A general framework to support cost-efficient fecal egg count methods and study design choices for large-scale STH deworming programs-monitoring of therapeutic drug efficacy as a case study.

BACKGROUND: Soil-transmitted helminth (STH) control programs currently lack evidence-based recommendations for cost-efficient survey designs for monitoring and evaluation. Here, we present a framework to provide evidence-based recommendations, using a case study of therapeutic drug efficacy monitoring based on the examination of helminth eggs in stool. METHODS: We performed an in-depth analysis of the operational costs to process one stool sample for three diagnostic methods (Kato-Katz, Mini-FLOTAC and FECPAKG2). Next, we performed simulations to determine the probability of detecting a truly reduced therapeutic efficacy for different scenarios of STH species (Ascaris lumbricoides, Trichuris trichiura and hookworms), pre-treatment infection levels, survey design (screen and select (SS); screen, select and retest (SSR) and no selection (NS)) and number of subjects enrolled (100-5,000). Finally, we integrated the outcome of the cost assessment into the simulation study to estimate the total survey costs and determined the most cost-efficient survey design. PRINCIPAL FINDINGS: Kato-Katz allowed for both the highest sample throughput and the lowest cost per test, while FECPAKG2 required both the most laboratory time and was the most expensive. Counting of eggs accounted for 23% (FECPAKG2) or ≥80% (Kato-Katz and Mini-FLOTAC) of the total time-to-result. NS survey designs in combination with Kato-Katz were the most cost-efficient to assess therapeutic drug efficacy in all scenarios of STH species and endemicity. CONCLUSIONS/SIGNIFICANCE: We confirm that Kato-Katz is the fecal egg counting method of choice for monitoring therapeutic drug efficacy, but that the survey design currently recommended by WHO (SS) should be updated. Our generic framework, which captures laboratory time and material costs, can be used to further support cost-efficient choices for other important surveys informing STH control programs. In addition, it can be used to explore the value of alternative diagnostic techniques, like automated egg counting, which may further reduce operational costs. TRIAL REGISTRATION: ClinicalTrials.gov NCT03465488.

Identifying thresholds for classifying moderate-to-heavy soil-transmitted helminth intensity infections for FECPAKG2, McMaster, Mini-FLOTAC and qPCR.

The World Health Organization (WHO) has defined moderate-to-heavy intensity (M&HI) infections with soil-transmitted helminths (Ascaris lumbricoides, Trichuris trichiura and the two hookworms, Ancylostoma duodenale and Necator americanus) based on specific values of eggs per gram of stool, as measured by the Kato-Katz method. There are a variety of novel microscopy and DNA-based methods but it remains unclear whether applying current WHO thresholds on to these methods allows for a reliable classification of M&HI infections. We evaluated both WHO and method-specific thresholds for classifying the M&HI infections for novel microscopic (FECPAKG2, McMaster and Mini-FLOTAC) and DNA-based (qPCR) diagnostic methods. For this, we determined method-specific thresholds that best classified M&HI infections (defined by Kato-Katz and WHO thresholds; reference method) in two multi-country drug efficacy studies. Subsequently, we verified whether applying these method-specific thresholds improved the agreement in classifying M&HI infections compared to the reference method. When we applied the WHO thresholds, the new microscopic methods mainly misclassified M&HI as low intensity, and to a lesser extent low intensity infection as M&HI. For FECPAKG2, applying the method-specific thresholds significantly improved the agreement for Ascaris (moderate → substantial), Trichuris and hookworms (fair → moderate). For Mini-FLOTAC, a significantly improved agreement was observed for hookworms only (fair → moderate). For the other STHs, the agreement was almost perfect and remained unchanged. For McMaster, the method-specific thresholds revealed a fair to a substantial agreement but did not significantly improve the agreement. For qPCR, the method-specific thresholds based on genome equivalents per ml of DNA moderately agreed with the reference method for hookworm and Trichuris infections. For Ascaris, there was a substantial agreement. We defined method-specific thresholds that improved the classification of M&HI infections. Validation studies are required before they can be recommended for general use in assessing M&HI infections in programmatic settings.

Diagnostic performance of a single and duplicate Kato-Katz, Mini-FLOTAC, FECPAKG2 and qPCR for the detection and quantification of soil-transmitted helminths in three endemic countries.

BACKGROUND: Because the success of deworming programs targeting soil-transmitted helminths (STHs) is evaluated through the periodically assessment of prevalence and infection intensities, the use of the correct diagnostic method is of utmost importance. The STH community has recently published for each phase of a deworming program the minimal criteria that a potential diagnostic method needs to meet, the so-called target product profiles (TPPs). METHODOLOGY: We compared the diagnostic performance of a single Kato-Katz (reference method) with that of other microscopy-based methods (duplicate Kato-Katz, Mini-FLOTAC and FECPAKG2) and one DNA-based method (qPCR) for the detection and quantification of STH infections in three drug efficacy trials in Ethiopia, Lao PDR, and Tanzania. Furthermore, we evaluated a selection of minimal diagnostic criteria of the TPPs. PRINCIPAL FINDINGS: All diagnostic methods showed a clinical sensitivity of ≥90% for all STH infections of moderate-to-heavy intensities. For infections of very low intensity, only qPCR resulted in a sensitivity that was superior to a single Kato-Katz for all STHs. Compared to the reference method, both Mini-FLOTAC and FECPAKG2 resulted in significantly lower fecal egg counts for some STHs, leading to a substantial underestimation of the infection intensity. For qPCR, there was a positive significant correlation between the egg counts of a single Kato-Katz and the DNA concentration. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the diagnostic performance of a single Kato-Katz is underestimated by the community and that diagnostic specific thresholds to classify intensity of infection are warranted for Mini-FLOTAC, FECPAKG2 and qPCR. When we strictly apply the TPPs, Kato-Katz is the only microscopy-based method that meets the minimal diagnostic criteria for application in the planning, monitoring and evaluation phase of an STH program. qPCR is the only method that could be considered in the phase that aims to seek confirmation for cessation of program. TRIAL REGISTRATION: ClinicalTrials.gov NCT03465488.

Therapeutic efficacy of albendazole against soil-transmitted helminthiasis in children measured by five diagnostic methods.

BACKGROUND: Preventive chemotherapy (PC) with benzimidazole drugs is the backbone of soil-transmitted helminth (STH) control programs. Over the past decade, drug coverage has increased and with it, the possibility of developing anthelmintic resistance. It is therefore of utmost importance to monitor drug efficacy. Currently, a variety of novel diagnostic methods are available, but it remains unclear whether they can be used to monitor drug efficacy. In this study, we compared the efficacy of albendazole (ALB) measured by different diagnostic methods in a head-to-head comparison to the recommended single Kato-Katz. METHODS: An ALB efficacy trial was performed in 3 different STH-endemic countries (Ethiopia, Lao PDR and Tanzania), each with a different PC-history. During these trials, stool samples were evaluated with Kato-Katz (single and duplicate), Mini-FLOTAC, FECPAKG2, and qPCR. The reduction rate in mean eggs per gram of stool (ERR) and mean genome equivalents / ml of DNA extract (GERR) were calculated to estimate drug efficacy. PRINCIPAL FINDINGS AND CONCLUSIONS: The results of the efficacy trials showed that none of the evaluated diagnostic methods could provide reduction rates that were equivalent to a single Kato-Katz for all STH. However, despite differences in clinical sensitivity and egg counts, they agreed in classifying efficacy according to World Health Organization (WHO) guidelines. This demonstrates that diagnostic methods for assessing drug efficacy should be validated with their intended-use in mind and that other factors like user-friendliness and costs will likely be important factors in driving the choice of diagnostics. In addition, ALB efficacy against STH infections was lower in sites with a longer history of PC. Yet, further research is needed to identify factors that contribute to this finding and to verify whether reduced efficacy can be associated with mutations in the ß-tubulin gene that have previously been linked to anthelmintic resistance. TRIAL REGISTRATION: ClinicalTrials.gov NCT03465488.

Comprehensive evaluation of stool-based diagnostic methods and benzimidazole resistance markers to assess drug efficacy and detect the emergence of anthelmintic resistance: A Starworms study protocol.

BACKGROUND: To work towards reaching the WHO goal of eliminating soil-transmitted helminth (STH) infections as a public health problem, the total number of children receiving anthelmintic drugs has strongly increased over the past few years. However, as drug pressure levels rise, the development of anthelmintic drug resistance (AR) is more and more likely to appear. Currently, any global surveillance system to monitor drug efficacy and the emergence of possible AR is lacking. Consequently, it remains unclear to what extent the efficacy of drugs may have dropped and whether AR is already present. The overall aim of this study is to recommend the best diagnostic methods to monitor drug efficacy and molecular markers to assess the emergence of AR in STH control programs. METHODS: A series of drug efficacy trials will be performed in four STH endemic countries with varying drug pressure (Ethiopia and Brazil: low drug pressure, Lao PDR: moderate drug pressure and Tanzania: high drug pressure). These trials are designed to assess the efficacy of a single oral dose of 400 mg albendazole (ALB) against STH infections in school-aged children (SAC) by microscopic (duplicate Kato-Katz thick smear, Mini-FLOTAC and FECPAKG2) and molecular stool-based diagnostic methods (quantitative PCR (qPCR)). Data will be collected on the cost of the materials used, as well as the time required to prepare and examine stool samples for the different diagnostic methods. Following qPCR, DNA samples will also be submitted for pyrosequencing to assess the presence and prevalence of single nucleotide polymorphisms (SNPs) in the ß-tubulin gene. These SNPs are known to be linked to AR in animal STHs. DISCUSSION: The results obtained by these trials will provide robust evidence regarding the cost-efficiency and diagnostic performance of the different stool-based diagnostic methods for the assessment of drug efficacy in control programs. The assessment of associations between the frequency of SNPs in the ß-tubulin gene and the history of drug pressure and drug efficacy will allow the validation of these SNPs as a marker for AR in human STHs. TRIAL REGISTRATION: The trial was retrospectively registered the 7th of March 2018 on Clinicaltrials.gov (ID: NCT03465488).

The Mini-FLOTAC technique for the diagnosis of helminth and protozoan infections in humans and animals.

This protocol is an extension to: Nat. Protoc. 5, 503-515 (2010); doi: 10.1038/nprot.2009.235; published online 25 February 2010The FLOTAC is a sensitive, accurate, and precise technique for the diagnosis of protozoan and helminth infections in humans and animals. However, it requires centrifugation, and hence might be out of reach in resource-constrained settings. As an extension of the original FLOTAC protocol, this protocol describes the Mini-FLOTAC technique, a logical evolution of FLOTAC conceived to perform multivalent, qualitative, and quantitative diagnosis of helminth and protozoan infections in human and animal feces, and urine. This has been found to be of most use in the processing of large numbers of samples with rapid laboratory workup, and for veterinary applications directly on-farm. In addition to the Mini-FLOTAC apparatus, we describe the use of the Fill-FLOTAC, a closed system used to facilitate the performance of the first four consecutive steps of the Mini-FLOTAC technique: fecal sample collection and weighing, homogenization, filtration, and filling of the Mini-FLOTAC chambers. Processing of an individual sample using this protocol requires ∼12 min.

The comparison of FLOTAC, FECPAK and McMaster techniques for nematode egg counts in cattle.

Three methods, FLOTAC, FECPAK and McMaster were compared for accuracy and sensitivity for counting numbers of nematode eggs in faeces of naturally infected cattle with high or low nematode egg counts. Only FLOTAC gave positive results for 12 replicates from pooled samples with low egg counts making it more sensitive than FECPAK (67%) and McMaster (41.7%). FLOTAC resulted in generally higher egg counts and lower coefficients of variation than the other two methods used. The reliability of FECPAK and McMaster is depended on the area under the slide counted. All three methods can be used for making decisions whether to treat but FLOTAC or Mini-FLOTAC should be used for faecal egg count reduction tests when lower egg counts are present.

Mini-FLOTAC, a new tool for copromicroscopic diagnosis of common intestinal nematodes in dogs.

BACKGROUND: The most common intestinal nematodes of dogs are Toxocara canis, hookworm and Trichuris vulpis. The present study was aimed to validate a new copromicroscopic technique, the Mini-FLOTAC and to compare its diagnostic efficiency and sensitivity with four other copromicroscopic techniques: direct smear, tube flotation, Wisconsin method and the FLOTAC dual technique. FINDINGS: Two experiments were performed. In the first, faecal positive samples collected from 59 stray asymptomatic dogs, of which 21 were naturally infected with ancylostomidae, 13 naturally infected with T. canis and 25 naturally infected with T. vulpis were used to validate the Mini-FLOTAC technique. The second experiment was performed on faecal samples randomly selected from 38 stray asymptomatic dogs to compare the diagnostic efficiency and sensitivity of the different techniques. Samples were fixed with 5% formalin; sodium chloride and zinc sulphate were used for flotation solutions because they performed best for detecting and quantifying intestinal nematode eggs in dogs. Mini-FLOTAC and FLOTAC were the most efficient and sensitive techniques and they gave higher EPG and higher numbers of positive samples in both the experiments, for all three parasites. CONCLUSIONS: As Mini-FLOTAC does not require centrifugation it is a very promising technique for counting helminth eggs in dog faeces.

Comparison of three copromicroscopic methods to assess albendazole efficacy against soil-transmitted helminth infections in school-aged children on Pemba Island.

BACKGROUND: The diagnostic accuracy of three faecal egg count techniques (Kato-Katz, McMaster and FLOTAC) to assess albendazole efficacy against soil-transmitted helminth (STH) infections was compared. METHODS: The study is registered with Current Controlled Trials [identifier: ISRCTN90088840]. During September-November 2009, 304 school-aged children on Pemba Island, Tanzania, were screened and those infected with Ascaris lumbricoides, hookworm or Trichuris trichiura were treated with a single dose of albendazole (400 mg). Twenty-one days post-treatment, children provided a single stool sample which was examined using the same diagnostic methods. All stool samples were divided into two aliquots and one was fixed in 5% formalin and examined using FLOTAC and McMaster approximately 6 months after collection. RESULTS: Using fresh stool samples, comparable prevalences were demonstrated for the three methods at baseline (90-92.2% for T. trichiura, 41.1-52.8% for hookworm, 32.9-37.2% for A. lumbricoides); FLOTAC was the most sensitive method at baseline and follow-up. Albendazole showed high cure rate (CR) against A. lumbricoides (90-97%), moderate CR against hookworm (63-72%) and very low CR against T. trichiura (6-9%), regardless of the technique used. Egg counts (eggs per gram) at baseline were similar for A. lumbricoides and for hookworm among the three methods, and higher using McMaster and Kato-Katz compared with FLOTAC for T. trichiura. All methods were similar for hookworm and A. lumbricoides egg reduction rate (ERR) estimation, but Kato-Katz indicated a significantly higher ERR than McMaster and FLOTAC for T. trichiura. Preserved stool samples revealed consistently lower FECs at baseline and follow-up for all STHs. CONCLUSION: Further development and validation of standard protocols for anthelminthic drug efficacy evaluation must be pursued.

FLOTAC for the diagnosis of Hymenolepis spp. infection: proof-of-concept and comparing diagnostic accuracy with other methods.

Hymenolepis nana is the most common cestode parasitizing humans, yet it is under-diagnosed. We determined the optimal flotation solution (FS) for the diagnosis of this intestinal parasite with the FLOTAC method, and compared its diagnostic accuracy with an ether-concentration technique and the Kato-Katz method. Zinc sulphate (specific gravity 1.20) proved to be the best-performing FS. Using this FS, we detected 65 H. nana infections among 234 fixed fecal samples from Tajik and Sahrawi children (prevalence 27.8 %). The ether-concentration technique detected 40 infections (prevalence 17.1 %) in the same samples. Considering the combined results as a reference, the sensitivities of FLOTAC and ether-concentration were 95.6 % and 58.8 %, respectively. The Kato-Katz method resulted in a prevalence of only 8.7 %. In terms of eggs per gram of stool, a significantly (P <0.05) higher value was obtained with the FLOTAC and Kato-Katz techniques compared to ether-concentration. In another study carried out in China, the FLOTAC method detected six Hymenolepis diminuta infections in 302 fecal samples, whereas five samples were found positive with the Kato-Katz technique. We conclude that FLOTAC is an accurate coprodiagnostic technique for H. nana and H. diminuta, two species which join a growing list of intestinal parasites that can be reliably diagnosed by this technique.

Comparison of faecal techniques including FLOTAC for copromicroscopic detection of first stage larvae of Angiostrongylus vasorum.

Angiostrongylus vasorum is a metastrongylid nematode that resides in the pulmonary arteries and the right heart chambers. In dogs, infection results in respiratory, bleeding and neurological disorders and further clinical signs. In the present study, FLOTAC was evaluated for the detection of first-stage larvae (L1) of A. vasorum in canine faecal samples. This technique is based on the counting of parasitic stages (eggs, larvae, oocysts and cysts) in chambers after spinning of faecal samples onto a surface. In a first step, nine flotation solutions were evaluated using faeces of two experimentally infected dogs. Zinc sulphate (specific gravity (s.g.) 1.2) and zinc sulphate plus potassium iodomercurate (s.g. 1.45) gave good results. However, with the latter technique, the larvae were slightly deformed. Subsequently, FLOTAC, using zinc sulphate, was compared through a randomisation technique with McMaster, flotation in tube and Baermann-Wetzel technique. The mean larvae per gramme (LPG) obtained by the FLOTAC for both dogs was significantly higher (P < 0.05) than those obtained by the other three techniques (the means of the other techniques all lie below the 95% CI of the mean LPG of the FLOTAC technique). In addition, the FLOTAC results were consistent across replicates with only Poisson (or random) variation between individual replicates. The other techniques appear to be less consistent with evidence of extra-Poisson variation in at least one of the two dogs across the replicates within each technique. The FLOTAC technique may contribute to an improvement of the ability to diagnose canine lungworm infections and represent a valuable alternative for larval counting of A. vasorum in faecal samples, especially following transport or storage where there may be limited larvae viability, and larval migration techniques cannot be used.

Fasciola hepatica: comparison of the sedimentation and FLOTAC techniques for the detection and quantification of faecal egg counts in rats.

We compared the sedimentation and FLOTAC techniques for the detection and quantification of Fasciola hepatica eggs in faecal samples obtained from 120 experimentally-infected rats before intervention, and in 42 rats after drug administration. Additionally, the average time for a single test was determined. A single FLOTAC showed a higher sensitivity (92.6%) than 2, 4 and 8 sedimentation readings (63.0-85.2%) for detecting F. hepatica eggs in rat faeces post-treatment. On average, it took 21 min to prepare and examine a single FLOTAC, whereas 114 min were needed for the sedimentation method including the reading of 8 slides. In both treated and untreated rats, the sedimentation method resulted in higher mean faecal egg counts (FECs) than FLOTAC (P<0.05). In view of the high sensitivity and efficiency, the FLOTAC technique holds promise for experimental work in the F. hepatica-rat model. Additional research is needed to determine the reasons for the observed differences in FECs.

A single FLOTAC is more sensitive than triplicate Kato-Katz for the diagnosis of low-intensity soil-transmitted helminth infections.

Accurate diagnostic tools are pivotal for patient management and surveillance of helminth control programmes, particularly in the current era of preventive chemotherapy. Three consecutive stool samples were obtained from 279 schoolchildren from Zanzibar, an island where anthelminthic drugs have been administered on a large scale for more than a decade. All stool samples were examined with the Kato-Katz method. Additionally, one sample per child was preserved in sodium acetate-acetic acid-formalin solution, and examined with the FLOTAC technique. Considering the pooled results of both methods as diagnostic 'gold' standard, the observed prevalences of Trichuris trichiura, hookworm and Ascaris lumbricoides were 63.4, 35.8 and 22.9%, respectively. The sensitivity of examining a single stool sample by FLOTAC for diagnosing T. trichiura, hookworm and A. lumbricoides was 88.7, 83.0 and 82.8%, respectively. Lower sensitivities were observed for Kato-Katz even after examining three stool samples: 71.8, 46.0 and 70.3%, respectively. Kato-Katz revealed considerably higher infection intensities than FLOTAC. The kappa agreement between a single FLOTAC and triplicate Kato-Katz was 0.63 for diagnosing A. lumbricoides and 0.50 for T. trichiura, but only 0.30 for hookworm. The high sensitivity of FLOTAC holds promise for patient management, monitoring soil-transmitted helminth transmission and endpoint(s) of control at the population level.