Modelling the long-acting administration of anti-tuberculosis agents using PBPK: a proof of concept study.

SETTING: Anti-tuberculosis formulations necessitate uninterrupted treatment to cure tuberculosis (TB), but are characterised by suboptimal adherence, which jeopardises therapeutic efficacy. Long-acting injectable (LAI) formulations or implants could address these associated issues. OBJECTIVE: niazid, rifapentine, bedaquiline and delamanid-in adults for treatment for latent tuberculous infection (LTBI). DESIGN: PBPK models were developed and qualified against available clinical data by integrating drug physicochemical properties and in vitro and population pharmacokinetic data into a mechanistic description of drug distribution. Combinations of optimal dose and release rates were simulated such that plasma concentrations were maintained over the epidemiological cut-off or minimum inhibitory concentration for the dosing interval. RESULTS: The PBPK model identified 1500 mg of delamanid and 250 mg of rifapentine as sufficient doses for monthly intramuscular administration, if a formulation or device can deliver the required release kinetics of 0.001-0.0025 h-1 and 0.0015-0.0025 h-1, respectively. Bedaquiline and isoniazid would require weekly to biweekly intramuscular dosing. CONCLUSION: We identified the theoretical doses and release rates of LAI anti-tuberculosis formulations. Such a strategy could ease the problem of suboptimal adherence provided the associated technological complexities for LTBI treatment are addressed.

Seroepidemiology of Toxoplasma in a coastal region of Haiti: multiplex bead assay detection of immunoglobulin G antibodies that recognize the SAG2A antigen.

Toxoplasma gondii is a globally distributed parasitic protozoan that infects most warm-blooded animals. We incorporated a bead coupled with recombinant SAG2A protein into our Neglected Tropical Disease (NTD) multiplex bead assay (MBA) panel and used it to determine Toxoplasma infection rates in two studies in Haiti. In a longitudinal cohort study of children aged 0-11 years, the infection rate varied with age reaching a maximum of 0·131 infections/year in children aged 3 years [95% confidence interval (CI) 0·065-0·204]. The median time to seroconversion was estimated to be 9·7 years (95% CI 7·6-∞). In a cross-sectional, community-wide survey of residents of all ages, we determined an overall seroprevalence of 28·2%. The seroprevalence age curve from the cross-sectional study also suggested that the force of infection varied with age and peaked at 0·057 infections/year (95% CI 0·033-0·080) at age 2·6 years. Integration of the Toxoplasma MBA into NTD surveys may allow for better estimates of the potential burden of congenital toxoplasmosis in underserved regions.

Ultrafast strain-induced current in a GaAs Schottky diode.

Picosecond acoustic pulses generated by femtosecond laser excitation of a metal film induce a transient current with subnanosecond rise time in a GaAs/Au Schottky diode. The signal consists of components due to the strain pulse crossing the edge of the depletion layer in the GaAs and also the GaAs/Au interface. A theoretical model is presented for the former and is shown to be in very good agreement with the experiment.

Quantification of Cryptosporidium parvum oocysts in mouse fecal specimens using immunomagnetic particles and two-color flow cytometry.

Although single-color flow cytometry has been shown to be more sensitive than fluorescence microscopy for the quantification of Cryptosporidium parvum oocysts, this method has not been optimized. Monoclonal antibody OW50, specific to the cell wall of oocysts, was conjugated to superparamagnetic particles, to fluorescein isothiocyanate, and to r-phycoerythrin. The oocysts were then double stained with the fluorochrome-labeled OW50 and were placed in tubes with known numbers of highly fluorescent polystyrene beads, allowing quantification of the oocysts without dependence on acquired sample volume by flow cytometry. Data from 2-color flow cytometry using logical gating of the oocysts and beads showed a linear relationship between dilutions of a purified oocyst suspension and the mean numbers of oocysts detected (r2 = 1.00). An average of 15 purified oocysts/ml were counted in a dilution with a theoretical concentration of 12 oocysts/ml. Known numbers of purified oocysts were seeded into normal mouse fecal specimens, captured by OW50-labeled immunomagnetic particles, eluted with 5% potassium dichromate at low pH, and double stained with fluorochrome-labeled OW50. By flow cytometry, the mean recovery was 43.1% (+/-8.3%), and as few as 133 oocysts were detected. The captured and eluted oocysts were infective in neonatal BALB/c mice. This 2-color flow cytometry method, used in conjunction with the capture and elution of oocysts by and from immunomagnetic particles, provides a powerful tool for not only the quantification and purification of C. parvum oocysts from different sources but also for the characterization of oocysts in vitro and in vivo.

Cross-sensitivity and the anticonvulsant hypersensitivity syndrome.

The anticonvulsant hypersensitivity syndrome is a rare complication that occurs with the use of antiepileptic medications. Although phenytoin is the most common culprit, carbamazepine and phenobarbital are known to cause a similar reaction. A familial occurrence has been reported. We present a case of the anticonvulsant hypersensitivity syndrome to emphasize the importance of recognizing the multiple clinical components of the syndrome and to raise awareness of the cross-sensitivity among anticonvulsants metabolized via arene oxide metabolites.

Detection by enzyme immunoassay of serum immunoglobulin G antibodies that recognize specific Cryptosporidium parvum antigens.

Human infection with Cryptosporidium parvum usually elicits characteristic immunoglobulin G (IgG), IgA, and IgM antibody responses against two sporozoite surface antigens with apparent molecular masses of approximately 27 and 17 kDa. We have determined that these two antigens are actually complex families of related antigens. We have developed two new enzyme-linked immunosorbent assays (ELISAs) for the detection and quantitation of serum IgG antibodies against both antigens. The assays utilize a recombinant form of the 27-kDa antigen and a partially purified native fraction isolated from sonicated whole oocysts that contains 17-kDa antigen. An immunoblot assay previously developed in our laboratory served as the reference, or "gold standard," seroassay for the assessment of the new ELISAs. Positive responses with the recombinant-27-kDa-antigen ELISA were correlated with the immunoblot results for the 27-kDa antigen, with a sensitivity and specificity of 90 and 92%, respectively. Similarly, positive responses with the partially purified native-17-kDa-antigen ELISA correlated with the immunoblot results for the 17-kDa antigen, with a sensitivity and specificity of 90 and 94%, respectively. For both ELISAs the median IgG antibody levels for serum sets collected during outbreaks of waterborne C. parvum infection were at least 2.5-fold higher than the levels determined for a nonoutbreak set. Using the immunoblot as the "gold standard," the new ELISAs were more specific and, in the case of the 27-kDa-antigen ELISA, more sensitive than the crude oocyst antigen ELISA currently in use. These assays will be useful in future epidemiologic studies.

Flow cytometric analysis of microsporidia belonging to the genus Encephalitozoon.

Flow cytometry was used in the identification of human microsporidia belonging to the genus Encephalitozoon. Microsporidian spores of Encephalitozoon hellem, E. cuniculi, and E. intestinalis were propagated in axenic cultures of monkey kidney E6 cells, purified with Percoll, and exposed to homologous and heterologous rabbit antiserum and monoclonal antibody prepared against E. hellem spores. After reaction to goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to fluorescein isothiocyanate, fluorescence histograms from gated data on light-scatter profiles showed that rabbit anti-E. hellem serum was reactive to E. hellem spores but also had cross-reactivity to spores of E. cuniculi and E. intestinalis. On the other hand, fluorescence histograms showed that rabbit anti-E. cuniculi and rabbit anti-E. intestinalis sera were reactive with homologous spores only. Monoclonal antibody prepared against E. hellem reacted only with spores of E. hellem. Neither the polyclonal antibodies nor the monoclonal antibodies reacted with Cryptosporidium parvum oocysts. Fluorescence histograms of spores treated with 10% formalin also showed reactivity, but the number of events in the most intense peaks of fluorescence was fewer (7 to 42%, depending on species) than the number of events in the most intense peaks of fluorescence for nontreated spores. By flow cytometry, formalin-treated and nontreated spores of Encephalitozoon were identified to the species level by using gated data on light-scatter profiles and analyzing the fluorescence histograms from the indirect immunofluorescence of the spores. Once a procedure is established for the isolation of Encephalitozoon spores from clinical specimens, identification of spores by flow cytometry may be useful not only for diagnosis but also for epidemiologic studies.

A two-year follow-up survey of antibody to Cryptosporidium in Jackson County, Oregon following an outbreak of waterborne disease.

To estimate the duration of Cryptosporidium-specific antibody, a Western blot assay measured antibody in paired sera from 124 residents of Jackson County, Oregon collected 0.5 and 2.5 years after the end of an outbreak in Talent, Jackson County. The outcome measure was the intensity of antibody responses, (which may approximate to a titre), to 27-kDa and 15/17-kDa antigens. Intensity of response to the 27-kDa antigen(s) declined to 54% of the 1992 value while responses to a 15/17-kDa antigen(s) remained close to the initial values. Increasing age of the donor predicted higher intensity of antibody to the 15/17-kDa antigen(s) in both the initial (P = 0.004) and follow-up (P = 0.038) surveys. No relationship was observed between age and antibody intensity for the 27-kDa antigen(s) during either survey (P > 0.10). Both the initial and follow-up surveys showed significant elevations in antibody intensity for Talent residents, possibly indicating a high endemic rate of infection/re-infection or high levels of chronic infection.

Comparisons of ELISA and Western blot assays for detection of Cryptosporidium antibody.

A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurred in Talent, Oregon 6 months earlier. The ELISA, which tested for combined IgG, IgA and IgM, and the WB, which tested separately for IgG and IgA, detected an almost twofold increase in serological response for persons who consumed Talent drinking water during the previous 11 months. The increases, however, were statistically significant (P < 0.05) only for the WB. The identification of serological evidence of infection, using sera collected 6 months after the end of the outbreak in a population not selected because of cryptosporidiosis-like illness, suggests that assays of Cryptosporidium-specific IgG and IgA may assist in estimating the magnitude of asymptomatic infections in the population.

The antibody response to 27-, 17-, and 15-kDa Cryptosporidium antigens following experimental infection in humans.

Previous studies have suggested that persons infected with Cryptosporidium parvum develop antibody responses to 27-, 17-, and 15-kDa C. parvum antigens. Studies of volunteers infected with Cryptosporidium species provided an opportunity to evaluate the relationship between antibody reactivity to these antigens and infection outcome. As monitored by immunoblot, increases in specific antibody reactivity were more prevalent among volunteers who developed signs and symptoms of cryptosporidiosis (n = 11) than among asymptomatic infected (n = 7; P = .05) or oocyst-negative volunteers (n = 11; P = .02). Volunteers with preexisting IgG antibody to the 27-kDa antigen excreted fewer oocysts than volunteers without this antibody (P = .003). IgG reactivity to the 17-kDa antigens and IgM reactivity to the 27-kDa antigens were higher at day 0 for asymptomatic infected persons than for those who developed symptoms (P = .03 and P = .04, respectively). These results suggest that characteristic antibody responses develop following C. parvum infection and that persons with preexisting antibodies may be less likely to develop illness.

Ultrastructure, immunofluorescence, western blot, and PCR analysis of eight isolates of Encephalitozoon (Septata) intestinalis established in culture from sputum and urine samples and duodenal aspirates of five patients with AIDS.

Microsporidia are ancient, intracellular, eukaryotic protozoan parasites that form spores and that lack mitochondria. Currently, as many as eight species included under six genera are known to infect humans, mostly patients with AIDS. Among these, Enterocytozoon bieneusi, the agent of gastrointestinal (GI) disease, is the most frequently identified microsporidian in clinical laboratories in the United States. Encephalitozoon (Septata) intestinalis, the agent that causes a disseminated infection including infection of the GI tract, is the second most frequently identified microsporidian parasite. In spite of this, not many isolates of E. intestinalis have been established in culture. We describe here the continuous cultivation of eight isolates of E. intestinalis obtained from different samples including the urine, sputum, and duodenal aspirate or biopsy specimens from five AIDS patients originating from California, Colorado, and Georgia. The specific identification was made on the bases of ultrastructural, antigenic, and PCR analyses.

Enzyme-linked immunoelectrotransfer blot analysis of a cryptosporidiosis outbreak on a United States Coast Guard cutter.

Symptoms consistent with an outbreak of cryptosporidiosis (diarrhea, vomiting, nausea, and abdominal cramps) occurred on a U.S. Coast Guard cutter within 0-18 days after the cutter filled its tanks with Milwaukee, Wisconsin city water in March 1993. At three-weeks postdocking (PD), the suspected water was removed, and serum samples and stool specimens were collected from 47 of the 58 crew members, as well as questionnaire data on their water consumption and symptoms aboard the cutter. At 10-weeks PD and/or at 28-weeks PD, additional serum specimens were collected. Intensitometric data from enzyme-linked immunoelectrotransfer blot (EITB) were obtained on IgA responses to a 17-kD antigen group, IgM responses to a 27-kD antigen group, and IgG responses to 27-, 17-, and 15-kD antigen groups extracted from oocysts. In addition, IgG responses to crude oocyst antigens were obtained by ELISA. Based on reported symptoms, EITB results, and stool examination, the crew members were classified as confirmed (10), probable (10), suspected (22), and noncases (16). Of the 10 confirmed cases (all symptomatic) and the 10 probable cases (eight symptomatic) whose stools were positive and negative, respectively, for Cryptosporidium oocysts by microscopy, all showed changes in EITB intensities to the antigen groups and were considered EITB positive. The remaining 38 crew members, 22 suspected cases (all symptomatic), and 16 noncases (all asymptomatic), if tested, had negative stool examinations and were considered EITB negative. Of the 10 confirmed cases, only four showed a significant change in IgG responses (P < 0.05) between three-weeks PD and follow-up serum specimens by ELISA. Crew members considered confirmed cases consumed significantly more water (P < or = 0.005) aboard the cutter than noncases. Crew members considered EITB positive consumed more water (P < or = 0.04) than crew members considered EITB negative while there was no significant difference in water consumption (P > or = 0.19) between crew members considered ELISA positive and ELISA negative. Using the EITB, the observation of changes in intensity of IgA responses to the 17-kD antigen group, IgM responses to the 27-kD antigen group, and IgG responses to the 27- 17-, and 15-kD antigen groups from C. parvum oocysts between acute and convalescent serum specimens appears useful for immunodiagnosis of Cryptosporidium infection and for prospective epidemiologic studies designed to monitor infection risk.

A case of Morgagni hernia presenting as pneumonia.

Morgagni-type diaphragmatic hernias are rare. The overwhelming majority are discovered in children who usually present with pneumonia or sepsis. We report an unusual case of a 57-yr-old woman with a Morgagni hernia presenting with pulmonary symptoms. Complicating the clinical picture, the pneumonia delayed the definitive diagnosis until a lateral chest X-ray study revealed loops of bowel in the right lower lung fields. It is important to entertain abdominal etiologies in the differential diagnosis of a thoracic density.

Proliferative responsiveness of lymphocytes from Cryptosporidium parvum-exposed mice to two separate antigen fractions from oocysts.

We assessed lymphoproliferative responsiveness of lymphocytes from the spleen and lymph nodes of inbred neonatal SWR/J H-2q mice at various times postinoculation (PI) using Cryptosporidium parvum oocysts. The lymphocytes were cultured in vitro with a water-soluble (SO) and a water-insoluble (IN) antigen fraction. The IN fraction was prepared by solubilizing particulates, the sediment obtained after centrifuging disrupted oocysts, in urea. Both fractions were characterized using silver stain and enzyme-linked immunoelectrotransfer blot (EITB) with hyperimmune rabbit anti-oocyst serum, monoclonal antibody specific to a 23-kD antigen, and serum from patients with symptoms of cryptosporidiosis. The EITB showed that the antigens in the IN fraction differed both quantitatively and qualitatively from those in the SO fraction. Lymphocytes from lymph nodes of exposed mice cultured with the SO fraction exhibited a significant (P < 0.05) and antigen-specific response compared with those from unexposed mice at days 10, 19, 22, and 28 PI. The response to the IN fraction of lymphocytes from lymph nodes of exposed mice was not as consistent as that to the SO fraction but showed a significant (P < 0.05) and antigen-specific response at days 10 and 19 PI. No significant response occurred when splenic lymphocytes were cultured with SO or IN fractions. These results show that lymphocytes from lymph nodes of mice exposed to Cryptosporidium parvum oocysts proliferate when cultured in vitro with soluble or particulate antigens prepared from oocysts.

Internment and ministry: A dialogue with Joseph Kitagawa.

This dialogue presents a profile of the late Joseph Kitagawa-a renowned scholar of the history of religions (Religionswissenschaft). It focuses on comparative religion and philosophy, as well as several other important issues related to his distinguished career as an Episcopal priest and dean of the Divinity School of the University of Chicago. They are: his experience of American concentration camps during World War II; Christian atheism and new theological models; concepts of time in Oriental and Occidental faiths; depth-psychology and contemporary ministry; and Paul Tillich's significance for the pastoral counseling movement.

Karl menninger: A centennial tribute.

This tribute primarily focuses on Karl Menninger's literary skills as a preacher. His last two books,Sparks and Whatever Became of Sin? are used as examples of what Reinhold Niebuhr called "vital prophetic Christianity." They clearly illustrate Dr. Menninger's homiletical style. These evaluations are augmented by personal conversations with him during which he strongly encouraged pastoral counselors to refocus their attention on the sermon as a basic resource for prevention and hope.

Isoenzyme comparison of axenic Giardia lamblia strains.

We obtained isoenzyme patterns by polyacrylamide gradient gel electrophoresis (PGGE) of water-soluble protein fractions prepared from trophozoites of 11 axenic G. lamblia strains. The strains were isolated from animals and humans (both symptomatic and asymptomatic) from various geographic locations. Isoenzymes were also separated by isoelectric focusing. Of 12 enzymes attempted, eight exhibited well-defined and reproducible isoenzyme patterns by PGGE, based on which the strains were grouped into four zymodemes. Although the 11 strains were grouped into four zymodemes based on PGGE, no correlation between zymodeme and the known characteristics of the strains existed. Thus, a high degree of characteristic sharing appears to occur among genetically different G. lamblia strains.

Isolation of two Giardia lamblia (WB strain) clones with distinct surface protein and antigenic profiles and differing infectivity and virulence.

To determine the relationship between antigenic profiles and pathogenicity among Giardia lamblia clones (WB strain), trophozoites were cloned by the technique of limiting dilution. The phenotype of each clone was determined by an indirect immunofluorescence test using a polyclonal rabbit anti-G. lamblia trophozoite serum made against the parent strain. Two clones were chosen for further studies: a highly fluorescent clone, F+, in which more than 95% of the trophozoites fluoresced, and a low-fluorescence clone, F-, in which fewer than 5% fluoresced. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and enzyme-linked immunotransfer blot studies of the membrane fractions of the two clones and parent strain revealed differences in both the total protein and antigenic profiles. A serum cytotoxicity test with the polyclonal serum showed that the F+ clones were more susceptible to immobilization and killing, while the majority of cells of the F- clones were resistant to such killing. Assessment of the infectivity of the two clones in the Mongolian gerbil animal model indicated that the F- clone more readily initiated infections, produced more cysts, had a higher intestinal trophozoite load, and produced a more severe clinical syndrome, while the F+ clone was less phenotypically stable in vivo and in some cases took longer to be cleared from the intestine.

Purification and characterization of Giardia lamblia antigens in the feces of Mongolian gerbils.

In a recent study, we identified Giardia lamblia 65- and 70-kDa antigens in the feces of infected Mongolian gerbils. The 65-kDa antigen was from a strain isolated from a human with symptoms of giardiasis, and the 70-kDa antigen was from a strain isolated from a human with no symptoms of giardiasis. In this study, we used preparative electrophoresis and electroelution techniques to purify these antigens to a degree which showed a single discrete protein band on silver-stained polyacrylamide gels. By enzyme-linked immunoelectrotransfer blot, common epitopes on the 65- and 70-kDa antigens were indicated by their cross-reactivity with rabbit anti-65-kDa and anti-70-kDa sera. By indirect immunofluorescence assay, the cysts and trophozoites of the two strains cross-reacted with these sera. Of seven lectins tested, only concanavalin A bound to the 70-kDa antigen, suggesting a glycoprotein, and it possessed a low isoelectric point as assessed by preparative isoelectric focusing. Molecular mass estimations of these antigens by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis were similar to the 65- and 70-kDa estimations obtained by native polyacrylamide gradient gel electrophoresis. Although the 65- and 70-kDa antigens proved to be resistant to 100 degrees C heat and stable in storage for up to 25 months at -20 degrees C, neither appeared to be the same as a fecal G. lamblia antigen with similar molecular mass found by other investigators. This suggests that variable G. lamblia antigens may be found in the feces of infected humans.