Learning from our mistakes: using key opportunities to remove the perverse incentives that help drive antibiotic resistance.

Introduction: Governments need to do far more to help curb the emergence and transmission of antibiotic resistance and help protect the efficacy of any new antibiotics that come to the market. Industry is an important stakeholder that must be brought on-board such efforts given its influence on the direction and scale of antibiotic sales. Financial incentives supporting industry R&D of novel antibiotics should structurally remove the drivers of superfluous sales and encourage access to newer antibiotics where infections are otherwise resistant to treatment. Indeed, the use of public money provides an important opportunity to prioritize these public health goals within market structures such that we both adequately reward industry for their efforts and prolong antibiotic efficacy for as long as possible.Areas covered: This work discusses possible financial 'pull' incentives that fully delink the reward paid to the developer from unit sales, examining their primary advantages and limitations.Expert opinion: Pharmaceutical companies need to be rewarded generously for their efforts to develop new, badly needed antibiotics. But the current marketplace does not provide a sustained financial lure and its reliance on unit-sales for profitability jeopardizes the efficacy of antibiotics both new and old. Fully delinked models can make antibiotic R&D more financially appealing and create a market environment that is far less threatening to public health.

Preserving the 'commons': addressing the sustainable use of antibiotics through an economic lens.

BACKGROUND: As the growth of antibiotic resistance has resulted in large part from widespread use of antibiotics, every effort must be made to ensure their sustainable use. AIMS: This narrative review aims to assess the potential contribution of health economic analyses to sustainable use efforts. SOURCES: The work draws on existing literature and experience with health economic tools. CONTENT: The study examines some of the weaknesses in the health, regulatory, and industry arenas that could contribute to inappropriate or suboptimal prescribing of antibiotics and describes how economic analysis could be used to improve current practice by comparing both costs and health outcomes to maximize societal wellbeing over the longer-term. It finds that economic considerations underpinning current antibiotic prescribing strategies are incomplete and short-termist, with the result that they may foster suboptimal use. It also stresses that perverse incentives that drive antibiotic sales and inappropriate prescribing practices must be dis-entangled for sustainable use policies to gain traction. Finally, payment structures can be used to re-align incentives and promote optimal prescribing and sustainable use more generally. In particular, eliminating or altering reimbursement differentials could help steer clinical practice more deliberately towards the minimization of selection pressure and the resulting levels of antibiotic resistance. IMPLICATIONS: This work highlights the need for appropriately designed cost-effectiveness analyses, incentives analysis, and novel remuneration systems to underpin sustainable use policies both within and beyond the health sector.

Comparing the cost-effectiveness of linezolid to trimethoprim/sulfamethoxazole plus rifampicin for the treatment of methicillin-resistant Staphylococcus aureus infection: a healthcare system perspective.

OBJECTIVE: Few industry-independent studies have been conducted to compare the relative costs and benefits of drugs to treat methicillin-resistant Staphylococcus aureus (MRSA) infection. We performed a stochastic cost-effectiveness analysis comparing two treatment strategies-linezolid versus trimethoprim-sulfamethoxazole plus rifampicin-for the treatment of MRSA infection. METHODS: We used cost and effectiveness data from a previously conducted clinical trial, complementing with other data from published literature, to compare the two regimens from a healthcare system perspective. Effectiveness was expressed in terms of quality-adjusted life-years (QALYs). Several sensitivity analyses were performed using Monte Carlo simulation, to measure the effect of potential parameter changes on the base-case model results, including potential differences related to type of infection and drug toxicity. RESULTS: Treatment of MRSA infection with trimethoprim-sulfamethoxazole plus rifampicin and linezolid were found to cost on average €146 and €2536, and lead to a gain of 0.916 and 0.881 QALYs, respectively. Treatment with trimethoprim-sulfamethoxazole plus rifampicin was found to be more cost-effective than linezolid in the base case and remained dominant over linezolid in most alternative scenarios, including different types of MRSA infection and potential disadvantages in terms of toxicity. With a willingness-to-pay threshold of €0, €50 000 and €200 000 per QALY gained, trimethoprim-sulfamethoxazole plus rifampicin was dominant in 100%, 96% and 85% of model iterations. A 95% discount on the current purchasing price of linezolid would be needed when it goes off-patent for it to represent better value for money compared with trimethoprim-sulfamethoxazole plus rifampicin. CONCLUSIONS: Combined treatment of trimethoprim-sulfamethoxazole plus rifampicin is more cost-effective than linezolid in the treatment of MRSA infection.

An Sau3 AI restriction endonuclease isoschizomer from Bacillus cereus.

The isolation and characterization of a restriction endonuclease from Bacillus cereus IOC 243 are described. The enzyme recognizes the palindromic sequence 5'-G(met-A,A)TC-3' as determined by PEI chromatography of pancreatic DNase, snake venom phosphodiesterase digestion products of labelled fragments, analysis of restriction digests from normal and N6-methyladenine-free DNA and direct sequence analysis of cloned fragments. The staggered cleavage products with 5' -terminal pGATC extensions are efficiently labelled with polynucleotide kinase and are easily cloned into BamHI sites. The enzyme, denoted Bce243, is thus an isoschizomer of Sau3AI. Its use and potential advantages in substituting Sau3AI. are discussed.

Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi.

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.

Stage specific gene expression precedes morphological changes during Trypanosoma cruzi metacyclogenesis.

The transformation of epimastigotes to metacyclic trypomastigotes of the Trypanosoma cruzi clone Dm 28c has been studied in an in vitro system consisting of artificial triatomine urine supplemented with newborn calf serum. The comparison of morphological data with gene expression products, as judged by the proteins synthesized during differentiation, has shown that stage specific gene activation precedes by far the morphological changes of differentiating cells. Immunoprecipitation of differentiating cell antigens with a trypomastigote stage specific antiserum has shown that although the morphological differentiation process takes six days to be completed, epimastigotes start to express the Mr 86 000 and the 78 000 trypomastigote antigens within the first 12 h of induction.

Characterization of tubulin genes in Trypanosoma rangeli.

Tubulin genes in Trypanosoma rangeli, the only trypanosome besides T. cruzi to infect humans in America, are organized in homogeneous, alternate alpha and beta gene tandem repeats of 3.8 kb. The basic repeat was cloned, mapped and partially sequenced. In contrast to most other eukaryotes, where tubulin genes are scattered throughout the genome, trypanosomatids so far studied are characterized by tandem arrangements of these genes with the genus Trypanosoma displaying an alternating alpha- and beta-tubulin tandem repeat.

In vitro differentiation of Trypanosoma cruzi under chemically defined conditions.

Metacyclic trypomastigotes of Trypanosoma cruzi have been obtained in chemically defined axenic culture. The differentiating medium, composed of artificial triatomine urine supplemented with proline, allows high yields of metacyclic trypomastigotes after 72-h incubation of T. cruzi cells at 27 degrees C. Morphological differentiation of the parasites is gradual under these chemically defined conditions and is preceded by the expression of stage-specific polypeptides. The yield of in vitro-induced metacyclic trypomastigotes depends upon the age of the epimastigote culture, the size of the inoculum and the depth of the medium. Metacyclic trypomastigotes differentiated in vitro from the Dm 28c clone of T. cruzi are both resistant to complement lysis and to macrophage digestion. They are able to infect mice with an efficiency similar to that obtained for natural metacyclic trypomastigotes obtained from triatomine excreta.

Schizodeme analysis of Trypanosoma cruzi stocks from South and Central America by analysis of PCR-amplified minicircle variable region sequences.

Kinetoplast DNA (kDNA) was isolated from 56 stocks of Trypanosoma cruzi isolated from human patients, animals and insects from Brazil, Venezuela, Colombia and Costa Rica. Comparison of the patterns of digested kDNA on acrylamide gels led to the grouping of several stocks into two schizodemes. Schizodeme analysis was also performed using a set of 330-bp fragments representing all the variable regions of the minicircle DNA molecules, which were obtained by PCR amplification of the kDNA using conserved region primers. The results of this analysis were consistent with the analysis using total kDNA, but the more informative restriction profiles allowed the construction of additional schizodemes. In addition, two oligomers were generated from variable region sequences of cloned minicircles from a Y and a Cl strain, and these were used as schizodeme-specific probes to detect homologous sequences in the amplified minicircle DNAs. The results indicate that a combination of restriction enzyme fingerprinting and hybridization of amplified variable region minicircle DNA with schizodeme-specific probes can be used for both sensitive detection and classification of T. cruzi.

Peculiar sequence organization of kinetoplast DNA minicircles from Trypanosoma cruzi.

The sequences of two minicircles from the kinetoplast DNA of the CL strain and one of the Y strain of Trypanosoma cruzi are reported. These 1.4 kb molecules have a peculiar sequence organization, the most distinctive feature being the occurrence of a 120 bp sequence repeated four times, located at 0, 90, 180 and 270 degrees along each circle. We have termed these conserved regions in this species 'minirepeats'. Minirepeats have a 3-fold higher concentration of cytosine residues in comparison with the variable regions and contain the universal 12-mer motif GGGGTTGGTGTA present in all sequenced minicircles and which was shown to be involved in DNA replication. A consensus sequence of T. cruzi minirepeats was determined using the 20 minirepeats present in five known T. cruzi minicircle sequences. This consensus sequence contains regions which have been remarkably well preserved in strains which show great biological diversity. In addition a low level of intraminicircle sequence similarity was also observed within the variable region, but this similarity did not extend between strains. The abundance of conserved minirepeat sequences containing invariant restriction sites in T. cruzi cells may prove valuable for the development of new direct diagnostic methods for Chagas' disease based on DNA probe technology.

Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area.

The feasibility of using DNA amplification by the polymerase chain reaction (PCR) for specific detection of Trypanosoma cruzi in human blood specimens was investigated. One hundred blood samples were collected in an endemic area of Minas Gerais, Brazil. They were submitted to DNA extraction and PCR amplification with kinetoplast DNA-specific primers using a simplified boiling procedure that linearized most minicircle molecules without the aid of chemical reagents. Samples that gave negative results were checked for possible inhibition of amplification using primers derived from a human-specific sequence, and those showing some level of inhibition were retested after a new DNA extraction. Of 86 patients previously diagnosed as chagasic by serologic techniques, 83 were positive in our PCR test (sensitivity = 96.5%), including all the xenodiagnosis-positive patients and 21 (87.5%) of 24 xenodiagnosis-negative individuals. In addition, four of six patients with doubtful serologic results were confirmed as positive by PCR. Our results suggest that the PCR may be a useful complement to serology in the diagnosis of Chagas' disease, and that it is the most powerful technique available for parasite detection in patients with chronic disease.

High correlation between Chagas' disease serology and PCR-based detection of Trypanosoma cruzi kinetoplast DNA in Bolivian children living in an endemic area.

The detection of Trypanosoma cruzi kinetoplast DNA by polymerase chain reaction (PCR) amplification is a potentially powerful tool for the parasitological diagnosis of Chagas' disease. We have applied this technique in a field situation in Bolivia, where 45 children from a primary school were subjected to serological testing, buffy coat analysis and PCR diagnosis. 26 of the 28 serology-positive individuals were also positive by PCR. In addition, two serology-negative children gave a positive result by PCR, including one who was positive in the buffy coat test. These results suggest that PCR detection of T. cruzi DNA in blood can be a very useful complement to serology in Chagas' disease diagnosis in Bolivia.

Changes in the isoenzyme and kinetoplast DNA patterns of Trypanosoma cruzi strains induced by maintenance in mice.

Culture forms of thirteen Trypanosoma cruzi strains from 4 zymodemes and 9 schizodemes were inoculated and kept by successive passages in C3H mice. The strains were initially from the following zymodemes: 3 from A, 3 from B, 4 from C and 2 from D and 1 from AB mixed zymodemes. After approximately 18 months maintenance the parasites were isolated by hemoculture and again typed according to their isoenzyme and kinetoplast DNA patterns. The zymodeme A strains kept their initial patterns; from the 3 zymodeme B strains, two kept the initial patterns and one changed to zymodeme A; from the 4 zymodeme C, two kept the initial pattern and two changed to zymodeme B; from the 2 zymodeme D strains, one kept the initial pattern and one changed to zymodeme A. The strain from AB mixed zymodeme was reduced to zymodeme. A. The zymodeme changes were accompanied by schizodeme changes. Although not simultaneously, in one T. cruzi strain the parasitemia change was followed by zymodeme and schizodeme changes. The results showed that prolonged maintenance of T. cruzi in mice by successive passages alters the isoenzyme and k-DNA patterns of some strains and that these alterations tend to move towards zymodeme A, suggesting a selective effect of mice over these T. cruzi populations.

Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener.

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28 degrees C in liver infusion-tryptose (LIT) medium is 58 +/- 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Grace's medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10(3) parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopurinol is inefficient in the treatment of mice experimental infection. The clone infects mammalian cultured cells and performs the complete intracellular cycle at 33 and 37 degrees C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24S alpha ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed.

Restriction endonucleases from microorganisms isolated in Brazil: an isoschizomer of HaeIII from a thermophilic Bacillus sp.

1. The isolation and characterization of a restriction endonuclease from a thermophilic strain of Bacillus is described. 2. The enzyme recognizes the palindromic sequence 5'...GGCC...3' as determined by PEI-cellulose chromatography of pancreatic DNAse and snake venom phosphodiesterase digestion products of labelled DNA fragments, analysis of restriction digests and direct sequence analysis. 3. The enzyme, denominated BspBR, is an isoschizomer of HaeIII and BspRI.

Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral and cutaneous leishmaniasis.

Leishmania parasites were isolated from humans and canines in foci of cutaneous and visceral leishmaniasis. After in vitro cultivation the parasites were examined by the following biochemical techniques: (i) restriction analysis of kinetoplast DNA (kDNA) also known as schizodeme analysis (Morel et al., 1980); (ii) zymodeme analysis (Barret et al., 1980); by agarose gel electrophoresis and (iii) isoelectricfocusing in polyacrylamide gels. The strains of cutaneous and visceralizing leishmanias studied could be differentiated by schizodeme analysis, using the endonuclease MspI, into three complexes agreeing with those accepted for human New World leishmaniasis. In the municipality of Rio de Janeiro, isolates from a focus of cutaneous leishmaniasis were identified as L. braziliensis braziliensis and from a focus of visceral leishmaniasis were identified as L. donovani by zymodeme characterization. Identical restriction enzyme profiles of kDNA from human and canine isolates indicated that in the cutaneous focus at Jacarepaguá, Rio de Janeiro, the same strain was probably circulating in both the canine and human populations. This suggests a possible role for dogs as a reservoir host for L. braziliensis braziliensis. In addition, our results confirm the importance of dogs as reservoirs in visceral leishmaniasis. The stability of the electrophoretic patterns of restriction digest ("fingerprints") of Leishmania kDNA as well as differences in the sensitivity of the techniques used were demonstrated. Strains from widely different geographical areas as well as strains maintained in vivo and in vitro showed identical kDNA restriction patterns, while strains showing similar banding patterns by enzyme electrophoresis could be differentiated by schizodeme analysis. These results demonstrate the usefulness of an integrated biochemical approach in the identification of Leishmania.

Paleoparasitology: perspectives with new techniques.

Paleoparasitology is the study of parasites found in archaeological material. The development of this field of research began with histological identification of helminth eggs in mummy tissues, analysis of coprolites, and recently through molecular biology. An approach to the history of paleoparasitology is reviewed in this paper, with special reference to the studies of ancient DNA identified in archaeological material.