Genetic basis of multidrug resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from diarrheic calves in Egypt

Up to this date, nothing is known about the molecular basis of antimicrobial resistance in Salmonella isolated from animals in Africa. Therefore, this study was carried out to screen the incidence of multidrug-resistant (MDR) strains of Salmonella from neonatal calf diarrhea in Egypt and also to characterize the molecular basis of this resistance. Nine unique Salmonella isolates were obtained from 220 fecal samples, and six of these showed multidrug resistance phenotypes and harbored at least two antimicrobial resistance genes. Four were Salmonellaenterica serovar Typhimurium and two were S.enterica serovar Enteritidis. Class 1 integrons were identified in all MDR Salmonella isolates. The identified gene cassettes within class 1 integrons were as follows; aminoglycoside adenyltransferase type A (aadA1, aadA2 and aadA5), which confer resistance to streptomycin and spectinomycin, and dihydrofolate reductase gene cassettes (dfrA1, dfrA15 and dfrA15), which confer resistance to trimethoprim. A class 2 integron containing dfrA1-sat2-aadA1 gene cassettes was identified in only one isolate of S. enterica serovar Enteritidis. The β-lactamase-encoding gene, bla_TEM-1, was identified in five isolates and the extended-spectrum β-lactamase-encoding genes, bla_CMY-2 and bla_SHV-12, were identified in S. enterica serovar Typhimurium. Furthermore, the plasmid-mediated quinolone resistance genes, qnrB, qnrS and aac(6′)-Ib-cr, were also identified. To the best of our knowledge, this is the first report of qnrS in S. enterica serovar Enteritidis, qnrB in S. enterica serovar Typhimurium, and aac(6′)-Ib-cr in Salmonella of animal origin. Also, this is the first report of the molecular characterization of antimicrobial resistance in Salmonella isolated from animals in Africa.     

Characterization of antimicrobial resistance and integrons among Escherichia coli isolated from animal farms in Eastern China

A total of 182 Escherichia coli isolates from animals, environment and workers of dairy cattle, swine and chicken farms in Shandong which locates in Eastern China, were investigated for antimicrobial resistance as well as prevalence and the transfer mechanisms of integrons. The results revealed isolates from swine and chicken farm exhibited high levels of resistance to antimicrobial agents. The positive rate of gene cassette of class 1 integron in dairy cattle, swine and chicken farms was 5%, 20% and 41.94%, respectively. Only four isolates possessed class 2 integron, all of which were from chicken farm. Nine distinct cassette arrays were detected and two novel gene cassette arrays yheSΔ-yheR-kefBΔ and chrAΔ-sul1-qacEΔ1-orf5-aadA5-dfrA17 were identified in class 1 integron for the first time. Class 1 integrons were found to be located mostly in both chromosomal and conjugative plasmid through southern hybridization and conjugation. PFGE revealed clonal relatedness among the isolates from different sources, especially within the same farm. The results confirmed the antimicrobial resistance and prevalence of integrons were strongly associated with the selection pressure of antimicrobial agents, and resistance genes in animal farms were probably spread by both vertical and horizontal transfer.     

In vivo experimental drug resistance study in Trypanosoma vivax isolates from tsetse infested and non-tsetse infested areas of Northwest Ethiopia

Ethiopia, particularly in the Northwest region, is affected by both tsetse fly and non-tsetse fly transmitted trypanosomosis with a significant impact on livestock productivity. The control of trypanosomosis in Ethiopia relies on either curative or prophylactic treatment of animals with diminazene aceturate (DA) or isometamidium chloride (ISM), respectively. However, since these two trypanocides have been on the market for more than 40 years, this may have resulted in drug-resistance. Therefore, in vivo drug resistance tests on two Ethiopian isolates of Trypanosoma vivax were completed, one from an area where tsetse flies are present and one from an area where tsetse flies are not present. Twenty four cattle (Bos indicus) aged between 6 and 12 months, purchased from a trypanosome-free area (Debre Brehan: Northcentral Ethiopia) and confirmed to be trypanosome-negative, were randomly assigned into four groups of six animals, which were infected with T. vivax isolated from a tsetse-infested or non-tsetse infested area, and in each case treated with curative doses of DA or ISM. Each animal were inoculated intravenously 3 × 10⁶ trypanosomes from donor animals. Parasitaemia became patent earlier in infections with non-tsetse T. vivax (∼7 days post-infection) than tsetse (∼14 days post-infection). Both groups were treated at the highest peak parasitaemia with DA or ISM and nine cattle, four with non-tsetse T. vivax (two ISM- and two DA-treated) and five with tsetse T. vivax (three ISM- and two DA-treated) showed relapses of parasitaemia. Moreover, treatment did not improve diagnostic host markers of trypanosome infections in these animals. In conclusion, in vivo drug tests indicated the presence of resistant parasites (>20% of treated animals in each group relapsed) against recommended doses of both available trypanocidal drugs.     

临床痢疾志贺菌整合子检测及耐药基因盒序列分析

目的 检测印度东部1988、1995和2002年部分临床分离痢疾志贺菌中细菌耐药关系密切的1、2、3类整合酶基因及整合子携带的耐药基因盒的分布,分析整合子系统对志贺菌耐药的影响.方法 纸片扩散法检测实验菌株对药物的敏感性;应用PCR方法对16株临床耐药菌株进行1、2、3类整合酶基因(intI)筛选,对阳性样本可变区基因盒序列进行鉴定分析.结果 所有16株菌均耐4种及4种以上药物,包括β-内酰胺类、氨基糖苷类、四环素类、磺胺类、氯霉素类和喹诺酮类.13株菌检出1类整合酶基因,全部菌株含2类整合酶基因,即发现同时存在两种整合子结构菌株,未检测到3类整合酶基因.1类整合酶插入基因盒以blaara30-aadAl基因家族为主,分别对β-内酰胺类抗生素、链霉素、壮观霉素耐药;2类整合酶插人基因盒以dfrAl-satl组合为主,分别对甲氧苄氨嘧啶、链丝菌素耐药,同时在4株菌中发现dfrAl-satl-aadAl基因盒组合.结论 2类整合子普遍存在于临床志贺菌中.整合子与志贺菌的多重耐药具有密切相关性.     

Defining the origin of Plasmodium falciparum resistant dhfr isolates in Senegal

We previously reported a high baseline prevalence of mutations in the dhfr and dhps genes of Plasmodium falciparum throughout Senegal. The highest prevalence of the triple dhfr pyrimethamine associated mutations were found in isolates obtained in the western part of the country near the capital city of Dakar. In this study, we sought out to determine the relatedness of dhfr wild type and mutated strains by analyzing three microsatellite regions upstream of the dhfr locus. Twenty-six of the 31 wild type strains had a unique microsatellite pattern. In contrast, of the 17 isolates containing the triple mutation in dhfr, 11 had an identical microsatellite pattern. Diverse geographical isolates in Senegal containing the triple dhfr mutation have arisen from a limited number of ancestral strains. In addition, we demonstrate that these isolates have shared ancestry with the previously reported triple mutation haplotype found in Tanzania, South Africa, and southeast Asia. This common ancestry may have implications for the malaria control strategy for reducing the spread of sulfadoxine-pyrimethamine resistance in Senegal and elsewhere in Africa.     

Molecular characterization of human isolates of Giardia duodenalis from Ethiopia

Giardia duodenalis, a flagellated protozoan, represents a common cause of gastroenteritis in Ethiopia, however very little information is available on the epidemiology and transmission routes of this pathogen, and a genetic characterization of the parasite has never been attempted in this country. The aim of this study was the genetic analysis of human isolates of G. duodenalis collected in different localities across the country, both from urban and rural areas. A fragment of the beta-giardin gene was amplified by nested PCR and analyzed by restriction and sequence analyses. Of the 59 isolates examined, 31 (52%) were typed as assemblage A and 13 (22%) as assemblage B. A strong correlation between the presence of symptoms and infection with assemblage B was observed. The remaining 15 (25%) isolates were typed as mixed infections by PCR-RFLP, specifically, A+F (in seven isolates) and A+B (in eight isolates). Sequencing of the A+F products confirmed the presence of assemblage F in three isolates, whereas the remaining four were identified as assemblage A. The detection of assemblage F, a cat-specific assemblage that to date has not been associated with human infections, was not able to be confirmed by the analysis of two commonly used markers (small subunit ribosomal RNA and triosephosphate isomerase). The analysis of the one isolate that was successfully amplified with the glutamate dehydrogenase primers unambiguously identified it as G. duodenalis, yet it was distinct from the established A and F sequences; thus the exact genetic identity of these isolates remains unclear.     

Inhibitory effects of emodin,baicalin, schizandrin and berberine on hef A gene: Treatment of Helicobacter pylori-induced multidrug resistance

AIM: To investigate the inhibitory effects of emodin, baicalin, etc. on the hefA gene of multidrug resistance(MDR) in Helicobacter pylori(H. pylori).METHODS: The double dilution method was used to screen MDR H. pylori strains and determine the minimum inhibitory concentrations(MICs) of emodin, baicalin, schizandrin, berberine, clarithromycin, metronidazole, tetracycline, amoxicillin and levofloxacin against H. pylori strains. After the screened MDR stains were treated with emodin, baicalin, schizandrin or berberine at a 1/2 MIC concentration for 48 h, changes in MICs of amoxicillin, tetracycline, levofloxacin, metronidazole and clarithromycin were determined.MDR strains with reduced MICs of amoxicillin were selected to detect the hefA mR NA expression by realtime quantitative PCR.RESULTS: A total of four MDR H. pylori strains were screened. Treatment with emodin, baicalin, schizandrin and berberine significantly decreased the MICs of amoxicillin and tetracycline against some strains, decreased by 1 to 2 times, but did not significantly change the MICs of clarithromycin, levofloxacin, and metronidazole against MDR strains. In the majority of strains with reduced MICs of amoxicillin, hef A m RNA expression was decreased; one-way ANOVA(SPSS 12.0) used for comparative analysis, P < 0.05.CONCLUSION: Emodin, baicalin, schizandrin and berberine significantly decreased the MICs of amoxicillin and tetracycline against some H. pylori strains, possibly by mechanisms associated with decreasing hefA mR NA expression.     

Biting behavior and Plasmodium infection rates of Anopheles arabiensis from Sille, Ethiopia

The man-biting behavior and Plasmodium infection rates of anopheline mosquitoes were investigated in Sille, a hyperendemic malarious area in southern Ethiopia. Seven Anopheles species were identified from all night landing collections, conducted from 18:00 to 06:00h between October 2001 and August 2002. The predominant species was Anopheles arabiensis (55.8%), followed by Anopheles coustani (31.5%), Anopheles pharoensis (9.5%), Anopheles funestus (2.2%), Anopheles nili (0.5%), Anopheles marshallii (0.4%) and Anopheles demeilloni (0.2%). Dissection of A. arabiensis showed an average parous rate of 73.2%. A large proportion of the parous mosquitoes were caught biting in the latter part of the night. Malaria sporozoite rates were determined by ELISA for A. arabiensis, with 0.5% (4/796) infective with Plasmodium falciparum and 1.76% (14/796) with Plasmodium vivax; there were no mixed infections. From our small sample of sporozoite positives we found no association between biting behavior and sporozoite infection status.     

Prevalence of plasmid-mediated quinolone resistance genes among ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from blood cultures in Korea

OBJECTIVES:

To analyze the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from patients at a tertiary care hospital in Korea.

METHODS:

A total of 102 nonduplicate isolates of ciprofloxacin-intermediate or ciprofloxacin-resistant E coli (n=80) and K pneumoniae (n=22) from blood cultures were obtained. The qnr (qnrA, qnrB, qnrS), aac(6′)-Ib-cr, qepA and oqxAB genes were detected using polymerase chain reaction (PCR) and confirmed using direct sequencing. To determine whether the PMQR-positive plasmid was horizontally transferable, conjugation experiments were performed.

RESULTS:

Of the 102 isolates, 81 (79.4%) had one or more PMQR genes; these consisted of 59 (73.8%) E coli and 22 (100%) K pneumoniae isolates. The qnr genes were present in 15 isolates (14.7%): qnrB4 was detected in 10.8% and qnrS1 was detected in 3.9%. The aac(6′)-Ib-cr, qepA and oqxAB genes were detected in 77.5%, 3.9% and 10.8%, respectively. In conjugation experiments, PMQR genes were successfully transferred from seven (8.6%) isolates. The range of minimum inhibitory concentrations of ciprofloxacin for these seven transconjugants increased to 0.5 mg/L to 1 mg/L, which was 16- to 33-fold that of the recipient E coli J53 bacteria.

CONCLUSIONS:

PMQR genes were highly prevalent among ciprofloxacin-nonsusceptible E coli and K pneumoniae from blood cultures in the authors’ hospital. Therefore, it is necessary to monitor for the spread of PMQR genes of clinical isolates and to ensure careful antibiotic use in a hospital setting.     

Therapeutic efficacy of chloroquine and chloroquine plus primaquine for the treatment of Plasmodium vivax in Ethiopia

Plasmodium vivax is the second most important cause of morbidity in Ethiopia. There is, however, little information on P. vivax resistance to chloroquine and chloroquine plus primaquine treatment although these drugs have been used as the first line treatment for over 50 years. We assessed the efficacy of standard chloroquine and chloroquine plus primaquine treatment for P. vivax infections in a randomized open-label comparative study in Debre Zeit and Nazareth in East Shoa, Ethiopia.A total of 290 patients with microscopically confirmed P. vivax malaria who presented to the outpatient settings of the two laboratory centers were enrolled: 145 patients were randomized to receive CQ and 145 to receive CQ + PQ treatment. Participants were followed-up for 28-157 days according to the WHO procedures. There were 12 (6.5%) lost to follow-up patients and 9 (3.1%) withdrawals. In all, 96% (277/290) of patients were analysed at day 28. Baseline characteristics were similar in all treatment groups. In all, 98.6% (275/277) of patients had cleared their parasitemia on day 3 with no difference in mean parasite clearance time between regimens (48.34 ± 17.68, 50.67 ± 15.70 h for the CQ and CQ + PQ group, respectively, P = 0.25). The cumulative incidence of therapeutic failure at day 28 by a life-table analysis method was 5.76% (95% CI: 2.2-14.61) and 0.75% (95% CI: 0.11-5.2%) in the CQ and CQ + PQ group, respectively (P = 0.19). The relapse rate was 8% (9/108) for the CQ group and 3% (4/132) for the comparison group (P = 0.07). The cumulative risk of relapse at day 157 by a life-table method was 61.8% (95% CI: 20.1-98.4%) in the CQ group, compared with 26.3% (95% CI: 7.5-29.4%) in the CQ + PQ group (P = 0.0038).The study confirms the emergence of CQ and PQ resistance/treatment failure in P. vivax malaria in Ethiopia. Although treatment failures were detected, they were similar between the treatment groups. We recommend regular monitoring and periodic evaluation of the efficacy of these antimalarial drugs in systematically selected sentinel sites to detect further development of resistance and to make timely national antimalarial drug policy changes.     

Spatial and temporal variations of malaria epidemic risk in Ethiopia: factors involved and implications

The aim of this study was to describe spatial and temporal variations in malaria epidemic risk in Ethiopia and to examine factors involved in relation to their implications for early warning and interpretation of geographical risk models. Forty-eight epidemic episodes were identified in various areas between September 1986 and August 1993 and factors that might have led to the events investigated using health facility records and weather data. The study showed that epidemics in specific years were associated with specific geographical areas. A major epidemic in 1988 affected the highlands whereas epidemics in 1991 and 1992 affected highland-fringe areas on the escarpments of the Rift Valley and in southern and north-western parts of the country. Malaria epidemics were significantly more often preceded by a month of abnormally high minimum temperature in the preceding 3 months than based on random chance, whereas frequency of abnormally low minimum temperature prior to epidemics was significantly lower than expected. Abnormal increases of maximum temperature and rainfall had no positive association with the epidemics. A period of low incidence during previous transmission seasons might have aggravated the events, possibly due to low level of immunity in affected populations. Epidemic risk is a dynamic phenomenon with changing geographic pattern based on temporal variations in determinant factors including weather and other eco-epidemiological characteristics of areas at risk. Epidemic early warning systems should take account of non-uniform effects of these factors by space and time and thus temporal dimensions need to be considered in spatial models of epidemic risks.     

Association between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from Thailand

The relationship between the in vitro susceptibility of Plasmodium falciparum isolates to the quinoline antimalarials chloroquine (CQ), mefloquine (MQ), and quinine (QN), and pfcrt and pfmdr1 gene polymorphisms were investigated. Field isolates (110 samples) were collected from various endemic areas of Thailand throughout 2002-2004. The pfcrt 76T allele was identified in 109 isolates (99.1%) while pfcrt 76K was found in a single (0.9%) isolate. The pfmdr 86N, 86Y, and the combination (86N + 86Y) alleles were identified in 83 (75.5%), 22 (20%), and 5 (4.5%) isolates, respectively. The pfmdr1 1042N, 1042D alleles and a mixture (1042N + 1042D) of the alleles were found in 94 (85.5%), 12 (10.9%) and 4 (3.6%) isolates, respectively. The pfmdr1 1246Y allele was detected in a single (0.9%) isolate. The pfmdr1 gene polymorphisms (86-1042-1246) was grouped into seven haplotypes as follows: N-N-D (68 isolates; 61.2%), Y-N-D (22 isolates; 19.8%), N-D-D (11 isolates; 9.9%), N-D-Y (1 isolate; 0.9%), N/Y-N-D (4 isolates; 3.6%), N-N/D-D (3 isolates; 2.7%), and N/Y-N/D-D (1 isolate; 0.9%). Eight different combinations of pfcrt-pfmdr1 genotypes were observed. Only one CQ-, MQ- and QN-sensitive isolate was found at the Thai-Laos border and no cases of QN resistance were found in this study.     

Quinoline resistance associated polymorphisms in the pfcrt, pfmdr1 and pfmrp genes of Plasmodium falciparum in Iran

Plasmodium falciparum resistance to chloroquine (CQ) has been documented in Iran since the early 1980s and has since gradually increased. Iran is therefore reviewing its national drug policy for malaria control. We describe the prevalence of single nucleotide polymorphisms (SNP) associated with quinoline drug resistance in south eastern Iran. Pre-treatment blood from patients with uncomplicated but symptomatic P. falciparum infection was analysed. Polymorphisms at codons 76, 152, 163 and 220 of the pfcrt gene (chloroquine resistance transporter) and at codons 86, 184, 1034, 1042 and 1246 of the pfmdr1 gene (multidrug resistance) were determined by PCR-RFLP and sequencing. In addition, SNPs on a recently described multidrug resistance protein (pfmrp) and a microsatellite (MS-4760) in the pfnhe-1 (sodium hydrogen exchanger) gene associated with quinoline and quinine resistance, respectively, were investigated for the first time in field samples not from Thailand. pfcrt 76T was found in 99% and pfmdr1 86Y in 72% of the samples. pfmrp 191H and 437S associated with decreased quinoline response were found to be absolutely linked at a frequency of 13.6%. The pfnhe-1 MS-4760 one repeat allele associated to quinine response in vitro was also detected. Sequencing of the pfcrt 72-76 haplotype revealed that SVMNT was the most common allele as previously observed in India. This suggests that pfcrt found in the Iranian P. falciparum population may have the same origin as in the P. falciparum populations in India but different from that normally found in south east Asia. In conclusion, the frequencies of quinoline resistance associated gene polymorphisms in this region suggest a population that has been significantly selected for by the long use of CQ.     

Genotyping of Giardia duodenalis from human and animal samples from Brazil using beta-giardin gene: a phylogenetic analysis

Giardia duodenalis is one of the major diarrhea agents in human and animals distributed worldwide, and present high levels of genetic diversity, showing seven genotypes: A, B, C, D, E, F, and G. Only Assemblages A and B have been detected in humans and in a wide range of other mammalians hosts, whereas the remaining Assemblages (C-G) are host-specific. Molecular characterization of cysts of human and animal origin are useful to address the co-circulate isolates between these host, and represents an objective means to evaluate zoonotic infection hypothesis. In the present work the G. duodenalis genotypes were characterized by restriction fragment length polymorphisms and DNA sequencing analysis of PCR products of the beta-giardin gene. The cysts were collected in the city of Rio de Janeiro, in Brazil, from a population composed by humans (n=366, 310 children and 56 adults), domestic animals (n=11) from a municipal daycare center in the surroundings of a slum and neighborhood medium-high class domestic animals (n=18). Parasitological exams were developed in human fecal samples. Parasites were found in 60% (186/310) and 66% (37/56) of the samples from children and adults, respectively. Among children's samples, 27.7% (86/310) were positive for G. duodenalis. Only 1.7% (1/56) of the adults was positive for this parasite. In general a total of 87 fecal samples (86 from children and 1 from adult) from all population studied were positive for G. duodenalis, and 62 of these were subjected to molecular analysis using a PCR that amplified a fragment of the beta-giardin gene. Sixty samples were typed as genotype A1, two as genotype A2 and genotype B was not encountered. Among domestic animals samples (n=29), eight (seven dogs and one cat) from the slum community were identified as genotype A1, and all control samples (n=18) were negative in the molecular assay. The host-specific genotypes C, D and, F were not found. In this study we described single case of G. duodenalis infection associated with a child and her dog and both isolates characterized as genotype A1. Despite the low incidence, this data suggest the putative existence of a zoonotic cycle of G. duodenalis in the studied population.     

Alfalfa benefits from Medicago truncatula: the RCT1 gene from M. truncatula confers broad-spectrum resistance to anthracnose in alfalfa

Alfalfa is economically the most important forage legume worldwide. A recurrent challenge to alfalfa production is the significant yield loss caused by disease. Although knowledge of molecular mechanisms underlying host resistance should facilitate the genetic improvement of alfalfa, the acquisition of such knowledge is hampered by alfalfa's tetrasomic inheritance and outcrossing nature. However, alfalfa is congeneric with the reference legume Medicago truncatula, providing an opportunity to use M. truncatula as a surrogate to clone the counterparts of many agronomically important genes in alfalfa. In particular, the high degree of sequence identity and remarkably conserved genome structure and function between the two species enables M. truncatula genes to be used directly in alfalfa improvement. Here we report the map-based cloning of RCT1, a host resistance (R) gene in M. truncatula that confers resistance to multiple races of Colletotrichum trifolii, a hemibiotrophic fungal pathogen that causes anthracnose disease of alfalfa. RCT1 is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant R genes and confers broad-spectrum anthracnose resistance when transferred into susceptible alfalfa plants. Thus, RCT1 provides a novel resource to develop anthracnose-resistant alfalfa cultivars and contributes to our understanding of host resistance against the fungal genus Colletotrichum. This work demonstrates the potential of using M. truncatula genes for genetic improvement of alfalfa.     

Role of pfmdr1 mutations on chloroquine resistance in Plasmodium falciparum isolates with pfcrt K76T from Papua New Guinea

The N86Y mutation in pfmdr1 is reported to play an additional role for the chloroquine resistance in Plasmodium falciparum isolates. However, not much has been done to clarify whether this mutation augments the level of chloroquine resistance in the isolates harboring pfcrt K76T mutation. We compared the in vitro chloroquine efficacy between pfcrt K76T mutant parasites with or without N86Y mutation from Papua New Guinea. A total of 57 isolates (4% sensitive, 14% borderline, and 82% resistant) were successfully tested in vitro for chloroquine sensitivity. We found a slightly higher effective concentration of chloroquine needed to inhibit P. falciparum by 50% (mean EC50=107 nM) in isolates with the pfcrt K76T+pfmdr1 N86Y than that in isolates with the pfcrt K76T+pfmdr1 N86 (EC50=88 nM), but this difference was not statistically significant. A significant non-random association was observed between the pfcrt K76T and pfmdr1 N86Y alleles. Our results suggest that the pfmdr1 N86Y mutation plays a compensatory role to chloroquine-resistant isolates under a chloroquine pressure while it may also augment the level of chloroquine resistance in the K76T parasites to a small extent.     

Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine-pyrimethamine resistance in malaria-infected patients from Nigeria

Mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes have been used as means to predict treatment failure to sulfadoxine-pyrimethamine (SP) and for monitoring/surveillance of resistance to the drug in many areas where malaria is endemic. However, patients responses to treatment are significantly dependent on factors like host immunity profile of treated patients. In order to investigate the relationship between molecular markers of SP resistance, host immunity and clinical outcome, the association between pre-treatment dhfr and dhps genotypes, age and treatment outcomes was evaluated in 109 children treated with SP for acute uncomplicated malaria in Ibadan, Nigeria. Seventy-three percent of the children were cured with the drug, while 27% failed treatment after 28 days of follow-up. All children infected with parasites harboring less than two dhfr/dhps mutations were cured with SP. The dhfr triple (Asn-108/Ile-51/Arg-59) mutants or the dhps double mutants (Gly-437/Glu-540) were independently associated with SP treatment failure in children aged less than 5 years, but not in older children. The dhfr and dhps quintuple mutant (dhfr triple mutant+dhps double mutant) was the genotype most strongly associated with SP treatment failure (OR=24.72, 95%CI=8.24-74.15) in both younger and older children.     

Occurrence of extended spectrum beta-lactamase enzymes in clinical isolates of Klebsiella species from Harar region, eastern Ethiopia

Extended Spectrum beta-Lactamases (ESBLs) producer and multidrug resistant Klebsiella spp. are becoming a major nosocomial pathogen globally. There are no documented reports yet on the occurrence of ESBL enzymes in Klebsiella spp. species from Ethiopia. This study was undertaken to isolate and determine the occurrence of ESBLs and multi-drug resistant Klebsiella spp. in different clinical samples obtained from patients. A cross-sectional survey was conducted in four different hospitals of Harar region (Hiwot Fana, Misrak-Arbegnoch, Police and Army) from December 2003 to February 2004. Three hundred and eighty four clinical specimens (202 sputum, 164 urine and 18 pus) were collected from patients admitted in different wards. Antimicrobial susceptibilities were performed on 57 clinical isolates by standard disk diffusion procedures against eight antimicrobial agents. The ESBLs detection was made by using cefotaxime and ceftazidime alone and in combination with clavulanate. A total of 57 (15%) Klebsiella spp. were isolated from 384 patients. Of the 57 isolates, 33 (58%) were from sputum, 18 (31.5%) from urine and 6 (10.5%) from pus. Of the 57 Klebsiella spp., 54 (94.7%) were identified as K. pneumoniae and 3 (5.3%) as K. oxytoca. Resistance was found against cephalosporins [cefotaxime (39.0%), cefoxitin (39.0%), ceftazidime (40.0%), ceftriaxone (40.0%), cephalothin (42.0%)], chloramphenicol (70.0%), gentamicin (61.0%) and trimethoprim-sulphamethoxazole (65.0%). Analyzed Klebsiella isolates were characterized also by a high degree of multi-resistance (67.0%). In 19/57 (33.3%) of the Klebsiella isolates, ESBL production was detected. Rates of detection of ESBL producers were 42.1, 26.3, 26.3 and 5.3% in Hiwot-Fana, Misrak-Arbegnoch, Police and Army hospitals, respectively. Multi-drug resistant isolates were more prevalent among the ESBLs producers (95.0%) than non-producers (53.0%) (p=0.24). In conclusion, our results show that awareness of ESBL production by Klebsiella spp. is clinically important. In the absence of infection control measures, ESBL producing organisms readily pass horizontally from patient to patient. These strains also transiently colonize the hands of hospital staff members, thereby facilitating patient-to-patient transmission of the organism.     

PCR and microsatellite analysis of diminazene aceturate resistance of bovine trypanosomes correlated to knowledge,attitude and practice of livestock keepers in South-Western Ethiopia

African Animal Trypanosomosis is threatening the agricultural production and cattle breeding more severely than any other livestock disease in the continent, even more since the advent of drug resistance. A longitudinal study was conducted from November 2012 to May 2013 in the Ghibe valley to evaluate diminazene aceturate (DA) resistance and assess livestock owner's perception of trypanocidal drug use. Four Peasant Associations (PAs) were purposively selected and the cattle randomly sampled in each PAs. At the beginning of the study (t₀), 106 bovines positive for trypanosomes by the haematocrit centrifugation technique (HCT) and 119 negative control animals were recruited for six months follow-up using HCT, 18S-PCR-RFLP, DpnII-PCR-RFLP and microsatellite analysis. Prevalence of trypanosomosis was 18.1% based on the HCT technique and the mean PCV value was 23.6 ± 5.1% for the 587 sampled cattle. Out of the 106 HCT positive, 64 (60.4%) were positive for the presence of trypanosomes using the 18S-PCR-RFLP. Species detection showed 38 (59.4%) Trypanosoma congolense savannah, 18 (28.1%) Trypanosoma vivax, 5 (7.8%) Trypanosoma theileri and 3 (4.7%) T. congolense Kilifi. Among the T. congolense savannah samples, 31 (81.6%) showed a DA resistant RFLP profile, 2 (5.3%) a mixed profile and 5 did not amplify using the DpnII-PCR-RFLP. A positive HCT had a significant effect on PCV (p < 0.001) with the mean PCV value equal to 24.4 ± 0.2% in the absence of trypanosomes and to 20.9 ± 0.3% in the presence of trypanosomes. PCV increased significantly (p < 0.001) with 4.4 ± 0.5% one month after treatment. All T. congolense savannah type were analyzed using microsatellite markers TCM1, TCM3 and TCM4. The main events were new infections (40.0%) and relapses (37.5%) with cures lagging at 22.5%. In 10 purposively selected PAs a semi-structured questionnaire was used. The average herd size was the highest in Abelti PA (6.7 ± 1.8 TLU) and the mean herd size was statistically different (p = 0.01) in the 10 PAs. Trypanosomosis was designated as the main disease affecting cattle by 97% of the respondents. DA was used by 95.5% of the farmers though more than half of them (51.9%) were not familiar with isometamidium (ISM). There was a trend to overdose young small animals and to underdose large ones. Oxen were treated very frequently (nearly 20 times/year) and calves almost never. To improve the situation in the Ghibe valley, extension messages should be delivered to promote a rational drug use, improved livestock management and the application of strategic vector control methods.