Xiang Y. Han

Detection of Gliotoxin in Experimental and Human Aspergillosis

ABSTRACT Gliotoxin was measured in the lungs (mean, 3,976 ± 1,662 ng/g of tissue) and sera (mean, 36.5 ± 30.28 ng/ml) of mice with experimentally induced invasive aspergillosis (IA), and levels decreased with antifungal therapy. Gliotoxin could also be detected in the sera of cancer patients with documented (proven or probable) IA.

Rapidly Growing Mycobacteria Clinical and Microbiologic Studies of 115 Cases

We analyzed clinical and microbiologic features of 115 cases involving rapidly growing mycobacteria (RGM) isolated at the University of Texas M.D. Anderson Cancer Center, Houston (2000-2005) and identified by 16S ribosomal RNA gene sequencing analysis. At least 15 RGM species were included: Mycobacterium abscessus (43 strains [37.4%]), Mycobacterium fortuitum complex (33 strains [28.7%]), and Mycobacterium mucogenicum (28 strains [24.3%]) most common, accounting for 90.4%. Most M abscessus (32/43) were isolated from respiratory sources, whereas most M mucogenicum (24/28) were from blood cultures. Antimicrobial susceptibility tests showed that M abscessus was the most resistant species; M mucogenicum was most susceptible. From blood and catheter sources, 46 strains (40.0%) were isolated; 44 represented bacteremia or catheter-related infections. These infections typically manifested high fever (mean temperature, 38.9 degrees C), with a high number of RGM colonies cultured. All infections resolved with catheter removal and antibiotic therapy. Six strains (M abscessus and M fortuitum only) were from skin, soft tissue, and wound infections. There were 59 strains from respiratory sources, and 28 of these represented definitive to probable infections. Prior lung injuries and coisolation of other pathogenic organisms were common. Overall, 78 RGM strains (67.8%) caused true to probable infections without direct deaths.

A New Mycobacterium Species Causing Diffuse Lepromatous Leprosy

Mycobacterium leprae causes leprosy. M leprae strains collected worldwide have been genetically clonal, which poorly explains the varying severity and clinical features of the disease. We discovered a new Mycobacterium species from 2 patients who died of diffuse lepromatous leprosy (DLL). The Mycobacterium was purified from heavily infected, freshly frozen autopsy liver tissue followed by DNA extraction in 1 case. Paraffin-embedded skin tissue was used for DNA extraction in another case. Six genes of the organism were amplified by polymerase chain reaction, sequenced on cloning or from amplicons, and analyzed. Significant genetic differences with M leprae were found, including a 2.1% divergence of the 16S ribosomal RNA (rRNA) gene, a highly conserved marker of bacterial evolution, and 6% to 14% mismatches among 5 less conserved genes. Phylogenetic analyses of the genes of 16S rRNA, rpoB, and hsp65 indicated that the 2 most related organisms evolved from a common ancestor that had branched from other mycobacteria. These results and the unique clinicopathologic features of DLL led us to propose Mycobacterium lepromatosis sp nov. This species may account for some of the clinical and geographic variability of leprosy. This finding may have implications for the research and diagnosis of leprosy.

Rapid and accurate identification of mycobacteria by sequencing hypervariable regions of the 16S ribosomal RNA gene.

We developed a method to identify mycobacteria by sequencing hypervariable regions of the polymerase chain reaction-amplified 16S ribosomal RNA gene. This method is nearly specific for mycobacteria and uses positive culture from liquid or solid medium without the needfor lengthy subculture. It shortens identification time to 3 days, which is much faster than the conventional biochemical method (mean, 8 weeks). It applies to all mycobacteria (approximately 100 species), unlike current nucleic acid hybridization methods, which probe only 4 species. The identifications are the same or are species specific for the well-characterized mycobacteria (59/68 [87%]) or more accurate for recently proposed species (9/68 [13%]). The method requires a single sequencing reaction, which is efficient and cost-effective. Therefore, this method is clinically and academically useful.

Viridans streptococci isolated by culture from blood of cancer patients: Clinical and microbiologic analysis of 50 cases

ABSTRACT Clinical and microbiologic studies of 50 cases of viridans streptococcal bacteremia in cancer patients were performed. The bacteria were identified to species level by sequencing analysis of the 16S rRNA gene. At least nine Streptococcus spp. were found, including S. mitis (25 strains, 50.0% of 50); currently unnamed Streptococcus spp. (11 strains); S. parasanguis (five strains); S. anginosus (three strains); S. salivarius (two strains); and one strain each of S. gordonii, S. sanguis, S. sobrinus, and S. vestibularis. There were no S. oralis strains. Among 11 antibiotics of nine classes tested, no resistance to vancomycin, linezolid, or quinupristin-dalfopristin was seen. Resistance to penicillin (MIC, 4 to 12 μg/ml) was noted only among S. mitis strains (28.0%, 7/25) and not non-S. mitis strains (0/25) (P = 0.004). Significantly more S. mitis strains than non-S. mitis strains were resistant to fluoroquinolones and to ≥3 classes of antibiotics. Isolation of quinolone-resistant organisms was associated with the prior usage of quinolones (P = 0.002). Quantitative blood cultures showed that the strains resistant to levofloxacin or gatifloxacin were associated with higher colony counts than were their corresponding nonresistant strains. The young and elderly patients also had higher levels of bacteremia caused predominantly by S. mitis. Septic shock was present in 17 (34.0% of 50) patients, and 13 of those cases were caused by S. mitis (P = 0.007). These results suggest that S. mitis is the most common cause of viridans streptococcal bacteremia in cancer patients and is more resistant to antibiotics than other species.

Clinical Significance and Epidemiologic Analyses of Mycobacterium avium and Mycobacterium intracellulare among Patients without AIDS

ABSTRACT The clinical significance and prevalence of Mycobacterium avium and Mycobacterium intracellulare were analyzed in a cohort of 7,472 patients who, from 1999 to 2003, sought care at the University of Texas M.D. Anderson Cancer Center, Houston, and had cultures performed for mycobacteria. Patients were stratified for age, sex, and underlying diseases, and bacteria were identified by 16S rRNA gene sequencing. M. avium was isolated in 62 (0.83%) of 7,472 patients and M. intracellulare in 65 (0.87%). Clinically, only 10 of the 62 (16.2%) patients with M. avium had probable to definite evidence of infection, whereas the majority (83.8%) had weak evidence of infection. Sex and age did not affect the isolation or infection of M. avium. Hematological tumors predisposed to M. avium colonization but not infection. In contrast, 41 of the 65 (63.1%) patients with M. intracellulare had probable to definite infection, a level much higher than those with M. avium (P < 0.001). M. intracellulare was more prevalent in women (1.33% of 3,311) than in men (0.50% of 4,161) (P < 0.001), and underlying diseases had no effect in women. Men with lung cancer had a higher prevalence (1.37%) than men without (0.34%) (4.0-fold; P < 0.001), but it was similar to that in women. A marked age trend for the isolation of M. intracellulare among women was noted: 0.27% (1-fold) for ages of <50 years, 0.85% (3.1-fold) for ages 50 to 59 years, 1.50% (5.6-fold) for ages 60 to 69 years, and 3.74% (13.9-fold) for ages ≥70 years (trend, P < 0.001). The combined rate for women ≥50 was 1.86% (95% confidence interval [1.30 to 2.42%]) (6.9-fold). Together, these results suggest that, among non-AIDS patients, M. intracellulare is more pathogenic and tends to infect women increasingly beyond menopause (age ≥50 years) regardless of underlying disease. The prevalence rate of 1.86% in postmenopausal women suggests the need to further investigate the public health significance of M. intracellulare.

Bacteriologic Characterization of 36 Strains of Roseomonas Species and Proposal of Roseomonas mucosa sp nov and Roseomonas gilardii subsp rosea subsp nov

We used a polyphasic approach (sequencing analysis of the 16S ribosomal RNA gene and phenotypic analyses) to characterize 36 strains of Roseomonas species isolated from blood. Five strains, represented by strain MDA5176 (M.D. Anderson Cancer Center), were identified as Roseomonas gilardii. One strain belonged to Roseomonas genomospecies 4. The 22 strains represented by strain MDA5527 showed significant differences genotypically and phenotypically with R gilardii and other Roseomonas species and represented a new Roseomonas species; Roseomonas mucosa sp nov was proposed to denote its prominent mucoid, almost runny colonies. Eight strains, represented by strain MDA5605, had minor differences with R gilardii and displayed obvious pink to red colonies; Roseomonas gilardii subsp rosea subsp nov was proposed. For subspecies differentiation, R gilardii was proposed to be R gilardii subsp gilardii subsp nov. Unique patterns of biochemical reactions were established for these Roseomonas species, which may assist routine identification of these organisms. All 36 strains and 2 American Type Culture Collection strains were susceptible to amikacin and ciprofloxacin but resistant to cefepime and ceftazidime. They also were frequently susceptible to imipenem and ticarcillin-clavulanate but far less susceptible to ceftriaxone, trimethoprim-sulfamethoxazole, and ampicillin. R mucosa strains were most resistant, whereas R gilardii subsp gilardii strains were most susceptible.

Cryptococcosis in patients with cancer

Records of 31 patients with cancer who did not have known human immunodeficiency virus infection and who developed culture-proven cryptococcosis during the period of 1989-1999 (incidence of 18 cases per 100,000 admissions) were retrospectively reviewed. Several presentations of cryptococcosis were seen, including pulmonary in 19 patients (13 of which were symptomatic), disseminated in 6, meningeal in 3, and other, less common manifestations in 3. Hematologic malignancy (in 20 patients [65%]) was the most common underlying disease. Lymphopenia was present in 19 patients (61%). Previous steroid use was noted in 16 patients (51%). The diagnosis of cryptococcosis was rarely suspected; lung and brain malignancy were frequent initial impressions. Cryptococcosis was diagnosed postmortem in only 2 cases (6%). In cases of both pulmonary and meningeal cryptococcosis, the yield of invasive diagnostic procedures was good. Antifungal treatment was heterogeneous, but only 18% of patients who received it had treatment failure. Fluconazole monotherapy was successful in 92% of patients. In conclusion, cryptococcosis is rare in patients with cancer and appears to have a relatively good diagnostic yield and therapeutic outcome.

Diagnosis of Invasive Mold Infection by Real-Time Quantitative PCR

We report the design and evaluation of a quantitative real-time polymerase chain reaction (PCR) assay to diagnose invasive mold infection (IMI) by detecting mold DNA in the serum. This assay detected 200 fg to 20 ng (5-log range) mold DNA and permitted a cutoff of 110 fg (3 genomes). Human or candidal DNA was not amplified. Specificity also was demonstrated by negative results in all 35 patients (76 serum samples) with unlikely IMI at the cutoff. For patients with possible, probable, and documented IMI diagnosed by a combination of clinical, microbiologic, and histologic criteria, this real-time PCR showed positivity in 40% (12/30), 68% (19/28), and 85% (11/13) cases, respectively, in testing of multiple serum samples. The overall serum positivity rate for these patients was 15.1% (73/483). Quantitative analysis of the positive serum samples estimated the bodily circulating mold burden to be 1.6 x 10(5) genomes (5.3 ng) by geometric mean with 4.2 x 10(7) genomes (1,400 ng) the highest. These results suggest that for the diagnosis of IMI, this real-time PCR may be a promising alternative to other invasive methods. Further evaluation is underway.

Clinical significance of Roseomonas species isolated from catheter and blood samples: Analysis of 36 cases in patients with cancer

This report analyzes 36 cases of bacteremia or catheter-related infection caused by Roseomonas species, a group of pink, slimy, waterborne, gram-negative coccobacilli. The causative species included the newly described Roseomonas mucosa (22 cases [61%]) and Roseomonas gilardii subspecies rosea (8 cases [22%]) and known species R. gilardii subspecies gilardii (5 cases [14%]) and Roseomonas genomospecies 4 (1 case [3%]). Twenty-nine (81%) of the cases were symptomatic, with fever being the most common symptom (in 27 [75%] of the cases). Twenty (56%) of the infections were monomicrobic. Six cases (17%) involved persistent catheter colonization, and 5 of these cases required removal of the catheter to clear the infection. All infections resolved, most with empirical antibiotic treatment. A summary of the antibiotic susceptibility pattern of these strains and other reported series show that Roseomonas species are consistently susceptible to amikacin and imipenem and frequently susceptible to ciprofloxacin and ticarcillin, but essentially nonsusceptible to ceftazidime and cefepime. This result may guide future therapy for infections due to Roseomonas species.