Davide Ianniello

Mini-FLOTAC, an Innovative Direct Diagnostic Technique for Intestinal Parasitic Infections: Experience from the Field

Background Soil-transmitted helminths and intestinal protozoa infection are widespread in developing countries, yet an accurate diagnosis is rarely performed. The aim of this study was to evaluate the recently developed mini–FLOTAC method and to compare with currently more widely used techniques for the diagnosis of intestinal parasitic infections in different settings. Methodology/Principal Findings The study was carried out in Dharamsala, Himachal Pradesh, India, and in Bukumbi, Tanzania. A total of 180 pupils from two primary schools had their stool analyzed (n = 80 in Dharamsala and n = 100 in Bukumbi) for intestinal parasitic infections with three diagnostic methods: direct fecal smear, formol-ether concentration method (FECM) and mini-FLOTAC. Overall, 72% of the pupils were positive for any intestinal parasitic infection, 24% carried dual infections and 11% three infections or more. The most frequently encountered intestinal parasites were Entamoeba coli, Entamoeba histolytica/dispar, Giardia intestinalis, hookworm, (and Schistosoma mansoni, in Tanzania). Statistically significant differences were found in the detection of parasitic infections among the three methods: mini-FLOTAC was the most sensitive method for helminth infections (90% mini-FLOTAC, 60% FECM, and 30% direct fecal smear), whereas FECM was most sensitive for intestinal protozoa infections (88% FECM, 70% direct fecal smear, and 68% mini-FLOTAC). Conclusion/Significance We present the first experiences with the mini-FLOTAC for the diagnosis of intestinal helminths and protozoa. Our results suggest that it is a valid, sensitive and potentially low-cost alternative technique that could be used in resource-limited settings — particularly for helminth diagnosis.

Comparison of individual and pooled faecal samples in sheep for the assessment of gastrointestinal strongyle infection intensity and anthelmintic drug efficacy using McMaster and Mini-FLOTAC.

A field study was conducted to validate pooled faecal samples in sheep for the assessment of gastrointestinal (GI) strongyle infection intensity (faecal egg count - FEC) and anthelmintic drug efficacy (FEC reduction - FECR). Ten sheep farms located in the Campania region of southern Italy were selected for the study. In each farm, individual faecal samples from 20 adult sheep (when possible) were collected, before (D0) and after (D14) an anthelmintic treatment with albendazole. For each farm and at each time point (D0 and D14) the faecal samples were examined individually and as pools. Specifically, three different pool sizes (5, 10 and 20 individual sheep samples) and three different analytic sensitivities (namely 10 using Mini-FLOTAC; 15 and 50 using the two variants of McMaster - McM15 and McM50) were compared for FEC and FECR using individual and pooled faecal samples. GI strongyle intensity (eggs per gram of faeces - EPG) of pooled samples correlated positively with mean EPG of individual samples, with very high correlation coefficients (ranging from 0.94 to 0.99) across the 3 different pool sizes and analytic sensitivities. Mini-FLOTAC was more sensitive compared to the two variants of McMaster (McM15 and McM50) for the diagnosis of GI strongyles in sheep (100% vs 88.5% vs 75.9%) and resulted in significant higher FEC compared to both McM15 and McM50, with a mean difference in egg counts of approximately 90 EPG (p<0.001). The drug efficacy results showed that FECR was higher than 98% at most farms independently of the pool size and analytic sensitivity. With the exception of two farms, FECR was 100% when calculated for individual animals and across the different pool size and analytic sensitivities. In conclusion, the present study highlighted that pooling ovine faecal samples is a rapid procedure that holds promise as a valid strategy for assessing GI strongyles FEC and FECR in sheep.

A comparison of the FECPAK and Mini-FLOTAC faecal egg counting techniques.

Faecal egg counts (FECs) are used for detecting and quantifying nematode infections and are the basis for determining drug efficacy and anthelmintic resistance in faecal egg count reduction tests (FECRTs). Currently, several FEC techniques are available for detecting and quantifying eggs of sheep nematodes. A comparison of the egg counts from the FECPAK (with a minimum detection limit of 30 eggs per gram (epg)) and Mini-FLOTAC (with a minimum detection limit of 5 epg) showed better diagnostic performance with Mini-FLOTAC in terms of measurement error (level of over- or under-estimation of FEC) and precision (variability in FEC). A tendency to under-estimate FEC was observed with the FECPAK particularly at egg densities of less than 500 epg. It is concluded that Mini-FLOTAC is a reliable diagnostic tool offering reduced measurement error and a higher level of precision.

How long can stool samples be fixed for an accurate diagnosis of soil-transmitted helminth infection using mini-FLOTAC?

Background Kato-Katz is a widely used method for the diagnosis of soil-transmitted helminth infection. Fecal samples cannot be preserved, and hence, should be processed on the day of collection and examined under a microscope within 60 min of slide preparation. Mini-FLOTAC is a technique that allows examining fixed fecal samples. We assessed the performance of Mini-FLOTAC using formalin-fixed stool samples compared to Kato-Katz and determined the dynamics of prevalence and intensity estimates of soil-transmitted helminth infection over a 31-day time period. Methodology The study was carried out in late 2013 on Pemba Island, Tanzania. Forty-one children were enrolled and stool samples were subjected on the day of collection to a single Kato-Katz thick smear and Mini-FLOTAC examination; 12 aliquots of stool were fixed in 5% formalin and subsequently examined by Mini-FLOTAC up to 31 days after collection. Principal Findings The combined results from Kato-Katz and Mini-FLOTAC revealed that 100% of children were positive for Trichuris trichiura, 85% for Ascaris lumbricoides, and 54% for hookworm. Kato-Katz and Mini-FLOTAC techniques found similar prevalence estimates for A. lumbricoides (85% versus 76%), T. trichiura (98% versus 100%), and hookworm (42% versus 51%). The mean eggs per gram of stool (EPG) according to Kato-Katz and Mini-FLOTAC was 12,075 and 11,679 for A. lumbricoides, 1,074 and 1,592 for T. trichiura, and 255 and 220 for hookworm, respectively. The mean EPG from day 1 to 31 of fixation was stable for A. lumbricoides and T. trichiura, but gradually declined for hookworm, starting at day 15. Conclusions/Significance The findings of our study suggest that for a qualitative diagnosis of soil-transmitted helminth infection, stool samples can be fixed in 5% formalin for at least 30 days. However, for an accurate quantitative diagnosis of hookworm, we suggest a limit of 15 days of preservation. Our results have direct implication for integrating soil-transmitted helminthiasis into transmission assessment surveys for lymphatic filariasis.

Parasitic infections on the shore of Lake Victoria (East Africa) detected by Mini-FLOTAC and standard techniques

BACKGROUND Helminths and protozoa infections pose a great burden especially in developing countries, due to morbidity caused by both acute and chronic infection. The aim of our survey was to analyze the intestinal parasitic burden in communities from Mwanza region, Tanzania. METHODS Subjects (n=251) from four villages on the South of Lake Victoria have been analyzed for intestinal parasites with direct smear (DS), formol-ether concentration method (FECM) and the newly developed Mini-FLOTAC technique; urinary schistosomiasis was also assessed in a subsample (n=151); symptoms were registered and correlation between clinic and infections was calculated by chi-squared test and logistical regression. RESULTS Out of the subjects screened for intestinal and for urinary parasites, 87% (218/251) were found positive for any infection, 69% (174/251) carried a helminthic and 67% (167/251) a protozoan infection, almost half of them had a double or triple infection. The most common helminths were hookworms, followed by Schistosoma mansoni and Schistosoma haematobium. Among protozoa, the most common was Entamoeba coli followed by Entamoeba histolytica/dispar and Giardia intestinalis. Mini-FLOTAC detected a number of helminth infections (61.7%) higher than FECM (38.6%) and DS (17.9%). Some positive associations with abdominal symptoms were found and previous treatment was negatively correlated with infection. CONCLUSION Despite the limited size of the examined population the current study indicates a high prevalence of intestinal parasitic infection in Bukumbi area, Tanzania, and Mini-FLOTAC showed to be a promising diagnostic tool for helminth infections. This high parasitic burden calls for starting a regular deworming programme and other preventive interventions in schools and in the community.

Freezing parasites in pre-Himalayan region, Himachal Pradesh: Experience with mini-FLOTAC.

BACKGROUND Helminths and protozoa infections pose a great burden especially in developing, countries, due to morbidity caused both by acute and chronic infections. Data on distribution of intestinal parasitic infections among the native and expatriates populations in Himachal Pradesh are scarce. The aim of our survey was to analyze the intestinal parasitic burden in communities from Dharamsala, Kangra district, in clinical and public health settings. We also field-tested the mini- FLOTAC, an innovative diagnostic device. METHODS Subjects referring to the Tibetan Delek Hospital for abdominal discomfort and all children of the Tibetan Primary School in Dharamsala were screened for intestinal parasitic infections with direct smear, formol-ether concentration (FEC) method and mini-FLOTAC, their clinical history was recorded, and correlations between clinical symptoms and infections analyzed. RESULTS 152 subjects were screened for intestinal parasites, of which 72 subjects in the outpatients department (OPD) (36 expatriates and 36 natives) and 80 in the school. 60% of schoolchildren and 57% of OPD patients were found positive for any infection, the most represented were protozoa infections (50%), whereas helminthic infections accounted only for 13% and 20% in OPD patients and schoolchildren, respectively. The most prevalent among helminths was Ascaris lumbricoides (11%). Giardia intestinalis was more present among schoolchildren than the OPD patients (20% vs 6%) and E. histolytica/dispar was more prevalent among the OPD patients (42%) than the school children (23%). Correlations were found between nausea and loose or watery stools and parasitic infections, particularly in expatriates, whereas schoolchildren, despite being as infected as adults, were completely asymptomatic. Mini-FLOTAC detected higher number of helminth infections whereas FEC method was more accurate for the diagnosis of protozoa. CONCLUSIONS This study presents an accurate snapshot of intestinal parasitic infections in Dharamsala, and their high prevalence calls for more awareness and control measures. Mini-FLOTAC is a promising and simple technique for the diagnosis of helminth infections.

Parasitic infections detected by FLOTAC in zoo mammals from Warsaw, Poland

The aim of this study was to estimate the occurrence of intestinal parasites in groups of mammals kept in the Warsaw zoological garden (Poland). 71 pools of fecal samples were analyzed using the FLOTAC techniques. 48% of animals were positive and 47% of positivities showed multiple infections. Toxocara cati (71.4%) was found in felines; marsupials were infected with Coccidia (90%). Giardia spp. (24.0%), Blastocystis spp. (12.3%), Iodamoeba spp. (10.0%), Enterobius vermicularis (6.0%) and Entamoeba coli (3.3%) were found in primates. Gastrointestinal strongyles (60.5%) were prevalent in ruminants which resulted positive also to Coccidia (Eimeria spp. = 50.0%), Trichuris spp. (25.0%) and Nematodirus (14.0%). Strongyles (34.0%) were the most frequent parasites in monogastric herbivores, followed by Parascaris equorum (17.0%). None of the animals showed any symptom associated with gastrointestinal parasitic infections. According to our results the need to prevent, diagnose, control, and treat intestinal parasitism trough specific control programs is mandatory for animal welfare in order to limit the spread of parasitic infections in animals and humans.

Innovative tools for the diagnosis of Echinococcus granulosus in definitive hosts

The aim of this study was to develop and validate an innovative protocol for the diagnosis of Echinococcus granulosus and other Taeniidae in dogs. For this purpose, three experiments were performed, using faecal samples from naturally infected dogs. Firstly, the FLOTAC technique was calibrated with five flotation solutions: saturated sodium chloride (specific gravity, s.g. = 1.20), zinc sulphate (s.g. = 1.35), zinc chloride (s.g. = 1.45), Breza (s.g. = 1.30) and modified Breza (s.g. = 1.40). Then, FLOTAC was compared with four techniques of flotation in centrifuge using: zinc sulphate (s.g. = 1.20), Breza (s.g. = 1.30), modified Breza (s.g. = 1.40), and zinc chloride (s.g. = 1.45). Finally, four different protocols of DNA extraction were compared for Taeniidae identification: QIAamp Tissue Kit and QIAamp Stool from eggs; QIAamp Stool and Wizard Magnetic Purification System for Food from faeces. FLOTAC with zinc sulphate was the most efficient method to detect Taeniidae eggs, showing highest mean of eggs per gram (EPG) of faeces. The QIAamp Stool, using eggs concentrated by FLOTAC, was the best method for DNA extraction. The combination of these protocols provided the highest number of positive samples with PCR, i.e., 47/50 (94.0%). The three negative samples showed a low faecal egg count (2 EPG) below the detection limit (4 EPG) of the protocol. From sequencing of the 47 positive samples: 6 samples were identified as E. granulosus sensu stricto (s.s.), 28 as Taenia hydatigena and 6 as T. pisiformis. A co-infection between different genera of Taeniidae was found in 7 samples.

The recovery of added nematode eggs from horse and sheep faeces by three methods

BackgroundNematode infections in horses are widespread across the world. Increasing levels of anthelmintic resistance, reported worldwide in equine parasites, have led to the creation of programs for the control of nematodes based on faecal egg counts (FEC). To improve nematode egg counting in equine faecal samples and establish whether the matrix of equine faeces or the eggs affect the counts, the analytical sensitivity, accuracy and precision of Mini-FLOTAC (combined with Fill-FLOTAC), McMaster and Cornell-Wisconsin techniques were compared. Known numbers of eggs extracted from equine or ovine faeces were added to egg free ovine and equine faeces to give counts of 10, 50, 200 and 500 eggs per gram (EPG) of faeces.ResultsThe Cornell-Wisconsin significantly underestimated egg counts and McMaster showed a low analytical sensitivity, revealing 100% of sensitivity only for concentrations greater than 200 EPG. EPG values detected by Mini-FLOTAC did not differ significantly from expected counts at any level of egg density.ConclusionsMini-FLOTAC combined to Fill-FLOTAC which provides an accurate method of weighing without need for a balance and filtering out debris, could be used for FEC on the farm as well as in the laboratory.