COMPARISON OF DIFFERRENT LABORATORY METHODS FOR DIAGNOSIS OF CRYPTOSPORIDIUM

The present study was carried on to compare several methods for identification of Cryptosporidium oocysts sp. which conclude modified acid fast stain, Sheather's flotation solutions, Ritchi formal-ether sedimentation solutions, noval stain and iodine technique. The modified acid-fast stain and its combination with formal – ether technique gave the best demonstration 100%. The most reliable technique for identification of oocysts was formal-ether concentration combination with modified acid fast stain (100%), followed by using modified acid fast stain only (97%), sucrose flotation technique (90%) novel staining technique (85%), formal ether concentration (84%), and iodine technique (69%) respectively.

First report of cryptosporidiosis among Iraqi children

Of 240 Iraqi children under five years old with a primary diagnosis of gastroenteritis and dehydration, 22 were excreting Cryptosporidium oocysts in their stools. This is the first reported occurrence of cryptosporidiosis among Iraqi schoolchildren. Samples were collected and examined by the modified Ziehl-Neelsen method between March and November 1994. The frequency distribution of Cryptosporidium among 114 positive cases with intestinal parasites was 18.3%. The boy-to-girl ratio was 1:2 among the Cryptosporidium-positive children. Most patients complained of abdominal colic and watery diarrhoea and were clinically dehydrated. The associated symptoms were fever and vomiting. Cryptosporidium and Giardia appeared to be associated

Use of Oxidative Stress Responses to Determine the Efficacy of Inactivation Treatments on Cryptosporidium Oocysts

Cryptosporidium oocysts are known for being very robust, and their prolonged survival in the environment has resulted in outbreaks of cryptosporidiosis associated with the consumption of contaminated water or food. Although inactivation methods used for drinking water treatment, such as UV irradiation, can inactivate Cryptosporidium oocysts, they are not necessarily suitable for use with other environmental matrices, such as food. In order to identify alternative ways to inactivate Cryptosporidium oocysts, improved methods for viability assessment are needed. Here we describe a proof of concept for a novel approach for determining how effective inactivation treatments are at killing pathogens, such as the parasite Cryptosporidium. RNA sequencing was used to identify potential up-regulated target genes induced by oxidative stress, and a reverse transcription quantitative PCR (RT-qPCR) protocol was developed to assess their up-regulation following exposure to different induction treatments. Accordingly, RT-qPCR protocols targeting thioredoxin and Cryptosporidium oocyst wall protein 7 (COWP7) genes were evaluated on mixtures of viable and inactivated oocysts, and on oocysts subjected to various potential inactivation treatments such as freezing and chlorination. The results from the present proof-of-concept experiments indicate that this could be a useful tool in efforts towards assessing potential technologies for inactivating Cryptosporidium in different environmental matrices. Furthermore, this approach could also be used for similar investigations with other pathogens.

Assessment of Parasitological Water Quality from House Kitchens and Desalination Plants Filters in Gaza Strip

This study was conducted to assess parasitological water quality from house kitchens and desalination plants filters in Gaza Strip. A total of 420 samples were collected from the five Governorates of Gaza Strip; 300 samples were collected from 100 houses and 120 samples were collected from 40 desalination plants. All samples were examined using direct wet mount, acid fast stain and Polymerase Chain Reaction. The randomlly distributed questionnaire included questions regarding economic and social factors, water sources, reported symptoms and public health. Results revealed that only Cryptosporidium spp. oocysts were detected in eight of drinking water samples 1.9% (8/420). Eight samples were positive when using acid-fast stain (Cryptosporidium spp.) in Reverse osmosis (RO) house filters. Using PCR to identify Cryptosporidium spp. (C. parvum and C. hominis), only one sample (0.24%) was positive for C. parvum while there are no positive samples for C. hominis. The occurrence of Cryptosporidium oocysts in the investigated water supplies may require the water utilities and water authorities in Gaza Strip to apply additional monitoring, treatment and/or watershed controls for safe drinking water.

Occurrence of Cryptosporidium Oocysts in Leisure Pools in the UK, 2017, and Modelling of Oocyst Contamination Events

Cryptosporidium is a major cause of diarrhoea outbreaks linked to swimming pools, but little is known about the frequency of contamination. The primary aim was to investigate the occurrence and concentration, through sampling and modelling, of Cryptosporidium oocysts in leisure pools. Secondary aims were to compare detections with operational parameters, provide the evidence-base for guidance, and improve sampling capacity and interpretation for public health investigations. Up to 1000 L pool water was sampled during swim sessions once weekly for 10 weeks from 8th August 2017 at six volunteer pools. Oocysts were detected by microscopy in 12/59 (20%) pool water samples, at least once in each pool; 8/12 (66%) detections were in August when bather loads were highest. At three pools, 1 L filter backwash was sampled weekly and oocysts were detected in 2/29 (7%) samples, following detections in pool water. The probabilities of a bather contaminating the pool ranged from 1 in 1000 to over 1 in 10,000. Monte Carlo analysis showed that when high bather numbers caused contamination on over 70% of days, multiple events per day were more likely than single events. In these generally well-managed leisure pools, Cryptosporidium risk related to high bather loads. We conclude that public awareness campaigns for bather hygiene, and reminding pool operators of current guidance for managing faecal accidents, should be ahead of peak swim season.

Prevalence and Some Predisposing Factors Associated with Cryptosporidium Infections in Domestic Poultry in Enugu State, Nigeria

Cryptosporidium is a zoonotic protozoan parasite that is of major public health and veterinary concerns. This study determined the prevalence and some predisposing factors associated with Cryptosporidium infection in domestic poultry kept in households and in the major live bird markets in the study area. Atotal of 314 fresh faecal samples were collected from domestic poultry in the randomly selected homes that keep poultry and live bird market in the study area. They were examined using the Formol-ether sedimentation method. Faecal smears were then stained by the Ziehl Neelson technique and examined under light microscopy. Cryptosporidium was identified using the staining characteristics of the oocysts. Results were analysed by descriptive statistics and GraphPad prism statistical package version 5.2.Cryptosporidium oocysts were detected in the faeces of 90 (28.7%) of the 314 poultry sampled. There was a strong association (p < 0.05) between the presence of Cryptosporidium oocysts and age, sex, breed and faecal consistency. However, there was no significant association (p > 0.05) between the presence of Cryptosporidium oocysts and locality as well as the type of management practice used in keeping the birds. This study suggests that domestic poultry in Enugu State harbour and shed Cryptosporidium oocysts in the environment. The shedding of this oocyst in the environment is of zoonotic importance especially in poultry that do not show clinical signs and therefore were not treated. Hence, constituting a public health risk, especially to immune-compromised humans, considering that almost every household keep chicken for food or income and the poultry dung is popularly used to cultivate vegetables eaten by humans and animals in the study area. Keywords: Cryptosporidium, Poultry, Prevalence, Risk Factor

Improved Cryptosporidium case findings using immunofluorescent microscopy on concentrated stool

Background: Diarrhoea is a major cause of morbidity in Cape Town, South Africa, and mortality is attributed to a failure to recognize the severity of the condition. Cryptosporidium and Giardia are increasingly recognized as important causes of diarrhoea in Africa however, suboptimal diagnostic techniques may lead to underappreciation of their significance. Our objectives are to compare the diagnostic yield of direct immunofluorescent antigen (DFA) microscopy on concentrated stool samples for Cryptosporidium and Giardia, with the current approach of wet mount microscopy for Giardia and auramine fluorescent stain for Cryptosporidium on unconcentrated stool.Methodology: Stool specimens (n=104) received at our hospital laboratory for routine microbiological investigations were used for the study. Direct wet-mount auramine-phenol fluorescent microscopy (auramine) detection of Cryptosporidium oocysts and wet mount iodine microscopy for Giardia detection, were performed on unconcentrated stool samples, while DFA stain for simultaneous detection of Cryptosporidium and Giardia was performed on sodium-acetate formalin concentrated stool samples. The diagnostic yields of the tests were compared using the MEDCALC® version 18.0Results: Of the 104 stool specimens received for microbiological analysis, only 66 (63.5%) had specific Cryptosporidium requests while 38 (36.5%) had no Cryptosporidium specific requests. Of the 66 specimens, 9 (13.6%) were positive for Cryptosporidium oocysts with DFA while only 1 (1.5%) was positive with auramine staining (p=0.013). The one auramine-positive specimen was also positive by DFA. Auramine stain microscopy gave a sensitivity of 11.1% (95%CI: 0.28-48.25%) and specificity of 100% (95%CI: 93.7%-100%) when compared to DFA. Of the 38 stool specimens without specific Cryptosporidium request, DFA yielded 5 (13.2%) additional positive results. Taken together, Cryptosporidium was detected in 14/104 (13.5%; 95%CI: 8.36–21.7%) specimens and only 1 of 14 (7.1%) specimens with the current routine laboratory testing approach. Giardia was detected by DFA in 3/104 (0.9%) specimens, while direct iodine wet mount microscopy did not yield any positive results (0%). All 3 Giardia-positive specimens had Cryptosporidium oocysts detected by DFA.Conclusion: These data suggest that a large proportion of Cryptosporidium cases remain undetected by the laboratory due to suboptimal testing methods, and failure by clinicians to specifically request for Cryptosporidium detection. There is need to periodically assess the effectiveness of diagnostic microbiology laboratory approaches to diarrhoea, and access to improved diagnostic laboratory techniques will contribute to more accurate differential diagnosis and a broadened understanding of local aetiology of diarrhoea diseases in Africa. Keywords: Cryptosporidium, Giardia, diarrhoea, stool concentration, DFA, microscopy French Title: Amélioration des découvertes de cas de Cryptosporidium à l'aide de la microscopie immunofluorescente sur des selles concentrées Contexte: La diarrhée est une cause majeure de morbidité au Cap, en Afrique du Sud, et la mortalité est attribuée à l'incapacité de reconnaître la gravité de la maladie. Cryptosporidium et Giardia sont de plus en plus reconnus comme des causes importantes de diarrhée en Afrique, cependant, des techniques de diagnostic sous-optimales peuvent conduire à une sous-estimation de leur importance. Nos objectifs sont de comparer le rendement diagnostique de la microscopie à antigène immunofluorescent direct (DFA) sur des échantillons de selles concentrées pour Cryptosporidium et Giardia, avec l'approche actuelle de la microscopie à montage humide pour Giardia et la coloration fluorescente auramine pour Cryptosporidium sur des selles non concentrées.Méthodologie: Des échantillons de selles (n=104) reçus au laboratoire de notre hôpital pour des examens microbiologiques de routine ont été utilisés pour l'étude. La détection directe par microscopie fluorescente auramine-phénol à montage humide (auramine) des oocystes de Cryptosporidium et la microscopie à l'iode à montage humide pour la détection de Giardia, ont été effectuées sur des échantillons de selles non concentrées, tandis que la coloration DFA pour la détection simultanée de Cryptosporidium et de Giardia a été réalisée sur de l'acétate de sodium formaline concentré échantillons de selles. Les rendements diagnostiques des tests ont étécomparés à l'aide de MEDCALC® version 18.0Résultats: Sur les 104 échantillons de selles reçus pour l'analyse microbiologique, seuls 66 (63,5%) avaient des demandes spécifiques de Cryptosporidium tandis que 38 (36,5%) n'avaient pas de demandes spécifiques de Cryptosporidium. Sur les 66 échantillons, 9 (13,6%) étaient positifs pour les oocystes de Cryptosporidium avec DFA tandis que seulement 1 (1,5%) était positif avec coloration à l'auramine (p=0,013). Le seul échantillon positif à l'auramine était également positif au DFA. La microscopie à l'auramine a donné une sensibilité de 11,1% (IC 95%: 0,28-48,25%) et une spécificité de 100% (IC 95%: 93,7% -100%) par rapport au DFA. Sur les 38 échantillons de selles sans demande spécifique de Cryptosporidium, le DFA a donné 5 (13,2%) résultats positifs supplémentaires. Pris ensemble, Cryptosporidium a été détecté dans 14/104 (13,5%; IC à 95%: 8,36–21,7%) et seulement 1 des 14 échantillons (7,1%) avec l'approche actuelle des tests de routine en laboratoire. Giardia a été détecté par DFA dans 3/104 (0,9%) échantillons, tandis que la microscopie directe à l'iode sur monture humide n'a donné aucun résultat positif (0%). Les 3 échantillons positifs à Giardia avaient des oocystes deCryptosporidium détectés par DFA.Conclusion: Ces données suggèrent qu'une grande proportion des cas de Cryptosporidium ne sont pas détectés par le laboratoire en raison de méthodes de test sous-optimales et de l'échec des cliniciens à demander spécifiquement la détection de Cryptosporidium. Il est nécessaire d'évaluer périodiquement l'efficacité des approches de laboratoire de microbiologie diagnostique pour la diarrhée, et l'accès à des techniques de laboratoire de diagnostic améliorées contribuera à un diagnostic différentiel plus précis et à une compréhension élargie de l'étiologie locale des maladies diarrhéiques en Afrique. Mots clés: Cryptosporidium, Giardia, diarrhée, concentration des selles, DFA, microscopie

Occurrence of Cryptosporidium Oocysts and Helminth Ova on Dried Crayfish (Procambarus clarkii) Sold in Kaduna State, Nigeria

Cryptosporidium spp. and helminths are responsible for diarrhoal illness in humans and many other animals. The transmission routes of these parasites suggest a risk for human infection through contaminated foods. In order to determine the occurrence of Cryptosporidium oocysts and helminth ova on dried crayfish (Procambarus clarkii) sold in Zaria and Kaduna Central market, Kaduna State, 100 crayfish samples were examined using the modified Ziehl-Neelsen staining technique for Cryptosporidium oocyst and the flotation technique for helminth ova. The measurements of the oocysts from the positive samples were estimated by using a calibrated microscope eyepiece. The prevalence of Cryptosporidium oocysts in dried crayfish was 20.0 % while the location-based prevalence was 43.3 %, 5.0 %, and 12.0 % for Sabo, Samaru and Kaduna central market, respectively. Micrometry revealed that the oocysts size ranged from 3.68 µm to 4.7 µm. Also, the prevalence of helminth ova in dried crayfish was 19 %, while the specific prevalence based on location showed a higher prevalence in Sabo (30.0 %) than in Kaduna central market (18.0 %) and Samaru (5.0 %). The helminth eggs recovered from this study included: Toxocara spp. (4.0 %), Ascaris spp. (7.0 %), Trichuris spp. (4.0 %), Schistosoma spp. (2.0 %), Ancylostoma spp. (1.0 %), and Capillaria spp. (1.0 %). The analysis of the questionnaires revealed that 71 (71.0 %) of the respondents ate uncooked cray-fish and 44.0 % used their bare hands to handle the cray-fish. There was no statistically significant association (P > 0.05) between the prevalence of Cryptosporidium oocysts or helminth ova and the locations sampled. This study has shown that dried crayfish obtained from markets within the study area were contaminated with parasite ova and oocysts, thus public enlightenment on the dangers of the consumption of raw or undercooked dried crayfish should be well publicized.