in vitro culture and isoenzyme analysis of giardia lamblia

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years. Figure 2.1 illustrates age distribution for these samples. The majority of the children (68%) were between the ages 2-5 years. Five of the 78 were obtained from adults while no ages were indicated in the hospital records in 9 samples. Age distribution for Giardia positive sam pies (from children) retrieved from the KEH laboratory (n=64) .. 8 0 7 0 60 ~ 5 0 .;;; 40 o ~ 30 2 0 1 0 o 0-2 3 m 2 - 5 Y 6 - 1 2 Y age 1_ % po s itiv e 1 Fig.2.1 Illustrates the percentage age distribution for 64 of 78 patients whose faecal samples (submitted to KEH microbiology laboratory between January 1996 and December 1997) were harbouring Giardia. Approximately 69% of the patients were children aged between 2 and 5 years. The percentage of Giardia positive samples ranged between 0,36 (July 1996) and 4,11 (May 1997) as shown in Table 2. 4 and Giardia was not detected from samples submitted during the months of May and October 1996. It should be noted, that many confounding factors in this sample population do not allow for determination of real prevalence: (i) the submitted samples were obtained from patients who came from different geographical areas because KEH is a referral hospital for many faroutlying areas (ii) the sample population comprised of a mixture of adults and children (iii) direct microscopic screening is known to be less sensitive than the concentration methods, therefore real prevalence would be underestimated. 51
Table 2. 4. Monthly summary of stools screened (from the KEH Micr?biology lab) and the percentages of Giardia positive samples over a two-year penod. January 2,83 460 0,82 363 February 0,62 325 0,99 302 March 1,08 278 0,83 240 April 0,8 251 0,91 222 May ° 291 4,11 219 June 0,37 273 2,21 226 July 0,36 276 0,95 211 August 0,48 210 0,61 163 September 0,89 226 2,8 179 October ° 248 0,39 258 November 0,67 298 1,65 242 December 0,72 278 0,9 207 Figure 2.2 indicates the monthly pattern of Giardia positive samples from the KEH laboratory. There were more positive samples in 1997 than in 1996, however this was not statistically significant (p=O.67). This increase was inexplicable because all the technical parameters were constant during the 2-year collection period. Seasonal variation (in terms of rainfall patterns) would be difficult to measure because of the diversity of geographical location of the source patients. 52
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IN VITRO CULTURE AND ISOENZYME ANAL
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(j)edicated to my {ate parents :No
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the positive specimens by sucrose g
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Declaration This study represents o
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ACKNOWLEDGEMENTS The author wishes
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Table of Contents Page CHAPTER 1 -
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4.2.1. Animal Sourcing and Care ...
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APPENDIX 8 List of enzyme systems u
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List of Tables Page Table 1.1 Commo
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List of Plates Page Plate 2.1 An eo
Page 21 and 22: ABBREVIATIONS AND UNITS OF MEASUREM
Page 23 and 24: agi/is in 1882. In 1914, Alexeieff
Page 25 and 26: unique about Giardia in humans, whi
Page 27 and 28: To-date, use of the different speci
Page 29 and 30: Differentiation into the cyst stage
Page 31 and 32: 1.3 Transmission 1.3.1 Human hosts
Page 33 and 34: Owen, 1984) 1.3. 2 The Zoonosis of
Page 35 and 36: 1.4 Distribution The morbidity and
Page 37 and 38: children by Schutte et al. in 1981
Page 39 and 40: of (a) the enormous absorptive capa
Page 41 and 42: Ig M (100% of patients), IgG (70%),
Page 43 and 44: chymotrypsin) between different iso
Page 45 and 46: impact on the physical growth of 29
Page 47 and 48: Figure 1.3. Transmission Electron m
Page 49 and 50: among children between the ages of
Page 51 and 52: where immunocompromised mice have p
Page 53 and 54: up to 4 distinct genotypes within e
Page 55 and 56: in initiation of infection (excysta
Page 57 and 58: In order to address these and many
Page 59 and 60: multiple stool examinations in 4 of
Page 61 and 62: 2.2 MATERIALS AND METHODS 2.2.1 Spe
Page 63 and 64: Table 2.1. Semi-quantitative assess
Page 65 and 66: 2.2.3.2 The modified Zinc sulphate
Page 67 and 68: Preliminary work with five samples
Page 69 and 70: % Viability = unstained cysts/total
Page 71: Table 2.3. Prevalence of Giardia in
Page 75 and 76: spp (59%). Other parasites less fre
Page 77 and 78: Observations • A large proportion
Page 79 and 80: Plate 2.1. An eosin-stained prepara
Page 81 and 82: 2.4 DISCUSSION In terms of yield, i
Page 83 and 84: examination of wet preparations. Th
Page 85 and 86: ecognised (on unstained preparation
Page 87 and 88: cultivation in vitro (Kasprzak & Ma
Page 89 and 90: 3.2 MATERIALS AND METHODS Three exc
Page 91 and 92: 3.2.3 The modified Acid Pepsin meth
Page 93 and 94: • After 15 minutes incubation: Ma
Page 95 and 96: Plate 3.2 (b) A completely excysted
Page 97 and 98: Twenty-four batches of cysts (71 %
Page 99 and 100: 3.3.3 The modified Acid Pepsin excy
Page 101 and 102: 3.4 DISCUSSION Since our source of
Page 103 and 104: an unreliable indicator of viabilit
Page 105 and 106: propagating Giardia for subsequent
Page 107 and 108: 4.2 MATERIALS AND METHODS 4.2.1. An
Page 109 and 110: A litter comprising of 7 C57BU6 mic
Page 111 and 112: for attached trophozoites and maint
Page 113 and 114: days pi. However, accurate quantifi
Page 115 and 116: Table 4.4. Summary of the inoculati
Page 117 and 118: not be infected in comparison with
Page 119 and 120: A proportionately higher percentage
Page 121 and 122: These findings strongly suggest tha
Page 123 and 124:
CHAPTERS IN VITRO CULTURE OF GIARDI
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Diamond et al.'s (1978) medium. He
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Majewska, 1985) United States (Nash
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were evaluated. 5,2,1,2 Biosate Bio
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Table 5.1 Outlines a relative semi-
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dislodged by chilling the culture t
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Table 5.2. Sensitivity to Ciproflox
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Table 5.3. Summary results of in vi
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Once established in culture for a l
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Plates 5.3 and 5.4 Confluent growth
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difficult to produce viable culture
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dominance of certain genotypes unde
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Cryopreservation and successful ret
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adjusted to 5°C per minute from am
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6.3 RESULTS Trophozoites have been
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Table 6.1. A longevity record of sa
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Diamond (1995) stated that an impor
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into two categories. One group cont
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technique to the first local (South
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for Giardia Iysates. These included
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4. To exclude the presence of bacte
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Plate 7.1 Banding pattern of 8 seri
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PGM activity was displayed by Giard
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Plate 7.6. Hexokinase Lane 1. Unino
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Plate 7.8. Phosphogluconate dehydro
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stably expressed between different
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techniques, which would allow diffe
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Belosevic M, Faubert GM, MacLean JD
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Farthing MJG, Mata L, Urrutia JJ, K
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Heidelberg pp.397-398. Hiatt RA, Ma
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patients with X-linked agammaglobul
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Press, Calgary. pp 57-58. Nash TE,
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of Entamoeba histolytica in a group
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Wolfe M. S.1979. Giardiasis. Sympos
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APPENDICES APPENDIX 1 Information t
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Unayo imvume yokunqaba ukungenela l
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Appendix 3 Excystation and culture
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Appendix 4 Results of excystation a
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Appendix 5 Results of in vitro (aci
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Key to Appendix 5 *Replicated inocu
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Appendix 7 Preparation of culture m
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Appendix 8 List of enzyme systems u
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Appendix 9 Appendix (9a) Preparatio
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Appendix 10. Quantitative descripti
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