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.

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.

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.

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.

Kinetics of the in vitro gelation of a sickling haemoglobin from Hog deer (Axis porcinus).

In order to distinguish the molecular mechanisms involved in the polymerisation of animal and human sickling haemoglobins, the gelation properties of hog deer (Axis porcinus) haemoglobin have been studied. Continuous monitoring of viscosity and minimum gelling concentration measurements of hog deer haemolysates were made over a range of pH, temperature, ionic strengths and in the presence of urea and tris(hydroxymethyl)methylamine. The inhibition of gelling caused by lowering the pH or increasing ionic strength and the abolition of the reversible endothermic nature of gelation by urea suggest that electrostatic interactions predominate in polymerisation but that weak hydrogen and hydrophobic bonds may also be present. Kinetic viscosity data demonstrated a pre-gelation lag phase, dependent on ahigh power of the haemoglobin concentration, similar to the nucleation of human haemoglobin S (HbS). The results indicate similarities in the kinetics of hog deer polymerisation with those of HbS, but major differences in the type of intermolecular attractions involved.

Minisatellites corresponding to the human polycore probes 33.6 and 33.15 in the genome of the most 'primitive' known eukaryote Giardia lamblia.

DNA fingerprinting has been widely used for genetic characterization and individual recognition in a range of species, from man and other mammals down the evolutionary scale to some lower eukaryotic parasites. These techniques utilise repetitive elements first characterised in the human genome, known as minisatellites, which display extensive allelic variability. Few biological or biochemical characteristics have been found that distinguish isolates of Giardia lamblia (Gl), or their apparent variations in virulence. We have characterized 21 Gl isolates in axenic culture using DNA fingerprinting with the human minisatellite probes, 33.6 and 33.15. Up to 12 variable bands per isolate were recognized in the size range of 2.5 to 15 kb by Southern blot hybridization of restriction endonuclease-digested Gl DNA. Most isolates demonstrated a distinct banding pattern or DNA fingerprint. The results suggest that this method may provide a basis for the detailed genotypic characterization of Gl which will be amenable to computer and statistical analysis for use in studies of virulence and epidemiology. Also, as Gl occupies a unique phylogenetic position as a member of the earliest known divergence from the eukaryotic line of descent, this study may provide a useful model for the study of other important eukaryotic pathogens, as it is rapidly becoming apparent that minisatellites are ubiquitous components of eukaryotic genomes.

Efficient translation and polyribosome binding of 125I-labelled rabbit globin messenger ribonucleoprotein.

Rabbit polyribosomal globin messenger ribonucleoprotein (mRNP) was labelled under mild conditions, using 125I and Iodogen, in the protein moiety so that the fate of mRNA-associated proteins could be followed during translation. 125I-mRNP was shown to retain functional activity in the nuclease-treated reticulocyte lysate translation system under optimal labelling conditions. Polyribsome binding of 125I-mRNP and its sensitivity to cycloheximide indicated a functional- and translation-dependent binding of mRNP proteins. The results constitute a successful and direct approach to the study of mRNA-associated proteins in translational control.

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.

Modulation of pristane-induced arthritis by mycobacterial antigens.

Several prominent mycobacterial protein antigens involved in antibody and T cell responses have been identified as members of highly conserved heat shock protein families. In particular, immune responses to the mycobacterial 65 kD heat shock protein (hsp65) have been implicated in the pathogenesis of autoimmune diseases both in experimental animal models and in man. Additionally, hsp65 has been shown to modulate the course of autoimmune disease in such experimental animal systems. In this report, we have examined the synthesis of heat shock proteins by a fast growing mycobacterial strain, M. vaccae, in heat stressed cultures and used the pristane induced arthritis model to investigate the immunoprophylactic and immunotherapeutic potential of heat killed M. vaccae. Heat shock of M. vaccae cultures at 48 degrees C demonstrated a 43-fold increase in hsp65 over that expressed at 37 degrees C. It is therefore suggested that heat killed M. vaccae contains sufficient hsp that can be presented in the context of appropriate adjuvant properties for use as an effective immunomodulatory agent. Immunisation experiments with M. vaccae revealed that protection or exacerbation of pristane induced arthritis was dependent on the dose (given in an oil or aqueous suspension), route and time of immunisation. In addition, it was demonstrated that the development of arthritis correlated with high levels of agalactosyl IgG and that "protected" animals had significantly depressed levels.

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.

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.

Development of an in vitro model of Toxoplasma gondii cyst formation.

Toxoplasma gondii tissue cyst reactivation is a major pathogenic mechanism in ocular toxoplasmosis, disease associated with AIDS and organ transplantation. The mechanisms associated with cyst formation and reactivation have not been elucidated. The complexity of studying these issues in animal models has led to the development of in vitro tissue culture strategies for cyst formation. In the present study we have adopted the human embryonic lung fibroblast (HEL) as the host cell and have compared the cyst forming abilities of eight clinical isolates. We describe by transmission electron microscopy and quantitative light microscopy the development of cysts in vitro. The numbers of in vitro cysts increased with time for all isolates. Cyst cultures were stabilised by manipulation of the free parasite load, an observation not previously recorded. Thus, in this paper we describe a viable model for the analysis of the mechanisms of Toxoplasma cyst development.

Protein synthesis is shutdown in dormant Mycobacterium tuberculosis and is reversed by oxygen or heat shock.

Oxygen-limiting conditions are critical to the survival of the bacteria in tuberculosis. Mycobacterium tuberculosis can survive anaerobiosis in vitro for long periods of time only after a gradual transition to a microaerophilic stationary phase. The underlying mechanism behind stationary phase adaption needs to be elucidated. The protein profiles of Mycobacterium tuberculosis during long-term stationary phase growth and under strict anaerobic incubation were monitored by [35S]methionine labelling, SDS-PAGE and fluorography. These experiments have established that protein synthesis gradually decreased over 50 days in the long-term stationary phase cultures which were considered to be microaerophilic. There was an 80% linear decrease in the level of total protein synthesis during the first 40 days of microaerophilic growth and then the rate of protein synthesis faded quickly. For the first time we have shown that total protein synthesis shutdown occurred when bacilli were incubated under further anaerobic conditions. Viability, estimated by cfu counts, remained constant during stationary phase growth and under anaerobic incubation. Furthermore, when oxygen was introduced into the anaerobic culture, protein synthesis restarted. Also heat shock at 45 degrees C, 48 degrees C and 50 degrees C rapidly induced protein synthesis in stationary and anaerobic cultures. These data indicate that dormant bacteria shut down protein synthesis but remain responsive to specific stimuli which restore protein synthesis. In addition the dormant bacilli induced by anaerobiosis developed more heat resistance than nondormant organisms.

Recognition of tissue cyst-specific antigens in reactivating toxoplasmosis.

Current serological tests do not discriminate between asymptomatic latent Toxoplasma gondii infection and reactivating toxoplasmosis, but timely therapeutic intervention before the development of symptoms would lead to major reductions in morbidity and permanent disability. This study developed a new enzyme-linked immunosorbent assay (ELISA) for antibody to T. gondii tissue cyst antigens and screened tissue cyst antigens by Western blot analysis to test the hypothesis that antibody recognition of T. gondii tissue cyst-derived antigen is a good indicator of reactivation disease. A total of 187 sera was tested by Sabin-Feldman dye test and tissue cyst ELISA, AIDS patients and patients with ocular disease were considered separately, as the exposure to parasite antigens may be different in these two groups. The dye test did not discriminate between immunocompetent and immunocompromised T. gondii seropositive patients or between active and quiescent toxoplasmosis. Tissue cyst ELISA demonstrated a raised specific antibody response in immunocompetent T. gondii seropositive patients and in quiescent HIV positive sera. These data support th view that the tissue cyst population is in a state of dynamic equilibrium. It is proposed that, in the immunocompetent host, tissue cyst development and rupture are under some degree of immune control, but that in the immunocompromised host this equilibrium is disturbed and reactivation disease results. Data from patients with reactivating ocular toxoplasmosis demonstrate that tissue cyst-specific antibody levels are not different in active and quiescent disease and indeed they are not significantly different from immunocompetent T. gondii seronegative sera. In the Western blot analysis of 57 HIV positive patient sera, eight antigens (65, 57, 49, 47, 36, 28, 26 and 18 kDa) were consistently recognised by one third or more of the sera tested, but no single antigen was diagnostic of quiescent or active toxoplasmosis. It is concluded that tissue cyst-derived antigens are not a reliable serological marker of reactivating toxoplasmosis.

Quantification of cytomegalovirus DNA in blood specimens from bone marrow transplant recipients by the polymerase chain reaction.

A nested PCR system for cytomegalovirus (CMV) DNA in blood specimens from bone marrow transplant recipients is described, in which the biotinylated tritium-labelled product from the second round of PCR is quantified using streptavidin-coated fluorometric Scintillation Proximity Assay (SPA) beads (Amersham, UK). This assay has been compared with a PCR procedure based on limiting-dilution, in which the end-point is determined visually following electrophoresis in agarose gel. The two systems were shown to be equivalent in sensitivity and specificity on testing stored serial blood samples from six CMV antibody-positive allogeneic bone marrow transplant patients who developed viraemia as detected by conventional methods of virus isolation in tissue culture.

A polymerase chain reaction to detect a spliced late transcript of human cytomegalovirus in the blood of bone marrow transplant recipients.

A reverse transcription (RT) nested polymerase chain reaction (PCR) procedure is described for detecting RNA to a spliced late gene (SLG) of human cytomegalovirus (CMV), the product of which (175 bp) is easily differentiated in agarose gels from the product when the target is unspliced viral RNA or DNA (258 bp). The SLG-RT-PCR has been compared against a semi-quantitative PCR for CMV DNA in buffy-coat specimens collected weekly after bone marrow transplantation from 3 patients and against the results of culturing these specimens for CMV both by conventional virus isolation, based on the detection of cytopathic effect, and by the early detection of infected cells by staining with virus-specific monoclonal antibodies. The detection of CMV RNA by SLG-RT-PCR correlated well with the detection of infective virus but only when the results of both culture methods were combined, in that neither culture method alone was as sensitive as the SLG-RT-PCR. The presence of SLG RNA in the circulation is of value as a marker of active CMV infection.

New strategies in microbiological diagnosis.

With the advent of the polymerase chain reaction (PCR), molecular biology is at last poised to enter the clinical microbiology laboratory. We describe this technique, and review its present and future applications in the diagnosis of infectious disease, with particular emphasis on its potential in diagnostic bacteriology. We discuss the suitability of different sequences as targets for DNA amplification. The disadvantages of PCR as a diagnostic strategy are covered, and current technical problems with the method are surveyed. We briefly mention two alternative strategies--the transcript-based amplification system and replicatable RNA reporter systems based on the Q beta replicase.

Antibodies to gut protozoa in commercial immunoglobulin preparations.

Using parasite agglutination, indirect immunofluorescence, enzyme-linked immunosorbent assay, and immunoblotting, Giardia lamblia specific antibody was detected in 5 commercially available immunoglobulin (IgG) preparations. IgG antibodies to Entamoeba histolytica were either present in very low titre or were absent. Immunoblotting showed that anti-G. lamblia antibodies were detected towards a range of Giardia proteins, 25-200 kDa. These findings raise the possibility that pooled human IgG preparations could be evaluated in the treatment of chronic giardiasis which is refractory to conventional therapy.

Application of the polymerase chain reaction to the diagnosis of human toxoplasmosis.

Toxoplasmosis may cause significant damage to the developing fetus and is a life-threatening opportunistic infection in immunocompromised persons. Serological investigation is unreliable, while isolation of the parasite is time consuming and may lack sensitivity. We have developed a system for detecting Toxoplasma gondii based on the amplification of the P30 gene using sequential rounds of PCR and nested primers. The clinical value of this technique was assessed by the investigation of a range of tissues taken from pregnant women, fetuses, neonates, AIDS patients and organ graft recipients. The PCR assay produced more positive reactions than isolation of the parasite by means of cell culture or animal inoculation. Extended autoradiography was found to be more sensitive than stained agarose gels for detecting the PCR product. Systematic contamination of PCR reactions was avoided but it was not possible to exclude sporadic contamination in certain cases. Detection of specific DNA is of clinical value in the investigation of the pregnant woman in order to assess the risk of transplacental passage of infection and in the fetus and neonate to identify congenital toxoplasmosis. Even so, PCR findings must be interpreted with caution because of the risk of a sample being contaminated. PCR may be the investigation of choice when brain biopsy is performed on a patient with AIDS and when toxoplasmosis associated with bone marrow transplantation is suspected.