Confirmatory identification of Neisseria gonorrhoeae by slide coagglutination

Neisseria gonorrhoeae was identified by the Phadebact gonococcus test, a rapid slide coagglutination technique, and the results obtained were compared with those obtained by conventional methods (Gram stain morphology, oxidase reaction, and carbohydrate utilization tests) for the confirmatory identification of gonococci. Of 308 clinical isolates examined, the coagglutination procedure correctly identified 97.8% of the isolates tested as N. gonorrhoeae and 93.9% of other bacteria as not N. gonorrhoeae. The coagglutination procedure also identified 29 laboratory strains correctly as not N. gonorrhoeae. The slide coagglutination test is easy to perform and offers a valuable alternative to other techniques for the confirmatory identification of N. gonorrhoeae.     

The enrichment and early detection of Streptococcus pyogenes in skin lesions, using colistin, oxolinic acid, Todd Hewitt broth and latex agglutination

An improved selective broth, colistin, oxolinic acid, Todd Hewitt broth (COTHB) for the isolation of Streptococcus pyogenes and other streptococci from swabs is described. Staphylococci, coryneforms and Gram negative organisms are inhibited in COTHB inoculated with swabs from skin lesions. Streptococcus pyogenes could be detected by a fluorescent antibody method and by the Streptex and Phadebact streptococcal grouping methods after 6 and 24 h incubation. Other streptococci of groups B, C and G, were also detected. Streptex was the most sensitive and specific method for the detection of streptococci of groups A, B, C and G in this broth. The method detected 78% of these streptococci in COTHB after 6 h incubation and 93% after 24 h incubation. Of the Strep. pyogenes isolated, 82% were detected in the broth by the Streptex method.     

The enrichment and early detection of Streptococcus pyogenes in skin lesions, using colistin, oxolinic acid, Todd Hewitt broth and latex agglutination

An improved selective broth, colistin, oxolinic acid, Todd Hewitt broth (COTHB) for the isolation of Streptococcus pyogenes and other streptococci from swabs is described. Staphylococci, coryneforms and Gram negative organisms are inhibited in COTHB inoculated with swabs from skin lesions. Streptococcus pyogenes could be detected by a fluorescent antibody method and by the Streptex and Phadebact streptococcal grouping methods after 6 and 24 h incubation. Other streptococci of groups B, C and G, were also detected. Streptex was the most sensitive and specific method for the detection of streptococci of groups A, B, C, and G in this broth. The method detected 78% of these streptococci in COTHB after 6 h incubation and 93% after 24 h incubation. Of the Strep. pyogenes isolated, 82% were detected in the broth by the Streptex method.     

Rapid identification of pathogenic neisserias using the Identicult-Neisseria test

A rapid enzymatic method using chromogenic substrates for the rapid identification of pathogenic neisseria (Identicult-Neisseria, Scott Laboratories Inc., CA, USA) was tested in parallel with the rapid carbohydrate utilization test (RCUT) and the Phadebact Monoclonal GC Test against 198 consecutive clinical isolates of oxidase-positive Gram-negative diplococci (118 Neisseria gonorrhoeae , 76 N. meningitidis and four N. lactamica ). On initial testing the Identicult-Neisseria gave a 95% overall concordance (97.5% N. gonorrhoeae , 90.8% N. meningitidis ). with the RCUT and Phadebact tests; the corresponding figures after repeat testing were 98% overall concordance (98.3% N. gonorrhoeae , 97.4% N. meningitidis ). Two of the three strains of N. gonorrhoeae mis-identified as N. meningitidis on primary testing were also mis-identified on repeat testing. Seven strains of N. meningitidis were mis-identified on initial testing (six as Moraxella catarrhalis and one as N. lactamica ) and two on repeat testing (both as Mor. catarrhalis ). We conclude that the Identicult-Neisseria is not sufficiently reliable for the culture confirmation of gonococci and meningococci.     

Determination of O and Vi antigens in typhoid fever in the slide coagglutination reaction

Investigations with a view to the development and trial of a slide coagglutination test system for the detection of specific Salmonella typhi antigens have been made. As a result, diagnostic agents with sensitivity to group D Salmonella lipopolysaccharides and Vi-antigen, equal to 1 X 10(-5) to 1 X 10(-6) g/l, have been obtained. Specimens of saliva, urine and fecal filtrates from 61 adult patients with bacteriologically confirmed typhoid fever and 54 practically healthy persons have been studied. The coagglutination test has been positive with specimens from 90 +/- 4% of typhoid fever patients and, within the first 5 days of the disease, with those from 85 +/- 7% of such patients. The slide coagglutination test with saliva specimens has been found to be more informative than that with urine specimens. The data obtained in these investigations indicate that the slide coagglutination test is highly sensitive and specific, which offers good prospects for its use as a simple, economic and demonstrative method for the early tentative diagnosis of typhoid fever.     

Rapid serological identification of Vibrio vulnificus by anti-H coagglutination

Staphylococcus aureus Cowan 1 cells were armed with anti-flagellar (anti-H) antibody produced in rabbits immunized with flagellar core protein prepared from Vibrio vulnificus. This reagent was assessed by coagglutination for its capacity to agglutinate and identify V. vulnificus. A species-specific H antigen is expressed in the core proteins of the polar flagella of V. vulnificus. Of 435 V. vulnificus isolates identified bacteriologically, 432 (99.3%) were agglutinated in the slide test within 2 min after the addition of the anti-V. vulnificus H coagglutination reagent. Other than Vibrio pelagius, the reagent did not agglutinate 19 heterologous Vibrio spp. tested, including 290 V. cholerae, 22 V. mimicus, 395 V. parahaemolyticus, and 16 V. fluvialis isolates recovered from seafood and the marine environment. The serological resolution of the coagglutination reaction was enhanced if the organism under test was suspended in 0.1 M Tris buffer-0.1 mM EDTA-1.0% Triton X-100 (TET) for 24 h before serological examination. The TET buffer also increased the sensitivity of the coagglutination reaction 100-fold over that for isolates suspended in 0.3% formalinized phosphate-buffered saline before testing. The anti-H coagglutination test is a rapid, serologically specific, and inexpensive procedure for identifying V. vulnificus one step beyond primary isolation.     

Rapid serological identification of Vibrio vulnificus by anti-H coagglutination.

Staphylococcus aureus Cowan 1 cells were armed with anti-flagellar (anti-H) antibody produced in rabbits immunized with flagellar core protein prepared from Vibrio vulnificus. This reagent was assessed by coagglutination for its capacity to agglutinate and identify V. vulnificus. A species-specific H antigen is expressed in the core proteins of the polar flagella of V. vulnificus. Of 435 V. vulnificus isolates identified bacteriologically, 432 (99.3%) were agglutinated in the slide test within 2 min after the addition of the anti-V. vulnificus H coagglutination reagent. Other than Vibrio pelagius, the reagent did not agglutinate 19 heterologous Vibrio spp. tested, including 290 V. cholerae, 22 V. mimicus, 395 V. parahaemolyticus, and 16 V. fluvialis isolates recovered from seafood and the marine environment. The serological resolution of the coagglutination reaction was enhanced if the organism under test was suspended in 0.1 M Tris buffer-0.1 mM EDTA-1.0% Triton X-100 (TET) for 24 h before serological examination. The TET buffer also increased the sensitivity of the coagglutination reaction 100-fold over that for isolates suspended in 0.3% formalinized phosphate-buffered saline before testing. The anti-H coagglutination test is a rapid, serologically specific, and inexpensive procedure for identifying V. vulnificus one step beyond primary isolation.     

Secondary selective enrichment of salmonellae from naturally contaminated specimens by using a selective motility system.

A selective motility medium was used as a secondary selective enrichment medium to examine specimens naturally contaminated with salmonellae. The medium, incubated at 37 degrees C, was inoculated from either selenite brilliant green sulfa enrichment broth or Müller-Kauffman tetrathionate broth, both of which had been incubated at 42 degrees C. The use of the selective motility medium resulted in an increase in the number of positive specimens from 65 and 74% to 80 and 82%, when inoculated at 24 and 48 h, respectively, from tetrathionate broth. Tetrathionate broth, when used singly, was significantly better than selenite brilliant green sulfa broth, which detected 55% of positive specimens at both 24 and 48 h. The use of the selective motility medium of Harper and Shortridge (J. Hyg. 67: 181--186, 1969) for the further examination of specimens culturally negative on primary selective enrichment is advocated.     

Secondary selective enrichment of salmonellae from naturally contaminated specimens by using a selective motility system.

A selective motility medium was used as a secondary selective enrichment medium to examine specimens naturally contaminated with salmonellae. The medium, incubated at 37 degrees C, was inoculated from either selenite brilliant green sulfa enrichment broth or Müller-Kauffman tetrathionate broth, both of which had been incubated at 42 degrees C. The use of the selective motility medium resulted in an increase in the number of positive specimens from 65 and 74% to 80 and 82%, when inoculated at 24 and 48 h, respectively, from tetrathionate broth. Tetrathionate broth, when used singly, was significantly better than selenite brilliant green sulfa broth, which detected 55% of positive specimens at both 24 and 48 h. The use of the selective motility medium of Harper and Shortridge (J. Hyg. 67: 181--186, 1969) for the further examination of specimens culturally negative on primary selective enrichment is advocated.     

Improved Isolation of Salmonellae from Naturally Contaminated Meat Products by Using Rappaport-Vassiliadis Enrichment Broth

A total of 454 specimens of meat products were examined for salmonellae by using five procedures of enrichment. The use of a selective motility medium, inoculated from enrichment in Muller-Kauffmann broth, resulted in an increase in the number of positive specimens. However, simple enrichment in Rappaport-Vassiliadis broth, after preenrichment, was more sensitive and specific for recovering salmonellae than the selective motility medium-Muller-Kauffmann broth method.     

A rapid coagglutination test for the detection and serogrouping of Legionellae

The applicability of coagglutination for the rapid detection and serogrouping of Legionellae has been investigated. The coagglutination reaction is carried out with the aid of self-made preparations of protein A containing staphylococci, sensitized with specific antibodies against the antigens of L. pneumophila (serogroups I to 6), L. bozemanii and L. micdadei. Preliminary heating of Legionella suspensions at 100% C for 15 min was used to prevent cross coagglutination reactions and ensure greater safety of laboratory personnel during the performance of the test. The results obtained demonstrate a high specificity of coagglutination. With the aid of the coagglutination reactions it has been shown that L. pneumophila strains isolated in Bulgaria belong to serogroup I. The coagglutination method is characterized by its rapidity, simplicity and feasibility. It is a useful and convenient means for the rapid detection and serogrouping of Legionellae.     

A rapid method of grouping beta-hemolytic streptococci using extemporaneous coagglutination

Extemporaneous coagglutination procedure for the serological grouping of beta-hemolytic streptococci is reported. Streptococcal group antigens were extracted with nitrous acid. 250 strains of groups A, B, C, F and G streptococci were tested with this method. An agreement of 100% was found between this method and the Lancefield capillary precipitation procedure. Extemporaneous coagglutination method was found to be rapid, reliable, easy and economical and could be adopted in any routine diagnostic laboratory.     

Selected enrichment broths for recovery of Campylobacter jejuni from foods.

We attempted to shorten the required time for enrichment broth culture for the isolation of Campylobacter jejuni. Enrichment broths described by Doyle and Roman and Park and Stankiewicz and one developed during this study were compared for ability to isolate C. jejuni from raw chicken carcasses. Our medium was a modification of that of Doyle and Roman with the addition of filter-sterilized FBP (0.2% ferrous sulfate, 0.025% sodium metabisulfite, 0.05% sodium pyruvate), 0.1% sodium lauryl sulfate, and 0.075% agar. Initially, laboratory strains were employed in the development of this medium. Subsequently, an indigenous load of C. jejuni obtained from chickens was used to compare media. Isolation rate comparisons were as follows: direct plating, 40%; Doyle and Roman broth, 45% at 7 h and 61% at 16 h; Park and Stankiewicz broth, 53% at 7 h and 60% at 16 h; our broth, 48% at 7 h and 50% at 16 h. In addition to having the highest isolation rate, the enrichment broth of Doyle and Roman showed greatest selectivity. Our inoculation method of indigenous bacteria provided a controlled means for comparison of isolation procedures.     

Cost-effectiveness of blood agar for isolation of mycobacteria

Background

Mycobacterium species are grown using specific media that increase laboratory cost, thus hampering their diffusion in resource-limited countries. Preliminary data suggested that versatile blood agar may be also used for mycobacterial culture.

Methodology

We examined the growth of 41 different Mycobacterium species on 5% blood agar. Over a 24-month period we analysed isolation of mycobacteria after parallel inoculation of clinical specimens into both a reference automated system (BACTEC 9000 MB broth) and 5% blood agar slant tubes, after NaOH decontamination, and compared the cost of performing 1,000 analyses using these two techniques.

Conclusions

Mycobacterium reference species cultured on blood agar, with the exception of Mycobacterium ulcerans. Inoculation of 1,634 specimens yielded 95 Mycobacterium isolates. Blood agar performed significantly more efficiently than BACTEC 9000 MB broth (94 vs 88 isolates, P = 0.03). Decontamination of Candida albicans in 5 specimens by addition of amphotericin B in blood agar yielded one more M. tuberculosis isolate that could not be isolated in BACTEC broth. Uneven distribution of time to culture positivity for M. tuberculosis had a median (range) of 19±5 days using blood agar and 26±6 days using BACTEC 9000 MB broth. Cost for 1,000 analyses in France was estimated to be of 1,913 euros using the blood agar method and 8,990 euros using the BACTEC 9000 MB method. Blood agar should be regarded as a first-line medium for culturing Mycobacterium species. It saves time, is cost-effective, is more sensitive than, and at least as rapid as the automated method. This is of particular importance for resource-limited countries in which the prevalence of tuberculosis is high.     

Validation of Morphometric Analyses of Small-Intestinal Biopsy Readouts in Celiac Disease

Background

Assessment of the gluten-induced small-intestinal mucosal injury remains the cornerstone of celiac disease diagnosis. Usually the injury is evaluated using grouped classifications (e.g. Marsh groups), but this is often too imprecise and ignores minor but significant changes in the mucosa. Consequently, there is a need for validated continuous variables in everyday practice and in academic and pharmacological research.

Methods

We studied the performance of our standard operating procedure (SOP) on 93 selected biopsy specimens from adult celiac disease patients and non-celiac disease controls. The specimens, which comprised different grades of gluten-induced mucosal injury, were evaluated by morphometric measurements. Specimens with tangential cutting resulting from poorly oriented biopsies were included. Two accredited evaluators performed the measurements in blinded fashion. The intraobserver and interobserver variations for villus height and crypt depth ratio (VH:CrD) and densities of intraepithelial lymphocytes (IELs) were analyzed by the Bland-Altman method and intraclass correlation.

Results

Unevaluable biopsies according to our SOP were correctly identified. The intraobserver analysis of VH:CrD showed a mean difference of 0.087 with limits of agreement from −0.398 to 0.224; the standard deviation (SD) was 0.159. The mean difference in interobserver analysis was 0.070, limits of agreement −0.516 to 0.375, and SD 0.227. The intraclass correlation coefficient in intraobserver variation was 0.983 and that in interobserver variation 0.978. CD3⁺ IEL density countings in the paraffin-embedded and frozen biopsies showed SDs of 17.1% and 16.5%; the intraclass correlation coefficients were 0.961 and 0.956, respectively.

Conclusions

Using our SOP, quantitative, reliable and reproducible morphometric results can be obtained on duodenal biopsy specimens with different grades of gluten-induced injury. Clinically significant changes were defined according to the error margins (2SD) of the analyses in VH:CrD as 0.4 and in CD3⁺-stained IELs as 30%.     

Influence of an extended incubation period on values of minimum inhibitory concentrations of antibiotics in Stenotrophomonas maltophilia clinical strains

In 106 Stenotrophomonas maltophilia clinical strains the susceptibility to 19 kinds of antibiotics was tested by the broth dilution micromethod at 24 h and 48 h incubation. Isolated strains demonstrated the lowest frequency of resistance to cotrimoxazole (7.5% of resistant strains at 24 h incubation and 18.9% at 48 h), ofloxacin (13.2% and 30.2%), ciprofloxacin (19.8% and 50.9%) and to cefoperazone/sulbactam (20.8% and 37.7%). The smallest growth of the number of resistant strains after extended incubation was recorded in gentamicin (by 10.4%), ceftazidime (by 11.3%) and cotrimoxazole (by 11.4%). On the contrary, the largest growth of resistance was demonstrated in cefoperazone and ciprofloxacin (by 31.1%). Average values of the growth of minimum inhibitory concentrations (MICs) were lowest in ciprofloxacin and ofloxacin (2.3 times) and highest in piperacillin/tazobactam (4.5 times) and piperacillin (5.0 times). As far as the stability of MIC is concerned, the largest occurrence of strains with the MIC growth doubled as a maximum was found in ceftazidime (78.4%), ofloxacin (76.1%) and ciprofloxacin (75.3%), the smallest in piperacillin/tazobactam (43.2%) and piperacillin (38.9%). The importance of incubation extended to 48 h during the testing of S. maltophilia strains was noted for correctly setting their susceptibility to antibiotics.     

Comparison of the BACTEC System with Blind Subculture for the Detection of Bacteremia

One thousand blood specimens were cultured in BACTEC vials containing modified Columbia broth in aerobic, anaerobic, and hypertonic formulations. Radiometric readings and subcultures were performed on aerobic and hypertonic vials at 24 h and 7 days, and on anaerobic vials at 48 h and 7 days. Significant numbers of false-positive BACTEC readings were obtained. Although all positive cultures were eventually detected by the BACTEC, approximately 20% of blood specimens yielding positive subcultures at 24 h did not give positive BACTEC readings until 48 h.     

Delayed secondary enrichment for the isolation of salmonellae from broiler chickens and their environment.

Specimens collected from six broiler flocks were cultured for salmonellae by three methods. (i) For direct enrichment, the specimen was homogenized, and 1 ml of the homogenate was inoculated into tetrathionate-brillant green broth; (ii) for preenrichment, liquid specimens and homogenates were incubated at 37 degrees C, and on the next day 1 ml was inoculated into tetrathionate-brillant green broth; and (iii) for delayed secondary enrichment, incubated preenrichment cultures were held at room temperature for 7 to 10 days and then subcultured to fresh tetrathionate-brilliant green broth. All tetrathionate-brilliant green broth cultures were incubated at 42 degrees C for 24 to 48 h before plating. Significantly more isolations of salmonellae were obtained by delayed secondary enrichment than by direct enrichment or preenrichment. Salmonellae were isolated from 417 of 2,283 (18.3%) samples of litter, intestinal contents, and feces cultured by all three methods. Of these positive specimens, direct enrichment detected 208 (49.9%), preenrichment detected 282 (67.6%), and delayed secondary enrichment detected 373 (89.4%). Of 896 specimens of swabs and rinse fluids that were cultured by preenrichment and delayed secondary enrichment, 259 (28.9%) yielded salmonellae. Delayed secondary enrichment detected 254 (98.1%) of these, and preenrichment detected 147 (56.8%). A total of 23 serotypes of salmonellae were identified. The greater effectiveness of delayed secondary enrichment for the isolation of salmonellae was not likely due to the selection of certain serotypes or to an increased inhibition of competing flora.     

贝莱斯芽孢杆菌TMQ-KSL-1分离鉴定及其发酵液对番茄根结线虫的生防作用

镰刀菌(Fusarium spp.)和根结线虫(Meloidogyne spp.)都是植物的重要病原物,这两种病原物在寄主植物中存在着非常复杂的互作关系,可导致严重的植物土传病害。为探寻对番茄根结线虫病害具有高效防治作用的优良菌株,本研究以禾谷镰刀菌(Fusarium graminearum)为靶标病菌,采用平板稀释涂布法从多年种植番茄的设施大棚土壤中分离和筛选到一株抑菌效果较好的生防细菌菌株TMQ-KSL-1,根据形态特征、生理生化特性和16S rRNA基因测序对该菌株进行鉴定;测定不同浓度的发酵液及发酵上清液对根结线虫卵孵化率以及根结线虫二龄幼虫死亡率的影响,通过盆栽实验分析其发酵液对根结线虫病害的防治效果。结果表明,菌株TMQ-KSL-1具有较强的杀线虫活性,其发酵液和发酵上清液处理48 h线虫卵孵化抑制率分别为94.76%和90.72%;处理24 h番茄根结线虫二龄幼虫的校正死亡率分别为100%和97.37%;菌株TMQ-KSL-1发酵液100倍稀释液、200倍稀释液对番茄根结线虫病害防治效果分别为59.54%和12.14%,且100倍液处理防效与阿维菌素500倍液处理防效(6...     

Evaluation of the PathoTec ?Rapid I-D System” and Two Additional Experimental Reagent-Impregnated Paper Strips

The PathoTec "Rapid I-D System" and two experimental test strips for ornithine decarboxylase and beta-galactosidase have been evaluated for accuracy and ability to identify 1,252 members of the Enterobacteriaceae obtained from fresh clinical specimens. Accuracy of identification with the commercially available test system was 94.7%; this level increased to 98.5% with the addition of the two experimental test strips. Average individual accuracy of the 12-strip test system on a side-by-side basis with similar conventional procedures was 98%. In addition, 103 gram-negative nonfermentors were accurately grouped. The PathoTec System was applicable to 95% of the primary isolation plates used and provided biochemical data within 4 h after inoculation. The conventional test procedures were applicable to 100% of the primary isolation plates used and produced data within 48 h.