Inhibition controls for qualitative real-time PCR assays: are they necessary for all specimen matrices?

A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤ 1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue.

Open lung biopsy in immunocompromised patients.

From January 1978 through December 1980, ninety-five immunocompromised patients underwent diagnostic open lung biopsy (OLB) at the Mayo Clinic, Rochester, Minn. A specific causative diagnosis was made in 77 patients (81%); 12 patients (13%) had more than one specific causative diagnosis. Of the 77 patients with a specific causative diagnosis, 52 patients (68%) had infections, 19 patients (24%) had malignant pulmonary disease, 12 patients (15%) had cytotoxic lung disease, ten patients (13%) had interstitial fibrosis (not related to cytotoxins), and one patient (1%) had vasculitis. In 36 (47%) of the 77 patients with a specific causative diagnosis, the diagnosis was made, by frozen sections or stains, within three hours of OLB. In 35 additional patients (45%), the diagnosis was established within 24 hours. Twelve patients (13%) had minor complications of OLB; no deaths were attributed to the OLB procedure.

Optimal testing parameters for blood cultures.

The effects of volume of blood, number of consecutive cultures, and incubation time on pathogen recovery were evaluated for 37,568 blood cultures tested with the automated BACTEC 9240 instrument (Becton Dickinson Diagnostic Instrument Systems) at a tertiary care center over the period of 12 June 1996 through 12 October 1997. When the results for this study were compared with previous data published for manual broth-based blood culture systems and patient samples obtained in the 1970s and 1980s, the following were found: (1) the percentage increase in pathogen recovery per milliliter of blood is less, (2) more consecutive blood culture sets over a 24-h period are required to detect bloodstream pathogens, and (3) a shorter duration of incubation is required to diagnose bloodstream infections. Guidelines developed in the 1970s and 1980s for processing and culturing blood may require revision.

Trimethoprim/sulfamethoxazole-resistant Nocardia asteroides causing multiple hepatic abscesses. Successful treatment with ampicillin, amikacin, and limited computed tomography-guided needle aspiration.

Hepatic abscesses are rarely encountered in disseminated Nocardia infections. Sulfonamides alone or trimethoprim/sulfamethoxazole is often efficacious in treating infections caused by Nocardia asteroides. In vitro resistance of N. asteroides to trimethoprim/sulfamethoxazole is occasionally present. The patient described in this report had disseminated nocardiosis initially manifesting as multiple subcapsular hepatic abscesses. In vitro susceptibility studies demonstrated resistance to trimethoprim/sulfamethoxazole. Subsequent treatment with ampicillin and amikacin in conjunction with computed tomography-guided needle aspiration of several of the hepatic abscesses, surgical drainage of a right pleural empyema, and eventual discontinuation of use of corticosteroids resulted in cure of the infection.

Polymicrobial cholangitis and Kaposi's sarcoma in blood product transfusion-related acquired immune deficiency syndrome.

Before presenting to the Mayo Clinic, a 24-year-old white woman had received 35 transfusions of blood products over a 72-hour period in February 1981. Two and one half years later, the diagnosis of polymicrobial cholangitis (Cryptosporidium, Candida albicans, and Klebsiella pneumoniae) was established. Further evaluation demonstrated profound helper T lymphocyte suppression, disseminated Mycobacterium avium-intracellular infection with mycobacteremia, and Kaposi's sarcoma of lymphoid tissue, confirming a diagnosis of acquired immune deficiency syndrome (AIDS). This case represents an unusual infectious complication of AIDS. Additionally, this is believed to be the first report of Kaposi's sarcoma occurring in a patient with AIDS associated with blood product transfusion.

Relevance and risk factors of enterococcal bacteremia following liver transplantation.

To analyze the clinical characteristics of and identify specific risk factors for enterococcal bacteremia following liver transplantation, we performed a study in 405 consecutive liver transplantation recipients prophylaxed with a selective bowel decontamination regimen. Seventy enterococcal bacteremias in 52 patients were identified. Enterococcus faecalis (50) outnumbered Enterococcus faecium isolates (18), and 49% of enterococcal bacteremias were polymicrobial. Biliary tree complications were present in 34% of enterococcal bacteremias. Of the 15 deaths (29%) among the patients with enterococcal bacteremia, 4 were directly associated with enterococcal bacteremia. In a multivariate analysis, Roux-en-Y choledochojejunostomy (P=0.005), a cytomegalovirus-seropositive donor (P=0.013), prolonged transplantation time (P=0.02), and biliary stricturing (P=0.016) were identified as significant risk factors. Other risk factors identified in a univariate analysis included primary sclerosing cholangitis (P=0.009) and symptomatic cytomegalovirus infection (P=0.008). Enterococcal bacteremia is a frequent infectious complication in liver transplantation recipients receiving selective bowel decontamination. Its association with cytomegalovirus and biliary tree abnormalities suggest specific areas for prophylactic intervention.

Retinopathy associated with enterococcus enteropathy in the neonatal rat.

PURPOSE: Preretinal neovascularization has been previously observed in neonatal rats with spontaneously occurring diarrhea. This neovascularization appears analogous to retinopathy of prematurity (ROP), which occurs in human neonates. A new enterococcus species, designated Enterococcus rattus, has been isolated from the duodenum of these rats. In the present controlled study, the effect of the enteropathy induced by this organism on the retinal vasculature in the neonatal rat was further investigated. METHODS: One hundred fifty newborn Sprague-Dawley rats were randomly assigned to 6 expanded litters (n = 25). On the second day of life, animals were gavaged with either 100 microl of E. rattus suspension (1.0 X 10(7) colony forming units, inoculated group, n = 100 rats) or 100 microl saline (control group, n = 50 rats). All rats were raised in room air and were killed on day 13 of life. Duodenal and blood samples were cultured. The retinal vasculature was assessed using fluorescent microscopy and ADPase staining in a masked manner. Two additional inoculated litters and one control litter were studied for evaluation of arterial blood gases and validation of the grading method for preretinal neovascularization. RESULTS: One hundred percent of rats in the inoculated group developed severe diarrhea and had duodenal cultures positive for E. rattus compared with 0% in the control group. Preretinal neovascularization similar to ROP occurred in 55% of rats in the inoculated group compared with 2% in the control group (P = 0.001). Retinal vascular areas were reduced in the inoculated group (mean +/- SD, 89% +/- 5% versus 96% +/- 2%; P < 0.001). Rats in the inoculated group demonstrated severe growth retardation (final weight, 9.7 +/- 2.2 versus 16.7 +/- 2.7 g, P < 0.001). Inoculated animals also experienced acidosis (pH 7.31 +/- 0.06 versus 7.39 +/- 0.06 control, P = 0.04). CONCLUSIONS: A previously undescribed enterococcal enteropathy was associated with preretinal neovascularization similar to ROP in the neonatal rat. This supports an independent role for factors other than inspired oxygen in the development of ROP.

Two cases of non-O157:H7 Escherichia coli hemolytic uremic syndrome caused by urinary tract infection.

Escherichia coli serotype O157:H7 is a leading cause of diarrhea and hemolytic uremic syndrome (HUS). Because of the limitations of current diagnostic techniques, the prevalence of non-O157:H7 Shiga toxin-producing E coli strains is not known. We describe two patients with HUS in whom no E coli O157:H7 was demonstrable in stool cultures. On culture of the urine, the first patient was found to have E coli O113:H21 strain, and the second patient had E coli O6:H1 serotype. Shiga toxin production (stx2) by the O113:H21 isolate was confirmed. The first patient required 15 days of peritoneal dialysis and subsequently recovered renal function. At last follow-up, serum creatinine was 0.9 mg/dL. The second patient had preservation of renal function throughout the acute illness with serum creatinine of 0.5 mg/dL. The clinical presentation, bacteriology, course, and outcome as well as epidemiologic implications of the increasing number of patients with E coli urinary tract infections associated with HUS are discussed. These cases illustrate the need to investigate patients with nondiarrheal HUS for infection with Shiga toxin-producing E coli of the non-O157 strain variety.

Conventional and genetic laboratory tests used to guide antimicrobial therapy.

Detection of antimicrobial resistance is important so that clinicians can make rational decisions about optimal antimicrobial therapy for their patients. During the past decade, new types of antimicrobial resistance have emerged, some of which present new challenges for the clinical microbiology laboratory. In most cases, conventional culture-based testing methods continue to be useful. In other situations in which the organism responsible for infection grows slowly (for example, Mycobacterium tuberculosis), culture methods are technically difficult (such as for human immunodeficiency virus), or genotypes are inconsistently expressed (for instance, methicillin resistance in staphylococci), genetic susceptibility testing methods may offer special advantages. Determining serum concentrations of antimicrobial agents may be useful both to ensure adequacy of treatment and to prevent toxicity. In this review, methods are described for conventional and genetic tests used to guide antimicrobial therapy.

Serologic testing for human immunodeficiency virus antibodies.

Familiarity with available serologic tests for antibodies to human immunodeficiency virus (HIV) has become increasingly important in a wide variety of clinical settings. Enzyme-linked immunosorbent assay (ELISA) commercial kits are most often used as Enzyme-linked immunosorbent assay (ELISA) commercial kits are most often used as screening tests, and Western blot techniques are used for confirmation of positive results. ELISA specificity and sensitivity exceed 98%; the predictive value of a positive test varies from 2% for a weakly positive test in a low-prevalence population to 99% for a strongly positive test in a high-risk group. Confirmatory Western blot testing identifies antibodies with affinity for specific HIV antigens. Indeterminate Western blot antibody patterns necessitate subsequent testing or alternative methods for interpretation. A "window" period of up to 3 or more months follows acute HIV infection before seropositivity occurs.

Trimethoprim-sulfamethoxazole.

The antimicrobial combination of trimethoprim and sulfamethoxazole is active in vitro against a variety of gram-positive and gram-negative bacteria. Clinically, it is useful for treatment and prophylaxis of various infections of the genitourinary tract and certain infections of the respiratory and gastrointestinal tracts. Trimethoprim-sulfamethoxazole by itself or in combination with other antimicrobial agents is indicated for most Nocardia asteroides infections. It is the antimicrobial agent of choice for Pneumocystis carinii pneumonia. The drug is relatively nontoxic in patients who do not have acquired immunodeficiency syndrome (AIDS), and it is available in oral and intravenous forms. The native compounds and the metabolites of trimethoprim and sulfamethoxazole are excreted primarily in the urine. When the creatinine clearance decreases to less than 30 ml/min, the dosage of trimethoprim-sulfamethoxazole should be adjusted.

Antiviral agents.

Amantadine is well established as the preferred antiviral agent for the prophylaxis of influenza A and may also be beneficial therapeutically when used early in the course of the disease. Idoxuridine is applicable only in the treatment of herpetic keratitis. Currently, acyclovir is the most effective agent for the treatment of herpes simplex and varicella-zoster virus infections. Ribavirin has recently been released for use in aerosol form for severe respiratory syncytial virus infections that occur in infants and young children. Vidarabine, which previously was the drug of choice in the treatment of severe herpetic infections, has now been replaced by the more effective acyclovir. Ganciclovir, an experimental agent, has shown promise against cytomegalovirus infections in patients who have undergone kidney or liver transplantation, but its effects are only temporary in patients who have undergone bone marrow transplantation and patients with acquired immunodeficiency syndrome (AIDS) who have cytomegalovirus infections.

Tetracyclines, chloramphenicol, erythromycin, and clindamycin.

The tetracyclines are effective in the treatment of Chlamydia, Mycoplasma pneumoniae, and rickettsial infections and may also be used for gonococcal infections in patients unable to tolerate penicillins. These drugs may cause gastrointestinal irritation, photo-toxic dermatitis, diarrhea, vestibular damage, and hepatotoxicity in pregnant women. Chloramphenicol is used primarily for anaerobic infections, Haemophilus influenzae meningitis, and typhoid fever. The most important toxic effect of chloramphenicol is bone marrow suppression, which can be dose related or idiosyncratic. Erythromycin is the drug of choice for the treatment of infections caused by M. pneumoniae, Legionella species, group A beta-hemolytic streptococci, and Streptococcus pneumoniae. The frequency of serious untoward effects associated with the use of erythromycin is low; epigastric distress may occur. Clindamycin is active against Bacteroides fragilis and other anaerobic microorganisms. Pseudomembranous enterocolitis has developed in as many as 10% of patients taking this drug. The use of clindamycin should be discontinued promptly if diarrhea occurs.

Antiviral agents.

Only a few agents with antiviral activity are available for routine clinical use. Amantadine hydrochloride is effective in the prophylaxis of influenza A. In addition, accumulated evidence shows that amantadine has some therapeutic effect when used early in the course of an influenza A infection. Idoxuridine and adenine arabinoside have found application as topical agents in the treatment of herpes simplex keratitis. Adenine arabinoside has also been approved for the treatment of disseminated infections due to herpes zoster and herpes simplex. Acyclovir sodium has been approved as a topical agent in the treatment of limited mucocutaneous herpes simplex viral infections in immunosuppressed patients and of initial episodes of genital herpes simplex infections in patients with normal immunity. Ribavirin, an experimental agent with a wide spectrum of activity in vitro, has not fulfilled expectations in clinical trials. Because of the eradication of smallpox, methisazone has become obsolete as a prophylactic agent in smallpox.

Trimethoprim-sulfamethoxazole.

The antimicrobial combination of trimethoprim and sulfamethoxazole is active in vitro against various gram-positive and gram-negative bacteria. Clinically, it is useful for prophylaxis and treatment of selected infections of the genitourinary, respiratory, and gastrointestinal tracts. Trimethoprim-sulfamethoxazole by itself or in combination with other antimicrobial agents is indicated for most Nocardia asteroides infections and is the antimicrobial agent of choice for Pneumocystis carinii pneumonia. The drug is relatively nontoxic in patients who do not have the acquired immunodeficiency syndrome (AIDS) and is available in both oral and intravenous forms. The native compounds and the metabolites of trimethoprim and sulfamethoxazole are excreted primarily in the urine. When the creatinine clearance is less than 30 ml/min, the dosage of trimethoprim-sulfamethoxazole should be adjusted.

Tetracyclines, chloramphenicol, erythromycin, and clindamycin.

The tetracyclines are active in vitro against many urinary tract pathogens such as Chlamydia, Mycoplasma pneumoniae, Brucella, rickettsiae, and Nocardia. Chloramphenicol is used primarily for anaerobic infections, Haemophilus influenzae meningitis, and infections due to Salmonella typhi. Erythromycin is active in vitro against M. pneumoniae, Legionella spp., Streptococcus pneumoniae, and group A beta-hemolytic streptococci; it may also be used as prophylactic therapy for subacute bacterial endocarditis and for recurrence of acute rheumatic fever in patients who are allergic to penicillin. Clindamycin should be used primarily for the treatment of anaerobic infections. The tetracyclines may cause gastrointestinal upset; phototoxic dermatitis; hepatitis, especially in pregnant women; discoloration of the teeth and bone dysplasia in the human fetus and in children; and superinfections, especially oral and anogenital candidiasis. The tetracyclines should be used with caution in patients with renal insufficiency. The most important toxic effect of chloramphenicol is bone marrow suppression, which is dose related or idiosyncratic. The incidence of undesirable side effects associated with the use of erythromycin is low; gastrointestinal irritation is the most common, and cholestatic hepatitis may occur with the use of erythromycin estolate. Pseudomembranous colitis is the most important toxic effect associated with the use of clindamycin.

Trimethoprim-sulfamethoxazole.

The antimicrobial combination of trimethoprim and sulfamethoxazole is active in vitro against a variety of gram-positive and gram-negative bacteria. Clinically, it is useful for treatment and prophylaxis of various infections of the genitourinary tract and certain infections of the respiratory and gastrointestinal tracts. Trimethoprim-sulfamethoxazole by itself or in combination with other antimicrobial agents is effective for most Nocardia asteroides infections. It is the antimicrobial agent of choice for Pneumocystis carinii pneumonia. The drug is relatively nontoxic and available in oral and intravenous forms. The native compounds and the metabolites of trimethoprim and sulfamethoxazole are excreted primarily in the urine. When the creatinine clearance decreases to less than 30 ml/min, the dosage of trimethoprim-sulfamethoxazole should be adjusted.

Prosthetic joint infection due to Staphylococcus lugdunensis.

Staphylococcus lugdunensis, a coagulase-negative staphylococcus, is being increasingly recognized as the cause of serious infections. We report 2 cases of total knee arthroplasty infection caused by S lugdunensis. S lugdunensis frequently produces a clumping factor that can result in a positive slide (short) coagulase test result. If the microbiology laboratory does not use the tube coagulase (long) test to confirm the slide coagulase test result, the organism may be misidentified as Staphylococcus aureus. S lugdunensis is more virulent than other coagulase-negative staphylococci and in many clinical situations behaves like S aureus, further increasing the confusion. However, S lugdunensis differs from S aureus in that it is susceptible to most antibiotics. This fact may alert the microbiology laboratory or the clinician that the isolate is likely not S aureus and prompt further testing of a specific isolate. Accurate identification of S lugdunensis isolates facilitates studies to define the epidemiology and pathogenesis of prosthetic joint infection due to S lugdunensis and delineates optimal medical and surgical therapies.

Laboratory methods for the diagnosis of disseminated histoplasmosis: clinical importance of the lysis-centrifugation blood culture technique.

Disseminated histoplasmosis, an uncommon disease that is usually fatal if not treated, is being recognized with increasing frequency in immunosuppressed patients. We retrospectively reviewed the laboratory methods used to establish the diagnosis of disseminated histoplasmosis in 28 patients examined at our institution. Tissue stains provided the initial diagnosis in 13 of the patients (46%). Serologic tests (complement fixation or immunodiffusion) were positive in 25 patients (89%); the frequencies of positive serologic results were similar in immunocompetent and in immunosuppressed patients. Blood cultures were positive in 20 of the patients (71%) and provided the initial diagnosis in 7 (25%); 5 of these 7 cases (71%) were detected with use of the lysis-centrifugation method. Furthermore, the recovery time for this blood culture method was less than that for a conventional blood culture method (brain-heart infusion biphasic technique). The lysis-centrifugation blood culture technique is an important laboratory method for the diagnosis of disseminated histoplasmosis.

Infective endocarditis due to unusual or fastidious microorganisms.

Infective endocarditis due to fastidious microorganisms is commonly encountered in clinical practice. Some organisms such as fungi account for up to 15% of cases of prosthetic valve infective endocarditis, whereas organisms of the HACEK group (Haemophilus parainfluenzae, H. aphrophilus, and H. paraphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) cause 3% of community-acquired cases of infective endocarditis. Special techniques are necessary to identify these microorganisms. A history of contact with mammals or birds may suggest infection caused by Coxiella burnetii (Q fever), Brucella species, or Chlamydia psittaci. A nosocomial cluster of postsurgical infective endocarditis may be caused by Legionella species or Mycobacterium species. If risk factors that are commonly associated with fungal infections (cardiac surgical treatment, prolonged hospitalization, indwelling central venous catheters, and long-term antibiotic use) are present, fungal endocarditis is possible. Patients with endocarditis and a history of periodontal disease or dental work in whom routine blood cultures are negative might have infection due to nutritionally variant streptococci or bacteria of the HACEK group. Communication between the microbiologist and the clinician is of crucial importance for identification of these microorganisms early during the course of the infection before complications such as embolization or valvular failure occur. In this article, we review the microbiologic and clinical features of these organisms and provide recommendations for diagnosis and treatment.