Assessment of the Viability of Adult Onchocerca Volvulus in Tissue Sections using Enzyme Histochemical Demonstration of Lactate Dehydrogenase Activity

Abstract

Current methods of assessing the viability of adult Onchocercs volvulus in drug evaluations are mainly based on in-vitro and histological techniques. The in-vitro method is efficient in detecting live and dead worms while the histological method is more efficient in determining the number of worms in the nodules. Despite their capabilities in some areas, these techniques have certain methodical deficiencies which often lead to inaccurate interpretation of results or marked variations between individuals assessing the viabilities of worms in the same nodules.The deficiencies associated with these techniques are partly responsible for the delay in developing safe and effective drugs for the treatment of onchocerciasis. Based on principles underlying lactate production and tetrazolium reduction tests used in in-vitro techniques for testing the viabilities of adult worms, an enzyme histochemical staining technique was employed to determine the presence of lactate dehydrogenase (LDH) in adult O. volvulus tissue sections and then used to determine their viabilities. Eleven onchocerciasis infected volunteers donated a total of sixty-one O. volvulus nodules for this study. Out of the total number of nodules donated, unfixed cryostat sections were obtained from thirty-two of the nodules (including two nodules which were heated in distilled water at 60°C to obtain heat-killed worms). The sections were incubated in a substrate containing sodium DL-lactate, nicotinamide adenine dinucleotide (NAD), magnesium chloride and nitro blue tetrazolium compound (NBT) at 37°C for an hour. The same nodules were fixed in phosphate buffered formalin after the cryostat sections were obtained from them and processed by the paraffin method to obtain paraffin processed sections which were stained by the haematoxylin and eosin technique. The other twenty-nine O. Volvulus nodules from the total number of nodules obtained from the volunteers (including two heat-killed nodules) were fixed in phosphate-buffered formalin and paraffin processed sections obtained from them and stained by the haematoxylin and eosin technique. Light microscopic examination of the three sets of sections revealed that worm-sections which were morphologically well preserved in their paraffin sections showed the presence of LDH activity in their corresponding cryostat sections and therefore were adjudged to be sections of live worms. On the other hand worm-sections which were morphologically degenerated in paraffin sections did not show any LDH activity in their cryostat sections and were adjudged to be sections of dead worms. This observation implied that the presence of LDH activity in worm sections were indications that the sections belonged to live worms and those sections which did not show enzyme activity belonged to dead worms.There was no significant difference between the enzyme technique and histology in determining the number of adult worms in the nodules examined. This suggested that the enzyme histochemical method has the same advantage that the histological method have over the in-vitro technique in determining the number of worms in the nodules. All the three worms detected in the heat-killed nodules were morphologically well preserved in their paraffin sections while their cryostat sections did not show enzyme activity. They were therefore alive histologically but declared dead by the enzyme technique indicating that the enzyme method is capable of detecting dead worms which are unable to maintain the activity of their enzymes even without any significant morphological degenerations. Since degenerative processes that must show on histology to determine dead worms occur several months after the death of the worm, the study indicates that the enzyme method may be able to detect the death of adult worms much earlier than the histological method. This is regarded as a possible advantage of the enzyme technique over the histological method, however, this assertion needs to be confirmed on a larger sample size. The results showed that, the enzyme histochemical technique can be used as a more reliable alternative to the histological technique in determining the viability of adult 0. volvulus during drug evaluations. It can also be used along with the histological technique in order to obtain early results during drug trials to be followed by histological results which often takes several weeks to process. Comparative analysis of the two sets of paraffin processed sections confirmed that statistical comparisons of the results from different groups of nodules are acceptable for drug investigations.