EVALUATING ONCHOCERCIASIS USING PARASITOLOGICAL, ENTOMOLOGICAL AND SLIDE AGGLUTINATION TECHNIQUE IN KACHIA, KADUNA STATE, NIGERIA
EVALUATING ONCHOCERCIASIS USING PARASITOLOGICAL, ENTOMOLOGICAL AND SLIDE AGGLUTINATION TECHNIQUE IN KACHIA, KADUNA STATE, NIGERIA
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Date
2015-07
Authors
OSUE, Hudu Okankhamame
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Abstract
It has become necessary to assess the ongoing community directed treatment with ivermectin (CDTI) of onchocerciasis after 18 years of commencement of intervention. This thesis compared baseline and post-treatment data generated in 1994 and 2011 from sample populations (n=531) and (n=593) respectively in six sentinel onchocerciasis meso-endemic communities. Secondly, development and evaluation of a novel Onchocerca volvulus slide flocculation test (Ov-SFT) was undertaken. Parasitological analyses of skin snips and engorged blackflies caught by human bait were dissected and examined for microfilaria. Eye and skin clinical examination were performed. In this study, the levels of onchocercal specific serum antibodies reacting with sodium dodecyl sulphate (SDS) extracted low molecular weight (LMW) antigens prepared from adult female worms of O. volvulus after collagenase digestion of nodules were assessed at post-control. The Ov-SFT post-treatment data were compared with that of enzyme linked immunosorbent (ELISA) baseline data for the study population. Eight tissue homogenates from rat organs (kidney, liver, heart, spleen, muscle, brain, lungs and testes) were screened for flocculation potential using confirmed onchocerciasis positive and negative sera. The skin microfilaria status of patients were validated using first internal transcribed spacer (ITS1) of ribosomal DNA (rDNA) that lies within 18S and 5.8S gene region in polymerase chain reaction (PCR) amplification of DNA templates extracted from skin biopsies (n=66). The DNA extracted from fragments of O. volvulus adult worms from two different nodules and template from three non-infected persons served as positive and negative controls, respectively. A cohort (n=445) of volunteered participants (75%) were examined at both visits. The annual village treatment, 11(64.7%) and population treatment compliance was 92.6% with a range between 88.7 to 100%. Overall mean number of treatment was 2.9±1.6 with a range, 2.0±1.2-3.3±0.6. Post-treatment skin microfilarial prevalence and palpable nodule occurrence were significantly
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reduced from 144 (27.0%) and 77 (15%) to 0 (0%) and 4 (0.7%), respectively compared with baseline data (P<0.05 t-test of unpaired data). There were significant decrease in cases of papular onchodermatitis from 51 (9.1%) to 2 (0.04%) and onchocercal inducible ocular lesions from 31 (5.8%) to 12 (2.1%), P<0.05. Cases of glaucoma 8 (1.4%) and blindness 6 (1.05%) remained unchanged. Those with visual acuity of ≥6/24 in one or both eyes had increased from 26 (4.9%) to 198 (33.45%) with a range 16 (17.4%) – 47 (61.8%) in the villages. The significant increases in cases of cataract 169 (28.5%); pterygium 157 (26.5%) and acute senilis 165 (27.9%) were strongly positively correlated with increase in age (R2= 0.898 - 0.949). The liver homogenate proved to be the highest, followed by the kidney and heart. 33 (50.76%) of the sera (n= 65) tested positive for onchocerciasis. Profile of antibody titre ranged from 1:2, 1 (1.54%) to 1:32, 13(20%). Three out of the five malaria positive cases were Ov-SFT sero-reactive compared to 9 (63.5%) of the 14 malaria negative. Nanodrop spectrophotometer analyses of DNA concentration of samples and control at 260 nanometer (nm) wave length were between 1.5 to 24.5 nanogram (ng). The 260/280nm ratios were between 1.45 and 1.68 compared to 105 and 10.5ng, and 1.65 and 1.44 nm for control, respectively. Out of the 66 DNA samples analysed using ITS1 primer sequences, 4 (6.1%); (two males and two females) were amplified. Two positive samples had single band of 344 base pairs (bp), while two gave double bands amplification products (amplicons) of 344 and 500bp molecular weight in an agarose gel electropherogram. The 4 amplicons gave DNA nucleotide sequences of between 629 and 639 sequence length. One of the two positive controls gave different amplicons of molecular weight that falls within expected O. volvulus 344 bp and other sympatric filarial that lies between 312, 305 and 301bp for Mansonnela ozzardi, M. perstan and Wuchereria bancroft, respectively. Whether the DNA amplified were from dead or living mf remained unknown. The ITS1 PCR amplification of DNA gave greater positive predictive values than skin snip microscopy for microfilaria. Despite varied
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compliance to ivermectin treatment, active disease transmission and progression of onchocerciasis induced skin and ocular clinical manifestations have been halted. Ivermectin treatment had no impact on existing cases of glaucoma and blindness, which remained unchanged but halted development of new cases. The Ov-SFT is simple, sensitive, fast and required less skill; it should be suitable for screening exposure to infection. Further validation to establish its specificity using related parasites is required. The need for an algorithm for assessing onchocerciasis treatment control involving antigen-specific antibody screening, molecular diagnosis and parasitological confirmation cannot be overemphasized
Description
A THESIS SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES,
AHMADU BELLO UNIVERSITY, ZARIA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD
OF A
DOCTOR OF PHILOSOPHY IN MICROBIOLOGY
DEPARTMENT OF MICROBIOLOGY,
FACULTY OF SCIENCE,
AHMADU BELLO UNIVERSITY, ZARIA, NIGERIA
Keywords
EVALUATING ONCHOCERCIASIS,, PARASITOLOGICAL,, ENTOMOLOGICAL,, SLIDE AGGLUTINATION TECHNIQUE,, KACHIA,, KADUNA STATE,, NIGERIA