Diversity in a variable-number tandem repeat from Yersinia pestis.

We have identified a tetranucleotide repeat sequence, (CAAA)(N), in the genome of Yersinia pestis, the causative agent of plague. This variable-number tandem repeat (VNTR) region has nine alleles and great diversity (calculated as 1 minus the sum of the squared allele frequencies) (diversity value, 0.82) within a set of 35 diverse Y. pestis strains. In contrast, the nucleotide sequence of the lcrV (low-calcium-response) gene differed only slightly among these strains, having a haplotype diversity value of 0.17. Replicated cultures, phenotypic variants of particular strains, and extensively cultured replicates within strains did not differ in VNTR allele type. Thus, while a high mutation rate must contribute to the great diversity of this locus, alleles appear stable under routine laboratory culture conditions. The classic three plague biovars did not have single identifying alleles, although there were allelic biases within biovar categories. The antiqua biovar was the most diverse, with four alleles observed in 5 strains, while the orientalis and mediaevalis biovars exhibited five alleles in 21 strains and three alleles in 8 strains, respectively. The CAAA VNTR is located immediately adjacent to the transcriptional promoters for flanking open reading frames and may affect their activity. This VNTR marker may provide a high-resolution tool for epidemiological analyses of plague.

PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: the presence of multiple Bacillus anthracis strains in different victims.

An outbreak of human anthrax occurred in Sverdlovsk, Union of Soviet Socialists Republic (now Ekaterinburg, Russia) in April 1979. Officials attributed this to consumption of contaminated meat, but Western governments believed it resulted from inhalation of spores accidentally released from a nearby military research facility. Tissue samples from 11 victims were obtained and methods of efficiently extracting high-quality total DNA from these samples were developed. Extracted DNA was analyzed by using PCR to determine whether it contained Bacillus anthracis-specific sequences. Double PCR using "nested primers" increased sensitivity of the assay significantly. Tissue samples from 11 persons who died during the epidemic were examined. Results demonstrated that the entire complement of B. anthracis toxin and capsular antigen genes required for pathogenicity were present in tissues from each of these victims. Tissue from a vaccination site contained primarily nucleic acids from a live vaccine, although traces of genes from the infecting organisms were also present. PCR analysis using primers that detect the vrrA gene variable region on the B. anthracis chromosome demonstrated that at least four of the five known strain categories defined by this region were present in the tissue samples. Only one category is found in a single B. anthracis strain.

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates.

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five polymorphisms differed by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations.

Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers.

Bacillus anthracis causes anthrax and represents one of the most molecularly monomorphic bacteria known. We have used AFLP (amplified fragment length polymorphism) DNA markers to analyze 78 B. anthracis isolates and six related Bacillus species for molecular variation. AFLP markers are extremely sensitive to even small sequence variation, using PCR and high-resolution electrophoresis to examine restriction fragments. Using this approach, we examined ca. 6.3% of the Bacillus genome for length mutations and ca. 0.36% for point mutations. Extensive variation was observed among taxa, and both cladistic and phenetic analyses were used to construct a phylogeny of B. anthracis and its closest relatives. This genome-wide analysis of 357 AFLP characters (polymorphic fragments) indicates that B. cereus and B. thuringiensis are the closest taxa to B. anthracis, with B. mycoides slightly more distant. B. subtilis, B. polymyxa, and B. stearothermophilus shared few AFLP markers with B. anthracis and were used as outgroups to root the analysis. In contrast to the variation among taxa, only rare AFLP marker variation was observed within B. anthracis, which may be the most genetically uniform bacterial species known. However, AFLP markers did establish the presence or absence of the pXO1 and pXO2 plasmids and detected 31 polymorphic chromosomal regions among the 79 B. anthracis isolates. Cluster analysis identified two very distinct genetic lineages among the B. anthracis isolates. The level of variation and its geographic distribution are consistent with a historically recent African origin for this pathogenic organism. Based on AFLP marker similarity, the ongoing anthrax epidemic in Canada and the northern United States is due to a single strain introduction that has remained stable over at least 30 years and a 1,000-mile distribution.

Displacement of an uncemented acetabular component after dislocation of a total hip prosthesis. A case report.

Review of the literature reveals few reports of complications encountered with noncemented acetabular components; most concern problems with screw or cup placement, component wear or migration, or disassembly of modular components. No reports involving the displacement of a noncemented acetabular component were found. This is a case report of a patient in whom a noncemented acetabular component was dislodged after the closed reduction of a dislocated total hip prosthesis 4.5 weeks after surgery. In light of this case, the authors believe these reductions should be performed under general anesthesia with fluoroscopic guidance. Care must be taken at surgery to ream sufficiently and obtain proper cup fit and position. Finally, the authors recommend bicortical screw fixation to provide maximum contact and rigid fixation in the early postoperative period.

Pathologic fractures of the humerus.

In a study of 57 actual or impending pathologic fractures of the humerus in 52 patients with inoperable cancer treated between 1972 and 1982, we retrospectively reviewed the charts for analysis and comparison of the functional result and pain relief afforded by the various treatments used. Function of the extremity and relief of pain were each graded as excellent, good, fair, or poor using a modification of Perez's rating system. Seven pathologic fractures were treated nonoperatively. These patients generally had only fair pain relief and a poor functional result. Forty-six pathologic fractures were treated with intramedullary fixation using a Rush rod (n = 16), a Küntscher rod (n = 29), or an Ender rod (n = 1); the Neer endoprosthesis was used in four patients. Thirty-one patients received radiation to the humerus. There were seven operative complications, the most common (n = 3) being prominence of an intramedullary rod at the insertion site which required a second minor procedure for advancement of the rod. From this series, we conclude that any patient who has a pathologic fracture or impending fracture of the humerus and a predicted survival of six weeks or more is likely to benefit from rigid internal fixation with an appropriately selected device, adjunctive use of methylmethacrylate, and postoperative local irradiation therapy as needed.

Medial synovial shelf plica syndrome. Treatment by intraplical steroid injection.

Medial synovial shelf plica syndrome is caused by acquired thickening and inflammation of a commonly present residual embryonic synovial fold. Treatment with a local injection into the plica and surrounding synovium of a steroid and a long-acting local anesthetic was tested in a series of 30 patients to see if more involved and expensive treatment could be avoided. Thirty-one knees were studied and treated prospectively; rigid criteria for making the diagnosis were maintained. Twenty-two (73%) patients had complete relief of pain and full return to activity; five patients had some amelioration of their symptoms and partial return to activity; and three patients had poor results. Two of the poor results were secondary to errors in diagnosis, and one was secondary to the presence of mature fibrosis confirmed during subsequent arthroscopic resection. As a control, ten patients were injected with long-acting local anesthetic alone. In all ten, symptoms were relieved only for the duration of the anesthetic. Intraplical steroid injection appears to be a reasonable, prudent, initial step in the treatment of medial synovial shelf plica syndrome. For competitive athletes, it provides very short morbidity and the ability to return to full practice and participation in a very short period of time.

Erdheim-Chester disease. A case report.

In a 57-year-old woman dizziness, staggering, altered mental status, and bilateral pain and cramping in the lower extremities developed. Extensive investigation yielded evidence of renal and long-bone lesions, with the latter being primarily metaphyseal and diaphyseal. Needle biopsy of the tibial marrow revealed the presence of lipid-laden histiocytes, osteosclerosis, and osteoclasts. A diagnosis of Erdheim-Chester disease was made; this is the 16th known case of the disease. The patient responded well to steroid therapy but later was readmitted and ultimately died of extensive infiltrates in the kidneys and pancreas. Erdheim-Chester disease, an extremely rare multisystem histiocytic disorder, has been characterized as a lipid (cholesterol) granulomatosis. Renal, cardiac, and pulmonary involvement are noted in most cases; the roentgenographic signs of skeletal involvement, when present, are pathognomonic.

Anterior cruciate-deficient knees: a review of the literature.

Injury to the anterior cruciate ligament may lead to functional instability, meniscal injury, and premature degenerative changes of the knee or, if treatment and rehabilitation are carried out carefully, to a functionally stable knee with no premature degenerative changes. Unfortunately, the complex anatomy of the three fiber bundles, consisting of multiple collagenous strands, which constitutes the anterior cruciate ligament combined with variation of injuries to the ligament, with or without injury to other structures of the knee, makes any standardization of surgical treatment difficult. In this paper, we review the biomechanics of the knee, the anatomy and vascularization of the anterior cruciate ligament, the healing characteristics of ligamentous material, and the types of surgical repair that have appeared in recent articles. Those repairs can be divided into direct repair, extraarticular substitution and intraarticular augmentation, freeze-dried fascia lata allografts, and temporary supportive prosthesis such as carbon fiber. Essential to the success of any surgical repair of the anterior cruciate-deficient knee is a prolonged, carefully organized rehabilitation program that allows adequate healing and strengthening of the ligament before it is used normally again. The patient's clear understanding of the importance of a prolonged rehabilitation is best established before the surgical procedure is done.

Intraosseous metastatic breast cancer treatment with internal fixation and study of survival.

The treatment of 31 actual or pending pathologic fractures of the long bones with open reduction and internal fixation with methylmethacrylate in 25 patients with intraosseous metastatic breast cancer is reported. Twenty hips, eight femurs, and three humeri were involved. On the basis of Wilcoxon survival curves, average life expectancy was unaltered by this treatment. However, relief of pain and overall function were significantly improved by rigid internal fixation; 74% of results were rated good (23 cases); 13% fair (four cases); and 13% poor (four cases). Three patients died within three weeks of operation; the fourth poor result was a fixation failure and was repaired with good results. Thus, the efficacy and success of open reduction and internal fixation with methylmethacrylate were confirmed, even in bones with widespread involvement. The procedure is recommended for any patient with fracture or impending fracture of the long bones secondary to metastatic breast cancer, as long as that patient's predicted survival is greater than six weeks after postoperative recovery.

Fracture-dislocation of the shoulder in a 32-month-old child.

A case of fracture-separation and dislocation of the proximal humeral epiphysis, a rare injury, is reported. The patient, a 32-month-old girl suspected of being an abused child, was reported to have fallen from her crib 2 days earlier. She had a painful, swollen, slightly ecchymotic left shoulder, which she held in the neutral position and refused to move. Radiographs showed a healing, nondisplaced radial fracture and fracture-separation of the proximal humeral epiphysis, which was dislocated into an anterior subglenoid location. Surgery under general anesthesia showed the humeral head to lie in a subglenoid position and the distal fragment of the separated epiphysis to have penetrated the posterior lateral capsule. The reduction was stabilized with a single, percutaneous, smooth Kirschner wire and shoulder immobilized in a Velpeau cast for 3 weeks. At 2 year follow-up, the patient has painless, full range of motion, the epiphysis is open, and the length of the humerous appears normal.