A Seroepidemiological Survey of Toxocariasis among Eosinophilia Patients in Chungcheongnam-do

There have been several reports on the relationship between toxocariasis and eosinophilia, but all have been limited to the areas of Seoul or Gangwon-do. In the present study, we investigated the seroprevalence of toxocariasis among eosinophilia patients in Chungcheongnam-do, the central district of Korea. Among the 101 patients tested, 51 (50.5%) were identified as positive by Toxocara ELISA, and 46 (45.5%) were confidently diagnosed with toxocariasis because of absence of any other cause of eosinophilia. Whereas 22 of 42 seropositive patients (52.3%) had a recent history of consuming raw livers, especially the cow liver, only 1 of 25 seronegative patients (4%) had done so (P<0.01). From these results, we could confirm that toxocariasis is related to eosinophilia, and infer that ingestion of raw cow liver plays a vital role in the transmission of toxocariasis in Chungcheongnam-do.     

Transmission of Toxocara canis via ingestion of raw cow liver: a cross-sectional study in healthy adults

The aim of this study is to ascertain the relationship between ingestion of raw cow liver and Toxocara canis infection. A total of 150 apparently healthy adults were divided into 2 groups; 1 group consisted of 86 adults with positive results of Toxocara ELISA, and the other group of 64 adults with negative results. One researcher collected the history of ingestion of raw cow liver within 1 year and recent history of keeping dogs. Among 86 seropositive adults for T. canis, 68 (79.1%) had a recent history of ingestion of raw cow liver. Multivariate statistical analysis showed that a recent ingestion of raw cow liver and keeping dogs were related to an increased risk of toxocariasis (odds ratios, 4.4 and 3.7; and 95% confidence intervals, 1.9-10.2 and 1.2-11.6, respectively). A recent history of ingestion of raw cow liver and keeping dogs was significantly associated with toxocariasis.     

Seroprevalence of Toxocara antibodies among patients suspected of ocular toxocariasis in Slovenia

Ocular toxocariasis named also ocular larva migrans is caused by larvae of the roundworm Toxocara spp. The purpose of this study was to find out the seroprevalence of Toxocara antibodies in patients suspected of ocular toxocariasis. Between January 2001 and December 2003, sera from 239 ocular patients, aged 3 to 80 years, were examined by ELISA and confirmed by Western blot test. Out of the 239 patients, 172 (72%) were seronegative and 67 (28%) were Toxocara seropositive; 95% CI (22-34%). The median age of Toxocara seropositive patients was 37.6 years. There was no significant difference in the number of Toxocara positive sera between the younger age group (< or = 14 years) and the older age group (> 14 years), p > 0.05. A high rate of Toxocara seropositivity in ocular patients should alert the ophthalmologists in Slovenia to include toxocariasis in the differential diagnosis of eye diseases more frequently.     

A familial case of visceral toxocariasis due to consumption of raw bovine liver

We present 3 adult cases of visceral toxocariasis from the same family, who each consumed thin slices of raw bovine liver weekly, and developed eosinophilia and multiple small lesions in their livers and lungs. Serological examinations using the larval excretory–secretory product of Toxocara canis strongly indicated infection with Toxocara species larvae. The patients responded well to treatment with albendazole. Ingestion of raw liver from paratenic animals is considered to be a common transmission route of human toxocariasis, especially in adults.     

Pulmonary Toxocariasis Mimicking Invasive Aspergillosis in a Patient with Ulcerative Colitis

A 45-year-old-male who had underlying ulcerative colitis and presented with fever and dry cough. Initially, the patient was considered to have invasive aspergillosis due to a positive galactomannan assay. He was treated with amphotericin B followed by voriconazole. Nevertheless, the patient deteriorated clinically and radiographically. The lung biopsy revealed eosinophilic pneumonia, and ELISA for Toxocara antigen was positive, leading to a diagnosis of pulmonary toxocariasis. After a 10-day treatment course with albendazole and adjunctive steroids, the patient recovered completely without any sequelae. Pulmonary toxocariasis may be considered in patients with subacute or chronic pneumonia unresponsive to antibiotic agents, particularly in cases with eosinophilia.     

Clinical characteristics that distinguish eosinophilic organ infiltration from metastatic nodule development in cancer patients with eosinophilia

ABSTRACT: BACKGROUND: When new space-occupying lesions are observed together with peripheral blood eosinophilia in patients diagnosed with cancer, the possibility of eosinophilic organ involvement should be differentiated from metastasis of primary cancer, since a misdiagnosis could lead to unnecessary chemotherapy. The aim of this study is to identify the clinical characteristics of eosinophilic organ involvement that distinguish it from distant metastasis in patients with primary cancer. METHODS: The medical records of 43 cancer patients who developed hepatic or pulmonary nodules with peripheral blood eosinophilia between January 2005 and February 2010 in the Asan Medical Center (Seoul) were reviewed. Eosinophilic infiltration and distant metastasis were identified on the basis of pathological findings and radiological features. Fisher's exact test, chi2 test or Mann-Whitney test were used for statistical analysis. RESULTS: In total, 33 patients (76 %) were diagnosed with eosinophilic infiltration, 5 (12 %) with cancer metastasis and 5 (12 %) had undetermined diagnoses. Compared to the patients with metastases, the patients with eosinophilic infiltration were significantly more likely to have serology indicating a parasitic infection, a history of eating raw food, high serum levels of total IgE, normal liver function, normal C-reactive protein levels, a normal erythrocyte sedimentation rate, and fewer and smaller nodules. The most common underlying malignancy in the eosinophilic organ infiltration group was stomach cancer. Physicians tended to neglect the eosinophilia in patients with a history of cancer. CONCLUSIONS: Several clinical characteristics of eosinophilic organ infiltration distinguish it from cancer metastasis. Physicians should make greater efforts to determine the causes of organ involvement with peripheral blood eosinophilia, especially in cancer patients.     

Serodiagnosis of Toxocariasis by ELISA Using Crude Antigen of Toxocara canis Larvae

Toxocariasis is a worldwide zoonosis caused by larvae of ascarid nematodes of dogs or cats, Toxocara canis or T. cati. Diagnosis of human toxocariasis currently relies on serology that uses T. canis excretory-secretory antigen to detect specific IgG antibodies by ELISA. We investigated the serodiagnostic efficacy of ELISA using crude antigen of T. canis larvae (TCLA). Serum specimens of 64 clinically confirmed toxocariasis, 115 healthy controls, and 119 other tissue-invading helminthiases were screened by ELISA using TCLA. The ELISA using TCLA showed 92.2% (59/64 patient samples) sensitivity and 86.6% (103/119) specificity. Its positive diagnostic predictivity was 78.7% and negative predictivity was 97.8%. No serum of healthy controls reacted but that of anisakiasis (45.5%), gnathostomiasis (19.2%), clonorchiasis (15.8%), sparganosis (11.1%), and cysticercosis (6.3%) cross-reacted. Immunoblot analysis on TCLA recognized antigenic proteins of 28- and 30-kDa bands in their dominant protein quantity and strong blotting reactivity. The present results indicate that the ELISA using our TCLA antigen is acceptable by the sensitivity and specificity for serodiagnosis of human toxocariasis. ELISA with TCLA is recommended to make differential diagnosis for patients with any sign of organ infiltration and eosinophilia.     

Seroprevalence of toxocariasis among healthy people with eosinophilia

The aim of this study is to determine the Toxocara seropositive rate among healthy people with eosinophilia. A total of 97 people residing in Seoul who were healthy and whose blood eosinophilia was over 10%, as shown by regular health check-ups in 2004, were subjected to this study. Their sera were tested by immunoblotting and ELISA with the antigen of larval Toxocara canis excretory-secretory (ES) protein. Sixty-five sera were band-positive (67.0%). The seropositve control sera were positive to band sizes of 66 kDa, 56 kDa, 32 kDa, and 13 kDa. In ELISA, 63 sera (65.0%) were positive to T. canis ES protein. There was no significant correlation between the IgG ELISA titer and the level of eosinophilia (r = 0.156, P = 0.156). As there were insufficient data to determine whether there were cross-reactions with other helminthic infections, or whether atopy occurred, further studies are required to verify the cause of the seropositive reactions against T. canis ES antigen. Toxocariasis seropositivity is suggested to be the major cause of eosinophilia, since the Toxocara seroprevalence among Korean rural adults was shown to be approximately 5%.     

Clinical Features and Course of Ocular Toxocariasis in Adults

Purpose

To investigate the clinical features, clinical course of granuloma, serologic findings, treatment outcome, and probable infection sources in adult patients with ocular toxocariasis (OT).

Methods

In this retrospective cohort study, we examined 101 adult patients diagnosed clinically and serologically with OT. Serial fundus photographs and spectral domain optical coherence tomography images of all the patients were reviewed. A clinic-based case-control study on pet ownership, occupation, and raw meat ingestion history was performed to investigate the possible infection sources.

Results

Among the patients diagnosed clinically and serologically with OT, 69.6% showed elevated immunoglobulin E (IgE) levels. Granuloma in OT involved all retinal layers and several vitreoretinal comorbidities were noted depending on the location of granuloma: posterior pole granuloma was associated with epiretinal membrane and retinal nerve fiber layer defects, whereas peripheral granuloma was associated with vitreous opacity. Intraocular migration of granuloma was observed in 15 of 93 patients (16.1%). Treatment with albendazole (400 mg twice a day for 2 weeks) and corticosteroids (oral prednisolone; 0.5–1 mg/kg/day) resulted in comparable outcomes to patients on corticosteroid monotherapy; however, the 6-month recurrence rate in patients treated with combined therapy (17.4%) was significantly lower than that in patients treated with corticosteroid monotherapy (54.5%, P = 0.045). Ingestion of raw cow liver (80.8%) or meat (71.2%) was significantly more common in OT patients than healthy controls.

Conclusions

Our study discusses the diagnosis, treatment, and prevention strategies for OT. Evaluation of total IgE, in addition to anti-toxocara antibody, can assist in the serologic diagnosis of OT. Combined albendazole and corticosteroid therapy may reduce intraocular inflammation and recurrence. Migrating feature of granuloma is clinically important and may further suggest the diagnosis of OT. Clinicians need to carefully examine comorbid conditions for OT. OT may be associated with ingestion of uncooked meat, especially raw cow liver, in adult patients.     

Reduced cognitive function in children with toxocariasis in a nationally representative sample of the United States

Toxocariasis has recently been recognised as a potentially important neglected infection in developed countries, particularly those that experience substantive health disparities such as the United States. Given a relatively high prevalence of infection, an association between Toxocara infection and cognitive function may elucidate an important mechanism by which toxocariasis could contribute significantly to morbidity while still remaining hidden and, thus, neglected. To assess the potential relationship between toxocariasis and cognitive function, this investigation measured differences in components of both the Wechsler Intelligence Scale for Children-Revised (WISC-R) and the Wide Range Achievement Test-Revised (WRAT-R) in children seropositive and in children seronegative for Toxocara antibodies in the Third National Health and Nutrition Examination Survey, a large, nationally-representative survey of the United States population. Seropositive children scored significantly lower on the WISC-R and WRAT-R compared with the seronegative children. Moreover, this relationship was independent of socioeconomic status, ethnicity, gender, rural residence, cytomegalovirus infection and blood lead levels. These results identify an important association that may reflect morbidity attributable to a genuine neglected infection. Nevertheless, longitudinal data are required to confirm an etiological connection between toxocariasis and cognitive function, as well as the true population attributable risk for toxocariasis and its chronic sequelae.     

The first human case of Trichinella spiralis infection in Korea

Three cases of human infection by Trichinella spiralis were first confirmed by detecting encysted larvae in the biopsied muscle in December 1997, in Korea. The patients were one 35- and two 39-year-old males residing in Kochang-gun, Kyongsangnam-do. They had a common past history of eating raw liver, spleen, blood and muscle of a badger, Meles meles melanogenys, and complained of high fever, facial and periorbital edema, and myalgia. Hematologic and biochemical examinations revealed leukocytosis and eosinophilia, and highly elevated levels of GOT, GPT, LDH and CPK. In the gastrocnemius muscle of a patient, roundly coiled nematode larvae were detected. The larvae measured 0.775-1.050 (av. 0.908) mm in length, and 0.026-0.042 (av. 0.035) mm in maximum width. The specific IgG antibody levels in three patients' sera were significantly higher when compared with those of normal controls. The patients were treated with flubendazole and albendazole for 15-30 days, and discharged at 13-34 days post-admission. From the above findings, it was confirmed that T. spiralis is present in Korea, and the badger plays a role of as the natural host.     

The frequency of Toxocara infection in mental retarded children

Human toxocariasis is commonly seen in places where stray and Toxocara canis-infected dog population is high. There is a strong correlation between frequency of Toxocara infection, life style, and infection risk. Institutionalization of mental retarded patients increases to risk of toxocariasis. In this study, we aimed at investigating the frequency of Toxocara infection among children with mental retardation not requiring institutionalization. The study included 96 cases, who had educatable mental retardation and 85 healthy subjects who comprised the control group. Anti-Toxocara IgG or IgM antibodies were investigated in all serum samples, using ELISA method. The frequency of Toxocara infection was found significantly higher in mental retarded cases than in those in the control group (18.8% and 7.1% respectively) (p < 0.05). There was no significant difference between mental retarded children and the control group in terms of mean age, age groups, gender, owning dogs and cats and duration of their ownership, socio-economic level and behavioural factors, and personal hygiene (p > 0.05). We did not find any significant difference between Toxocara seropositive and seronegative mental retarded children in terms of demographic factors and epidemiological factors that could increase the risk of Toxocara infection (p > 0.05). The present study is the first seroprevalence study carried out with a mental retarded group not requiring institutionalization. Determination of high frequency of Toxocara infection suggests that these subjects constitute a risk factor for Toxocara infection, which may be attributed to their behavioural patterns.     

Serologic Survey of Toxoplasmosis in Seoul and Jeju-do, and a Brief Review of Its Seroprevalence in Korea

Knowledge of the prevalence of human Toxoplasma gondii infection is required in the Republic of Korea. In this study, we surveyed the seroprevalence of T. gondii infection and analyzed the risk factors associated with seropositivity among residents in 2 administrative districts; Seoul and the island of Jeju-do, which have contrasting epidemiologic characteristics. Sera and blood collected from 2,150 residents (1,114 in Seoul and 1,036 in Jeju-do) were checked for IgG antibody titers using ELISA and for the T. gondii B1 gene using PCR. In addition, participants completed a questionnaire that solicited information on gender, age, occupation, eating habits, history of contact with animals, and travel abroad. The T. gondii B1 gene was not detected in all residents examined. However, ELISA showed 8.0% (89 of 1,114 sera) positive for IgG antibodies against T. gondii in Seoul and 11.3% (117 of 1,036 sera) in Jeju-do. In both districts, the positive rates were higher in males than in females, and those 40-79 years of age showed higher rates than other ages. In Seoul, residents older than 70 years of age showed the highest positive rate, 14.9%, whereas in Jeju-do the highest prevalence, 15.6%, was in those in their sixties. The higher seropositive rate in Jeju-do than in Seoul may be related to eating habits and occupations. The present results and a review of related literature are indicative of an increased seroprevalence of T. gondii in Korea in recent years.     

Seroprevalence of Fusobacterium varium in ulcerative colitis patients in Japan

The etiology of ulcerative colitis (UC) is unknown, while an exacerbating factor of this disease is associated with infectious agents. Recently, Fusobacterium varium has been found in the mucosa of a significant number of patients with UC. The aim of this study was to estimate the prevalence of F. varium infection based on serology, evaluate the relationship between F. varium seropositivity and UC, and determine the clinical characteristics of infected UC individuals. Seropositive patients were determined by immunoblotting with F. varium ATCC 8501 antigen. We also identified cross-reactive protein spots by peptide mass mapping analysis. These protein spots showed putative caseinolytic protease protein, putative translation elongation factor G, and putative enolase. Immunoblotting with F. varium antigen revealed signals with sera from 45 (40.2%) of the 112 UC patients and 20 (15.6%) of the 128 healthy controls, respectively ( P <0.01). In terms of disease activity, seropositive UC patients were more likely to have clinically severe disease than seronegative UC patients. Disease location in seropositive patients was more extensive than the seronegative patients. In conclusion, F. varium is a predominant infection in the UC population and is a potential pathogen of UC.     

A seroepidemiological survey for toxocariasis in apparently healthy residents in Gangwon-do,Korea

We investigated the sero-prevalence of toxocariasis among healthy Korean adults in 1999. A total of 314 sera from normal inhabitants in Whachon-gun, Gangwondo, Korea was examined for specific antibody levels against excretory-secretory products of second stage larvae of Toxocara (TES). The presence of cross-reactions with other helminthiases such as cysticercosis, paragonimiasis, sparganosis or clonorchiasis was also checked by specific IgG ELISA. Sera showing positive reaction against TES were also tested by IgG immunoblot and by IgE ELISA. Out of 314 subjects, 16 was found to be positive by TES IgG ELISA and immunoblot, among whom 12 were also positive by TES IgE ELISA. Among the 16 seropositive samples, two sera showed positive reaction against Paragonimus and sparganum antigen, respectively. These results inferred that cross-reactions were negligible between toxocariasis and other helminthiases. Toxocariasis seroprevalence among Korean rural adults was detected to be approximately 5%.     

Larval toxocariasis and its clinical manifestation in childhood in the Slovak Republic

Results are presented of 90 children aged 1-15 years hospitalized with toxocariasis. Blood count analysis and laboratory examination were done by routine clinical laboratory methods. Anti-Toxocara antibodies were detected in the serum of patients using an ELISA method. Demographic analysis of the children's families exposed to the risk of disease allowed estimation of age-specific rates for clinical toxocariasis. The probability of toxocaral infection and the intensity of its clinical manifestations in children are determined by the epidemiology of this zoonosis and by the risk factors in the family. The presence of high titres of specific IgG antibodies in all age categories correlates with the clinical manifestations of toxocariasis. The highest admission rate is in the age categories of 3-5 years (43.3%) and 6-10 years (36.7%). Laboratory findings show that the most conspicuous changes occur in the age category 1-5 years. The high percentage of seropositive dog-keeping and puppy-breeding families and the possibility of infection with repeated doses of larvae stimulate eosinophilia, which prevails in children under the age of five years. We present the percentage of patients whose parameters showed deviations from the reference values for a particular age category. Analyses of laboratory indices and of clinical manifestations will contribute to the accuracy of diagnosis and effectiveness of treatment of this disease.     

Cutaneous Gnathostomiasis with Recurrent Migratory Nodule and Persistent Eosinophilia: a Case Report from China

The present study reports a human case of cutaneous gnathostomiasis with recurrent migratory nodule and persistent eosinophilia in China. A 52-year-old woman from Henan Province, central China, presented with recurrent migratory reddish swelling and subcutaneous nodule in the left upper arm and on the back for 3 months. Blood examination showed eosinophila (21.2%), and anti-sparganum antibodies were positive. Skin biopsy of the lesion and histopathological examinations revealed dermal infiltrates of eosinophils but did not show any parasites. Thus, the patient was first diagnosed as sparganosis; however, new migratory swellings occurred after treatment with praziquantel for 3 days. On further inquiring, she recalled having eaten undercooked eels and specific antibodies to the larvae of Gnathostoma spinigerum were detected. The patient was definitely diagnosed as cutaneous gnathostomiasis caused by Gnathostoma sp. and treated with albendazole (1,000 mg/day) for 15 days, and the subsequent papule and blister developed after the treatment. After 1 month, laboratory findings indicated a reduced eosinophil count (3.3%). At her final follow-up 18 months later, the patient had no further symptoms and anti-Gnathostoma antibodies became negative. Conclusively, the present study is the first report on a human case of cutaneous gnathostomiasis in Henan Province, China, based on the past history (eating undercooked eels), clinical manifestations (migratory subcutaneous nodule and persistent eosinophilia), and a serological finding (positive for specific anti-Gnathostoma antibodies).     

Electrocardiographic Abnormalities in Trypanosoma cruzi Seropositive and Seronegative Former Blood Donors

Background

Blood donor screening leads to large numbers of new diagnoses of Trypanosoma cruzi infection, with most donors in the asymptomatic chronic indeterminate form. Information on electrocardiogram (ECG) findings in infected blood donors is lacking and may help in counseling and recognizing those with more severe disease.

Objectives

To assess the frequency of ECG abnormalities in T.cruzi seropositive relative to seronegative blood donors, and to recognize ECG abnormalities associated with left ventricular dysfunction.

Methods

The study retrospectively enrolled 499 seropositive blood donors in São Paulo and Montes Claros, Brazil, and 483 seronegative control donors matched by site, gender, age, and year of blood donation. All subjects underwent a health clinical evaluation, ECG, and echocardiogram (Echo). ECG and Echo were reviewed blindly by centralized reading centers. Left ventricular (LV) dysfunction was defined as LV ejection fraction (EF)<0.50%.

Results

Right bundle branch block and left anterior fascicular block, isolated or in association, were more frequently found in seropositive cases (p<0.0001). Both QRS and QTc duration were associated with LVEF values (correlation coefficients −0.159,p<0.0003, and −0.142,p = 0.002) and showed a moderate accuracy in the detection of reduced LVEF (area under the ROC curve: 0.778 and 0.790, both p<0.0001). Several ECG abnormalities were more commonly found in seropositive donors with depressed LVEF, including rhythm disorders (frequent supraventricular ectopic beats, atrial fibrillation or flutter and pacemaker), intraventricular blocks (right bundle branch block and left anterior fascicular block) and ischemic abnormalities (possible old myocardial infarction and major and minor ST abnormalities). ECG was sensitive (92%) for recognition of seropositive donors with depressed LVEF and had a high negative predictive value (99%) for ruling out LV dysfunction.

Conclusions

ECG abnormalities are more frequent in seropositive than in seronegative blood donors. Several ECG abnormalities may help the recognition of seropositive cases with reduced LVEF who warrant careful follow-up and treatment.     

Toxocariasis in humans: clinical expression and treatment dilemma

A new scheme of clarifying clinical forms of toxocariasis is proposed to include: (i) systemic forms: classical VLM and incomplete VLM; (ii) compartmentalized forms: ocular and neurological toxocariasis; (iii) covert toxocariasis; and (iv) asymptomatic toxocariasis. The following markers are helpful in defining clinical forms namely, patient characteristics and history, clinical symptoms and signs, positive serology, eosinophilia and increased levels of IgE. Amongst the available drugs albendazole is the most commonly used, although other benzimidazole compounds have a similar efficacy. The recommended dose of albendazole is 15 mg kg(-1) body weight daily for 5 days and in some cases with VLM syndrome the treatment needs to be repeated. An evaluation of treatment efficacy can be made by observing a rise in eosinophilia within a week followed by any improvement in clinical symptoms and signs, lower eosinophilia and serological tests taken over a period of at least 4 weeks. In addition to clinical rationales for the specific treatment of VLM and OLM, preventive treatment needs to be considered bearing in mind the increasing risk of larvae localizing in the brain during the course of an infection. To reduce migration of Toxocara larvae a single course of albendazole is suggested in cases where eosinophilia and serology are at least moderately positive.     

Toxocara Seropositivity, Atopy and Wheezing in Children Living in Poor Neighbourhoods in Urban Latin American

Background

Toxocara canis and T. cati are parasites of dogs and cats, respectively, that infect humans and cause human toxocariasis. Infection may cause asthma-like symptoms but is often asymptomatic and is associated with a marked eosinophilia. Previous epidemiological studies indicate that T. canis infection may be associated with the development of atopy and asthma.

Objectives

To investigate possible associations between Toxocara spp. seropositivity and atopy and childhood wheezing in a population of children living in non-affluent areas of a large Latin American city.

Methods

The study was conducted in the city of Salvador, Brazil. Data on wheezing symptoms were collected by questionnaire, and atopy was measured by the presence of aeroallergen-specific IgE (sIgE). Skin prick test (SPT), total IgE and peripheral eosinophilia were measured. Toxocara seropositivity was determined by the presence of anti-Toxocara IgG antibodies, and intestinal helminth infections were determined by stool microscopy.

Findings

Children aged 4 to 11 years were studied, of whom 47% were seropositive for anti-Toxocara IgG; eosinophilia >4% occurred in 74.2% and >10% in 25.4%; 59.6% had elevated levels of total IgE; 36.8% had sIgE≥0.70 kU/L and 30.4% had SPT for at least one aeroallergen; 22.4% had current wheezing symptoms. Anti-Toxocara IgG was positively associated with elevated eosinophils counts, total IgE and the presence of specific IgE to aeroallergens but was inversely associated with skin prick test reactivity.

Conclusion

The prevalence of Toxocara seropositivity was high in the studied population of children living in conditions of poverty in urban Brazil. Toxocara infection, although associated with total IgE, sIgE and eosinophilia, may prevent the development of skin hypersensitivity to aeroallergens, possibly through increased polyclonal IgE and the induction of a modified Th2 immune reaction.