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.

Thrombocytopenia among extremely low birth weight neonates: data from a multihospital healthcare system.

OBJECTIVES: Thrombocytopenia is common in neonatal intensive care units (NICU), with 18 to 35% of patients developing this problem before hospital discharge. It might be even more common among extremely low birth weight neonates (ELBW, < or = 1000 g birth weight). However, little is known about thrombocytopenia in the ELBW population. We sought to determine the incidence, timing, causes, platelet transfusions given, and outcomes of thrombocytopenia among ELBW neonates. STUDY DESIGN: We performed a cohort analysis of all 284 ELBW neonates born during 2003 and 2004 cared for in any of the Intermountain Healthcare level III NICUs. RESULTS: Multiple platelet counts were obtained in all 284 (range, 4 to 441 platelet counts/patient). Of the 284, 208 (73%) had one or more platelet counts < or =150 000/microl. Most were detected during the first days of life; 80% were detected during the first week and only 20% were detected thereafter. Thrombcytopenia was more common among the smallest patients; 85% incidence among those < or =800 g, 60% among those 801 to 900 g, and 53% among those 901 to 1000 g. Platelet transfusions were given to 129 of the 208 thrombocytopenic neonates. More than 90% were given prophylactically (the patient was not bleeding). The mortality rate among those that received platelet transfusions was twice that of those that received no platelet transfusions (P < 0.01). In 48% of cases, the cause of the thrombocytopenia went undiagnosed. The most common explanations were being small for gestational age or delivered to a hypertensive mother, DIC, bacterial infection, fungal infection, and necrotizing enterocolitis, respectively. CONCLUSIONS: We observed thrombocytopenia among ELBW neonates at a rate more than twice that reported among the general NICU population. Much remains to be discovered about the etiology and best treatments of thrombocytopenia among ELBW neonates.

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.

Pattern of daily weights among low birth weight neonates in the neonatal intensive care unit: data from a multihospital health-care system.

OBJECTIVES: We sought to generate a contemporary postnatal weight grid for low birth weight (LBW, <2500 g) neonates using actual data, not mathematically derived idealized weight curves. To do this, we collected electronic data from all neonatal intensive care units (NICU) patients weighing 400-2600 g at birth, during 30 consecutive months (January 2003-June 2005) in all four NICU's within a single health system, Intermountain Health Care (IHC). METHODS: A deidentified limited data set was collected from electronic IHC records. Data were limited to the birth weights and all subsequent daily weights, until discharge, death, or transfer to a non-IHC facility. Lines were drawn connecting the exact daily means of body weight for patients in 200 g birth weight groupings, ranging from 400 to 2600 g. We assessed differences in growth patterns predicted by this grid vs three previously published NICU longitudinal weight grids. RESULTS: All recorded weights were electronically extracted for 1813 consecutive patients with dates of birth from January 1, 2003 through June 30, 2005. The daily weights of each patient totaled over 48,000 individual data points. The new grid differed from previously published grids, in that the new grid showed; (1) no predicted postnatal weight loss among neonates in categories<900 g birth weight, (2) less initial weight loss among neonates 900-2500 g, (3) fewer days to regain birth weight among neonates

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.

Cutaneous side-effects in cancer patients treated with the antiepidermal growth factor receptor antibody C225.

BACKGROUND: C225 is an antibody to the epidermal growth factor receptor (EGFR), and inhibits growth of various tumour cells. The antibody is currently being used as a therapeutic agent in several clinical trials of patients with carcinomas. Objectives To determine and investigate the cutaneous side-effects in cancer patients treated with C225. Methods We clinically examined 10 patients treated with C225, and performed immunohistochemical and in situ hybridization studies on skin biopsies. Results The most common cutaneous reaction to C225 therapy was the development of an acneiform follicular eruption, which was most pronounced on the face, chest and upper back and typically manifested a week after the onset of treatment. The consistency of the morphology and timing of the clinical findings in 10 different patients following monotherapy with C225 strongly suggested a direct biological effect of the antibody. Additional dermatological side-effects included focal areas of tender paronychial inflammation of toes and fingers and small aphthous ulcers of the oral mucosa. Serial punch biopsies of chest skin before and after treatment (at 8 days) revealed two main reaction patterns: a superficial dermal inflammatory cell infiltrate surrounding hyperkeratotic and ectatic follicular infundibula, and a suppurative superficial folliculitis. In two biopsies focal intraepidermal acantholysis was found. Microbiological cultures failed to reveal an infectious aetiology. Immunohistochemical and in situ hybridization studies on a subset of the biopsies showed an increase in the expression of p27Kip1 in epidermal keratinocytes after treatment with C225. Conclusions Our findings support the concept that p27Kip1 plays a part in the in vivo regulation of follicular and epidermal homeostasis by EGFR.

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.

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.

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.

Rotavirus outbreak on a pediatric oncology floor: possible association with toys.

BACKGROUND: Several outbreaks of rotavirus gastroenteritis have occurred in hospitals and day care centers. In the spring of 1997, an outbreak of rotavirus occurred on our pediatric unit. Aggressive infection control measures were instituted, and potential lapses in infection control were assessed. METHODS: Memorial Sloan-Kettering Cancer Center is a 434-bed cancer hospital in New York City. The pediatric unit is a 42-bed ward with both bone marrow transplant patients and non-bone marrow transplant oncology patients. Nosocomially acquired rotavirus was defined as diarrhea, vomiting, or gastrointestinal upset with onset 48 hours or more after hospital admission, accompanied by a positive enzyme immunoassay for rotavirus antigen. RESULTS: Between February 24 and April 4, 1997, 8 patients on the pediatric unit had nosocomial rotavirus. Aggressive infection control measures were instituted. Patients with rotavirus were cohorted and placed on contact precautions (strict handwashing, gloves, and gown). Investigation by the infection control team revealed that communal toys in the playroom were not being cleaned according to the weekly protocol. CONCLUSIONS: An outbreak of nosocomial rotavirus occurred on our pediatric oncology unit. Shared toys may have served as fomites in the transmission of rotavirus.

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.