Linda A. Dunn

Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

ABSTRACTTrichomoniasis is the most common, sexually transmitted infection. It is caused by the flagellated protozoan parasite Trichomonas vaginalis. Symptoms include vaginitis and infections have been associated with preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDS and cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved, effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and cross-resistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, with some cases remaining unresolved. The mechanism of metronidazole resistance in T. vaginalis from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasing metronidazole pressure. In the latter situation, hydrogenosomal function which is involved in activation of the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal of drug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintain their resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded as a laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. vaginalis appears to be on the increase and improved surveillance of treatment failures is urged.

Efficacy of antigiardial drugs

The flagellated protozoa Giardia duodenalis is the most commonly detected parasite in the intestinal tract of humans. Infections with the parasite result in diarrhoeal disease in humans and animals, with infants at risk from failureto-thrive syndrome. The incidence of giardiasis worldwide may be as high as 1000 million cases. Current recommended treatments include the nitroheterocyclic drugs tinidazole, metronidazole and furazolidone, the substituted acridine, quinacrine, and the benzimidazole, albendazole. Paromomycin is also used in some situations, and nitazoxanide is proving to be useful. However, treatment failures have been reported with all of the common antigiardial agents, and drug resistance to all available drugs has been demonstrated in the laboratory. In addition, clinical resistance has been reported, including cases where patients failed both metronidazole and albendazole treatments. The identification of new antigiardial drugs is an important consideration for the future, but maintaining the usefulness of the existing drugs is the most cost-effective measure to ensure the continued availability of antigiardial drugs.

5-Nitroimidazole Drugs Effective against Metronidazole-Resistant Trichomonas vaginalis and Giardia duodenalis

ABSTRACT Metronidazole (Mz)-resistant Giardia and Trichomonas were inhibited by 1 of 30 new 5-nitroimidazole drugs. Another five drugs were effective against some but not all of the Mz-resistant parasites. This study provides the incentive for the continued design of 5-nitroimidazole drugs to bypass cross-resistance among established 5-nitromidazole antiparasitic drugs.

Potential strategies utilised by papillomavirus to evade host immunity.

Summary: The co‐evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non‐specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus‐induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced lo PV antigens, resting keratinocytes (KC) appear resistant to interferon‐γ‐enhanced mechanisms of cytotoxic T‐lymphocyte (CTL)‐mediated lysis, and expression of PV antigens by resting KC can tolerise PV‐specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV‐induced proliferative skin lesions for months to years, even in immunocompetent hosts.

Pyruvate:ferredoxin oxidoreductase and thioredoxin reductase are involved in 5-nitroimidazole activation while flavin metabolism is linked to 5-nitroimidazole resistance in Giardia lamblia

OBJECTIVES The mechanism of action of, and resistance to, metronidazole in the anaerobic (or micro-aerotolerant) protozoan parasite Giardia lamblia has long been associated with the reduction of ferredoxin (Fd) by the enzyme pyruvate:ferredoxin oxidoreductase (PFOR) and the subsequent activation of metronidazole by Fd to toxic radical species. Resistance to metronidazole has been associated with down-regulation of PFOR and Fd. The aim of this study was to determine whether the PFOR/Fd couple is the only pathway involved in metronidazole activation in Giardia. METHODS PFOR and Fd activities were measured in extracts of highly metronidazole-resistant (MTR(r)) lines and activities of recombinant G. lamblia thioredoxin reductase (GlTrxR) and NADPH oxidase were assessed for their involvement in metronidazole activation and resistance. RESULTS We demonstrated that several lines of highly MTR(r) G. lamblia have fully functional PFOR and Fd indicating that PFOR/Fd-independent mechanisms are involved in metronidazole activation and resistance in these cells. Flavin-dependent GlTrxR, like TrxR of other anaerobic protozoa, reduces 5-nitroimidazole compounds including metronidazole, although expression of TrxR is not decreased in MTR(r) Giardia. However, reduction of flavins is suppressed in highly MTR(r) cells, as evidenced by as much as an 80% decrease in NADPH oxidase flavin mononucleotide reduction activity. This suppression is consistent with generalized impaired flavin metabolism in highly MTR(r) Trichomonas vaginalis. CONCLUSIONS These data add to the mounting evidence against the dogma that PFOR/Fd is the only couple with a low enough redox potential to reduce metronidazole in anaerobes and point to the multi-factorial nature of metronidazole resistance.

A new-generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia lamblia in vitro

The 5-nitroimidazole (NI) compound C17, with a side chain carrying a remote phenyl group in the 2-position of the imidazole ring, is at least 14-fold more active against the gut protozoan parasite Giardialamblia than the 5-NI drug metronidazole (MTR), with a side chain in the 1-position of the imidazole ring, which is the primary drug for the treatment of giardiasis. Over 10 months, lines resistant to C17 were induced in vitro and were at least 12-fold more resistant to C17 than the parent strains. However, these lines had ID(90) values (concentration of drug at which 10% of control parasite ATP levels are detected) for MTR of >200 microM, whilst lines induced to be highly resistant to MTR in vitro have maximum ID(90) values around 100 microM (MTR-susceptible isolates typically have an ID(90) of 5-12.8 microM). The mechanism of MTR activation in Giardia apparently involves reduction to toxic radicals by the activity of pyruvate:ferredoxin oxidoreductase (PFOR) and the electron acceptor ferredoxin. MTR-resistant Giardia have decreased PFOR activity, which is consistent with decreased activation of MTR in these lines, but C17-resistant lines have normal levels of PFOR. Therefore, an alternative mechanism of resistance in Giardia must account for these super-MTR-resistant cells.

Tolerance or Immunity to a Tumor Antigen Expressed in Somatic Cells Can Be Determined by Systemic Proinflammatory Signals at the Time of First Antigen Exposure

Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers.

Microsatellite polymorphism in the sexually transmitted human pathogen Trichomonas vaginalis indicates a genetically diverse parasite

Given the growing appreciation of serious health sequelae from widespread Trichomonas vaginalis infection, new tools are needed to study the parasites genetic diversity. To this end we have identified and characterized a panel of 21 microsatellites and six single-copy genes from the T. vaginalis genome, using seven laboratory strains of diverse origin. We have (1) adapted our microsatellite typing method to incorporate affordable fluorescent labeling, (2) determined that the microsatellite loci remain stable in parasites continuously cultured for up to 17 months, and (3) evaluated microsatellite marker coverage of the six chromosomes that comprise the T. vaginalis genome, using fluorescent in situ hybridization (FISH). We have used the markers to show that T. vaginalis is a genetically diverse parasite in a population of commonly used laboratory strains. In addition, we have used phylogenetic methods to infer evolutionary relationships from our markers in order to validate their utility in future population analyses. Our panel is the first series of robust polymorphic genetic markers for T. vaginalis that can be used to classify and monitor lab strains, as well as provide a means to measure the genetic diversity and population structure of extant and future T. vaginalis isolates.

Impaired parasite attachment as fitness cost of metronidazole resistance in Giardia lamblia

ABSTRACT Infections with the diarrheagenic protozoan pathogen Giardia lamblia are most commonly treated with metronidazole (Mz). Treatment failures with Mz occur in 10 to 20% of cases and Mz resistance develops in the laboratory, yet clinically, Mz-resistant (Mzr) G. lamblia has rarely been isolated from patients. To understand why clinical Mzr isolates are rare, we questioned whether Mz resistance entails fitness costs to the parasite. Our studies employed several newly generated and established isogenic Mzr cell lines with stable, high-level resistance to Mz and significant cross-resistance to tinidazole, nitazoxanide, and furazolidone. Oral infection of suckling mice revealed that three of five Mzr cell lines could not establish infection, while two Mzr cell lines infected pups, albeit with reduced efficiencies. Failure to colonize resulted from a diminished capacity of the parasite to attach to the intestinal mucosa in vivo and to epithelial cells and plastic surfaces in vitro. The attachment defect was related to impaired glucose metabolism, since the noninfectious Mzr lines consumed less glucose, and glucose promoted ATP-independent parasite attachment in the parental lines. Thus, resistance of Giardia to Mz is accompanied by a glucose metabolism-related attachment defect that can interfere with colonization of the host. Because glucose-metabolizing pathways are important for activation of the prodrug Mz, it follows that a fitness trade-off exists between diminished Mz activation and reduced infectivity, which may explain the observed paucity of clinical Mzr isolates of Giardia. However, the data also caution that some forms of Mz resistance do not markedly interfere with in vivo infectivity.

Metronidazole resistance in Trichomonas vaginalis from highland women in Papua New Guinea

BACKGROUND The prevalence of the sexually transmissible protozoan parasite Trichomonas vaginalis in the highlands of Papua New Guinea (PNG) has been reported to be as high as 46% and although not previously studied in Papua New Guinea, clinical resistance against metronidazole (Mz), the drug most commonly used to treat trichomoniasis, is well documented worldwide. This study was primarily aimed at assessing resistance to Mz in T. vaginalis strains from the Goroka region. METHODS Consenting patients presenting at the Goroka Base Hospital Sexually Transmitted Diseases (STD) Clinic and local women were asked to provide two vaginal swabs: one for culturing of the parasite; and one for polymerase chain reaction detection of T. vaginalis, Chlamydia trachomatis and Neisseria gonorrhoeae. T. vaginalis isolates were assayed for Mz susceptibility and a selection was genotyped. RESULTS The prevalence of T. vaginalis was determined to be 32.9% by culture and polymerase chain reaction of swabs among 82 local women and patients from the STD clinic. An unexpectedly high level of in vitro Mz resistance was determined with 17.4% of isolates displaying unexpectedly high resistance to Mz. The ability to identify isolates of T. vaginalis by genotyping was confirmed and the results revealed a more homogeneous T. vaginalis population in Papua New Guinea compared with isolates from elsewhere. CONCLUSION T. vaginalis is highly prevalent in the Goroka region and in vitro Mz resistance data suggest that clinical resistance may become an issue.