Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin.

Pneumocystis carinii specific DNA sequences have been cloned from the experimental rat model. The sequence of the gene coding for the large subunit of mitochondrial ribosomal RNA has been used to construct P. carinii specific oligonucleotide primers for the polymerase chain reaction. These oligonucleotides produced amplification of specific sequences from both P. carinii infected rat and human lung samplings, but none from a range of other organisms including potential pulmonary pathogens. Comparison of the sequence of amplified products from the infected rats and humans demonstrated limited but consistent differences between P. carinii from these two hosts and allowed for the construction of a human specific internal oligonucleotide. The application of the specific oligonucleotides for DNA amplification and subsequent Southern hybridisation affords extremely sensitive and specific detection of P. carinii in human samples, which may be applicable to both epidemiological research and clinical studies.

Comparison of DNA amplification and immunofluorescence for detecting Pneumocystis carinii in patients receiving immunosuppressive therapy.

Two studies were performed to compare the sensitivity of DNA amplification with immunofluorescence for the detection of Pneumocystis carinii in asymptomatic normal and immunosuppressed subjects receiving no anti-Pneumocystis chemoprophylaxis. In the first study, immunofluorescence and silver stains were used to examine 12 induced sputa and 12 bronchoalveolar lavage specimens from 24 normal control subjects; induced sputa from 20 renal transplant recipients; and induced sputa from 11 patients with fibrosing alveolitis. All specimens were negative for P carinii using both stains, apart from one renal patient in whom 2 P carinii cysts were seen by immunofluorescence alone. In the second study, DNA amplification and immunofluorescence were used to examine induced sputa from 3 groups of 10 control, renal, and heart/lung transplant recipients. All 30 specimens were negative for P carinii by immunofluorescence. However, 3 renal and 2 heart/lung patients were positive for P carinii by DNA amplification alone. One of these patients developed P carinii pneumonia 6 weeks after sputum induction. DNA amplification is a more sensitive technique than immunofluorescence for detecting P carinii. P carinii colonization occurs in asymptomatic organ transplant recipients, but not in normal individuals.

Identification of porcine Pneumocystis carinii as a genetically distinct organism by DNA amplification.

DNA was amplified from lung samples from three piglets infected with Pneumocystis carinii, using oligonucleotide primers designed to the P. carinii mitochondrial large subunit ribosomal RNA gene. The nucleotide sequence of the amplification product was determined and indicated lack of sequence variation among these pig-derived P. carinii samples at this locus. The data showed that porcine P. carinii was genetically distinct from P. carinii isolated from other mammalian host species.

Granulomatous Pneumocystis carinii pneumonia: DNA amplification studies on bronchoscopic alveolar lavage samples.

Three HIV positive subjects presented with symptoms and radiographic changes suggestive of Pneumocystis carinii pneumonia. Methenamine silver staining of bronchoscopic alveolar lavage (BAL) fluid was negative (from one sample in one patient and two samples in the other two patients). Open lung biopsy was performed because of uncertain clinical progress and diagnosis; all three patients were found to have multiple pulmonary granulomata encasing numerous P carinii organisms. DNA amplification, using P carinii specific oligonucleotides, was performed on stored bronchoscopic BAL samples. P carinii specific amplification product was detected by ethidium bromide staining after electrophoretic separation on agarose gel in one case, and by the more sensitive technique of oligohybridisation in all three cases. In granulomatous P carinii pneumonia organisms are rarely identified in bronchoscopic alveolar lavage samples using histochemical staining, but are detectable by DNA amplification, although not at levels which can be readily distinguished from low, subclinical infection.

Clusters of Pneumocystis carinii pneumonia: analysis of person-to-person transmission by genotyping.

Genotyping at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon was performed on isolates of P. carinii sp. f. hominis from three clusters of P. carinii pneumonia among eight patients with haematological malignancies and six with HIV infection. Nine different ITS sequence types of P. carinii sp. f. hominis were identified in the samples from the patients with haematological malignancies, suggesting that this cluster of cases of P. carinii pneumonia was unlikely to have resulted from nosocomial transmission. A common ITS sequence type was observed in two of the patients with haematological malignancies who shared a hospital room, and also in two of the patients with HIV infection who had prolonged close contact on the ward. In contrast, different ITS sequence types were detected in samples from an HIV-infected homosexual couple who shared the same household. These data suggest that person-to-person transmission of P. carinii sp. f. hominis may occur from infected to susceptible immunosuppressed patients with close contact within hospital environments. However direct transmission between patients did not account for the majority of cases within the clusters, suggesting that person-to-person transmission of P. carinii sp. f. hominis infection may be a relatively infrequent event and does not constitute the major route of transmission in man.

Genetic heterogeneity in Pneumocystis carinii: an introduction.

Molecular techniques have played an important role in demonstrating a high level of heterogeneity among the different types of organisms which are collectively known as Pneumocystis carinii. Genetic heterogeneity has been observed in Pneumocystis organisms isolated from different host species, suggesting that Pneumocystis infection is host-species specific. On the basis of this genetic divergence a provisional trinomial nomenclature has been adopted, in which different types of Pneumocystis organisms are designated a 'special form'. Multiple special forms of Pneumocystis have been detected in some host species, for example in infected rat and ferret lungs, but not in human lungs. Lower levels of genetic heterogeneity have been observed within each Pneumocystis special form, and in particular in human-derived Pneumocystis. Analysis of the genetic heterogeneity of populations of Pneumocystis is contributing to the understanding of the epidemiology and pathophysiology of this infection.

Genetic heterogeneity in human-derived Pneumocystis carinii.

Genetic heterogeneity has been observed among isolates of human-derived Pneumocystis carinii (P. carinii sp. f. hominis). DNA sequence analysis has been shown to be informative in distinguishing between isolates of P. carinii sp. f. hominis. Single base polymorphisms have been observed in the genes encoding the mitochondrial large subunit ribosomal RNA, the mitochondrial small subunit ribosomal RNA and the AROM protein. The highest level of genetic variation has been found at the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA operon. Typing of isolates of P. carinii sp. f. hominis has enabled the examination of the frequency of different types of P. carinii sp. f. hominis in distinct populations. It has also facilitated studies on the acquisition and transmission of P. carinii sp. f. hominis infection.

PCR for detecting Pneumocystis carinii in clinical or environmental samples.

Since Pneumocystis carinii cannot be cultured in vitro, the introduction of polymerase chain reaction (PCR) has been an enormous advantage for research purposes. It is now possible to detect P. carinii in specimens containing low numbers of organisms where conventional detection methods using microscopic examination of histochemical stains has been insufficient. PCR has been used to detect P. carinii in bronchoalveolar lavage, induced sputum, spontaneous expectorates, oropharyngeal gargles, nasopharyngeal aspirates, serum, blood and in environmental samples. The use of PCR will enable the study of the epidemiology of P. carinii infection by detecting the organism in environmental samples, permitting molecular typing and thereby the study of the transmission of the organism. Furthermore PCR will facilitate studies on the response to therapy, studies monitoring for the emergence of drug resistant strains of P. carinii and in the diagnosis of P. carinii pneumonia in noninvasive specimens, in patients unable to undergo more invasive diagnostic procedures.

Infection with Pneumocystis carinii is prevalent in healthy Gambian children.

Pneumocystis pneumonia is rarely identified in the many immunosuppressed individuals with acquired immune deficiency syndrome (AIDS) and malnutrition in Africa. To test whether infection with Pneumocystis carinii occurs in the continent we conducted a comparative serological study, measuring by enzyme-linked immunosorbent assay antibodies to the parasite in 150 healthy young individuals from both Britain and the Gambian savanna. The prevalence of significant titres of antibody to P. carinii steadily increased with age and included more than 70% of both populations by 8 years of age. Infection with P. carinii is, therefore, common in the Gambia. Thus opportunistic pneumocystis pneumonia may be an important but largely unrecognized disease in the continent, though its impact is probably diminished by the prevalence of fatal tuberculous infection, particularly in the AIDS population.

PIP: The prevalence of sero-positivity toward Pneumocystis carinii parasite antigens was determined in 150 British subjects aged 3 months-40years, and in 150 Gambians of the same age age range. The Gambians were from Katabas villages north of the Gambian river. The sera were tested with an ELISA assay developed from parasites and cysts isolated from rats, which were previously shown to share many antigens with organisms from humans. IgG titers reactive with P. carinii increased with age in the British and Gambian subjects, with essentially no positives at ages 3-6 months, to a peak of 70% positive in both groups by age 8 years. British children and parents were questioned about history of pneumonia illness, with negative findings. There are very few reports of P. carinii infections, judging from sputum and bronchoscopic lavage, in African AIDS patients. 2 cases of Cryptococcus have been reported, but tuberculosis is a common and rapidly lethal infection in African AIDS patients.

The use of lipid-coated microbubbles as a delivery agent of 7beta-hydroxycholesterol in a radiofrequency lesion in the rat brain.

OBJECTIVE: This laboratory has previously described the aggregation of intravenously administered lipid-coated microbubbles (LCM) around tumors and areas of injury. 7Beta-hydroxycholesterol has been used to inhibit astrocytic proliferation in nervous system injury models. The compound has been given by direct infusion, by epidural catheter, or in liposomes (delivered stereotactically to the injury site). In this article, we report the use of LCM to deliver 7beta-hydroxycholesterol to a radiofrequency injury site in the rat cerebrum. METHODS: First, the ability of LCM to target the thermal lesion in the rat brain was characterized using a lipid-soluble fluorescent dye 3,3-dioctadecyloxacarbocyanine perchlorate. Then, the effectiveness of this delivery system in suppression of glial proliferation was measured by glial fibrillary acidic protein immunoreactivity. RESULTS: Glial fibrillary acidic protein immunoreactivity was significantly reduced when 7beta-hydroxycholesterol was administered via LCM but not alone, suggesting that astrocytic proliferation would correspondingly be diminished. CONCLUSION: LCM were assessed as a delivery vehicle for 7beta-hydroxycholesterol in a rat brain radiofrequency lesion and found to be efficient in reducing astrogliosis, as measured by glial fibrillary acidic protein immunoreactivity.

The affinity of lipid-coated microbubbles to maturing spinal cord injury sites.

OBJECTIVE: This laboratory has demonstrated that lipid-coated microbubbles (LCMs) effectively aggregate and deliver chemotherapeutic drugs into rat brain tumor cells and antigliosis agents into maturing rat brain injury sites. In this study, we report the affinity of tail vein-injected LCMs to the injured rat spinal cord by a compressive lesion to the upper thoracic region. METHODS: The accumulation of LCMs in the injured spinal cord was analyzed by labeling it with a lipid-soluble fluorescent dye, 3,3'-dioctadecyloxacarbocyanine perchlorate. Indices of glial fibrillary acidic protein were measured concomitantly with 3,3'-dioctadecyloxacarbocyanine perchlorate-labeled LCMs using confocal microscopy. RESULTS: There was no aggregation of LCMs accumulated 1 and 6 hours after injury; however, when given 2, 4, and 7 days after injury, LCMs showed a clear affinity for the injured region. LCM aggregation shifted from the central necrotic area of the injury on postinjury Day 2 and postinjury Day 4 to the white matter among glial fibrillary acidic protein-positive astrocytes by postinjury Day 7. CONCLUSION: Affinity of LCMs for spinal cord injury sites may be mediated in the early stages after injury by proliferating macrophages in the necrotic center, and then in later stages by glial fibrillary acidic protein-positive astrocytes in adjacent white matter. These findings suggest a potential for using LCMs as a delivery vehicle to concentrate lipid-soluble agents in spinal cord injury sites.

Redislocation of the shoulder during the first six weeks after a primary anterior dislocation: risk factors and results of treatment.

BACKGROUND: After an anterior dislocation, shoulder instability may occur with disruption of the soft-tissue or osseous restraints, leading to early redislocation. The aim of the present study was to clarify the risk factors for this complication within the first six weeks after a first-time anterior traumatic dislocation and to assess the outcome of treatment with immediate operative stabilization. METHODS: A three-year, prospective, observational cohort study of 538 consecutive patients with a first-time anterior dislocation of the shoulder was carried out. Reassessment of shoulder function was performed at a dedicated shoulder clinic, and suspected early redislocations were assessed with additional radiographs. All medically fit patients with a confirmed acute redislocation were treated with repeat closed reduction under anesthesia. Patients with unstable reductions were treated operatively. Functional and radiographic assessment of outcome was carried out during the first year after dislocation. RESULTS: Seventeen (3.2%) of the 538 patients sustained an early redislocation within the first week after the original dislocation. Patients at increased risk of early redislocation included those who sustained the original dislocation as the result of a high-energy injury (relative risk = 13.7), those who had a neurological deficit (relative risk = 2.0), those in whom a large rotator cuff tear occurred in conjunction with the dislocation (relative risk = 29.8), those in whom the original dislocation was associated with a fracture of the glenoid rim (relative risk = 7.0), and those who had a fracture of both the glenoid rim and the greater tuberosity (relative risk = 33.5). Following operative reconstruction, the outcome at one year after the injury was favorable in terms of function, general health, and radiographic findings. None of the patients had a redislocation or symptoms of instability at one year. CONCLUSION: All patients who have substantial pain, a visible shoulder deformity, or restriction of movement at one week after reduction of a first-time dislocation should be evaluated with repeat radiographs to exclude a redislocation. Patients in whom this complication develops usually have either (1) severe disruption of the soft-tissue envelope due to a large rotator cuff tear or (2) disruption of the normal osseous restraints to dislocation due to either an isolated fracture of the glenoid rim or fractures of both the glenoid rim and the greater tuberosity. Early operative stabilization is justified for patients in whom the dislocation is associated with these coexisting conditions and who have evidence of gross instability.

The role of physiotherapy and clinical predictors of outcome after fracture of the distal radius.

The capacity for physiotherapy to improve the outcome after fracture of the distal radius is unproven. We carried out a randomised controlled trial on 96 patients, comparing conventional physiotherapy with a regime of home exercises. The function of the upper limb was assessed at the time of removal of the plaster cast and at three and six months after injury. Factors which may predict poor outcome in these patients were sought. Grip strength and hand function did not significantly differ between the two groups. Flexion and extension of the wrist were the only movements to improve with physiotherapy at six months (p = 0.001). Predictors of poor functional outcome were malunion and impaired function before the fracture. These patients presented with pain, decreased rotation of the forearm and low functional scores at six weeks. Our study has shown that home exercises are adequate rehabilitation after uncomplicated fracture of the distal radius, and routine referral for a course of physiotherapy should be discouraged. The role of physiotherapy in patients at high risk of a poor outcome requires further investigation.

Biomechanics of thoracic discectomy.

The thoracic spine is a structurally unique region that renders it uniquely susceptible to thoracic disc herniation. Surgical management strategies are complicated, in part, by the regional anatomical and biomechanical nuances. Surgical approaches include posterior, posterolateral, and anterior routes. Each is associated with specific indications and contraindications. The biomechanical principles and safe anatomical trajectories must be considered in the surgical decision-making process. These issues are discussed in the pages that follow.

Genotypic variation in Pneumocystis jirovecii isolates in Britain.

BACKGROUND: Pneumocystis jirovecii is the cause of Pneumocystis pneumonia (PCP) in immunosuppressed humans. Asymptomatic colonisation with P jirovecii may occur in patients with minor immunosuppression or chronic lung disease. The aim of this study was to describe the molecular epidemiology of P jirovecii in Britain over a period of 12.5 years. METHODS: Between January 1989 and July 2001 161 samples of P jirovecii were obtained from patients with PCP (n = 119), patients colonised by P jirovecii (n = 35), and from air spora (n = 6). Genotyping of samples was performed at the mitochondrial large subunit rRNA (mt LSU rRNA). RESULTS: Genotype 1 (38%) was the most frequently identified genotype: genotypes 2 (26.6%), 3 (20.3%), and 4 (5%) were less common. Mixed infection (more than one genotype) was identified in 10% of samples. While genotype 1 was the most frequently detected type in both patients with PCP and those colonised by P jirovecii (38% and 42%, respectively), these groups differed in the relatively lower rate of detection of genotype 4 (2% v 17%) and the higher detection of mixed infection in those with PCP (13% v 3%). Detection of specific genotypes of P jirovecii was associated with the patient's place of residence (p = 0.02). There was no association between specific genotypes and severity of PCP as measured by arterial oxygen tension (p = 0.3). CONCLUSIONS: The evidence of clustering of specific genotypes with patient's postcode of residence is consistent with the hypothesis of person to person transmission of P jirovecii via the airborne route. The lack of association between specific mt LSU rRNA genotypes and severity of PCP suggests that this locus is not implicated in the virulence of the organism.

DNA sequences identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in samples of air spora.

Samples of ambient air collected with three different types of spore traps in a rural location were examined for the presence of Pneumocystis carinii by screening for P. carinii-specific DNA sequences by DNA amplification. Eleven spore trap samples were analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of P. carinii f. sp. carinii and P. carinii f. sp. hominis. The samples were collected over a 3-year period during the months of May to September, with a range of sampling times from 9 to 240 h. One air sample from an animal facility housing P. carinii-infected rats was also examined. P. carinii-specific amplification products were obtained from samples from each of the spore traps. The amplification products from eight air samples were cloned and sequenced. The majority of the recombinants from each of these samples had sequences identical to those of P. carinii f. sp. carinii and P. carinii f. sp. hominis, and a number of clones had single-base differences. These data suggest that sequences identical to those of P. carinii f. sp. carinii and P. carinii f. sp. hominis can be detected in samples of air collected in a rural location and that P. carinii may be a component of the air spora of rural Oxfordshire.