Tropheryma whipplei infection (Whipple's disease) in a patient after liver transplantation.

Whipple's disease (WD) is a rare infection caused by the bacterium Tropheryma whipplei that can affect multiple organs and most commonly occurs in the immunocompetent host. Only 3 cases of WD have been reported in the setting of immunosuppression for organ transplantation. Here, we report the first case of WD, to our knowledge, in a patient after liver transplantation with comorbid graft-versus-host-disease. We discuss the diagnostic challenges in this setting and the value of electron microscopy and in situ hybridization methods for confirming the infection. WD may be under-diagnosed in immunosuppressed transplant patients because the disease can present with atypical clinical and histological features that suggest other conditions.

Deciphering the aetiology of a mixed fungal infection by broad-range PCR with sequencing and fluorescence in situ hybridisation.

Simultaneous infections with multiple fungi may be misinterpreted as monomicrobial infections by current diagnostics with ramifications for the choice of antimicrobial agents that may impact patient outcomes. The application of molecular methods on tissue samples may be useful to decipher the aetiology of mixed fungal infections. We present a leukaemic patient who died from sepsis due to candidaemia. The postmortem examination documented fungal elements in lung tissue. Fungal DNA was amplified from the lung sample by broad-range PCR assays targeting the 28S ribosomal RNA gene or the internal transcribed spacer 2 (ITS-2). Fluorescence in situ hybridisation (FISH) using differentially labelled fungal probes was applied on the tissue. Sequencing identified the PCR amplicons as Aspergillus fumigatus (28S assay) and Candida tropicalis (ITS-2 assay). As a chromatogram suggested mixed amplicons, the Isentio ripseq(®) tool for in silico analysis was applied and confirmed the presence of both amplicons in the PCR products of both assays. FISH confirmed the presence of Aspergillus and Candida within the infectious process, a prerequisite for inferring a causal relationship with the infection. The combination of broad-range PCR with sequence analysis and FISH applied on tissue samples is a powerful approach to identify the aetiology of invasive fungal infections, including mixed infections.

The impact of a change in antibacterial prophylaxis from ceftazidime to levofloxacin in allogeneic hematopoietic cell transplantation.

Antibiotic prophylaxis has been used during the initial phases of myeloablative hematopoietic cell transplantation (HCT) for more than two decades. However, the optimal regimen in terms of both cost and clinical effectiveness is unclear. We retrospectively compared the clinical and microbiological impact of a change in antibiotic prophylaxis practice from ceftazidime (n=216 patients with HCT in 2000-2002) to levofloxacin (n=219 patients, August 2002-2005) in patients receiving myeloablative conditioning. Levofloxacin prophylaxis was associated with fever and a change in antibiotics during neutropenia, but this strategy was not associated with any adverse outcomes. Patients receiving levofloxacin had lower rates of significant bacteremia than did those receiving ceftazidime (day 100, 19.2 vs 29.6%, P=0.02). The use of levofloxacin was associated with lower antibiotic acquisition costs. There was no deleterious impact caused by levofloxacin prophylaxis on survival, emergence of antibiotic resistance, detection of Clostridium difficile Ag in stool specimens, incidence of viridans group streptococcal bacteremia or Pseudomonas infections. There was a trend toward lower rates of bacteriuria, wound and bacterial respiratory infections in the levofloxacin than in the ceftazidime group, but these differences were not statistically significant. These data support the use of levofloxacin as prophylaxis in myeloablative allogeneic HCT when prophylaxis is used.

Comparison of three methodologies for the determination of pulmonary fungal burden in experimental murine aspergillosis.

Quantitative culture, quantitative PCR and the galactomannan enzyme immunoassay (EIA) were compared for their ability to determine the pulmonary fungal burden in a murine model of invasive aspergillosis. Quantitative culture of specimens containing hyphae under-represented the absolute fungal burden in established infection when compared with the two other methods. The best correlation was observed between the two non-culture methods. Higher variability was observed with the galactomannan EIA when compared with quantitative PCR. Collectively, these data suggest that quantitative PCR is the preferred method for determination of the pulmonary fungal burden in experimental aspergillosis.

Localization of Tropheryma whippelii rRNA in tissues from patients with Whipple's disease.

Whipple's disease is caused by a cultivation-resistant bacterium, Tropheryma whippelii. Ultrastructural studies of intestinal biopsy specimens from patients with Whipple's disease have shown that intracellular and extracellular bacteria are present, but the preferred site of growth is unknown. Tissue sections from 8 patients with Whipple's disease and from 19 healthy control subjects were analyzed by use of fluorescence in situ hybridization and laser scanning confocal microscopy, to determine the location of rRNA that would indicate the presence of metabolically active bacteria. T. whippelii rRNA was most prevalent near the tips of intestinal villi, in the lamina propria, just basal to epithelial cells. Most of the bacterial rRNA signal appeared to be located between cells and did not colocalize with the human intracellular protein vimentin. The location of bacterial rRNA in tissues from patients with Whipple's disease provides evidence that bacteria are growing outside cells and suggests that T. whippelii is not an obligate intracellular pathogen.

Tropheryma whippelii DNA is rare in the intestinal mucosa of patients without other evidence of Whipple disease.

BACKGROUND: Little is known about the pathogenesis of Whipple disease, the reservoirs of Tropheryma whippelii, and the proportion of persons harboring the bacterium without "classic" intestinal abnormalities. OBJECTIVE: To assess the presence of T. whippelii in patients undergoing upper endoscopy for a variety of indications. DESIGN: Prospective and routine diagnostic examination of patients. SETTING: Three academic medical centers in California; Minnesota; and Heidelberg, Germany. PATIENTS: 342 patients undergoing endoscopy for evaluation of dyspepsia or possible peptic ulcer (group A, 173 patients), malabsorption (group B, 37 patients), or clinical suspicion of Whipple disease (group C, 132 patients). MEASUREMENTS: Small-intestinal biopsy specimens were tested by polymerase chain reaction for T. whippelii DNA and examined for histopathologic abnormalities. RESULTS: All patients with negative histologic findings also had negative results for T. whippelii DNA. CONCLUSIONS: T. whippelii occurs only rarely in intestinal mucosa that lacks histopathologic evidence of Whipple disease. The human small intestinal mucosa is an unlikely reservoir for this organism.

Microbial ecology of human skin in health and disease.

Cultivation of human skin reveals numerous bacteria and at least one fungus to be normal inhabitants of this ecosystem; however, most of our knowledge about the microbiology of human skin was acquired decades ago. Modern techniques employing nucleic acid-based microbial identification methods demonstrate the limitations of cultivation for appreciating microbial diversity in many ecosystems. The application of modern molecular methods to the study of skin may offer new perspectives on the resident microfora, and new insights into the causes of antibiotic responsive dermatologic conditions, such as acne and rosacea.

Rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites.

Rhinosporidium seeberi, a microorganism that can infect the mucosal surfaces of humans and animals, has been classified as a fungus on the basis of morphologic and histochemical characteristics. Using consensus polymerase chain reaction (PCR), we amplified a portion of the R. seeberi 18S rRNA gene directly from infected tissue. Analysis of the aligned sequence and inference of phylogenetic relationships showed that R. seeberi is a protist from a novel clade of parasites that infect fish and amphibians. Fluorescence in situ hybridization and R. seeberi- specific PCR showed that this unique 18S rRNA sequence is also present in other tissues infected with R. seeberi. Our data support the R. seeberi phylogeny recently suggested by another group. R. seeberi is not a classic fungus, but rather the first known human pathogen from the DRIPs clade, a novel clade of aquatic protistan parasites (Ichthyosporea).

Absence of Kaposi's sarcoma-associated herpesvirus DNA in bacillary angiomatosis-peliosis lesions.

Bartonella henselae and B. quintana induce an unusual vascular proliferative tissue response known as bacillary angiomatosis (BA) and bacillary peliosis (BP) in some human hosts. The mechanisms of Bartonella-associated vascular proliferation remain unclear. Although host factors probably play a role, microbial coinfection has not been ruled out. Because of the vascular proliferative characteristics noted in both Kaposi's sarcoma (KS) and BA and occasional colocalization of KS and BA, the possibility was explored that KS-associated herpesvirus (KSHV) might be associated with BA lesions. Tissues with BA and positive and negative control tissues were tested for the presence of KSHV DNA by a sensitive polymerase chain reaction assay. Only 1 of 10 BA tissues, a splenic biopsy, was positive in this assay; this tissue was from a patient with concomitant KS of the skin. Thus, KSHV is probably not involved in the vascular proliferative response seen in BA-BP.

Infectious agents and the etiology of chronic idiopathic diseases.

At the end of the nineteenth century, the field of microbiology was born, and the infectious nature of many previously unexplained diseases was illuminated as powerful new technology was applied. At the end of the twentieth century, the etiology of myriad chronic diseases remains unexplained. We have argued that many of these diseases have clinical, epidemiological, and pathological features that suggest a role for microbes in their pathogenesis. Although definitive evidence of microbial disease causation is lacking, we believe that new technologies, such as sequence-based microbial identification, will successfully be applied to many of these chronic idiopathic diseases in the near future. As novel pathogens and previously described pathogens are revealed as the causative agents for some of these conditions, new diagnostic, preventive, and therapeutic modalities may emerge, transforming some diseases from idiopathic and chronic, to infectious and curable.

Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate.

Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with Escherichia coli was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted E. coli 16S rRNA gene was achieved by adding 5 microliter of undiluted processed sample DNA to a 50-microliter PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance.

PCR analysis of T. whippelii DNA in a case of Whipple's disease: effect of antibiotics and correlation with histology.

A 58-yr-old man developed severe weight loss, arthralgias, and diarrhea. Endoscopic examination of the stomach and duodenum revealed thickened folds of duodenal mucosa. Biopsy of the gastric mucosa was negative, whereas duodenal biopsy revealed blunted epithelial villi and PAS-positive foamy macrophages within the lamina propria. Bacilli typical of those associated with Whipple's disease were found by electron microscopy. The diagnosis was confirmed by polymerase chain reaction (PCR) assay, which detected a portion of the 16S ribosomal RNA gene sequence corresponding to the Whipple bacillus (Tropheryma whippelii) in duodenum, stomach, and liver biopsies before therapy. T. whippelii DNA was eliminated from all tissues tested within 3 months of starting antibiotic treatment, but the histological improvement lagged behind the clinical and molecular evidence of improvement.

AIDS-related disseminated histoplasmosis in San Francisco, California.

The published reports of patients with the acquired immunodeficiency syndrome (AIDS) with disseminated histoplasmosis come mostly from institutions located in endemic areas for histoplasmosis, where disease is thought to occur by either primary infection or reactivation. The characteristics of reactivation disease are not well delineated. We describe the clinical features of reactivation disseminated histoplasmosis in 46 residents of San Francisco, California, with AIDS who did not report recent travel to an area endemic for histoplasmosis. Patients presented with illness lasting days to months, manifested most frequently by fever, chills, sweats, cough or dyspnea, gastrointestinal complaints, malaise, and weight loss. Physical examination and imaging studies were notable for hepatosplenomegaly, lymphadenopathy, or abnormal pulmonary findings in more than half of patients. Laboratory studies revealed a high rate of cytopenia, elevated serum lactate dehydrogenase levels, abnormal liver function test values, respiratory alkalosis with hypoxemia, and a median CD4 lymphocyte count of 36 x 10(9) per liter. The clinical presentation of reactivation disseminated histoplasmosis in patients with AIDS living in San Francisco is similar to that of disseminated histoplasmosis reported in patients with AIDS living in endemic areas. Reactivation disseminated histoplasmosis should be considered in any AIDS patient with a low CD4 lymphocyte count, a febrile illness, and a history of travel or residence in an endemic area.

Diagnosis and monitoring of Whipple disease by polymerase chain reaction.

BACKGROUND: Whipple disease is a chronic, multisystem disorder associated with infection with Tropheryma whippelii, an organism that has not yet been grown on artificial media. In some cases, the diagnosis of Whipple disease is uncertain if it is based on histology alone. Although antibiotic regimens of various durations have been used, the disease recurs in about one third of cases. No test for cure is available. OBJECTIVE: To develop a test that is more sensitive and specific than histologic examination to diagnose Whipple disease and monitor the effects of antibiotic therapy. DESIGN: Retrospective, laboratory-based evaluations of stored tissue specimens. PATIENTS: 30 patients with clinically diagnosed, histologically confirmed Whipple disease and 8 patients in whom Whipple disease was clinically suspected but who did not have definitive histologic evidence. MEASUREMENTS: Pretreatment and post-treatment biopsy specimens of the small bowel and lymph node were tested by polymerase chain reaction for the presence of T. whippeli DNA. RESULTS: Results on PCR were positive in 29 of the 30 specimens from patients with histologically confirmed disease (sensitivity, 96.6%; specificity, 100%) and in 7 of the 8 specimens from patients in whom disease was clinically suspected. Small-bowel biopsy specimens were obtained after treatment from 17 patients (median duration of follow-up, 119 months); specimens from 12 of these patients had positive results on PCR. When these cases were correlated with therapeutic outcome, it was found that 7 of the 12 patients had clinical relapse during subsequent follow-up or had never responded to treatment (positive predictive value, 58% [95% CI, 28% to 85%]). In contrast, none of the 5 patients whose post-treatment biopsy specimens had negative results on PCR had relapse (negative predictive value, 100% [CI, 48% to 100%]; P = 0.044). No correlation was found between post-treatment histology and clinical outcome (P > 0.2). CONCLUSIONS: Polymerase chain reaction is highly sensitive and specific when used to confirm the diagnosis of Whipple disease, to identify inconclusive and suspicious cases, and to monitor response to therapy. A negative result on PCR may predict a low likelihood of clinical relapse; a positive test result that remains positive despite therapy may be associated with a poor clinical outcome. Histopathologic evaluation of post-treatment specimens does not predict clinical cure or relapse.

Tularemia presenting as community-acquired pneumonia. Implications in the era of managed care.

A case of pleuropulmonary tularemia was diagnosed by sputum culture and serologic studies in a patient who did not have classic epidemiological risks for tularemia. The patient had atypical pneumonia when initially seen and his condition slowly improved with antibiotic therapy that included erythromycin lactobionate. The diagnosis of tularemia was delayed because the gram-negative rod isolated from the patient's sputum was initially not speciated in an effort to reduce laboratory costs.

Central nervous system abscesses due to Coccidioides species.

Meningitis occurs in one-third to one-half of patients with disseminated coccidioidomycosis, but mass lesions have rarely been described; these lesions are usually found at autopsy. We report six cases of disseminated coccidioidomycosis with central nervous system (CNS) abscesses. Four patients had cerebellar involvement, and two had spinal cord involvement. Four patients were diabetic, and two subsequently died. Review of the literature on CNS coccidioidomycosis indicated that parenchymal brain involvement occurs in 1%-33% of cases, and < 40 cases with mass lesions have been reported since 1905. Almost all patients were male and had other active disseminated foci of coccidioidomycosis. In approximately one-third of all cases, meningitis was absent. Brain lesions may be superficial or deep and multiple or single. In the absence of meningitis, serology of cerebrospinal fluid is negative. Hematogenous origin appears to be more common than direct extension from the meninges. Spinal cord involvement is rare. Diabetes was present in several cases, thus suggesting a vascular predisposition. We hope our experience will increase awareness of this entity, which appears to be more common than previously appreciated, and will facilitate diagnosis.