Search

We report here the use of novel “sloppy” molecular beacon probes in homogeneous PCR screening assays in which thermal denaturation of the resulting probe-amplicon hybrids provides a characteristic set of amplicon melting temperature (Tm) values that identify which species is present in a sample. Sloppy molecular beacons possess relatively long probe sequences, enabling them to form hybrids with amplicons from many different species despite the presence of mismatched base pairs. By using four sloppy molecular beacons, each possessing a different probe sequence and each labeled with a differently colored fluorophore, four different Tm values can be determined simultaneously. We tested this technique with 27 different species of mycobacteria and found that each species generates a unique, highly reproducible signature that is unaffected by the initial bacterial DNA concentration. Utilizing this general paradigm, screening assays can be designed for the identification of a wide range of species. A classic approach for determining the identity of a bacterial species is to employ PCR to exponentially amplify a selected segment of a 16S rRNA gene (26), utilizing a pair of “universal primers” that bind to highly conserved sequences at the ends of the target region, and to then use a sophisticated technique to identify a species-specific sequence in the middle of the resulting amplicons (12). These identification methods include nucleotide sequence analysis (27) and electrophoretic determination of the sizes of fragments produced by incubation of the amplicons with selected restriction endonucleases (17). However, these techniques are time consuming, costly, and labor intensive.

Summary Background Human metapneumovirus is a recently discovered RNA virus that typically causes respiratory disease in children. It has been linked to severe lower airway disease in hematopoietic stem cell and solid-organ transplant recipients. hMPV infection in a large population of patients with underlying cancer and varying degrees of immunosuppression has not been reported. We sought to characterize hMPV infection in patients with cancer. Methods Review of all cases of hMPV infection from two seasons (2005–6 and 2006–7) detected by DFA and/or real-time PCR at MSKCC, a tertiary cancer center in New York City. Results Among MSKCC patients with cancer, 51 (2.7%) of 1899 patients were positive for hMPV, including 3.2% with hematologic neoplasm and 1.7% with solid tumors. More children (4.5%) were positive than adults (2.2%). PCR detected twice as many cases as DFA. Cough and fever were common complaints. The longest shedding period was 80 days. 40 patients received radiographic evaluation; of these, 22 showed abnormalities including patchy (11), ground glass (5), and interstitial infiltrates (4). Conclusions hMPV causes a nonspecific respiratory illness and was found in more than 2% of all tested persons with cancer. PCR detected substantially more cases than DFA. Unlike previous reports, we observed no fatalities due to hMPV, including 22 HSCT recipients with the infection.

Background. Viridans streptococcal bacteremia (VSB) after allogeneic hematopoietic stem cell transplantation (HSCT) is associated with substantial mortality. Prevention of this serious complication is therefore a high priority. The objective of this study was to evaluate the effect of early vancomycin administration on rates and outcomes of VSB. Methods. We analyzed the effect of early vancomycin on the incidence of VSB in a cohort of 430 consecutive HSCTs performed during the period of 1 January 1998 to 30 September 2002. The primary end point was time to diagnosis of VSB. Early vancomycin was defined as ≥2 doses of vancomycin between days -7 through +7 after HSCT or diagnosis of VSB, whichever occurred first. Risk factors for VSB were identified in univariate and multivariate Cox proportional hazard models. Results. The incidence of VSB in the cohort was 7.4%. The incidence of VSB in patients who did not receive early vancomycin was 24.8%, compared with 0.3% in patients who did (P

Introduction In 1995, a report from Dublin, Ireland described atypical, germ-tubeand chlamydospore-positive yeast isolated most often fi-om HIV-infected patients who had oral candidiasis (1). The yeast formed a homogenous cluster by DNA fingerprinting which was different from that of other Candida species, including the other germtubeand chlamydospore-positive yeast, Candida aibicans. The authors proposed the name Candida dubliniensis for this novel species. Since then, a series of reports have documented the prevalence and epidemiology, clinical manifestations, virulence, phenotypic and genotypic characteristics, and antimicrobial susceptibility test results of C. dubliniensis. The present review summarizes those findings and notes the experience with C. dubliniensis at Memorial Sloan-Kettering Cancer Center (MSKCC) in New York City.

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.

To the Editor: The immunosuppressive agent alemtuzumab is a DNA-derived, humanized monoclonal antibody directed against the panlymphocyte, cell-surface antigen CD52 (1). The drug is approved for the treatment of refractory B-cell chronic lymphocytic leukemia (2) and also has been used after stem cell (3) and organ transplantations (4). Alemtuzumab causes profound and prolonged lymphocyte depletion, which results in a variety of complications involving infections (5). However, mycobacteria have rarely been reported to cause infection after alemtuzumab treatment. We describe infections with Mycobacterium haemophilum, a fastidious nontuberculous mycobacterium, in 2 patients who experienced cutaneous lesions while they received alemtuzumab.

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.

A resurgence of tuberculosis has occurred in recent years in the United States and abroad. Deteriorating public health services, increasing numbers of immigrants from countries of endemicity, and coinfection with the human immunodeficiency virus (HIV) have contributed to the rise in the number of cases diagnosed in the United States. Outbreaks of resistant tuberculosis, which responds poorly to therapy, have occurred in hospitals and other settings, affecting patients and health care workers. This review covers the pathogenesis, epidemiology, clinical presentation, laboratory diagnosis, and treatment of Mycobacterium tuberculosis infection and disease. In addition, public health and hospital infection control strategies are detailed. Newer approaches to epidemiologic investigation, including use of restriction fragment length polymorphism analysis, are discussed. Detailed consideration of the interaction between HIV infection and tuberculosis is given. We also review the latest techniques in laboratory evaluation, including the radiometric culture system, DNA probes, and PCR. Current recommendations for therapy of tuberculosis, including multidrug-resistant tuberculosis, are given. Finally, the special problem of prophylaxis of persons exposed to multidrug-resistant tuberculosis is considered.

Mycobacterium haemophilum is emerging as a pathogen of immunocompromised patients particularly those with AIDS and organ transplants. Infection has also occurred in healthy children. Adults usually present with cutaneous manifestations, septic arthritis or occasionally pneumonia. Children have perihilar, cervical or submandibular adenitis. The organism grows on mycobacterial media supplemented with ferric ammonium citrate or hemin, incubated at 30 degrees C to 32 degrees C, two to three weeks after inoculation. The most active antimicrobial agents in vitro are amikacin, ciprofloxacin, clarithromycin, rifabutin and rifampin. Development of resistance to the rifamycins has been demonstrated after patients were treated for several months with several antimycobacterial agents, including the rifamycins. Treatment for several months with at least two agents demonstrated to have low MICs for the organism has been shown to be effective.

Mycobacterium haemophilum is an emerging opportunistic pathogen, and since 1989, infections caused by this organism have been identified more frequently in the New York City area than in any other region of the United States. A DNA fingerprinting method, based on restriction fragment length polymorphisms (RFLPs) was developed. A genomic library of M. haemophilum isolate 1A was constructed; screening the library yielded a recombinant strain that incorporated a genetic element present in multiple copies in the M. haemophilum genome. This clone was used to produce a probe for RFLP analyses of PvuII digests of genomic DNA. We used this probe to determine the RFLP patterns of 43 clinical isolates of M. haemophilum from 28 patients. A total of six distinct patterns were observed. Two patterns, designated types 1 and 2, accounted for 91% of the infections in patients from the New York City area. Two isolates from Arizona had identical patterns but were distinct from those of New York isolates, and an isolate from Israel, the type strain, had another distinct pattern (type 6). The type 6 pattern was also seen in a recent isolate from Norway. All of the type 1 isolates and 60% of the type 2 isolates were recovered from patients with AIDS in the New York City area. This molecular subtyping method should provide a useful tool for epidemiological studies and may help identify the associated risk factors, vehicles, and possible reservoirs of this newly emerging pathogen.

In May 1991, an article in a New York newspaper was entitled "HIV Victims' New Foe--Haemophilum: Rare but Deadly Microbe Remains a Mystery" (1). The article noted that "Marie, a once-beautiful 32-yr-old New Jersey suburbanite with AIDS, is covered from head to toe with huge purple scars that occasionally erupt in painful oozing sores. She routinely has fevers of more than 101°F. She walks with a limp because a deadly organism has eaten a hole in a bone in her left leg." The purple sores, originally thought to be the lesions of Kaposi's sarcoma, were eventually found to be caused by infection with Mycobacterium haemophilum Pus from lesions in the left leg contained the mycobacterium. During 1990 and 1991 at Memorial Sloan-Kettering Cancer Center (MSKCC), we observed four cases of M. haemophilum infection and presented them at Intercity Infectious Disease Rounds. Physicians from two other hospitals in New York City described their own cases. Laboratory directors in the greater New York area were called, and a total of five additional cases were disclosed. In May 1991, we received a call from Dr. Robert C. Good, Chief of the Respiratory Diseases Branch of the Centers for Disease Control (CDC), who stated that he had received a call from a New York reporter who wondered what the "haemophilum" organism was. Investigations by the New York City Health Department and the CDC were begun. In addition to the four cases of M. haemophilum infection seen at MSKCC (2), at least nine additional cases of suspected M. haemophilum infection occurred in the New York City area within the past 2 yr (3), and 17 additional cases of M. haemophilum infection were reported in journals between 1976 and 1991 (4, 5). A summary of the four MSKCC cases and the 17 previously reported culture proven cases is presented in Table 1.

Four cases of meningitis due to beta-hemolytic non-A, non-D streptococci among adult patients with neoplastic disease are reported. All four patients had head or neck tumors for greater than or equal to 4 years, and all had undergone surgery for these tumors. Three of four patients had received local radiation therapy. None of the patients were neutropenic. One patient died. A review of the literature revealed that most patients with non-A, non-D streptococcal meningitis had disruption of the normal barrier protecting the CNS due to trauma, surgery, or the presence of a tumor, or had extensive exposure to animals or an underlying medical disease. Infection with non-A, non-D streptococci should be considered in any patient with meningitis who has a tumor of the head or neck and who has undergone surgery and/or radiation therapy.

A significant increase in the use of vascular access devices has changed the spectrum of organisms causing bacteremia and fungemia at Memorial Sloan-Kettering Cancer Center. This paper documents the 1988 laboratory experience with bacteremia and fungemia and contrasts some of that data with information obtained in 1984. In 1988, 439 tunnelled-catheters and 355 ports were inserted in patients; 2,778 organisms were subsequently recovered from 933 episodes of bacteremia and fungemia. Fifty-percent of the episodes of bacteremia and fungemia were vascular access device-related. Compared to 1984, the relative incidence of bacteremia due to gram-positive organisms increased from 33 to 43%, polymicrobic cultures increased from 24 to 27%, and the number of organisms with colony counts greater than 100 cfu/ml increased from 24 to 44%. In 1988, device-related sepsis was often caused by Acinetobacter spp., Bacillus spp., Corynebacterium spp., pseudomonads other than Pseudomonas aeruginosa, and coagulase-negative staphylococci. Infection was also caused by species of flavobacteria, Micrococcus, and Rhodotorula. Efforts required for identification of many of the newer pathogens have escalated material and personnel costs.

We describe a 12-year-old boy with acute myeloid leukemia who developed pleuropericarditis while he was neutropenic and was receiving intravenously administered antibiotic and antifungal therapy for pneumonia. A KOH preparation of the purulent material from an extensive diagnostic and therapeutic pleuropericardial drainage procedure revealed multiple irregularly septate hyphae, and cultures yielded the organism Pythium insidiosum. After completing a 12-month course of intravenously administered liposomal amphotericin B (AmBisome; Fujisawa Healthcare) and itraconazole, the patient remained alive, in clinical remission, and symptom free.

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.

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.

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.

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.

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 Bsm AI and Nsp BII, 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.

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 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.

The diagnostic mycobacterial laboratory of the 1990s must respond to a change in the clinical spectrum of mycobacterial infections brought about by an increase in the number of patients who are immunocompromised, are indigent, or have temporary or permanent implanted devices. Emerging pathogens such as Mycobacterium haemophilum and Mycobacterium genavense, multidrug-resistant strains of Mycobacterium tuberculosis, and catheter infections with rapidly growing mycobacteria are examples of new issues. Fortunately, new methods for detection and identification of microbes have been or are being developed. Procedures that, when applied directly to clinical specimens or actively growing cultures, dramatically reduce the time to diagnosis of mycobacterial infections include radiometric broth, lysis-centrifugation, and biphasic systems for specimen culture, and DNA probes, high-performance liquid chromatography, DNA hybridization, restriction fragment length polymorphism, and gene amplification for organism detection and identification. At present, in vitro antimicrobial susceptibility tests are most helpful in guiding treatment of infections caused by M. tuberculosis and rapidly growing mycobacteria.

Four cases of infection with Mycobacterium haemophilum occurred at a single hospital in a seven-month period. Only 22 cases have been reported since 1976. All four patients were immunocompromised; two had AIDS and two were the first known recipients of allogeneic bone marrow transplants (BMT) to develop the infection. One BMT recipient died of Mycobacterium haemophilum pneumonia. The organism requires hemin or ferric ammonium citrate and incubation of media at 30 degrees C for optimum growth. Clinicians and microbiologists should consider infection with Mycobacterium haemophilum, particularly when specimens are from immunocompromised patients with unexplained illness and/or when acid-fast bacilli are seen on smear.

An unusual case of disseminated Nocardia brasiliensis infection is presented. The patient, who had been receiving chronic dexamethasone therapy for 4 years, had pneumonia and septic arthritis of the left knee due to N. brasiliensis. To our knowledge, this is the first report from the United States of a synovial joint infection with this organism. Disseminated disease due to N. brasiliensis is infrequently reported; it is most often seen in the immunocompromised patient and is often unresponsive to therapy.

With increased use of surgically implanted silastic central venous catheters, there has been an increase in the recovery from blood cultures at Memorial Sloan-Kettering Cancer Center (New York) of environmental and skin organisms including the red yeast Rhodotorula. From 1985 through 1989, 23 patients had catheter-related Rhodotorula sepsis. All 23 patients had indwelling central venous catheters that had been in place from 1 to 22 months (average, 9.3 months) prior to the detection of fungemia. All patients had blood drawn both through the catheter and from a peripheral source, and only one patient had a peripheral blood culture positive for Rhodotorula. Colony counts of yeast from the catheter cultures often exceeded 100 (15 patients) and even 1,000 (seven patients) cfu/mL of blood. Thirteen of the patients were treated with antifungal therapy and had the catheter removed, and five patients received antifungal therapy without catheter removal (suggesting that compulsory removal of the catheter may not always be required). Five patients had the catheter removed without antifungal therapy. All patients survived the fungemic episode and experienced no recurrence of the infection.

A case of Flavobacterium meningosepticum bacteremia complicating the course of a patient with leukemia is described. The patient was treated successfully when rifampin was added to the antibiotic therapy. Unusual organisms should be considered as agents of infection in immunocompromised hosts and susceptibility testing with drugs not commonly employed for gram-negative rods should be performed in complicated cases.

Blood cultures obtained with a lysis-centrifugation (L-C) system and a conventional two-bottle broth system were compared for the recovery of bacteria and yeasts from 7,000 cultures. The L-C system recovered significantly more total organisms, Escherichia coli, and Candida spp. and detected more patients with bacteremia and fungemia due to members of the family Enterobacteriaceae and yeasts. The broth system recovered significantly more streptococci and detected significantly more low-level Pseudomonas bacteremias. Polymicrobic bacteremia and fungemia were detected equally well by either culture system. Aerobic organisms grew equally well on blood, chocolate, or brain heart infusion agar plates used for L-C inoculation. A total of 82% of colony counts measured no more than 10 CFU/ml of blood, and it was at these low levels that enhanced detection of organisms by either system was observed. The L-C system isolated organisms and detected yeasts more rapidly than did the broth system. Contaminants occurred in 8.2% of L-C cultures and 1.9% of broth cultures. Low colony counts on L-C plates occurred for both Staphylococcus epidermidis contamination and septicemia.

The Mycobacterium avium complex, only rarely described as an invasive pathogen in humans, has recently been reported to frequently cause disseminated disease in patients with the acquired immune deficiency syndrome. Between February 1981 and February 1984 at Memorial Sloan-Kettering Cancer Center, 30 patients with acquired immune deficiency syndrome, 3 patients with leukemia, and 2 patients with congenital severe combined immunodeficiency syndrome developed disseminated M. avium complex infection. Mycobacteria were often found in multiple sites both antemortem and postmortem. Blood cultures were a reliable method for detecting disseminated infection, and the new lysis blood culture systems provided an efficient technique for determining the number of organisms per milliliter of blood. Acid-fast stains and cultures of fecal specimens were also helpful in diagnosing infection. Most of the mycobacteria were serovar 4 (77%), and most (86%) produced a deep yellow pigment. All isolates were susceptible to standard concentrations of clofazimine, cycloserine, and ansamycin, but tended to be resistant to isoniazid, streptomycin, ethambutol, ethionamide, and rifampin.

A case of pneumonia caused by Cunninghamella bertholletiae is described in a patient with chronic lymphatic leukemia. The species of Cunninghamella, a genus in the order Mucorales, are characterized by the formation of conidia on the surface of an inflated conidiophore (vesicle). C. bertholletiae, not C. elegans, is the most appropriate name for this fungus. This zygomycete was resistant by in vitro sensitivity testing to amphotericin B. The source of the infection is not known.

The potential clinical value of colony counts determined by the lysis-centrifugation blood culture method was studied by reviewing the records of eight patients with persistent septicemia in whom colony counts were available on at least 3 days. Colony counts of the five patients who survived decreased steadily as the patients improved. One of the three patients who died had counts repeatedly below 1.0 CFU/ml while she was clinically stable and higher counts when her condition deteriorated. Two patients died despite decreasing colony counts. One was improving and died unexpectedly of an unrelated cause; the other died of candidiasis, but declining serial arabinitol/creatinine ratios suggested a partial response to therapy. In addition, septicemia related to infected intravenous catheters was documented by demonstrating large differences in colony counts determined simultaneously from two different sites in two patients and by demonstrating a precipitous drop in CFU per milliliter after removal of the infected catheter in one patient. Routine availability of colony counts appears to be an important advantage of the lysis-centrifugation method.

Debaryomyces hansenii was isolated from a cystic lesion in the distal tibia of a healthy 23-year-old woman. Ascospores were demonstrated when the organism was incubated at 25 but not at 30 degrees C. Electron microscopy was necessary to demonstrate the warty surface of the ascospore wall. Growth was inhibited in vitro by low concentrations of amphotericin B, 5-fluorocytosine, miconazole, and ketoconazole. Four previous cases of infection caused by Debaryomyces spp. and one caused by the related fungus Torulopsis candida were reviewed.

A case of catheter-associated fungemia caused by Fusarium chlamydosporum is described in a patient with lymphocytic lymphoma. The fungus, which has been isolated from soil but not reported to cause human infection, characteristically produces microconidiophores that are polyphialides bearing microconidia that are spindle-shaped but never globose. Results of in vitro antimicrobial susceptibility tests depended on the test conditions used.

The first report describing a blood culture technique based on lysis and centrifugation appeared in 1976 (6). During the next few years improvements were made in the system, and in the early 1980s the Isolator system was introduced by DuPont Co. (Wilmington, Del.). There has since been a number of evaluations of the Isolator 7.5 ml, 10 ml, and 1.5 ml systems (2-5, 8, 13, 19). The evaluations have included comparisons of the Isolator to broth systems for the recovery and time to detection of bacteria and fungi, and the interpretation of colony counts from the Isolator system. The various Isolator systems have been compared to a variety of broth systems including conventional broth bottles with 50mL, 100 mL, and biphasic fills with Columbia, tryptic soy, and Brain Heart Infusion broth; the Roche Septi-Chek (Roche Diagnostics, Nutley, N.J.) bottles; and BACTEC (Johnston Laboratories, Towson, Md.) resin and nonresin broth media. I would like to summarize these findings and include my own opinion as to our observations and those of others. The evaluations represent a wide variety of study protocols, patient populations, media, etc., thus they must be interpreted with some caution. For example, at this institution, many of our patients are immunosuppressed, approximately 50% are receiving antibiotics at the time the blood culture is taken, and approximately 50% of blood cultures are taken through indwelling catheters. These circumstances may predispose our studies to different results from those of a large general hospital. A summary of the studies suggests, in general, that neither Isolator nor broth has consistently provided the greatest sensitivity for all organism groups. Thus for optimal recovery of a variety of aerobic and anaerobic bacteria, yeasts and molds, at least two blood culture systems should be used. In several studies, Isolator recovered more yeast and staphylococci, and the broth systems recovered more streptococci and anaerobes. We have compared Isolator to conventional Columbia broth, BACTEC 6B-7D, and BACTEC 16B-17D media. Of the three broth media combinations, the BACTEC resin media was the most effective and recovered aerobic and facultative anaerobic bacteria and yeasts as efficiently as did the Isolator system. Both Isolator and resin media remove antibiotics, thus in a hospital where many patients have received antibiotics before culture, these systems may be preferred. Recovery of yeasts has often been favored by the Isolator system, and Streptococcus pneumoniae has been recovered more often by the broth systems. Several large studies referenced earlier and individual case reports (18, 20) also showed that Candida spp., Histoplasma capsulatum, and Cryptococcus neoformans were detected more often and sooner on an agar surface from the Isolator system or broth subcultures than from broth media alone. Perhaps increased yeast recovery on agar is due to the aerobic metabolism of the yeast. Thus when fungemia is suspected, the Isolator system may be the blood culture system of choice. For those using only broth systems for detecting fungemia, terminal subcultures of the broth should be performed. Decreased yield of S. pneumoniae by the Isolator system may be caused by lysis of the bacteria by saponin in the Isolator tube. Saponin, used to lyse red blood cells in the Isolator system, was shown in 1933 to lyse pneumococci that had been sensitized with cholesterol and ergosterol (16). Organisms have been detected at approximately the same time with both the Isolator and broth systems. Colonies on Isolator plates may more readily provide inoculum for identification and antibiotic tests, and the presence of different colonial morphologies on Isolator plates may indicate polymicrobic infection earlier than would a broth system. These potential advantages, however, may not necessarily translate to earlier reports of important information. The availability of blood culture information depends, in addition to the system used, on the hours of operation of the laboratory, culture sampling times, the use of rapid procedures for organism identification, and the timely ability to communicate the results to the clinician. In our laboratory, initially more contaminants were recovered from the Isolator than by broth systems. These results suggested that cultures should be processed with minimum air flow and that hooded environments should be used. We now inoculate Isolator sediment to agar plates and examine those plates daily inside a SteriLGARD biosafety hood. We do not routinely report the presence of one or two staphylococci colonies on Isolator plates if these organisms are present only in that culture. The number of colony-forming units of organism per milliliter of blood (CFU/ml) can readily be determined by the Isolator system. How important is this information? Interest in quantifying organisms in the blood as an aid in the diagnosis and management of septicemia is not new. In 1985 I reviewed 52 years of blood culture quantitation (14) and found that many papers showed that colony counts have been used in studies of the diagnosis and treatment of endocarditis, the relationship between magnitude of bacteremia and mortality, and the identification of sites of infection in adults, children, and neonates. In most of these studies, labor-intensive pourplate techniques have been used for quantitation of organisms, and these cumbersome procedures have tended to discourage routine determinations of colony counts in septicemia. Introduction of the Isolator system has caused a renewed interest in the quantitation of bacteria and fungi in

A case of Nocardia asteroides pneumonia in a patient with the acquired immunodeficiency syndrome who was intolerant of sulfadiazine is described. On cefuroxime, the patient had a complete resolution of his Nocardia pneumonia. Disk-diffusion and broth microdilution antibiotic susceptibility testing (MIC less than or equal to 2 micrograms/ml) strongly supported the use of cefuroxime as treatment in this patient. Susceptibility testing with newer cephalosporins should be considered for all significant Nocardia isolates.

M. avium complex is a common cause of widely disseminated infection among AIDS patients. The origin of the infection appears to be the gastrointestinal tract. Stool smears and cultures for acid-fast bacilli as well as blood cultures are sensitive diagnostic tests. Quantitative blood culture methods are useful in enumerating organisms in the peripheral blood and organisms have been shown to be present in leukocytes. Our patients have been treated with a variety of antimicrobial agents but without a sustained remission of MAC disease. The remarkable load of organisms seen and these patients' inability to produce high levels of MAC antibody is further evidence of the extraordinary effect the AIDS disease has on T-cell as well as B-cell immune function.

D-Arabinitol is a five-carbon polyol that is produced by many fungi. Detection of the metabolite has been reported in serum from patients with invasive candidiasis. We studied the production and assimilation of arabinitol by 46 clinical isolates of yeast species. Cultures of isolates of Candida albicans (9 strains), Candida tropicalis (12 strains), Candida parapsilosis (13 strains), Candida krusei (4 strains), Candida pseudotropicalis (3 strains), Torulopsis glabrata (3 strains), and Cryptococcus neoformans (2 strains) were assayed by gas-liquid chromatography. Yeast cells were cultured at 34 degrees C in yeast nitrogen base with 3.0 g of glucose per liter. At 1.5- to 3-h intervals, cells were counted and glucose and arabinitol were measured in media filtrates. The levels of arabinitol in cultures with 7.5 X 10(6) yeast cells per ml were compared. The mean concentrations of the metabolite in C. albicans, C. tropicalis, C. parapsilosis, and C. pseudotropicalis cultures wee 14.1, 1.6, 8.4, and 5.5 micrograms/ml, respectively. No arabinitol was detected in cultures of C. krusei, T. glabrata, or C. neoformans.

A case of septicemia caused by Neisseria meningitidis serogroup 29E (Z') in a patient with plasma cell leukemia is described. The patient developed disseminated intravascular coagulation, had a cardiopulmonary arrest, and died. The effects of altered immune function leading to a predisposition to meningococcal infections are discussed.

Blood cultures processed with a lysis-centrifugation (Isolator) system and a radiometric (BACTEC 13A) broth system were compared for the recovery of Mycobacterium avium-M. intracellulare from patients with acquired immunodeficiency syndrome. Forty-nine isolates of M. avium-M. intracellulare were recovered by both systems, 9 were recovered by the Isolator system only, and 12 were recovered by the BACTEC system only. Average times to detection were 16 and 14 days for the Isolator and BACTEC systems, respectively. There was no significant difference between the two blood culture systems in sensitivity or time to detection.

Considerable changes have occurred during the 1980s in the clinical nature and diagnosis of bacteremia and fungemia in the immunocompromised patient. Cancer patients with prolonged neutropenia, many with indwelling catheters, and AIDS patients with both T-cell and B-cell deficiencies have changed the spectrum of organisms causing septicemia. There has been a shift to infection with gram-positive bacteria, including mycobacteria, and water-borne organisms, including Acinetobacter spp. and Pseudomonas spp. New blood culture systems, including a lysis-centrifugation system and radiometric methods utilizing resin broth media, remove antagonistic antimicrobial agents, and the lysis-centrifugation system routinely provides quantitation of organisms from the blood. Quantitation has been used to identify sources of infection, to differentiate contamination from true infection, and to monitor the course of antibiotic treatment.

We compared three methods for identifying clinical yeast isolates: Abbott Quantum II, API 20C, and a modified BBL Minitek system. The API 20C and modified Minitek systems agreed on the identification of 243 of 245 yeasts (99.2%). The Quantum II system correctly identified 197 (80.4%), incorrectly identified 19 (7.8%), and did not identify 29 (11.8%) of the yeasts. Most of the misidentifications with the Quantum II occurred because assimilation or biochemical results were false-positive. Sixteen different species of yeasts and 16 different Quantum II substrates contributed to the discrepancies. On retesting with the Quantum II, 31% of the discrepant strains were correctly identified, while the remaining 69% were incorrectly identified or were not identified. Erroneous biochemical and assimilation results were also noted with yeasts that were correctly identified by the Quantum II system.

Two hundred episodes of fungemia that occurred at Memorial Sloan-Kettering Cancer Center between January 1, 1978, and June 30, 1982, are reviewed and compared with those seen from 1974 through 1977. The total number of episodes of fungemia per year increased by 30.6%, episodes per 100 new lymphoma and solid tumor patients increased by 73% and 95%, respectively, and episodes per 100 new leukemia patients decreased by 50%. Fungemia also occurred earlier during hospitalization, and embolic skin lesions were a common early sign of Candida tropicalis fungemia. Mortality was not significantly different with and without amphotericin B therapy in fungemic patients with leukemia, lymphoma, or aplastic anemia (51 of 70 vs. 21 of 24) or solid tumors (29 of 36 vs. 29 of 43); however, some patients appeared to benefit from combination therapy with amphotericin B and flucytosine. The prevalence of disseminated candidiasis at autopsy was the same in treated (11 of 15) and untreated (8 of 11) patients with leukemia, lymphoma, and aplastic anemia, but it was significantly lower in treated (none of 8) than in untreated (5 of 11) patients with solid tumors. Fungal infection remains an important cause of morbidity and mortality in patients with cancer, particularly neutropenic patients with leukemia and lymphoma who are receiving broad-spectrum antibiotic therapy, patients receiving total parenteral nutrition, and patients with solid tumors in whom abdominal surgery is performed [1-23]. A recent review of fungemic patients seen at our hospital between 1974 and 1977 documented a marked increase in the frequency of fungemia and a high mortality rate. Of particular note was that mortality was high whether or not amphotericin B was given; 81% of patients treated with amphotericin B for fungemia due to Candida albicans or Candida tropicalis died despite therapy [14]. Partly because of results such as these, our approach to therapy has changed. We now give am

Routine terminal aerobic subcultures of macroscopically negative blood culture bottles were evaluated during a 15-month period when 30,000 blood cultures were processed. Each blood culture set consisted of a vented and an unvented 50-ml broth bottle. Forty-eight pathogens and 47 contaminants were isolated only from terminal subcultures. Twenty-two of the significant isolates were yeasts (usually recovered from vented bottles), and 10 were Pseudomonas aeruginosa (usually recovered from unvented bottles). Blood cultures that were positive by terminal subculture provided clinically relevant information in many cases, whether other blood cultures were positive or not. Microbiology laboratories, particularly those in hospitals where yeasts and P. aeruginosa are commonly isolated from blood specimens, should evaluate carefully the need for terminal subcultures of blood culture bottles before abandoning their use.

In the immunosuppressed patient the usual hallmarks of infection, such as leukocytosis and antibody response, may be absent; thus the microbiology laboratory plays a fundamental role in the diagnosis of infection. Methods used to demonstrate microorganisms in a specimen submitted to the laboratory include visualization techniques, culture, and non-cultural methods involving immunologic, immunochemical, and nucleic acid probe methodologies. Because infections in the immunosuppressed patient may be caused by unusual organisms whose identification requires special techniques, close communication between the physician and the laboratory is important. New technologies allow the clinical microbiology laboratory to gather important diagnostic information more readily. When these results are delivered rapidly to physicians via computerized information systems, care of the immunosuppressed patient is significantly enhanced.