New insights about cross-reactive epitopes of six trypanosomatid genera revealed that Crithidia and Leptomonas have antigenic similarity to L. (L.) chagasi

We investigated whether ELISA using crude antigens from insect and plant trypanosomatids, which are non-pathogenic and easily cultivated in large scale, has the same positivity data as Leishmania (Leishmania) chagasi, the etiological agent of human visceral leishmaniasis (VL) or canine leishmaniasis (CanL), or as Trypanosoma cruzi, the etiological agent of Chagas disease (CD). The antigens from Crithidia fasciculata, Crithidia luciliae, and Leptomonas seymouri showed 100% cross-reactivity with VL and CanL samples, with no statistically titers differences from L. (L.) chagasi, however, 34% (17/50) of VL samples revealed higher titers using the insect trypanosomatids than the homologous antigen. On the other hand, antigens from Strigomonas culicis, Angomonas deanei, and Phytomonas serpens showed low cross-reactivity with VL and CanL samples. The sera from patients with American tegumentary leishmaniasis showed low levels of cross-reactivity with all trypanosomatids investigated, even with L. (L) chagasi, without titers dissimilarity among them. These parasites were also worthless as antigen source for detection of CD cases, which required homologous antigens to reach 100% positivity. This study showed, by ELISA, that crude extract of Crithidia and Leptomonas have epitopes similar to L. (L.) chagasi, which supports the idea of using them as antigens source for the serodiagnosis of visceral leishmaniasis.     

The life cycle of Ascocotyle (Phagicola) longa (Digenea: Heterophyidae), a causative agent of fish-borne trematodosis

The complete life cycle of the trematode Ascocotyle (Phagicola) longa (Digenea: Heterophyidae) is elucidated by natural observation validated by experimental infections. The natural first intermediate host of A. (P.) longa, an agent of human heterophyiasis in Brazil, is the cochliopid snail Heleobia australis (new first intermediate host). Metacercariae were found encysted in the body musculature, heart, stomach, liver, kidney, spleen, gonads and mesentery of mullets Mugil liza. Hamsters Mesocricetus auratus were experimentally infected with metacercariae of A. (P.) longa obtained from the mullets, and the adults recovered were used to infect the snails H. australis. Rediae and cercariae of A. (P.) longa are described for the first time. The ultrastructure of the tegument of A. (P.) longa shows a change in spination pattern from the cercaria with single-pointed spines to the metacercaria and adult with multipointed, brush-shaped spines. The life cycle of A. (P.) longa is related to estuaries and coastal lagoons where the recruitment of mugilid juveniles occurs. The high prevalence (100%) of A. (P.) longa encysted in the mullets examined within the urban area of Rio de Janeiro indicates the potentially great public health impact of the consumption of raw mullets.     

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.     

First human cases of Leishmania (Viannia) naiffi infection in Ecuador and identification of its suspected vector species

Epidemiological surveillance of leishmaniasis was conducted in a northern Amazonian region of Ecuador, in which cutaneous leishmaniasis cases were recently reported. Sand flies were captured in the military training camp, and the natural infection of sand flies by Leishmania species was examined. Out of 334 female sand flies dissected, the natural infection by flagellates was microscopically detected in 3.9% of Lutzomyia yuilli yuilli and 3.7% of Lutzomyia tortura, and the parasite species were identified as Endotrypanum and Leishmania (Viannia) naiffi, respectively. After the sand fly surveillance, specimens from cutaneous leishmaniasis (CL) patients considered to have acquired the infection in the training camp area were obtained, and the infected parasite species were identified as L. (V.) naiffi. The present study reported first cases of CL caused by L. (V.) naiffi infection in Ecuador. In addition, a high ratio of infection of Lu. tortura by L. (V.) naiffi in the same area strongly suggested that Lu. tortura is responsible for the transmission of L. (V.) naiffi in this area.     

Phlebotomus papatasi exposure cross-protects mice against Leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate

Leishmania parasites are inoculated into host skin together with sand fly saliva and multiple exposures to uninfected sand fly bites protect mice against Leishmania infection. However, sand fly vectors differ in composition of the saliva and therefore the protection elicited by their salivary proteins was shown to be species-specific. On the other hand, the optimal vaccine based on sand fly salivary proteins should be based on conserved salivary proteins conferring cross-reactivity. In the present study we therefore focused on cross-protective properties of saliva from Phlebotomus papatasi and Phlebotomus duboscqi, the two natural vectors of Leishmania major. Two groups of mice exposed to bites of P. papatasi and two control, non-immunized groups were infected with L. major promastigotes along with either P. papatasi or P. duboscqi salivary gland homogenate. All mice were followed for the development of Leishmania lesions, parasite burdens, specific antibodies, and for production of NO, urea, or cytokines by peritoneal macrophages. Protection against Leishmania infection was observed not only in exposed mice challenged with homologous saliva but also in the group challenged with P. duboscqi saliva. Comparing both exposed groups, no significant differences were observed in parasite load, macrophage activity, or in the levels of anti-L. major and anti-P. papatasi/P. duboscqi antibodies. This is the first study showing cross-protection caused by salivary antigens of two Phlebotomus species. The cross-protective effect suggests that the anti-Leishmania vaccine based on P. papatasi salivary proteins might be applicable also in areas where L. major is transmitted by P. duboscqi.     

Cutaneous leishmaniasis caused by Leishmania (L.) major infection in Sindh province, Pakistan

Leishmaniasis is endemic in Pakistan and is wide-spread throughout the country. Polymerase chain reaction (PCR) was performed to identify the Leishmania species present in cutaneous leishmaniasis (CL) patients from new endemic areas of the central part of Sindh province, Pakistan. The PCR primers used were designed for the identification and differentiation of Leishmania (Leishmania) major and Leishmania (Leishmania) tropica species, and PCR bands at 620 and 830 bp of the parasite-specific kinetoplast DNA sequences was identified for L. (L.) major and L. (L.) tropica, respectively. Among a total of 144 DNA samples purified from the skin biopsies of clinically suspected CL patients, 108 (75%) were positive for PCR amplification. Out of the 108 cases, 105 (97.2%) were determined to be positive for L. (L.) major infection, and 3 (2.8%) were positive for L. (L.) tropica infection. It was concluded that CL caused by L. (L.) major is the main source of infection in the central part of Sindh province in Pakistan. This rapid screening technique could be used for the diagnosis of a large number of samples from skin lesions, which commonly contain other bacterial and fungal infections.     

Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi

We evaluated the antifilarial activity of 6 flavonoids against the human lymphatic filarial parasite Brugia malayi using an in vitro motility assay with adult worms and microfilariae, a biochemical test for viability (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-reduction assay), and two animal models, Meriones unguiculatus (implanted adult worms) and Mastomys coucha (natural infections). In vitro, naringenin and hesperetin killed the adult worms and inhibited (>60%) MTT-reduction at 7.8 and 31.2 μg/ml concentration, respectively. Microfilariae (mf) were killed at 250-500 μg/ml. The half maximal inhibitory concentration (IC₅₀) of naringenin for motility of adult females was 2.5 μg/ml. Flavone immobilized female adult worms at 31.2 μg/ml (MTT > 80%) and microfilariae at 62.5 μg/ml. Rutin killed microfilariae at 125 μg/ml and inhibited MTT-reduction in female worms for >65% at 500 μg/ml. Naringin had adulticidal effects at 125 μg/ml while chrysin killed microfilariae at 250 μg/ml. In vivo, 50 mg/kg of naringenin elimiated 73% of transplanted adult worms in the Meriones model, but had no effect on the microfilariae in their peritoneal cavity. In Mastomys, the same drug was less effective, killing only 31% of the naturally acquired adult worms, but 51%, when the dose was doubled. Still, effects on the microfilariae in the blood were hardly detectable, even at the highest dose. In summary, all 6 flavonoids showed antifilarial activity in vitro, which can be classed, in a decreasing order: naringenin > flavone = hesperetin > rutin > naringin > chrysin. In jirds, naringenin and flavone killed or sterilized adult worms at 50 mg/kg dose, but in Mastomys, where the parasite produces a patent infection, only naringenin was filaricidal. Thus naringenin and flavone may provide a lead for design and development of new antifilarial agent(s). This is the first report on antifilarial efficacy of flavonoids.     

Anisakis/Ascaris IgE ratio improves specificity for the diagnosis of Anisakis simplex sensitization in travellers and immigrants

Anisakis simplex is a fish parasite responsible for human infection and is able to induce IgE-mediated reactions with several clinical manifestations. Laboratory diagnosis of Anisakis allergy is based on the detection of specific IgE using parasite whole antigen. Unfortunately, these diagnostic tools detect cross-reactivities with other nematodes and micro-organisms leading to low specificity of the diagnostic tests. The aim of this retrospective study was to assess the diagnostic value of specific IgE to Anisakis for diagnosis of A. simplex-sensitization in native Spanish residents (IMM, n = 766) and subjects coming from tropical and sub-tropical geographic areas (TRO, n = 233). Since Ascaris is the human parasite most closely related to Anisakis, specific IgE to Ascaris was also determined to assess Anisakis cross-reaction with other nematodes and the diagnostic value of Anisakis/Ascaris IgE ratio for Anisakis allergy was examined. IMM and TRO groups showed similar specific IgE to Anisakis levels, while TRO had higher levels of specific IgE to Ascaris than IMM group (p = 0.001). ROC curve analysis determined that an Anisakis specific IgE threshold of 0.71 kU/L yielded 93% and 82% specificities in IMM and TRO groups, respectively. A cut-off value ≥4.4 for Anisakis/Ascaris IgE ratio increased specificity to 95% for samples having IgE to Ascaris ≥0.35. In conclusion, the ratio of specific IgE to Anisakis and Ascaris improved remarkably the specificity and this parameter easily obtained from the commercially available system could be useful in the diagnosis of hypersensitivity to A. simplex.     

Asociación en un mismo plásmido de blaVIM-1 y qnrS2 en Klebsiella pneumoniae y Klebsiella oxytoca aisladas en Sevilla

Background

Combined resistance to quinolones and β-lactams is common in Enterobacteriaceae. The appearance in enterobacteria coding for metallo-β-lactamases and determinants of plasmid-mediated quinolone resistance are an emerging problem in our country.

Methods

The susceptibility was determined by E-test. The resistance genes were detected by PCR and the corresponding plasmids were characterised.

Results

This study describes 2 strains (1 Klebsiella oxytoca, 1 Klebsiella pneumoniae) carrying the genes qnrS2 and blaVIM-1 in a transferable plasmid of 70-Kb isolated in surveillance cultures at the University Hospital Virgen Macarena in Seville.

Conclusion

This is the first combination of qnrS2 and bla_VIM-1 on the same non-typeable plasmid isolated in our centre.     

Distribution of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles in Plasmodium vivax isolates from Thailand

The analysis of prevalence and distribution of pvdhfr and pvdhps mutations were performed in 169 samples collected from patients with Plasmodium vivax infection who attended the malaria clinics in the provinces along the three international borders of Thailand (Thai-Myanmar, Thai-Cambodian, and Thai-Malaysian borders). SNP-haplotypes of the pvdhfr at amino acid positions 13, 33, 57, 58, 61, 117, and 173 and of the pvdhps at positions 383 and 553 were examined by nested PCR-RFLP. Significant differences in the prevalence and distribution of pvdhfr and pvdhps combination alleles were observed in P. vivax isolates collected from all the three border areas. The most prevalent combination alleles were triple mutant pvdhfr 57L/58R/117T alleles/double wild-type pvdhps alleles (n = 18), double mutant pvdhfr 58R/117N alleles/double wild-type pvdhps alleles (n = 10), and triple mutant pvdhfr 58R/61M/117N alleles/double wild-type pvdhps alleles (n = 52) or with single mutant pvdhps 383G allele (n = 28), respectively. These information on prevalence and patterns of pvdhfr and pvdhps polymorphisms obtained from the present study suggest the presence of SP pressure on P. vivax isolates in Thailand which could be linked to the introduction of malaria from neighboring countries. Results did not support the application of SP for P. vivax control program in Thailand as well as the neighboring countries.     

Diarylheptanoid compounds from Alnus nepalensis express in vitro and in vivo antifilarial activity

A large number of medicinal plants remain to be explored for antifilarial compounds. In the present study a crude methanolic extract of leaves of Alnus nepalensis, chloroform- and n-butanol-partitioned fractions from the crude extract and 6 bioactivity-guided isolated compounds including two new diarylheptanoid from the fractions were assayed for microfilaricidal, macrofilaricidal and female worm sterilizing activity using the lymphatic filariid Brugia malayi in in vitro and in vivo systems. In vitro, the crude methanolic extract exerted better microfilaricidal (LC100: 15.63 μg/ml, IC50: 6.00 μg/ml) than macrofilaricidal (LC100: >250; IC50: 88 μg/ml) activity whereas chloroform and n-butanol fractions were more macrofilaricidal (LC100: 125 and 31.25 μg/ml; IC50: 13.14 and 11.84, respectively) than microfilaricidal (LC100: 250–500 μg/ml, IC50: 44.16 μg/ml). In addition, n-butanol fraction also caused 74% inhibition in MTT reduction potential of the adult worms. In vivo (doses: crude: 100–200 mg/kg; fractions: 100 mg/kg, i.p. × 5 days) the chloroform fraction exerted >50% macrofilaricidal activity whereas methanolic extract and n-butanol fraction produced 38–40% macrofilaricidal action along with some female sterilizing efficacy. Of the 5 diarylheptanoid compounds isolated, alnus dimer, and (5S)-5-hydroxy-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-3-heptanone were found to show the most potent with both macrofilaricidal (LC100: 15.63 μg/ml, IC50: 6.57–10.31 μg/ml) and microfilaricidal (LC100: 31.25–62.5 μg/ml, IC50: 11.05–22.10 μg/ml) activity in vitro. These findings indicate that the active diarylheptanoid compounds may provide valuable lead for design and development of new antifilarial agent(s).     

Taxonomic assessment of Anopheles crawfordi and An. dangi of the Hyrcanus Group of subgenus Anopheles in Vietnam

Anopheles dangi, introduced as a new species of the Hyrcanus Group of subgenus Anopheles in an illustrated dichotomous key for the identification of the Anopheles mosquitoes of Vietnam published in 1987, was distinguished from Anopheles crawfordi based on the presence of a humeral pale spot on the base of the costal vein of the wing. However, this character has been known to occur occasionally in An. crawfordi. To determine whether An. dangi is distinct from An. crawfordi, we analyzed nucleotide sequences of the COI, COII and Cyt-b genes of mtDNA and the D3 gene of rDNA obtained from specimens collected in south-central Vietnam that were identified as An. dangi and An. crawfordi based on the presence or absence, respectively, of a humeral pale spot. Maximum Likelihood and Bayesian analyses of the sequences showed a low mean genetic distance of 0.004 for specimens identified as An. crawfordi and 0.008 for those identified as An. dangi. The mean genetic distance between the two nominal species was 0.006, compared with 0.077 for any group versus the outgroup taxa Anopheles dirus and Anopheles minimus, and the specimens of the two forms clustered in a single strongly supported clade. Consequently, An. dangi is merely a morphological variant of An. crawfordi and is deemed to be a synonym of that nominal species.     

Laboratory and field evaluation of neem (Azadirachta indica A. Juss) and Chinaberry (Melia azedarach L.) oils as repellents against Phlebotomus orientalis and P. bergeroti (Diptera: Psychodidae) in Ethiopia

The study evaluated the efficacy of neem (Azadirachta indica A. Juss.) and Chinaberry (Melia azedarach L.) seed oils as repellents against laboratory and field populations of some sandflies in Ethiopia. In the laboratory, concentrations of 2% and 5% neem oil in coconut oil tested against Phlebotomus orientalis (vector of visceral leishmaniasis) provided 96.28% (95% CI = 95.60-96.97) protection up to a mean time of 7 h and 20 min and 98.26% (95% CI = 93.46-104. 07) protection up to 9 h, respectively. Similarly, M. azedarach oil at 2% concentration produced 95.13% (95% CI = 90.74-99.52) protection for the same duration (7 h and 20 min), while the 5% oil gave 96.20 (95% CI = 86.98-105.41) protection for 8 h and 20 min against the same species with no significant difference in percentage protection between the two oils at 2% and 5% concentrations. In the field tests with only neem oil (A. indica) against field populations of P. orientalis and P. bergeroti, similar high level of repellencies were recorded with about the same duration of protection. Application of both neem and Chinaberry oils can be safe and low-cost means of personal protection against sandfly bites in endemic areas of Ethiopia, if the community is advised and encouraged to grow the plants abundantly.     

Molecular evidence of misidentification of Anopheles minimus as Anopheles fluviatilis in Assam (India)

Anophelesminimus s.l. and Anophelesfluviatilis s.l., two closely related taxa, are reported vectors of malaria in Assam state of India. We determined the DNA sequences of morphologically identified A. minimus s.l. and A. fluviatilis s.l. collected from the Kamrup district in Assam, for two rDNA loci—internal transcribed spacer 2 (ITS2) and D3 domain of 28S rDNA (28S-D3). Analysis of rDNA data revealed that the sequences of both the morphologically identified A. minimus s.l. and A. fluviatilis s.l. from Assam are identical, homologous to the sequences of A. minimus s.s. (former species A) and different from that of all the reported members of the Fluviatilis Complex (species S, T and U). This indicates that A. fluviatilis s.l. being reported in Kamrup district, Assam, in low density, mostly during January to April, is actually a hypermelanic and seasonal variant of A. minimus. It was also found that the banding pattern on chromosome arm 2 (which bears species-diagnostic inversions for the Fluviatilis Complex) of A. minimus and of A. fluviatilis s.l. from Assam is homosequential with A. fluviatilis species U suggesting that probably previously described A. fluviatilis U from Assam were also A. minimus.     

Anthelmintic activity of benzimidazole derivatives against Toxocara canis second-stage larvae and Hymenolepis nana adults

The anthelmintic activity of 11 benzimidazole derivatives (A1-A11) and 2 thioureides N,N′-disubstituted (B1-B2) was determined. Each compound and albendazole was tested in vitro against Toxocara canis larvae and in vivo against Hymenolepis nana adult. Compounds A1-A6 and B1-B2 were designed as albendazole prodrugs. Compounds A8-A11 were designed as direct analogues of A7, which had previously proved to be an effective agent against Fasciola hepatica. Results of the in vitro screening showed that A6 was more active than albendazole at 0.18 μM (relative mobility 40% and 80%, respectively). Whereas that the in vivo evaluation against H. nana, compounds A7-A11 demonstrated significant activity in terms of removing cestode adults in the range of 88-97%, displaying better efficacy than albendazole (83%).     

The effects of the fungus Metarhizium anisopliae var. acridum on different stages of Lutzomyia longipalpis (Diptera: Psychodidae)

The control of Visceral Leishmaniasis (VL) vector is often based on the application of chemical residual insecticide. However, this strategy has not been effective. The continuing search for an appropriate vector control may include the use of biological control. This study evaluates the effects of the fungus Metarhizium anisopliae var. acridum on Lutzomyia longipalpis. Five concentrations of the fungus were utilized, 1 × 10⁴ to 1 × 10⁸ conidia/ml, accompanied by controls. The unhatched eggs, larvae and dead adults previously exposed to fungi were sown to reisolate the fungi and analysis of parameters of growth. The fungus was subsequently identified by PCR and DNA sequencing. M. anisopliae var. acridum reduced egg hatching by 40%. The mortality of infected larvae was significant. The longevity of infected adults was lower than that of negative controls. The effects of fungal infection on the hatching of eggs laid by infected females were also significant. With respect to fungal growth parameters post-infection, only vegetative growth was not significantly higher than that of the fungi before infection. The revalidation of the identification of the reisolated fungus was confirmed post-passage only from adult insects. In terms of larvae mortality and the fecundity of infected females, the results were significant, proving that the main vector species of VL is susceptible to infection by this entomopathogenic fungus in the adult stage.     

dSir2 and longevity in Drosophila

The silent information regulator 2 (Sir2 or Sirtuin) family of proteins is highly conserved and has been implicated in the extension of longevity for several species. Mammalian Sirtuins have been shown to affect various aspects of physiology including metabolism, the stress response, cell survival, replicative senescence, inflammation, the circadian rhythm, neurodegeneration, and even cancer. Evidence in Drosophila implicates Sir2 in at least some of the beneficial effects of caloric restriction (CR). CR delays age-related pathology and extends life span in a wide variety of species. Here we will review the evidence linking Drosophila Sir2 (dSir2) to longevity regulation and the pathway associated with CR in Drosophila, as well as the effects of the Sir2 activator resveratrol and potential interactions between dSir2 and p53.     

Paediatric hepatocellular carcinoma due to somatic CTNNB1 and NFE2L2 mutations in the setting of inherited bi-allelic ABCB11 mutations

Hepatocellular carcinoma (HCC) rarely occurs in childhood. We describe a patient with new onset of pruritus at 8 months of age who at 17 months of age was found to have a 2.5 cm HCC. To delineate the possible genetic basis of this tumour, we performed whole exome sequencing (WES) of the germline DNA and identified two novel predictably deleterious missense mutations in ABCB11, encoding bile salt export pump (BSEP), confirmed in the parental DNA as bi-allelic and inherited. Although inherited ABCB11 mutations have previously been linked to HCC in a small number of cases, the molecular mechanisms of hepatocellular carcinogenesis in ABCB11 disease are unknown. WES of the HCC tissue uncovered somatic driver mutations in the beta-catenin (CTNNB1) and nuclear-factor-erythroid-2-related-factor-2 (NFE2L2) genes. Moreover, clonality analysis predicted that the CTNNB1 mutation was clonal and occurred earlier during carcinogenesis, whereas the NFE2L2 mutation was acquired later. Interestingly, background liver parenchyma showed no inflammation or fibrosis and BSEP expression was preserved. This is the first study to identify somatic CTNNB1 and NFE2L2 mutations in early childhood arisen in the setting of inherited bi-allelic ABCB11 mutations. Rapid WES analysis expedited this child’s diagnosis and treatment, and likely improved her prognosis.