Microbiology and Infection Control

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Cryptosporidium and cryptosporidiosis.
(1990) Moodley, Dhayendre.; Jackson, Terry F. H. G.
Cryptosporidium parvum can cause debilitating disease in immunocompetent persons with cholera-like symptoms characterised by self-limiting, profuse diarrhoea; on the other hand asymptomatic infection with this organism frequently occurs. However, in immunocompromised patients, the disease is more severe and is lifethreatening. A pivotal aspect of the present survey was a comparative assessment of four commonly used staining techniques (viz. modified Ziehl-Neelsen, safranin-methylene blue, auramine phenol fluorescence and Sheather's sucrose flotation) for the detection and identification of Cryptosporidium oocysts. The Sheather's flotation method proved to be superior to the other three procedures which were not only less sensitive but also less specific. A modification of the Sheather's flotation technique was developed for use with diarrhoeal stools; this was found to be simple, reliable, costeffective and the least time consuming of the above methods; this was used exclusively in a subsequent survey of the association of Cryptosporidium infection with diarrhoea in hospitalised children. Although previous epidemiological surveys of cryptosporidiosis have been conducted in South Africa standardised methods have not been employed. This initial assessment of diagnostic techniques therefore provided a tool for accurately assessing the importance of Cryptosporidium as a causative organism of diarrhoea. In an extensive study performed on children younger than 10 years old, who were hospitalised with a primary diagnosis of diarrhoea at King Edward VIII Hospital, it was found that 9,0% (111/1229) were passing Cryptosporidium oocysts; this was the second most common enteric pathogen. In 72% (80/111) of patients with Cryptosporidium infections it was the only pathogen. The prevalence of cryptosporidiosis was highest during the months of February, March, April and May; direct correlation between the rainfall in the Durban area and the prevalence of cryptosporidiosis was demonstrated (r = 0,6125). Cryptosporidium infection was more prevalent in the 4-6 month age group (p = 0,001). The fact that Cryptosporidium infections may be symptomatic in some individuals and asymptomatic in others, suggests that strain differences in respect of pathogenic potential may occur. A prerequisite to the investigation of strain differences was to increase parasite numbers; both in vivo and in vitro culture techniques were employed. Culture in chicken embryos failed to increase the parasite population and only limited areas of the chorio-allantoic membranes showed a few developmental stages. Cell cultures proved to be more suitable for Cryptosporidium growth and parasite numbers increased proportionally with duration in culture. Attempts at infecting suckling Balb/c mice were unsuccessful; however experimental infection of immunosuppressed adult rats facilitated the examination of various developmental stages of the parasite. Isoenzyme electrophoresis is an excellent method for demonstrating polymorphism in many species. Of the five enzyme systems that were tested, glucose phosphate isomerase, malic enzyme and phosphoglucose dehydrogenase proved to be the most promising. The electrophoresis of lysates, prepared from oocysts, in an agarose gel system was found to give adequate and reproducible resolution of isoenzyme patterns. Isoenzyme polymorphism could be demonstrated in oocysts harvested from the stools of four children. Such polymorphism has not been described previously and indicates a more extensive study to investigate strain differences, and to correlate these with the clinical histories of infected subjects. This approach may be invaluable in elucidating the pathogenesis of Cryptosporidium infections in man.
In vitro culture and isoenzyme analysis of giardia lamblia.
(1999) Kwitshana, Zilungile L.; Jackson, Terry F. H. G.; Sturm, Adriaan Willem.
Giardia lamblia, an enteric protozoan parasite, infects a large number of individuals worldwide. In South Africa prevalences ranging between 4 and 63% are documented, however, the impact of giardiasis is underreseached in this country. Giardia infections vary from asymptomatic carriage or a self-limiting acute symptomatic illness to chronic, debilitating malabsorption syndrome. The factors responsible for development of symptomatic versus asymptomatic infection are poorly understood. It is believed by some that host factors determine the clinical outcome of infection. On the other hand, the possibility of the existence of pathogenic and non-pathogenic strains (a situation akin to Entamoeba spp.) remains to be explored. One requirement for investigation of the potential contribution of strain differences to pathogenecity of infection is establishment of laboratory cultures of different strains isolated from symptomatic and asymptomatic patients. The present study was undertaken to develop and modify existing methods for: (i) establishment of laboratory cultures of Giardia trophozoites from excystation of faecal cysts, (ii) long-term maintenance and cryopreservation of the cultures and (iii) preliminary characterisation methodology. One thousand and twenty-three stool specimens were collected from day care centres, hospital wards and Hlabisa hospital laboratory. A further 6246 were retrieved from the Microbiology Laboratory at King Edward VIII Hospital and screened by direct wet preparation. Giardia was detected by light microscopy following formol-ether concentration (127 of 1023 samples) or direct examination of wet preparations (78 of 6246 samples). Cysts were purified from the positive specimens by sucrose gradient separation. Viability was assessed by a dye-exclusion method (eosin). Three in vitro excystation techniques were employed in an attempt to obtain trophozoites for initiation and establishment of viable cultures thereof. Culture conditions were optimised using two reference strains of Giardia, WB & H7 (obtained from the National Institutes of Health, USA). The percentage excystation ranged between 0-42% with all the in vitro methods of excystment. Excysted trophozoites remained viable in TYI-S-33 culture medium for periods ranging between 12-72 hours or up to 9 days, and gradually died, hence viable trophozoite cultures could not be established. Some culture initiates (overall 65%) were lost through overwhelming bacterial and!or fungal contaminants. An animal model was subsequently set up in which C57BL/6 and Praomys (Mastomys) coucha mice were used for in vivo excystation experiments. 1-3 day old suckling mice were intragastrically injected with 10,5 -cysts/ ml in 0,1 ml distilled water. Trophozoites were retrieved from the stomachs of infected mice 7-10 days after inoculation and cultivated in TYI-S-33 medium. Six local isolates were axenised using the in vivo excystation method. They have been maintained for more than 15 months in culture after stabilates and Iysates of confluent growths had been cryopreserved in Liquid Nitrogen. Successful (100%) retrieval of the cryopreserved cultures has been achieved. Seven isoenzyme electrophoresis systems have been set up and optimised. Reproducible results were obtained in six of the enzymes. Some differences in banding patterns of the enzymes were demonstrated.

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Browsing Microbiology and Infection Control by Author "Jackson, Terry F. H. G."