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ABSTRACTEpidemiologic research requires identification of Chlamydia trachomatisserovars and detection of mixed infection. Antibody-based serotyping is unworkable when specimens are urine or vaginal swabs. We developed a reverse dot blot (RDB) to screen for multiple serotypes in these specimens. RDB yielded the predicted results on all artificially mixed samples and on seven of eight clinically mixed samples.

Significant evidence suggests that Chlamydia pneumoniaehas a major role in occlusive vascular disease. Vascular access thrombosis in chronic hemodialysis patients is a frequent problem; the underlying pathological state is stenosis caused by endothelial hyperplasia. There is presently no literature concerning C pneumoniaein vascular access thrombosis. We embarked on a study to evaluate the possible role of C pneumoniaein access failure. Ten consecutive patients with thrombosed polytetrafluoroethylene (PTFE) conduit arteriovenous fistulae undergoing surgical thrombectomy and revision were studied. We sought to detect C pneumoniaeusing both culture and polymerase chain reaction (PCR) methods. An excisional biopsy of the stenotic vein segment just above the anastomosis with the PTFE graft was obtained at surgery. Vein samples weighing at least 30 mg were aseptically placed in transport media and stored at 4°C for up to 24 hours. The samples then were sonicated, inoculated in Hep-2 cell culture vials containing confluent monolayers, and passaged three times over 2 weeks. Detection was by direct fluorescent antibody staining. Both culture and PCR were performed in an active chlamydia research laboratory. None of the 10 samples was positive for C pneumoniaeby culture or PCR. Based on our preliminary pilot study, we do not believe C pneumoniaehas a major role in endothelial hyperplasia and consequent graft loss in the hemodialysis patients we studied.

ABSTRACTThe first genus- and subgenus-specific fluorescent oligonucleotide probes for in situ staining of Acanthamoebaare described. Sequences of these phylogeny-based probes complement the 18S rRNA and the gene encoding it (18S rDNA). The genus-specific probe (GSP) is a fluorescein-labeled 22-mer specific forAcanthamoebaas shown here by its hybridization to growing trophozoites of all 12 known Acanthamoeba18S rDNA sequence types and by its failure to hybridize with amoebae of two other genera (Hartmannella vermiformisand Balamuthia mandrillaris), two human cell lines, and two bacteria (Pseudomonas aeruginosaand Escherichia coli). The sequence type T4-specific probe (ST4P) is a rhodamine-labeled 30-mer specific for Acanthamoeba18S rDNA sequence type T4, as shown here in hybridization tests with trophozoites of all 12 sequence types. T4 is the subgenus group associated most closely withAcanthamoebakeratitis (AK). GSP also was tested with corneal scrapings from 17 patients with a high index of clinical suspicion of AK plus 5 patient controls. GSP stained both trophozoites and cysts, although nonspecific cyst wall autofluorescence also was observed. Results could be obtained with GSP in 1 to 2 days, and based on results from cell culture tests, the probe correctly detected the presence or absence of Acanthamoebain 21 of 24 specimens from the 22 patients. The use of GSP with cultured trophozoites and cysts from corneal scrapings has illustrated the suitability of using fluorescent oligonucleotide probes for identification of the genusAcanthamoebain both environmental and clinical samples. In addition, the use of ST4P with cultured amoebae has indicated the potential of oligonucleotide probes for use in subgenus classification.

ABSTRACTOur laboratory recently recovered Chlamydia trachomatisin tissue culture from a urogenital specimen which tested negative by commercial plasmid-based PCR. Immunotyping and omp1sequencing identified the isolate as a serovar E isolate. Further investigation by PCR and Southern hybridization indicated that this isolate lacks the chlamydial cryptic plasmid.

The bacterial genus Rickettsiais traditionally divided into three biotypes, the spotted fever group (SFG), the typhus group (TG), and the scrub typhus group (STG) based on vector host and antigenic cross-reactivity. DNA sequence data were gathered from the 16S ribosomal RNA gene of several SFG and TG species. Comparative sequence analysis shows that: i) all species of Rickettsiaare closely related, exhibiting 0.3–2.6% sequence divergence; ii) although there are identifiable clusters corresponding to the SFG and TG, species of Rickettsiafall into more than two distinct phylogenetic groups; iii) the tick-borne species Rickettsia belliiand Rickettsia canadadiverged prior to the schism between the spotted fever and typhus groups; iv) the newly described AB bacterium is clearly a member of Rickettsia, but its phylogenetic placement within the genus is problematic; v) the mite-borne Rickettsia akari, the tick-borne Rickettsia australisand the recently described flea-borne ELB agent form a loose cluster that cannot be definitively associated with either the TG or the traditional SFG cluster. This latter Glade may represent a unique group(s) distinct from the main cluster of spotted fever and typhus group species. The divergence of Rickettsiawas an ancient event within the α-subclass of the proteobacteria. The sequence divergence between Rickettsiaand Ehrlichia, the closest known genus to Rickettsia, is nearly equal to the sequence divergence between Rickettsiaand all other α-subclass proteobacterial taxa included in the analysis. When Rickettsiawas compared to a representative group of the α-subclass, twenty-eight nucleotide sites were identified which uniquely characterize the 16S rRNA sequences of all species of Rickettsia. The approximate time of divergence between the various species of Rickettsia, estimated from the bacterial 16S rRNA molecular clock, coincides with the approximate divergence time of the hard body ticks which are the arthropod hosts of many Rickettsia. Thus, the possibility of coevolution between these intracellular bacteria and their tick hosts exists.

ABSTRACTPhylogenetic analysis was utilized to investigate biological relationships (tissue tropism, disease presentation, and epidemiologic success), as evidenced by coevolution, among human strains ofChlamydia trachomatis. Nucleotide sequences ofomp1, the gene encoding the major outer membrane protein (MOMP) of C. trachomatis, were determined for 40 strains representing 11 serovars. These data were combined with availableomp1sequences from GenBank for an analysis encompassing a total of 69 strains representing 17 serovars infecting humans. Phylogenetic analysis of the nucleotide and inferred amino acid sequences showed no evolutionary relationships among serovars that corresponded to biological or pathological phenotypes (tissue tropism, disease presentation, and epidemiologic success). In addition, no specific residues that may have evolved to play a role in determining biologically relevant characteristics of chlamydia, such as tissue specificity, disease presentation, and epidemiologic success, were apparent in the MOMP. These results suggest that variation in MOMP may have arisen from a need to be diverse in the presence of immune pressure rather than as a function of pathogenicity. Therefore, the role of MOMP in disease pathogenesis and infection may be passive, and it may not be the major ligand responsible for directing infection of various human cell types.

ABSTRACTPrevious epidemiological studies of Chlamydia trachomatisfrequently have required expansion of isolates in tissue culture. The possibility that C. trachomatis omp1might undergo mutation during such expansion has not been examined systematically. We found no differences in the omp1sequences from 10 clinical specimens before and after 20 in vitro passages.