Summary StatementConventional culture techniques have poor sensitivity for detecting bacteria growing in biofilms, which can result in under-diagnosis of infections. Sonication of biofilm colonised orthopaedic biomaterials can render bacteria in biofilm more culturable, thereby improving diagnosis of orthopaedic implant infections.IntroductionProsthetic joint infection (PJI) is a potentially devastating complication in arthroplasty. Biofilm formation is central to PJI offering protection to the contained bacteria against host defence system and antimicrobials. Orthopaedic biomaterials generally have a proclivity to biofilm colonisation. Conventional culture technique has a low sensitivity for detecting bacteria in biofilm. Sonication can disrupt bacteria biofilms aggregations and dislodge them from colonised surfaces, rendering them culturable and consequently improve the diagnosis of otherwise culture-negative PJI. We investigated the effect of ultrasonication on biofilms adherent to poylmethylmethacryla...

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 beta-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.

Introduction: Recent changes in pneumococcal immunization schedules for both children and the elderly could have profound effects on pneumococcal carriage and invasive disease in the elderly, but there is very little background data on carriage in this population. The aims of this study were to evaluate methods for the assessment of pneumococcal carriage in an elderly population (≥ 65 years) and to establish the prevalence and dynamics of pneumococcal carriage in this group.

Since the 1970s many tissue banks have been testing allograft heart valves (HVs) for Mycobacterium tuberculosis (MTB). Donor selection for low risk of tuberculosis (TB) was introduced in the 1980s and appears to have reduced the risk of TB transmission. Regulatory guidance does not specify testing for TB, but does exclude donors with a recent history of TB. This survey of HV international bank practices revealed variations in donor selection, testing and processing of valves. Participant banks (from Europe and the USA) reported that over a period of 15 years, HV tissues from 38,413 donors were banked and 32,289 donors were tested for TB, none being positive. HV-associated tissue from 27,840 donors was stained and underwent microscopy; none of these were positive for acid-fast bacilli (AFB). Non-tuberculosis mycobacteria (NTBM) were detected by culture on 24 HVs. It is recommended that HV banks employ donor selection to exclude donors at risk of TB, to culture material for mycobacteria, and to investigate potential sources when clusters of NTBM are found to facilitate corrective and preventative actions.

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.

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 ofM 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 ofRFLP fingerprints for long term local surveillance and large epidemiological studies. (Jr Clin Pathol: Mol Pathol 1995;48:M133-M135)

The degree of risk of cross-infection of patients via lung function testing equipment has yet to be quantified. Based on current evidence, elaborate precautions are not justified for the majority of patients attending the laboratory, but attention to appropriate routine cleaning and disinfection protocols is important. Disinfection and sterilization can be achieved by a variety of methods, although chemical methods should be used with caution. Identification of factors increasing the susceptibility or infectivity of particular patients is important in determining appropriate precautions. Where patients are known to be infectious or are immunocompromized, additional precautions such as using a barrier filter may be appropriate. However, because of cost constraints, the routine use of barrier filters is difficult to justify based on current evidence of minimal cross-infection associated with lung function equipment. Until further studies have been conducted to quantify the degree of risk of cross-infection that lung function test equipment poses, the recommendations given in this review provide a practical approach to dealing with this problem.

The frequency of carriage of antibiotic-resistant bacteria in healthy 7- and 8-year-old children in Bristol was studied. Children born in Avon between 1 April 1991 and 31 December 1992, attending the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC) 7 year follow-up clinic, formed the study population. Carriage was estimated using mouth and stool samples. None of 105 children on whom information was available had received tetracycline, chloramphenicol, ciprofloxacin or an extended-spectrum cephalosporin in the previous year. Staphylococcus aureus was isolated from mouthwashes from 200 (37.1%) of 539 children sampled. Six (3%) of the isolates were resistant to chloramphenicol or tetracycline and four (2%) were methicillin resistant. Haemophilus spp. were isolated from 369 (72%) of 513 samples and 63 (17%) were ampicillin resistant, 49 (13.3%) were erythromycin resistant and seven (1.9%) were tetracycline resistant. Branhamella catarrhalis was isolated from 333 (74%) of 450 samples. Twenty-eight (8.4%) were erythromycin resistant and 14 (4.2%) strains were tetracycline resistant. Group A beta-haemolytic streptococci were isolated from 17 of 507 children sampled. One (5.9%) was tetracycline resistant. Stool samples were returned from 335 (62%) of 539 children from whom they were requested. Eleven per cent of samples yielded Gram-negative bacilli with high-level resistance to chloramphenicol, which was frequently linked to resistance to ampicillin, spectinomycin and streptomycin. Isolates demonstrating resistance to the third-generation cephalosporin ceftazidime were recovered from 17 subjects (3.2%). Six (35%) of 17 isolates possessed extended-spectrum beta-lactamases. Healthy children carry bacteria resistant to antibiotics to which children are not usually exposed. Resistance to ceftazidime, chloramphenicol and tetracycline may be co-selected by exposure to other antibiotics used in children or may be acquired from family members, pets, other children or food. These results suggest that antibiotic-resistant bacteria are widely disseminated and may be acquired by children before exposure to specific selection pressure.

Ciprofloxacin-resistant gonococci have been isolated from patients in the United Kingdom since 1993. Until recently, evidence has suggested that the majority of infections are not endemic but have been acquired abroad. In October 1999, increasing numbers of ciprofloxacin resistant isolates of the non-requiring auxotype were reported in Oldham and Rochdale (Northwest England). These and similar isolates from elsewhere in England and Wales were genetically characterized using a simplified opa-typing method (a non-radioactive PCR–RFLP method targeting the opa family of genes). Of 73 isolates studied, 24 had unique opa-types (10 from infections acquired abroad), whilst the remaining 49 were indistinguishable (none were known to be acquired abroad). This cluster included 31 isolates from Oldham and Rochdale, 16 from elsewhere in the north of England, and 2 from Southern England and South Wales with known epidemiological links to cases from Manchester and Rochdale respectively. This study illustrates the potential for spread of an antibiotic resistant clone of N. gonorrhoeae both locally and nationally and demonstrates that endemic acquisition of ciprofloxacin-resistant gonococci is now a significant problem in the United Kingdom.

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 36 days (range 3-248 days) 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.

In penicillinase-producing Neisseria gonorrhoeae (PPNG), resistance to penicillin may be mediated by one of several related plasmids of different sizes. These include the Asian, African and Rio/Toronto plasmids. Identification of these plasmids provides useful epidemiological information, but has necessitated plasmid purification and gel analysis. We have developed a rapid, simple multiplex polymerase chain reaction (PCR) which discriminates between the beta-lactamase resistance plasmids that are frequently found in strains of N. gonorrhoeae. Amplicons of 1191, 958 and 650 bp were produced from strains containing the African, Asian and Rio/Toronto plasmids, respectively, whilst no products resulted from non-PPNG strains harbouring the cryptic, conjugative or tetracycline resistance plasmids. PCR analysis of 123 strains of PPNG identified 60 strains with African, 16 strains with Asian and 47 strains with Rio/Toronto plasmids and showed complete agreement with the standard plasmid analysis.

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.

OBJECTIVE: To examine the epidemiology of the tetM gene in Neisseria gonorrhoeae strains with high level resistance to tetracycline (TRNG) using a polymerase chain reaction (PCR) assay. METHODS: A single tube PCR was developed which distinguishes between the American and Dutch variants of the tetM gene. Between 1988 and 1995, 518 strains of TRNG (tetracycline MIC > 8.mg/l) were referred to the Gonococcus Reference Unit by other laboratories or isolated from routine swabs taken at local clinics. The strains were analysed for plasmid content, auxotype, serovar, and the tetM gene type. Travel details of the patients were determined by a questionnaire. RESULTS: A PCR product was obtained from all TRNG examined. 387 TRNG strains produced a 778 bp PCR product (American type tetM) and 131 produced a 443 by PCR product (Dutch type tetM). Infections acquired in the United Kingdom contributed 57% of the TRNG strains included in this study; 82% of these carried the American type of tetM. The number of UK acquired TRNG received by the GRU increased each year except 1993--from four strains received in 1990 to 92 in 1995. After the United Kingdom, Caribbean and African countries contributed most strains, with 56 and 60 TRNG acquired in each area respectively. All strains originating in Africa, except one from South Africa, contained the American type tetM. Infections caught in Nigeria and Kenya contributed most strains (15 and 14 respectively). The TRNG originating from Caribbean countries comprised 36% Dutch tetM type. Infections caught in Jamaica accounted for 82% of the Caribbean strains. All 35 TRNG strains originating in the Far East contained the Dutch type tetM. 25 of the Far East strains were also penicillinase producing (PPNG). Infections originating in Indonesia accounted for 49% of the Far East strains but these belonged to 12 different auxotype/serovar combinations. A geographical variation in the type of penicillinase coding plasmids found in PPNG/TRNG was also detected. CONCLUSIONS: These data suggest that the Dutch type tetM may have originated in the Far East and the American type in the African continent. Subsequent spread has resulted in a heterogeneous distribution of TRNG types in other parts of the world. At completion of the survey the numbers of TRNG imported each year from the major overseas sources had reached a plateau while UK contracted TRNG continued to rise providing evidence for the establishment of endemic TRNG strains in the United Kingdom.

PURPOSE:To identify an antimicrobial factor previously demonstrated in rabbit aqueous humour.METHODS:Rabbit aqueous humour was fractionated by a multi-stage process involving anion-exchange and size-exclusion liquid chromatography. The antimicrobial effect of aqueous humour fractions upon Staphylococcus aureus were evaluated in an in vitro model. The components of aqueous humour fractions mediating an antimicrobial effect were investigated by SDS-PAGE.RESULTS:A single peptide of molecular weight approximately 8 kDa was identified which mediated an antimicrobial effect upon Staphylococcus aureus. Attempts to identify the peptide have been unsuccessful.CONCLUSIONS:Rabbit aqueous humour contains an unidentified peptide that mediates an antimicrobial effect upon Staphylococcus aureus. If such a peptide is present in human aqueous humour it may contribute to the apparent resistance to bacterial infection manifest in the anterior chamber.

ABSTRACT We have developed a PCR-based method for the subspecific discrimination of Aspergillus fumigatus types by using two primers designed to amplify the intergenic spacer regions between ribosomal DNA transcription units. The method permitted the reproducible discrimination of 11 distinct DNA types among a total of 119 isolates of A. fumigatus collected from patients and from the environment of a bone marrow transplantation (BMT) unit over a three-year period. Ten DNA types of A. fumigatus were isolated from patients in the BMT unit; eight of these types were also found in the hospital environment, and six of these were present in the unit itself. Thirteen BMT patients developed infection with one of three DNA types some months after these had first been found in the environment of the unit. In other instances, the same DNA types of A. fumigatus were isolated from BMT patients that were later recovered from the environment of the unit. Several DNA types of A. fumigatus were found in the hospital environment over an 18-month period. Molecular typing of multiple isolates of A. fumigatus , obtained from postmortem tissue samples, showed that one patient was infected with a single DNA type, but two others had up to three different DNA types. Our findings suggest that A. fumigatus infection in BMT recipients may be nosocomial in origin and underline the need for careful environmental monitoring of units in which high-risk patients are housed.