Background
Strongyloidiasis is frequently under diagnosed since many infections remain asymptomatic and conventional diagnostic tests based on parasitological examination are not sufficiently sensitive. Serology is useful but is still only available in reference laboratories. The need for improved diagnostic tests in terms of sensitivity and specificity is clear, particularly in immunocompromised patients or candidates to immunosuppressive treatments. This review aims to evaluate both conventional and novel techniques for the diagnosis of strongyloidiasis as well as available cure markers for this parasitic infection.

http://www.iacld.ir/DL/elm/thelaboratorydiagnosisandfollowupofStrongyloidiasisasystematicreview.pdf

The laboratory diagnosis and follow up of strongyloidiasis: a systematic review.

The majority of the 30-100 million people infected with Strongyloides stercoralis, a soil transmitted intestinal nematode, have subclinical (or asymptomatic) infections. These infections are commonly chronic and longstanding because of the autoinfective process associated with its unique life cycle. A change in immune status can increase parasite numbers, leading to hyperinfection syndrome, dissemination, and death if unrecognized. Corticosteroid use and HTLV-1 infection are most commonly associated with the hyperinfection syndrome. Strongyloides adult parasites reside in the small intestine and induce immune responses both local and systemic that remain poorly characterized. Definitive diagnosis of S. stercoralis infection is based on stool examinations for larvae, but newer diagnostics - including new immunoassays and molecular tests - will assume primacy in the next few years. Although good treatment options exist for infection and control of this infection might be possible, S. stercoralis remains largely neglected.

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Human infection with Strongyloides stercoralis and other related Strongyloides species.

Although Strongyloides stercoralis often causes chronic and clinically asymptomatic infection, the number of Strongyloides parasites can increase substantially in immunocompromised hosts, leading to hyperinfection, dissemination, and death if unrecognized [1]. Early recognition of S. stercoralis infection is challenging because of scanty and intermittent excretion of larvae in chronically infected immunocompetent hosts [2]. Despite this, the mainstay of diagnostic testing for S. stercoralis infection has been stool examination, although more recently ELISAs have been used to measure antibodies to crude larval antigen. Serologic approaches to the diagnosis of S. stercoralis infection, however, have been hampered by poor specificity, reliance on crude parasite extracts, and the time needed to perform the assays [3–5].

A major drawback to ELISA-based diagnosis of S. stercoralis infection has been a reliance on crude antigen that must be prepared by isolating worms from the feces of heavily infected patients or experimental animals. Thus, investigators have turned to recombinant antigens, which can be purified easily and produced in large amounts [6]. Indeed, a 31-kDa recombinant antigen (termed NIE) derived from an S. stercoralis L3 cDNA library provided the basis for an ELISA that approaches the sensitivity and specificity of the crude antigen–based ELISA [5]. An attractive alternative to ELISA-based methods, luciferase immunoprecipitation systems (LIPS), has been successfully applied to the characterization of antibody responses to Pneumocystis jirovecii, HIV, and hepatitis viruses [7]. LIPS is a relatively straightforward technology for identifying serum containing antigen-specific antibodies and for generating quantitative antibody response profiles. Briefly, this approach involves fusion of a protein antigen to the enzyme reporter Renilla luciferase (Ruc), expression of the Ruc-antigen fusion in mammalian COS cells, immobilization of the Ruc-antigen fusion on protein A/G beads, and quantitation of antigen-specific antibody by the addition of a coelenterazine substrate and the measurement of light production [8]. This assay represents a major improvement over ELISA technology in that it produces a low background often with a 7-log dynamic range, thereby generating values with substantial separation between negative and positive antibody responses. The low background and high signal seen in the LIPS method can be credited, in part, to the use of a solution-phase immunoprecipitation assay that allows detection of a large number of conformational epitopes. The use of mammalian cells produces antigens free of contaminating bacterial proteins. An additional advantage of LIPS is that, once the Ruc-antigen constructs are made, relatively little time is needed to perform the assay.

Recently, we reported the use of Ruc-antigen fusion proteins, produced in COS1 monkey kidney cells, in an immunoprecipitation assay to measure human antibody responses to tumor-associated proteins [8] and to a variety of infectious agents [7]. In this study, we have broadened the application of LIPS to the diagnosis and monitoring of S. stercoralis infection. To develop a more rapid, specific, and standardized assay, we first developed a LIPS assay based on IgG (or IgG4) antibody to NIE and compared it with a standard NIE ELISA. Our data, generated using serum samples from S. stercoralis–infected patients, filaria-infected patients, and uninfected control subjects, demonstrated the clear benefit of the LIPS method. Moreover, when a second antigen, S. stercoralis immunoreactive antigen (SsIR), was used in combination with NIE in the LIPS format, we found an even greater degree of sensitivity and specificity. Finally, we assessed the ability of LIPS to evaluate the success of treatment in the follow-up of S. stercoralis–infected patients.

A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection

Strongyloidiasis: A Neglected Tropical Disease

The serodiagnosis of Strongyloides stercoralis infection by enzyme-linked immunosorbent assays based on crude antigen (CrAg-ELISA), while useful, has been limited by the reliance on crude parasite extracts. Newer techniques such as the luciferase immunoprecipitation system assay (LIPS), based on a 31-kDa recombinant antigen (termed NIE) from S. stercoralis and/or the recombinant antigen S. stercoralis immunoreactive antigen (SsIR), or the NIE-ELISA have shown promise in controlled settings. We compared each of these serologic assays in individuals from both regions of the world in which S. stercoralis is endemic and those in which it is not. A comprehensive stool evaluation (sedimentation concentration, Baermann concentration with charcoal cultures, agar plate, and Harada-Mori) and four different serologic techniques using CrAg-ELISA or recombinant NIE-ELISA as well as LIPS using NIE alone or in combination with a second recombinant antigen (NIE/SsIR-LIPS) were compared among individuals with parasitologically proven infection (n = 251) and healthy controls from regions of the world in which the infection is nonendemic (n = 11). Accuracy was calculated for each assay. The prevalence of S. stercoralis infection was 29.4% among Argentinean stool samples (n = 228). Sedimentation concentration and Baermann were the most sensitive stool-based methods. NIE-LIPS showed the highest sensitivity (97.8%) and specificity (100%) of the serologic assays. The calculated negative predictive value was highest for both the NIE-LIPS and CrAg-ELISA (>97%) irrespective of disease prevalence. No cross-reactivity with soil-transmitted helminths was noted. NIE-LIPS compares favorably against the current CrAg-ELISA and stool evaluation, providing additional accuracy and ease of performance in the serodiagnosis of S. stercoralis infections irrespective of disease prevalence.

https://cvi.asm.org/content/cdli/17/10/1624.full.pdf

Improved diagnosis of Strongyloides stercoralis using recombinant antigen-based serologies in a community-wide study in northern Argentina.

Characterization of a recombinant immunodiagnostic antigen (NIE) from Strongyloides stercoralis L3-stage larvae☆

A new recombinant protein (NIE) for immunodiagnosis of human Strongyloides infection has 13% to 18% amino acid identity with antigen 5 insect venom allergen, but the C-terminal segment of NIE showed highest identity with Ves v 5 (yellow jacket) and Pol a 5 (paper wasp). A rabbit polyclonal anti-NIE antibody identified a single band of NIE antigen as well as bands of Pol a 5 and Ves v 5 antigens, and mouse anti-Pol a 5 and anti-Ves v 5 sera reacted with recombinant NIE antigen by Western blot. A cyanogen bromide-digested C-terminal fragment of NIE was reactive with mouse anti-Ves v 5 and Pol a 5 antibodies as well as with rabbit anti-NIE serum. Although IgE and IgG antibodies from pooled sera from Strongyloides-infected patients reacted with Pol a 5 and Ves v 5 recombinant antigens on immunoblots, neither antigen inhibited human IgG reaction with NIE antigen in a competitive enzyme-linked immunosorbent assay.

Varatharajalu Ravi

Papers

Characterization of a recombinant immunodiagnostic antigen (NIE) from Strongyloides stercoralis L3-stage larvae☆

Strongyloides stercoralis recombinant NIE antigen shares epitope with recombinant Ves v 5 and Pol a 5 allergens of insects.