Utilization review of the use of BACTEC PLUS high-volume blood culture bottles.

The BACTEC PLUS 26 (NR26) (Becton Dickinson, Towson, Md.) high-volume blood culture bottle replaced the less expensive smaller-volume NR6A bottle in our hospital. An audit carried out several months after their introduction revealed that only 17.5% of the NR26 bottles received the required blood volume. Several audits and educational programs were required in order to achieve a compliance rate of > 60%.     

Rapid Cytomegalovirus pp65 Antigenemia Assay by Direct Erythrocyte Lysis and Immunofluorescence Staining

A rapid cytomegalovirus (CMV) pp65 antigenemia assay with direct erythrocyte lysis (DL) with 0.8% NH₄Cl, followed by indirect immunofluorescence staining (IF), was evaluated with 82 blood samples from renal transplant recipients, and the results were compared to those of the conventional antigenemia assay with dextran sedimentation and two-cycle alkaline phosphatase, anti-alkaline phosphatase staining (DS-APAAP). The DL-IF modification gave a higher leukocyte yield compared to DS-APAAP (75.4 versus 54.9%; P < 0.05), with similar leukocyte viability rates of >95%. The DL-IF methodology involved fewer technical steps, and the assay time was shortened from 5 h to less than 3 h. Nineteen of the 82 samples concordantly tested positive for pp65 antigenemia by both assays, and the readings showed a good correlation (r = 0.996; P < 0.01). No discordant results were observed. We conclude that the CMV pp65 antigenemia assay by this novel DL-IF modification is technically simpler, cheaper, and less time-consuming but yields results comparable to those of the conventional DS-APAAP assay. The shortened assay time and increased capacity to handle more samples confer distinct advantages in the rapid diagnosis and prompt treatment of CMV disease in immunosuppressed patients.     

Autoantibodies directed against the amino-terminal domain I of human calpastatin (ACAST-DI Ab) in connective tissue diseases. High levels of ACAST-DI Ab are associated with vasculitis in lupus

To identify new autoantibody populations in patients with rheumatic diseases, a cDNA expression library was immunoscreened with a rheumatoid arthritis (RA) patient's serum which contains autoantibodies binding to uncharacterized polypeptides by Western-blotting. One clone encoding the amino-terminal region (Nt) [domain L and half of domain I] of human calpastatin was selected. Different fragments of the selected cDNA were prepared and the corresponding recombinant polypeptides were produced by in vitro translation and analysed by Western blotting. Most RA sera bound to recombinant amino-terminal region and domain I but not to domain L. This prompted us to use a recombinant polypeptide corresponding to the domain I of calpastatin as the antigen in a solid-phase ELISA to test sera from patients with various systemic rheumatic diseases and healthy controls.Anti-calpastatin domain I antibodies (ACAST-DI Ab), were detected by ELISA in RA, systemic lupus erythematosus (SLE), Sj?gren's syndrome and control sera at respective frequencies of 10, 9, 0 and 1%. These Ab did not have prognostic value in early RA; high levels were significantly associated with vasculitis in SLE. Antibodies reacting with the calpastatin amino-terminal region are produced during systemic rheumatic diseases and are predominantly directed against domain I. High levels of these Ab may constitute a marker of vasculitis in SLE.     

The first clinical isolate of Legionella parisiensis, from a liver transplant patient with pneumonia.

A bluish white autofluorescent strain of Legionella was isolated from the tracheal aspirate of a female liver transplant patient who developed hospital-acquired pneumonia. This strain had biochemical characteristics compatible with those of L. cherrii, L. anisa, and L. parisiensis and could not be differentiated from L. bozemanii and L. parisiensis by the direct fluorescent-antibody assay. Phylogenetic analysis of partial 16S rRNA gene sequences of this strain (ATCC 700174) revealed the closest homology to the species L. parisiensis (99.5%). An L. parisiensis species-specific profile was also identified by a random amplified polymorphic DNA technique. This is the first report of L. parisiensis isolation from humans.     

Detection and Identification of Actinobacillus pleuropneumoniae Serotype 5 by Multiplex PCR

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10² CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.     

Bilateral simultaneous tubal sextuplets: pregnancy after in-vitro fertilization--embryo transfer following salpingectomy

The presence of a damaged tube has been suggested in recent studies to have a negative effect on in-vitro fertilization (IVF) outcome. Performing bilateral salpingectomy prior to IVF to maximize pregnancy rates may also result in unnecessary surgery. This case is also an example of the occurrence of interstitial pregnancy after salpingectomy. This unusual type of ectopic pregnancy must be kept in mind when evaluating a patient suspected of a possible early abnormal gestation after assisted reproductive technolologies.      

Congenital infection by human cytomegalovirus with a 65 bp deletion in the morphological transforming region II

Summary Human cytomegalovirus (HCMV) infection is an important cause of neonatal death. Using primers derived from sequences within the morphological transforming region II (mtrII), HCMV DNA was amplified by polymerase chain reaction (PCR) from fixed tissues of infants who had died of congenital HCMV infection. In one neonate, HCMV DNA with reduction in the expected size was detected in the liver, spleen, kidney, adrenal, und thyroid tissues by gel electrophoresis. Nucleotide sequencing of the PCR product revealed a 65bp frame-shift deletion within the 79 amino acid open reading frame (ORF) of themtrII. Based upon this observation, it is likely that viral genomic rearrangement involving themtrII may occur in some cases of congenital HCMV infection.     

Pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (SARS)

BACKGROUND: Severe acute respiratory syndrome (SARS) became a worldwide outbreak with a mortality of 9.2%. This new human emergent infectious disease is dominated by severe lower respiratory illness and is aetiologically linked to a new coronavirus (SARS-CoV). METHODS: Pulmonary pathology and clinical correlates were investigated in seven patients who died of SARS in whom there was a strong epidemiological link. Investigations include a review of clinical features, morphological assessment, histochemical and immunohistochemical stainings, ultrastructural study, and virological investigations in postmortem tissue. RESULTS: Positive viral culture for coronavirus was detected in most premortem nasopharyngeal aspirate specimens (five of six) and postmortem lung tissues (two of seven). Viral particles, consistent with coronavirus, could be detected in lung pneumocytes in most of the patients. These features suggested that pneumocytes are probably the primary target of infection. The pathological features were dominated by diffuse alveolar damage, with the presence of multinucleated pneumocytes. Fibrogranulation tissue proliferation in small airways and airspaces (bronchiolitis obliterans organising pneumonia-like lesions) in subpleural locations was also seen in some patients. CONCLUSIONS: Viable SARS-CoV could be isolated from postmortem tissues. Postmortem examination allows tissue to be sampled for virological investigations and ultrastructural examination, and when coupled with the appropriate lung morphological changes, is valuable to confirm the diagnosis of SARS-CoV, particularly in clinically unapparent or suspicious but unconfirmed cases.     

Factors associated with successful referral for clinical care of drug users with chronic hepatitis C who have or are at risk for HIV infection

The objective of this study was to determine outcomes of referring drug users (DUs) with chronic hepatitis C for clinical evaluation and care. Two hundred twenty-eight persons with detectable hepatitis C virus RNA were given expedited referrals for evaluation and possible treatment of hepatitis C from a prospective study cohort of current and former opiate-addicted DUs. Four outcomes were analyzed: accepted referral, arrived for clinical evaluation, had liver biopsy, and received treatment. One hundred twenty-seven participants (56%) accepted referral, of whom 54 (43%) arrived for evaluation. Of these participants, 12 (22%) had liver biopsy, and 4 (7%) were treated. Multivariate logistic regression revealed that HIV-infected DUs were significantly less likely to accept referral (adjusted odds ratio [O(Radj)], 0.51; 95% confidence interval [CI], 0.30-0.88), and older participants were more likely to keep an appointment (O(Radj), 1.06/y; 95% CI, 1.00-1.12). Of HIV-seropositive participants, those with a history of injection were more likely to accept referral (O(Radj), 3.60; 95% CI, 1.08-11.96), and those with higher HIV load (O(Radj), 0.50/log10; 95% CI, 0.26-0.94) and Hispanic ethnicity (O(Radj), 0.26; 95% CI, 0.07-0.89) were less likely to keep an appointment. Despite expedited referrals for hepatitis C care, only a few participants received an evaluation, and even far fewer were treated. Because increasingly effective treatment is available, better methods are urgently needed to improve evaluation and treatment of HCV-infected DUs, including those coinfected with HIV.