|
 
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| REVIEW ARTICLE |
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| Year : 2014 | Volume
: 7
| Issue : 1 | Page : 5-12 |
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Obstacles in the diagnosis of Strongyloides stercoralis
Ashraf S Metwally, Sara A Abdel-Rahman
Department of Medical Parasitology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
| Date of Submission | 10-Sep-2013 |
| Date of Acceptance | 02-Jun-2014 |
| Date of Web Publication | 25-Sep-2014 |
Correspondence Address:
Sara A Abdel-Rahman
MD, Department of Medical Parasitology, Faculty of Medicine, Zagazig University
Egypt
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/1687-7942.139685
Infection by strongyloides stercoralis is currently recognized as a worldwide health problem, particularly in developing countries. Many cases of S. stercoralis infection may be over-looked if only diagnostic conventional methods are used. Additionally, the failure to detect larvae in a single stool sample does not necessarily indicate the absence of infection due to irregular output of the larvae. Thus, repeated examinations of feces using more sensitive parasitological methods are highly required to confi rm diagnosis. Although, strongyloidiasis is mostly an asymptomatic infection but it is important to detect latent S. stercoralis infection to avoid the possible complications which may occur after administration of immunosuppressive drugs especially for patients in endemic areas who are at risk. Up to date a specifi c and sensitive diagnostic test is lacking. Keywords: autoinfection, diagnosis, filariform larvae, larva currens, Strongyloides stercoralis, strongyloidiasis
How to cite this article:
Metwally AS, Abdel-Rahman SA. Obstacles in the diagnosis of Strongyloides stercoralis
. Parasitol United J 2014;7:5-12 |
| Introduction | |  |
Strongyloidiasis is an intestinal infection in humans caused by the nematode Strongyloides stercoralis. It is distributed in the tropical and subtropical regions where the warm climates are suitable for parasite survival [1],[2] . The parasite was first reported in 1876 in French soldiers working in Vietnam. It took nearly 50 years for the complete elucidation of the complex life cycle after the discovery of the parasite [3] because of the rare and characteristic feature of autoinfection that occurs in its life cycle. As cited by Concha et al. [1] , strongyloidiasis was first described in 1926 by Fulleborn. The first reports of disseminated infection or hyperinfection date back to 1966 when two studies independently documented the occurrence of fatal strongyloidiasis associated with immunosuppression [4],[5] .
The infection is acquired by walking barefooted on infected soil. Filariform larvae can penetrate the human skin, travel to the bloodstream, and reach the lung. After ascending the tracheobronchial tree, they arrive in the small intestine, where they evolve to adult stages, and fertilized females begin oviposition in the intestinal wall. Rhabditoid larvae emerging from these eggs may differentiate into infectious L3 stage after a few days in the environment or develop into autoinfective filariform stage (aL3) in the host intestine, the latter being able to penetrate through the bowel mucosa or perianal skin overinfecting the host [1],[6] .
Because of the ability of the parasite to replicate within the host (as a result of its unique autoinfective cycle), it causes a chronic condition that may be lifelong. A variety of clinical presentations may occur, varying from asymptomatic patients who are the majority to a condition of hyperinfection with potentially life-threatening dissemination of larvae in immunocompromised patients [7] .
Strongyloidiasis is one of the most difficult parasitic diseases to diagnose because of a general absence of distinctive ova in stool specimens, a low parasite load, rarity of diagnostic rhabditiform larvae in stool, and asymptomatic presentation or nonspecific symptoms particularly in uncomplicated patients. Hence, it remains an underestimated public health problem. The current estimate of 30-100 million infected persons in the world dates back to the review articles published between 1989 and 1996 and is cited by most subsequent studies. These figures were mostly based on surveys that aimed at defining the prevalence of parasitic infections, without using adequate diagnostic techniques for S. stercoralis [8] . Although there is no gold standard for diagnosing S. stercoralis, several diagnostic methods have been compared to detect its presence, including stool examination, modified Baermann's technique, stool culture on a nutrient agar plate, enzyme-linked immunosorbent assay (ELISA), serum indirect fluorescent antibody test, PCR, and gastrointestinal aspirate or biopsy. However, all of these techniques have problems with sensitivity, specificity, or availability in endemic areas [9],[10] .
This review focuses on the obstacles confronting the diagnosis of strongyloidiasis, including confusing clinical manifestations, as well as various parasitological, serological, and molecular-based techniques. Early diagnosis of the disease ensures reduction in morbidity and mortality especially in individuals at high risk of acquiring the infection and in immunodeficient patients.
| Clinical picture | | |
Strongyloidiasis can manifest as a wide spectrum of clinical features ranging from asymptomatic disease, disease with mild initial symptoms, disease with chronic symptoms, and acute exacerbation, with hyperinfection or dissemination of larvae involving the respiratory and gastrointestinal systems or multiple organ systems, respectively. The initial symptoms occur soon after entry of the infective filariform larvae into the human host from its environmental migration sites. In normally healthy individuals, the infection is usually asymptomatic with low minimal and intermittent larval excretion. Although fatal hyperinfection or dissemination can occur, asymptomatic strongyloidiasis is the most common form of the disease [1] .
Acute manifestations
In acute infections, there is a wide spectrum of clinical features. These include cutaneous (larva currens), characterized by a migratory, serpiginous, urticarial rash [11],[12] ; pulmonary (cough and tracheal irritation); and gastrointestinal symptoms including diarrhea, abdominal discomfort, nausea, and anorexia [12],[13],[14] . Abdominal bloating is the most common complaint when malabsorption ensues. The radiograph findings are similar to those of tropical sprue, including increased diameter of the small intestine lumen, generalized hypotonia, and edema [15] . The diagnosis of acute strongyloidiasis requires a high index of suspicion, as patients present with no distinctive clinical features and ancillary laboratory imaging and endoscoping findings are often nonspecific [16] . Hence, when evaluating a patient with suspected acute strongyloidiasis, other parasitic conditions should also be considered, including acute schistosomiasis, ascariasis, amebiasis, and hookworm infections with Ancylstoma duodenale or Necator americanus [17] .
Chronic manifestations
The ability of S. stercoralis to establish a cycle of autoinfection within the host results in delayed presentation of the disease, often decades after primary infection, thus complicating the diagnosis. Chronic infections are often asymptomatic, but when symptoms occur they are usually mild, episodic, and prolonged including nausea, vomiting, diarrhea, constipation, weight loss, or cutaneous reactions [18] . Malabsorption syndrome and eosinophilia may occur, both of which may also be associated with other parasitic diseases such as ascariasis, hookworm infection, giardiasis, and schistosomiasis. In most patients, the intensity of symptoms correlates with the digestive parasitic burden [19] .
Hyperinfection syndrome
In the case of immunosuppression such as that induced by human T-cell lymphocytotropic virus type 1 (HTLV-1) infection and corticosteroid or cytotoxic chemotherapy, an uncontrolled life cycle can take place leading to the so-called hyperinfection syndrome and even to dissemination of larvae through the body, the latter being associated with a very bleak prognosis [20],[21] . In hyperinfection and dissemination, complete disruption of the mucosal patterns, ulcerations, and paralytic ileus have been observed. In the presence of dissemination, pulmonary involvement may lead to bilateral edema and patchy, often rapidly changing, infiltrates [22],[23] . Bacteremia is a common complication of hyperinfection syndrome. It is caused by the leakage of gut flora from bowels damaged by filariform larvae to the bloodstream with subsequent secretion into the host circulation [24] . The enteric bacteria are also carried by invasive L3 larvae on their outer surfaces. This can result in septicemia, pneumonia, meningitis, and disseminated bacterial infection in many parts of the body including the lungs, liver, heart, lymph nodes, gall bladder, kidneys, pancreas, and brain [25] . Petechiae and purpura have also been reported in disseminated cases as a result of larval migration through vessel walls, promoting hemorrhage [26] . Hyperinfection is usually associated with autoinfection in which the number of worms increases tremendously and becomes detectable in the extraintestinal regions especially the lungs [27],[28] . Thus, it is essential to diagnose strongyloidiasis in patients coming from endemic areas, notably those with mild or no symptomatic forms before the initiation of any kind of immunosuppressive treatment [29] .
| Diagnosis of strongyloidiasis | | |
Strongyloidiasis is suspected in patients with a classical triad of urticaria, abdominal pain, and diarrhea [26] . Although associated eosinophilia is considered indicative of the disease, the fact that it is frequently mild and nonspecific may hinder preliminary diagnosis [30] . Up to date, definitive diagnosis of strongyloidiasis is usually made on the basis of detection of larvae in the stool [19],[31] .
Parasitological diagnosis
It has been shown that a single stool examination fails to detect larvae in up to 70% of patients. Repeated examinations of stool specimens are required to improve the chances of finding the excreted forms. In some studies, diagnostic sensitivity increases to 50% with three stool examinations and can reach 100% if seven serial stool samples are examined [32],[33] , which may not be feasible in all situations. Besides direct fecal smears examination, several parasitological methods have been used to concentrate the detection of larvae in stool samples, including the Baermann technique, Harada-Mori filter paper culture, and APC [30, 34, 35]. Concentrating the stool with formalin-ethyl acetate increases the yield, but dead individual larvae are more difficult to discern at low magnification [34],[36] .
Direct wet mount
It is considered the most widely used method for identifying the larvae in a stool specimen [37] . This method is easy, less time consuming, and inexpensive, allowing direct visualization of the larvae. However, it has a main disadvantage in that the preparation dries within a few minutes rendering it unreadable. Hence, examination of sealed fresh preparations is required [38],[39] . It is important to note that failure to detect larvae in a stool examination is a hindering obstacle because it does not necessarily indicate the unequivocal absence of the infection [11],[12] . In addition, the output of larvae is irregular as for other digestive nematodes. This phenomenon can lower the sensitivity of direct parasitological methods.
Formol-ether concentration method
Concentration of the stool sample may be resorted to improve the sensitivity of direct examination. This method is one of the most commonly used methods for detecting intestinal parasites in stool [40],[41] . Young et al. [42] reported that the sensitivity of formol-ether concentration method (79.1%) is quite higher than that of direct wet smear method (50.3%), which agrees favorably with other similar studies [43],[44] .
Baermann's technique
First described in 1917, it is a cheap and simple technique based on the ability of S. stercoralis to enter a free-living cycle of development [45] . It is considered as one of the most sensitive tests available for the assessment of the presence of S. stercoralis larvae in fecal samples [46] . However, controversial results achieved for Baermann's concentration in comparison with APC ranged from 48.5 to 100% [47],[48] . An improved modification of this technique was proposed in which the funnel is substituted by a test tube that is closed with a perforated rubber stopper containing a pipette tip. This tube containing the suspected fecal suspension is inverted over another one containing 6 ml of saline solution, and both are incubated at 37°C for at least 2 h. The saline solution from the second tube is centrifuged and the pellet is observed microscopically [49] .
Harada-Mori technique
This is a filter paper culture method that utilizes water tropism of Strongyloides larvae for their concentration [50] . A filter paper smeared with the stool sample is inserted into a 15 ml tube containing warm distilled water. The tube is checked daily by withdrawing a small amount of fluid from its bottom and examining it microscopically [30, 46, 51]. Former sensitivities of 24 and 50.5% were reported for Harada-Mori culture compared with other conventional methods. Although it seems to have greater sensitivity compared with direct fecal smear or formalin-ethyl acetate concentration technique [52] , the detection rate is inferior compared with the Baermann or agar plate methods [51],[53] . Furthermore, it is rarely deployed as a standard procedure in clinical parasitological laboratories [3] . In some opinions, a disadvantage of this technique is that it may fail to detect S. stercoralis-positive samples due to water evaporation from the culture tube giving rise to false-negative results [54] .
Agar plate culture
By stool culture, the in vitro reproduction of the environmental cycle with release of new larvae may be more sensitive. Stool (3 g) is placed in a sterile plastic Petri dish containing ˜5 ml of nutrient agar and is incubated at 32°C. As larvae crawl over the agar, they carry bacteria with them creating visible tracks. Plates are checked daily for the presence of crawling marks [55] . Early in the 1990s, some groups reported the APC as a superior method (1.6-6 times more sensitive) compared with traditional methods such as direct smear or formol-ether concentration [56],[57] . A comparative study that used more than 1300 stool samples and four different methods of stool examination (direct fecal smear, formalin-ethyl acetate concentration, Harada-Mori culture, and APC) found the APC method to be 96% sensitive [58] . In another study, the APC method sensitivity also recorded 95% [59] . Hence, APC had the highest sensitivity among the conventional techniques [60],[61],[62] . In chronic infections, the sensitivity of this method might not be satisfactory. In the study by Sato et al. [63] the detection rate of APC was still less than 60% when only one sample was tested. Thus, the value of repeated stool examinations to increase the diagnostic yield using APC or other techniques is widely accepted, as it has been demonstrated to result in increased sensitivity [32],[48] . However, the culture technique has the disadvantage of being time consuming and presents a safety risk for laboratory staff. The time required for conclusive results (3-5 days) may also delay the onset of treatment [49],[64] .
Combination of these diagnostic techniques was found to increase the detection rate of S. stercoralis [65] . This was approved in several studies as the Harada-Mori culture had the lowest capacity for the detection of S. stercoralis (9.6%), but when combined with formalin-ether acetate centrifugation, Baermann method, and APC, the detection rate increased to 15.7% [58] . In another study, the combination of methods also increased the detection rate from 10.4% by the Baermann concentration method alone to 25.5% when combined with two coprocultures [10] .
String test
The string test used for sampling duodenal contents proved to be a reliable method for the diagnosis of S. stercoralis. Briefly, a nylon thread coiled inside a lined gelatin capsule is swallowed and the capsule is delivered to the stomach and duodenum. Thereafter, the line is pulled back with adhered bile-stained duodenal mucus. Goka et al. [66] compared fecal examination with duodenal fluid obtained by the string test in a group of patients with gastrointestinal symptoms, and showed better sensitivity of the latter. However, this invasive method should perhaps be recommended only in selected patients, such as immunosuppressed patients, to maximize the chance of detecting larvae when a prompt diagnosis is essential [67] .
Immunological diagnosis
To circumvent limitations of direct examination techniques, there is a great need for a highly specific and efficient serodiagnostic test for S. stercoralis diagnosis [3, 65, 68]. Several immunodiagnostic assays have been tested over the years, with limited success, including the immediate hypersensitivity reaction in skin to different somatic and excretory/secretory antigens. Although reported as a reliable skin test for the diagnosis of strongyloidiasis, its use was limited by cross-reactions with other nematode infections and the persistence of a positive skin test reaction after treatment. Moreover, in immunosuppressed patients, particularly those coinfected with HTLV-1, the immediate hypersensitivity reaction might be reduced leading to a lower sensitivity in this test. Therefore, it was concluded that intradermal skin testing is not a realistic option for routine diagnosis [69],[70] . An ELISA test for detecting specific serum IgG against a crude extract of the filariform larvae of S. stercoralis was attempted [63],[71] . The ELISA test proved to be 88% sensitive and 99% specific with positive and negative predictive values of 97 and 95%, respectively [72] . In another study, ELISA recorded 95% sensitivity, 29% specificity, and positive and negative predictive values of 30 and 95%, respectively [73] . Unfortunately, the demonstration of IgG antibodies, even when correct, does not distinguish between past and current infection. This is mainly because antibody levels remain detectable for years after antihelminthic treatment [74] . Moreover, there is the presence of cross-reactions especially in regions with high prevalence of intestinal parasites - for example, filariasis, ascariasis, and acute schistosomiasis [56] .
The demonstration of specific IgE response in human strongyloidiasis by ELISA has been used for its diagnosis [75] . It was signified that the circulating-specific IgE may be used as a marker to distinguish between the past and current infection of strongyloidiasis [76] . In addition, IgE-ELISA showed higher specificity and sensitivity than IgG-ELISA, presenting few cross-reactions with other intestinal parasites [75] . Several studies supported these findings and reported average sensitivity and specificity of 80 and 90.8%, respectively [68,75-77]. However, IgE anti-S. stercoralis may not be detected by ELISA because of the presence of excessive amounts of circulating IgE binding sites [78] . Moreover, patients under steroid therapy or coinfected with S. stercoralis and HTLV may have lower circulating-specific IgE [79] . On a practical basis, this test is unlikely to be available for wide use, because a constant supply of S. stercoralis filariform larvae is needed to obtain the crude antigen preparation [11],[12] .
The development of immunologic methods using modified or purified parasite antigens could improve the sensitivity and specificity of serodiagnostic methods for S. stercoralis infection [80] . In fact, nematode species are covered by a carbohydrate-rich glycocalyx that can be secreted and excreted as glycoconjugate antigens [81] . Such molecules have been shown to be involved in parasite survival, infection, and specific recognition by the cells [46, 82, 83]. The role of glycoprotein epitopes in increased antigenicity has been demonstrated in immunological tests to detect parasite antibodies [84],[85] . This was approved by several studies as the antibody reactions to S. stercoralis were found to increase in the presence of glycosylated epitopes. Thereby, the sensitivity of ELISA decreased from 76 to 72% for IgG and from 80 to 74% for IgE with antigen treatment by sodium metaperiodate, which destroyed the carbohydrate [77] . It was also found that analysis of the proteins on the surface of infective larvae is useful in the improvement of serodiagnosis. The most prominent antigenic proteins on the surface of infective larvae of S. stercoralis are 28, 31, and 41 kDa in size [76],[86] . In 2010, Ismail et al. [87] identified and characterized specific S. stercoralis antigenic epitopes and they concluded the significant use of 43, 41, 63, and 32 kDa bands. Two S. stercoralis recombinant antigens, 5a and 12a, which showed no cross-reactivity with serum samples from patients with filarial nematode infections, were identified; IgE and IgG4 antibodies against the 5a and 12a antigens were detected in strongyloidiasis patients [88] .
Screening of S. stercoralis complementary DNA library with affinity-purified antibodies against antigens p1, p4, and p5 has led to the identification of these antigens as oxoglutarate dehydrogenase, alkaline phosphatase, and isocitrate dehydrogenase, which can be used to detect S. stercoralis in multiple parasite infections [89] . A luciferase immunoprecipitation system using recombinant S. Stercoralis antigen to identify specific antibodies in serum was described. The assay had a better sensitivity (97%) and specificity (100%) than ELISA and did not cross-react with serum samples of filarial-infected patients. It can be performed rapidly (<2.5 h) and can detect changes in antibody response over time. An even faster version of this assay can be performed in less than 2 min and has the potential to be utilized as a rapid diagnostic test. This may be especially useful in critically ill patients suspected of having hyperinfection syndrome [90] .
ELISA has also been used to detect coproantigen of S. stercoralis in fecal samples from animal models [91] . El-Badry [92] developed an ELISA that is able to capture S. stercoralis coproantigen from infected patients without cross-reactions with the nematode Capillaria philippinensis or with the trematodes Schistosoma mansoni and Fasciola gigantica, although it was tested on only a very small number of human fecal samples. This could prove to be an easy and inexpensive technique, although more studies are needed on its performance for the diagnosis of strongyloidiasis.
Molecular diagnosis
A real-time PCR method targeting the small subunit of the rRNA gene was developed for specific detection of S. stercoralis DNA in fecal samples. In a study performed in 2009, it was found that real-time PCR could detect all the samples that were positive by the conventional methods and additional 18 samples that were negative by these methods [10] . In another study, real-time PCR identified all S. stercoralis-positive samples diagnosed by the parasitological techniques with a sensitivity of 100% and specificity of 94.8% [93] . Although PCR is the most expensive method, this technology is becoming available in developing countries and is a time-saving method [58] .
Diagnosis of hyperinfection
In patients with disseminated infection, the diagnosis is relatively easy due to high numbers of larvae that exist in the stool and are distributed in different parts of the body [94] . Once hyperinfection is considered, the examination of duodenal aspirate is very sensitive [95] . Microscopic examination of a single specimen of duodenal aspirate or mucus (using string test) was found to be more sensitive than wet mount analysis of stool samples for the detection of larvae [66] . In some patients, histological examination of duodenal or jejunal biopsy specimens may reveal S. stercoralis embedded in the mucosa [96] . The larvae can also be identified in wet preparations of sputum, broncho-alveolar lavage fluid, brushing, lung biopsies, or examination of pleural fluid [97] . Diagnosis through imaging is usually possible. Chest radiographs of some patients have shown infiltrates, which when present may be focal or diffuse or unilateral or bilateral. Lung consolidation and even abscess formation have also been reported [15] .
| Concluding remarks | | |
Obstacles in diagnosing strongyloidiasis are both clinical and diagnostic. Strongyloidiasis is a neglected disease, the prevalence of which might be underestimated in many countries because of lack of characteristic clinical features apart from larva currens. Its presentation is usually asymptomic or nonspecific particularly in uncomplicated patients.
The ability of S. stercoralis to establish a cycle of autoinfection within the host results in delayed presentation of the disease, often decades after primary infection, thus complicating the diagnosis.
Direct microscopic examination of stool specimens for rhabditiform larvae is the usual method of diagnosis. However, in chronic infection, larval excretion may be low and fluctuating. For this reason, microscopic observation is not sensitive enough and multiple stool specimens need to be analyzed to increase the sensitivity of the test. It has been reported that a single stool examination only detects larvae in as much as 30% of the patients.
Other parasitological techniques aiming to increase the sensitivity of fecal examination, such as Baermann's technique, Harada-Mori technique, or APC, have the disadvantages of being time consuming and presenting a safety risk for laboratory staff.
Serology is a useful tool but has the disadvantages of cross-reactivity with other nematode infections and difficulty in distinguishing recent from past (and cured) infections. However, most of the serological techniques have problems with sensitivity, specificity, or availability in endemic areas.
In population-based studies, it is widely believed that stool examination generally underestimates the prevalence, whereas serological examination generally overestimates it.
In recent years, the detection of parasite DNA in fecal samples using real-time PCR proved to be a sensitive and specific method for the diagnosis of S. stercoralis, but it is expensive and not adaptable for wide use because laboratory facilities are often limited.
The difficulty in calculating diagnostic efficiency parameters can be attributed to the absence of a definitive gold standard for diagnosing S. stercoralis infection.
| Acknowledgements | | |
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