Objectives: To determine the activity of newer triazoles against strains of Histoplasma capsulatum resistant to fluconazole.Methods: Susceptibility testing was performed on 17 paired pre- and post-treatment H. capsulatum isolates from patients with AIDS who failed fluconazole.Results: The median MICs of fluconazole, voriconazole, and posaconazole and ravuconazole for the pre-treatment isolates were 1 mg/L, 0.015 mg/L and < 0.007 mg/L, respectively. A 4-fold or greater increase in the MIC of fluconazole and voriconazole was observed in 12 and 7 of the post-treatment isolates, respectively; the median fold increases in MIC were 8 and 2.1, respectively. No MIC increases were observed for posaconazole and ravuconazole. One pair of isolates exhibiting reduced susceptibility was examined in more detail. A single amino acid substitution (at tyrosine 136) was identified in the active site of the CYP51 protein from the post-treatment isolate, which is presumed to be responsible for reduced susceptibility to voriconazole and fluconazole, analogous to recent observations in Candida albicans.Conclusions: These findings support careful monitoring for relapse in patients receiving voriconazole treatment for histoplasmosis, particularly in those who were previously treated with fluconazole.

Use of a pulmonary model of histoplasmosis in CD4/CD8 lymphocyte depleted animals offers an opportunity to study pathogenesis in a setting resembling AIDS. Flow cytometric analysis demonstrated that administration of anti-CD4 and anti-CD8 antibodies reduced CD4 and CD8 T cell subsets in the lungs, lymph nodes and spleen. Depletion of these cells transformed the infection from a self-limited course in normal mice to a progressive, fatal course in CD4/CD8 depleted mice. CD4 depletion alone had a lesser effect on survival, but increased fungal burden, while CD4/CD8 depletion had the greatest effect. Histopathologic studies showed marked differences in the inflammatory response between the dually depleted animals and the non-depleted controls. In conclusion, the course of histoplasmosis in CD4/CD8 depleted animals is progressive and fatal, resembling that observed in immunosuppressed patients. This model appears suitable for investigation of immunity to H. capsulatum, and should be useful for evaluation of treatment in the immunocompromised host.

Summary: The relationship of immunity to Histoplasma capsulatum and CD4 count in HIV-1-infected patients is unknown. Samples of blood from people with HIV infection and from HIV-negative volunteers were assessed for immune responsiveness to the histoplasmin antigen using proliferation and interferon-γ production as indicators of immunity. Results of histoplasmin skin tests, lymphoproliferative responses (LPR), and interferon-γ production were positive in 9 of 20 (45%) HIV-negative controls, and in vitro measurements agreed highly with skin test reactivity. Among HIV-1-infected patients with recent histoplasmosis, skin test results were positive in none, LPR results were positive in 14%, and interferon-γ production in 18%. Among HIV-1-infected patients with CD4 counts between 200 and 500 cells/mm3, LPR was positive in 8% and interferon-γ production in 33%, and among those with CD4 counts >500 cells/mm3, LPR was positive in 31% and interferon-γ production in 46%. In conclusion, immune responsiveness to H. capsulatum was depressed in HIV-1infected persons with CD4 counts between 200 and 500 cells/mm3, but approached normal in those with CD4 counts >500 cells/mm3.

Histoplasmosis is a common infection in endemic regions of North and Latin America, causing a broad spectrum of clinical findings. The diagnosis may be missed or delayed because histoplasmosis is not considered in the differential. A battery of serologic and mycologic tests may be used for the diagnosis, but each has advantages and limitations. Antigen detection may be particularly helpful for making a rapid diagnosis in patients with more extensive infection. The purpose of this review is to provide a comprehensive discussion of the role of antigen detection in the diagnosis of histoplasmosis, to provide the clinician and laboratory worker with a fuller understanding of the benefits and limitations of this useful laboratory method. This report is based soley upon the experience at the Histoplasmosis Reference Laboratory, and can not be used in interpretation of results of Histoplasma antigen testing done at other laboratories.

CD40 ligand-CD40 ligation is important in the development of T-cell-mediated immune responses. The purpose of this study was to examine the role of CD40L in recovery from histoplasmosis using a murine model of intratracheally induced infection. B6C3F1 mice were infected intratracheally with Histoplasma capsulatum yeast and monitored for clearance of the organism from the lungs and spleen. CD40L treatment was begun on either day -2 or +2 post inoculation and continued until day 14 in CD4-depleted animals and from day -2 to day +4 in non-immunosuppressed animals. Amphotericin B treatment was begun four days following inoculation and given every other day for 10 days. CD40L reduced fungal burden by less than one log when started two days before infection but did not act synergistically with low-dosage amphotericin B (0.2 mg kg(-1) qod) in CD4 depleted mice. Low-dose amphotericin B, CD40L, and the combination of the two failed to lower the fungal burden in a second experiment using a more virulent isolate of the same strain of H. capsulatum in CD4-depleted mice. Furthermore, CD40L did not increase the concentrations of IFN-gamma, IL-12 or IL-10 in the lungs or spleens of infected animals. In summary, CD40L had minimal or no effect on the course of infection in this murine model of histoplasmosis.

The outcome of central nervous system (CNS) histoplasmosis is often unfavorable. Although fluconazole plays an integral role in treatment of fungal meningitis, its role in the treatment of histoplasmosis is hampered by reduced activity and potential development of resistance. A murine model of CNS histoplasmosis was used to evaluate the hypothesis that a combination of amphotericin B and fluconazole therapy would be superior to amphotericin B monotherapy. Groups of B6C3F(1) mice were infected by injection of Histoplasma capsulatum into the subarachnoid space. The addition of fluconazole hindered the antifungal effect of amphotericin B, as determined by measurement of fungal burden, suggesting antagonism in the brain. Fluconazole was less effective as a single agent than was amphotericin B, despite the greater penetration of fluconazole into brain tissues. The hypothesis that amphotericin B-fluconazole combination therapy would be superior to amphotericin B monotherapy for treatment of CNS histoplasmosis was not supported by this study.

ABSTRACT Antifungal treatment reduces the concentration of Histoplasma antigen in blood and urine, supporting a hypothesis that antigen clearance could be used to compare the activity of new agents for treating histoplasmosis. In separate trials in patients with AIDS, clinical response was similar with itraconazole (85%) and fluconazole (74%). Fungal blood cultures at week 4, however, were negative in a significantly higher proportion of patients treated with itraconazole (92.3%) than in those treated with fluconazole (61.9%) ( P = 0.017). Baseline antigen concentrations were similar in the two groups: serum, P = 0.7235; and urine, P = 0.1360. After 4 weeks of treatment, the decline in antigen from baseline in serum was similar in the two treatment groups ( P = 0.5237), as it was in urine ( P = 0.4679). At week 12, the decline in antigen from baseline in serum also was similar in the two groups ( P = 0.4911) and in urine ( P = 0.2786). More rapid clearance of fungemia suggests that itraconazole is more effective than fluconazole in treating histoplasmosis. This study demonstrates that clearance of fungemia is a better measure of antifungal effect than clearance of antigen.

In sequential clinical trials of treatment for histoplasmosis in patients with acquired immunodeficiency syndrome, therapy with fluconazole failed in a higher proportion of patients than did therapy with itraconazole. To determine the cause for failure with fluconazole, antifungal susceptibility testing that used modified National Committee on Clinical Laboratory Standards procedures was performed on all baseline and failure isolates. Failure occurred more frequently in patients with baseline isolates with fluconazole minimum inhibitory concentrations (MICs) > or =5 microg/mL versus lower MICs; 29% versus 3%, respectively. There was at least a 4-fold increase in fluconazole MIC in the isolates from 10 (59%) of 17 patients for whom paired pretreatment and failure or relapse isolates were available. Cross-resistance to itraconazole was not seen. In conclusion, fluconazole is less active than itraconazole for Histoplasma capsulatum and induces resistance during therapy, which accounted for treatment failure in some patients.

ABSTRACTA murine model of intratracheally induced histoplasmosis in immunocompromised B6C3F1mice was used to evaluate a new triazole antifungal agent, posaconazole. This compound was previously shown to be comparable to amphotericin B and superior to itraconazole for the treatment of histoplasmosis in immunocompetent mice. The current study used mice that were depleted of T lymphocytes by intraperitoneal injection of anti-CD4 and anti-CD8 monoclonal antibodies beginning 2 days before infection and continuing at 5-day intervals until completion of the study. Groups of B6C3F1mice that were depleted of CD4 and CD8 T cells were infected with an inoculum of 104Histoplasma capsulatumyeasts. All mice receiving posaconazole at 1 or 0.1 mg/kg of body weight/day, amphotericin B at 2 mg/kg every other day (qod), or itraconazole at 75 mg/kg/day survived to day 29. Only 60% of mice receiving itraconazole at 10 mg/kg/day and none receiving amphotericin B at 0.2 mg/kg qod survived to that date. Fungal burdens were determined at day 14 of infection, 1 day after discontinuation of therapy. Quantitative colony counts andHistoplasmaantigen levels in lung and spleen tissues declined following treatment with amphotericin B at 2 mg/kg qod, posaconazole at 5 and 1 mg/kg/day, and itraconazole at 75 mg/kg/day but not in mice treated with amphotericin B at 0.2 mg/kg qod or itraconazole at 10 mg/kg/day. Posaconazole at 0.1 mg/kg/day reduced fungal colony counts and antigen levels in spleens but not in lungs. This study shows posaconazole activity for the treatment of histoplasmosis in immunosuppressed animals.

ABSTRACT Twenty clinical isolates of Histoplasma capsulatum were tested for their in vitro susceptibilities to caspofungin in comparison to those to amphotericin B by following National Committee for Clinical Laboratory Standards guidelines for yeasts. The mean MICs were 16.6 μg/ml (range, 8 to 32 μg/ml) for caspofungin and 0.56 μg/ml (range, 0.5 to 1.0 μg/ml) for amphotericin B. Survival experiments used a 10 5 dose in a pulmonary challenge model with B6C3F 1 mice. All mice that received amphotericin B at 2 mg/kg of body weight every other day (q.o.d.), 30% of mice that received caspofungin at 8 mg/kg/day, and 20% of mice that received caspofungin at 4 mg/kg/day survived to day 15, while mice that received caspofungin at 2 mg/kg/day and all control mice that received the vehicle died by day 14. Amphotericin B at 2 mg/kg q.o.d. markedly reduced the fungal burden in the lungs and spleens, as measured by Histoplasma antigen detection techniques and quantitative cultures, for each comparison. Caspofungin at 10 mg/kg twice a day (b.i.d.) did not reduce the fungal burden, as measured by antigen detection techniques, but slightly reduced the levels of fungi in both the lungs and spleens, as determined by quantitative cultures. Caspofungin at 5 mg/kg b.i.d. did not affect fungal burden. Overall, caspofungin had only a slight effect on survival or fungal burden.

ABSTRACT Nikkomycin Z was tested both in vitro and in vivo for efficacy against Histoplasma capsulatum . Twenty clinical isolates were tested for susceptibility to nikkomycin Z in comparison to amphotericin B and itraconazole. The median MIC was 8 μg/ml with a range of 4 to 64 μg/ml for nikkomycin Z, 0.56 μg/ml with a range of 0.5 to 1.0 μg/ml for amphotericin B, and ≤0.019 μg/ml for itraconazole. Primary studies were carried out by using a clinical isolate of H. capsulatum for which the MIC of nikkomycin Z was greater than or equal to 64 μg/ml. In survival experiments, mice treated with amphotericin B at 2.0 mg/kg/dose every other day (QOD) itraconazole at 75 mg/kg/dose twice daily (BID), and nikkomycin Z at 100 mg/kg/dose BID survived to day 14, while 70% of mice receiving nikkomycin Z at 20 mg/kg/dose BID and none of the mice receiving nikkomycin Z at 5 mg/kg/dose BID survived to day 14. All vehicle control mice died by day 12. Fungal burden was assessed on survivors. Mice treated with nikkomycin Z at 20 and 100 mg/kg/dose BID had significantly higher CFUs per gram of organ weight in quantitative cultures and higher levels of Histoplasma antigen in lung and spleen homogenates than mice treated with amphotericin B at 2.0 mg/kg/dose QOD or itraconazole at 75 mg/kg/dose BID. Studies also were carried out with a clinical isolate for which the MIC of nikkomycin Z was 4 μg/ml. All mice treated with amphotericin B at 2.0 mg/kg/dose QOD; itraconazole at 75 mg/kg/dose BID; and nikkomycin Z at 100, 20, and 5 mg/kg/dose BID survived until the end of the study at day 17 postinfection, while 30% of the untreated vehicle control mice survived. Fungal burden assessed on survivors showed similar levels of Histoplasma antigen in lung and spleen homogenates of mice treated with amphotericin B at 2.0 mg/kg/dose QOD; itraconazole at 75 mg/kg/dose BID; and nikkomycin Z at 100, 20, and 5 mg/kg/dose BID. The three surviving vehicle control mice had significantly higher antigen levels in lung and spleen than other groups ( P < 0.05). The efficacy of nikkomycin Z at preventing mortality and reducing fungal burden correlates with in vitro susceptibility.

ABSTRACT A murine model of intratracheally induced histoplasmosis was used to evaluate a new triazole antifungal agent, Schering (SCH) 56592, for treatment of histoplasmosis. MICs were determined for SCH 56592, amphotericin B, and itraconazole by testing yeast-phase isolates from 20 patients by a macrobroth dilution method. The MICs at which 90% of the isolates are inhibited were for 0.019 μg/ml for SCH 56592, 0.5 μg/ml for amphotericin B, and ≤0.019 μg/ml for itraconazole. Survival studies were done on groups of 10 B6C3F 1 mice with a lethal inoculum of 10 5 . All mice receiving 5, 1, or 0.25 mg of SCH 56592 per kg of body weight per day, 2.5 mg of amphotericin B per kg every other day (qod), or 75 mg of itraconazole per kg per day survived to day 29. Only 44% of mice receiving 5 mg of itraconazole/kg/day survived to day 29. Fungal burden studies done in similar groups of mice with a sublethal inoculum of 10 4 showed a reduction in CFUs and Histoplasma antigen levels in lung and spleen tissue in animals treated with 2 mg of amphotericin B/kg qod, 1 mg of SCH 56592/kg/day, and 75 mg of itraconazole/kg/day, but not in those treated with lower doses of the study drugs (0.2 mg of amphotericin B/kg qod, 0.1 mg of SCH 56592/kg/day, or 10 mg of itraconazole/kg/day). Serum drug concentrations were measured 3 and 24 h after the last dose in mice (groups of five to seven mice), each treated for 7 days with SCH 56592 (10 and 1 mg/kg/day) and itraconazole (75 and 10 mg/kg/day). Mean levels measured by bioassay were as follows: SCH 56592, 10 mg/kg/day (2.15 μg/ml at 3 h and 0.35 μg/ml at 24 h); SCH 56592, 1 mg/kg/day (0.54 μg/ml at 3 h and none detected at 24 h); itraconazole, 75 mg/kg/day (22.53 μg/ml at 3 h and none detected at 24 h); itraconazole, 10 mg/kg/day (1.33 μg/ml at 3 h and none detected at 24 h). Confirmatory results were obtained by high-pressure liquid chromatography assay. These studies show SCH 56592 to be a promising candidate for studies of treatment of histoplasmosis in humans.

PURPOSE The purpose of these examinations was to monitor changes, in the prevalence of dental fluorosis. METHODS In February 1992 and December 1994, children who were residents of one of three communities with varying levels of fluoride in their communal water supply were examined for dental fluorosis. Since some children were available at both examination periods, it was also possible to determine changes in the incidence of dental fluorosis. RESULTS The prevalence of fluorosis increased by approximately 14%, 20%, and 6% in the negligibly, optimally, and 4X optimally fluoridated communities, respectively. In the negligibly and optimally fluoridated communities, the incidence of dental fluorosis increased by 12% and 7%, respectively. In the 4X optimally fluoridated community, all the children examined had evidence of fluorosis at both examinations. CONCLUSION Fluoride continues to be the primary therapeutic agent for the prevention of dental caries in adults and children. With the downward adjustment in the fluoride supplement schedule, continued monitoring of the prevalence of dental fluorosis in young children is needed to determine if any additional steps are even necessary to restrict fluoride intake during the years that enamel formation is occurring.