The stepwise selection for ketoconazole resistance induces upregulation of C14-demethylase (CYP51) in Leishmania amazonensis

Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.

Primary resistance of HIV to antiretrovirals among individuals recently diagnosed at voluntary counselling and testing centres in the metropolitan region of Recife, Pernambuco

Determining the prevalence and type of antiretroviral (ARV) resistance among ARV-naïve individuals is important to assess the potential responses of these individuals to first-line regimens. The prevalence of primary resistance and the occurrence of recent infections among individuals with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) were identified among recently diagnosed patients at five sexually transmitted disease/AIDS testing and counselling centres in the metropolitan region of Recife (RMR), Pernambuco, Brazil, between 2007-2009. One-hundred and eight samples were analysed using the Calypte® BED assay. Males predominated (56%), as did patients aged 31-50 years. Twenty-three percent presented evidence of a recent HIV infection. The median CD4+ T lymphocyte count was 408 cells/mm³ and the median viral load was 3.683 copies/mL. The prevalence of primary resistance was 4.6% (confidence interval 95% = 1-8.2%) based on criteria that excluded common polymorphisms in accordance with the surveillance drug resistance mutation criteria. The prevalence of resistance to non-nucleoside reverse transcriptase, nucleoside/nucleotide reverse transcriptase and protease inhibitors were 3.8%, 1.5% and 0.8%, respectively. Fifty-seven percent of strains were from clade B, 37.7% were clade F and 3.1% were clade C; there were no statistically significant differences with respect to resistance between clades. Recent infection tended to be more common in men (p = 0.06) and in municipalities in the south of the RMR (Jaboatão dos Guararapes and Cabo de Santo Agostinho) (p = 0.046). The high prevalence of recent infection and the high prevalence of non-B strains in this poor Brazilian region merit further attention.

Evolution of insecticide resistance in non-target black flies (Diptera: Simuliidae) from Argentina

Black flies, a non-target species of the insecticides used in fruit production, represent a severe medical and veterinary problem. Large increases in the level of resistance to the pyrethroids fenvalerate (more than 355-fold) and deltamethrin (162-fold) and a small increase in resistance to the organophosphate azinphos methyl (2-fold) were observed between 1996-2008 in black fly larvae under insecticide pressure. Eventually, no change or a slight variation in insecticide resistance was followed by a subsequent increase in resistance. The evolution of pesticide resistance in a field population is a complex and stepwise process that is influenced by several factors, the most significant of which is the insecticide selection pressure, such as the dose and frequency of application. The variation in insecticide susceptibility within a black fly population in the productive area may be related to changes in fruit-pest control. The frequency of individuals with esterase activities higher than the maximum value determined in the susceptible population increased consistently over the sampling period. However, the insecticide resistance was not attributed to glutathione S-transferase activity. In conclusion, esterase activity in black flies from the productive area is one mechanism underlying the high levels of resistance to pyrethroids, which have been recently used infrequently. These enzymes may be reselected by currently used pesticides and enhance the resistance to these insecticides.

Toxicity of non-pyrethroid insecticides against Triatoma infestans (Hemiptera: Reduviidae)

Triatoma infestans (Klug) is the main vector of Chagas disease, which is a public health concern in most Latin American countries. The prevention of Chagas disease is based on the chemical control of the vector using pyrethroid insecticides. In the last decade, different levels of deltamethrin resistance have been detected in certain areas of Argentina and Bolivia. Because of this, alternative non-pyrethroid insecticides from different chemical groups were evaluated against two T. infestans populations, NFS and El Malá, with the objective of finding new insecticides to control resistant insect populations. Toxicity to different insecticides was evaluated in a deltamethrin-susceptible and a deltamethrin-resistant population. Topical application of the insecticides fenitrothion and imidacloprid to first nymphs had lethal effects on both populations, producing 50% lethal dose (LD50) values that ranged from 5.2-28 ng/insect. However, amitraz, flubendiamide, ivermectin, indoxacarb and spinosad showed no insecticidal activity in first instars at the applied doses (LD50 > 200 ng/insect). Fenitrothion and imidacloprid were effective against both deltamethrin-susceptible and deltamethrin-resistant populations of T. infestans. Therefore, they may be considered alternative non-pyrethroid insecticides for the control of Chagas disease.

Fitness evaluation of two Brazilian Aedes aegypti field populations with distinct levels of resistance to the organophosphate temephos

In Brazil, decades of dengue vector control using organophosphates and pyrethroids have led to dissemination of resistance. Although these insecticides have been employed for decades against Aedes aegypti in the country, knowledge of the impact of temephos resistance on vector viability is limited. We evaluated several fitness parameters in two Brazilian Ae. aegypti populations, both classified as deltamethrin resistant but with distinct resistant ratios (RR) for temephos. The insecticide-susceptible Rockefeller strain was used as an experimental control. The population presenting the higher temephos resistance level, Aparecida de Goiânia, state of Goiás (RR95 of 19.2), exhibited deficiency in the following four parameters: blood meal acceptance, amount of ingested blood, number of eggs and frequency of inseminated females. Mosquitoes from Boa Vista, state of Roraima, the population with lower temephos resistance level (RR95 of 7.4), presented impairment in only two parameters, blood meal acceptance and frequency of inseminated females. These results indicate that the overall fitness handicap was proportional to temephos resistance levels. However, it is unlikely that these disabilities can be attributed solely to temephos resistance, since both populations are also resistant to deltamethrin and harbour the kdr allele, which indicates resistance to pyrethroids. The effects of reduced fitness in resistant populations are discussed.

Susceptibility and resistance to deltamethrin of wild and domestic populations of Triatoma infestans (Reduviidae: Triatominae) in Bolivia: new discoveries

Bolivia is a high-endemic country for Chagas disease, for which the principal vector is Triatoma infestans (Triatominae). This is a mainly domestic species that is also found in the wild environment. Recently, an increasing number of studies have shown the importance of Triatominae resistance to insecticides, especially in Bolivia. Data regarding the susceptibility/resistance of wild and domestic populations of T. infestans to deltamethrin are presented. For the first time, domestic populations of the department of Santa Cruz were tested, showing low resistance. Although most of the wild populations were found to be susceptible to deltamethrin, three populations from three departments showed a mortality rate of less than 100%. This result is emphasised here.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

Shigella in Brazilian children with acute diarrhoea: prevalence, antimicrobial resistance and virulence genes

Diarrhoeal disease is still considered a major cause of morbidity and mortality among children. Among diarrhoeagenic agents, Shigella should be highlighted due to its prevalence and the severity of the associated disease. Here, we assessed Shigella prevalence, drug susceptibility and virulence factors. Faeces from 157 children with diarrhoea who sought treatment at the Children's Hospital João Paulo II, a reference children´s hospital in Belo Horizonte, state of Minas Gerais, Brazil, were cultured and drug susceptibility of the Shigella isolates was determined by the disk diffusion technique. Shigella virulence markers were identified by polymerase chain reaction. The bacterium was recovered from 10.8% of the children (88.2% Shigella sonnei). The ipaH, iuc, sen and ial genes were detected in strains isolated from all shigellosis patients; set1A was only detected in Shigella flexneri. Additionally, patients were infected by Shigella strains of different ial, sat, sen and set1A genotypes. Compared to previous studies, we observed a marked shift in the distribution of species from S. flexneri to S. sonnei and high rates of trimethoprim/sulfamethoxazole resistance.

Transmitted human immunodeficiency virus-1 drug resistance in a cohort of men who have sex with men in Belo Horizonte, Brazil - 1996-2012

The presence of transmitted human immunodeficiency virus (HIV)-1 drug-resistance (TDR) at the time of antiretroviral therapy initiation is associated with failure to achieve viral load (VL) suppression. Here, we report TDR surveillance in a specific population of men who have sex with men (MSM) in Belo Horizonte, Brazil. In this study, the rate of TDR was evaluated in 64 HIV-infected individuals from a cohort of MSM between 1996-June 2012. Fifty-four percent had a documented recent HIV infection, with a seroconversion time of less than 12 months. The median CD4+T lymphocyte count and VL were 531 cells/mm3and 17,746 copies/mL, respectively. Considering the surveillance drug resistance mutation criteria, nine (14.1%) patients presented TDR, of which three (4.7%), five (7.8%) and four (6.2%) had protease inhibitors, resistant against nucleos(t)ide transcriptase inhibitors and against non-nucleoside reverse-transcriptase inhibitors mutations, respectively. Two of the patients had multi-drug-resistant HIV-1. The most prevalent viral subtype was B (44, 68.8%), followed by subtype F (11, 17.2%). This study shows that TDR may vary according to the population studied and it may be higher in clusters of MSM.

Comparison of the infectivity of Trypanosoma cruzi insect-derived metacyclic trypomastigotes after mucosal and cutaneous contaminative challenges

Trypanosoma cruzi infects humans when infected triatomine vector excreta contaminate breaks in skin or mucosal surfaces. T. cruzi insect-derived metacyclic trypomastigotes (IMT) invade through gastric mucosa after oral challenges without any visible inflammatory changes, while cutaneous and conjunctival infections result in obvious local physical signs. In this study we compared the infectivity of T. cruzi IMT in mice after cutaneous and oral contaminative challenges simulating natural infections. The 50% infective dose (ID50) for oral challenge was 100 fold lower than the ID50for cutaneous challenge, indicating that oral mucosal transmission is more efficient than cutaneous transmission.

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

Prevalence of plasmid-mediated quinolone resistance determinants among oxyiminocephalosporin-resistant Enterobacteriaceae in Argentina

High quinolone resistance rates were observed among oxyiminocephalosporin-resistant enterobacteria. In the present study, we searched for the prevalence of plasmid-mediated quinolone resistance (PMQR) genes within the 55 oxyiminocephalosporin-resistant enterobacteria collected in a previous survey. The main PMQR determinants were aac(6')-Ib-cr and qnrB, which had prevalence rates of 42.4% and 33.3%, respectively. The aac(6')-Ib-crgene was more frequently found in CTX-M-15-producing isolates, while qnrB was homogeneously distributed among all CTX-M producers.

Evidence of an increased incidence of day 3 parasitaemia in Suriname: an indicator of the emerging resistance of Plasmodium falciparum to artemether

The emerging resistance to artemisinin derivatives that has been reported in South-East Asia led us to assess the efficacy of artemether-lumefantrine as the first line therapy for uncomplicated Plasmodium falciparum infections in Suriname. This drug assessment was performed according to the recommendations of the World Health Organization in 2011. The decreasing number of malaria cases in Suriname, which are currently limited to migrating populations and gold miners, precludes any conclusions on artemether efficacy because adequate numbers of patients with 28-day follow-up data are difficult to obtain. Therefore, a comparison of day 3 parasitaemia in a 2011 study and in a 2005/2006 study was used to detect the emergence of resistance to artemether. The prevalence of day 3 parasitaemia was assessed in a study in 2011 and was compared to that in a study in 2005/2006. The same protocol was used in both studies and artemether-lumefantrine was the study drug. Of 48 evaluable patients in 2011, 15 (31%) still had parasitaemia on day 3 compared to one (2%) out of 45 evaluable patients in 2005/2006. Overall, 11 evaluable patients in the 2011 study who were followed up until day 28 had negative slides and similar findings were obtained in all 38 evaluable patients in the 2005/2006 study. The significantly increased incidence of parasite persistence on day 3 may be an indication of emerging resistance to artemether.

Acoustic communication in insect disease vectors

Acoustic signalling has been extensively studied in insect species, which has led to a better understanding of sexual communication, sexual selection and modes of speciation. The significance of acoustic signals for a blood-sucking insect was first reported in the XIX century by Christopher Johnston, studying the hearing organs of mosquitoes, but has received relatively little attention in other disease vectors until recently. Acoustic signals are often associated with mating behaviour and sexual selection and changes in signalling can lead to rapid evolutionary divergence and may ultimately contribute to the process of speciation. Songs can also have implications for the success of novel methods of disease control such as determining the mating competitiveness of modified insects used for mass-release control programs. Species-specific sound “signatures” may help identify incipient species within species complexes that may be of epidemiological significance, e.g. of higher vectorial capacity, thereby enabling the application of more focussed control measures to optimise the reduction of pathogen transmission. Although the study of acoustic communication in insect vectors has been relatively limited, this review of research demonstrates their value as models for understanding both the functional and evolutionary significance of acoustic communication in insects.

Circadian rhythms in insect disease vectors

Organisms from bacteria to humans have evolved under predictable daily environmental cycles owing to the Earth’s rotation. This strong selection pressure has generated endogenous circadian clocks that regulate many aspects of behaviour, physiology and metabolism, anticipating and synchronising internal time-keeping to changes in the cyclical environment. In haematophagous insect vectors the circadian clock coordinates feeding activity, which is important for the dynamics of pathogen transmission. We have recently witnessed a substantial advance in molecular studies of circadian clocks in insect vector species that has consolidated behavioural data collected over many years, which provided insights into the regulation of the clock in the wild. Next generation sequencing technologies will facilitate the study of vector genomes/transcriptomes both among and within species and illuminate some of the species-specific patterns of adaptive circadian phenotypes that are observed in the field and in the laboratory. In this review we will explore these recent findings and attempt to identify potential areas for further investigation.