Diagnostic accuracy of a prototype rapid chlamydia and gonorrhoea recombinase polymerase amplification assay: a multicentre cross-sectional preclinical evaluation.

OBJECTIVES: Rapid and accurate sexually transmitted infection diagnosis can reduce onward transmission and improve treatment efficacy. We evaluated the accuracy of a 15-minute run-time recombinase polymerase amplification-based prototype point-of-care test (TwistDx) for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). METHODS: Prospective, multicentre study of symptomatic and asymptomatic patients attending three English sexual health clinics. Research samples provided were additional self-collected vulvovaginal swab (SCVS) (female participants) and first-catch urine (FCU) aliquot (female and male participants). Samples were processed blind to the comparator (routine clinic CT/NG nucleic acid amplification test (NAAT)) results. Discrepancies were resolved using Cepheid CT/NG GeneXpert. RESULTS: Both recombinase polymerase amplification and routine clinic NAAT results were available for 392 male and 395 female participants. CT positivity was 8.9% (35/392) (male FCU), 7.3% (29/395) (female FCU) and 7.1% (28/395) (SCVS). Corresponding NG positivity was 3.1% (12/392), 0.8% (3/395) and 0.8% (3/395). Specificity and positive predictive values were 100% for all sample types and both organisms, except male CT FCU (99.7% specificity (95% confidence interval (CI) 98.4-100.0; 356/357), 97.1% positive predictive value (95% CI 84.7-99.9; 33/34)). For CT, sensitivity was ≥94.3% for FCU and SCVS. CT sensitivity for female FCU was higher (100%; 95% CI, 88.1-100; 29/29) than for SCVS (96.4%; 95% CI, 81.7-99.9; 27/28). NG sensitivity and negative predictive values were 100% in FCU (male and female). CONCLUSIONS: This prototype test has excellent performance characteristics, comparable to currently used NAATs, and fulfils several World Health Organization ASSURED criteria. Its rapidity without loss of performance suggests that once further developed and commercialized, this test could positively affect clinical practice and public health.

Genome sequencing and characterization of an extensively drug-resistant sequence type 111 serotype O12 hospital outbreak strain of Pseudomonas aeruginosa.

A series of extensively drug-resistant isolates of Pseudomonas aeruginosa from two outbreaks in UK hospitals were characterized by whole genome sequencing (WGS). Although these isolates were resistant to antibiotics other than colistin, we confirmed that they are still sensitive to disinfectants. The sequencing confirmed that isolates in the larger outbreak were serotype O12, and also revealed that they belonged to sequence type ST111, which is a major epidemic strain of P. aeruginosa throughout Europe. As this is the first reported sequence of an ST111 strain, the genome was examined in depth, focusing particularly on antibiotic resistance and potential virulence genes, and on the reported regions of genome plasticity. High degrees of sequence similarity were discovered between outbreak isolates collected from recently infected patients, isolates from sinks, an isolate from the sewer, and a historical isolate, suggesting that the ST111 strain has been endemic in the hospital for many years. The ability to translate easily from outbreak investigation to detailed genome biology by use of the same data demonstrates the flexibility of WGS application in a clinical setting.

Moxifloxacin and pyrazinamide susceptibility testing in a complex case of multidrug-resistant tuberculosis.

Multidrug-resistant tuberculosis (MDR-TB) is a public health problem of global concern. It is critical that drug susceptibility testing (DST) methods accurately predict clinical response. We present a patient with a challenging case of MDR-TB with additional resistance to quinolones and pyrazinamide. Treatment with a regimen including high-dosage moxifloxacin, based on additional genotypic and phenotypic DST, produced excellent results. This case highlights the possibility of treatment with high-dose fluoroquinolones despite apparent bacterial resistance to these agents. Improved DST methods are necessary for both agents. Development of genotypic approaches may offer a susceptibility profile rapidly, enabling early introduction of individualised treatments.

Prevalence and clinical correlations of genetic subtypes of Giardia lamblia in an urban setting.

The clinical significance of different genetic subtypes or assemblages of Giardia lamblia is uncertain. Cases of giardiasis in south-west London between 1999 and 2005 were studied, comparing molecular-typing results with clinical and epidemiological findings from routine surveillance. We identified 819 cases, of whom 389 returned surveillance questionnaires. A subset of 267 faecal samples was submitted for typing by sequencing of the triose phosphate isomerase (tpi) and ribosomal RNA genes, and/or a separate duplex PCR of the tpi gene. Typing was successful in 199 (75%) samples by at least one of the molecular methods. Assemblage A accounted for 48 (24%) samples and Assemblage B for 145 (73%); six (3%) were mixed. Both assemblages had similar seasonality, age distribution and association with travel. Clinical features were available for 59 successfully typed cases: both assemblages caused similar illness, but Assemblage A was significantly more frequently associated with fever than Assemblage B.

Whole genome analysis using microarrays.

The development of microarray technology has allowed the genomes of mycobacteria to be directly compared to identify DNA regions that differ between strains due to deletion, insertion, or sequence divergence. The use of microarrays in comparative genomics has proved to be a valuable tool for comparing both mycobacterial species and strains. We describe here the methodology for comparing two mycobacterial DNA samples by microarray hybridization, from labeling and slide preparation, to DNA microarray analysis options. Further developments in microarray design and methodology promise to ensure that microarrays remain an important resource for comparative genomic studies in the future.

The Use of Rap-Pcr in Studying Mycobacterium Tuberculosis Intracellular Gene During Macrophage Infection

Mycobacterium tuberculosis is the second leading cause of death from infectious agent. This study sought to detect M. tuberculosis genes; which were specifically expressed; or upregulated during intracellular infection of J774 murine macrophages; as such genes may be potential targets for novel drug action. J774 murine macrophage cell line was infected with M. tuberculosis (H37Rv strain) at 10:1 multiplicity of infection (MOI). RNA was differentially extracted from M. tuberculosis infecting J774 macrophage cell line. The control in this case was RNA from extracellular broth grown bacteria. Approximately 50 ng of RNA from intracellular derived bacteria and extracellular derived bacteria (control) were subjected to random arbitrarily primed PCR (RAP-PCR) using 50 primer combinations. Eleven differential RAP-PCR products were observed. All RAP-PCR products were cloned into pCRr2.1 and sequenced in order to determine the identity of the products. Four of the eleven products were derived from macrophage genes and another 4 products were derived from the M. tuberculosis rRNA genes (three 23S and one 16S rRNA). The 3 remaining RAP-PCR products were found to be mycobacterial genes other than ribosomal genes. The three products were genes encoding enzyme involving in a shikimate pathway; a putative carboxyphosphonoenolpyruvate phosphonomutase and a serine protease with homology to HtrA. Of the 3 mycobacterial genes other than ribosomal genes detected; none were specifically expressed during intracellular infection but bacilli

Mycobacterium tuberculosis DNA repair in response to subinhibitory concentrations of ciprofloxacin.

OBJECTIVES: To investigate how the SOS response, an error-prone DNA repair pathway, is expressed following subinhibitory quinolone treatment of Mycobacterium tuberculosis. METHODS: Genome-wide expression profiling followed by quantitative RT (qRT)-PCR was used to study the effect of ciprofloxacin on M. tuberculosis gene expression. RESULTS: Microarray analysis showed that 16/110 genes involved in DNA protection, repair and recombination were up-regulated. There appeared to be a lack of downstream genes involved in the SOS response. qRT-PCR detected an induction of lexA and recA after 4 h and of dnaE2 after 24 h of subinhibitory treatment. CONCLUSIONS: The pattern of gene expression observed following subinhibitory quinolone treatment differed from that induced after other DNA-damaging agents (e.g. mitomycin C). The expression of the DnaE2 polymerase response was significantly delayed following subinhibitory quinolone exposure.

Inactivation of polyketide synthase and related genes results in the loss of complex lipids in Mycobacterium tuberculosis H37Rv.

AIMS: Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. METHODS AND RESULTS: Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirmed to be within the M. tuberculosis open reading frames for tesA (a probable thioesterase), drrB (predicted ABC transporter), pks11 (putative chalcone synthase) and pks6 (polyketide synthase). The first three of these transposon mutants were unable to generate PDIM and the fourth lacked novel polar lipids. CONCLUSIONS: Mycobacterium tuberculosis can be cultivated in vitro without the involvement of certain lipid synthesis genes, which may be necessary for in vivo pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of transposon mutants is a new functional genomic approach for the eventual definition of the mycobacterial 'lipidome'.

Molecular epidemiology of tuberculosis in London 1995-7 showing low rate of active transmission.

BACKGROUND: Tuberculosis notification rates for London have risen dramatically in recent years. Molecular typing of Mycobacterium tuberculosis has contributed to our understanding of the epidemiology of tuberculosis throughout the world. This study aimed to assess the degree of recent transmission of M tuberculosis in London and subpopulations of the community with high rates of recent transmission. METHODS: M tuberculosis isolates from all persons from Greater London diagnosed with culture positive tuberculosis between 1 July 1995 and 31 December 1997 were genetically fingerprinted using IS6110 restriction fragment length polymorphism (RFLP) typing. A structured proforma was used during record review of cases of culture confirmed tuberculosis. Cluster analysis was performed and risk factors for clustering were examined in a univariate analysis followed by a logistic regression analysis with membership of a cluster as the outcome variable. RESULTS: RFLP patterns were obtained for 2042 isolates with more than four copies of IS6110; 463 (22.7%) belonged to 169 molecular clusters, which ranged in size from two (65% of clusters) to 12 persons. The estimated rate of recent transmission was 14.4%. Young age (0-19 years) (odds ratio (OR) 2.65, 95% confidence interval (CI) 1.59 to 4.44), birth in the UK (OR 1.55, 95% CI 1.04 to 2.03), black Caribbean ethnic group (OR 2.19, 95% CI 1.15 to 4.16), alcohol dependence (OR 2.33, 95% CI 1.46 to 3.72), and streptomycin resistance (OR 1.82, 95% CI 1.15 to 2.88) were independently associated with an increased risk of clustering. CONCLUSIONS: Tuberculosis in London is largely caused by reactivation or importation of infection by recent immigrants. Newly acquired infection is also common among people with recognised risk factors. Preventative interventions and early diagnosis of immigrants from areas with a high incidence of tuberculosis, together with thorough contact tracing and monitoring of treatment outcome among all cases of tuberculosis (especially in groups at higher risk of recent infection), remains most important.

Early detection of cytomegalovirus (CMV) infection in bone marrow transplant patients by reverse transcription-PCR for CMV spliced late gene UL21.5: a two site evaluation.

BACKGROUND: Bone marrow transplant (BMT) patients at risk of developing cytomegalovirus (CMV) pneumonitis are identified routinely by the early detection of virus in blood. For early diagnosis of CMV infection, the RNA-based approach demonstrates advantages when compared with the current CMV antigen and DNA detection methods. OBJECTIVES: We have evaluated our previously developed reverse transcription-polymerase chain reaction (RT-PCR) to a spliced late CMV gene (SLG; J. Virol. Methods 56 (1996), 139) to monitor CMV infection in BMT patients at two clinical sites. The diagnostic value of the SLG RT-PCR was compared with the routine CMV antigen and DNA detection methods. STUDY DESIGN: Weekly blood samples from BMT patients were tested for CMV during the first 3 months post-transplant. The qualitative SLG RT-PCR, semiquantitative DNA PCR, and viral antigen tests were compared. The RNA and DNA PCR results were analysed in terms of their temporal relationship and consistency of CMV detection and compared with CMV infection diagnosed by viral antigen tests. RESULTS: Of the 101 BMT recipients studied, 25 developed CMV antigenemia and/or DNAemia resulting in symptomatic infection in two patients. All CMV PCR-positive patients were either CMV seropositive pretransplant or received marrow from seropositive donor. The highest incidence of CMV infection was seen in seropositive recipients (R+) irrespective of the donor's status. Detection of CMV infection by SLG RNA preceded CMV DNA detection by 0-2 weeks (median 1 week) and CMV antigen detection by 0-8 weeks (median 3 weeks). Once detected, the SLG RNA remained consistently positive before antiviral treatment was commenced. Both the SLG RNA and CMV DNA detection methods had the same clinical sensitivity, specificity, positive and negative predictive values of 100, 94, 80 and 100%, respectively. CONCLUSIONS: The RT-PCR for SLG RNA proved to be the earliest indicator of CMV infection in BMT patients demonstrating a sustained pattern of CMV detection during the 3 months post-transplant period. Although very similar in its diagnostic performance to CMV DNA PCR the SLG RNA RT-PCR does not require quantitation and provides an efficient and ongoing indication of active CMV infection.

Whole genome comparison of Campylobacter jejuni human isolates using a low-cost microarray reveals extensive genetic diversity.

Campylobacter jejuni is the leading cause of bacterial food-borne diarrhoeal disease throughout the world, and yet is still a poorly understood pathogen. Whole genome microarray comparisons of 11 C. jejuni strains of diverse origin identified genes in up to 30 NCTC 11168 loci ranging from 0.7 to 18.7 kb that are either absent or highly divergent in these isolates. Many of these regions are associated with the biosynthesis of surface structures including flagella, lipo-oligosaccharide, and the newly identified capsule. Other strain-variable genes of known function include those responsible for iron acquisition, DNA restriction/modification, and sialylation. In fact, at least 21% of genes in the sequenced strain appear dispensable as they are absent or highly divergent in one or more of the isolates tested, thus defining 1300 C. jejuni core genes. Such core genes contribute mainly to metabolic, biosynthetic, cellular, and regulatory processes, but many virulence determinants are also conserved. Comparison of the capsule biosynthesis locus revealed conservation of all the genes in this region in strains with the same Penner serotype as strain NCTC 11168. By contrast, between 5 and 17 NCTC 11168 genes in this region are either absent or highly divergent in strains of a different serotype from the sequenced strain, providing further evidence that the capsule accounts for Penner serotype specificity. These studies reveal extensive genetic diversity among C. jejuni strains and pave the way toward identifying correlates of pathogenicity and developing improved epidemiological tools for this problematic pathogen.

Pulmonary tuberculosis in Kumasi, Ghana: presentation, drug resistance, molecular epidemiology and outcome of treatment.

To assist implementation of tuberculosis (TB) control measures, knowledge of the disease characteristics in a community is essential. This study in Kumasi, Ghana, correlates the clinical presentation, microbiology, molecular epidemiology and clinical outcome of thirty consecutively diagnosed patients with new smear-positive pulmonary TB. Several important factors that potentially promote disease transmission in the community were identified: patients had prolonged duration of productive cough prior to diagnosis (mean=4.1 months; SD=2.1); the disease was typically advanced at presentation and Ziehl-Neelson sputum smears indicated a high bacterial load (80% graded > AFB++); home accommodation was overcrowded with a mean of 3.3 other persons sleeping in the same room as the patients at night. IS6110 restriction fragment length polymorphism (RFLP) fingerprinting of 25 isolated (23 Mycobacterium tuberculosis and 2 Mycobacterium africanum) from epidemiologically unrelated cases identified 3 identical strains and 3 clusters containing 2, 4 and 8 isolates of > or =80% similarity, suggesting high rates of disease transmission. A high prevalence of primary resistance to isoniazid was found (6 out 26; 23%) but resistance to rifampicin, pyrazinamide, ethambutol, streptomycin and ciprofloxacin was not detected. Smear coversion at 2 months and final outcome of treatment with short courses chemotherapy were independent of isoniazid resistance, but the rate of treatment default was unacceptably high (37%). High rates of disease transmission, primary isoniazid resistance and treatment default all indicate poor TB control. The use of rifampicin-containing short-course chemotherapy in this community must be accompanied by adequate resources and infrastructure to ensure very stringent treatment supervision to improve case-holding and reduce the risk of multi-drug resistance.

Human cytomegalovirus UL21.5 gene is expressed as an "early-late" gene in cultured human fibroblasts.

The human cytomegalovirus (CMV) UL21.5 gene encodes a secreted glycoprotein of unknown function. Both the UL21.5 protein and mRNA accumulate in abundance at late stages of infection making the RNA an attractive target for diagnosis of active CMV infection. The UL21.5 was originally described as a 'spliced late' gene (SLG) (Rawlinson and Barrell, J. Virol. 67, 5502 (1993)). However, we found that the the UL21.5 mRNA was detectable in CMV-infected patients before the onset of CMV DNA replication (Boriskin et al., J. Clin. Virol., in press). Here, we re-examined the UL21.5 mRNA kinetic class in CMV-infected human fibroblast culture using a RNAse protection assay and RT-PCR. The UL21.5 mRNA was detectable before the "true late" UL75 RNA, was resistant to a CMV DNA replication inhibitor but moderately sensitive to inhibitors of protein synthesis. In the presence of protein synthesis inhibitors the UL21.5 mRNA was detectable only by a nested reverse transcription - PCR (RT-PCR) with the bulk of it in unspliced form. This suggests that splicing factors for UL21.5 mRNA are encoded by the virus rather than by the cell. Our results indicate that UL21.5 should be defined as an "early-late" rather than a "late" (L) CMV gene.

Extraction of RNA from Intracellular Mycobacterium tuberculosis : Methods, Considerations, and Applications.

Pathogenicity in Mycobacterium tuberculosis may be thought of as a multifactorial process with both pathogen and host-response effector molecules contributing to the process of infection, leading either to immunopathology and disease or control of infection and long-term persistence. Little is known about this at a genetic level, but it is becoming recognized that bacterial virulence constitutes the correct temporal and spatial regulation of many genes that may be necessary for a particular phase in infection in response to specific environmental cues.

Detection of mRNA transcripts and active transcription in persistent Mycobacterium tuberculosis induced by exposure to rifampin or pyrazinamide.

Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.

Human cytomegalovirus genome sequences in lymph nodes.

Human cytomegalovirus (CMV) is a major cause of morbidity in heart and lung transplant patients, resulting from immunosuppression-mediated reactivation of latent CMV originating either from the transplanted tissue, or the recipient. We showed that out of eight donor/recipient pairs, the lymph nodes (LNs) of three donors and four recipients, all CMV seropositive, harboured CMV DNA at exceeding levels compared with those of matched blood samples, as well as CMV RNA otherwise undetectable in patients' blood. On follow-up, patients positive for CMV DNA and RNA in LNs developed viraemia 4 to 5 weeks earlier than those initially polymerase chain reaction-negative for CMV. Our results indicate that LN are a significant site for sequestration and persistence of CMV and that LN may be important in seeding of CMV-infected cells into the circulation.

Evaluation of reverse transcription-PCR and a bacteriophage-based assay for rapid phenotypic detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis.

New rapid phenotypic assays for the detection of rifampin resistance in Mycobacterium tuberculosis have recently been described, but most of these require liquid cultures, which reduces the utility of many tests in terms of turnaround times. In the United Kingdom, over 90% of rifampin-resistant isolates are also resistant to isoniazid, so rifampin resistance can be used as a sensitive marker for multidrug-resistant tuberculosis. In this study, two new rapid phenotypic assays were compared to the standard resistance ratio method on 91 clinical isolates of M. tuberculosis. One, the phage amplified biologically (PhaB) assay, has been described previously and is based on the inability of susceptible isolates of M. tuberculosis to support the replication of bacteriophage D29 in the presence of inhibitory doses of rifampin. The other employed reverse transcription (RT)-PCR to demonstrate a reduction in inducible dnaK mRNA levels in susceptible isolates treated with rifampin. After incubation for 18 h with 4 microg of rifampin per ml, the PhaB assay showed concordance with the resistance ratio method for 46 of 46 (100%) susceptible and 31 of 31 (100%) resistant isolates, while RT-PCR showed concordance for 46 of 48 (96%) susceptible and 35 of 36 (97%) resistant isolates. We believe these assays provide a reliable rapid means of susceptibility testing with a total turnaround time of only 48 h, although the PhaB assay is better in terms of its lower technical demand and cost and its applicability to tuberculosis susceptibility testing in developing countries.

Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family.

Mammalian natural resistance-associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe(2+), Mn(2+), Zn(2+), and Cu(2+). Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) also encode an Nramp homologue (Mramp). RNA encoding Mramp induces approximately 20-fold increases in (65)Zn(2+) and (55)Fe(2+) uptake when injected into Xenopus laevis oocytes. Transport is dependent on acidic extracellular pH and is maximal between pH 5.5 and 6.5. Mramp-mediated (65)Zn(2+) and (55)Fe(2+) transport is abolished by an excess of Mn(2+) and Cu(2+), confirming that Mramp interacts with a broad range of divalent transition metal cations. Using semiquantitative reverse transcription PCR, we show that Mramp mRNA levels in M. tuberculosis are upregulated in response to increases in ambient Fe(2+) and Cu(2+) between <1 and 5 microM concentrations and that this upregulation occurs in parallel with mRNA for y39, a putative metal-transporting P-type ATPase. Using a quantitative ratiometric PCR technique, we demonstrate a fourfold decrease in Mramp/y39 mRNA ratios from organisms grown in 5-70 microM Cu(2+). M. bovis BCG cultured axenically and within THP-1 cells also expresses mRNA encoding Mramp. Mramp exemplifies a novel prokaryotic class of metal ion transporter. Within phagosomes, Mramp and Nramp1 may compete for the same divalent cations, with implications for intracellular survival of mycobacteria.

Quantitation of Toxoplasma gondii DNA in a competitive nested polymerase chain reaction.

AIM: To quantify Toxoplasma gondii DNA using a specially constructed artificial template as competitor in a nested polymerase chain reaction (PCR). METHODS: The diagnostic assay was a nested PCR employing four primers that amplify part of the single copy gene for the P30 major surface antigen in T gondii. An artificial competitor containing the four primer binding sites was made first by creating a 216 bp deletion in the native 914 bp full length PCR product using restriction enzyme digestion, ligation of selected digestion fragments, and cloning the ligation product into an E coli plasmid vector for production. Competitive nested PCR using three different quantities of T gondii genomic DNA with four corresponding 10-fold dilutions of the artificial competitor was then performed, and the results visualised with agarose gel electrophoresis. A standard curve was drawn by plotting the T gondii to competitor ratio readings against log10 of the competitor readings. RESULTS: The band intensities on agarose gel showed quantitative amplification in competitive nested PCR. The amount of competitor required to achieve equal molar amounts of PCR products is calculated by reading off the value of the competitor where the T gondii to competitor ratio equals 1 on the standard curves. CONCLUSIONS: Competitive PCR is possible with a nested assay, and quantitative amplification is well preserved. The use of an artificial competitor containing the same primer binding sites as the target enables the absolute amount of T gondii DNA in unknown samples to be estimated. In addition, the competitor simultaneously serves as a control for detecting false negative results of failed reactions in individual assay runs.