Beatrice Barda

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.

Mini-FLOTAC and Kato-Katz: helminth eggs watching on the shore of lake Victoria

BackgroundOne of the challenges for monitoring helminth control programmes based on preventive chemotherapy is the lack of a copro-parasitological gold–standard method that combines good sensitivity with quantitative performance, low cost, and easy-to-learn technique.The aim of our study was to evaluate and compare, the WHO recommended quantitative diagnostic technique (Kato-Katz) and the Mini-FLOTAC.MethodsMini-FLOTAC is an innovative method based on floatation of helminths eggs with two different solutions (FS2 and FS7) using a close system (Fill-FLOTAC) with 5% fixative. Kato-Katz was performed following WHO recommendation. The study was carried out in a rural part of Tanzania, close to Lake Victoria, where the laboratory facilities are fairly scarce, and the basic technique used in the local laboratory (direct smear) was taken as reference standard.Results201 children were screened for intestinal helminths and 91% of them were found to be positive. The agreement among the three techniques was calculated with k Cohen coefficient and was fairly good (k = 0.4), although the Mini-FLOTAC results were more sensitive for hookworm (98%) with FS2, and for S.mansoni (90%) with FS7 followed by Kato-Katz (91% and 60% respectively) and direct smear (30% and 10% respectively). A good agreement was found between Mini-FLOTAC and Kato-Katz (k = 0.81) with FS7 (k = 0.76) for hookworm diagnosis and a fairly good one for S.mansoni diagnosis (k = 0.5). For both infections we had a poor agreement between the two quantitative techniques and the direct smear (k<0.3). Kato-Katz diagnosed a higher number of eggs (calculated by arithmetic mean) both for hookworm (455 vs 424 EPG) and for S.mansoni (71 vs 58 EPG) compared with the Mini-FLOTAC, but the differences were not significant (p = 0.4).ConclusionsMini-FLOTAC is a promising technique, comparable and as sensitive as the Kato-Katz, which is the recommended method in intestinal helminthology for monitoring helminth control programmes. A comparative advantage of the Mini-FLOTAC is that it comprises of a closed system with preserved samples that both protects the operators and allows subsequent examination of the samples. Further studies are needed to validate the mini-FLOTAC with other quantitative techniques (McMaster) and in different settings where other soil-transmitted helminths are also endemic.

StrongNet: An International Network to Improve Diagnostics and Access to Treatment for Strongyloidiasis Control

Strongyloidiasis is a disease caused by an infection with a soil-transmitted helminth that affects, according to largely varying estimates, between 30 million and 370 million people worldwide [1,2]. Not officially listed as a neglected tropical disease (NTD), strongyloidiasis stands out as particularly overlooked [3]. Indeed, there is a paucity of research and public health efforts pertaining to strongyloidiasis. Hence, clinical, diagnostic, epidemiologic, treatment, and control aspects are not adequately addressed to allow for an effective management of the disease, both in clinical medicine and in public health programs [4]. The manifold signs and symptoms caused by Strongyloides stercoralis infection, coupled with the helminth’s unique potential to cause lifelong, persistent infection, make strongyloidiasis relevant beyond tropical and subtropical geographic regions, where, however, most of the disease burden is concentrated. Indeed, strongyloidiasis is acquired through contact with contaminated soil, and the infection is, thus, primarily transmitted in areas with poor sanitation, inadequate access to clean water, and lack of hygiene. While the actual morbidity of chronically infected, immunocompetent individuals is subtle and difficult to appreciate [5], the particular importance of this parasitic worm is linked to its potential for maintaining lifelong autoinfections and causing a life-threatening hyperinfection syndrome in immunocompromised individuals [6]. Lack of point-of-care (POC) diagnostics and poor availability of, and access to, ivermectin (the current treatment of choice) are the two most significant bottlenecks that hinder effective management of the disease both in clinical and in public health settings. Examples of the management and importance of strongyloidiasis in two clinical contexts (in a tropical setting and a high-income country) and from a public health perspective are given in Boxes 1–3. Box 1. Individual Living in an Endemic Area with Diarrhea, Abdominal Pain, Pruritus, and Significant Dermatological Manifestations [10] A 43-year-old male farmer, living in the rural eastern part of Preah Vihear province, northern Cambodia, was diagnosed with a heavy Strongyloides stercoralis infection (924 and 478 larvae present in two Baermann examinations). Additionally, larvae and adult S. stercoralis were detected in Koga agar plate culture examinations of the stools. The patient was co-infected with hookworm and presented with abdominal pain, diarrhea, nausea, vomiting, fever, and a pronounced and persistent skin rash, which had been present with extensive itching for more than two years. The rash was observed on the back, chest, abdomen, and extremities and, due to frequent and intense scratching, showed signs of focal infection. Three weeks after treatment with a single oral dose of ivermectin (200 μg/kg) and a single oral dose of mebendazole, the patient’s rash had almost disappeared, and he was free of episodes of intensive itching.

Residual viraemia does not influence 1 year virological rebound in HIV-infected patients with HIV RNA persistently below 50 copies/mL

OBJECTIVES It is currently debated whether patients with residual viraemia are at higher risk of virological failure than those attaining <1 HIV RNA copy/mL. We therefore investigated the effect of residual viraemia on virological rebound. METHODS We used a prospective, non-interventional, single-centre, study. This analysis was based on HIV-infected patients with two consecutive HIV RNA viral loads (VLs) of <50 copies/mL as tested by Versant bDNA, followed by two HIV RNA VLs of <50 copies/mL as tested using the Versant kinetic PCR molecular system (kPCR; limit of quantification = 1 copy/mL). Virological rebound was defined as two consecutive HIV RNA values of >50 copies/mL after baseline, and the time to virological rebound was calculated using the Kaplan-Meier method. RESULTS There were 739 eligible patients; 446 (60.4%) had HIV RNA <1 copy/mL (group A) and 293 (39.6%) had residual viraemia (1-49 HIV RNA copies/mL; group B). After a follow-up (median 48.9 weeks), virological rebound occurred in four patients in group A (0.9%) and six patients in group B (2%); the time to virological rebound was similar in the two groups (log-rank test P = 0.231). CD4+ cell recovery (slope) was significantly less in the patients with residual viraemia; +14.3 (-7.7, 43.9) cells/mm(3) per year versus +21.2 (-2.5, 53.2) cells/mm(3) per year; P = 0.036. CONCLUSIONS Residual viraemia assessed by kPCR was not associated with virological rebound during 1 year of follow-up. However, the patients attaining <1 HIV RNA copy/mL showed a small but statistically significant improvement in CD4+ cell recovery.

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.

Evolution patterns of raltegravir-resistant mutations after integrase inhibitor interruption

The objective of this study was to address the evolution of human immunodeficiency virus type 1 (HIV-1) mutations resistant to the integrase inhibitor raltegravir after drug interruption. Thirteen HIV-1 infected patients undergoing virological failure due to the selection of raltegravir-resistant variants, who had interrupted raltegravir treatment, were enrolled. For all patients, the virological failure was associated with the selection of variants, with mutations conferring resistance to all of the drugs present in their regimens. Patients were prospectively monitored at baseline (raltegravir interruption) and every 4-24 weeks for clinical, virological and immunological parameters, including HIV-1 viraemia, CD4(+) T-cell counts, and sequence analysis of the HIV-1 integrase sequence. Reversion to the wild-type HIV-1 integrase sequence genotype was observed between 4 and 36 weeks after raltegravir withdrawal in eight out of the 13 patients. Reversion was not observed in three patients. In two patients, reversion was partial at week 24 from raltegravir interruption. These results highlight that in eight out of 13 patients under treatment with raltegravir and experiencing a virological failure, HIV-1 variants harbouring mutations associated with raltegravir resistance become undetectable after drug interruption within a few weeks (in some cases, very rapidly). This occurs under different therapy regimens and in patients receiving 3TC mono-therapy. In the other patients, complete reversion of the integrase sequence is not observed, and either primary or secondary resistance mutations are fixed in the replication competent viral population in vivo also for long time, suggesting that other factors may influence this dynamic process.

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.

Efficacy and Safety of Moxidectin, Synriam, Synriam-Praziquantel versus Praziquantel against Schistosoma haematobium and S. mansoni Infections: A Randomized, Exploratory Phase 2 Trial.

Background Schistosomiasis affects millions of people, yet treatment options are limited. The antimalarial Synriam (piperaquine 150 mg/arterolane 750 mg) and the anthelminthic moxidectin revealed promising antischistosomal properties in preclinical or clinical studies. Methodology We conducted two single-blind, randomized exploratory Phase 2 trials in Schistosoma mansoni and S. haematobium-infected adolescents in northern and central Côte d’Ivoire. Our primary endpoints were cure rates (CRs) and egg reduction rates (ERRs) based on geometric mean and safety. Each subject was asked to provide two stool samples (S. mansoni trial) for Kato-Katz analysis or three urine samples (S. haematobium trial) for urine filtration and one finger prick for malaria screening at baseline and follow-up. Participants were randomly assigned to either moxidectin, Synriam, Synriam plus praziquantel or praziquantel. Principal Findings 128 adolescents (age: 12–17 years) were included in each study. Against S. haematobium moxidectin and Synriam revealed low efficacy. On the other hand, Synriam plus praziquantel and praziquantel yielded CRs of 60.0% and 38.5% and ERRs of 96.0% and 93.5%, respectively. CRs observed in the treatment of S. mansoni were 13.0%, 6.7%, 27.0%, and 27.6% for moxidectin, Synriam, Synriam plus praziquantel and praziquantel, respectively. ERRs ranged from 64.9% (Synriam) to 87.5% (praziquantel). Conclusion/Significance Synriam and moxidectin show low efficacy against S. haematobium, hence an ancillary benefit is not expected when these drugs are used for treating onchocerciasis and malaria in co-endemic settings. Further studies are needed to corroborate our findings that moxidectin and Synriam show moderate ERRs against S. mansoni.

Human Trichuriasis: Diagnostics Update

Trichuris trichiura infection is part of the so-called neglected tropical diseases, given the little interest and resources spent in developing novel diagnostic tools and treatment to detect and fight this disease. One of the main neglected aspects of trichuriasis pertains to diagnostic methods, which are currently based on copro-parasitological methods and burdened by low sensitivity. This leads to different levels of underestimation of the real prevalence and morbidity caused by T. trichiura, in both public health and individual patient management. Only few new diagnostic methods showing good performance and affordability have been standardized and fine-tuned. Molecular-based diagnostics such as those based on the polymerase chain reaction are the main diagnostic tool for infectious diseases nowadays, but their use in parasitology is still limited owing to their high cost making them unaffordable in many countries where T. trichiura is endemic. Research and innovation are needed in order to develop new and accurate methods that are accessible for the diagnosis of this parasite and of other soil-transmitted helminths.