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Histoplasma capsulatum infection causes significant morbidity and mortality in human immunodeficiency virus-infected individuals, particularly those in countries with limited access to rapid diagnostics or antiretroviral therapies. The fungus easily disseminates in persons with AIDS, resulting in progressive disseminated histoplasmosis (PDH), which can progress rapidly to death if undiagnosed. The availability of a simple, rapid method to detect H. capsulatum infection in less developed countries where the infection is endemic would dramatically decrease the time to diagnosis and treatment of PDH. We have developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect PDH antigenuria in infected patients. The assay uses polyclonal antibodies against H. capsulatum as both capture and detection reagents, and a standard reference curve is included to quantify antigenuria and ensure reproducibility. We evaluated this assay using specimens collected from patients with AIDS and culture-proven histoplasmosis in a Guatemalan clinic (n ‫؍‬ 48), from healthy persons (n ‫؍‬ 83), and from patients with other, nonhistoplasmosis diseases (n ‫؍‬ 114). The ELISA demonstrated a sensitivity of 81% and a specificity of 95% in detecting H. capsulatum antigen in urine. This assay relies on simple technology that can be performed in institutions with limited resources. Use of this test will facilitate rapid diagnosis of PDH in countries where mortality is high, expediting treatment and likely reducing PDH-related mortality.

BackgroundInvasive fusariosis (IF) is a rare but often fatal fungal infection in immunosuppressed patients. In 2007, cases of IF above the expected epidemiologic baseline were detected in the hematology ward of a hospital in Rio de Janeiro, Brazil. Possible sources of infection were investigated by performing environmental sampling and patient isolate collection, followed by molecular typing. Isolates from dermatology patients with superficial fusariosis were included in the study for comparison to molecular types found in the community.MethodsEnvironmental sampling focused on water-related sources in and around the hematology ward. Initially, we characterized 166 clinical and environmental isolates using the Fusarium translation elongation factor 1α (EF-1α) genetic locus. Isolates included 68 collected from water-related sources in the hospital environment, 55 from 18 hematology patients, and 43 from the skin/nails of 40 outpatients seen at the hospital dermatology clinic. Multi-locus sequence typing was performed on Fusarium solani species complex (FSSC) species 1 and 2 isolates to investigate their relatedness further.ResultsMost of the hematology samples were FSSC species 2, with species type FSSC 2-d the most commonly isolated from these patients. Most of the outpatient dermatology samples were also FSSC 2, with type 2-d again predominating. In contrast, environmental isolates from water sources were mostly Fusarium oxysporum species complex (FOSC) and those from air samples mostly Fusarium incarnatum-equiseti species complex (FIESC). A third of the environmental samples were FSSC, with species types FSSC 1-a and FSSC 1-b predominating.ConclusionsFusarium isolate species types from hematology patient infections were highly similar to those recovered from dermatology patients in the community. Four species types (FSSC 1-a, 1-b, 2-d and 2-f) were shared between hematology patients and the environment. Limitations in environmental sampling do not allow for nosocomial sources of infection to be ruled out. Future studies will focus on environmental factors that may have influenced the prevalence of FSSC fusariosis in this hematology ward.

c Improved methods for the detection of Histoplasma capsulatum are needed in regions with limited resources in which the organism is endemic, where delayed diagnosis of progressive disseminated histoplasmosis (PDH) results in high mortality rates. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid inexpensive molecular diagnosis of this disease. Primers for LAMP were designed to amplify the Hcp100 locus of H. capsulatum. The sensitivity and limit of detection were evaluated using DNA extracted from 91 clinical isolates of known geographic subspecies, while the assay specificity was determined using DNA extracted from 50 other fungi and Mycobacterium tuberculosis. Urine specimens (n ‫؍‬ 6) collected from HIV-positive individuals with culture-and antigen-proven histoplasmosis were evaluated using the LAMP assay. Specimens from healthy persons (n ‫؍‬ 10) without evidence of histoplasmosis were used as assay controls. The Hcp100 LAMP assay was 100% sensitive and specific when tested with DNA extracted from culture isolates. The median limit of detection was <6 genomes (range, 1 to 300 genomes) for all except one geographic subspecies. The LAMP assay detected Hcp100 in 67% of antigen-positive urine specimens (4/6 specimens), and results were negative for Hcp100 in all healthy control urine specimens. We have shown that the Hcp100 LAMP assay is a rapid affordable assay that can be used to expedite culture confirmation of H. capsulatum in regions in which PDH is endemic. Further, our results indicate proof of the concept that the assay can be used to detect Histoplasma DNA in urine. Further evaluation of this assay using body fluid samples from a larger patient population is warranted.

Histoplasmosis is one of the most common and deadly opportunistic infections among persons living with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome in Latin America, but due to limited diagnostic capacity in this region, few data on the burden and clinical characteristics of this disease exist. Between 2005 and 2009, we enrolled patients ≥ 18 years of age with suspected histoplasmosis at a hospital-based HIV clinic in Guatemala City. A case of suspected histoplasmosis was defined as a person presenting with at least three of five clinical or radiologic criteria. A confirmed case of histoplasmosis was defined as a person with a positive culture or urine antigen test for . Demographic and clinical data were also collected and analyzed. Of 263 enrolled as suspected cases of histoplasmosis, 101 (38.4%) were confirmed cases. Median time to diagnosis was 15 days after presentation (interquartile range [IQR] = 5-23). Crude overall mortality was 43.6%; median survival time was 19 days (IQR = 4-69). Mycobacterial infection was diagnosed in 70 (26.6%) cases; 26 (25.7%) histoplasmosis cases were coinfected with mycobacteria. High mortality and short survival time after initial symptoms were observed in patients with histoplasmosis. Mycobacterial coinfection diagnoses were frequent, highlighting the importance of pursuing diagnoses for both diseases.

Exserohilum rostratum was the major cause of an outbreak of fungal infections linked to injections of contaminated methylprednisolone acetate. Because almost 14,000 persons were exposed to product that was possibly contaminated with multiple fungal pathogens, there was unprecedented need for a rapid throughput diagnostic test that could detect both E. rostratum and other unusual agents of fungal infection. Here we report development of a novel PCR test that allowed for rapid and specific detection of fungal DNA in cerebrospinal fluid (CSF), other body fluids and tissues of infected individuals. The test relied on direct purification of free-circulating fungal DNA from fluids and subsequent PCR amplification and sequencing. Using this method, we detected Exserohilum rostratum DNA in 123 samples from 114 case-patients (28% of 413 case-patients for whom 627 samples were available), and Cladosporium DNA in one sample from one case-patient. PCR with novel Exserohilum-specific ITS-2 region primers detected 25 case-patients with samples that were negative using broad-range ITS primers. Compared to fungal culture, this molecular test was more sensitive: of 139 case-patients with an identical specimen tested by culture and PCR, E. rostratum was recovered in culture from 19 (14%), but detected by PCR in 41 (29%), showing a diagnostic sensitivity of 29% for PCR compared to 14% for culture in this patient group. The ability to rapidly confirm the etiologic role of E. rostratum in these infections provided an important contribution in the public health response to this outbreak. In late September 2012, the Centers for Disease Control and Prevention (CDC) began to investigate an outbreak of fungal meningitis associated with epidural injections of contaminated methylprednisolone acetate solution purchased from a single compounding pharmacy (1-5). By 15 January 2013, 678 cases of fungal infections, including meningitis, spinal, paraspinal, or peripheral joint infections, had been reported from 19 states, and 44 people had died. Although Aspergillus fumigatus was isolated from the index patient (1), and several fungal species were identified in patients during the outbreak, the vast majority of laboratory-confirmed infections were caused by Exserohilum rostratum, which was detected in cerebrospinal fluid (CSF) and tissues of patients (3, 6).E. rostratum (teleomorph Setophaeria rostrata) is a dematiaceous (brown-black) filamentous fungus, which is common in the environment and occasionally causes opportunistic infections in humans (7-9). In healthy individuals, exposure to E. rostratum can cause allergic sinusitis or cutaneous and subcutaneous infections (7, 10, 11). Invasive fungal infections due to E. rostratum are rare and limited to persons with severely impaired immune systems (7, 10, 12). To our knowledge, no cases of fungal meningitis or encephalitis due to this fungus had been described prior to this outbreak. In addition, no genome or animal models had been developed to study E. rostratum, which also complicated the deve...

Resistance to first-line treatment agents in patients with Salmonella bacteremia is a concern for public health and for informing clinical decisions. Judicious antimicrobial use is crucial to limit resistance.

We validated an antigen capture enzyme-linked immunosorbent assay (ELISA) in Colombian persons with AIDS and proven histoplasmosis and evaluated the correlation between antigenuria and clinical improvement during follow-up. The sensitivity of the Histoplasma capsulatum ELISA was 86%, and the overall specificity was 94%. The antigen test successfully monitored the response to therapy. Histoplasmosis is a disease caused by the fungus Histoplasma capsulatum and is most frequently diagnosed in the American continent. In persons living with HIV/AIDS (PLWHA), an infection often develops into a clinical form called progressive disseminated histoplasmosis (PDH), which has a high mortality if not treated early (1, 2). PDH symptoms are nonspecific and may be similar to those of other infectious diseases, thus complicating diagnosis and treatment (1, 3). In Colombia and other countries in the Americas where the disease is endemic and where access to highly active antiretroviral therapy (HAART) is limited, histoplasmosis is a major cause of mortality (up to 30%) in PLWHA (4-9).Detection of circulating Histoplasma antigens in urine specimens by an antigen capture enzyme-linked immunoabsorbent assay (ELISA) is highly sensitive (95%), but this test is not generally available outside the United States (10, 11). To make such a test available to resource-challenged countries, a similar Histoplasma antigen capture ELISA was developed at the Centers for Disease Control and Prevention (CDC) (12) and was validated in a cohort of AIDS patients in Guatemala, demonstrating a sensitivity of 81% infections, 23 were identified as M. tuberculosis and 1 was identified as M. avium-Mycobacterium intracellulare; 4 could not be identified to the species level.