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Reproducibility of capsular serotypes of 55 consecutive clinical isolates of Klebsiella pneumoniae was evaluated by an indirect immunofluorescent-antibody technique previously described by Riser et al. (J. Clin. Pathol. 29: 296-304, 1976) Five colonies per specimen were examined for colony-to-colony variation, day-to-day variation, and reader-to-reader variation. Seventy-two reference strains were tested with each of 18 pools and 72 specific antisera prior to the clinical specimens to determine antiserum specificity and cross-reaction patterns. Lot-to-lot variation was examined with the reference strains. There was minimal lot-to-lot variation among the antisera tested. Ten antiserum pools required supplementation with individual antisera. The patterns of supplementation may vary from lot to lot. Colony-to-colony variation in intensity of immunofluorescence occurred, but there was no variation in serotype. These findings differ from previously reported colonial variation which occurred when API 20E biotypes were determined for individual colonies of K. pneumoniae directly from clinical specimens. Eighteen percent of clinical isolates studied gave cross-reactions when tested with the indicated specific antiserum. All but one of the cross-reactions were resolved with further dilutions. Day-to-day and reader-to-reader variations were minimal. The immunofluorescent-antibody technique is a reliable and reproducible method for capsular serotype determination. Capsular serotypes are less variable than API biotypes since colony-to-colony variation of serotype does not occur.

A case of Haemophilus paraphrophilus endocarditis successfully treated with ampicillin is described. The patient, a 24-year-old woman, had a prolapsed mitral valve. The organism was initially misidentified as H. parainfluenzae , which it closely resembles. H. paraphrophilus is distinguished by its requirement of 10% CO2 for growth on NaCl-free medium and its inability to ferment xylose.

The API (Analytab Products, Inc., New York, N.Y.) biotypes of 117 clinical isolates of Serratia marcescens were determined and fell into 13 different patterns. The O and H antigens were determined by tube agglutination, and 27 serotypes were identified. The biotype and serotype appeared to vary indepently. Serotyping and biotyping combined divided these isolates into 56 different types. There was a problem interpreting the end points for inositol fermentation and urease production, which could affect reproducibility of API biotypes. Biotyping is a simple way of screening for possible nosocomial outbreaks of S. marcescens.

Klebsiellamarker systems include determination of susceptibility patterns, serotype, bacteriocin susceptibility, bacteriophage susceptibility, biotype, and plasmid content, size and endonuclease fragment size. Susceptibility patterns are useful only if unusual patterns (ie, aminoglycoside resistance) occur. Serotypes are stable, reproducible markers. Over 90% of isolates can be serotyped and epidemiologically unrelated strains are widely distributed among the 72 standard types. Antisera are not available commercially except to types 1 to 6 and serotyping is expensive and time-consuming. Bacteriocin susceptibility typing is easier and cheaper but reproducibility is sometimes poor. Depending on the producer strains used, between 67% and 96% of strains are typable. As a single typing method, bacteriophage typing is not very sensitive. Only 70% of strains are typable and 20% are of a single type. There are two major problems with most biotyping systems: poor reproducibility and poor sensitivity. A high percent of strains is the same type. Plasmid analysis is technically the most complicated but is important if strains are aminoglycoside resistant. No one method is ideal, and characterization of isolates from an outbreak is best done by using several different marker systems.

Vibrio alginolyticus was isolated from two patients with leg wounds. Both had a history of recent exposure to ocean water.

Herpetic lesions of the genitalia may be confused clinically with other ulcerative, genital lesions. Direct immunofluorescence (FA) provides a rapid method of diagnosis, and the utility of this method for the diagnosis of genital ulcers was examined. One hundred and ten patients with genital lesions were examined by darkfield for syphilis and by FA and culture for herpes simplex virus (HSV) infections. Satisfactory samples were obtained from 102 patients, of which 81 were clinically suspected cases of HSV. Acetone-fixed slides of scrapings of ulcerative lesions were stained with conjugated antiserum prepared in rabbits against HSV type 2. HSV was isolated from 73% of specimens of suspected herpetic lesions, and 77% of these specimens were positive by FA. Nine percent were positive by FA only and these were not thought to represent false positives. Five percent were positive by culture only. A comparison of clinical diagnoses with laboratory findings revealed that 4% of the cases were misdiagnosed when only the clinical evaluation was considered. The data suggest that the inclusion of a diagnostic FA test for HSV along with the darkfield examination may be useful for differentiating the etiological agents of ulcerative, genital lesions.

Campylobacter fetus subspecies jejuni has recently been recognized as a significant cause of enteritis in humans. The organism has been previously postulated to cause illness by direct invasion of the mucosa of the gastrointestinal tract, but this has not been documented. Reported is a case of Campylobacter enteritis in which mucosal ulceration and crypt abscesses were seen on rectal biopsy, suggesting that the pathogenesis of Campylobacter fetus enteritis involves direct mucosal invasion.

Among the confirmed cases of viral meningitis and encephalitis, the most commonly diagnosed agents are enteroviruses, arboviruses, and herpes simplex virus. Definitive diagnosis of a viral infection often affects patient management, length of hospitalization, and antibiotic use. Laboratory diagnostic methods include microscopic examination of clinical specimens, virus culture, serologic studies, and immunologic detection of virus or viral antigens. Microscopic examination of cerebrospinal fluid is nonproductive, and, except for enteroviruses, culture almost always gives negative results. Viral culture of the cerebrospinal fluid, throat, and feces is the diagnostic method of choice for enteroviruses. Definitive diagnosis of herpes simplex virus encephalitis must be based on virus isolation from brain biopsy material. Arbovirus infection is diagnosed serologically. Although none of the newer rapid immunologic techniques is commercially available, some do hold great promise. These include measurement of virus-specific immunoglobulin M (IgM) and detection of viral antigens by enzyme or radioimmunoassay. A sensitive and specific procedure for early detection of herpes simplex virus antigens in cerebrospinal fluid would be of great benefit.

The occurence of multiple biotypes of Klebsiella pneumoniae within single specimens was determined in 59 clinical specimens. Biotyping was performed on five colonies of K. pneumoniae from each specimen, using the API 20E system (Analytab, Inc., New York) for identification of Enterobacteriaceae with strict adherence to the manufacturer's instructions. Multiple biotypes of K. pneumoniae were present in 31% (18) of the clinical specimens. Twenty-eight colonies representative of specimens with single and multiple biotypes were tested further for biotype reproducibility. Whereas genus and species identification was 100% reproducible, variation of one or more biochemical tests on serial transfers resulted in biotype reproducibility of only 64%. The greatest variation in biochemical tests occurred with urease (14%), indole production (10%) and citrate utilization (9%). Multiple biotypes in single specimens appear to be due to both inherent differences among the colonies in the specimen and variability in the system used to determine biochemical reactions. The presence of multiple biotypes limits the usefulness of biochemical typing for epidemiological surveilance of K. pneumoniae.

Two commercial sources of MRC-5 and primary rabbit kidney cell cultures were compared for sensitivity of herpes simplex virus isolation and cytopathic effect detection time. Forty-six of 78 previously positive and 50 of 234 fresh clinical specimens were positive and could be evaluated. Herpes simplex virus recovery from frozen specimens positive in at least one cell culture ranged from 85% in MRC-5 cells (M.A. Bioproducts) to 89% in primary rabbit kidney cells (M.A. Bioproducts). Herpes simplex virus recovery from fresh clinical specimens ranged from 94% in primary rabbit kidney cells (Flow Laboratories), MRC-5 cells (Flow Laboratories), and MRC-5 (M.A. Bioproducts) cells to 100% in primary rabbit kidney (M.A. Bioproducts) cells. Among the 96 positive specimens evaluated, no significant difference was found between commercially prepared MRC-5 and primary rabbit kidney cell cultures in either sensitivity or time to first detectable cytopathic effect. Cells from M.A. Bioproducts and Flow Laboratories were equally sensitive. Herpes simplex virus detection was dependent upon inoculating at least two cell culture tubes rather than if the cells were MRC-5 cells or primary rabbit kidney cells.

A gram-positive coccus, presently named Micrococcus mucilaginosus incertae sedis, was isolated from 14 blood cultures from a patient with endocarditis. The first positive blood culture was drawn 5 days after the patient underwent cardiac catheterization.

In the seven-month period from July 1975 through January 1976, 11 pediatric patients had Acinetobacter calcoaceticus var. anitratus cultured from blood; this organism had not been isolated from pediatric patients in the previous six months. In 10 of 11 patients, only the first of two cultures was positive. All patients recovered uneventfully, although only two were treated with appropriate antibiotics. Nine of 11 had been in mist tents at the time of the culture. Mist cultured from one tent contained the same organism found in the patient's blood culture. Eight of 10 patients, however, had blood for culture drawn from the same needle as samples for other blood work, compared with only three of 13 controls (p = .013); this represented a deviation from proper blood culture technique, and a mock trial confirmed contamination of blood cultures when technique was broken. Contamination by this organism occurred in the tent water reservoir and mist, and the nose and skin of the children were colonized. The hands of respiratory therapy technicians and blood-drawing personnel became contaminated while handling the mist tents. Thorough attention to hand-washing, tent sterilization, and technique in drawing blood cultures stopped the pseudo-epidemic.

Buffy-coat smears prepared from 599 blood samples, obtained simultaneously with blood for culture, were examined for bacteria. Each blood sample was quantitatively cultured on chocolate agar. Twenty-one blood cultures (3.6%) from 17 patients were positive. Two (11.7%) of the 17 patients had positive buffy-coat smears. One patient had two positive smears, for a total of three positive buffy-coat smears from 21 positive blood cultures. Chart reviews were performed for two patients with positive buffy-coat smears, three patients with positive blood cultures (containing the same organisms as the two study patients) but negative buffy-coat smears, and six additional patients who had had positive buffy-coat smears between 1974 and 1979. Of the eight patients with positive smears, six (75%) were infected with Gram-positive cocci, three of which wereStreptococcus pneumoniae. Mortality was 100%. (JAMA1981;245:357-359)