Lactobacillus acidophilus sepsis in a neonate.

Lactobacillus species are non-spore-forming, anaerobic, gram-positive rods that cause disease in immunocompromised adults. Few cases have been described in children. We present the case of a 2-month-old infant who apparently developed Lactobacillus acidophilus sepsis from an infected central venous catheter. Physicians should be aware that although Lactobacillus species rarely cause disease in children, they should be considered a possible pathogen when isolated from the blood of a newborn infant.

Identification of herpes simplex virus genital infection: comparison of a multiplex PCR assay and traditional viral isolation techniques.

Genital herpes simplex virus (HSV) is of major public health importance, as indicated by the marked increase in the prevalence of genital herpes over the past two decades. Viral culture has traditionally been regarded as the gold standard for diagnosis. In this study, we compared viral culture and the amplification of HSV DNA by the polymerase chain reaction (PCR) with respect to sensitivity, cost, clinical utility, and turnaround time. Patient sample swabs from 100 individuals were inoculated onto MRC-5 cells for isolation. Positive results were confirmed via a direct fluorescent antibody technique, and serotyping, when requested, was performed using HSV-1 and -2-type-specific sera. PCR techniques employed an extraction step of the same initial swab specimen, followed by PCR amplification, using a multiplex assay for HSV-1, 2 DNA. HSV-positive results were found in 32/100 samples via culture and in 36/100 samples via PCR. PCR-positive results yielded 16 (44%) patients infected with HSV-1 and 20 (56%) patients infected with HSV-2. Turnaround time for viral culture averaged 108 hours for positive results and 154 hours for negative results; PCR turnaround time averaged 24--48 hours. Laboratory cost using viral culture was $3.22 for a negative result and $6.49 for a positive result (including direct fluorescent antibody). Serotyping added $10.88 to each culture-positive test. Although laboratory costs for PCR were higher at $8.20/sample, reimbursement levels were also higher. We propose a multiplex PCR assay for diagnosis of HSV-1 and HSV-2 from patient swabs for use in a routine clinical laboratory setting. This assay offers increased sensitivity, typing, and improved turnaround time when compared with traditional viral culture techniques. Although it appears that PCR testing in a routine clinical laboratory setting is cost prohibitive compared with the case of nonserotyped viral culture, it may be very useful when clinical utility warrants distinguishing between HSV 1 and 2 and may be cost effective when reimbursement issues are examined.

Cord formation in BACTEC medium is a reliable, rapid method for presumptive identification of Mycobacterium tuberculosis complex.

Serpentine cord formation in BACTEC 12B medium was evaluated as a rapid method for the presumptive identification of M. tuberculosis complex. Kinyoun acid-fast stained smears were prepared from 666 positive BACTEC 12B bottles and examined for the presence or absence of serpentine cording. Cord formation had a sensitivity, specificity, positive predictive value, and negative predictive value of 89.2, 99.2, 98.5, and 94.2%, respectively. The evaluation of the presence of cord formation in BACTEC 12B medium is reliable and permits the rapid presumptive reporting of M. tuberculosis.

Comparison of virus culture and direct immunofluorescent staining of cytocentrifuged virus transport medium for detection of varicella-zoster virus in skin lesions.

Direct immunofluorescent staining of centrifuged viral transport medium (CVTM) was compared with conventional cell culture for the detection of varicella-zoster virus (VZV) in 87 dermal lesions from 84 patients. A total of 21 (24%) were positive for VZV; 8 (38%) of these were positive by culture and CVTM, 13 (62%) by CVTM alone, and none by culture only. Virus cultures were positive for VZV in an average of 9.1 days (range, 4-20 days). CVTM, using cytocentrifugation, is more sensitive and rapid than conventional cell culture for the detection of VZV in cutaneous specimens.

Comparison of MRC-5 and primary rabbit kidney cells for the detection of herpes simplex virus.

OBJECTIVE: To compare MRC-5 and primary rabbit kidney (PRK) cells to either cell monolayer alone for the recovery of herpes simplex virus (HSV). DESIGN: A total of 2476 specimens received for HSV culture during a 3-year period were cultured on MRC-5 and PRK cells. Detection rates and the time to first detection were determined for each cell type used. A cost analysis was also performed for the isolation and identification of HSV using both cell types, the MRC-5 cell line alone, and PRK cell culture alone. SETTING: Large, urban, tertiary-care, university-affiliated hospital. RESULTS: Of the 2476 specimens cultured for HSV, 535 (21.6%) were positive. The MRC-5 cell line detected 531 (99.3%), and the PRK cell culture detected 522 (97.6%) of the positive specimens. Thirteen HSV isolates were detected only in MRC-5 cells, and four were isolated only in PRK cells. Approximately 44% of the cultures were positive by day 1, 84% by day 2, and 98% by day 3, regardless of the cell type used. The total cost per culture was comparable for MRC-5 and PRK cells. CONCLUSIONS: There was no difference in the sensitivity or time to detection of HSV between PRK and MRC-5 cells either alone or in combination. Either cell type alone represents an efficient, cost-effective method for the isolation of HSV.

Quality evaluation of sputum specimens for mycobacterial culture.

The evaluation of sputum specimen quality before bacterial culture is an accepted practice. Traditionally, sputum received for mycobacterial culture has been processed regardless of specimen quality. In view of the increasing emphasis placed on the primary acid-fast smear, the effect of specimen quality and the contribution of the presence of neutrophils on subsequent smear and culture positivity for mycobacteria was assessed. A total of 873 sputa were evaluated and initially assigned a quality (Q) score of 1 (many squamous cells relative to neutrophils) to 3 (no squamous cells) based on criteria for specimen acceptability for routine bacterial culture. The percentage of specimens that were Q1, Q2, and Q3 were 46.8, 35.3, and 17.9, respectively. Most of the specimens received were Q1, and these specimens demonstrated the highest number of positive smears and cultures compared to Q2 or Q3 specimens. Thus, routine bacterial culture criteria were not helpful in assessing specimen quality for mycobacterial culture. The contribution of the presence of neutrophils to smear and culture positivity was assessed. A total of 724 sputa were evaluated for cellular composition: 665 (91.9%) specimens contained neutrophils, and 59 (8.1 %) did not contain neutrophils. A total of 51 (7.0%) primary smears and 121 (16.7%) cultures were positive for mycobacteria; 92.2% of the positive smears; and 90.1 % of the positive cultures were from specimens that contained neutrophils. Thus, screening sputum specimens for the presence of neutrophils provides an effective method to evaluate the acceptability of sputum for mycobacterial smear and culture, especially from patients in respiratory isolation.

In-vitro production of ethanol in urine by fermentation.

Driving while under the influence of alcohol (DUI) can lead to serious injuries to the intoxicated driver and surrounding individuals, in addition to revocation or suspension of driving privileges. The accuracy and interpretation of the testing procedures may be compromised if an individual's urine contains sugar, and either bacteria or yeast. Under these conditions, ethanol can be produced in vitro, producing a result that may be erroneously indicative of DUI. In this study three yeast species and six bacterial species were added to a blank urine sample devoid of any alcohol or sugar. Samples were incubated at 0, 25, and 35 degrees C for 24, 48, and 144 hours in the presence of one of four different sugars. Ethanol concentrations were assayed using an enzymatic alcohol dehydrogenase assay. Results showed that when glucose was used as a substrate, all yeast species (Candida albicans, Candida parapsilosis, and Candida sp. not albicans) and three bacterial species (Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis) were capable of producing ethanol while the other three (Enterococcus sp., Staphylococcus sp. not aureus, and Pseudomonas aeruginosa) were not. The rate of ethanol production is temperature dependent and can be inhibited by storage of samples at 0 degrees C or the use of approximately 1% sodium fluoroide as an antimicrobial agent. Many of these species were also able to use other substrates (sucrose, fructose, and galactose) to produce ethanol by fermentation.

Comparison of Crystal Enteric/Nonfermenter system, API 20E system, and Vitek AutoMicrobic system for identification of gram-negative bacilli.

A comparative evaluation of the Crystal Enteric/Nonfermenter system (Crystal; Becton Dickinson, Cockeysville, Md.), API 20E (API; bioMérieux Vitek, Inc., Hazelwood, Mo.), and the Vitek GNI card (Vitek; bioMérieux Vitek) was performed with 512 clinical isolates of gram-negative bacilli, including 381 members of the family Enterobacteriaceae and 131 nonenteric bacilli. With supplemental testing, API, Crystal, and Vitek correctly identified to the genus and species level 505 (98.6%), 489 (95.5%), and 494 (96.5%) of the 512 isolates, respectively. Supplemental testing, as specified by the manufacturer, was required to identify 119 (23.2%), 18 (3.5%), and 5 (1.0%) of the isolates with the three systems, respectively. Of the 381 isolates from the family Enterobacteriaceae, API and Crystal correctly identified 90.3 and 91.6% by 18 to 24 h without supplemental testing, respectively, and Vitek identified 92.4 and 96.1% following 10 and 18 h of incubation, respectively. Of the 131 nonenteric organisms, API and Crystal correctly identified 28.2 and 93.9% by 18 to 24 h without supplemental testing, respectively, and Vitek identified 84.0% by 10 h and 93.9% by 18 h. Errors in identification with each system were infrequent and appeared to be randomly distributed among the genera evaluated. The three systems were comparable in accuracy when either a weighted clinical laboratory profile of organisms or a group of selected isolates in a stress test sample was evaluated (P > 0.05). There were no significant differences between the three systems in their ability to identify either the isolates in the weighted group or those in the stress test (P > 0.05). Crystal compared favorably with API and Vitek, which have established track records in clinical laboratories, and is acceptable for the identification of members of the Enterobacteriaceae and nonenteric bacilli in a clinical microbiology laboratory.

Detection of acid-fast bacilli in concentrated primary specimen smears stained with rhodamine-auramine at room temperature and at 37 degrees C.

Many laboratory workers prefer the rhodamine-auramine method of staining acid-fast bacilli (AFB) in primary specimen smears rather than carbol fuchsin stains because the stain is more readily interpreted and yields greater sensitivity. The increasing incidence of AFB infections serves as an impetus to optimize the rhodamine-auramine stain. A total of 782 primary smears were evaluated blindly by the rhodamine-auramine method at both room temperature and 37 degrees C. Thirty-five smears (4.5%) were positive for AFB, 30 were positive by both methods, and 5 were positive at 37 degrees C only. Room temperature staining detected only 85.7% of the positive primary smears. Of the 30 smears positive by both methods, 13 (43.3%) had equal numbers of AFB on both smears, 13 (43.3%) had more AFB on the smear stained at 37 degrees C, and 4 (13.3%) had greater numbers of AFB on the smear stained at room temperature. No smears were positive only when stained at room temperature. The increasing diagnostic emphasis placed on the primary smear underscores the importance of optimizing AFB smear methods, and rhodamine-auramine staining at 37 degrees C enhances the detection of AFB compared with conventional staining at room temperature.

The comparative activity of fleroxacin, three other quinolones and eight unrelated antimicrobial agents.

The in vitro activity of fleroxacin, a new trifluorinated quinolone was evaluated against 432 bacterial isolates. Fleroxacin was 1- to 2-fold less active than ciprofloxacin and at least as active as ofloxacin and lomefloxacin against most members of the family Enterobacteriaceae. The MICs of fleroxacin for 90% of strains tested (MIC90) were < or = 0.25 micrograms/ml against all isolates of Enterobacteriaceae except Citrobacter freundii (MIC90, 4 micrograms/ml) and Serratia marcescens (MIC90, 2 micrograms/ml). Fleroxacin was as active as ciprofloxacin, ofloxacin and lomefloxacin against Pseudomonas spp, (MIC90 for all quinolones tested were > 8 micrograms/ml). Acinetobacter and Haemophilus influenzae were very susceptible to fleroxacin; however fleroxacin was 1-fold less active than lomefloxacin against Acinetobacter and at least 1-fold less active than ciprofloxacin or ofloxacin against H. influenzae. Methicillin-susceptible and -resistant strains of Staphylococcus epidermidis and methicillin-susceptible strains of S. aureus were very susceptible to fleroxacin, with an MIC90 < or = 1 microgram/ml (range 0.5-1 microgram/ml). Methicillin-resistant S. aureus and Staphylococcus spp. other than aureus and epidermidis were not susceptible to fleroxacin (MIC90 > 8 micrograms/ml). In addition, fleroxacin as well as ciprofloxacin, ofloxacin and lomefloxacin were inactive against Enterococcus spp. (MIC90 > 8 micrograms/ml). Streptococcus pneumoniae and S. pyogenes were resistant to both fleroxacin and lomefloxacin but were very susceptible to ciprofloxacin and ofloxacin. These results suggest that fleroxacin represents a valid therapeutic option in the treatment of infections caused by most Enterobacteriaceae and some species of staphylococcus.