Direct sequence evaluation of the major outer membrane protein gene variant regions of Chlamydia trachomatis subtypes D'', I'', and L2''.

The nucleotide sequences of variable segments (VS) 1, 2, and 4 for the major outer membrane protein gene (omp1) of Chlamydia trachomatis were determined for serologically defined subtypes D', I', and L2'. Asymmetric DNA amplification was used to produce single-stranded DNA for direct sequencing. Amino acid substitutions were detected in VS1, VS2, and VS4 for I', in VS2 for L2', and in VS4 for D'. DNA sequencing of omp1 variant regions may be an important method for evaluating the molecular epidemiology of Chlamydia spp.     

Campylobacter butzleri sp. nov. isolated from humans and animals with diarrheal illness.

Seventy-eight aerotolerant Campylobacter isolates were characterized phenotypically and by DNA hybridization (hydroxyapatite method at 50 and 65 degrees C). Two DNA relatedness groups were found. (i) Sixty-four strains belonged to aerotolerant Campylobacter DNA hybridization group 2. These organisms were isolated from humans, primarily with diarrheal illness, and animals on several continents. Strains were aerotolerant at 30 and 36 degrees C and catalase negative or weakly catalase positive, grew in media containing glycine and on MacConkey agar, were susceptible to nalidixic acid, and were resistant to cephalothin. The name Campylobacter butzleri sp. nov. is proposed for this group. (ii) DNA hybridization group 1 consisted of the type strain of Campylobacter cryaerophila and 13 additional strains isolated from 10 animals outside the United States and from three humans within the United States. This group was genetically diverse; five strains were closely related to the type strain of C. cryaerophila (DNA hybridization group 1A), and eight strains were more closely related to one another (DNA hybridization group 1B). Strains in DNA hybridization group 1B were phenotypically diverse, with two of eight strains resembling C. cryaerophila. The seven strains from DNA hybridization groups 1A and 1B which resembled C. cryaerophila and the C. cryaerophila type strain were aerotolerant only at 30 degrees C and catalase positive, did not grow in glycine or on MacConkey agar, were generally susceptible to nalidixic acid, and were resistant to cephalothin. The remaining six strains of DNA hybridization group 1B phenotypically resembled C. butzleri; however, they were generally catalase positive and susceptible to nalidixic acid and cephalothin. DNA hybridization group 1B is not designated as a separate species at this time since it cannot, with certainty, be separated genetically from C. cryaerophila or phenotypically from C. butzleri.     

Analysis of HL and O serotypes of Campylobacter strains by the flagellin gene typing system.

We recently developed a molecular typing system for Campylobacter jejuni and Campylobacter coli based on restriction fragment length polymorphism analysis of the flagellin gene,flaA (I.Nachamkin, K. Bohachick, and C.M. Patton, J. Clin. Microbiol. 31:1531-1536, 1993). We extended the typing system to 83 flagellin types (designated flaA-1,flaA-2, etc.) on the basis of analysis of 404 isolates of C. jejuni and C. coli including common serotypes isolated in the United States, a selection of less common serotypes, and serotype reference strains. Of the 295 strains previously shown to belong to common HL and O serotypes (C. M. Patton, M.A. Nicholson, S.M. Ostroff, A.A. Ries, I.K. Wachsmuth, and R.V. Tauxe, J. Clin. Microbiol. 31:1525-1530, 1993), six flaA types accounted for 53.6% of strains as follows: flaA-1, 21.7%; flaA-7, 14.9%; flaA-27, 5.1%; flaA-49, 4.4%; flaA-13, 3.7%; and flaA-21, 3.7%. Seventy-five percent of the strains were within 15 flaA types, 90% were within 30 flaA types, and all 295 strains were contained within 52 flaA types. Within each HL or O serotype, there usually were multiple flaA types. For 12 common HL serotypes and 7 common O serotypes, more than 50% of these isolates were a single flaA type. A database was developed by using commercially available restriction fragment length polymorphism analysis software (ProRFLP; DNA ProScan, Inc., Nashville, Tenn.) that should allow other investigators to perform typing with this system.     

Comparison of PCR-ELISA and galactomannan detection for the diagnosis of invasive aspergillosis

AIM: To compare PCR with galactomannan antigen detection for the diagnosis of invasive aspergillosis (IA). METHODS: We prospectively collected serial blood samples from haematological patients at risk of IA, and analysed their samples retrospectively for galactomannan (GM) antigen using the Platelia test and for aspergillus DNA using an in-house PCR-ELISA assay. Matched GM and PCR analyses were performed on 263 samples from 25 patients. Patients were classified for potential IA according to international consensus criteria, with five patients classified as positive (four proven, one probable) and 20 classified as negative (seven possible, 13 no evidence IA). RESULTS: All five patients with IA were positive by PCR with positive results in 24 of 82 samples, whereas three of five patients were positive by GM with four of 82 samples being positive. Three of 20 patients without IA were positive by PCR in 18 of 181 samples, whereas corresponding results for GM detection were one of 20 and one of 181, respectively. Adjustment of ELISA cut-off values and/or the requirement for two consecutive samples to be positive generated different results; however, lowering the positivity index (PI) for GM detection to 0.5 did not improve the sensitivity of the assay. Optimal results for PCR detection and GM were: 100% and 60% sensitivity, 85% and 95% specificity, 0.625 and 0.75 positive predictive value, and 1.0 and 0.8 negative predictive value, with a false-positive sample rate of 8 and 0.4%, positive likelihood ratio of 6.66 and 11.99 and negative likelihood ratio of 0 and 0.42, respectively. CONCLUSIONS: This PCR method is very sensitive for the diagnosis of IA but is associated with a moderate rate of false positives; the GM assay exhibited poor sensitivity but high specificity. Further evaluation of PCR assays for the diagnosis of IA and other invasive fungal infections is warranted.     

Comparison of selective media for primary isolation of Campylobacter fetus subsp. jejuni.

Three selective media, Skirrow, Butzler, and a modification of Butzler medium, were compared for the primary isolation of Campylobacter fetus subsp. jejuni. This organism was isolated from 87 of 347 specimens (72 from 240 dogs rectal swabs and 15 from 107 cats rectal swabs). The positive rate for dogs (30%) was twice as high as that for cats (14%). Skirrow and Butzler media were comparable in their isolation of C. fetus subsp. jejuni. A significantly higher rate of positive results was obtained with modified Butzler medium. The best combination of two media was that of modified Butzler and Skirrow media, which detected 98% of the isolates obtained. The percentage of Campylobacter-positive specimens was increased by 9% by holding primary isolation plates 72 h.     

Hydrosalpinges adversely affect markers of endometrial receptivity

While in-vitro fertilization (IVF) was initially developed in women with tubal factor infertility, recent clinical studies have suggested that the presence of hydrosalpinges lowers implantation and pregnancy rates. We postulated that these hydrosalpinges cause impaired endometrial receptivity. A total of 103 women with hydrosalpinges were prospectively evaluated, and compared with 55 infertile and 44 fertile controls. All women had endometrial biopsies during the window of implantation, analysed by conventional histological criteria, and also stained for three integrin markers of endometrial receptivity (alpha1beta1, alpha4beta1 and alpha vbeta3). Women with hydrosalpinges (cases) expressed significantly less of the alpha vbeta3 integrin compared with controls. There was no difference in expression of alpha1beta1 or alpha4beta1 among groups. A significantly greater number of cases had out of phase histology and missing alpha vbeta3 (type I defects) and absent integrin expression despite normal histological maturation (type II) defects, compared with controls. Of 20 women with impaired endometrial receptivity who were also biopsied after hydrosalpinx surgery, 70% demonstrated increased alpha vbeta3 expression. Seventy-seven percent of type I and 57% of type II defects were corrected postoperatively. Using markers of endometrial receptivity, this study demonstrates that inflammatory hydrosalpinges have an adverse effect on endometrial receptivity, which in some cases may be overcome by surgical treatment of the hydrosalpinx.      

Comparative baseline levels of mercury, Hsp 70 and Hsp 60 in subsistence fish from the Yukon-Kuskokwim delta region of Alaska

In subsistence fish; northern pike (Esox lucius), burbot (Lota lota), whitefish (Coregonus nelsoni), grayling (Thymallus arcticus) and sheefish (Stenodus lencichthys), we determined the Hsp 60 and Hsp 70 levels in 31 samples from adult fish gills. A dot-blot analysis using antibodies to either Hsp 70 or Hsp 60 showed the average Hsp 70 concentration was 9.1 microg/mg protein, while the average Hsp 60 concentration was 147.4 microg/mg protein. Mercury levels in muscle tissue in these fish averaged 0.382 ppm. Using a subset of samples (n = 24), we determined that the major component in the muscle of Alaskan subsistence fish was methyl mercury. No correlation was observed between Hsp 60 or Hsp 70 expression in gill tissue and mercury concentrations in muscle tissue. Hsp 60 and Hsp 70 protein levels in the gills were correlated.