Male factor as determinant of in-vitro fertilization outcome.

The effect of different semen parameters was evaluated in 200 consecutive couples in an in-vitro fertilization (IVF) programme. All semen analyses were performed on the native aliquot of semen which was subsequently prepared and used for in-vitro insemination. Morphology evaluation using strict criteria (kappa 0.46 and r = 0.565) was compared with progressive motile sperm density (kappa 0.37 and r = 0.333) and the conventional World Health Organisation (WHO) evaluation of morphology (kappa 0.31 and r = 0.378). Results show that morphology evaluation using strict criteria is the best predictor of IVF and density of progressively motile spermatozoa can be an optional method. The combined results of strict morphology and motile concentration progressively showed that if both parameters were below the cut-off points of 5% and 3 x 10(6)/ml respectively, the fertilization rate per oocyte was very low (18%). No pregnancies were achieved in this group. When both parameters were above the cut-off points, the fertilization rate per oocyte was high (72%) (P less than 0.005) and the pregnancy rate per embryo transfer was 27%. Predictive values indicate that morphology evaluation using strict criteria and the number of progressive motile spermatozoa can be used as patient selection criteria for infertility clinics.


Laparoscopic application of interceed (TC7).

Interceed (TC7) is a surgical adjuvant which has been shown to be efficacious in reducing adhesion formation in gynaecological pelvic surgery by laparotomy. In view of the increasing number of laparoscopic interventions and the concern with reducing post-operative adhesions, we evaluated the possibility of Interceed delivery through the ancillary trocars. Twenty-four patients underwent operative laparoscopy followed by the application of Interceed. No difficulties were noticed. Material sized from 1.9 x 2.5 to 3.8 x 5 cm was successfully delivered into the peritoneal cavity, while the duration of the procedure ranged from 2.5 to 6 min for each piece. In conclusion, it is feasible to consider application of Interceed during laparoscopic interventions. This approach to delivery requires a short operative time, minimal instrumentation and enhanced ease of application.


Improved development of human embryos in vitro by a human oviductal cell co-culture system.

This study was undertaken to determine the effect of co-culture with human oviductal cells on human embryos. Spare embryos from gamete intra-Fallopian transfer (GIFT), pronuclear stage transfer (PROST) and in-vitro fertilization/embryo transfer (IVF/ET) programmes were either cultured in serum-supplemented Earle's balanced salt solution alone, or co-cultured in the same solution with oviductal cells from the pronuclear stage (day 1 post-insemination) or two- to four-cell stage (day 2 post-insemination). The co-cultured embryos appeared to have a higher developmental potential (higher rate of blastocyst formation and lower fragmentation rate), although there was no statistical difference in their rate of development, degree of fragmentation and stages attained, when compared with conventionally cultured embryos. The percentage of hatching blastocysts was significantly higher (P less than 0.05, Fisher's exact test) for embryos co-cultured from day 1 post-insemination (38%) than for embryos which had not been co-cultured (7%). The blastocyst hatching rate for embryos co-cultured from day 2 post-insemination was 15%. It was therefore concluded that co-culture of human embryos with oviductal cells could improve the development of the embryos in vitro. The degree of improvement was more pronounced when the co-culture started at an earlier stage.


The influence of solubilized porcine zona pellucida protein on the binding capacity of human spermatozoa.

The acrosome reaction, sperm-zona pellucida binding, sperm-oolemma binding/fusion and subsequent fertilization are known to be influenced by homologous as well as heterologous follicular fluid and zona pellucida protein. In this study, the effect was investigated of different concentrations of solubilized porcine zona pellucida protein on the zona binding potential of human spermatozoa under hemizona assay conditions. Human spermatozoa incubated with 617 and 142 micrograms/ml porcine zona pellucida protein showed a statistically significant increase in zona binding when compared with control spermatozoa (106.5 +/- 18.0 versus 60.9 +/- 29.0, P less than 0.02 and 111.0 +/- 26.6 versus 63.0 +/- 25.5, P less than 0.0001, respectively). Concentrations of 67 micrograms/ml porcine zona pellucida protein did not show a significant increase in zona binding (78.7 +/- 21.7 versus 66.7 +/- 25.4, P greater than 0.05). Control zona binding values for different experiments did not differ significantly (60.9 +/- 29.0; 63.0 +/- 25.5; and 66.7 +/- 25.4, P greater than 0.6). In conclusion, it seems likely that a factor(s) present in the porcine zona pellucida might play a beneficial role during human sperm-oocyte binding. The results of the study might be used in future investigations to manipulate gamete interaction to such an extent that improved fertilization rates can be accomplished.


Increased pregnancy failure rates after clomiphene following assisted reproductive technology.

The obstetric outcome of 1941 in-vitro fertilization (IVF) and 1436 gamete intra-Fallopian transfer (GIFT) pregnancies reported from 25 units in Australia and New Zealand have been reviewed. Recently, gonadotrophin-releasing hormone analogues (GnRHa) have replaced clomiphene as part of many ovarian stimulation protocols. Clinical abortion rates after clomiphene (24.4% for IVF; 23.0% for GIFT) were not significantly higher than after GnRHa (20.7% for IVF; 17.9% for GIFT) when IVF and GIFT data were considered separately. However, the abortion rate for combined IVF and GIFT was significantly higher after clomiphene than after GnRHa. This pattern was found for most maternal age groups and causes of infertility although differences were not significant in all categories. The combined IVF and GIFT ectopic pregnancy rate of 6.7% for clomiphene was significantly higher than 4.1% for GnRHa. Because the mechanism of action of clomiphene for oocyte recruitment during folliculogenesis means that GnRHa cannot be used with clomiphene, luteinizing hormone (LH) levels are higher in clomiphene cycles than in GnRHa cycles. Clomiphene itself could cause the increase in pregnancy wastage or increased levels of LH during follicule genesis associated with the use of clomiphene may cause the observed pregnancy failures.


Births in Israel resulting from in-vitro fertilization/embryo transfer, 1982-1989: National Registry of the Israeli Association for Fertility Research.

Our objective was to describe the characteristics of pregnancies, deliveries and children at birth following in-vitro fertilization (IVF) and related technologies in Israel, from 1982 to 1989. A national survey with collaboration from all IVF units in the public hospitals was designed and data were collected on individual patients. Comparison of results was made with data from a national delivery census and from other national IVF registries. During the period covering this survey, 1149 deliveries resulted in 1475 newborns; 98% of deliveries occurred following conventional IVF and embryo transfer (IVF-ET), 2% after in-vivo fertilization and gamete intra-Fallopian transfer. Following IVF-ET, 23% of the pregnant women were hospitalized due to a complication of pregnancy and 47.3% of the deliveries were by Caesarean section (41% if multiple births are omitted). The male to female birth ratio was 1.07:1.0; 23.6% of the deliveries were multiple births, 28.6% of deliveries were pre-term and the median length of gestation decreased with multiple births. At delivery, 23.8% of newborns weighed less than 2500 g. The incidence of low birthweight newborns was significantly higher in multiple births. The ratio of perinatal mortality (22.8/1000), double the incidence found in a national census (13/1000), increased dramatically with multiple births (12.7, 24.5 and 75.8/1000 for singleton, twins and triplets respectively). The incidence of a major congenital malformation was 2.2%, no higher than in the general population. A survey of published national IVF registries from Australia and New Zealand, Great Britain, USA and France showed much similarity in all aspects of pregnancy outcome following IVF and related technologies.(ABSTRACT TRUNCATED AT 250 WORDS)


Successful treatment with methotrexate of five vital interstitial pregnancies.

Five patients with vital, unruptured interstitial pregnancies of less than 3 cm maximum diameter were treated successfully with methotrexate and leucovorin rescue. Four pregnancies showed cardiac activity. Diagnosis was established with transvaginal ultrasonography in all patients. The human chorionic gonadotrophin serum levels were measured to monitor the effectiveness of therapy. This is the first publication on methotrexate treatment for interstitial twin pregnancy and the first on instillation of methotrexate after puncture and aspiration of interstitial pregnancy. In all cases, total and uneventful regression of trophoblast tissue was achieved. No adverse reactions were observed. The advantages and drawbacks of these therapeutic approaches are discussed. Methotrexate appears to be an effective medical non-surgical treatment for unruptured interstitial pregnancy with or without cardiac activity, and preserves reproductive potential.


Incidence and significance of unequal gestational sac diameter or embryo crown-rump length in twin pregnancy.

The incidence of differences in gestational sac diameter and crown-rump length, measured at the time of the first ultrasound, in which at least one gestational sac or crown-rump length could be visualized, were analysed retrospectively in 260 twin pregnancies in which one or both fetuses were delivered at term. The difference in gestational sac diameter averaged 1.2 +/- 0.1 mm for pregnancies which ended in twin births, compared to 2.0 +/- 0.3 mm when pregnancy ended in single births (P less than 0.02). The difference in crown-rump length averaged 2.4 +/- 0.6 mm for pregnancies which ended in a single birth, compared to 0.9 +/- 0.1 mm for twin births (P = 0.02). Disparities of greater than or equal to 3 mm in gestational sac diameter (P less than 0.05) or crown-rump length (P less than 0.001) were associated with an embryo loss rate greater than or equal to 50%. The disparity in gestational sac diameter (P less than 0.04) and crown-rump length (P less than 0.01) was smaller in pregnancies resulting from assisted reproductive technologies, compared with pregnancies resulting from coitus or insemination. Differences in gestational sac diameter and crown-rump length in early pregnancy were unrelated to differences in birth weight, length or sex.


Embryo reduction in multifetal pregnancies using saline injection: comparison between the transvaginal and the transabdominal approach.

A total of 30 patients with multifetal pregnancies, all resulting from treatment with superovulatory agents or assisted reproductive techniques, underwent embryo reduction. All patients had three or more fetuses (one sextuplet, two quintuplets, seven quadruplets and 20 triplets). The procedure was carried out using intra-embryonal injection of 0.9% sodium chloride solution. Embryo reduction was carried out via the transabdominal approach in 10 patients, performed at 11-12 weeks of gestation, and via the transvaginal route in 20 other patients, at 8-10 weeks of gestation. In the transabdominal group, one patient aborted following repeated attempts at embryo reduction while the other nine gave birth to healthy newborns (eight twins and one triplet). In the transvaginal group, four pregnancies are currently ongoing (all beyond 28 weeks of gestation), 14 pregnancies resulted in a delivery of at least one live newborn (13 twins and one singleton), one patient had a late abortion at 24 weeks' gestation and another was delivered at 27 weeks' gestation due to severe pre-eclampsia. Transvaginal ultrasound-guided needle procedures are commonly practised in most in-vitro fertilization units. The employment of this route for embryo reduction, performed at an earlier gestational age and with the use of a non-toxic substance such as 0.9% saline solution, is advocated.


New vancomycin disk diffusion breakpoints for enterococci. The National Committee for Clinical Laboratory Standards Working Group on Enterococci.

Since 1988, when the first vancomycin-resistant enterococcus was described, several descriptions of failures of disk diffusion breakpoints to detect low-level vancomycin resistance (MICs, 8 to 32 micrograms/ml) have been published. A four-laboratory collaborative study was undertaken to establish more accurate breakpoints for the disk test. Mueller-Hinton agar was used to perform dilution testing (in three laboratories) and disk diffusion testing (in all laboratories). Results were determined at 18, 24, and 48 h, and zones of inhibition were read using both transmitted and reflected light. One hundred organisms (35 Enterococcus faecalis, 55 E. faecium, and 10 E. gallinarum or E. casseliflavus isolates) were selected to represent vancomycin-susceptible and -resistant phenotypes. Interlaboratory agreement of agar dilution MICs was better at 24 h (91 to 94% within +/- 1 dilution) than at 18 h (76% within +/- 1 dilution). Therefore, 24-h agar dilution MIC results were used as the reference. For disk diffusion, it was critical to note the presence of a haze or colonies inside the zone when interpreting the test, since this correlated better with the results of the agar dilution test. The presence of a haze or inner colonies was best detected by reading the zones with transmitted light and incubating the plates for a full 24 h. When plotted against 24-h agar dilution MICs, breakpoints of </= 14 mm (resistant), 15 to 16 mm (intermediate), and >/= 17 mm (susceptible) resulted in 58 minor errors (14.5% of total values) and 5 very major errors (2.2% of resistant values or 1.3% of total values). No major errors were seen. Results of repeat testing using a common lot of Mueller-Hinton agar showed 52 minor errors (13.3%) and 4 major errors (4.2% of susceptible values of 1.0% pf total values) but no very major errors. It is recommended that any haze or colonies within the zone be taken into account when determining zones of inhibition and that an MIC test be performed for strains with intermediate zones if vancomycin is being considered for treatment.


Relationship between frequency of infectious human immunodeficiency virus type 1-harboring cells and kinetics of viral replication: a simple procedure for quantitation of infectious virus-carrying cells in blood samples.

Statistical analysis of a limiting dilution assay (LDA) showed that the occurrence of infectious human immunodeficiency virus type 1 (HIV-1)-harboring cells in serially diluted samples of peripheral blood mononuclear cells (PBMCs) of HIV-1-seropositive patients fits the model describing a single-hit Poisson distribution. This observation led to the discovery that there is a direct correlation (r = 0.957) between the number of HIV-1-positive cells and the time when viral culture produces 1 ng of the HIV-1 p24 gag protein per ml. Frequency estimates based on this relationship were highly accurate (P less than 0.01) within the first 15 days of viral culture, which consisted of coculture of 10(6) normal PBMCs with the equivalent number of test PBMCs. This approach was less cumbersome than LDA and was sensitive enough to detect a single infectious HIV-1-harboring cell among as many as 320,000 cells. The values obtained for 57 patients agreed well with the data in the literature and showed that the frequencies of infectious cells in PBMCs reflect the advancement in the clinical stage, being 1/38,000, 1/11,000, and 1/7,000 for asymptomatic patients (Centers for Disease Control [CDC] group II/III), patients with AIDS-related complex (CDC group IVa), and patients with AIDS (CDC group IVb/c), respectively. A nearly 10-fold disparity in mean frequencies was observed when these values were correlated with the numbers of CD4-positive cells (1/9,000, 1/1,500, and 1/300, respectively, for asymptomatic patients, patients with AIDS-related complex, and patients with AIDS). The described method provides a simple means of determining infectious HIV-1-positive cells in blood samples.


Evaluation of a monoclonal antibody-based latex agglutination test for diagnosis of cryptococcosis: comparison with two tests using polyclonal antibodies.

Cryptococcal antigen detection has become a routine biological test performed for patients with AIDS. The poor prognosis of cryptococcosis explains the need for reliable tests. We evaluated the performances of a newly commercialized agglutination test that uses a monoclonal antibody specific for cryptococcal capsular polysaccharide (Pastorex Cryptococcus; Sanofi-Diagnostics Pasteur, Marnes-la-Coquette, France) and compared them with those of tests that use polyclonal immune sera (Cryptococcal Antigen Latex Agglutination System, Meridian Diagnostics, Inc., Cincinnati, Ohio; and Crypto-LA, International Biological Labs Inc., Cranbury, N.J.). The sensitivities and specificities of the tests were compared by using purified polysaccharides and yeast suspensions. Clinical specimens (131 serum samples, 41 cerebrospinal fluid samples, 34 urine samples, and 19 bronchoalveolar lavage samples) from 87 human immunodeficiency virus-positive subjects with (40 patients) and without (47 patients) culture-proven cryptococcosis were retrospectively tested during a blinded study. The effect of pronase treatment of samples was assessed for Pastorex Cryptococcus and the Cryptococcal Antigen Latex Agglutination System, and the antigen titers were compared. Our results show that (i) during the screening, concordance among the three tests was 97%; (ii) the use of pronase enhanced both the sensitivities and specificities of the Pastorex Cryptococcus test; (iii) titers agreed for 67% of the cerebrospinal fluid samples and 60% of the serum samples; and (iv) cryptococcosis was detected equally well with Pastorex Cryptococcus and with the other tests, whatever the infecting serotype (A, B, or D). The meaning of in vitro sensitivity and the relationship between titers and sensitivity are discussed. The results show that Pastorex Cryptococcus is a rapid and reliable test for the detection of cryptococcal antigen in body fluids and suggest that kits cannot be used interchangeably to monitor antigen titers in patients.


Effects of ascorbic acid on Chlamydia trachomatis infection and on erythromycin treatment in primary cultures of human amniotic cells.

Ascorbic acid (vitamin C) is an essential nutrient for humans. It may also be needed by Chlamydia trachomatis, an intracellular bacterium. We investigated the effects of vitamin C on the growth of C. trachomatis E/UW-5/Cx in a primary culture of human amniotic epithelial cells. The results showed that vitamin C enhances C. trachomatis infection at concentrations of 0.2, 0.6, and 1.2 mg/dl (P less than 0.001). These three concentrations represent the in vivo concentrations of deficiency, normal, and overload levels in serum, respectively. The enhancement was dose dependent. However, the growth of C. trachomatis was inhibited at vitamin C concentrations of 120 and 1,200 mg/dl. The inhibitory effect of erythromycin against C. trachomatis was shown to be reduced in the presence of vitamin C at the three concentrations tested (P less than 0.025-0.001), and MICs were four times greater (1.6 versus 0.4 micrograms/ml). Human amniotic cells were tolerant to vitamin C concentrations of up to 1,200 mg/dl. The results show that vitamin C may be an important nutrient for C. trachomatis and that incorporation of vitamin C in the culture medium may enhance the isolation and propagation of C. trachomatis in cell cultures.


Improvement of simultaneous detection of antibodies to Gag and envelope antigens of human T-lymphotropic virus type I by western immunoblot assay.

To determine seropositivity for human T-lymphotropic virus type I (HTLV-I), we attempted to improve the detection system that uses antibody to HTLV-I Env in Western immunoblotting (WB) by adding an envelope glycoprotein (gp46) purified from the culture fluid of HTLV-I-producing cells by immunoaffinity chromatography and gel chromatography. In this WB, 177 of 179 serum samples showing seropositivity in an indirect immunofluorescence assay showed positive reactions to the gp46 envelope antigen as well as to p19, p24, and p53 Gag antigens. The remaining two samples showed negative reactions to p24. False-positive results were not found for 533 indirect immunofluorescence assay-negative serum samples, although one band to p19 or p24 was observed in 46 of the 533 samples. These 46 samples did not react to p53 and gp46, suggesting that these samples belonged to the indeterminate group in accordance with the criteria proposed by the World Health Organization. Therefore, this improved WB can be used for the confirmation of seropositivity.


Comparison of four different enzyme-linked immunosorbent assays for serological diagnosis of Salmonella enteritidis infections in experimentally infected chickens.

The program for the eradication of Salmonella enteritidis from chickens in The Netherlands is based on bacteriological examination of breeding flocks. There is a great need for a specific and sensitive serological screening test. For that purpose, we developed four different enzyme-linked immunosorbent assays (ELISAs), i.e., an indirect ELISA with S. enteritidis flagellin, an indirect ELISA with S. enteritidis lipopolysaccharide, a double-antibody sandwich blocking ELISA that uses monoclonal antibodies against S. enteritidis flagellin (GM-DAS blocking ELISA), and a double-antibody sandwich ELISA that uses monoclonal antibodies against S. enteritidis lipopolysaccharide. In the present study, we compare the results of those ELISAs with sera from experimentally infected 1-day-old chickens and with sera and eggs from experimentally infected laying hens. Experimental infections were induced with strains of S. enteritidis phage types 1 and 2, S. typhimurium, and S. panama. Sera were collected up to days 44 and 39 after infection from 1-day-old chickens and laying hens, respectively. Only the GM-DAS blocking ELISA was able to discriminate between S. enteritidis infections and infections with the other serotypes. This ELISA had both a sensitivity and a specificity of 100% for all serum samples from experimentally infected chickens. A field study is in progress to evaluate whether this test can be implemented in the Dutch S. enteritidis eradication program.


Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system.

A test based on the polymerase chain reaction (PCR) was developed for the detection of the Mycobacterium tuberculosis complex in clinical samples. In this test, a 245-bp sequence of the insertion element IS986 was amplified and detected by agarose gel electrophoresis in the presence of ethidium bromide and by Southern blot and dot blot hybridization by using a 188-bp digoxigenin-labeled probe. We tested clinical specimens from 227 patients suspected of having tuberculosis. These included 102 cerebrospinal fluid, 48 sputum, 18 pleural fluid, 5 bronchoalveolar lavage, 18 blood, 7 pus, 8 bone marrow, and 6 urine samples and 15 tissue biopsy specimens. We also tested sputum samples from 75 patients with diseases other than tuberculosis. Sputum samples were first decontaminated, and all samples were treated with proteinase K-detergent solution to extract the DNA. Part of each sample was spiked with M. tuberculosis to provide a semiquantitative assay and to control for the loss of mycobacteria or interference with the PCR which may cause false-negative results. One femtogram of M. tuberculosis DNA could be detected. PCR was positive for all 32 culture-positive (for M. tuberculosis) and Ziehl-Neelsen staining (ZN)-positive samples, 10 of 12 culture-positive and ZN-negative samples, and all 4 culture-negative and ZN-positive samples. PCR detected M. tuberculosis complex bacteria in 35 of 178 culture- and ZN-negative samples. Clinical data supported the diagnosis of tuberculosis in the majority of the 35 patients from whom those samples were obtained.


Detection of Babesia bigemina-infected carriers by polymerase chain reaction amplification.

A SpeI-AvaI fragment (0.3 kbp) from pBbi16 (a pBR322 derivative containing a 6.3-kbp Babesia bigemina DNA insert) was subcloned into the pBluescript phagemid vector and was sequenced by the dideoxy-mediated chain termination method. Two sets of primers were designed for the polymerase chain reaction (PCR) assay. Primer set IA-IB was used to amplify a 278-bp DNA fragment, and primer set IAN-IBN was used to prepare a probe directed to a site within the PCR-amplified target DNA. Digoxigenin-dUTP was incorporated into the probe during the amplification reaction. PCR amplification of target DNA obtained from in vitro-cultured B. bigemina and nucleic acid hybridization of amplified product with the nonradioactive DNA probe showed that a 278-bp fragment could be detected when as little as 100 fg of parasite genomic DNA was used in the assay. A fragment of similar size was amplified from genomic DNAs from several B. bigemina isolates but not from DNAs from Babesia bovis, Anaplasma marginale, or six species of bacteria or bovine leukocytes. Similarly, the PCR product could be detected in DNA samples purified from 200 microliters of blood with a parasitemia of as low as 1 in 10(8) cells and which contained an estimated 30 B. bigemina-infected erythrocytes. By a direct PCR method, B. bigemina DNA was amplified from 20 microliters of packed erythrocytes with a calculated parasitemia of 1 in 10(9) cells. With the analytical sensitivity level of the PCR-DNA probe assay, six cattle with inapparent, 11-month chronic B. bigemina infection were found to be positive. No PCR product was observed in bovine blood samples collected from a splenectomized, A. marginale-infected bovine, a 4-year chronic B. bovis-infected animal, or 20 uninfected cattle from Missouri which were subjected to amplification. The PCR-DNA probe assay was shown to be sensitive in detecting latently infected cattle. The specificity and high analytical sensitivity of the test provide valuable tools for performing large-scale epidemiological studies.


Latex agglutination-negative methicillin-resistant Staphylococcus aureus recovered from neonates: epidemiologic features and comparison of typing methods.

An unusual strain of methicillin-resistant Staphylococcus aureus (MRSA) was repeatedly isolated from infants in a newborn special care unit (NBSC) and a newborn intensive care unit. Between January 1989 and March 1990, approximately 100 isolates from infected or colonized infants were recovered. Surveillance cultures taken during this time revealed a 20% colonization rate, which was defined as recovery of MRSA from the nares, umbilicus, or groin. Isolates were identified as S. aureus by tube coagulase reactivity and heat-stable nuclease production but were unreactive in a latex agglutination assay. Representative isolates that were collected during the outbreak and that were found to share the latex agglutination assay-negative phenotype were compared by antibiogram (12 isolates), bacteriophage typing (20 isolates), capsular polysaccharide typing (30 isolates), and plasmid as well as chromosomal DNA analyses (20 isolates). All isolates known to be associated with the outbreak had nearly identical antibiograms and were notably susceptible to clindamycin. Staphylococcal bacteriophage typing was not useful in determining the relatedness of the isolates, since the majority were nontypeable. Plasmid pattern analysis revealed one large plasmid (approximately 100 kb) of equivalent size among the isolates. Capsular polysaccharide typing revealed that 14 of 30 isolates tested were type 5. Isolates identified in children at two other hospitals in the city which were also unreactive by the latex agglutination assay and clindamycin susceptible had plasmid and antibiogram patterns identical to those of isolates from the NBSC. Pulsed-field gel electrophoresis of restriction enzyme-digested genomic DNAs from the outbreak isolates demonstrated identical patterns which could be clearly differentiated from those of other unrelated MRSA. The strain from the NBSC is, therefore, unique and underscores the need for caution in interpreting the latex agglutination reactivities of MRSA isolates.


Pyrazinamidase, CR-MOX agar, salicin fermentation-esculin hydrolysis, and D-xylose fermentation for identifying pathogenic serotypes of Yersinia enterocolitica.

We evaluated several simple laboratory tests that have been used to identify pathogenic serotypes of Yersinia enterocolitica or to indicate the pathogenic potential of individual strains. A total of 100 strains of Y. enterocolitica were studied, including 25 isolated during five outbreak investigations, 63 from sporadic cases, and 12 from stock cultures. The pyrazinamidase test, which does not depend on the Yersinia virulence plasmid, correctly identified 60 of 63 (95% sensitivity) strains of pathogenic serotypes and 34 of 37 (92% specificity) strains of nonpathogenic serotypes. Salicin fermentation-esculin hydrolysis (25 degrees C, 48 h) correctly identified all 63 (100% sensitivity) strains of the pathogenic serotypes and 34 of 37 (92% specificity) strains of the nonpathogenic serotypes. The results of the pyrazinamidase and salicin-esculin tests disagreed for only 7 of the 100 strains of Y. enterocolitica, and these would require additional testing. Congo red-magnesium oxalate (CR-MOX) agar determines Congo red dye uptake and calcium-dependent growth at 36 degrees C, and small red colonies are present only if the strain contains the Yersinia virulence plasmid. This test has proven to be extremely useful for freshly isolated cultures, but only 15 of 62 strains of pathogenic serotypes that had been stored for 1 to 10 years were CR-MOX positive. None of the 16 strains of Y. enterocolitica serotype O3 fermented D-xylose, so this test easily differentiated strains of this serotype, which now appears to be the most common in the United States. Although antisera that can actually be used to serotype strains of Y. enterocolitica are not readily available, the four simple tests described above can be used to screen for pathogenic serotypes.


Genotyping Naegleria spp. and Naegleria fowleri isolates by interrepeat polymerase chain reaction.

All six Naegleria species recognized to date were studied by interrepeat polymerase chain reaction (PCR). Priming at repeat sequences, which are known to be variable among eukaryotes, yielded electrophoretic DNA banding patterns that were specific for any single species. With a single PCR and simple gel electrophoresis, species determination could be performed in less than 1 day. Unambiguous discrimination between the pathogen N. fowleri and nonpathogenic Naegleria species appeared to be possible. Analysis of DNAs obtained from 20 separate isolates of N. fowleri revealed that geographic variation of the genetic fingerprints rarely occurs. All but 3 of 20 isolates of N. fowleri which were investigated showed identical banding patterns; for two isolates from New Zealand and one from Australia, a limited number of additional bands was detected, independent of the PCR primers used. These data corroborate previous findings on the genetic stability of pathogenic N. fowleri.


Detection of binding antibodies to native and recombinant human immunodeficiency virus type 1 envelope glycoproteins following recombinant gp160 immunization measured by flow cytometry and enzyme immunoassays. The AIDS Vaccine Clinical Trials Network.

The ability of antibody induced by vaccination with recombinant gp160 (rgp160) to bind to native and recombinant human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins was measured. Thirty-three HIV-1-seronegative healthy adult volunteers were injected four times with 40 or 80 micrograms of an HIV-1LAV envelope glycoprotein candidate vaccine per dose. The vaccine consisted of rgp160 produced in insect tissue culture cells infected with a recombinant baculovirus which contains the gp160 gene from the HIV-1LAV strain. By using a flow cytometric indirect immunofluorescence assay (FIFA) to detect vaccine-induced antibody to native envelope glycoprotein expressed by target cells infected with HIV-1IIIB, sera from 9 of the 33 vaccinees were positive. These included sera from eight vaccinees which stained HIV-1IIIB-infected cells and sera from two vaccinees which stained target cells infected with HIV-1MN, a heterologous virus strain. None of the sera stained cells infected with the HIV-1RF strain. Envelope glycoprotein-binding antibody was more frequently detectable in an enzyme-linked immunosorbent assay (ELISA) by using rgp160 compared with that which was detectable in the FIFA with uninfected target cells which were pulsed with rgp160 antigen. Positive correlations were observed between the rgp160 FIFA and a whole-virus-lysate enzyme immunoassay, between the rgp160 FIFA and the rgp160 ELISA, and between the rgp160 ELISA and the whole-virus-lysate enzyme immunoassay. The ability of sera from some volunteers who received rgp160 vaccine to bind to HIV-1-infected cells suggests that further studies with this vaccine should be done.


Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples.


Comparison of polymerase chain reaction and culture for detection of Borrelia burgdorferi in naturally infected Peromyscus leucopus and experimentally infected C.B-17 scid/scid mice.

Culture and the polymerase chain reaction (PCR) were compared for detection of Borrelia burgdorferi infection in wild-caught Peromyscus leucopus and experimentally inoculated C.B-17 scid/scid (severe combined immunodeficient) mice. PCR targeted highly conserved regions of the ospA gene and could detect one to five cultured organisms and 10 to 50 copies of molecularly cloned ospA DNA. Organs (kidney, spleen, and urinary bladder) and/or ear biopsy samples were obtained from 108 captured P. leucopus mice, and tissues were obtained from 7 experimentally inoculated mice. A simple sample-processing procedure with proteinase K and detergent treatment was used in the PCR analysis. Overall, B. burgdorferi was detected in 29 of 108 (27%) P. leucopus mice by culture and in 31 of 108 (29%) mice by PCR. As assessed by the kappa statistic, agreement between PCR and culture was high for ear and bladder (kappa = 0.80 and 0.65, respectively) and low for kidney and spleen (kappa = 0.37 and 0.03, respectively). While concordant results were obtained from 98 animals, PCR detected B. burgdorferi from 6 additional mice for which cultures were negative and culture detected B. burgdorferi from 4 animals which were PCR negative. Further phenol-chloroform extraction of DNA in a limited number of samples improved the sensitivity of PCR compared with that of culture. These results indicate that PCR may be as sensitive as culture for detecting B. burgdorferi in ear samples and that PCR analysis is suitable for establishing the infection status of animals in mark-release-recapture studies.


Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris.

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.


Detection of Mycoplasma pneumoniae in clinical samples from pediatric patients by polymerase chain reaction.

The polymerase chain reaction (PCR) technique was used to detect Mycoplasma pneumoniae DNA in clinical samples (nasopharyngeal aspirations or bronchoalveolar lavages) obtained from 100 children, 1 month to 16 years old. PCR allowed the detection of M. pneumoniae DNA from 20 out of the 100 patients studied. In 16 cases, PCR positivity was associated with acute respiratory symptomatology. For five PCR-positive patients, a positive culture or a serological response evidenced acute M. pneumoniae infections. A lack of antibody response was observed particularly with immunocompromised children and infants less than 12 months old. The amount of M. pneumoniae DNA in the PCR was estimated in a semiquantitative way by comparison of its hybridization signal with those obtained for 100, 10, and 1 color-changing unit (CCU) of the M. pneumoniae FH strain. Small amounts (less than or equal to 10(2) CCU/ml) of M. pneumoniae were found in samples from asymptomatic patients, while larger amounts (greater than or equal to 10(2) to greater than or equal to 10(4) CCU/ml) were found for 8 out of 10 patients with acute pneumonia.


Description and evaluation of the semiautomated 4-hour rapid ID 32 Strep method for identification of streptococci and members of related genera.

The rapid ID 32 Strep system (bioMérieux, La Balme les Grottes, France) is a new system which allows the identification in 4 h of most streptococci and members of related genera encountered in medical and veterinary bacteriology. Four hundred thirty-three isolates first identified by conventional methods and belonging to the genera Streptococcus, Lactococcus, Enterococcus, Aerococcus, Gemella, Leuconostoc, Erysipelothrix, Gardnerella, and Listeria were tested. Overall, rapid ID 32 Strep correctly identified 413 (95.3%) of the strains, with 109 (25.1%) requiring extra tests for complete identification. Sixteen strains (3.7%) were not identified, and 4 (1.0%) were misidentified. The rapid ID 32 Strep system is a suitable alternative for rapid identification of members of the genus Streptococcus and of related genera.


Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen.

An internal protein was purified from cell extracts of Brucella melitensis B115 by a combination of preparative isoelectric focusing and high-performance size exclusion chromatography. The protein has an apparent molecular mass of 230 kDa as determined by size exclusion chromatography. The protein was resolved to a single band of 20 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein had an isoelectric point of 4.9. The N-terminal sequence of the 20-kDa protein was determined. The 20-kDa protein has been identified as antigen A-2 with a previously described anti-antigen A-2 serum (B. Stemshorn, K. Nielsen, and B. Samagh, Can. J. Comp. Med. 45:77-81, 1981). Antigen A-2 reacted with sera from infected sheep in immunoblotting and may be useful in developing diagnostic tests for brucellosis.


Detection of pathogenic Yersinia enterocolitica by polymerase chain reaction and digoxigenin-labeled polynucleotide probes.

Yersinia enterocolitica is widespread in nature, but only a few bioserotypes are involved in human infections. Pigs are considered to be the major reservoirs of pathogenic strains. It is essential to have an accurate and rapid method for the detection of pathogenic yersiniae. To achieve this objective, 19-base synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) to detect the ail gene (which is conserved only in pathogenic strains) in strains of Y. enterocolitica and related species originating from pigs or pork products. Digoxigenin-labeled probes derived from the ail, inv, and yst genes were also evaluated on these strains. The PCR amplified a 273-bp fragment of the ail gene involved in eukaryotic cell invasion and serum resistance. The PCR detected template DNA only in strains of Y. enterocolitica traditionally classified as human pathogens but not in biotype 1A strains and related species. Other members of the family Enterobacteriaceae were also negative for the target gene. The digoxigenin-labeled ail probe gave identical results to the PCR. By use of this nonisotopic method, inv-homologous DNA was detected only among yersiniae, except for Y. ruckeri. Although all pathogenic serotypes of Y. enterocolitica were positive for the heat-stable enterotoxin yst gene, two strains of biotype 1A, one Y. intermedia strain, and six other species of the Enterobacteriaceae were also positive. Our results support the notion that pigs constitute an important reservoir of pathogenic Y. enterocolitica and that the inv-homologous sequence is Yersinia specific.


Initial testing of a novel urine culture device.

The Diaslide urine culture device consists of a hinged case containing two opposing agar media separated by a sampler with a handle at one end and two bent sampler tips at the opposite end. The tips of the sampler are first dipped into the urine. The sampler is then pulled out through the casing, simultaneously inoculating both agar surfaces with a streaking dilution. As a result, individual colonies can be observed even when bacterial concentrations exceed 10(6) CFU/ml. The number of colonies on the Diaslide correlated linearly with CFU per milliliter as determined by dilution plating. The clinical performance of the Diaslide was compared with those of ordinary dipslides and conventional cultures with a sample of 473 prescreened hospital urine specimens. The sensitivity, specificity, and positive predictive value of Diaslide versus those of culture at the 10(4)-CFU/ml cutoff level were 97.5, 98.3, and 98.3%, respectively, compared with 98.8, 95.7, and 97.2%, respectively, for dipslide versus culture. Similar results were found at the 10(5)-CFU/ml cutoff level. Only 5.5% of the Diaslides required subculturing, compared with 14.7 and 9.4% of the dipslides and conventional cultures, respectively. The Diaslide proved more convenient than an ordinary dipslide for sampling low volumes of urine. These data suggest that the Diaslide is a simple, effective device for culturing of urine specimens.


In vitro antimicrobial susceptibility testing of Borrelia burgdorferi: a microdilution MIC method and time-kill studies.

The susceptibility of Borrelia burgdorferi, the causative agent of Lyme borreliosis, to various antimicrobial agents varies widely among published studies. These differences are probably due in part to variations in susceptibility testing techniques and growth endpoint determinations. We developed a microdilution method for determining the MICs of antibiotics against B. burgdorferi. The method incorporated BSK II medium, a final inoculum of 10(6) cells per ml, and a 72-h incubation period and was found to be simple and highly reproducible. A variety of antibiotics and strains of B. burgdorferi and one strain of Borrelia hermsii were examined by this method. MICs of penicillin, ceftriaxone, and erythromycin for the B31 strain of B. burgdorferi were 0.06, 0.03, and 0.03 microgram/ml, respectively. We compared the MICs obtained by the microdilution method with those obtained by a macrodilution method using similar criteria for endpoint determinations and found the values obtained by both methods to be in close agreement. To further investigate the bactericidal activities of penicillin, ceftriaxone, and erythromycin against strain B31, we used subsurface plating to determine MBCs and we also performed time-kill studies. The MBCs of penicillin, ceftriaxone, and erythromycin were 0.125, 0.03, and 0.06 micrograms/ml, respectively. Time-kill curves demonstrated a greater than or equal to 3-log10-unit killing after 72 h with penicillin, ceftriaxone, and erythromycin; ceftriaxone provided the greatest reduction in CFU. The described methods offer a more standardized and objective approach to susceptibility testing of B. burgdorferi.


Identification of Mycobacterium avium complex strains and some similar species by high-performance liquid chromatography.

Strains of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium xenopi, and Mycobacterium gordonae were identified by high-performance liquid chromatography (HPLC) analysis of mycolic acids as bromophenacyl esters. HPLC criteria were used to develop a flow chart identification scheme, which was evaluated in our laboratory with a set of 234 strains representing five species and a hitherto undescribed species. Correct identifications of M. gordonae and M. xenopi were easily made. Flow chart differentiation of M. avium, M. intracellulare, and M. scrofulaceum was done with 97.9, 97.5, and 89.2% accuracies, respectively. Independent evaluation of the flow chart at a separate laboratory demonstrated an overall identification accuracy of 97% for M. avium complex. Strains that have been described biochemically as being intermediate between M. avium-M. intracellulare and M. scrofulaceum were identified as one or the other of these known species. Strains which were negative with the species-specific radioactive probe for M. avium complex but which were positive with the nonradioactive SNAP X probe were usually identified as M. intracellulare and M. scrofulaceum but rarely as M. avium.


Purification of listeriolysin O and development of an immunoassay for diagnosis of listeric infections in sheep.

A protein of 58,000-Da molecular mass was purified from the supernatant fluid of a dialysis sac culture of Listeria monocytogenes by cation-exchange chromatography. The purified protein, homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and possessing the characteristics of listeriolysin O (LLO), was used to develop an indirect enzyme-linked immunosorbent assay. Anti-LLO antibodies were shown to be consistently produced in sheep after experimental challenge with L. monocytogenes serovar 4b. The assay also successfully detected and measured specific anti-LLO antibodies in the sera of silage-fed sheep among which listeric enteritis and abortions had occurred.


Comparison of five methods, including the PDM Epsilometer test (E test), for antimicrobial susceptibility testing of Pseudomonas aeruginosa.

The antimicrobial susceptibilities of 100 clinical isolates of Pseudomonas aeruginosa to six antipseudomonal antibiotics were tested by five methods: the National Committee for Clinical Laboratory Standards (NCCLS) methods for broth microdilution, agar dilution, and agar disk diffusion; the Vitek Automicrobic System method (Vitek Systems, Hazelwood, Mo.); and the PDM Epsilometer test (E test) (AB Biodisk, Solna, Sweden). The E test results showed excellent correlation with agar dilution results, with over 90% agreement within 1 doubling dilution between the E test and reference agar dilution MICs for all antimicrobial agents tested. The E test results also showed good correlation with the results from the reference agar disk diffusion method, with 90 to 99% complete agreement and 100% essential agreement on categories for all antibiotics tested (essential agreement is the agreement obtained when minor discrepancies are ignored). Comparison of categories with the E test and broth microdilution methods, using the broth microdilution method as the reference method, gave only 59% complete agreement for gentamicin, with 28 minor discrepancies and 13 very major discrepancies. Some discrepancies were observed between results from the E test and broth methods for gentamicin, with the broth microdilution and Vitek methods giving higher MICs than the E test and other methods using agar. The most recent NCCLS guidelines for broth dilution testing have reduced the recommended levels of cation supplementation, which may enhance future agreement between results for the aminoglycosides and P. aeruginosa on broth and on agar. We found that the E test offers a simple, labor-efficient, and accurate method for MIC determination on an agar medium.


Comparison of the Sceptor Pseudomonas Plus MIC Panel with agar dilution for susceptibility testing of Pseudomonas aeruginosa.

The antimicrobial susceptibilities of 100 clinical isolates of Pseudomonas aeruginosa to gentamicin, amikacin, tobramycin, ticarcillin, piperacillin, and ceftazidime were determined by using the Sceptor system (BBL Microbiology Systems, Cockeysville, Md.), and the results were compared with those obtained using the National Committee for Clinical Laboratory Standards reference agar dilution method. Excellent correlation was observed for the aminoglycosides, with greater than 95% agreement within 1 doubling dilution of the reference agar dilution MIC, while ticarcillin and piperacillin showed lower percent agreement values of 91 and 88%, respectively.


Identification of Streptococcus pneumoniae with a DNA probe.

The Accuprobe Streptococcus pneumoniae Culture Identification Test (Gen-Probe, Inc.) was evaluated with 172 isolates of S. pneumoniae and 204 nonpneumococcal isolates. The sensitivity and specificity of the Accuprobe test were 100%. Optimum results were obtained when four or more discrete colonies were selected for testing. The Accuprobe test was determined to be an accurate and rapid method for identification of S. pneumoniae.


Identification of Pseudomonas aeruginosa by using a disk of phenanthroline and 9-chloro-9-[4-(diethylamino)phenyl]-9,10-dihydro-10-phenylacridine hydrochloride and by cell agglutination testing with monoclonal antibodies.

To establish a simple identification procedure for Pseudomonas aeruginosa, we developed a disk consisting of phenanthroline and 9-chloro-9-[4-(diethylamino)phenyl]-9,10-dihydro-10- phenylacridine hydrochloride (PC disk). Nine of 248 P. aeruginosa isolates and all other oxidase-positive gram-negative isolates (143 isolates) had clear inhibition zones around the PC disk. Of these nine P. aeruginosa isolates, seven produced a blue, blue-green, or green pigment on Mueller-Hinton agar after 24 h of incubation. The sensitivity and specificity of the PC disk susceptibility test in combination with pyocyanin production were 99 and 100%, respectively. Ten P. aeruginosa isolates showed no agglutination reactions with monoclonal antibodies against P. aeruginosa (96% sensitivity and 100% specificity).


Vibrio-associated gastroenteritis in the lower Cross-River Basin of Nigeria.

A total of 120 Vibrio species were isolated from 588 patients with acute diarrheal disease during an outbreak of gastrointestinal tract infections at different locations in the lower Cross River Basin of Nigeria. Vibrio cholerae O1, biotype El Tor, serotype Ogawa, was the prominent organism isolated from the Vibrio-associated diarrheal cases. During the 3 months of study, V. cholerae non-O1 was recovered from 10 patients while Vibrio parahaemolyticus was recovered from 19 patients. The significance of this study is the recognition that there is an ecological niche which supports V. cholerae non-O1 and V. parahaemolyticus in the Cross River Basin of Nigeria.


Seroprevalence of enteric and nonenteric adenoviruses in Bangladesh.

Single serum samples obtained from infants between 0 and 24 months of age admitted to a diarrheal disease hospital in Bangladesh were tested for the presence of adenovirus-specific immunoglobulin G (IgG) and IgA antibodies by using enzyme immunoassay and neutralizing antibodies to adenovirus types 2, 40, and 41. IgG antibodies were more prevalent than IgA antibodies, and neutralizing activity to enteric adenovirus was found in serum samples from 50% of infants who had reached 2 years of age.


Optimum use of selective plated media in primary processing of respiratory tract specimens from patients with cystic fibrosis.

A total of 258 respiratory tract specimens from patients with cystic fibrosis were inoculated onto nine different plated media, and the rates of recovery of potential pathogens were compared. Media included sheep blood agar, enriched chocolate agar, MacConkey agar for gram-negative bacilli, chocolate agar containing bacitracin for Haemophilus spp., bromcresol green agar for yeasts, cetrimide agar for Pseudomonas spp., sheep blood agar containing colistin and nalidixic acid for gram-positive cocci, mannitol salt agar for Staphylococcus aureus, and oxidation-fermentation agar containing 300 U of polymyxin B per ml and 2 U of bacitracin per ml (OF-PBL medium) for Pseudomonas cepacia. With two exceptions, all of these media proved useful in recovering potential pathogens from respiratory tract specimens from patients with cystic fibrosis. The two exceptions were cetrimide agar and colistin-nalidixic acid-supplemented sheep blood agar, which were found to be superfluous. In addition, the results of this study further delineated the prevalence of selected bacteria and fungi in respiratory tract secretions from patients with cystic fibrosis. In rank order of frequency of isolation, we recovered isolates of Pseudomonas aeruginosa, Haemophilus parainfluenzae, Candida albicans, S. aureus, Haemophilus influenzae, molds, members of the family Enterobacteriaceae, yeasts other than Candida albicans, miscellaneous gram-negative bacilli, beta-hemolytic streptococci, P. cepacia, and Streptococcus pneumoniae.


Time to detection of positive BacT/Alert blood cultures and lack of need for routine subculture of 5- to 7-day negative cultures.

Consecutive BacT/Alert blood cultures which were instrument negative following a 7-day incubation were subcultured. Eighteen (0.2%) of 11,476 bottles had growth on subculture. Eleven of these eighteen isolates were considered contaminants on the basis of the identity of the organism and lack of other positive blood cultures from the same patient. In addition, analysis of time to instrument detection for approximately 2,900 positive blood cultures indicates that 5 or 6 days of incubation is sufficient for the routine detection of clinically significant organisms from BacT/Alert blood cultures. These data indicate that subculture of 5- to 7-day instrument-negative BacT/Alert blood culture bottles is not necessary.


Septicemia with Ewingella americana.

Ewingella americana was isolated from two blood cultures from a 75-year-old male after cholecystectomy. The characteristics of this strain are compared with the reported biochemical characteristics of 44 American strains. Previously described infections and pseudoinfections with E. americana are reviewed.


Colony morphotype on Sabouraud-triphenyltetrazolium agar: a simple and inexpensive method for Candida subspecies discrimination.

A new method of Candida subspecies discrimination on Sabouraud-triphenyltetrazolium agar is reported. Five hundred sixty-two strains of Candida and Torulopsis glabrata, previously identified by conventional mycological methods, were studied. Each strain received a three-letter code and a number based on its colonial morphology. Sixteen morphotypes were found for Candida albicans, 6 were found for Candida parapsilosis, 4 were found for both Candida guilliermondii and Candida krusei, and 12 were found for Candida tropicalis. None of the 56 T. glabrata strains studied grew on this agar. A reproducibility of 95% was found for C. albicans. The simplicity and low cost could make this method useful for typing Candida spp.


Polymerase chain reaction for detection of Leptospira spp. in clinical samples.

A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema pallidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative.


Growth of Porphyromonas gingivalis, Treponema denticola, T. pectinovorum, T. socranskii, and T. vincentii in a chemically defined medium.

A chemically defined medium, OMIZ (Oral Microbiology and Immunology, Zürich)-W1 was developed. Medium OMIZ-W1 supports the long-term proliferation of a wide range of oral anaerobes, including representative strains of four Treponema species and Porphyromonas gingivalis. High concentrations of ascorbic acid and ammonium ions proved to be important for the growth of these organisms. T. denticola CD-1 grew in the absence of polyamines and long-chain fatty acids, T. pectinovorum and T. socranskii required polyamines, whereas T. vincentii depended on both polyamines and lecithin for growth. Specific requirements for purines and/or pyrimidines were detected, and these requirements could be used to distinguish Haemophilus-Actinobacillus group organisms. Some strains of P. gingivalis grew without vitamin K, while others were not satisfied by menadione but required its precursor 1,4-dihydroxy-2-naphthoic acid. Protoporphyrin IX or hemin equally satisfied the porphyrin requirements of P. gingivalis and Bacteroides forsythus, whereas ferrous sulfate was more efficiently used as a source of iron than was hemin. The cellular cohesiveness of P. gingivalis increased with high concentrations of hemin in the growth medium. Prevotella intermedia, B. forsythus, and several strains of P. gingivalis were more fastidious and required a protein or serum supplement to grow in medium OMIZ-W1.


Evaluation of a cold-adapted influenza B/Texas/84 reassortant virus (CRB-87) vaccine in young children.

A cold-adapted (ca) influenza B reassortant virus vaccine that contained the six internal RNA segments from influenza B/Ann Arbor/1/66 ca virus and the neuraminidase and hemagglutinin genes from wild-type influenza B/Texas/1/84 virus was evaluated in children ranging in age from 8 months to 14 years. The children were vaccinated intranasally with doses ranging from 10(3.2) to 10(6.2) 50% tissue culture infective doses (TCID50). Thirty children were seropositive, and 26 were seronegative. Thirty-three children participated as unvaccinated controls. The vaccine was well tolerated by both seronegative and seropositive children. The amount of virus required to infect 50% of seronegative children was approximately 10(4.5) TCID50. Vaccine viruses recovered from airway secretions retained temperature-sensitive and cold-adapted characteristics. The results of this study indicate that the vaccine virus, influenza B/Texas/84 ca reassortant virus, is attenuated, immunogenic, and phenotypically stable when given to young seronegative children.


Confirmatory polymerase chain reaction testing for Chlamydia trachomatis in first-void urine from asymptomatic and symptomatic men.

First-void urine specimens from 683 men (592 without symptoms) were tested for Chlamydia trachomatis by a polymerase chain reaction (PCR) with KL1 and KL2 plasmid primers and by a Chlamydiazyme enzyme immunoassay (EIA). Thirty-seven specimens were confirmed to be positive by using the EIA blocking reagent and a second set of plasmid primers (T1 and T2). By comparing unconfirmed PCR results (KL1 and KL2 primers only) with the blocked Chlamydiazyme EIA results, the sensitivity and specificity of PCR were 100% (37 of 37 specimens) and 99.5% (643 of 646 specimens), respectively. Three additional specimens were negative by EIA but positive by PCR and were confirmed to be positive with primers T1 and T2. Two of the three specimens were from men with symptoms. The confirmatory PCR assay performed equally well in detecting positive specimens from symptomatic (31 of 31) and asymptomatic (9 of 9) men. Comparison of confirmatory testing of first-void urine specimens by PCR and EIA showed that PCR was 100% sensitive (40 of 40 specimens) and that the EIA was 92.5% sensitive (37 of 40 specimens) but that the assays were equally specific (100%).


Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes.

We describe a novel flow cytometric method for quantifying opsonophagocytosis and killing of Staphylococcus aureus in cell-rich plasma obtained after dextran sedimentation of erythrocytes. To analyze opsonophagocytosis, phagocytes were labeled with a phycoerythrin-conjugated monoclonal antibody and were incubated with viable staphylococci containing carboxyfluorescein as a vital fluorescent dye. Phagocytosing cells assumed a dual, orange-green fluorescence. The relative numbers of bacteria associating with phagocytes could be determined by quantifying the decrease of free green fluorescent particles. A parallel incubation of fluorescent bacteria with unlabeled cell-rich plasma was performed to assess phagocytic killing. Blood cells were lysed with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate. This detergent spared viable bacteria, and residual green fluorescent particles were counted. The decrease in the number of these particles relative to the controls yielded the degree of killing. At bacteria-to-phagocyte ratios of 1:1 and 10:1, approximately 36 and 75% of the phagocytes participated in opsonophagocytosis, respectively. Over 90% of the staphylococci were phagocyte associated after 30 to 60 min. Killing rates were on the order of 66% +/- 12% and 80% +/- 7% after 1 and 2 h of incubation, respectively. These numbers, which were confirmed by colony countings, were significantly lower than those reported in the majority of past reports.


Cell-mediated and humoral immune responses induced by scarification vaccination of human volunteers with a new lot of the live vaccine strain of Francisella tularensis.

Tularemia is a disease caused by the facultative intracellular bacterium Francisella tularensis. We evaluated a new lot of live F. tularensis vaccine for its immunogenicity in human volunteers. Scarification vaccination induced humoral and cell-mediated immune responses. Indications of a positive immune response after vaccination included an increase in specific antibody levels, which were measured by enzyme-linked immunosorbent and immunoblot assays, and the ability of peripheral blood lymphocytes to respond to whole F. tularensis bacteria as recall antigens. Vaccination caused a significant rise (P less than 0.05) in immunoglobulin A (IgA), IgG, and IgM titers. Lymphocyte stimulation indices were significantly increased (P less than 0.01) in vaccinees 14 days after vaccination. These data verify that this new lot of live F. tularensis vaccine is immunogenic.


The plastic envelope method, a simplified technique for culture diagnosis of trichomoniasis.

Although culture of Trichomonas vaginalis is more sensitive than wet mounts in the diagnosis of trichomoniasis, the lack of convenience of culture prevents it from being widely used. To improve the acceptability of diagnosis by culture, a plastic envelope method (PEM) was devised. PEM permits both immediate examination and culture in one self-contained system. The medium consists of dry ingredients that are reconstituted with water before use. The effectiveness of immediate examinations by PEM was compared with that of wet mounts, and the effectiveness of culture by PEM was compared with that of culture in Trichomonas Medium No. 2 (Oxoid). Of 710 vaginal secretion specimens from symptomatic and asymptomatic women that were tested by the four methods, 62 (9%) were positive for T. vaginalis. The sensitivity was 66% by wet mount, 66% by immediate examination by PEM, 89% by cultures in Oxoid medium, and 97% by culture by PEM. The two culture methods had equivalent sensitivities but were significantly (P less than 0.0001) more sensitive than the two immediate methods. The combined immediate examination by PEM plus culture was more convenient to use than wet mounts plus culture in Oxoid medium. The long shelf-life of PEM's dry medium and its anticipated low cost are additional advantages.


Evaluation of an immunoenzymatic assay detecting specific anti-Toxocara immunoglobulin E for diagnosis and posttreatment follow-up of human toxocariasis.

In order to complete the immunodiagnosis of human toxocaral disease, an immunoenzymatic assay with excretory-secretory antigens from Toxocara canis larvae was developed for the detection of specific immunoglobulin E (sIgE enzyme-linked immunosorbent assay [ELISA]). The specificity of the assay was evaluated in patients presenting with various allergic or helminthic diseases. The sensitivity was assessed in patients exhibiting clinical and biological symptoms indicative of toxocariasis, serodiagnosis of which was made by the Western blot (WB; immunoblot) procedure that used the same antigen as that used in the sIgE ELISA but that detected specific IgG. The value of the sIgE ELISA for posttreatment follow-up was tested in two groups of patients: one group was treated with diethylcarbamazine; the other group was not treated with DEC. Results showed that the specificity and sensitivity of the sIgE ELISA were moderate. Thus, the sIgE ELISA appeared to be insufficient for properly ensuring the serodiagnosis of toxocariasis when it is used alone. However, sIgE ELISA might be an interesting complementary method for the detection of specific IgG. It was the only assay that was found to be positive in sera from some hypereosinophilic patients. sIgE ELISA values decreased significantly among the patients treated with DEC, indicating that this test would be useful for posttreatment follow-up assessment.


Development of oligonucleotide primers and probes against structural and regulatory genes of human immunodeficiency virus type 1 (HIV-1) and their use for amplification of HIV-1 provirus by using polymerase chain reaction.

The polymerase chain reaction is a powerful technique for amplifying a few copies of double-stranded genetic material to millions of copies in a few hours. The sensitivity and specificity of the polymerase chain reaction technique depend to some extent on the nucleotide sequences of the oligonucleotide primer pair used in the amplification. We report new oligonucleotide primers and probes which can be used for the amplification and detection of human immunodeficiency virus type 1 provirus sequences of not only structural but also regulatory genes. These primers are very sensitive and specific and can be used for the detection of African and North American strains of human immunodeficiency virus type 1.


Broth microdilution testing of Haemophilus influenzae with haemophilus test medium versus lysed horse blood broth. Canadian Haemophilus Study Group.

Broth microdilution testing of 702 community-acquired isolates of Haemophilus influenzae from across Canada was performed with both Mueller-Hinton broth supplemented with 3% lysed horse blood broth (LHB) (BBL Microbiology Systems, Cockeysville, Md.) and haemophilus test medium (HTM). The prevalence of beta-lactamase production was found to be 26% with no regional variation. MICs determined with LHB tended to be higher than those with HTM, but interpretive errors due to these differences were observed only rarely with trimethoprim-sulfamethoxazole (n = 5), cefaclor (n = 8), and cefamandole (n = 3). The interobserver variability in MIC determinations was found to be greater when LHB was used than when HTM was used. There was no difference in intraobserver variability between the two medium formulations. beta-Lactamase-positive isolates developed false resistance to amoxicillin-clavulanate 2 weeks after microdilution panels of both types of medium were stored at -20 degrees C but not when panels were stored at -70 degrees C. In conclusion, this study supports the use of HTM rather than LHB for sensitivity testing of H. influenzae because of its lower rate of interobserver variability and its ability to support the growth of these organisms, which is comparable to that of LHB.


Neisseria spp. and AIDS.

Neisseria meningitidis from various serogroups and two commensal neisseriae (N. sicca and N. perflava) were isolated from 15 patients at various stages of human immunodeficiency virus infection in this clinical and bacteriological study. The cases were grouped into the following three classes: (i) infections with an N. meningitidis strain of a serogroup known to be pathogenic (A, B, or C) and apparently independent of the human immunodeficiency virus infection, (ii) infections with a N. meningitidis strain of a serogroup which is normally either commensal or poorly pathogenic (serogroups Y, X, Z, and Z,29E), (iii) pulmonary and disseminated infections occurring in the course of the clinical evolutionary stage of AIDS, in two cases of which commensal neisseriae (N. sicca and N. perflava) were isolated from blood cultures.


Development and testing of a nonradioactive DNA oligonucleotide probe that is specific for Vibrio cholerae cholera toxin.

An alkaline phosphatase-labeled oligonucleotide DNA probe (CTAP) that was specific for the cholera toxin gene (ctxA) was identified. All cholera toxin-producing strains of Vibrio cholerae, regardless of serotype, hybridized with the CTAP probe, while nontoxigenic strains from either environmental sources or from deletion or substitution mutations did not hybridize. Unlike the whole-gene probes for either ctxA or for the heat-labile toxin or Escherichia coli (eltA), this 23-base sequence did not hybridize with E. coli or with vibrios other than V. cholerae that produce related toxins. By using CTAP to identify colonies grown on nonselective medium, V. cholerae was enumerated at concentrations of 10(3) to 10(7)/g from stool samples of volunteers who had ingested V. cholerae O1 strain 569B. CTAP provides a specific and sensitive tool for diagnosis and environmental monitoring of cholera toxin-producing V. cholerae.


Endocarditis caused by Blastoschizomyces capitatus and taxonomic review of the genus.

Blastoschizomyces capitatus Salkin, Gordon, Samsonoff et Rieder was found to be the etiologic agent of endocarditis in a patient with a prosthetic mitral valve. Cultures inoculated with peripheral blood and portions of the valve yielded B. capitatus. Examination of stained tissue sections revealed the presence of fungal filaments morphologically consistent with this organism. The salient characteristics of B. capitatus and the factors contributing to its recognition as a distinct taxon are described.


Serological detection of Helicobacter pylori by a flow microsphere immunofluorescence assay.

A flow cytometric immunofluorescence assay (FMIA) for the detection of immunoglobulin G antibodies to Helicobacter pylori was developed. A multicomponent antigen was prepared and used to coat carboxylated polystyrene microspheres for reaction with patient sera followed by fluorescein isothiocyanate-labelled goat anti-human immunoglobulin G. The reacted microspheres were collected with a flow cytometer, and fluorescence was quantitated relative to the cutoff value provided by pooled sera from patients in whom H. pylori could not be demonstrated by culture or histology. Serum samples from 28 H. pylori-positive patients and 27 H. pylori-negative patients were tested by FMIA. Additionally, an in-house enzyme-linked immunosorbent assay (ELISA) employing the same antigen preparation and a commercially available ELISA were used to assay the patient population. Both the FMIA and in-house ELISA were 100% sensitive and 89% specific with positive and negative predictive values of 90 and 100% and no equivocal results. The commercial ELISA was 96% sensitive and 89% specific with positive and negative predictive values of 90 and 96% and five equivocal results. FMIA provides a rapid, inexpensive, and easily performed means for serodiagnosis of H. pylori.


Enzymatically active Peptostreptococcus magnus: association with site of infection.

Fifty-four strains of Peptostreptococcus magnus (11 were recovered from abdominal infections, 18 were from nonpuerperal breast abscesses, and 21 were from diabetic foot infections; the type strain and three other strains were from the American Type Culture Collection, Rockville, Md.) and the type strain of Peptostreptococcus micros were tested for their ability to produce various enzymes, including catalase, hippurate hydrolase, serine dehydratase, threonine dehydratase, collagenase, gelatinase, alkaline phosphatase, and esterase C4. The data were analyzed by cluster analysis. The results showed that all but one strain could be assigned to either of two distinct, valid clusters. The first cluster of 11 strains was composed of strains that were relatively inactive, having produced one or two of the eight strain-dependent enzymes. The second was a large cluster of strains (n = 43) that were considerably more active, all having produced at least three enzymes; the vast majority of strains (89%) produced four or more enzymes. The unclustered strain produced one enzyme that was different from that produced by the strains in the first cluster. The chi 2 test of homogeneity applied to the clustering solution indicated that greater enzyme activity was significantly associated with the site of infection (P less than 0.001). The more enzymatically active P. magnus strains were recovered significantly more often from nonpuerperal breast abscesses and diabetic foot infections than they were from abdominal infections. These results may provide insight into the nature of certain polymicrobial soft tissue infections and suggest that (i) P. magnus may participate more in nonpuerperal breast and diabetic foot infections than in abdominal infections and that (ii) peptostreptococcal production of proteolytic enzymes may have an important adjunctive effect on the pathogenesis of certain soft tissue infections.


Outbreak of recurrent abdominal cramps associated with Arcobacter butzleri in an Italian school.

In the autumn of 1983, an outbreak of recurrent abdominal cramps occurred in a nursery and primary school in the Rovigo area in Italy. None of the 10 affected children had diarrhea. An atypical Campylobacter-like organism was isolated from feces in all cases. Conventional enteropathogens were searched for but not detected. The Campylobacter-like organism was identified as Arcobacter butzleri by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and cellular fatty acid analysis. Its identity was confirmed by DNA-DNA hybridizations versus Arcobacter reference strains. All of the preserved outbreak strains have identical protein profiles and phenotypic characteristics and belong to serogroup 1 of the Lior serotyping scheme on the basis of slide agglutination of crude and absorbed antisera of A. butzleri reference strains versus heat-labile antigens of live bacteria. These data point to an epidemiological relationship. The successive timing of the cases suggests person-to-person transmission.


Highly sensitive immunoassay for direct diagnosis of viral hemorrhagic septicemia which uses antinucleocapsid monoclonal antibodies.

An antigen capture enzyme-linked immunosorbent assay (ELISA) based on the detection of the viral nucleocapsid (anti-N system) was developed for the diagnosis of viral hemorrhagic septicemia. Four monoclonal antibodies directed against the viral nucleocapsid were produced; they all recognized the four viral hemorrhagic septicemia virus (VHSV) serotypes. Three of these monoclonal antibodies were used in a new antigen capture ELISA. The efficiency of the anti-N system in detecting purified and crude viruses as well as the virus in infected-organ extracts and infected blood was compared with that of a recently described antigen capture ELISA based on the detection of viral envelope glycoprotein Gp (anti-G system). For the detection of purified virus, the anti-N system was found to be as sensitive as the anti-G system (detection limit, 1 ng of total viral protein per ml), but the anti-N system was much more sensitive than the anti-G system for the detection of crude VHSV I (detection limits, 1 x 10(4) PFU/ml versus 5 x 10(5) PFU/ml). In organ extracts, VHSV I could be detected by both systems 3 days postinfection. The signal for the assay of VHSV I in blood 24 h postinfection was higher with the anti-N system than the anti-G system. Furthermore, VHSV I could be detected in 80% of the brain samples of surviving trout by the anti-N system and also by the anti-G system, but with a lower signal. In conclusion, we have developed a highly sensitive immunoassay for VHSV I that is more rapid and easier to perform than the currently used plaque assay.


Blood spot screening and confirmatory tests for syphilis antibody.

We developed a blood spot test for syphilis antibody using enzyme-linked immunosorbent assay (ELISA) technology. Dried blood was eluted by buffered saline or, for a supplementary confirmatory test, by treponemal-antibody test diluent. Eluates were diluted in an absorption buffer (Calypte Biomedical, Berkeley, Calif.) and added to plate wells coated with cardiolipin antigen (ADI Diagnostics, Toronto, Ontario, Canada). The wells were washed and treated sequentially with an immunoglobulin G conjugate, buffer washes, and enzyme substrate. Substrate conversion was measured photometrically, and specimen reactivity was determined by reference to nonreactive controls. The optimum test protocol was established by tests of serum and plasma. The serum ELISA specificity with normal specimens was 98.9%. The sensitivity with sera from patients with undefined syphilis was 97.4%, that with sera from patients with documented primary and secondary disease was 100%, and that with sera from patients with early and late latent disease was 95.7%. The specificity of the spot test with donor blood was 94.2%, and its specificity with newborn blood was 94.9%. The sensitivity with 25 spots spiked with reactive sera was 96%. The seroprevalence rates for parturient women in one hospital were 6.01% according to spot tests of sera from 599 newborns and 6.81% according to Rapid Plasma Reagin tests of 499 maternal serum specimens. Seventy percent of infants born to 50 seropositive women were reactive by either the newborn spot or the Rapid Plasma Reagin serum test. The results show that blood spots may be used in seroprevalence or serodiagnostic studies, especially to identify women who are infected or to identify possible cases of congenital infection. The test provides for studies of children and adults when routine venipuncture and serum handling and storage are problematic.


Early detection of antibody to human immunodeficiency virus type 1 by using an antigen conjugate immunoassay correlates with the presence of immunoglobulin M antibody.

Sequential plasma samples obtained from 16 individuals who seroconverted were tested for the presence of antibody to human immunodeficiency virus type 1 (HIV-1) by an antigen conjugate enzyme immunoassay (EIA) and a conventional antibody conjugate assay. In 11 of these individuals, the antigen conjugate assay detected antibody to HIV-1 2 to 11 days (mean, 5.5 days) earlier than the antibody conjugate assay. In 11 individuals, HIV-1 p24 antigen was detected a median of 6.5 days (range, 3 to 14 days) prior to positivity by the antigen conjugate EIA. Using class-specific probes, we determined the profiles of immunoglobulin M (IgM), IgG, and IgA antibodies for each individual and correlated these profiles with the EIA signals from both assays. In general, the appearance of IgM exhibited a peak at about 1 week postseroconversion, which was followed by gradually declining levels. Absorbance levels for IgG antibody, however, rose steadily and reached a plateau after 3 to 5 weeks. The levels of IgA were generally low and variable. In contrast to the progressive increase in EIA absorbance observed by the antibody conjugate assay, the antigen conjugate assay displayed a rapid early rise in absorbance which generally coincided with the transient expression of IgM antibody. The subsequent gradual increase coincided with rising levels of IgG. Because the configuration of the antigen conjugate EIA allows for an increased sensitivity for IgM compared with that for other classes of immunoglobulins, these results suggest that earlier detection of antibody to HIV-1 is due to the detection of IgM antibody during the early phase of seroconversion.


Antimicrobial susceptibility patterns of common and unusual species of enterococci causing infections in the United States. Enterococcal Study Group.

We collected 705 isolates of enterococci (1 per patient) from cultures of a variety of anatomic sites from patients at eight tertiary-care hospitals in six geographic regions of the United States. A total of 632 (90%) Enterococcus faecalis, 58 (8%) E. faecium, 5 E. gallinarum, 4 E. avium, 3 E. casseliflavus, 1 E. raffinosus, and 1 E. hirae isolate and 1 biochemical variant of E. faecalis were identified; 606 (86%) of these isolates were associated with clinical infections. The most common sites of isolation were the urinary tract (402 [57%]), nonsurgical wounds (94 [13%]), the bloodstream (74 [10%]), and surgical wounds (62 [9%]). High-level resistance to gentamicin or streptomycin or both was detected in 265 (38%) of the isolates. We identified two E. faecalis isolates resistant to vancomycin (MICs, 32 and 128 micrograms/ml) and 11 beta-lactamase-producing E. faecalis isolates. E. faecium isolates were significantly more resistant than E. faecalis isolates to penicillin, ampicillin, piperacillin, imipenem, and ciprofloxacin (P less than 0.001). The MICs for the 15 non-E. faecalis, non-E. faecium enterococci indicated variable resistance to ciprofloxacin and the penicillins. Antimicrobial susceptibility patterns vary among species of enterococci, and these organisms, while commonly resistant to high-level aminoglycosides, can also acquire resistance to vancomycin or the ability to produce beta-lactamase. Because of these diverse antimicrobial resistance mechanisms, successful treatment and control of enterococcal infections with current antimicrobial agents are becoming increasingly difficult.


Phenotypic variation of Staphylococcus epidermidis isolated from a patient with native valve endocarditis.

Two colonial variants of Staphylococcus epidermidis were isolated from the valvular tissue of a patient with native valve endocarditis. In addition to differing in colonial morphology, the two variants differed in hemolysis on blood-containing media, in adherence capacity, and in the expression of certain enzymes. Under suitable conditions, both variants were themselves capable of phenotypic variation, although they differed in the rate at which variants were generated. The variants yielded identical profiles on restriction endonuclease analysis of plasmid DNA and pulsed-field gel electrophoresis of whole-cell DNA. This report suggests a possible role for phenotypic variation in coagulase-negative staphylococcal virulence. Congo red agar would be an excellent medium for studying the contribution of variation to the virulence of these organisms.


M protein gene typing of Streptococcus pyogenes by nonradioactively labeled oligonucleotide probes.

A new approach for the typing of Streptococcus pyogenes is described. Oligonucleotide probes of 30 nucleotides in length were derived from currently known sequences of the N-terminal regions of M protein genes (emm genes). The oligonucleotides were labeled with digoxigenin-dUTP and hybridized to dot-blotted genomic DNA from 116 group A streptococcal strains of serotypes M-1, M-2, M-3, M-5, M-6, M-12, M-18, M-19, M-24, and M-49. Hybridization reactions were visualized with a chemiluminescent substrate. In comparison with conventional serological typing of expressed M proteins, the binding of the probes to the corresponding emm genes exhibited 100% sensitivity and specificity. The results emphasize the high degree of type-specific conservation of the N-terminal regions of emm genes from reference strains and epidemiologically unrelated U.S. and European clinical isolates. The existence of two distinct genetic subgroups among eight investigated M-49 strains was unequivocally shown by hybridization assays and further confirmed by nucleotide sequence data obtained from four selected M-49 strains. Because oligonucleotide probes are relatively easy to prepare, easy to handle, and known to give consistent interlaboratory results, the "oligotyping" technique appears to offer potential advantages over conventional serological typing methods.


Legionella lansingensis sp. nov. isolated from a patient with pneumonia and underlying chronic lymphocytic leukemia.

A Legionella-like organism, strain 1677-MI-H, was isolated from the bronchoscopy washings of a patient with pneumonia who had a 2-year history of progressive, chronic lymphocytic leukemia. The growth characteristics, cellular fatty acids, and ubiquinone content of the isolate were consistent with those for Legionella spp. The isolate was serologically distinct in the slide agglutination test with absorbed antisera. DNA hybridization studies showed that strain 1677-MI-H (ATCC 49751) represents a new Legionella species which is named Legionella lansingensis.


High-performance liquid chromatography patterns of Mycobacterium gordonae mycolic acids.

An important class of fatty acids contained in the cell envelopes of Mycobacterium organisms is the group of high-molecular-weight, long-chain, alpha-branched, beta-hydroxylated mycolic acids. By using standard saponification techniques and derivatization of the acids to their p-bromophenacyl esters, it is possible to differentiate them by high-performance liquid chromatography. Mycolic acid chromatograms of 63 clinical isolates of Mycobacterium gordonae were compared with conventional biochemical methods for identification. The data show two distinct pattern types for this species, only one of which has been elaborated in the literature by using this protocol. Laboratory workers who intend to use this method as a clinical tool need to be aware that these two pattern types exist.


Restriction endonuclease analysis and ribotyping differentiate genital and nongenital strains of Bacteroides ureolyticus.

Thirty-three clinical isolates from male nongonococcal urethritis and 28 isolates from soft tissue infections and ulcers were identified as Bacteroides ureolyticus by conventional bacteriological tests and were compared with five reference strains of the species. Whole-cell proteins from these clinical isolates and the reference strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The majority of the strains from the two sources could be divided into five different groups, named phenons I to V; phenons I to IV have been described previously by others, while phenon V has been described recently by us. Digestion of chromosomal DNA from 16 of the clinical isolates (including strains representative of each of the five SDS-PAGE phenons) and the five reference strains was attempted with restriction endonucleases EcoRI, PstI, SmaI, and HindIII. After electrophoresis in agarose gels, good digestion was observed with HindIII only, and 12 different banding patterns (restriction endonuclease analysis [REA] profiles) were obtained for the 19 strains digested; one nongonococcal urethritis isolate and one reference strain did not show any digestion. From the agarose gels, HindIII-digested fragments of DNA were transferred to nylon membranes by use of vacuum blotting and subjected to hybridization with 32P-labelled 16S-23S rRNA from Escherichia coli. The resultant pattern of bands (ribotypes), which depends on the restriction fragment length polymorphisms in the rRNA genes, was used as a measure of genomic variation within the species. In total, 13 different ribotypes were obtained for the 19 strains. For some strains, good correlation was achieved among the SDS-PAGE phenons, REA profiles, and ribotypes. However, for others, REA analysis and ribotyping were able to discriminate between strains which shared the same SDS-PAGE phenon. Interestingly, these two techniques of DNA characterization were able to differentiate between isolates from the genital tract and those associated with soft tissue infections and ulcers.


Detection of lipoarabinomannan as a diagnostic test for tuberculosis.

A coagglutination technique was established for the detection of lipoarabinomannan of Mycobacterium tuberculosis in human serum samples and evaluated for its utility in the diagnosis of tuberculosis at the Instituto Nacional de Enfermedades Respiratorias in Mexico City. The test had a sensitivity of 88% in patients with sputum-smear-positive active pulmonary tuberculosis. The sensitivity in patients with active pulmonary tuberculosis negative for acid-fast bacilli in sputum was 67%. Less favorable results were obtained for patients with AIDS and tuberculosis, with a sensitivity of 57%. The specificity in control patients with lung diseases different from tuberculosis and in healthy subjects was 100%. The positive predictive value was 100%, and the negative predictive value for patients with sputum-positive active pulmonary tuberculosis was 97%. The results of this study suggest that the detection of lipoarabinomannan is an accurate test for the diagnosis of pulmonary tuberculosis.


Antigenic and molecular characterization of bat rabies virus in Europe.

The predominant role of Eptesicus serotinus in the epizootic of bat rabies in Europe was further outlined by the first isolation of the rabies virus from this species in France. The distribution of the virus was studied in naturally infected E. serotinus bats at the time of death and suggested that the papillae of the tongue and the respiratory mucosa may play a role in virus production and excretion. The analysis of 501 French rabies virus isolates from various animal species by antinucleocapsid monoclonal antibodies indicated that transmission of the disease from bats to terrestrial animals is unlikely. The antigenic profile of two isolates from French bats corresponded to that of European bat lyssavirus type 1 (EBL1). Comparisons of 12 different isolates from bats with antinucleocapsid and antiglycoprotein monoclonal antibodies and by direct sequencing of the polymerase chain reaction amplification product of the N gene indicated that EBL1, EBL2, Duvenhage virus (serotype 4 of lyssavirus), and the European fox rabies virus (serotype 1) are phylogenetically distant. They formed four tight genetic clusters named genotypes. EBL1 was shown to be antigenically and genetically more closely related to Duvenhage virus than to EBL2. We propose that EBL1 and EBL2 constitute two distinct genotypes which further serologic characterization will probably classify as new serotypes. We also report a simple method for the rapid characterization of EBL based on the digestion of the polymerase chain reaction product of the N gene by three restriction endonucleases.


Identification of Mycobacterium tuberculosis and Mycobacterium avium-M. intracellulare directly from primary BACTEC cultures by using acridinium-ester-labeled DNA probes.

Identification of members of the Mycobacterium tuberculosis complex and the M. avium-M. intracellulare complex (MAC) directly from primary BACTEC cultures was evaluated by using acridinium-ester-labeled DNA probes (AccuProbe; GenProbe, Inc., San Diego, Calif.). In preliminary experiments, blood present in samples was found to interfere with the assay because of nonspecific chemiluminescence, which was measured in relative light units (RLUs). There was a direct relationship between the age of the culture and the number of nonspecific RLUs. A protocol using 1% sodium dodecyl sulfate-5 mM EDTA to treat BACTEC broth cultures which, with specimens containing blood, gave on the average a ninefold reduction in nonspecific chemiluminescence was developed. By using this treatment protocol, 120 specimens were tested directly from BACTEC broth cultures with an AccuProbe for the M. tuberculosis complex and/or the MAC. In order to establish the background of the specimen, the patient sample was assayed without probe. The criteria for the inclusion of BACTEC cultures in the evaluation were a growth index of greater than or equal to 100 and a positive smear for acid-fast bacilli directly from the BACTEC broth. For the 120 cultures tested, if a hybridization result of greater than or equal to 30,000 RLUs was considered positive, the sensitivities for detecting the M. tuberculosis complex and the MAC were 47 and 90%, respectively, with a specificity of 100% for both. However, if a ratio of the RLUs obtained with the MAC or the M. tuberculosis complex probe to those obtained with the specimen background of >/= 20 was considered positive, this gave 77% sensitivity and 100% specificity for BACTEC cultures containing M. tuberculosis complex isolates and 96% sensitivity and 100% specificity for those growing MAC isolates.


Biochemical and antigenic properties of Streptococcus bovis isolated from pigeons.

Biochemical and serological properties of 60 strains of Streptococcus bovis isolated from healthy pigeons and from pigeons that died from S. bovis septicemia were determined. On the basis of the hemolysis of bovine erythrocytes, the production of polysaccharides on saccharose-containing media, and the fermentation of mannitol, inulin, trehalose, and L-arabinose, the isolates were classified in five biotypes and two subbiotypes. Slide agglutination and microagglutination tests using monospecific rabbit antisera allowed the classification of all isolates in five serotypes.


Evidence that Lo's mycoplasma (Mycoplasma fermentans incognitus) is not a unique strain among Mycoplasma fermentans strains.

Mycoplasma fermentans incognitus has attracted much interest either as a cofactor for the progression of AIDS or as a pathogenic agent in non-AIDS-related diseases (S.-C. Lo, M. S. Dawson, P. B. Newton III, M. A. Sonoda, J. W.-K. Shih, W. F. Engler, R. Y.-H. Wang, and D. J. Wear, Am. J. Trop. Med. Hyg. 41:364-376, 1989; S.-C. Lo, M. S. Dawson, M. Wong, P. B. Newton III, M. A. Sonoda, W. F. Engler, R.Y.-H Wang, J. W.-K. Shih, H. J. Alter, and D. J. Wear, Am. J. Trop. Med. Hyg. 41:601-616, 1989; S.-C. Lo, J.W.-K. Shih, N.-Y. Yang, C.-Y. Ou, and R. Y.-H. Wang, Am. J. Trop. Med. Hyg. 40:213-226, 1989). In the present study, the genetic and serologic properties of the incognitus strain and other M. fermentans strains were compared. Furthermore, the replication of human immunodeficiency virus type 1 (HIV-1), determined by reverse transcriptase activity and HIV-1 p24 antigen level, in peripheral blood mononuclear cells was evaluated after stimulation with mycoplasma cell lysates. The psb-2.2 viruslike infectious agent DNA probe, used to identify the incognitus strain in the tissues of AIDS and non-AIDS patients by Lo et al. (Am. J. Trop. Med. Hyg. 41:364-376 and 40:213-226, 1989), showed reaction patterns similar to those of two M. fermentans strains isolated from cell cultures but not to that of the type strain PG18. Restriction enzyme patterns of the incognitus strain with EcoRI and HindIII were also similar to those of M. fermentans strains isolated from cell cultures. There were no remarkable differences in the immunoblot profiles between the incognitus strain and the other M. fermentans strains. These results suggest that the incognitus strain is not a unique strain among M. fermentans strains. Further, cell lysates of each M. fermentans strain could also enhance the replication of HIV-1 to a level similar to that of the incognitus strain as determined by the reverse transcriptase activity and the amount of the p24 antigen.