection of Cryptosporidium parvum and wet mount procedure for detection of Giardia intestinalis. Confirmation of samples that tested negative for the parasites was done using immunochromatographic assay. The enzyme-linkedimmunosorbent assay was used to screen for rotavirus. Rotavirus electrophoretypes from ELISA positive specimens were detected by polyacrylamide gel electrophoresis. Molecular characterization of rotavirus was conducted by reverse transcriptase-polymerase chain reaction using genotype specific primers that target VP4 and VP7. Selected PCR amplicons were sequenced and analyzed by Clustal W. Distance matrices were constructed by using the Kimura 2 parameter nucleotide substitution model in MEGA 6.06. Phylogenetic analysis was carried out by utilizing the neighbor joining model with 1000 bootstrap replicates and sequences were compared with reference strains from GenBank using the Basic Local Alignment Search Tool. Results: Prevalence rates of 20.5% (41/200), 16.5% (33/200) and 11.0% (22/200) in diarrhea cases were observed for Cryptosporidium parvum, Giardia intestinalis and rotavirus, respectively. Four percent (n=8) of diarrheic specimens had multiple infections. Peak infections of G. intestinalis and C. parvum were frequently observed during hot and rainy season, while peak prevalence for rotavirus occurred in April and July. For rotavirus, long electrophoretypes occurred more frequently (56.25%) than short electrophoretypes (25%). The most G/P combination observed was GIP[8] (7/15, 46.7%) followed by G2P[4] (2/15, 13%) and G3P[8] (1/15, 7%). One mixed strain, G1+G2P[4,8], was found in 13% (2/15) of case samples. Twenty percent of the specimens were non-typeable. Phylogenetic analysis of VP4 and VP7 sequences clustered rotavirus strains from Botswana within G1 lineages 1 and 2, G3-lineage 1, P[8] lineage 3 and P[4] lineage 5 together with Southern African strains, but distantly related to Rotarix. Conclusion: This study suggests that Cryptosporidium parvum, Giardia intestinalis and rotavirus are important pathogens in the burden of diarrhea in children below the age of five years in Botswana. Rotavirus strain diversity was similar to that previously observed worldwide. Findings in this study suggests that rotavirus surveillance is an important tool to assess the impact of the ongoing vaccination program. Monitoring of circulating rotavirus strains is essential for assessment of effectiveness of current vaccines in Botswana.