Future of diagnostic microbiology.

Diagnostic Microbiology is the tool that makes it possible to identify the exact etiology of infectious diseases and the most optimal therapy at the level of individual patients as well as communities. Conventional methods require time to grow the microbes in vitro under specific conditions and not all microbes are easily cultivable. This is followed by biochemical methods for identification which also require hours and sometimes days. Transport of the specimens under less than ideal conditions, prior use of antibiotics and small number of organisms are among the factors that render culture-based methods less reliable. Newer methods depend on amplification of nucleic acids followed by use of probes for identification. This mitigates the need for higher microbial load, presence of metabolically active viable organisms and shortens the time to reporting. These methods can be used to detect antibiotic resistance genes directly from the specimen and help direct targeted therapy. Since these methods will not fulfill all the diagnostic needs, a second approach is being used to shorten the time to identification after the organism has already grown. Mass spectrometry and bioinformatics are the tools making this possible. This review gives a historical perspective on diagnostic microbiology, discusses the pitfalls of current methodology and provides an overview of newer and future methods.

Granulocyte colony-stimulating factor dosing in pegylated interferon alpha-induced neutropenia and its impact on outcome of anti-HCV therapy.

Many patients with chronic hepatitis C (HCV) infection undergoing treatment with pegylated interferon-alpha (PEG-IFN-alpha) and ribavirin develop neutropenia requiring dose reduction or granulocyte colony-stimulating factor (G-CSF) supplement. We analysed the database of patients who completed treatment for chronic HCV infection between 2003 and 2006. Patients with absolute neutrophil counts below 1000 cells/microL were initiated on G-CSF (G-CSF group) while a matching group of patients who received anti-HCV treatment without developing neutropenia were used as a control group (non-G-CSF group). Patients on the G-CSF arm were divided into two subgroups based on the timing of G-CSF administration relative to PEG-IFN-alpha administration. Of the 163 patients with HCV infection, 30 patients received G-CSF, most of who were maintained on 300 microg of G-CSF once a week. Administration of G-CSF 2 days before or after each dose of PEG-IFN-alpha did not make a significant difference in the neutrophil counts. In the G-CSF arm, 23 of 30 patients (77%) had undetectable end-of-treatment viral response which was comparable with 27 of 30 in the control group (90%; P = 0.17). There was no statistically significant difference in the sustained viral response between the two groups (61%vs 76%, P = 0.18). In most patients PEG-IFN-alpha induced neutropenia improved with a once-a-week dose of G-CSF with a comparable virological outcome. Timing of G-CSF administration did not make any significant impact on the patient's neutrophil counts but was better tolerated when given 2 days apart from PEG-IFN-alpha.

Interactions of Candida albicans with other Candida spp. and bacteria in the biofilms.

AIMS: To study the interactions between Candida albicans and 12 other species of Candida and bacteria in biofilms. METHODS AND RESULTS: The number of cells within growing biofilms in a polystyrene tube model was measured after adding C. albicans to preformed biofilms of other micro-organisms and vice versa. It was also measured after simultaneous biofilm formation of C. albicans and other micro-organisms. The number of cells of C. albicans within the growing biofilms decreased significantly (P < 0.05) when the fungus was added to preformed biofilms of Candida spp. and bacteria except, with C. parapsilosis, Torulopsis glabrata and the glycocalyx producer Pseudomonas aeruginosa. When C. parapsilosis, Staphylococcus epidermidis (nonglycocalyx producer) or Serratia marcescens was added to preformed biofilms of C. albicans, the number of cells of these micro-organisms increased in the growing biofilms. CONCLUSIONS: Biofilms of C. albicans are capable of holding other micro-organisms and more likely to be heterogeneous with other bacteria and fungi in the environment and on medical devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of the heterogeneity of biofilm-associated organisms can influence treatment decisions, particularly in patients who do not respond to initial appropriate therapy.

Interaction between biofilms formed by Staphylococcus epidermidis and quinolones.

The interaction between pefloxacin, ciprofloxacin, norfloxacin, and ofloxacin and biofilms formed by Staphylococcus epidermidis (20 clinical isolates) was studied. In the presence of 1/2-MIC and 1/8-MIC of quinolones, the optical density of the biofilms was reduced to 22-24% and 65-74% of the controls, respectively. Treatment of preformed biofilms with quinolones in concentrations ranging from 12.5 microg/ml to 400 microg/mL caused reduction in the optical density of the adherent biofilms to 45-77% of the control. In an in vitro model of vascular catheter colonization, subinhibitory concentrations (12, 14 and 1/8 MIC) of fluoroquinolones reduced the number of adherent bacteria to 24-28%, 48-55% and 58-76% of the controls, respectively. The vascular catheter segments precolonized with Staphylococcus epidermidis for 24 h and exposed to the fluoroquinolones in 8-16 times MIC (100 microg/mL) for 2 h showed no growth of adherent cells. The activity of pefloxacin in reducing the bacterial adhesion and eradicating the preformed biofilms was demonstrated by scanning electron microscope. These data show that subinhibitory concentrations of ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin inhibit the adhesion of Staphylococcus epidermidis to plastic surfaces and vascular catheters. Higher concentrations of fluoroquinolones were able to eradicate the preformed biofilms on vascular catheters.

A comparison of two antimicrobial-impregnated central venous catheters. Catheter Study Group.

BACKGROUND: The use of central venous catheters impregnated with either minocycline and rifampin or chlorhexidine and silver sulfadiazine reduces the rates of catheter colonization and catheter-related bloodstream infection as compared with the use of unimpregnated catheters. We compared the rates of catheter colonization and catheter-related bloodstream infection associated with these two kinds of antiinfective catheters. METHODS: We conducted a prospective, randomized clinical trial in 12 university-affiliated hospitals. High-risk adult patients in whom central venous catheters were expected to remain in place for three or more days were randomly assigned to undergo insertion of polyurethane, triple-lumen catheters impregnated with either minocycline and rifampin (on both the luminal and external surfaces) or chlorhexidine and silver sulfadiazine (on only the external surface). After their removal, the tips and subcutaneous segments of the catheters were cultured by both the roll-plate and the sonication methods. Peripheral-blood cultures were obtained if clinically indicated. RESULTS: Of 865 catheters inserted, 738 (85 percent) produced culture results that could be evaluated. The clinical characteristics of the patients and the risk factors for infection were similar in the two groups. Catheters impregnated with minocycline and rifampin were 1/3 as likely to be colonized as catheters impregnated with chlorhexidine and silver sulfadiazine (28 of 356 catheters [7.9 percent] vs. 87 of 382 [22.8 percent], P<0.001), and catheter-related bloodstream infection was 1/12 as likely in catheters impregnated with minocycline and rifampin (1 of 356 [0.3 percent], vs. 13 of 382 [3.4 percent] for those impregnated with chlorhexidine and silver sulfadiazine; P<0.002). CONCLUSIONS: The use of central venous catheters impregnated with minocycline and rifampin is associated with a lower rate of infection than the use of catheters impregnated with chlorhexidine and silver sulfadiazine.

Factors influencing adherence of Candida spp. to host tissues and plastic surfaces.

Attachment of Candida spp. to host tissues and plastic surfaces is the first and a crucial step that initiates colonization by yeast cells and subsequent development of disseminated fungal infection. These infections are associated with high degree of morbidity, mortality and extra cost. Modern trends have focused not only on how best to treat but also on how to prevent Candida infections. To achieve this goal, the factors that influence the adherence of Candida spp. to biological and non biological surfaces have been studied. C. albicans adheres at a degree higher than that of the other Candida spp. and C. tropicalis adheres to a lesser extent. This may reflect the higher pathogenicity of C. albicans compared to the other Candida spp. Germinated C. albicans cells adhere to host tissue more readily than do yeast-phase. Sugars play an important role in the adherence of Candida spp. Overall, galactose was found to promote the adherence of Candida spp. to host tissues and plastic surfaces more than any other mono or disaccharide. Amino sugars on the other hand inhibit the adherence of the yeast cells. Divalent ions such as Ca2+ and Mg2+ promote the adherence of Candida spp. more than monovalent ions. Candida spp. express on their surface receptors, which interact with a wide variety of host proteins including fibrinogen, fibronectin, lamanin, and type I and IV collagen thus binding Candida spp. To glycoproteinaceous conditioning film at the blood-polymer interface. Coaggregation of Candida spp. with other bacteria promotes colonization of yeast cells to oral biofilm, host tissues, and to surfaces of the indwelling vascular catheters. These factors form the basis for the interference with the adherence of Candida spp.

Modulation of biofilms of Pseudomonas aeruginosa by quinolones.

The interaction between four fluoroquinolones (ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin) and biofilms of Pseudomonas aeruginosa in wells of microtiter plates and on segments of vascular catheters were studied in an in vitro model of vascular catheter colonization. Subinhibitory concentrations (one-half, one-fourth, and one-eight of the MIC) of the fluoroquinolones reduced the adherence of P. aeruginosa to 30 to 33, 44 to 47, and 61 to 67% of that of controls, respectively. The addition of high concentrations of the fluoroquinolones (12.5 and 400 micrograms/ml) to preformed biofilms (grown for 48 h at 37 degrees C) decreased the adherence of P. aeruginosa to 69 to 77 and 39 to 60% of that of controls, respectively. In an in vitro model of vascular catheter colonization, subinhibitory concentrations (one-half, one-fourth, and one-eight of the MIC) of fluoroquinolones reduced the number of adherent bacteria to 21 to 23, 40 to 46, and 55 to 70% of that of the controls, respectively. Scanning electron microscopy demonstrated a significant reduction in glycocalyx formation and adherent bacteria in the presence of pefloxacin at one-half to one-eight of the MIC. Vascular catheter segments precolonized with P. aeruginosa for 24 h and exposed to the fluoroquinolones at 4 to 25 times the MIC (50 micrograms/ml) for 2 h showed <5% growth of adherent cells compared with controls. No adherent organisms were cultured in the presence of 8 to 50 times the MIC (100 micrograms/ml). Scanning electron microscopy studies of preformed biofilms exposed to pefloxacin verified the results obtained by culture. These data show that subinhibitory concentrations of ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin inhibit the adherence of P. aeruginosa to plastic surfaces and vascular catheters. Clinically achievable concentrations of fluoroquinolones (50 to 100 micrograms/ml) were able to eradicate preformed biofilms on vascular catheters.

Biofilms in device-related infections.

The use of various medical devices including indwelling vascular catheters, cardiac pacemakers, prosthetic heart valves, chronic ambulatory peritoneal dialysis catheters and prosthetic joints has greatly facilitated the management of serious medical and surgical illness. However, the successful development of synthetic materials and introduction of these artificial devices into various body systems has been accompanied by the ability of microorganism to adhere to these devices in the environment of biofilms that protect them from the activity of antimicrobial agents and from host defense mechanisms. A number of host, biomaterial and microbial factors are unique to the initiation, persistence and treatment failures of device-related infections. Intravascular catheters are the most common devices used in clinical practice and interactions associated with these devices are the leading cause of nosocomial bacteremias. The infections associated with these devices include insertion site infection, septic thrombophlebitis, septicemia, endocarditis and metastatic abscesses. Other important device-related infections include infections of vascular prostheses, intracardiac prostheses, total artificial hearts, indwelling urinary catheters, orthopedic prostheses, endotracheal tubes and extended wear lenses. The diagnosis and management of biofilm-associated infections remain difficult but critical issues. Appropriate antimicrobial therapy is often not effective in eradicating these infections and the removal of the device becomes necessary. Several improved diagnostic and therapeutic modalities have been reported in recent experimental studies. The clinical usefulness of these strategies remains to be determined.

Tolerance of Staphylococcus epidermidis grown from indwelling vascular catheters to antimicrobial agents.

During a prospective study of indwelling vascular catheter-related infections, 134 isolates of Staphylococcus epidermidis were grown from 700 catheter tips. In vitro antimicrobial susceptibility testing of these isolates to oxacillin, vancomycin and ofloxacin was performed using the standard broth microdilution technique. These results were compared to those for the same organisms grown in biofilm before the addition of antimicrobial agents. In 96-well flat bottom microtiter plates, 10(4)-10(5) colony forming units of S. epidermidis in 0.1 ml broth were grown for 18 h at 37 degrees C, at which time a biofilm was observed for all isolates. Different concentrations of antimicrobial agents (0.1 ml) were then added to the plates. The plates were incubated for 18 h at 37 degrees C. Since MICs could not be estimated in these plates, all the wells were subcultured after mixing the biofilm with the broth. Minimum bactericidal concentrations (MBCs) were defined as 99.9% reduction in colony forming units. For organisms grown in suspension, 100% of the isolates were susceptible to vancomycin, 81% to ofloxacin and 40% to oxacillin. MBCs of susceptible isolates were within four-fold differences for vancomycin (53%), oxacillin (50%), and ofloxacin (51%). When grown as a biofilm, 78%, 93% and 71% of isolates had MBCs of > or = 2048 micrograms ml-1 of oxacillin, vancomycin and ofloxacin respectively. These data demonstrate the reduced bactericidal activity of antimicrobial agents against S. epidermidis in a biofilm and a simple method for its detection in the microbiology laboratory.

Blastomycosis infection of the hand.

Blastomycosis is an uncommon cause of infection in the hand. Fungal infection should be suspected in any case when expected improvement does not occur with appropriate antibiotic therapy and wound care. We describe a rare case of primary blastomycosis of the hand that progressed to severe soft tissue destruction and significant loss of hand function.

In vitro susceptibilities of rapidly growing mycobacteria to newer antimicrobial agents.

The in vitro antimicrobial susceptibilities of 42 isolates of rapidly growing mycobacteria (Mycobacterium fortuitum, M. chelonae, and Mycobacterium species [other than M. fortuitum and M. chelonae]) to nine quinolones, including newer agents, two new aminoglycosides, and an aminocyclitol (trospectomycin) were determined by a broth microdilution method. The new quinolones, PD 117596, PD 127391, and PD 117558, showed excellent in vitro activities against M. fortuitum (MICs for 90% of isolates [MIC90s], 0.06, 0.06, and 0.12 microgram/ml, respectively). The MIC90 of ciprofloxacin for M. fortuitum was 0.5 microgram/ml. Only 14 to 28% of isolates of M. chelonae were susceptible to various quinolones. Most isolates of all three species were susceptible to the new aminoglycosides SCH 21420 and SCH 22591. The MIC90s of trospectomycin were 8 micrograms/ml for M. chelonae, 32 micrograms/ml for Mycobacterium species, and > 64 micrograms/ml for M. fortuitum.

In vitro antimicrobial susceptibilities of Nocardia species.

The in vitro activities of various quinolones, two new aminoglycosides, a new cephamycin analog (cefmetazole) and a new spectinomycin analog (trospectomycin), imipenem, and trimethoprim-sulfamethoxazole against 26 isolates of Nocardia asteroides, 7 isolates of N. brasiliensis, and 6 isolates of N. caviae were determined by a broth microdilution method. The three new quinolones, PD 117558, PD 117596 and PD 112739, inhibited 90% of N. asteroides at 1 to 2 micrograms/ml, and two new aminoglycosides, SCH 21420 and SCH 22591, inhibited 90% of N. asteroides at 2 to 4 micrograms/ml. Among the beta-lactams, cefmetazole was more active than imipenem. N. brasiliensis and N. caviae isolates were also very susceptible to the three quinolones (MICs for 50% of the isolates, 0.25 to 1 microgram/ml) and the two aminoglycosides (MICs for 50% of the isolates, 1 to 2 micrograms/ml). Cefmetazole was moderately active against N. brasiliensis, whereas imipenem showed poor activity against both of these species.

Comparative efficacies of cilofungin (Ly121019) and amphotericin B against disseminated Candida albicans infection in normal and granulocytopenic mice.

The efficacies of cilofungin (Ly121019), a semisynthetic lipopeptide antifungal agent, and amphotericin B in the treatment of disseminated candidiasis in normal and neutropenic mice were compared. In mice infected with 2 x 10(6) CFU of Candida albicans, treatment with cilofungin in twice-daily doses of 25 or 35 mg/kg of body weight by intraperitoneal injection for 10 days gave survival rates of 83 and 90%. In contrast, there was 97% mortality in infected controls receiving 2 x 10(6) CFU intravenously and 93% survival in mice treated with 1 mg of amphotericin B per kg once a day. Mice rendered granulocytopenic by the administration of cyclophosphamide showed survival rates of 83 and 80% when treated with 25 or 35 mg of cilofungin per kg for 10 days compared with 43% survival rate in mice treated with 1 mg of amphotericin B per kg (P = 0.0030 and P = 0.0080, respectively). Similar results were obtained when the two antifungal agents were administered for a period of 30 days. Administration of 25 or 35 mg of cilofungin per kg twice a day to granulocytopenic mice receiving 10(6) CFU of C. albicans gave survival rates of 93% and 93% compared with 53% survival with amphotericin B. With 15 mg of cilofungin per kg twice a day for 10 days, a survival rate of 43 to 50% was observed in both normal and granulocytopenic mice compared with 56 and 60%, respectively, when this dosage was continued for 30 days. Cilofungin eradicated C. albicans from the kidneys, spleens, and livers of surviving animals. No toxic effects were observed with any of the dosage regimens used. The clearance of C. albicans from the kidneys, spleens, livers, and brains in normal mice was studied following infection with 5 x 10(5) and 1 x 10(5) intravenously. The mice in the treatment groups received 25 mg of cilofungin per kg twice a day for 10 days. In 8 to 12 days, this treatment was able to clear the organisms from the kidneys, spleens, and livers of mice infected with 5 x 10(5) C. albicans. Mice infected with 10(5) C. albicans and treated with cilofungin (25 mg/kg) twice a day for 10 days had no organisms in the kidney, spleen, and liver at days 8, 2, and 8, respectively. There was 1-log-unit reduction in C. albicans counts in brain tissue from mice of one of the treated groups between 2 h and 2 days postinfection, after which the numbers of organisms remained the same until day 12. These data demonstrate the efficacy of cilofungin in the treatment of disseminated C. albicans infections in normal and granulocytopenic mice. The treatment regimen used in this study was able to clear C. albicans from the kidneys, spleen, and liver but not from brain tissue.

Gene therapy: a status report.

Gene therapy is a way to treat disease by transfer of genetic material into the cells of a diseased organism. Generally it is transferred in the form of one or few genes. There is a tremendous potential for application of this technique to the treatment of human hereditary diseases, particularly single gene disorders. Due to ethical and practical reasons, it is currently being used only as somatic cell therapy but it could be achieved by germline also. The major problem in gene therapy is achieving an efficient gene transfer and a persistent gene expression in appropriate somatic cells.

A comparison of imipenem to ceftazidime with or without amikacin as empiric therapy in febrile neutropenic patients.

BACKGROUND: Neutropenic patients with cancer are traditionally treated with empiric antibiotic combinations when they become febrile. The availability of broad-spectrum antibiotics such as ceftazidime and imipenem has made it possible to initiate therapy with a single agent (monotherapy). The objectives of this trial were to compare ceftazidime and imipenem as single agents for the therapy of febrile episodes in neutropenic patients and to ascertain whether the addition of an aminoglycoside (amikacin) to either of these agents would provide an advantage. METHODS: A prospective clinical trial was conducted in which eligible neutropenic patients with cancer were randomized to one of four treatment arms: ceftazidime alone; imipenem alone; ceftazidime plus amikacin; and imipenem plus amikacin. Efficacy analysis was done for 750 assessable episodes. A multivariate logistic-regression analysis was also performed to examine the unique contribution of various prognostic factors. RESULTS: The overall response rates were 76% with imipenem plus amikacin, 72% with imipenem, 71% with ceftazidime plus amikacin, and 59% with ceftazidime alone. Single-organism gram-positive infections occurred in 101 of 750 episodes. Without a change in antibiotics, the response rates were 50% with imipenem, 40% with imipenem plus amikacin, 39% with ceftazidime plus amikacin, and 38% with ceftazidime. Most responded to vancomycin or other antibiotics, and the mortality associated with gram-positive infections was only 5%. Regardless of the antibiotic regimen, the majority of uncomplicated gram-negative infections responded to therapy and the majority of complicated gram-negative infections failed to respond. Multivariate logistic-regression analysis showed that recovery of the neutrophil count was the most favorable prognostic factor in a patient's response to infection, whereas the presence of gram-positive infection, acute leukemia, pulmonary or enteric infection, and therapy with ceftazidime were unfavorable factors. CONCLUSIONS: Single-agent therapy with imipenem is as effective as more conventional combination antibiotic therapy for the empirical treatment of febrile episodes in neutropenic patients with cancer.

Infectious complications of hepatic artery catheterization procedures in patients with cancer.

A total of 353 hepatic artery catheterization procedures were carried out in 211 patients with cancer over a 1-year period (January-December 1988). The procedures included 49 embolizations in 32 patients, 123 chemoembolizations in 73 patients, and 181 chemoinfusions in 106 patients. The overall infection rate was 3.4%. Infectious complications occurred in 3.1% of patients undergoing hepatic artery embolization alone, 1.9% of patients undergoing hepatic artery chemoinfusion, and 4.1% of patients undergoing hepatic artery embolization followed by chemoinfusion. Four patients had infectious complications that included four episodes each of cholangitis, liver abscess, and septicemia. One patient developed a subphrenic abscess in addition to a liver abscess. Enteric gram-negative bacilli (aerobic and anaerobic) were isolated from all four patients. None of the patients had received prophylactic antibiotics. All patients responded to antimicrobial therapy and percutaneous drainage of abscesses.