Introduction

Infections with the soil-transmitted helminths (STHs) Trichuris trichiura, Ascaris lumbricoides, and hookworm affect more than 800 million people worldwide [1], among which almost 500 million are infected with T. trichiura. About one billion school-aged children live in countries where the prevalence of STHs is above 20 %, hence at risk of acquiring an infection [1]. However, prevalence of STHs is likely to be still widely underestimated, mainly because sensitive, reliable, and accurate diagnostic methods are lacking [2••]. STH infections are part of the so-called neglected tropical diseases (NTDs), with a burden of disease of more than 5 million disability-adjusted life years (DALYs) in 2010. Of these, 0.64 million were attributable to T. trichiura [3]. Moreover, the number of years lived with disability (YLD) attributable to T. trichiura infection has been estimated as 13 % of the total burden of STHs in 2010 [1].

There is little interest to invest in novel drugs and diagnostics for NTDs as these diseases, notwithstanding their wide distribution, hit mainly poor populations of middle-low income countries where there is no lucrative market. Between 2007 and 2013, US$22,418,733,255 was spent globally for research and drug development, of which US$6,222,526 (<0.03 %) was used for T. trichiura [4]. Data show that between 2000 and 2011, 37 out of 850 new therapeutic products (formulations, molecules, vaccines, and biological) were for NTDs, but only 1 % was actually approved [5]. The lack in research and development for novel, innovative therapeutic options is flanked by scarceness in new diagnostic tools. However, diagnostic accuracy is crucial since reliable data on prevalence and intensity of infection are needed for planning future interventions. Moreover, accurate diagnostic tools are essential to assess and monitor the efficacy of treatment [68]. Due to the absence of a standardized procedure, clinical trials on drug efficacy in endemic countries have been conducted utilizing a variety of parasitological methods, including molecular techniques, to detect egg reduction rate (ERR) and cure rate (CR) in T. trichiura-infected children [6, 9•, 10•, 11•]. The choice of the most appropriate diagnostic methods must take into account the accuracy of the method, the endemicity of T. trichiura infections, and the availability of resources including the cost of the diagnostics. In areas where STHs are endemic—mainly settings with limited resources—techniques with moderate sensitivity, like Kato-Katz, could be used for quantitative and qualitative diagnosis. On the other hand, in areas of low endemicity and those where control strategies approach to elimination and a robust surveillance system should be built up, more sensitive and specific tools are required moving from good-old direct copro-parasitological techniques toward more sophisticated albeit more expensive molecular or serological diagnostics [12••, 13, 14•]. Although of great importance, public health is not the only drive for research: Also individual patient management requires improved diagnostics. In addition, standard operating procedures to monitor drug efficacy and early detection of drug resistance need sensitive diagnostics to alert when drug-resistant strains are still below the threshold of clinical treatment failures [15].

The aim of this review is to present the state of art of the diagnostic tools currently applied for detection of T. trichiura with a special focus on recently developed techniques. We discuss different approaches for diagnosis of T. trichiura infection, highlighting innovations and new fields of research.

Current Diagnostic Methods Applied for T. Trichiura Infection

One of the first evaluation of quantitative diagnosis of a Trichuris infection before and after treatment measuring egg counts and worm burden dates back in 1950 [16], and still at present the diagnosis of intestinal helminth infections relies mainly on direct examination on the presence of eggs in stool samples in a quantitative or qualitative manner. Quantitative methods have the advantage to record the number of eggs per gram (EPG) of feces, a proxy measure of intensity of infection, which is an indicator directly associated with helminth morbidity.

It is worth highlighting that STHs do not release eggs at a constant rate [17]; therefore, multiple collection of fecal samples are needed to increase the sensitivity of the methods. As stated in a recent meta-analysis on sampling and its diagnostic power for STHs and schistosomes, however, oversampling has no benefit in terms of diagnostic sensitivity, workload, and costs. Collection of multiple samples is important to better estimate prevalence and cure rates, but not for accurately calculating infection intensity and ERR. [18•]. Nikolay et al. [2••] demonstrated that preparing and analyzing more than two Kato-Kato slides from one stool sample only slightly increases the sensitivity for T. trichiura detection (84.8 % two slides vs 90.5 % three slides).

Qualitative direct copro-parasitological techniques include the direct smear and formol-ether concentration techniques (Fig. 1) [19••, 20•, 21]. Used in parasitology since the 1970s, the former consists of microscopic examination of roughly 2 mg of fresh samples on a slide on the same day of collection [22]. The formol-ether concentration method (FECM) requires processing of 1–2 g of the sample by adding formol (10 % formaldehyde), ether, and a centrifugation step before analyzing under the microscope a few drops of the sample pellet on a slide [21] (Fig. 1). Both techniques are not quantitative, as the amount of sample is not weighed but roughly calculated, and the dilution factor is unknown. Hence, neither method is suitable for EPG calculation. As well, direct smear and FECM are both hindered by low-moderate sensitivity [20•, 23•] and, moreover, being qualitative techniques, their adoption in clinical trials to test drug efficacy and in epidemiological studies is far from ideal, yet still widely used [24, 25].

Fig. 1
figure 1

Diagnostic techniques for detection of T. trichiura. (1) Direct method; (2) formol-ether concentration method; (3) Kato-Katz thick smear; (4) McMaster method; (5) Mini-FLOTAC method; (6) FekPac technique

Another qualitative method used for STH diagnosis is the sedimentation technique after fixation with sodium acetate-acetic acid-formalin (SAF) [26]. Some authors compared SAF sedimentation with standard sedimentation technique. For the former, 2 g of feces was deposited in containers and diluted with 7 ml of SAF and set for spontaneous sedimentation; after 30 min, the sediment was placed on a slide and read under the microscope. Standard sedimentation is performed using tap water instead of SAF and letting the sample rest for 2 h instead of 30 min. The two methods showed good concordance for STHs (0.7–0.8) except for T. trichiura (0.3), apparently due to mistakes in homogenization [26].

These three methods (direct thick smear, FECM, and sedimentation), despite not being quantitative, allow a broader spectrum of diagnosis of the parasites potentially present in the sample. In fact, both protozoa cysts and helminth eggs as well as nematode larvae could be detected during the same examination.

More accurate diagnosis for T. trichiura eggs is achieved by quantitative methods: Kato-Katz thick smear method, McMaster method, FLOTAC and Mini-FLOTAC techniques (Fig. 1), and bio-molecular methods.

In more detail, Kato-Katz diagnostic method has been widely used in STH (including T. trichiura) public health and drug efficacy studies because (i) it has sufficient sensitivity in areas of high/moderate endemicity, (ii) it is relatively simple, (iii) most of the materials used can be recycled and utilized for several examinations, (iv) it is currently the recommended technique by the World Health Organization [19••]. This method is performed with fresh stools and can be briefly described as follows. First, a small amount of the sample is sieved and collected into a template based on a slide. The template contains a fixed amount of sample (41.7 mg). Subsequently, the sample is stained by pressing a layer of cellophane (which has been soaked overnight in a solution of malachite green and glycerol) on the small amount collected on the slide and exposing it to the dye for at least 30 min. The rationale of the method is that glycerol clears the fecal material while the malachite green stains the background but cannot penetrate the parasite wall, making the detection of eggs easier and clearer.

The McMaster technique is among the currently recommended direct methods for intestinal parasitic infection diagnosis [27••]. It is widely used in veterinary medicine and is now used in human parasitology as well [11•, 28•, 29]. This method is based on flotation of the eggs. In brief, 3 g of stool is filtered and diluted (1:30) with hypersaturated saline solution; the two flotation chambers of the McMaster slide are filled with the solution and after a few minutes are read under the microscope. Hypersaturated saline allows the eggs to float and separate from debris (Fig. 1).

More than 200 studies have been conducted over the past 5 years to compare the different techniques for T. trichiura diagnosis. The unstandardized use of different techniques is due to the lack of a gold standard for diagnosis of intestinal helminth infection, including T. trichiura [28•, 29, 30]. Sayasone et al. [21] recently carried out a comparison between FECM and triplicate Kato-Katz and demonstrated that for detection of T. trichiura infection, FECM is less sensitive than Kato-Katz (73 vs 95.9 %, respectively). These data were also confirmed by Funk et al. [31], who found a better performance of Kato-Katz when compared to methods like formol-ethyl acetate concentration and Midi Parasep. Levecke et al. [28•] conducted a comparison between Kato-Katz and McMaster in the diagnosis of T. trichiura and found a slightly better although not statistically significant performance of Kato-Katz.

One of the greater limitations of direct techniques is that most require fresh stool samples, which is problematic in large studies. Alfredo Fernandez et al. [32] recently compared the diagnostic performance of Kato-Katz method on fresh versus preserved samples after 6 months of SAF fixation. The prevalence of T. trichiura in preserved samples was statistically decreased (p < 0.01) compared to the fresh samples [32]. Hence, the need of a diagnostic method, which allows stool preservation, is crucial.

FLOTAC techniques are innovative methods imported from veterinary parasitology and based on flotation of the eggs with different flotation solutions that allow the separation between eggs and fecal debris [33•]. FLOTAC techniques have been tested and compared with the above mentioned methods and found to be more sensitive [2••, 10•], but few limitations have been highlighted. First of all, the procedure is fairly complex and long and, just as the FECM, requires a centrifugation step, and therefore constant power supply and a large centrifuge, which are not always available in low-resource settings [10•]. Second, due to flotation solutions limits, some debris may float together with the eggs, making the egg count difficult to perform; this could be avoided by adding a further step of clarification through ether [10•, 33•]. An advantage of the FLOTAC techniques, however, is that they can be performed on fixed samples, allowing the storage of faces for a few weeks [33•].

Mini-FLOTAC is an evolution of FLOTAC designed to be a simpler and more appropriate method to be used in resource-limited settings, and proved to be a good alternative to Kato-Katz and other direct methods (like McMaster and formol-ether concentration method) being both a quantitative and low-cost method (Fig. 1).

A recent study comparing different diagnostic tools for STH infections found a similar sensitivity for Kato-Katz and Mini-FLOTAC for T. trichiura detection [34•]. Mini-FLOTAC is a promising alternative to currently used methods, but, because its sensitivity is similar to the that of other methods, it has not yet replaced older approaches nor fully innovated the diagnostic scenario. However, in contrast to Kato-Katz, Mini-FLOTAC uses a closed system and is therefore safer for the operator, as it does not require manipulation of fresh sample and avoids the risk of contamination [34•]. In addition, fixation with 5 % formalin allows reading of the sample after at least 2 weeks after collection (even up to 30 days or longer for T. trichiura) with stable egg counts compared with the first examination [35•]. Preservation of samples might be helpful for field studies, including prevalence and epidemiological surveys and clinical trials, and for diagnosing STHs during Transmission Assessment Surveys for Lymphatic Filariasis [36, 37] where large amounts of stool samples cannot be screened immediately.

The above mentioned techniques are widely used in endemic countries for public health trials and single-patient management and diagnosis. Kato-Katz, Mc Master, and recently Mini-FLOTAC methods are also recommended in a standard protocol from WHO to monitor drug efficacy according to agreed thresholds of ERR [27••]. The main characteristics of these techniques together with some new highlights are summarized in Table 1.

Table 1 Main characteristics of different diagnostic techniques for detection of T. trichiura

When used during clinical trials, it is a good recommended practice that a subsample of the slides is randomly selected for quality control and re-examined by a different experienced laboratory technician, in order to guarantee high standard quality of the results [27••]. Around 10 % of all the slides read with any copro-parasitological method should be re-read; if the discordance found between the two readings is greater than 10 %, a third slide should be prepared and read by an independent expert. If still discordant, corrective measures should be taken (closer supervision and further training), and in the worst case, all samples should be re-analyzed. Speich et al. [38•] made a systematic review of quality control procedure of Kato-Katz slides in clinical trials conducted in Pemba Island, Tanzania, during the past 3 years. The false positive rate for T. trichiura was low (0.35 %) and the false negative rate was 1.94 %.

New Highlights

Novel technologies sustain the development of innovative tools for the diagnosis of STHs, including T. trichiura. The new diagnostics evolve either from refinement of methods from veterinary parasitology based on remote diagnosis and telemedicine, or from detection of copro-antigens and DNA-based methods.

The FecPakG2 is a new diagnostic technique recently developed for veterinary medicine in New Zealand [39•, 40]. Similar to McMaster and Mini-FLOTAC, this method is based on the flotation of eggs using hypersaturated saline. Its innovation lays in the use of a high-tech device for the examination of the slide. Briefly, once the solution is mixed with the sample, a well is filled with the sample and read by a camera connected to a tablet with internet, which sends the image to expert parasitologists. Once the experts have analyzed the picture of the sample, they return the result to the farmer or to the technician who processed the sample and took the picture. As long as there is internet connectivity, this method might be helpful especially in remote areas, where resources are limited, equipped laboratories are not easy to reach, or trained technicians are not available. The diagnostic technique is still under development so no conclusion can yet be drawn as to whether FecPakG2 is a suitable addition to the diagnostic tools for human STH infections.

On the same track of introducing electronic devices into direct diagnosis for T. trichiura is the mobile diagnosis, which is lately gaining interest among different disciplines [4143]. Bogoch et al. [44] used a smartphone as microscope and compared the performance of mobile and standard microscopy. Their results showed a good performance for A. lumbricoides and T. trichiura detection, but low sensitivity for hookworm. Moreover, images were not of good quality and often light infections were missed [44]. Bogoch and colleagues also tested a handheld light microscope for the diagnosis of STHs in comparison with standard microscope. They found that the new approach had not a better performance, showing sensitivity for all STHs of 69.4 versus 86.9 % of conventional microscopy; sensitivity for T. trichiura detection was 54.4 versus 79.4 % with conventional microscopy [45]. However, this approach worth improvement and further evaluation, as it matches new insights and technology with classical direct methods for parasitological diagnosis.

New attempts are made in order to render the stool analysis easier and user-friendly in large-scale examinations for public health purposes. Among them, many laboratories are now exploring the way of pooling samples. This approach is widely used in veterinary practice and consists of mixing a fixed weight (1 g each) of 10–60 samples together before analysis. This strategy has proved time- and cost-effective and showed a good correlation between pooled and individual egg counts for hookworm and T. trichiura infection intensity although not for A. lumbricoides egg counts [46•]. Rinaldi et al. [47] compared two diagnostic methods in the veterinary field (Mini-FLOTAC vs McMaster techniques) on pooled samples for gastrointestinal strongyles and found that pooling was positively correlated with single-sample diagnosis both at baseline and at follow-up after treatment to assess ERR.

Helminth copro-antigen detection has been tested in veterinary medicine as well, but not in human diagnostics so far. Briefly, ELISA tests were performed on experimentally infected fecal samples with Trichuris vulpis eggs yielding good results compared with flotation methods. Specificity and sensitivity were acceptable compared to other ELISA assay used for intestinal infections diagnosis [48]. Further studies are needed before immunoassays can be recommended for the diagnosis of T. trichiura.

Molecular biology could be the new frontier of intestinal parasite diagnosis. In the past decade, the application of polymerase chain reaction (PCR) has exponentially increased, underlying its potentials in the diagnostic field [49, 50]. Being adopted mainly in human virology and bacteriology, its use has been recently extended to human parasitology [51••, 52] and even for diagnosis of T. trichiura [53]. This technique has many advantages compared to direct microscopic methods used so far: It allows a more sensitive detection of the parasite, especially at low intensity of infection; it permits to differentiate between Trichuris species with eggs of very similar morphology [53]; and it appears to have a good correlation with fecal egg counts [51••, 54•]. It is worth to underline that the amount of DNA needed to run the test is small; therefore, the collection of stool samples is not dependent on its weight, which could be a limitation when multiple direct methods are performed. Moreover, the multiplex assay allows the detection of several parasites (such as Strongyloides stercoralis, other helminthes, and protozoa) within the same test, which is a great advantage in settings where multiparasitism occurs, and their epidemiology is not clearly known. Few data are available on post-treatment reliability of PCR detection. A large study conducted with 125 children in Ecuador demonstrated that detection of A. lumbricoides DNA was negative 21 days after treatment with albendazole [54•]. Moreover, with DNA being a stable molecule, isolates could be stored for many years after collection, a crucial step forward in case further analyses are needed, such as the research of different pathogens or drug resistance.

Although PCR method is usually not required for T. trichiura diagnosis (since, unlike hookworm species, these worm eggs have a peculiar shape and are easily distinguished from other nematodes’ eggs) [55], this sensitive method could be useful in low-prevalence settings where the aim is elimination of infection. Molecular methods like PCR and sequencing are promising tools not only for diagnostic purposes but also for their role in building up a robust surveillance system and for detection of drug-resistant strains. The main disadvantage of PCR is its high cost, as it requires not only expensive instruments but also constant power supply, specific reagents, and high cost maintenance.

T. trichiura is widely known to be less susceptible to current drugs than the other STHs [56]. This might be due to intrinsic tolerance or acquired parasitic resistance, triggered by overtreatment and the misuse of anthelminthic drugs. Strong monitoring and early detection of drug resistance rely on the development of sensitive and stable primers in order to detect resistance alleles at a threshold that is still manageable and reversible [15]. In this perspective, a few studies have been conducted on DNA sequencing in order to detect β-tubulin point mutations and single-nucleotide polymorphism (SNP), which might confer resistance to benzimidazole treatment [57••]. Hansen et al. [58] conducted a study on human and baboon’s infection performing PCR on both adults and eggs stadia of infection and did not find SNP and mutations at codon 168, 198, or 200 of the β-tubulin gene linked to resistance. These findings are in contrast with the above mentioned study by Diawara et al. [57••], who found a significant increase of frequencies of alleles with the point mutation in codon 200 in T. trichiura specimens, although drug efficacy was not assessed.

No clear pattern of resistance has been highlighted so far in human nematodes, and data are not consistent in suggesting a clear link between SNP and reduced drug efficacy. Due to this evidence, scientific research is currently running in both directions: on one side, tracking resistance markers and new and more sensitive diagnostic tools to detect T. trichiura infection, and on the other side, testing different drugs and drug combinations against T. trichiura [6, 38•, 59••].

Conclusion

From the above review on available and novel diagnostics for T. trichiura infection, it appears that much work needs to be done before reaching an optimal level for diagnosis. Even if some standardized operating procedures have been developed, sensitive yet affordable methods are still missing. Despite many different trials conducted in this area, this issue seems not yet been solved. The lack of a diagnostic gold standard and clear guidelines, along with wide variations prompted by resource limitations in diverse settings and different research objectives, not only jeopardizes coordination among researchers and laboratory personnel but also hampers comparison of findings obtained by various studies. The WHO is currently addressing this issue through the publication of a manual on standard recommended methods for diagnosis of intestinal parasites, available also on visual aids and platforms through the web. At present, efforts are being made to develop high-tech diagnostics based on sensitive molecular-based multiplex platform that could be eventually used as point-of-care tests in countries endemic for T. trichiura and other STH infections. Special attention should be given to developing sensitive diagnostic tools to monitor drug efficacy and prevent the resistance threat against the few good anthelminthic drugs available.