Polymorphism in the HASPB Repeat Region of East African Leishmania donovani Strains

Background/Objectives

Visceral leishmaniasis (VL) caused by Leishmania donovani is a major health problem in Ethiopia. Parasites in disparate regions are transmitted by different vectors, and cluster in distinctive genotypes. Recently isolated strains from VL and HIV-VL co-infected patients in north and south Ethiopia were characterized as part of a longitudinal study on VL transmission.

Methodology/Principal Findings

Sixty-three L. donovani strains were examined by polymerase chain reaction (PCR) targeting three regions: internal transcribed spacer 1 (ITS1), cysteine protease B (cpb), and HASPB (k26). ITS1- and cpb - PCR identified these strains as L. donovani. Interestingly, the k26 - PCR amplicon size varied depending on the patient''s geographic origin. Most strains from northwestern Ethiopia (36/40) produced a 290 bp product with a minority (4/40) giving a 410 bp amplicon. All of the latter strains were isolated from patients with HIV-VL co-infections, while the former group contained both VL and HIV-VL co-infected patients. Almost all the strains (20/23) from southwestern Ethiopia produced a 450 bp amplicon with smaller products (290 or 360 bp) only observed for three strains. Sudanese strains produced amplicons identical (290 bp) to those found in northwestern Ethiopia; while Kenyan strains gave larger PCR products (500 and 650 bp). High-resolution melt (HRM) analysis distinguished the different PCR products. Sequence analysis showed that the k26 repeat region in L. donovani is comprised of polymorphic 13 and 14 amino acid motifs. The 13 amino acid peptide motifs, prevalent in L. donovani, are rare in L. infantum. The number and order of the repeats in L. donovani varies between geographic regions.

Conclusions/Significance

HASPB repeat region (k26) shows considerable polymorphism among L. donovani strains from different regions in East Africa. This should be taken into account when designing diagnostic assays and vaccines based on this antigen.     

Phylogenetic characterisation of Taenia tapeworms in spotted hyenas and reconsideration of the “Out of Africa” hypothesis of Taenia in humans

The African origin of hominins suggests that Taenia spp. in African carnivores are evolutionarily related to the human-infecting tapeworms Taenia solium, Taenia saginata and Taenia asiatica. Nevertheless, the hypothesis has not been verified through molecular phylogenetics of Taenia. This study aimed to perform phylogenetic comparisons between Taenia spp. from African hyenas and the congeneric human parasites. During 2010–2013, 233 adult specimens of Taenia spp. were collected from 11 spotted hyenas in Ethiopia. A screening based on short DNA sequences of the cytochrome c oxidase subunit 1 gene classified the samples into four mitochondrial lineages designated as I–IV. DNA profiles of nuclear genes for DNA polymerase delta (pold) and phosphoenolpyruvate carboxykinase (pepck) showed that lineages II and III can be assigned as two independent species. Common haplotypes of pold and pepck were frequently found in lineages I and IV, suggesting that they constitute a single species. Morphological observations suggested that lineage II is Taenia crocutae, but the other lineages were morphologically inconsistent with known species, suggesting the involvement of two new species. A phylogenetic tree of Taenia spp. was reconstructed by the maximum likelihood method using all protein-coding genes of their mitochondrial genomes. The tree clearly demonstrated that T. crocutae is sister to T. saginata and T. asiatica, whereas T. solium was confirmed to be sister to the brown bear tapeworm, Taenia arctos. The tree also suggested that T. solium and T. arctos are related to two species of Taenia in hyenas, corresponding to lineages I + IV and III. These results may partially support the African origin of human-infecting Taenia spp., but there remains a possibility that host switching of Taenia to hominins was not confined to Africa. Additional taxa from African carnivores are needed for further testing of the “Out of Africa” hypothesis of Taenia in humans.     

The impact of asymptomatic helminth co-infection in patients with newly diagnosed tuberculosis in north-west ethiopia

Background

Areas endemic of helminth infection, tuberculosis (TB) and HIV are to a large extent overlapping. The aim of this study was to assess the impact of asymptomatic helminth infection on the immunological response among TB patients with and without HIV, their house hold contacts and community controls.

Methodology

Consecutive smear positive TB patients (n = 112), their household contacts (n = 71) and community controls (n = 112) were recruited in Gondar town, Ethiopia. Stool microscopy, HIV serology, serum IgE level, eosinophil and CD4 counts were performed and tuberculosis patients were followed up for 3 months after initiation of anti-TB treatment.

Results

Helminth co-infection rate was 29% in TB patients and 21% in both community control and household contacts (p = 0.3) where Ascaris lumbricoides was the most prevalent parasite. In TB patients the seroprevalence of HIV was 47% (53/112). Eosinophilia and elevated IgE level were significantly associated with asymptomatic helminth infection. During TB treatment, the worm infection rate of HIV+/TB patients declined from 31% (10/32) at week 0 to 9% (3/32) at week 2 of TB treatment, whereas HIV−/TB patients showed no change from baseline to week 2, 29% (13/45) vs. 22.2% (10/45). This trend was stable at week 8 and 12 as well.

Conclusion

One third of smear positive TB patients were infected with helminths. Eosinophilia and elevated IgE level correlated with asymptomatic worm infection, indicating an effect on host immunity. The rate of worm infection declined during TB treatment in HIV+/TB co-infected patients whereas no decline was seen in HIV−/TB group.     

High Parasitological Failure Rate of Visceral Leishmaniasis to Sodium Stibogluconate among HIV Co-infected Adults in Ethiopia

Background

Antimonials are still being used for visceral leishmaniasis (VL) treatment among HIV co-infected patients in East-Africa due to the shortage of alternative safer drugs like liposomal amphotericin B. Besides tolerability, emergence of resistance to antimonials is a major concern.

Objectives

This study was aimed at assessing the clinical outcome of VL-HIV co-infected patients when treated with sodium stibogluconate (SSG).

Methods

Retrospective patient record analysis of VL-HIV co-infected patients treated at a clinical trial site in north-west Ethiopia was done. Patients with parasitologically confirmed VL and HIV co-infection treated with SSG were included. The dose of SSG used was 20 mg Sb5 (pentavalent antimony)/kg and maximum of 850 mg Sb5 for 30 days. The clinical outcomes were defined based on the tissue aspiration results as cure or failure, and additionally the safety and mortality rates were computed.

Results

The study included 57 patients treated with SSG and by the end of treatment only 43.9% of patients were cured. The parasitological treatment failure and the case fatality rate were 31.6% and 14.0% respectively. SSG was discontinued temporarily or permanently for 12 (21.1%) cases due to safety issues. High baseline parasite load (graded more than 4+) was significantly associated with treatment failure (odds ratio = 8.9, 95% confidence interval = .5-51.7).

Conclusion

SSG is not only unsafe, but also has low effectiveness for VL-HIV patients. Safe and effective alternative medications are very urgently needed. Drug sensitivity surveillance should be introduced in the region.     

Visceral Leishmaniasis and HIV Coinfection in East Africa

Visceral Leishmaniasis (VL) is an important protozoan opportunistic disease in HIV patients in endemic areas. East Africa is second to the Indian subcontinent in the global VL caseload and first in VL-HIV coinfection rate. Because of the alteration in the disease course, the diagnostic challenges, and the poor treatment responses, VL with HIV coinfection has become a very serious challenge in East Africa today. Field experience with the use of liposomal amphotericin B in combination with miltefosine, followed by secondary prophylaxis and antiretroviral drugs, looks promising. However, this needs to be confirmed through clinical trials. Better diagnostic and follow-up methods for relapse and prediction of relapse should also be looked for. Basic research to understand the immunological interaction of the two infections may ultimately help to improve the management of the coinfection.     

Mathematics of a model for Zika transmission dynamics

Zika is a flavivirus transmitted to humans through either the bite of infected Aedes mosquitoes or sexual intercourse with infected individuals. In this paper, we present a mathematical model based on these two modes of transmission. Using the next-generation matrix method, a threshold parameter called basic reproduction number is determined. Sensitivity analysis of the basic reproduction number in terms of parameters involved in its formulation is discussed. A dynamically consistent nonstandard finite difference scheme is designed to replicate the properties of the continuous model. Numerical simulations of the nonstandard finite difference scheme that we have constructed show the number of infected humans due to sexual intercourse with Zika virus infectious individuals. The numerical simulations also support the theoretical analysis of the model.     

Molecular markers from a BAC contig spanning the Rdr1 locus: a tool for marker-assisted selection in roses

We constructed a BAC contig of about 300 kb spanning the Rdr1 locus for black spot resistance in Rosa multiflora hybrids, using a new BIBAC library from DNA of this species. From this contig, we developed broadly applicable simple sequence repeat (SSR) markers tightly linked to Rdr1, which are suitable for genetic analyses and marker-assisted selection in roses. As a source for the high molecular weight DNA, we chose the homozygous resistant R. multiflora hybrid 88/124-46. For the assembly of the BAC contig, we made use of molecular markers derived from a previously established R. rugosa contig. In order to increase the resolution for fine mapping, the size of the population was increased to 974 plants. The genomic region spanning Rdr1 is now genetically restricted to 0.2 cM, corresponding to a physical distance of about 300 kb. One single-stranded conformational polymorphism (SSCP) and one SSR marker cosegregate with the Rdr1-mediated black spot resistance, while one SSR and several cleaved amplified polymorphic sequence or SSCP markers are very tightly linked with one to three recombinants among the 974 plants. The benefits of the molecular markers developed from the R. multiflora contig for the genetic analysis of roses and the integration of rose genetic maps are discussed.     

Effect of a formulation of Bacillus firmus on root-knot nematode Meloidogyne incognita infestation and the growth of tomato plants in the greenhouse and nursery

Bacillus firmus, commercial WP formulation (BioNem) was evaluated against the root-knot nematode Meloidogyne incognita in a laboratory, greenhouse and under field conditions on tomato plants. In the laboratory tests, an aqueous suspension of BioNem at 0.5%, 1%, 1.5% and 2% concentration reduced egg hatching from 98% to 100%, 24-days after treatment. Treatment of second-stage juveniles with 2.5% and 3% concentration of BioNem, caused 100% inhibition of mobility, 24 h after treatment. In the green house trials, BioNem applied at 8 g/pot (1200 cc soil) planted with a tomato seedlings reduced gall formation by 91%, final nematode populations by 76% and the number of eggs by 45%. Consequently, plant height and biomass was increased by 71% and 50%, respectively, compared to the untreated control, 50-days after treatment application. Application of BioNem at 16 g/pot was phytotoxic to plants. In the field trails, BioNem applied at 200 and 400 kg ha⁻¹ was effective in reducing the number of galls (75-84%), and increased shoot height (29-31%) and weight (20-24%) over the untreated control, 45-days after treatment. Our results indicate that B. firmus is a promising microorganism for the biological control of M. incognita in tomato pots.     

Isolation of a partial sequence of a putative nucleotide sugar epimerase, which may involve in stamen development in cucumber (Cucumis sativus L.)

Sex determination is the most widely studied subject in cucumber. The sex of cucumber plants can be monoecious, hermaphrodite, gynoecious, androecious, or andromonoecious. Besides environmental factors, three major genes, F/f, M/m, and A/a mainly govern the sex types in cucumber. Regardless of their sex all floral buds are bisexual at the early bud stage. A stage specific arrest of either stamen or carpel leads to unisexual flower development. The possible downstream product of the interaction of the sex determining genes that may directly allow the growth or selectively arrest stamen or pistil is not yet identified. Therefore, in the current study, we performed suppression subtractive hybridization using floral buds from nearly isogenic gynoecious and hermaphrodite cucumber plants and identified for the first time a cDNA homologous to nucleotide sugar epimerase. The expression level of the isolated putative nucleotide sugar epimerase is weak in female floral buds but strong in bisexual and male flowers. The weak level of the putative nucleotide sugar epimerase may be an indication for its improper functioning, which may influence stamen development in cucumber plants.