John P. Leeming

Diagnosis of invasive aspergillosis in bone marrow transplant recipients by polymerase chain reaction

A nested polymerase chain reaction (PCR) test targeting Aspergillus spp. large ribosomal subunit genes was evaluated retrospectively on 175 serum samples from 37 bone marrow transplant recipients, 70% of whom received grafts from unrelated donors. Six patients had proven infection, seven had probable infection, and three had possible infection, using the revised EORTC case definitions. These 16 patients were all PCR positive (57 out of 93 samples tested). Two additional patients who did not fulfil current diagnostic criteria, but in whom invasive aspergillosis (IA) was thought clinically probable, were also PCR positive (five out of nine samples). Invasive aspergillosis was unlikely in the remaining 19 patients, four of whom were PCR positive on a single occasion (four out of 70 samples). Three samples were inhibitory to PCR. Sensitivity of PCR in diagnosing patients with IA was 100%, specificity was 79% and positive predictive value was 80%, using the criterion of a single positive result. If two positive results were required, these values were 81%, 100% and 100% respectively. The median duration of infection documented by PCR was 36u2003days (range 3–248u2003days) in 17 out of 18 patients (94%) who did not survive. Positive PCR results predated the institution of antifungal therapy in two‐thirds of patients. Four patients became PCR positive during pretransplant conditioning therapy.

Childhood empyema: limited potential impact of 7-valent pneumococcal conjugate vaccine

During 2003–2004, locally presenting pleural empyema cases in children increased 3-fold. Antigen analysis of empyema fluid identified Streptococcus pneumoniae in 27 of 29 cases for whom samples were available and capsular polysaccharide type 1 in 18 of these. Use of a conjugate vaccine without serotype 1 antigen would have had limited impact on this morbidity in our region.

Culture-Negative Childhood Empyema Is Usually Due to Penicillin-Sensitive Streptococcus pneumoniae Capsular Serotype 1

Two studies in the United States have shown that isolates of Streptococcus pneumoniae from children with empyema are more likely to be penicillin sensitive and of capsular serotype 1 than those from uncomplicated pneumococcal pneumonia (1, 4). n nIn the United Kingdom, childhood empyema is becoming more common (S. D. Playfor, A. R. Smyth, and R. J. Stewart, Letter, Thorax 52:932, 1977; J. H. M. Rees, D. A. Spencer, D. Parikh, and P. Weller, Letter, Lancet 349:402, 1997). Many cases have no definitive etiology, despite attempts at noncultural diagnosis by antigen detection, and are most likely due to previous antibiotic therapy. It is uncertain, therefore, if these culture-negative cases of childhood empyema are from the same population as the S. pneumoniae culture-positive ones or whether they are a separate and different problem. n nWe have examined pleural aspirates from 43 children (aged 6 months to 17 years; mean age, 6.2 years; median age, 6 years; 30 of whom were male) presenting with empyema thoracis over a 4-year period to the Freeman Hospital in Newcastle upon Tyne, a tertiary referral center for the North East of England. n nRoutine culture and a latex agglutination test for pneumococcal antigen (SLIDEX; BioMerieux, Marcy lEtoile, France) were performed with these specimens, after which the remaining material was digested with dithiothreitol. A 1-ml aliquot of the digest was centrifuged at 10,000 × g for 10 min, and the pellet was washed twice in phosphate-buffered saline (PBS). The pellet was resuspended in 12.5% Chelex-100 resin (Bio-Rad Laboratories Ltd.) and heated at 56°C for 30 min and then at 100°C for 15 min. The supernatant was then tested for the presence of S. pneumoniae DNA by a previously described real-time PCR assay targeting the pneumolysin gene (ply) (A. M. Kearns, R. Freeman, O. M. Murphy, P. R. Seiders, M. Steward, and J. Wheeler, Letter, J. Clin. Microbiol. 37:3434, 1999). Positive samples underwent a second real-time PCR assay targeting a conserved sequence of the PBP2B gene (pbp2b) (3) found in all strains of penicillin-susceptible S. pneumoniae (PSSP) for which the MIC of penicillin was <0.06 mg/liter. Finally, whenever sample volumes permitted, 250 μl of the digested sample was mixed with 750 μl of PBS and heated to 100°C for 10 min and then centrifuged for 2 min (21,000 × g). The supernatant was used in solid-phase indirect sandwich enzyme-linked immunosorbent assays (ELISAs) for the detection of type-specific pneumococcal capsular polysaccharides, with monoclonal antibodies to types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F donated by Wyeth Vaccines Research, West Henrietta, N.Y. n nTwo samples were culture positive. One of them, for which all tests for S. pneumoniae were negative, yielded Staphylococcus aureus. The other grew scanty Staphylococcus epidermidis, which was regarded as a skin contaminant. All pbp2b assays indicated “penicillin sensitivity.” Other results are found in Table u200bTable11. n n n nTABLE 1. n nResults of real-time PCR assays for pneumococcal DNA and ELISAs for 13 pneumococcal capsular serotype antigens on 43 pleural aspirates from children with empyema n n n nWe found evidence of S. pneumoniae infection in over 70% of culture-negative empyema fluids. The infecting strains were likely to have been highly penicillin sensitive, and at least 60% were of capsular serotype 1, which, in the United Kingdom, as in the United States, is not the commonest serotype in invasive pneumococcal disease (2). n nWe conclude that cases of culture-negative childhood empyema presenting at tertiary referral centers are not an etiologically distinct group, that childhood pneumococcal vaccines must include capsular serotype 1 antigen, and that early detection of S. pneumoniae capsular serotype 1 antigen in cases of childhood pneumonia may aid management and predict complications, especially empyema.

Treatment of Ocular Infections with Topical Antibacterials

Topically applied ophthalmic antibacterial preparations are widely used in the treatment of patients with superficial ocular infections. In addition, they are frequently used to augment treatment for intraocular infection administered systemically or via local instillation.Direct application delivers high concentrations of antimicrobial agents to the surface of the eye conveniently, quickly and with minimal systemic exposure to the agent. However, antibacterials are rapidly dissipated from the tear film and intraocular penetration of topical antibacterial agents is generally poor, necessitating intensive application for successful treatment of corneal infections. Therapeutic concentrations are rarely achieved at other sites in the eye.This article reviews what is known of the pharmacokinetics of topical ocular agents and how this information can be used to optimise ocular persistence and penetration and minimise systemic absorption of antibacterials. A review of the features of the most commonly employed topical antibacterials suggests that for the treatment of uncomplicated bacterial conjunctivitis there is little difference between the various agents in terms of clinical efficacy, although chloramphenicol should be used with care because of its potential haematological toxicity. Carefully considered therapy is imperative for bacterial keratitis; fortified β-lactam/aminoglycoside combinations are often used for these infections. The fluoroquinolones appear promising, but caution is necessary in treating keratitis of unknown aetiology with these agents alone because of inherent and emerging acquired resistance among Gram-positive bacteria.

Diagnosis of Invasive Pneumococcal Infection by Serotype-Specific Urinary Antigen Detection

ABSTRACT Widespread use of conjugate pneumococcal polysaccharide-protein vaccines may alter the spectrum of pneumococci producing invasive disease. Novel sensitive diagnostic methods would be valuable for monitoring the epidemiology of pneumococcal disease within populations and vaccine recipients. Ideally, these methods should allow determination of the serotype of the infecting clone. Serotype-specific enzyme-linked immunosorbent assays (ELISA) for 13 capsular polysaccharides (types 1, 3, 4, 5, 6A, 6B, 7A, 9V, 14, 18C, 19A, 19F, and 23F) were developed. Experiments with pure capsular polysaccharide demonstrated that the assays were sensitive (0.01 to 1.0 ng/ml) and specific. These assays were used to detect capsular polysaccharide in urine from 263 adult patients with proven (blood culture-positive) invasive pneumococcal disease and pneumonia of unknown etiology and from patients with positive blood cultures yielding bacteria other than pneumococci (control group). Among 76 patients with invasive pneumococcal disease from whom blood culture isolates had been serotyped, 62 (82%) had infections with pneumococci of serotypes represented in the ELISA panel. Capsular antigen matching the serotype of the blood culture isolate was detected in the urine of 52 of these patients, giving a sensitivity of 83.9% for the target serotypes. The tests were significantly more sensitive for urine from patients with pneumococcal pneumonia (89.8%) than for urine from patients with nonpneumonic invasive infection (61.5%; P < 0.05). Data from the control group indicated a specificity of 98.8%. These assays should prove valuable in epidemiological investigation of invasive pneumococcal infection in adults, particularly if combined with a sensitive C-polysaccharide detection assay to screen for positive samples.

Reservoirs of coagulase negative staphylococci in preterm infants.

This investigation was undertaken to determine the magnitude of, and interrelations between, reservoirs of coagulase negative staphylococci on infants skin at various sites (including sites used for insertion of intravascular catheters) and in faeces during the first six months of life. Sites with large numbers of coagulase negative staphylococci were identified by sampling 16 skin sites and stools from 20 preterm neonates at 8-30 days of life. A more detailed survey of numbers and types of coagulase negative staphylococci in stool and at six skin sites of 10 preterm infants was then performed over the first six months of life. Isolates of coagulase negative staphylococci were collected and characterised by speciation, antibiotic susceptibility profiling, and plasmid restriction fragment length polymorphism analysis. Large, relatively stable reservoirs were identified in the faeces, around the ear, and in the axilla and nares. Skin on the forearm and leg, sites at which peripheral catheters are frequently sited, carried small unstable numbers of coagulase negative staphylococci, which were usually indistinguishable from coagulase negative staphylococci isolated from other body sites on the same baby. Contamination of catheter insertion sites with coagulase negative staphylococci from reservoir sites on the same baby could explain these observations. These data suggest that interventions reducing cross-contamination between sites on the same baby might be as important in preventing coagulase negative staphylococcal bacteraemias as measures taken to prevent cross infection between babies. Procedures which are likely to result in heavy coagulase negative staphylococcal contamination of the hands of healthcare staff, such as changing soiled nappies, should receive particular attention.

Skin disinfection in preterm infants.

Greater care and a more thorough approach to intravenous catheter site disinfection may be important for the prevention of catheter related sepsis, especially with coagulase negative staphylocci in preterm infants. The efficacy of skin disinfection was evaluated in preterm infants using a skin swabbing technique after disinfectant exposure. In the first part of the study, 25 peripheral intravascular catheter sites were quantitatively sampled immediately after routine cannula insertion. Bacterial counts greater than 100 colony forming units/cm2 were observed from 10 (40%) sites. In the second part, sampling for bacterial colony counts was done after skin cleansing with various durations of exposure of chlorhexidine/alcohol swabs or povidone iodine. The overall mean reduction in bacterial colony counts after skin cleansing ranged from 90-99%. Skin sterilisation was achieved in 33-92% of cases. The use of two consecutive 10 second exposures resulted in a significantly improved reduction in colony counts compared with a single 10 second wipe. A longer 30 second exposure also resulted in a greater reduction of bacterial numbers compared with a shorter duration of 5 or 10 seconds. Repopulation of disinfected sites occurred within 48 hours. This effect was delayed by occluding the cleansed site with a semipermeable dressing. There were no significant differences between povidone iodine and the chlorhexidine swabs in reducing bacterial numbers. This study has demonstrated that a brief exposure with a premoistened disinfectant swab is not sufficient for complete elimination of resident skin flora of newborn infants. The use of two consecutive cleanings, or a longer duration of cleansing is recommended for more effective skin sterilisation.

A novel polymerase chain reaction assay to detect Mycoplasma genitalium

Aims: To design and validate a polymerase chain reaction (PCR) assay targeting the 16S rRNA gene of Mycoplasma genitalium. Methods: Primers were designed that were complementary to the 16S rRNA gene sequence of M genitalium. After optimisation of the reaction conditions, the PCR was tested against nine M genitalium strains, a dilution series of M genitalium DNA, and a panel of common microorganisms. The PCR was also challenged in parallel with a published assay against 54 urine specimens from men with urethritis. Results: The expected 341 bp product was produced on amplification of material from all M genitalium strains and from none of the other microorganisms tested. The lower limit of detection was 50 genome copies. The new assay detected M genitalium DNA in nine of 54 men with urethritis, in comparison with eight positive specimens detected with the alternative PCR. Conclusions: This novel PCR targeting the M genitalium 16S rRNA gene has been optimised and now provides a sensitive and specific alternative or addition to the available MgPa gene targeting assays.

Molecular approaches for the diagnosis and epidemiological investigation of Aspergillus infection.

Molecular techniques have been applied to the diagnosis of invasive aspergillosis and to investigate the ecology and epidemiology of Aspergillus. Recent advances in diagnosis include the development of PCRs targeting either panfungal or Aspergillus‐specific sequences, using whole blood or serum samples. When a sensitive PCR is used, invasive aspergillosis in bone marrow transplant patients can be detected several weeks before antigen tests become positive, and a positive PCR often pre‐dates the institution of antifungal therapy. The role of PCR in monitoring response to therapy in immunocompromised patients is unclear. No prospective studies have yet demonstrated that management incorporating PCR alters the poor outcome of invasive aspergillosis in immunocompromised patients. Molecular typing of Aspergillus fumigatus has shown wide geographical dispersal of indistinguishable strains. This, combined with the observation that multiple strains may be isolated from individual colonised patients with cystic fibrosis and from immunocompromised patients with disseminated disease, makes the elucidation of the epidemiology of aspergillosis relatively complex.

Comparison of random amplified polymorphic DNA with restriction fragment length polymorphism as epidemiological typing methods for Mycobacterium tuberculosis

Aim—To compare restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) methods for the epidemiological typing of Mycobacterium tuberculosis. Methods—Thirty one M tuberculosis cultures originating from patients in the Canton of Berne in Switzerland, which had previously been typed by RFLP, were subjected to RAPD analysis. Cultures were coded so that the investigators were blind to the RFLP results until RAPD analysis was complete. Results—The 31 cultures of M tuberculosis were divided into nine groups by RFLP and eight groups by RAPD. Generally there was good correlation between the groups identified by the two techniques, with the exception of strains that had only one copy of IS6110. Both methods subdivided isolates that were placed in a single group by the other method. Conclusions—RAPD analysis is quick, simple, and useful for the comparison of small numbers of isolates. RFLP is more reproducible and therefore better suited for the accumulation of RFLP fingerprints for long term local surveillance and large epidemiological studies.