The population impact of MRSA in a country: the national survey of MRSA in Wales, 1997.

Continuous data collection on all new isolates of MRSA via CoSurv has taken place in Wales since January 1996. In order to audit this data collection, and to address some of the issues that it does not include, a survey of MRSA was carried out. Questionnaires were completed by infection control teams. Rates were calculated using hospital throughput denominators. Results from the one-day prevalence survey, the two-week incidence survey, and the follow-up survey carried out on new MRSA patients identified in the incidence survey, are presented. Results were found to be broadly similar to those collected via routine surveillance. MRSA was found frequently and disproportionately in the elderly, with higher rates in male than female patients. The highest incidence of total and invasive MRSA was in males aged 75 and over (total: 12.5/1000 finished consultant episodes; invasive: 2.8/1000). Although there was a large community reservoir of MRSA, most appeared to have been acquired in hospital, since most patients had a history of hospitalization, often with multiple hospital admissions. Community-based isolates from cases with no hospital history tended to have been from ulcers. Prevalence and incidence of MRSA was relatively low compared with hospital throughput (mean prevalence: 2.4/100 occupied beds; mean incidence: 3.6/1000 finished consultant episodes), there was also quite large variation between sites, even when screening samples were removed. Patients with MRSA had strikingly long stays before isolation of the organism (prevalence survey: 39 days; incidence survey: 31 days) and highest incidence occurred in elderly care wards. The outcome survey showed that approximately half of the patients were treated with some type of antimicrobial therapy for MRSA. Decontamination therapy was associated with clearance of MRSA only when controlling for sex of the patient. The majority of patients were discharged still with MRSA, mostly to their own homes. The survey emphasizes the need to continue surveillance to detect any changes, to allow guidelines based on evidence to be developed and to monitor the effectiveness of such guidelines.

Resistance to methicillin in isolates of Staphylococcus aureus from blood and cerebrospinal fluid in Wales, 1993-1997.

Surveillance data for organisms isolated from blood cultures and cerebrospinal fluid (CSF) specimens has been gathered electronically in Wales since 1993. Over this period the proportion of total reported organisms from blood cultures and CSF represented by methicillin-resistant staphylococci (MRSA) has risen steadily. This has corresponded to a rise in rates of methicillin resistance amongst Staphylococcus aureus isolated from blood cultures and CSF from 4 to 43%. In certain age/gender groups in 1997, more than 50% of isolates of S. aureus were resistant to methicillin, suggesting that a change in empirical treatment may be necessary for suspected staphylococcal sepsis.

All Wales surveillance of methicillin-resistant Staphylococcus aureus (MRSA): the first year's results.

Over the last five years, hospitals in Wales have experienced difficulties with increasing numbers of isolates of methicillin-resistant Staphylococcus aureus (MRSA). Continuous total population surveillance of MRSA was introduced with the objectives of gaining an understanding of the extent and variation in time and place of its occurrence, the burden of disease and possible risk factors associated with its isolation and resistance to other antibiotics. All first isolates of MRSA from both hospital and community settings and all isolates of methicillin-sensitive Staphylococcus aureus (MSSA) associated with bacteraemia and cerebrospinal fluid (CSF) isolates detected in medical microbiology laboratories in Wales were collected via CoSurv, a set of interconnected data-base modules for communicable disease control. A data set was collected on each isolate and the patient associated with that isolate and compiled centrally at CDSC (Wales) for all-Wales analysis of the MRSA situation. Surveillance started in January 1996 and at the end of the first year, 2700 new isolates of MRSA had been reported from hospital and community settings, giving a rate of 92.43/100,000 population. The incidence of MRSA from bacteraemias and CSF was 5.20/100,000 compared with 12.70/100,000 for MSSA. MRSA from bacteraemia and CSF was significantly more commonly associated with male patients than MSSA. MRSA patients were significantly older. For all MRSA isolates, the highest reporting rate was in men aged 75+ (647.21/100,000). The highest incidence of invasive disease was also in men aged 75+ (45.69/100,000). Isolates from post-surgical patients were more likely to be involved in invasive disease (OR = 2.59), P < 0.001) than strains from other sources. The majority of isolates were resistant to at least two antibiotics in addition to methicillin, most frequently erythromycin and the fluoroquinolones. Very little resistance to fusidic acid, mupirocin or rifampicin was reported. Continuous total population surveillance has provided a minimum incidence of MRSA in Wales and has allowed a simple and intelligible picture of the problem to be determined, which has been fed back to hospitals to assist decisions on control.

Acute and long-term stability studies of deoxy hemoglobin and characterization of ascorbate-induced modifications.

The reaction of ascorbate with recombinant hemoglobin (rHb1.1) in the presence of differing partial pressures of oxygen was studied. In the presence of 15 000 ppm (1.5%) residual oxygen, ascorbate/oxygen-mediated reactions resulted in an increased rate of autoxidation, modification of the beta-globin, increased oxygen affinity and decreased maximum Hill coefficient. One of the observed modifications to the beta-globin was a 72 Da addition to its N-terminus. Detailed characterization indicates the modification was an imidazolidinone type structure. Thorough deoxygenation of the hemoglobin solution to <150 ppm of oxygen prior to addition of ascorbate was required to prevent these modifications. Addition of ascorbate to the deoxy hemoglobin (deoxyHb) at pH 8 induced aggregation, eventually leading to precipitation. No such precipitation was observed at pH 7. Long-term storage of the hemoglobin was carried out by addition of ascorbate to deoxyHb at pH 7. The level of methemoglobin remained at <2% for up to 1 year at 4 degreesC, with no detectable precipitation of the protein. Modifications similar to those observed by the acute studies were observed over the 1-year period and correlated with disappearance of the added ascorbate.

Postanginal septicaemia (Lemmiere's disease) complicated by haemophagocytosis.

We report a case of postanginal septicaemia complicated by bronchopneumonia and haemophagocytosis in a 19-year old male, presenting with severe thrombocytopenia. We believe that this is the first reported case of thrombocytopenia due to haemophagocytosis in this unusual condition.

A human recombinant haemoglobin designed for use as a blood substitute.

The need to develop a blood substitute is now urgent because of the increasing concern over blood-transmitted viral and bacterial pathogens. Cell-free haemoglobin solutions and human haemoglobin synthesized in Escherichia coli and Saccharomyces cerevisiae have been investigated as potential oxygen-carrying substitutes for red blood cells. But these haemoglobins cannot be used as a blood substitute because (1) the oxygen affinity in the absence of 2,3-bisphosphoglycerate is too high to allow unloading of enough oxygen in the tissues, and (2) they dissociate into alpha beta dimers that are cleared rapidly by renal filtration, which can result in long-term kidney damage. We have produced a human haemoglobin using an expression vector containing one gene encoding a mutant beta-globin with decreased oxygen affinity and one duplicated, tandemly fused alpha-globin gene. Fusion of the two alpha-globin subunits increases the half-life of this haemoglobin molecule in vivo by preventing its dissociation into alpha beta dimers and therefore also eliminates renal toxicity.

Was the loss of the D helix in alpha globin a functionally neutral mutation?

Proteins in the globin family are found in a variety of species from bacteria to man. From the many globin sequences known, evolutionary trees have been constructed showing that alpha and beta globins diverged from a common ancestor between 425 and 500 million years ago, after vertebrate species had appeared and roughly when sharks and bony vertebrates diverged. The alpha and beta globins assemble to form tetrameric haemoglobin, alpha 2 beta 2, which can switch between quaternary states having high and low oxygen affinity. This allows the protein to bind oxygen cooperatively and therefore efficiently transport oxygen from the lungs to respiring tissues. The alpha and beta globins have closely related tertiary structures, being alpha-helical proteins with similar haem-binding sites. Most globins consist of eight helices, designated A to H from the N terminus, connected by short nonhelical segments, but all known vertebrate alpha globins lack a D helix. Because the loss of this helix by alpha globin occurred shortly before tetrameric haemoglobin appeared, it might be a functionally important mutation required for a tetramer assembly or allostery. We have now tested this idea by engineering human haemoglobins containing beta subunits without a D helix and alpha subunits with a D helix. Both of these mutations have little effect on the oxygen-binding properties of the molecule. Thus it is possible that deletion of the D helix in the alpha subunit was caused by a neutral mutation.

Expression of fully functional tetrameric human hemoglobin in Escherichia coli.

Synthetic genes encoding the human alpha- and beta-globin polypeptides have been expressed from a single operon in Escherichia coli. The alpha- and beta-globin polypeptides associate into soluble tetramers, incorporate heme, and accumulate to greater than 5% of the total cellular protein. Purified recombinant hemoglobin has the correct stoichiometry of alpha- and beta-globin chains and contains a full complement of heme. Each globin chain also contains an additional methionine as an extension to the amino terminus. The recombinant hemoglobin has a C4 reversed-phase HPLC profile essentially identical to that of human hemoglobin A0 and comigrates with hemoglobin A0 on SDS/PAGE. The visible spectrum and oxygen affinity are similar to that of native human hemoglobin A0. The recombinant protein shows a reduction in Bohr and phosphate effects, which may be attributed to the presence of methionine at the amino termini of the alpha and beta chains. We have also expressed the alpha- and beta-globin genes separately and found that the expression of the alpha-globin gene alone results in a marked decrease in the accumulation of alpha-globin in the cell. Separate expression of the beta-globin gene results in high levels of insoluble beta-globin. These observations suggest that the presence of alpha- and beta-globin in the same cell stabilizes alpha-globin and aids the correct folding of beta-globin. This system provides a simple method for expressing large quantities of recombinant hemoglobin and allows facile manipulation of the genes encoding hemoglobin to produce functionally altered forms of this protein.

The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae.

A DNA probe encoding approximately 80% of the 18-kDa protein gene of Mycobacterium leprae was isolated and tested for specificity by assessing hybridization of the probe to genomic DNA from taxonomically related and unrelated DNA samples. The 360-base-pair (bp) probe was specific for M. leprae DNA and did not hybridize with genomic DNA from 18 species of bacteria nor with DNA from human, murine, and armadillo sources. Oligonucleotide primers were synthesized corresponding to the 5' and 3' ends of the 360-bp fragment to yield a fragment of similar size on amplification of M. leprae DNA by the polymerase chain reaction (PCR). A simple procedure for DNA extraction from M. leprae-infected tissues was developed that provided suitable template DNA for amplification. The PCR test was specific for M. leprae DNA from human and murine sources and detected M. leprae DNA in biopsies from leprosy patients and from control and uninfected human skin biopsy preparations seeded with as few as 100 M. leprae.

The synergistic action of pyrazolopyrimidines and pentavalent antimony against Leishmania donovani and L. braziliensis.

Pyrazolopyrimidines, particularly allopurinol, allopurinol riboside, and other purine analogues, show promise as experimental therapeutic compounds for the treatment of leishmaniasis. The combination of these agents with pentostam may produce an improved therapeutic effect. We report here on strong synergistic activity between pyrazolopyrimidines and pentavalent antimonials in a human macrophage tissue culture system infected with Leishmania donovani and L. braziliensis.

A tissue culture system for the growth of several species of Leishmania: growth kinetics and drug sensitivities.

We have developed a simple in vitro method of infecting a continuous human macrophage cell line (U937) with promastigotes of several species of Leishmania. These include L. braziliensis braziliensis, L. b. panamensis, L. donovani, L. mexicana mexicana, L. m. pifanoi, L. tropica, and L. major. The growth kinetics of these species are presented as well as drug sensitivity data. The U937 cell system can be used to determine drug efficacy and eliminates the need to use amastigotes from animal tissues to infect the tissue culture.

Growth of Leishmania donovani amastigotes in the continuous human macrophage cell line U937: studies of drug efficacy and metabolism.

We have developed a simple and reproducible system for infecting a human macrophage cell line (U937) with stationary-phase Leishmania donovani promastigotes. Four days after infection, greater than 90% of the promastigotes had transformed to amastigotes. The antileishmanial agents allopurinol riboside, formycin B, 9-deazainosine, and sodium stibogluconate effectively inhibited the growth of L. donovani amastigotes in this cell line. To study the capability of amastigotes in the U937 cell line to carry out biochemical reactions that could be monitored experimentally, we incubated the cells with radiolabeled 9-deazainosine. This purine analogue underwent metabolism in the amastigote phase similar to that occurring in the promastigote phase. This cell line should be useful for studies of parasite maturation and differentiation, parasite-human interactions, and antiparasitic drugs.

Mechanisms of action of pyrazolopyrimidines in Leishmania donovani.

We investigated the antileishmanial actions of the pyrazolopyrimidines allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine), thiopurinol (4-thiopyrazolo[3,4-d]pyrimidine), and aminopurinol (4-aminopyrazolo[3,4-d]pyrimidine). These compounds affect several metabolic processes. The first is the inhibition of GMP reductase by the IMP analogues allopurinol ribonucleoside monophosphate and thipurinol ribonucleoside monophosphate which reduces the organism's ability to synthesize ATP from guanine. Second, interconversion of adenine nucleotides to guanine nucleotides, is curtailed by the inhibition of IMP dehydrogenase by these same IMP analogues. Third, the IMP analogues reduce intracellular UTP content. The fourth affect is increased catabolism of RNA and consequent reduction of protein synthesis. This latter effect is due to the adenine nucleotide analogues aminopurinol ribonucleoside mono-, di-, and/or triphosphates, metabolic products of both allopurinol and aminopurinol.

Gamma interferon activates human macrophages to become tumoricidal and leishmanicidal but enhances replication of macrophage-associated mycobacteria.

Recombinant human gamma interferon (rIFN-gamma) was examined for its ability to activate human peripheral blood monocyte-derived macrophages to kill tumor cells and to affect the replication of two phylogenetically distinct intracellular pathogens, Mycobacterium tuberculosis and Leishmania donovani. Macrophages preincubated overnight with doses of rIFN-gamma from 5 to 500 U/ml killed [3H]thymidine-labeled mouse L929 tumor targets, as measured by the release of [3H]thymidine into the supernatant after 48 h. Counts of macrophages initially infected with leishmania promastigotes showed that rIFN-gamma-pretreated macrophages could both inhibit the replication of and kill the resulting intramacrophage amastigotes over a 7-day period. However, rIFN-gamma pretreatment of macrophages actually enhanced mycobacterial replication over a 5- to 7-day period, as assessed by (i) counting acid-fast bacilli or (ii) lysing macrophages to release bacteria and determining the numbers of viable units. Mycobacterial growth was not affected by rIFN-gamma in the absence of macrophages. rIFN-gamma pretreatment had opposite effects on the uptake of mycobacteria and leishmania. As many as 80% fewer activated macrophages ingested mycobacteria compared with controls, whereas 50% more activated macrophages were infected with leishmania. These results suggest that rIFN-gamma may interfere with the immune destruction of intracellular tubercle bacilli and that the mechanisms of immunity against mycobacteria and leishmania may differ.

Inosine analogs as chemotherapeutic agents for African trypanosomes: metabolism in trypanosomes and efficacy in tissue culture.

Certain purine analogs, the pyrazolopyrimidines, are effective chemotherapeutic agents against Leishmania spp. and Trypanosoma cruzi both in vitro and in some clinical models. Heretofore they have not been effective against the African trypanosomes; this suggested that these organisms were not comparable to the other pathogens with respect to their purine metabolism. We have studied the efficacy and metabolism of the pyrazolopyrimidine bases allopurinol and thiopurinol, their respective ribonucleosides, and the C-nucleosides formycin B and 9-deazainosine in Trypanosoma brucei subsp. gambiense and Trypanosoma brucei subsp. rhodesiense. The efficacy of these compounds was dependent on the purine content of the culture medium. The C-nucleosides were the most effective, with 90% effective doses for formycin B and 9-deazainosine of 0.01 and 2 micrograms/ml, respectively. Metabolism was the same in both the bloodstream and culture forms and identical to that reported for Leishmania spp. and T. cruzi. Both agents were phosphorylated to the ribonucleotide and then aminated to produce adenine nucleotide analogs. Growth inhibition studies were performed with three inosine analogs (allopurinol riboside, formycin B, and 9-deazainosine) on trypomastigotes grown in bone marrow tissue culture. Both C-nucleosides eradicated the infection at a concentration of 0.25 micrograms/ml. Unlike formycin B, 9-deazainosine is not known to be aminated by mammalian cells and appears to be relatively nontoxic in three different mammalian tissue culture systems. This nucleoside was very active against all pathogenic leishmaniae and trypanosomes investigated and is worthy of further study.

Efficacy of pyrazolopyrimidine ribonucleosides against Trypanosoma cruzi: studies in vitro and in vivo with sensitive and resistant strains.

Strains of Trypanosoma cruzi differ in their susceptibilities to and metabolism of pyrazolopyrimidines. Allopurinol riboside can control but not eliminate infections with a sensitive strain in both tissue culture and mice. Formycin B, which proved to be greater than 10-fold more effective on a weight basis, showed a similar strain specificity but could eliminate an infection with a sensitive strain from tissue culture. However, this drug, unlike allopurinol riboside, was converted to toxic analogues of adenosine mono-, di-, and triphosphate by uninfected tissue culture cells. Thiopurinol and its riboside were effective against all strains unless culture was performed in purine-defined medium. Thus formycin B and allopurinol riboside appear to be good models for the design of antitrypanosomal agents. Suitable modification of the molecule may provide an effective chemotherapeutic agent.

Purine metabolism in Leishmania donovani amastigotes and promastigotes.

Purine metabolism in Leishmania donovani amastigotes was found to be similar to that of promastigotes with the exception of adenosine metabolism. Adenosine kinase activity in amastigotes is approximately 50-fold greater than in promastigotes. Amastigotes deaminate adenosine to inosine through adenosine deaminase, an enzyme not present in promastigotes. Inosine is cleaved to hypoxanthine and phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase. Promastigotes cleave adenosine to adenine and deaminate adenine to hypoxanthine via adenase, an enzyme not present in amastigotes. Hypoxanthine is phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase.