Amplified fragment length polymorphism analysis of human clinical isolates of Mycobacterium haemophilum from different continents.

The role of the species Mycobacterium haemophilum as a pathogenic non-tuberculous microorganism is becoming better defined with the use of specific detection methods. However, epidemiological investigations of this species are still scarce. We analysed the genetic diversity of M. haemophilum by amplified fragment length polymorphism (AFLP) typing and compared isolates from different parts of the world. In total, 128 isolates, including 41 from the USA, 51 from Australia, 28 from Europe and eight from Israel were compared using AFLP methodology. Two restriction enzymes (MseI and EcoRI) and one selective primer were applied and provided a high discriminatory power. Clusters of isolates with identical AFLP patterns, which could indicate a possible common source, were observed from the Netherlands, New York and Australia. No clear clustering on the basis of continental origin was observed; however, types were restricted to geographical areas and not found on other continents. A high genetic stability within the species was demonstrated by the long-term existence of a single type.

Bacterial and fungal meningitis in patients with cancer.

OBJECTIVE: To analyze cases of bacterial and fungal meningitis in patients with cancer. METHODS: Retrospective chart review from 1993 to 2004 was performed of patients with cancer at our institution who had positive CSF bacterial or fungal culture. RESULTS: We identified 312 positive CSF cultures representing 175 unique presentations. Ninety-six cultures were deemed contaminants, leaving 79 cultures for analysis in 77 patients; 78% had prior neurosurgery. Organisms included 68% gram-positive cocci, 10% gram-positive bacilli, 14% gram-negative bacilli, 7% Cryptococcus, and 1% C. albicans. None had N. meningitidis or H. influenza. Two patients each had S. pneumoniae or L. monocytogenes. Five percent of presentations demonstrated the triad of fever, nuchal rigidity, and mental status changes. Seventy-five percent of presentations demonstrated CSF pleocytosis (> or = 10). Median CSF WBC count was 74 cells/mm(3). CSF protein was elevated and glucose was depressed in 71%. In neutropenic patients (n = 6), 4 had 0 to 1 CSF WBC/mm(3), and 2 had normal CSF. VP shunt infections were more likely to present with mental status changes. Thirty day mortality was 13%. CONCLUSIONS: Patients with cancer do not manifest symptoms of meningitis as often as patients without cancer and display a very different set of CSF organisms compared to a general population. The CSF inflammatory response is muted in patients with cancer with meningitis. Most patients with cancer with meningitis have had prior neurosurgery. Additionally, the organisms causing meningitis in the cancer population have shifted over time, with a decline in the organisms which typically infect immunocompromised hosts and an increase in gram-positive infections.

Preemptive diagnosis and treatment of Epstein-Barr virus-associated post transplant lymphoproliferative disorder after hematopoietic stem cell transplant: an approach in development.

Hematopoietic stem cell transplant (HSCT) recipients are at risk for Epstein-Barr virus (EBV)-associated, post transplant lymphoproliferative disorder (PTLD). Studies have suggested that early treatment may improve the outcome of patients with PTLD. Thus, significant attention has been focused on PCR-based approaches for preemptive (i.e., prior to clinical presentation) diagnosis. Reports from several transplant centers have demonstrated that HSCT recipients with PTLD generally have higher concentrations of EBV DNA in the peripheral blood than patients without PTLD. However, the PCR values of patients with PTLD typically span multiple orders of magnitude and overlap significantly with values from patients without PTLD. Thus, questions remain about the sensitivity and predictive value of these assays. Preemptive strategies using rituximab and/or EBV-specific cytotoxic T lymphocytes have been evaluated in patients with elevated EBV viral loads. We review the current literature, discuss our institutional experience and identify several areas of future research that could improve the diagnosis and treatment of this life-threatening disorder in HSCT recipients.

Pre- and post-engraftment bloodstream infection rates and associated mortality in allogeneic hematopoietic stem cell transplant recipients.

We report on bloodstream infection (BSI) rates, risk factors, and outcome in a cohort of 298 adult and pediatric hematopoietic stem cell transplantation (HSCT) recipients at Memorial Sloan-Kettering Hospital from September 1999 through June 2003. Methods. Prospective surveillance study. BSI rates are reported per 10,000 HSCT days. Date of engraftment is defined as the first of at least 3 consecutive dates of absolute neutrophil count >500/mm(3) after stem cell infusion. BSI severity grades: severe (intravenous antibiotics), life threatening (sepsis), or fatal (caused or contributed to death). Results. The incidence of pre- and post-engraftment BSI was 22% and 19.5%, respectively. Pre-engraftment highest rates were observed for viridans streptococci (58), Enterobacteriaceae (39), and Enterococcus faecium (34). Post-engraftment rates ranged from 0.2 to 2.9 without any predominant pathogen. In multivariate analyses, pre-engraftment BSI was associated with diagnosis of chronic myelogenous leukemia, age >18 years and peripheral blood stem cell graft; post-engraftment BSI was associated with acute graft-versus-host disease, neutropenia, and liver or kidney dysfunction. Attributable mortality was 12.5% and 1.7% for pre- and post-engraftment BSI, respectively. BSI fatality rates were 24% for viridans streptococci, 8% for E. faecium, 11% for Staphylococcus aureus, and 67% for Candida. Conclusions. Pre-engraftment BSI, especially by viridans streptococci and E. faecium, was associated with substantial attributable mortality. Post-engraftment BSI was a marker of post-transplant complications and rarely the primary cause of death.

Respiratory syncytial virus infection following hematopoietic stem cell transplantation.

Respiratory syncytial virus, one of the most common causes of respiratory infections in immunocompetent individuals, is frequently spread to recipients of HSCT by family members, other patients, and health care workers. In immunosuppressed individuals, progression from upper respiratory tract disease to pneumonia is common, and usually fatal if left untreated. We performed a retrospective analysis of RSV infections in recipients of autologous or allogeneic transplants. The incidence of RSV following allogeneic or autologous HSCT was 5.7% and 1.5%, respectively. Of the 58 patients with an RSV infection, 16 of 21 patients identified within the first post-transplant month, developed pneumonia. Seventy-two percent of patients received aerosolized ribavirin and/or RSV-IGIV, including 23 of 25 patients diagnosed with RSV pneumonia. In this aggressively treated patient population, three patients died of RSV disease, each following an unrelated HSCT.

Cytomegalovirus ventriculoencephalitis in a bone marrow transplant recipient receiving antiviral maintenance: clinical and molecular evidence of drug resistance.

We describe a case of CMV ventriculoencephalitis in a severely immunocompromised bone marrow transplant recipient who was receiving combination therapy with ganciclovir and foscarnet for treatment of viremia and retinitis. Analysis of sequential viral isolates recovered from the patient's cerebrospinal fluid suggested that disease developed because of the presence of viral resistance and, possibly, low tissue penetration of antiviral agents.

Mycobacterium haemophilum in immunocompromised patients.

Mycobacterium haemophilum, a recently described pathogen, can cause an array of symptoms in immunocompromised patients. To date, 90 patients with this infection have been described worldwide. We report our institution's experience with 23 patients who were treated from 1990 through 2000. Fourteen patients had undergone bone marrow transplantation, 5 were infected with human immunodeficiency virus, 3 had hematologic malignancies, and 1 had no known underlying immunosuppression. Clinical syndromes on presentation included skin lesions alone in 13 patients, arthritis or osteomyelitis in 4 patients, and lung disease in 6 patients. Although patients with skin or joint involvement had favorable outcomes, 5 of 7 patients with lung infection died. Prolonged courses of multidrug therapy are required for treatment. A diagnosis of M. haemophilum infection must be considered for any immunocompromised patient for whom acid-fast bacilli are identified in a cutaneous, synovial fluid or respiratory sample or for whom granulomas are identified in any pathological specimen.

Application of the Sherlock Mycobacteria Identification System using high-performance liquid chromatography in a clinical laboratory.

There is a growing need for a more accurate, rapid, and cost-effective alternative to conventional tests for identification of clinical isolates of Mycobacterium species. Therefore, the ability of the Sherlock Mycobacteria Identification System (SMIS; MIDI, Inc.) using computerized software and a Hewlett-Packard series 1100 high-performance liquid chromatograph to identify mycobacteria was compared to identification using phenotypic characteristics, biochemical tests, probes (Gen-Probe, Inc.), gas-liquid chromatography, and, when necessary, PCR-restriction enzyme analysis of the 65-kDa heat shock protein gene and 16S rRNA gene sequencing. Culture, harvesting, saponification, extraction, derivatization, and chromatography were performed following MIDI's instructions. Of 370 isolates and stock cultures tested, 327 (88%) were given species names by the SMIS. SMIS software correctly identified 279 of the isolates (75% of the total number of isolates and 85% of the named isolates). The overall predictive value of accuracy (correct calls divided by total calls of a species) for SMIS species identification was 85%, ranging from only 27% (3 of 11) for M. asiaticum to 100% for species or groups including M. malmoense (8 of 8), M. nonchromogenicum (11 of 11), and the M. chelonae-abscessus complex (21 of 21). By determining relative peak height ratios (RPHRs) and relative retention times (RRTs) of selected mycolic acid peaks, as well as phenotypic properties, all 48 SMIS-misidentified isolates and 39 (91%) of the 43 unidentified isolates could be correctly identified. Material and labor costs per isolate were $10.94 for SMIS, $26.58 for probes, and $42.31 for biochemical identification. The SMIS, combined with knowledge of RPHRs, RRTs, and phenotypic characteristics, offers a rapid, reasonably accurate, cost-effective alternative to more traditional methods of mycobacterial species identification.

Candida dubliniensis at a cancer center.

Candida dubliniensis, a germ tube-positive yeast first described and identified as a cause of oral candidiasis in patients with acquired immunodeficiency syndrome in Europe in 1995, has an expanding clinical and geographic distribution that appears to be similar to that of the other germ tube-positive yeast, Candida albicans. This study determined the frequency, clinical spectrum, drug susceptibility profile, and suitable methods for identification of this emerging pathogen at a cancer center in 1998 and 1999. Twenty-two isolates were recovered from 16 patients with solid-organ or hematologic malignancies or acquired immunodeficiency syndrome. Two patients with cancer had invasive infection, and 14 were colonized with fungus or had superficial fungal infection. All isolates produced germ tubes and chlamydospores at 37 degrees C, did not grow at 45 degrees C, and gave negative reactions with d-xylose and alpha-methyl-d-glucoside in the API 20 C AUX and ID 32 C yeast identification systems. Phenotypic identification was confirmed by molecular beacon probe technology. All isolates were susceptible to the antifungal drugs amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, and ketoconazole.

Control of influenza A on a bone marrow transplant unit.

In January 1998, an outbreak of influenza A occurred on our adult bone marrow transplant unit. Aggressive infection control measures were instituted to halt further nosocomial spread. A new, more rigorous approach was implemented for the 1998/99 influenza season and was extremely effective in preventing nosocomial influenza at our institution.

Rapid identification of Candida dubliniensis using a species-specific molecular beacon.

Candida dubliniensis is an opportunistic fungal pathogen that has been linked to oral candidiasis in AIDS patients, although it has recently been isolated from other body sites. DNA sequence analysis of the internal transcribed spacer 2 (ITS2) region of rRNA genes from reference Candida strains was used to develop molecular beacon probes for rapid, high-fidelity identification of C. dubliniensis as well as C. albicans. Molecular beacons are small nucleic acid hairpin probes that brightly fluoresce when they are bound to their targets and have a significant advantage over conventional nucleic acid probes because they exhibit a higher degree of specificity with better signal-to-noise ratios. When applied to an unknown collection of 23 strains that largely contained C. albicans and a smaller amount of C. dubliniensis, the species-specific probes were 100% accurate in identifying both species following PCR amplification of the ITS2 region. The results obtained with the molecular beacons were independently verified by random amplified polymorphic DNA analysis-based genotyping and by restriction enzyme analysis with enzymes BsmAI and NspBII, which cleave recognition sequences within the ITS2 regions of C. dubliniensis and C. albicans, respectively. Molecular beacons are promising new probes for the rapid detection of Candida species.

Disseminated toxoplasmosis following T cell-depleted related and unrelated bone marrow transplantation.

More than 95% of reported cases of disseminated toxoplasmosis following BMT have occurred following an unmodified transplant. Most have been fatal, diagnosed at autopsy and without antemortem institution of specific therapy. From 1989 to 1999, we identified 10 cases of disseminated toxoplasmosis, in 463 consecutive recipients of a T cell-depleted (TCD) BMT. Transplants were from an unrelated donor (n = 5), an HLA-matched sibling (n = 4) or an HLA-mismatched father (n = 1). In 40%, both the donor and recipient had positive IgG titers against T. gondii pre-transplant; in 30%, only the recipient was sero-positive. Three recipients of an unrelated TCD BMT developed toxoplasmosis despite both donor and host testing negative pretransplant. All 10 patients presented with high grade fever. CNS involvement ultimately occurred in seven patients, with refractory respiratory failure and hypotension developing in nine. Eight of 10 cases were found only at autopsy, involving the lungs (n = 7), heart (n = 5), GI tract (n = 5), brain (n = 8), liver and/or spleen (n = 5). The only survivor, treated on the day of presentation with fever and headache, was diagnosed by detection of T. gondii DNA by polymerase chain reaction (PCR) performed on the blood and spinal fluid. This study demonstrates the similar incidence of toxoplasmosis following TCD BMT and that reported post T cell-replete BMT, and underscores the need for rapid diagnostic tests in an effort to improve outcome.

Histologic reactions to cutaneous infections by Mycobacterium haemophilum.

Mycobacterium haemophilum is an emerging pathogen in immunocompromised patients. We report the clinical and histologic findings of 16 skin biopsies from 11 patients with culture-proven infections by M. haemophilum. The patients had leukemia or non-Hodgkin's lymphoma. Ten of them had undergone bone marrow transplantation. When the skin biopsy specimens were taken, a portion of the skin was simultaneously submitted to a microbiology laboratory for cultures. The remaining skin was processed routinely. Acid-fast bacilli were found in 11 of 16 lesions. The number of histologically detectable organisms was typically low: nine biopsies had fewer than three bacilli per 50 oil immersion fields. The most common histologic pattern was a mixed suppurative and granulomatous reaction (7 of 16 biopsies). Four biopsies showed well-formed epithelioid granulomas. Two showed necrosis, one of which was ulcerated. One lesion was a subcutaneous abscess. Two biopsies showed a mixed lichenoid and granulomatous dermatitis. In one of them, the granulomatous reaction was focal and small. One biopsy lacked a granulomatous tissue reaction altogether; it showed an interface dermatitis, a perivascular and periadnexal lymphocytic infiltrate, and necrotizing lymphocytic small vessel vasculitis. A subsequent biopsy from the same patient additionally showed a focal granulomatous reaction. Our observation that infections by M. haemophilum can present with nongranulomatous or pauci-granulomatous reactions without necrosis is of note. Failure to suspect mycobacterial infection in such reactions contributes to probable underreporting of M. haemophilum and to misdiagnoses. Furthermore, our findings emphasize the importance of simultaneous biopsies for culture and histology in immunocompromised patients.

Pulmonary nodule due to Mycobacterium haemophilum in an immunocompetent host.

We present a case of a pulmonary nodular lesion in an immunocompetent patient documented at open lung biopsy to be due to Mycobacterium haemophilum. This organism has recently been recognized as a cause of disease in immunocompromised patients, presenting predominantly as skin lesions, arthritis, and rarely pneumonia. Because this mycobacterium is fastidious and difficult to grow without the use of special media and conditions, our case raises the possibility that M. haemophilum could be an underrecognized cause of granulomatous pulmonary lesions and should be considered particularly in cases where smears for acid-fast bacteria are positive but cultures are negative.

Mycobacterium tuberculosis infection in a green-winged macaw (Ara chloroptera): report with public health implications.

Mycobacterium tuberculosis was isolated from the eyelid, skin, tongue, and lungs of a green-winged macaw (Ara chloroptera). Two persons living in the same household were culture positive for pulmonary tuberculosis 3 to 4 years before tuberculosis was diagnosed in the bird. Although humans have not been shown to acquire tuberculosis from birds, an infected bird may be a sentinel for human infection.

Mycobacterium haemophilum: microbiology and expanding clinical and geographic spectra of disease in humans.

Reports of the association of Mycobacterium haemophilum with disease in humans have greatly increased. At least 64 cases have now been reported, with symptoms ranging from focal lesions to widespread, systemic disease. The organism is now known to cause primarily cutaneous and subcutaneous infection, septic arthritis, osteomyelitis, and pneumonitis in patients who are immunologically compromised and lymphadenitis in apparently immunocompetent children. Underlying conditions in the compromised patients have included AIDS; renal, bone marrow, and cardiac transplantation; lymphoma; rheumatoid arthritis; marrow hypoplasia; and Crohn's disease. Reports have originated from diverse geographic areas worldwide. The epidemiology of M. haemophilum remains poorly defined; there appears to be a genetic diversity between strains isolated from different regions. The organism is probably present in the environment, but recovery by sampling has not been successful. M. haemophilum has several unique traits, including predilection for lower temperatures (30 to 32 degrees C) and requirement for iron supplementation (ferric ammonium citrate or hemin). These may in the past have compromised recovery in the laboratory. Therapy has not been well elucidated, and the outcome appears to be influenced by the patient's underlying immunosuppression. The organisms are most susceptible to ciprofloxacin, clarithromycin, rifabutin, and rifampin. Timely diagnosis and therapy require communication between clinician and the laboratory.

Mycobacterium genavense infections in pet animals.

Mycobacterium genavense, a recently reported cause of a wasting illness in patients with AIDS, was isolated from a cervical lymph mode from a dog with severe hind limb weakness and from trachael tissue from a parrot with acute onset respiratory distress. Physicians caring for immunocompromised patients should consider birds and dogs potential sources of M. genavense infection and submit appropriate specimens for culture.