The investigation of Strongyloides stercoralis seroprevalence in immunosupressed patients in Turkey

Background/aim In immunosuppressed patients, strongyloidiasis can be lifethreatening because of hyperinfection or dissemination. Therefore, diagnosis of S. stercoralis is important in immunosuppressed patients with chronic strongyloidiasis. In this study, our objective was to investigate the presence of S. stercoralis antibodies by an ELISA method in immunosuppressed patients. Materials and methods A total of 100 immunosuppressed patients’ sera were included in the study. Forty-two of the patients were receiving immunosuppressive therapies for cancer or being treated for hematopoietic malignancies, 38 of the patients were receiving immunosuppressive drugs for rheumatic diseases, 14 were receiving immunosuppressive therapies for liver transplantation. Two of the patients were being treated for HIV infection and 4 were being treated for hypogammaglobulinemia. As control group, 50 individuals without a known disease were included in the study. The presence of IgG antibodies against S. stercoralis was investigated with a commercial ELISA kit. Results S. stercoralis antibody test was positive in 4 of 100 (4%) sera from immunosuppressed patients. All control patients were negative for S. stercoralis. Conclusion Strongyloidiasis can be a lifelong chronic infection if not treated. In patients who are going to receive immunosuppressive therapy, it should be tested before treatment, as it can become a disseminated and life-threatening infectious disease.


Introduction
S. stercoralis is a soil-transmitted intestinal nematode that causes strongyloidiasis in humans. It is estimated that 30-100 million people are infected with S. stercoralis worldwide. Strongyloidiasis is endemic in tropical and subtropical areas especially where the sanitation conditions are poor (1).
The infection is acquired through contact with soil that is contaminated with free-living larvae. The larvae proceed via venous circulation, migrate to the lungs, are swallowed and reach the small intestine where they develop into adult parasites. Rhabditiform larvae are excreted in stool and transform into infective filariform larvae. The parasite can cause autoinfection which may lead persistent infections (1,2) .
Most of the S. stercoralis infections are asymptomatic. Symptomatic infection is usually characterized by gastrointestinal symptoms such as diarrhea and abdominal pain or skin manifestations like itching and urticaria (3).
S. stercoralis infection can be severe and lifethreatening in patients with immunosuppression. In this case, hyperinfection or dissemination of strongyloidiasis that leads to systemic sepsis, and multiorgan failure may develop. Hyperinfection syndrome is defined as accelerated autoinfection. Disseminated infection occurs when larvae migrate into organs other than the skin, gastrointestinal tract, or lungs. Predisposing risk factors for developing disseminated infection and hyperinfection syndrome are immunosuppressive therapies for immune-mediated disorders, infections with the human immunodeficiency virus (HIV), hematopoietic malignancies, and solid organ transplants (4).
Diagnosis of asymptomatic S. stercoralis infection in immunosuppressed patients is important to prevent life-threatening complications. If strongyloidiasis is diagnosed early, it is easily treatable with oral antihelmintic drugs (4). Because of the low parasite load and irregular larval output, diagnosis may be difficult by stool examination (5). Serological methods such as enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescent test (IFAT) and molecular tests may be alternative diagnostic methods for rapid detection of S. stercoralis infection (6).
In this study, our objective was to investigate the presence of S. stercoralis antibodies in the serum samples by using an ELISA method in immunosuppressed patients.

Patients
A total of 100 immunosuppressed patients' sera were included in the study. In this patient group ranging from 18 to 66 years of age, 52 patients were male and 48 were female. Forty-two of the patients were receiving immunosuppressive therapies for cancers such as lung cancer and breast cancer or being treated for hematopoietic malignancies such as lymphoma. Thirty-eight of the patients were receiving immunosuppressive drugs for rheumatic diseases such as ankylosing spondylitis and rheumatoid arthritis. Fourteen of the patients were receiving immunosuppressive therapies for liver transplantation. Two of the patients were being treated for HIV infection and four of the patients were being treated for hypogammaglobulinemia. As the control group, 50 individuals without a known disease, i.e. apparently healthy, and not receiving immunosuppressed treatment were included in the study. In the control group, there were 29 males and 21 females whose ages ranged from 19 to 63.

Blood samples
A total of 150 blood samples were collected. A hundred of them were from the patients that are shown in Table  and 50 were from the healthy subjects. Sera were separated from blood and stored at −20 °C until analysis.

ELISA testing
The presence of IgG antibodies against S. stercoralis in sera were investigated by a commercial ELISA kit (DRG Strongyloides IgG, USA) according to the instructions of manufacturers. In brief, serum samples were diluted 1:64 in dilution buffer and added antigen-coated wells. After being incubated at room temperature for 10 min, the wells were washed three times with the diluted wash buffer and 2 drops of enzyme conjugate were added to each well.
After incubation at room temperature for 5 min, the wells were washed again and 2 drops of chromogen were added. Following a 5 min of incubation, 2 drops of stop solution were added and the reaction was stopped. The results were read at 450 nm and 0.5 OD units and above were accepted as positive results. Each serum sample was examined twice at two different times. All tests were performed in the Parasitology Section of the Department of Medical Microbiology. The study was approved by the Ethical Committee of Hacettepe University.

Results
There was no difference between the two groups in terms of age and sex. Epidemiologic and sociodemographic features were similar.
Out of 100 patients, 24 were diagnosed with ankylosing spondylitis and 14 with rheumatoid arthritis. All of these rheumatology patients were receiving immunosuppressive agents. Four of them were positive for S. stercoralis antibodies. Two were diagnosed with ankylosing spondylitis and treated with etanercept, methotrexate, and prednisolone. Two patients had rheumatoid arthritis and were taking infliximab and methotrexate. None of these patients had symptoms, such as diarrhea, abdominal pain, nausea, vomiting, cutaneous lesions, or respiratory symptoms, that point to a parasitic infection. Eosinophil levels were within normal range in all sera-positive patients. There were 42 patients with malignancies in the study. Twenty-four patients were receiving chemotherapy for solid organ tumour and 18 for hematopoietic malignancies. None of them were positive for S. stercoralis antibodies. In two AIDS patients with diarrhea, S. stercoralis antibodies were negative. Fourteen of the patients were receiving immunosuppressive therapies for liver transplantation. S. stercoralis antibodies were not detected in any of them. All control patients were negative for S. stercoralis antibody (Table).  (4). It is reported that serological methods can be used as screening tests in immunosuppressed patients and the sensitivity of ELISA ranges from 80% to 100% (7,8).
In this preliminary study, our object was to evaluate the S. stercoralis serology for the management of probable chronic strongyloidiasis in patients with immunosuppresion.
Taking high doses of corticosteroids or antiinflammatory agents for rheumatoid diseases is a risk factor for developing severe strongyloidiasis (9) (12,13). In our study, S. stercoralis antibodies were found positive in rheumatology patients who were receiving immunosuppressive drugs. None of them had symptoms that point to a parasitic infection. Eosinophil levels were also within the normal range. In these patients, further investigations, such as Baermann's technique, stool culture, polymerase chain reaction, or endoscopic duodenal biopsy, may be required to detect possible S. stercoralis infection.
In oncology patients, diarrhea is one of the complications of chemotherapy and may be disregarded in terms of parasitic infection. There are reported cases of strongyloidiasis in patients with malignancies receiving immunosuppressive therapies (14). Zueter et al. studied 192 stool and serum samples collected from cancer patients who were receiving chemotherapy. They found that 1 (0.5%) sample was positive for S. stercoralis by microscopy, 3 (1.6%) by real-time PCR, 8 (4.2%) by IgG-ELISA. In patients with malignancies, immunosuppressive therapy can be the cause of fatal strongyloidiasis (15). Although we found no seropositivity in oncology patients in our study, it is important to follow up these individuals in terms of developing parasitic infection.
Strongyloidiasis is reported to be common in AIDS patients in endemic areas. Jongwutiwes et al. reported a case-control study for determining the prevalence and risk factors of S. stercoralis infection. They found that strongyloidiasis was seen more often in male patients with eosinophilia and with HIV infection (9). Despite the fact that prevalence rates of infection are higher in the individuals with HIV, some researchers report that hyperinfection syndrome is rarely seen in advanced HIV disease because of increased Th2 response which may protect against severe forms of strongyloidiasis (16)(17)(18)(19). In our study, there were two HIV-positive patients and they both had diarrhea. S. stercoralis antibodies were found negative in these individuals.
Several S. stercoralis infections in heart, kidney, intestine, liver, and stem cell transplant patients have been reported. In most of the cases, the infection was caused by reactivation of latent infections; in a few cases, it was acquired from the donors. Severe strongyloidiasis in transplant patients reveals high mortality rates (20)(21)(22)(23)(24). We did not detect S. stercoralis antibodies in patients receiving immunosuppressive therapies for liver transplantation.
In Turkey, the prevalence of S. stercoralis infection is reported between 0% and 4.4% based on stool examination (25). Aksoy Gökmen et al. investigated the incidence of S. stercoralis in individuals who live in rural areas and work in the garden; one of 281 volunteers (0.3%) was positive for S. stercoralis-IgG antibodies (26).
To our knowledge, this is the first study conducted to detect the S. stercoralis seroprevalance of immunosuppressed patients in Turkey. A total of 150 sera sample were tested for the presence of IgG antibodies against S. stercoralis. Four of 100 (4%) sera from immunosuppressed patients were positive. In the control group, all individuals were sera-negative. Limitations of this study are low number of patients and inadequacy of fecal tests due to poor compliance of patients to the repeated stool examinations. Further investigation with molecular techniques may be an alternative diagnostic tool for the detection of S. stercoralis in these patients.
Although there were no signs of parasitic involvement in our sera-positive patients, it might be beneficial to follow up these patients in terms of developing S. stercoralis infection. It is important to detect latent S. stercoralis infections before administering chemotherapy or before the onset of immunosuppression in patients at risk to prevent severe complications.