Safety, efficacy and pharmacokinetics of vedolizumab in patients with simultaneous exposure to an anti-tumour necrosis factor.

BACKGROUND: Data on combination-biologic treatment in (IBD) are still scant. AIM: To explore outcomes of patients co-exposed to anti-TNF and vedolizumab. METHODS: Patients starting vedolizumab having measurable anti-TNF levels after recently stopping adalimumab/infliximab ('VDZ-aTNF' group), were compared with control vedolizumab patients in a retrospective 1:2 matched case-control study. RESULTS: Seventy-five patients were included (25 VDZ-aTNF, 50 VDZ). Adverse events were experienced by 9/25 VDZ-aTNF compared to 13/50 VDZ patients (P = 0.4, follow-up 14 weeks in all). Week 14 clinical remission was attained in 10/25 (40%) of VDZ-aTNF patients versus 23/50 (46%) of VDZ patients (OR = 0.8, 95% CI 0.3-2.1, P = 0.6) and clinical response in 19/25 (76%) versus 39/50 (78%) respectively (OR = 0.9, 95% CI 0.3-2.7, P = 0.8). Corticosteroid-free remission and corticosteroid-free response were experienced by 30% and 54%, respectively, of the entire cohort, and were similar between the two groups. Vedolizumab drug concentrations at week 2, 6 and 14 were similar among VDZ-aTNF and VDZ patients (P > 0.5). Multi-variable analysis showed independent association of some vedolizumab drug-levels time-points with baseline albumin and weight, but not with anti-TNF co-exposure. In a prospective study of a separate cohort of patients starting infliximab (n = 12), the percentage of α4ß7+ memory T cells, slightly but nonsignificantly increased throughout weeks 0, 2 to 14 (26 ± 2.3%, 27.8 ± 2.9%, 29.5 ± 2.6% respectively, P = 0.06). CONCLUSIONS: Vedolizumab/anti-TNF co-exposure did not generate new safety signals during 14-weeks induction, nor did it reduce efficacy or alter vedolizumab pharmacokinetics. These observations may aid the design of future co-biologics trials and also suggest that a deliberate waiting-interval between anti-TNF cessation and subsequent vedolizumab initiation may not be warranted.

Early drug and anti-infliximab antibody levels for prediction of primary nonresponse to infliximab therapy.

BACKGROUND: Primary nonresponse, defined as lack of clinical benefit during the induction phase, occurs in up to 30% of IBD patients treated with infliximab. The mechanisms underlying primary nonresponse have not yet been clearly defined. AIM: To evaluate the association of early (week 2 and week 6) induction infliximab and anti-infliximab antibody levels with primary nonresponse. METHODS: A retrospective observational case-control study of inflammatory bowel disease patients treated with infliximab and followed at Sheba Medical Center between 2009 and 2016 was performed. Pre-infusion infliximab and antibodies to infliximab (ATI) levels were measured by our previously described drug-tolerant ELISA assay. RESULTS: Thirty-five primary nonresponders have been identified and matched with 105 primary responders (1:3 ratios). Both week 2 and week 6 infliximab levels were significantly lower among primary nonresponders compared to responders (week 2, 6: median level 7.2, 2.2 µg/mL vs 13.5, 9.5 µg/mL, P = .0019, P < .0001 respectively). Antibodies to infliximab appeared more frequently (either week 2 or 6, 68% vs 28% prevalence, P = .0004) and at higher levels in nonresponders compared to responders (week 2, 6: median ATI 7.3, 10.8 µg/mL-eq vs 3.8, 4.4 µg/mL-eq, P = .005, P = .008 respectively). Moreover, week 2 infliximab levels <6.8 µg/mL (AUC = 0.68, P = .002, sensitivity 50%, specificity 86%) and antibodies to infliximab levels >4.3 µg/mL-eq (AUC = 0.78, P = .0004, sensitivity 77%, specificity 71%) were predictive of primary nonresponse. Among the other clinical and demographic variables, higher baseline ulcerative colitis clinical score, infliximab monotherapy, prior adalimumab therapy and previous Crohn's disease-related surgeries were also associated with an increased risk of primary nonresponse. CONCLUSIONS: Infliximab levels below 6.8 µg/mL and antibodies to infliximab levels above 4.3 µg/mL-eq before the second infusion are associated with primary nonresponse, especially among Crohn's disease patients.

Re-phrasing the question: A simple tool for evaluation of adherence to therapy in patients with inflammatory bowel disease.

BACKGROUND: Non-adherence to medication in patients with inflammatory bowel disease (IBD) is a challenging problem which is often overlooked or under-estimated by the physician or denied by the patient. We aimed to examine if re-phrasing the wording of the question used by the physician could help in revealing more patients who are non-adherent, and for whom appropriate counseling may be instituted. METHODS: A cross-sectional questionnaire-based study of IBD patients treated in a tertiary center was conducted. Patients received a questionnaire detailing their treatments and disease course, as well as their perceptions about disease. Two forms of questions about adherence were deliberately placed in two separate parts of the questionnaire: One was 'are you taking your medications regularly as prescribed?' (Standard question), and the second, more emphatic question, was 'how often does it happen that you miss a drug dosing?' (Re-phrased question). The rate of non-adherence disclosed by each of these questions was compared. Sensitivity, specificity and predicative values were computed for each question against the conventional definition of non-adherence as taking of less than 80% of prescribed medication doses disclosed by any of the methods. Predictors of non-compliance and of denying non-compliance were also explored. RESULTS: Overall, 165 patients were included (49% female, mean age 33.7 ± 12.7 SD, median age 30 years, 29.6% with ulcerative colitis, 62.4% with Crohn's disease). Upon questioning, 50 (30.3%) of the patients admitted to non-adherence in the last month when asked by the emphatic re-phrased question format, compared with only 10 patients (6%) reporting non-adherence when asked directly by the standard question (OR 7.4, 95%CI 3.6-15.2, p < 0.001). Thus, a 'Do you take your medicine regularly' question format disclosed only 20% of genuinely non-compliant patients and had 16% sensitivity and 98.2% specificity for revealing non-adherence (PPV 80%, NPV 72.9%) compared with the reference re-phrased question. The leading cause for non-adherence was skepticism about drug efficacy or safety (20.5%), followed by vacation or weekend (15%), problems with prescription or pharmacy (13.5%) and forgetfulness (10%). No single demographic or clinical factor correlated with non-adherence. The only factor which correlated with higher probability for non-adherence was biological and combination treatment. CONCLUSION: Non-compliance with treatment is much more common than patients admit. Asking patients how often does it happen that they miss a drug dosing is a simple, practical tool which performs significantly better in disclosing non-adherence compared with asking patients if they take their medication as they should.

Dust mite induces multiple polar T cell axes in human skin.

BACKGROUND: House dust mite/HDM atopy patch test/APT elicits positive reactions in a high fraction of atopic dermatitis/AD and healthy individuals. Experimental systems for new-onset/chronic AD are needed to support rapid therapeutic development, particularly since animal models representing human AD are lacking. While HDM APT has been considered to simulate AD, its suitability to model AD's emerging Th2/Th22 phenotype with Th1 and Th17 components is unknown. OBJECTIVE: To assess whether HDM APT reproduces AD. METHODS: Positive HDM APTs (n = 15) from patients with and without AD were evaluated, using genomic and immunohistochemistry studies, against intrapersonal control skin. RESULTS: APT lesions showed higher T cell and dendritic cell infiltrates vs. CONTROLS: Seven hundred and forty-three up- and 326 downregulated genes were differentially expressed in HDM APT (fold change >2 and false discovery rate < 0.05), with increased expression of Th2, Th9, Th17/Th22 polar cytokines (i.e. IL-5, IL-13, IL-9, IL-17, IL-22). CONCLUSION: While HDM caused significant Th2 skewing, it also illustrated differences in Th2 induction and barrier defects; thus, HDM APT does not fully simulate AD. Given its widespread availability and sensitization rates, HDM may potentially be a useful tool that represents select aspects of AD, psoriasis, or contact dermatitis.

Addition of an immunomodulator can reverse antibody formation and loss of response in patients treated with adalimumab.

BACKGROUND: Anti-adalimumab antibodies (AAA) are associated with loss of clinical response (LOR). Addition of an immunomodulator has been shown to reverse immunogenicity and regain response with infliximab monotherapy. Similar data on adalimumab are lacking. AIM: To study the impact of immunomodulator addition on the emergence of AAA and LOR among adalimumab therapy patients. METHODS: The databases of three tertiary medical centres were reviewed to identify patients who developed AAA during adalimumab monotherapy with resultant LOR, and received an immunomodulator as a salvage combination therapy. All sera were prospectively analysed using previously described ELISA assays. Clinical response was determined using appropriate clinical scores. Elimination of AAA, designated as 'sero-reversal', elevation of drug levels and regained clinical response were the sought outcomes. RESULTS: Twenty-three patients (21 Crohn's disease, and 2 ulcerative colitis) developed AAA with subsequent LOR and were thereafter prescribed an immunomodulator as salvage therapy (thiopurine n = 14, methotrexate n = 9). Eleven patients (48%) underwent sero-reversal with gradual elimination of AAA, increase in drug trough levels and restoration of clinical response (median time to sero-reversal 5 months). In 12 patients (52%), immunogenicity and loss of response could not be reversed. There was no difference between responders and nonresponders in the type of immunomodulators used or baseline clinical characteristics. CONCLUSIONS: In almost half of inflammatory bowel disease patients developing anti-adalimumab antibodies and loss of response, established immunogenicity of adalimumab can be gradually reversed by the addition of immunomodulator therapy with restoration of a clinico-biological response. However, these observations need to be confirmed with larger studies.

Induction infliximab levels among patients with acute severe ulcerative colitis compared with patients with moderately severe ulcerative colitis.

BACKGROUND: Infliximab is effective as salvage therapy for patients with steroid refractory acute severe ulcerative colitis (UC). Although current data suggest that the pharmacokinetics of infliximab are influenced by inflammatory burden in patients with acute severe UC, data comparing infliximab trough levels in patients with acute severe UC vs. moderately severe UC are scarce. AIM: To compare infliximab trough and anti-infliximab antibody levels at a standard fixed time-point during induction between patients with acute severe and moderately severe UC. METHODS: A multi-centre retrospective study comparing infliximab drug and antibody levels 14 days after the first infusion in hospitalised acute severe UC versus out-patients with moderately severe UC was performed. RESULTS: Sixteen acute severe UC patients, hospitalised between 2010-2015 and refractory to intravenous corticosteroids, were treated with infliximab 5 mg/kg salvage therapy. They were compared to 16 moderately severe UC out-patient controls. Mean infliximab trough levels at day 14 were significantly lower in patients with acute severe UC compared to moderately severe UC (7.15 ± 5.3 vs. 14.4 ± 11.2 µg/mL, P = 0.007). Seven patients (three acute severe and four moderate severe UC) were primary nonresponders to infliximab induction therapy. Infliximab level at day 14 did not differ between responders and nonresponders (9.8 ± 9 vs. 12.1 ± 10.6 µg/mL, respectively, P = N.S.). However, week 2 median antibody-to-infliximab levels were numerically higher among primary nonresponders (3.4 ± 5.7 vs. 1.2 ± 4 µg/mL-eq, respectively, P = 0.06). CONCLUSIONS: Infliximab trough levels at day 14 were lower in patients with acute severe UC compared to moderately severe UC, possibly due to a higher inflammatory burden and/or increased drug clearance. However, drug levels at day 14 were not lower among nonresponders compared with responders. Controlled trials are warranted to examine whether an a-priori-intensified infliximab induction protocol will lead to an improved outcome in acute severe UC.

Undetectable anti-TNF drug levels in patients with long-term remission predict successful drug withdrawal.

BACKGROUND: Low drug levels are associated with emerging loss of response to anti-TNF. However, this may not be the case in patients with long-term remission. AIM: To investigate the outcome of anti-TNF discontinuation in patients with long-term remission and incidental undetectable drug levels. METHODS: A retrospective cohort study examining the duration of relapse-free survival in IBD patients in remission who discontinued infliximab or adalimumab having undetectable drug levels. RESULTS: Forty eight patients who discontinued anti-TNF while in remission and had available drug levels were identified in two centres in France and Israel (infliximab-treated 35, adalimumab-13, Crohn's disease 30, ulcerative colitis 18, mean treatment duration of 22.7 ± 12.4 months). Endoscopy/MRE before stopping showed absence of active inflammation in 40/42 (95%) of evaluated patients, while inflammatory biomarkers (CRP and/or Calprotectin) were completely normal in only 31/48 (65%) of patients. During 12 months median follow-up, relapse occurred in 16/20 (80%) of patients who stopped anti-TNF while having measurable drug levels compared with 9/28 (32%) of patients who had undetectable drug levels (OR: 8.4, 95% CI: 2.2-32, P = 0.002). Relapse-free survival after anti-TNF cessation was significantly longer in patients with absent drug compared to those with detectable drug (P < 0.001, log rank test). On multivariate analysis, a patient's decision to stop therapy was weakly associated and abnormal inflammatory biomarkers and detectable drug levels were both strongly and independently associated with a higher risk of relapse after drug discontinuation. CONCLUSION: Incidental finding of undetectable anti-TNF drug levels in patients with stable long-term deep remission may identify a subset of patients whose clinical remission is no longer dependent on anti-TNF treatment.

Regional seroprevalence of Cryptosporidium parvum-specific IgG of cats in the United States.

The objective of this study was to determine the regional prevalence of Cryptosporidium parvum-specific IgG in the sera of cats in the United States. The continental United States was partitioned into eight regional areas. Serum samples from 75 cats from each region were assayed for C. parvum-specific IgG using an indirect enzyme-linked immunosorbent assay (ELISA). Age, sex, breed, and indoor/outdoor status were examined as possible risk factors for developing a positive C. parvum-specific IgG antibody titer. The presence of gastro-intestinal signs and Toxoplasma gondii-specific IgG in the serum were also evaluated for association with C. parvum seropositivity. Of the 600 samples assayed, 50 (8.3%) were positive for C. parvum-specific IgG. Regional seroprevalence ranged from 1.3% in the mid-Atlantic states to 14.7% in the south-eastern states. The oldest group of cats (>10 years) had the highest seroprevalence (15.3%). The prevalence of C. parvum-specific IgG was higher among male (10.1%) than among female cats (6.9%), although, the difference was not statistically significant (p = 0.17). Seropositivity was not associated with pure-bred status. C. parvum-specific IgG antibodies was detected most frequently in T. gondii-specific IgG seropositive cats, outdoor cats, and cats with gastro-intestinal signs. These results suggest that cats in the United States are commonly exposed to C. parvum.

Enzyme-linked immunosorbent assay for the detection of Cryptosporidium parvum IgG in the serum of cats.

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium parvum IgG in the serum of cats. The ELISA was an indirect ELISA using soluble C. parvum oocyst antigens and a peroxidase-labeled anti-feline IgG secondary antibody. Sera from cats with Toxocara felis, Giardia spp., Aelurostrongylus abstrusus, Isospora felis, Isospora rivolta, Toxoplasma gondii, or Taenia spp. infections were assayed in specificity studies. Following optimization, the ELISA and fecal examination for oocysts were performed on samples from 170 client-owned or humane society source cats and 1 cat inoculated orally with C. parvum oocysts. Cryptosporidium parvum oocysts were detected in feces (4/170; 2.4%), and C. parvum IgG was detected in serum (26/170; 15.3%) from naturally exposed cats. The seroprevalence data suggest that some cats in the geographical area studied were exposed to C. parvum, but persistent oocyst shedding was less common. The ELISA is not useful for predicting oocyst shedding in individual cats.

Comparison of sequential, random, and hemacytometer methods for counting Cryptosporidium oocysts.

In animal and clinical studies of Cryptosporidium infection, counting Cryptosporidium oocysts in collected fecal samples is often critical to interpretation of results and validity of statistical analyses. In this experiment, 3 methods of counting oocysts derived from feces of infected mice were compared. These included sequential counting of oocysts in 25 adjacent microscopic fields, random counting of oocysts in 25 scattered microscopic fields, and hemacytometer counting of oocysts using standard procedures. Numerical results are expressed as the numbers of oocysts per microscopic field for sequential and random counting methods or per microliter for the hemacytometer method. Our results showed that the numbers of oocysts adjusted for volume and weight, or for volume alone, were similar for all 3 techniques. Linear relationships between the 3 methods allow use of linear regression to convert the number of oocysts counted by 1 method into those counted by another. This suggests that comparisons between different experiments in which Cryptosporidium oocysts are counted using any of these methods are both possible and valid.

Increased exposure to cryptosporidia among dairy farmers in Wisconsin.

Cryptosporidium infection is an important cause of diarrhea in humans and livestock; no effective therapy is known. A self-administered questionnaire and an ELISA were used to assess the risk of exposure to cryptosporidia among 70 dairy farmers and 50 who were not dairy farmers in Wisconsin. Dairy farmers (44.3%) were more likely to be seropositive for cryptosporidia than were other persons (24.0%; relative risk = 1.9). Among dairy farmers, age > or = 50 and use of a canister method of milking were associated with seropositive status. Among persons who were not dairy farmers, feeding or milking cows was associated with being seropositive. These findings suggest that dairy farmers and other persons who have contact with cattle are at greater risk of Cryptosporidium infection than are persons who do not have such contact. Identification and avoidance of farming practices associated with Cryptosporidium infection may reduce the risk of infection among dairy farmers.

Genetic analysis of HIV-1 isolates from Zambia and an expanded phylogenetic tree for HIV-1.

Geographic variation in the HIV-1 virus is extensive but incompletely documented. We herein report the first genetic characterization of HIV-1 isolates from Zambia. The genomic region encoding the GAG polyprotein has been compared among 22 Zambian isolates and 14 North American isolates using a combination of polymerase chain reaction (PCR) and DNA sequencing methods. The Zambian isolates were similar to one another but distinct from other HIV-1 isolates. They exhibited a characteristic PCR "fingerprint" wherein certain primer combinations were unable to amplify because of mispairing. The sequence of the complete gag gene of three isolates from Zambia has been determined, and phylogenetic tree analysis placed them in a branch distinct from other African isolates and North American isolates. The PCR procedure used here may be widely applicable for genetic characterization of HIV-1.

In vitro screening of therapeutic agents against Cryptosporidium: hyperimmune cow colostrum is highly inhibitory.

An in vitro model of Cryptosporidium parvum infection was developed utilizing an adherent human intestinal epithelial cell line HT29.74. The efficacy of potential immunologic therapy in the form of Cryptosporidium-specific hyperimmune bovine colostrum was evaluated for the ability to inhibit in vitro infection. Oocysts were purified from stool of chronically infected AIDS patients. Hyperimmune colostrum obtained from cows immunized with Cryptosporidium and nonimmune conventional colostrum were evaluated. oocysts (10(5)-10(6)) were pre-incubated with either hyperimmune colostrum, conventional colostrum, or saline as control, for 15 min at room temperature than applied to a 70% confluent monolayer of HT29.74 cells. Cryptosporidium schizonts were identified and counted per 1,000 HT29.74 cells under oil immersion after 24 h. In the presence of hyperimmune colostrum, parasite infection was inhibited by 82% (p less than 0.001), and the presence of conventional colostrum, infection was inhibited by 67% (p less than 0.001). Treatment with the soluble fraction of hyperimmune colostrum resulted in 69% inhibition (p less than 0.001) compared to the soluble fraction of conventional colostrum which resulted in only 17% inhibition (p = NS). In vitro Cryptosporidium parvum infection of the differentiated human enterocyte cell line HT29.74 is a viable method for screening immunologic therapies. Hyperimmune bovine colostrum was highly inhibitory of Cryptosporidium infection in vitro and its soluble fraction remained significantly inhibitory while the soluble fraction of conventional colostrum did not.

An alga-like organism associated with an outbreak of prolonged diarrhea among foreigners in Nepal.

An unidentified organism was found in the stools of 55 immunocompetent patients who presented to the CIWEC Clinic in Kathmandu, Nepal between June and November 1989. The microscopic features of the organism share characteristics of both coccidia and cyanobacteria species. From June 26, 1989 to November 17, 1989, 55 persons were identified as having the organism in at least one stool sample. The illness was characterized by prolonged watery diarrhea, anorexia, fatigue, and weight loss. The mean +/- SD duration of illness was 43 +/- 24 days (range 4-107). Thirty-four patients received a total of 78 courses of antimicrobial treatment (2.3 courses/patient). The mean +/- SD duration of illness in 34 treated patients was 46 +/- 24 days. In 14 untreated patients, the mean +/- SD duration of illness was 35 +/- 23 days. The organism is 8.0-9.0 microns in diameter, floats in Sheather's solution, and stains red with the modified acid-fast stain. Since the agent was closely associated with a prolonged, self-limited diarrheal illness, it could easily have been misdiagnosed as Cryptosporidium. The organism should be looked for in the stools of patients with persistent diarrhea and a history of foreign travel.

Cryptosporidium infection in an adult mouse model. Independent roles for IFN-gamma and CD4+ T lymphocytes in protective immunity.

Cryptosporidium is a protozoan parasite that can cause chronic life-threatening diarrhea in immunocompromised persons. Host immune responses are poorly understood, an impediment to development of effective therapy. In mice, normal adult BALB/c animals resist infection whereas chronic symptomatic cryptosporidiosis develops in adult nude mice and in neonatally infected BALB/c mice treated with anti-CD4 mAb. To define further the immune defects that allow mice to be infected with Cryptosporidium, adult BALB/c mice were treated with cytolytic anti-CD4 or anti-CD8 or with neutralizing anti-IFN-gamma or anti-IL-2 mAb. Chronic infection, manifested by continuous shedding of sparse but statistically significant numbers of oocysts, occurred with anti-CD4 +/- anti-CD8 mAb treatment although anti-CD8 mAb treatment alone did not allow infection. Treatment with anti-IFN-gamma mAb greatly enhanced oocyst shedding but infection was self-limited. Treatment with a combination of anti-CD4 and anti-IFN-gamma mAb permitted both chronic infection and shedding of large numbers of oocysts. Furthermore mice treated initially with anti-CD4 mAb showed a substantial increase in oocyst shedding when later treated with anti-IFN-gamma mAb; and mice treated initially with both mAbs showed a decline in oocyst shedding when anti-IFN-gamma mAb was stopped. Anti-IFN-gamma mAb treatment of congenitally athymic adult BALB/c mice led to an approximately a 75-fold increase in oocyst shedding. Treatment of adult BALB/c mice with anti-IL-2 mAb did not permit Cryptosporidium infection. These results suggest that redundant immunologic mechanisms limit Cryptosporidium infection such that both CD4+ cells and IFN-gamma are required to prevent initiation of infection whereas either alone can limit the extent (IFN-gamma) or duration (CD4+ T cells) of infection. They also suggest that production of IFN-gamma by a non-T cell contributes to host immunity.

Detection of Giardia lamblia antigen in children living in a Peruvian periurban shantytown (Pueblo Joven).

Stool microscopy and an enzyme-linked immunosorbent assay (ELISA) for Giardia lamblia antigen detection were compared for detecting G. lamblia in 30 Peruvian infants. Of 1,131 fecal specimens, G. lamblia was detected by ELISA alone in 44, by microscopy alone in 17, and by both methods in 91. In another group of 17 children negative for G. lamblia by stool microscopy, 6 had G. lamblia detected by ELISA or duodenal aspiration: 2 only by ELISA, 1 only by duodenal aspirate examination, and 3 by both examinations. The ELISA is useful for the detection of G. lamblia in fecal specimens but compared to stool microscopy does not significantly increase the detection of cases.

Genetic comparison of human immunodeficiency virus (HIV-1) isolates by polymerase chain reaction.

Amplification of DNA by polymerase chain reaction (PCR) is influenced by the homology of oligonucleotide primers with the DNA template. We have developed a procedure, termed anchored PCR, whereby nucleotide sequence alterations in the template can be directly related to the quantity of amplified product. Genetic variation in the human immunodeficiency virus HIV-1 has been studied using anchored PCR. In four field isolates of the virus, the 3'LTR was compared both by PCR analysis of DNA from virus cultures and DNA sequencing. DNA templates that matched the primers varied less than threefold in PCR product yield, whereas significant 3' end primer-template mispairing decreased PCR product 10- to 100-fold. Using these guidelines for genetic variability manifested through PCR, 40 PCR primers encompassing the GAG, ENV, and 3' LTR segments of the genome were used to compare sequential HIV-1 isolates form six patients. Some primers were apparently located in genomic regions without significant interisolate variability, as they yielded equivalent amounts of amplified DNA from all the isolates. The quantity of amplified DNA obtained with other primers varied 10- to 100-fold among patients, but was consistent for sequential isolates from an individual patient. Two African HIV-1 isolates were readily distinguished from a panel of North American isolates by the same method. Systematic classification of HIV-1 genetic variants may be possible by anchored PCR.

Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens.

Cryptosporidium sp. is a ubiquitous 4- to 6-micron protozoan parasite infecting the intestinal tract of humans. It causes mild to fulminant diarrhea in patients, especially immunocompromised persons, and it may be hard to detect by microscopic fecal examination. An indirect, double-antibody enzyme-linked immunosorbent assay (ELISA) was developed using specifically produced goat and rabbit antisera to detect Cryptosporidium antigens in human feces. Of 62 frozen stools from patients with cryptosporidiosis, as detected by at least two microscopic diagnostic techniques, 51 were positive by ELISA; all ELISA-negative specimens came from patients with fewer than five oocysts per 0.01 ml of concentrated fecal sample examined after modified acid-fast or fluorescent monoclonal antibody staining. A total of 182 specimens from persons without Cryptosporidium infection were negative by ELISA in 176 instances; 3 ELISA-positive specimens came from patients with cryptosporidiosis diagnosed earlier. The sensitivity of the assay was 82.3%, and specificity was 96.7%. The predictive value of a positive ELISA was 89.5%, and the predictive value of a negative ELISA was 94.2%. The ELISA was not affected by the presence of eight other intestinal parasites but was sometimes affected by repeated freezing and thawing of fecal specimens. All fecal specimens were heated to 100 degrees C for 2 min to reduce proteolytic enzyme activity, although the necessity of this step needs further evaluation. This first-generation ELISA is a simple, rapid, easily standardized test for Cryptosporidium antigens in stool samples which will be useful for diagnosis and for large-scale epidemiologic studies.

Association of serum antibodies to adherence lectin with invasive amebiasis and asymptomatic infection with pathogenic Entamoeba histolytica.

The recognition of the Entamoeba histolytica galactose-inhibitable adherence lectin by antibodies was studied using sera obtained from subjects in South Africa with an amebic liver abscess or asymptomatically pathogenic or nonpathogenic E. histolytica infection and from uninfected regional controls. In addition, sera from healthy American controls or Americans known to be infected with other parasites were studied. Of the 95 sera containing antibodies to total parasite protein, 95% demonstrated antibodies to the 170-kDa heavy subunit but not to the 35-kDa light lectin subunit. All sera (n = 253) were tested by ELISA for antibodies to lectin: 99% from liver abscess patients and all 4 from individuals asymptomatically infected with pathogenic E. histolytica were positive; all from the 40 healthy American controls and the 29 infected with other parasites were negative (P less than .01). The prevalence of serum anti-lectin antibodies was identical (25%) in asymptomatic South Africans with either a nonpathogenic infection or a negative stool culture for E. histolytica. Thus, the presence of serum antibodies to lectin seems to indicate current or prior invasive amebiasis or asymptomatic intestinal infection with pathogenic E. histolytica.