Baylisascaris procyonis: an emerging helminthic zoonosis.

Baylisascaris procyonis, a roundworm infection of raccoons, is emerging as an important helminthic zoonosis, principally affecting young children. Raccoons have increasingly become peridomestic animals living in close proximity to human residences. When B. procyonis eggs are ingested by a host other than a raccoon, migration of larvae through tissue, termed larval migrans, ensues. This larval infection can invade the brain and eye, causing severe disease and death. The prevalence of B. procyonis infection in raccoons is often high, and infected animals can shed enormous numbers of eggs in their feces. These eggs can survive in the environment for extended periods of time, and the infectious dose of B. procyonis is relatively low. Therefore, the risk for human exposure and infection may be greater than is currently recognized.

Permanence of Fungi-Fluor epifluorescence stain to detect microsporidia.

OBJECTIVE: Epifluorescence microscopy, a methodology for the screening of bodily fluids and tissue specimens for microsporidia species, was directed to evaluate the retention of epifluorescence of fixed and stained specimens over time. METHODS: Thirty samples of stool, bodily fluids, duodenal touch preparations, and biopsies, were tested for the retention of their epifluoresence using the Fungi-Fluor procedure. Specimens were examined under a 330- to 380-nm UV filter at the time of preparation, 3 wk later, and then at monthly intervals for 18 months. All specimens were reevaluated for the presence or absence of fluorescence and any decrement of fluorescence over time. No special preservation techniques were used on any of the slides. RESULTS: All 30 specimens maintained their epifluorescence from the time of slide preparation to 18 month later. No decrement in fluorescence was noted in any sample examined. Accuracy and ease of spore identification was maintained. CONCLUSIONS: Epifluorescence microscopy demonstrates the utility of this technique for archival study of microsporidia-containing specimens over prolonged periods of time.

Isolation and identification of nontuberculous mycobacteria from foods as possible exposure sources.

A variety of foods collected from local supermarkets and produce stands were examined as possible sources of nontuberculous mycobacterial exposure. Food samples were combined with sterile ultrapure water and manually shaken. To remove large particles, the suspensions were filtered through a sterile strainer, centrifuged, and the supernatants were discarded. The food pellets were stored at -75 degrees C. The pellets were treated with either oxalic acid or sodium hydroxide-sodium citrate solutions to reduce contamination by nonmycobacterial organisms. Decontaminated pellets were cultured on both Middlebrook 7H10C agar and Middlebrook 7H10C agar with supplemental malachite green. Plates were observed for growth at 2 and 8 weeks. Isolates demonstrating acid-fastness were identified to species using polymerase chain reaction and restriction enzyme analysis. Nontuberculous mycobacteria (NTM) were recovered from 25 of 121 foods. Six different species of NTM were isolated, the most predominant being Mycobacterium avium.

Therapy for human gastrointestinal microsporidiosis.

Gastrointestinal microsporidiosis is a major cause of diarrhea and wasting in persons with acquired immune deficiency syndrome (AIDS). Microsporidia demonstrate properties of both true eukaryotes and prokaryotes. The biology of microsporidia makes its elimination from the gastrointestinal tract therapeutically challenging. This organism depends greatly on the host for its energy needs and reproduction; microsporidial spores are impervious to the elements. Microsporidial infection of the gastrointestinal tract, principally with Enterocytozoon bieneusi and Encephalitozoon intestinalis in patients with AIDS has been treated with different medical regimens with variable success. The less common pathogen, E. intestinalis, responds well to albendazole, making it excellent first-line therapy, but such is not the case for E. bieneusi. None of the benzimidazoles has been demonstrated to be efficacious for E. bieneusi. On the other hand, E. bieneusi has shown excellent clinical therapeutic response to either direct action with fumagillin or its analogue, TNP-470, or indirectly by immune enhancement by suppression of the HIV virus with more aggressive, highly effective antiretroviral therapy. Further work is necessary to fully establish proper therapeutic protocols and manage side effects of the treatments. Other promising forms of therapy such as polyamine inhibitors and thalidomide demonstrate certain effectiveness in treatment of microsporidian in vitro (polyamine inhibitors) and in selected cases in vivo (thalidomide). Lack of either sufficiently suggestive or definitive human studies prevents the endorsement of these modes of therapy for treatment of gastrointestinal microsporidiosis at this time.

PCR comparison of Mycobacterium avium isolates obtained from patients and foods.

Mycobacterium avium is a cause of disseminated disease in AIDS patients. A need for a better understanding of possible sources and routes of transmission of this organism has arisen. This study utilized a PCR typing method designed to amplify DNA segments located between the insertion sequences IS1245 and IS1311 to compare levels of relatedness of M. avium isolates found in patients and foods. Twenty-five of 121 food samples yielded 29 mycobacterial isolates, of which 12 were M. avium. Twelve food and 103 clinical M. avium isolates were tested. A clinical isolate was found to be identical to a food isolate, and close relationships were found between two patient isolates and two food isolates. Relatedness between food isolates and patient isolates suggests the possibility that food is a potential source of M. avium infection. This study demonstrates a rapid, inexpensive method for typing M. avium, possibly replacing pulsed-field gel electrophoresis.

Comparison of large restriction fragments of Mycobacterium avium isolates recovered from AIDS and non-AIDS patients with those of isolates from potable water.

We examined potable water in Los Angeles, California, as a possible source of infection in AIDS and non-AIDS patients. Nontuberculous mycobacteria were recovered from 12 (92%) of 13 reservoirs, 45 (82%) of 55 homes, 31 (100%) of 31 commercial buildings, and 15 (100%) of 15 hospitals. Large-restriction-fragment (LRF) pattern analyses were done with AseI. The LRF patterns of Mycobacterium avium isolates recovered from potable water in three homes, two commercial buildings, one reservoir, and eight hospitals had varying degrees of relatedness to 19 clinical isolates recovered from 17 patients. The high number of M. avium isolates recovered from hospital water and their close relationship with clinical isolates suggests the potential threat of nosocomial spread. This study supports the possibility that potable water is a source for the acquisition of M. avium infections.

Modification of the clinical course of intestinal microsporidiosis in acquired immunodeficiency syndrome patients by immune status and anti-human immunodeficiency virus therapy.

The clinical course of 37 Enterocytozoon bieneusi-infected acquired immunodeficiency syndrome patients with diarrhea was studied. Parasite clearance was seen in 15 patients (40.5%). Clearance of E. bieneusi resulted in a 25-100% reduction in episodes of diarrhea, suggesting that microsporidia are true pathogens. Univariate and multivariate proportional hazards analyses revealed that peripheral blood CD4 cell counts > or = 100/mm3, the use of two or more antiretroviral medications, and use of a protease inhibitor were statistically associated with decreased time to clearance of E. bieneusi. Specific anti-microsporidial therapy (albendazole) was not associated with parasite eradication. Factors related to immunocompetence and human immunodeficiency virus suppression appeared to be important in the clearance of E. bieneusi.

Examination of the prevalence and seasonal variation of intestinal microsporidiosis in the stools of persons with chronic diarrhea and human immunodeficiency virus infection.

The epidemiology of human microsporidiosis is poorly understood and environmental factors affecting transmission of the organism have not been fully elucidated. Temporal variation in the prevalence of microsporidia in the stool of patients with human immunodeficiency virus (HIV) infection and diarrhea was studied to evaluate the role of water-borne transmission. From January 1993 to December 1996, 8,439 stools from HIV-infected individuals were examined for microsporidia spores in southern California. Yearly positivity rates were 8.8% in 1993, 9.7% in 1994, 6.6% in 1995, and 2.9% in 1996. An analysis for linear trend showed a statistically significant decrease in stool positivity rates over time (chi2 = 81.9, P = 0.001). No significant seasonal variation in the prevalence of microsporidiosis was seen over that time period. These results suggest the constant presence of microsporidia in the environment, rather than a seasonal association with recreational water use or seasonal contamination of the water supply, and a real decrease in yearly prevalence of microsporidia related diarrhea. Factors related to a progressive decrease in prevalence are subjects of future investigation.

Workup of gastrointestinal microsporidiosis.

Microsporidia, which are members of the phylum Microspora, are increasingly recognized as causing opportunistic infections in persons with immunodeficiency (e.g., AIDS). Diarrhea is the predominant clinical sign associated with infections by two Microsporidia, namely Enterocytozoon bieneusi and Encephalitozoon intestinalis (which was formerly named Septata intestinalis). Prevalence rates of microsporidiosis in persons with AIDS and chronic diarrhea range fron 7 to 50%. Transmission electron microscopy has been the gold standard by which to diagnose microsporidiosis and requires observing a polar filament which is the structure distinguishing Microsporidia from other organisms. Transmission electron microscopy is difficult, time-consuming, costly, relatively insensitive, and requires a great deal of expertise. As such, histochemical methods have been developed and improved for detecting Microsporidia. Diagnoses from stool specimens or enteric fluids can be made using the chitin-staining fluorochromes (e.g., Calcofluor White) and the modified trichrome stain which are highly sensitive, particularly when both are used. Immunofluorescent antibody staining methods are being developed to improve specificity, but reagents are not yet commercially available. Microsporidia can be detected most readily in tissue biopsies by Gram stain, Giemsa stain, or immunofluorescent antibody. Polymerase chain reaction methods are in their infancy for application, but should prove to be particularly sensitive and specific in the future.

Comparative efficacy of topical ciprofloxacin for treating Mycobacterium fortuitum and Mycobacterium chelonae keratitis in an animal model.

Mycobacterium fortuitum and M. chelonae are the two most common causes of nontuberculous mycobacterial keratitis, and they may be difficult to differentiate at diagnosis. Mycobacterium fortuitum is generally more sensitive to ciprofloxacin in vitro than is M. chelonae. Using a rabbit model, we compared the efficacy of topical ciprofloxacin (3 mg/ml) against M. chelonae keratitis to its efficacy against M. fortuitum keratitis. After four days of therapy, ciprofloxacin significantly reduced the number of both organisms in treated eyes compared to untreated control eyes (both P values < .001). Mean culture ratios (colony-forming units in treated eye divided by colony-forming units in untreated eye for each rabbit) were used to compare efficacy between groups. When all treated animals were considered, no significant difference was found between groups (P = .13). When outlier values were excluded, ciprofloxacin was more effective against M. fortuitum than M. chelonae (P = .01). When treated and untreated eyes were compared after therapy in the M. fortuitum group, ciprofloxacin treatment was associated with a reduction in mean stromal infiltrate area (P = .03) and in the tendency to form satellite lesions (P = .07). A clinical effect was not observed in the M. chelonae group. Although ciprofloxacin is effective against both organisms, it appears to be less effective against M. chelonae than M. fortuitum in vivo.

Recovery of Cyclospora organisms from patients with prolonged diarrhea.

A newly recognized protozoan human parasite, Cyclospora has been incriminated as the cause of prolonged diarrhea. Five patients had episodes of diarrhea accompanied by nausea, weight loss, and/or low-grade fever for 10-45 days. Multiple fecal samples fixed in sodium acetate-acetic acid-formalin contained spherical organisms measuring 8-10 microns in diameter; a modified concentration technique was used to detect them. The sediment was examined by direct microscopy and autofluorescence, and the identification was confirmed by acid-fast stain. All patients had visited either Mexico or Thailand. The presence of Cyclospora organisms in these patients shows that these can be etiologic agents of traveler's diarrhea in both immunocompetent and immunocompromised hosts. Fecal specimens from patients with unexplained diarrhea should be routinely examined for their presence.

Comparison of topical antibiotics for treating Mycobacterium fortuitum keratitis in an animal model.

The efficacy of three topical antibiotic treatments for Mycobacterium fortuitum (strain ATCC-6841) keratitis were compared in rabbits. Rabbits were treated with ciprofloxacin (3 mg/ml) or clarithromycin (20 mg/ml) or a combination of amikacin (100 mg/ml) and vancomycin (50 mg/ml). All three treatments significantly reduced the number of organisms in treated eyes compared to untreated, control eyes (all P values < .001). No significant difference in treatment efficacy was found between the three treatment groups (all P values > or = .48), although ciprofloxacin (3 mg/ml) was more effective than clarithromycin (20 mg/ml) after excluding outliers (P = .01). All treatments stabilized or reduced the size of stromal infiltrates after four days of therapy, whereas infiltrates continued to enlarge in untreated eyes. These results suggest that topical clarithromycin, topical ciprofloxacin, and combined amikacin and vancomycin may all be clinically useful for treating M. fortuitum keratitis. Both clarithromycin and ciprofloxacin were better tolerated than combined amikacin and vancomycin. This study supports the further development of clarithromycin, a new macrolide antibiotic, as a topical drug for treatment of M. fortuitum keratitis.