Ex Vivo Host and Parasite Response to Antileishmanial Drugs and Immunomodulators

Background

Therapeutic response in infectious disease involves host as well as microbial determinants. Because the immune and inflammatory response to Leishmania (Viannia) species defines the outcome of infection and efficacy of treatment, immunomodulation is considered a promising therapeutic strategy. However, since Leishmania infection and antileishmanial drugs can themselves modulate drug transport, metabolism and/or immune responses, immunotherapeutic approaches require integrated assessment of host and parasite responses.

Methodology

To achieve an integrated assessment of current and innovative therapeutic strategies, we determined host and parasite responses to miltefosine and meglumine antimoniate alone and in combination with pentoxifylline or CpG 2006 in peripheral blood mononuclear cells (PBMCs) of cutaneous leishmaniasis patients. Parasite survival and secretion of TNF-α, IFN-γ, IL-10 and IL-13 were evaluated concomitantly in PBMCs infected with Luc-L. (V.) panamensis exposed to meglumine antimoniate (4, 8, 16, 32 and 64 μg Sb^V/mL) or miltefosine (2, 4, 8, 16 and 32 μM HePC). Concentrations of 4 μM of miltefosine and 8 μg Sb^V/mL were selected for evaluation in combination with immunomodulators based on the high but partial reduction of parasite burden by these antileishmanial concentrations without affecting cytokine secretion of infected PBMCs. Intracellular parasite survival was determined by luminometry and cytokine secretion measured by ELISA and multiplex assays.

Principal Findings

Anti- and pro-inflammatory cytokines characteristic of L. (V.) panamensis infection were evaluable concomitantly with viability of Leishmania within monocyte-derived macrophages present in PBMC cultures. Both antileishmanial drugs reduced the parasite load of macrophages; miltefosine also suppressed IL-10 and IL-13 secretion in a dose dependent manner. Pentoxifylline did not affect parasite survival or alter antileishmanial effects of miltefosine or meglumine antimoniate. However, pentoxifylline diminished secretion of TNF-α, IFN-γ and IL-13, cytokines associated with the outcome of infection by species of the Viannia subgenus. Exposure to CpG diminished the leishmanicidal effect of meglumine antimoniate, but not miltefosine, and significantly reduced secretion of IL -10, alone and in combination with either antileishmanial drug. IL-13 increased in response to CpG plus miltefosine.

Conclusions and Significance

Human PBMCs allow integrated ex vivo assessment of antileishmanial treatments, providing information on host and parasite determinants of therapeutic response that may be used to tailor therapeutic strategies to optimize clinical resolution.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Development of Assays Using Hexokinase and Phosphoglucomutase Gene Sequences That Distinguish Strains of Leishmania tropica from Different Zymodemes and Microsatellite Clusters and Their Application to Palestinian Foci of Cutaneous Leishmaniasis

Background/Objectives

Palestinian strains of L.tropica characterized by multilocus enzyme electrophoresis (MLEE) fall into two zymodemes, either MON-137 or MON-307.

Methodology/Principle Findings

Assays employing PCR and subsequent RFLP were applied to sequences found in the Hexokinase (HK) gene, an enzyme that is not used in MLEE, and the Phosphoglucomutase (PGM) gene, an enzyme that is used for MLEE, to see if they would facilitate consigning local strains of L.tropica to either zymodeme MON-137 or zymodeme MON-307. Following amplification and subsequent double digestion with the restriction endonucleases MboI and HaeIII, variation in the restriction patterns of the sequence from the HK gene distinguished strains of L.tropica, L.major and L.infantum and also exposed two genotypes (G) among the strains of L.tropica: HK-LtG1, associated with strains of L.tropica of the zymodemes MON-137 and MON-265, and HK-LtG2, associated with strains of L.tropica of the zymodemes MON-307, MON-288, MON-275 and MON-54. Following amplification and subsequent digestion by the restriction endonuclease MboI, variation in the sequence from the PGM gene also exposed two genotypes among the strains of L.tropica: PGM-G1, associated only with strains of L.tropica of the zymodeme MON-137; and PGM-G2, associated with strains of L.tropica of the zymodemes MON-265, MON-307, MON-288, MON-275 and MON-54, and, also, with six strains of L.major, five of L.infantum and one of L.donovani. The use of the HK and PGM gene sequences enabled distinction the L.tropica strains of the zymodeme MON-137 from those of the zymodeme MON-265. This genotyping system ‘correctly’ identified reference strains of L.tropica of known zymodemal affiliation and also from clinical samples, with a level of sensitivity down to <1 fg in the case of the former and to 1 pg of DNA in the case of the latter.

Conclusions/Significance

Both assays proved useful for identifying leishmanial parasites in clinical samples without resource to culture and MLEE.     

Efficacy of Ciprofloxacin for Treatment of Cholera Associated with Diminished Susceptibility to Ciprofloxacin to Vibrio cholerae O1

Objective

We identified a poor clinical response to treatment of cholera with a single 1 g dose of ciprofloxacin, a standard treatment for cholera.

Methods

To determine reasons for the poor response and better therapeutic approaches we examined the minimal inhibitor concentration (MIC, n = 275) and disc-diffusion zone sizes (n = 205) for ciprofloxacin and nalidixic acid of V. cholerae O1 strains isolated in Bangladesh from 1994 to 2012, and reexamined data from 161patients infected with Vibrio cholerae O1 recruited in four clinical trials who received single- or multiple-dose ciprofloxacin for treatment of cholera and compared their clinical response to the V. cholerae O1 susceptibility.

Results

Although all 275 isolates of V. cholerae O1 remained susceptible to ciprofloxacin using standard MIC and disc-diffusion thresholds, the MIC⁹⁰ to ciprofloxacin increased from 0.010 in 1994 to 0.475 μgm/ml in 2012. Isolates became frankly resistant to nalidixic with the MIC⁹⁰ increasing from 21 μgm/ml in 1994 to >256 μgm/ml and 166 of 205 isolates from 1994 to 2005 being frankly resistant using disc-diffusion testing. Isolates resistant to nalidixic acid by disc-diffusion testing had a median ciprofloxacin MIC of 0.190 μgm/ml (10^th-90^th centiles 0.022 to 0.380); nalidixic acid-susceptible isolates had a median ciprofloxacin MIC of 0.002 (0.002 to 0.012).The rate of clinical success with single-dose ciprofloxacin treatment for nalidixic acid-susceptible strains was 94% (61 of 65 patients) and bacteriologic success 97% (63/65) compared to 18% (12/67) and 8% (5/67) respectively with nalidixic acid-resistant strains (P<0.001 for both comparisons). Multiple-dose treatment with ciprofloxacin had 86% and 100% clinical and bacteriologic success rates respectively in patients infected with nalidixic acid-susceptible strains of V. cholerae O1 compared to clinical success 67% and bacteriologic success 60% with nalidixic acid-resistant strains.

Conclusions

Single-dose ciprofloxacin is not effective for treating cholera caused by V. cholerae O1 with diminished susceptibility to ciprofloxacin, and nalidixic acid disc-diffusion testing effectively screens for such isolates.     

Fluoroquinolone Resistance Mechanisms of Shigella flexneri Isolated in Bangladesh

Objective

To investigate the prevalence and mechanisms of fluoroquinolone resistance in Shigella species isolated in Bangladesh and to compare with similar strains isolated in China.

Methods

A total of 3789 Shigella isolates collected from Clinical Microbiology Laboratory of icddr,b, during 2004–2010 were analyzed for antibiotic susceptibility. Analysis of plasmids, plasmid-mediated quinolone-resistance genes, PFGE, and sequencing of genes of the quinolone-resistance-determining regions (QRDR) were conducted in representative strains isolated in Bangladesh and compared with strains isolated in Zhengding, China. In addition, the role of efflux-pump was studied by using the efflux-pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP).

Results

Resistance to ciprofloxacin in Shigella species increased from 0% in 2004 to 44% in 2010 and S. flexneri was the predominant species. Of Shigella spp, ciprofloxacin resistant (Cip^R) strains were mostly found among S. flexneri (8.3%), followed by S. sonnei (1.5%). Within S. flexneri (n = 2181), 14.5% were resistance to ciprofloxacin of which serotype 2a was predominant (96%). MIC of ciprofloxacin, norfloxacin, and ofloxacin were 6–32 mg/L, 8–32 mg/L, and 8–24 mg/L, respectively in S. flexneri 2a isolates. Sequencing of QRDR genes of resistant isolates showed double mutations in gyrA gene (Ser⁸³Leu, Asp⁸⁷Asn/Gly) and single mutation in parC gene (Ser⁸⁰Ile). A difference in amino acid substitution at position 87 was found between strains isolated in Bangladesh (Asp⁸⁷Asn) and China (Asp⁸⁷Gly) except for one. A novel mutation at position 211 (His→Tyr) in gyrA gene was detected only in the Bangladeshi strains. Susceptibility to ciprofloxacin was increased by the presence of CCCP indicating the involvement of energy dependent active efflux pumps. A single PFGE type was found in isolates from Bangladesh and China suggesting their genetic relatedness.

Conclusions

Emergence of fluoroquinolone resistance in Shigella undermines a major challenge in current treatment strategies which needs to be followed up by using empirical therapeutic strategies.     

Multilocus microsatellite typing (MLMT) of strains from Turkey and Cyprus reveals a novel monophyletic L. donovani sensu lato group

Background

New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic.

Methodology/Principal Findings

The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey.

Conclusion

The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring and controlling the spread of leishmaniasis in this region.     

Enzymatic polymorphism and phylogenetic relationships in Leishmania Ross, 1903 (Sarcomastigophora: Kinetoplastida): a case study in Colombia

Leishmaniasis is widespread in Colombia and is found in 30 of 32 Departments. More than 200 infection zones have been reported from different regions, which vary from sea-level to an altitude of 2,300 m along the Atlantic Coast, Pacific coast, Amazon basin, Cauca and Magdalena valleys. We report 76 Leishmania stocks isolated from humans, dogs and phlebotomine hosts. Isoenzyme electrophoresis revealed 16 zymodemes, which could be divided into four phylogenetic complexes, i.e., L. braziliensis, L. amazonensis, L. guyanensis/panamensis and L. infantum. Three zymodemes became integrated into the subgenus Leishmania and the other zymodemes into the subgenus Viannia. Cutaneous infections were due to the L. braziliensis (9.2%) and L. guyanensis/panamensis (85.54%) complexes. Mucous secondary involvement was due to the L. braziliensis and L. guyanensis/panamensis complexes. In this work the specific status of L. (V.) guyanensis and L. (V.) panamensis is discussed.     

Detection of Antibiotic Resistance in Leprosy Using GenoType LepraeDR, a Novel Ready-To-Use Molecular Test

Background

Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and to second-line drugs (fluoroquinolones) was described worldwide. Since Mycobacterium leprae is not growing in vitro, phenotypic susceptibility testing requires a one year experiment in the mouse model and this is rarely performed. Genetics on antibiotic resistance provide the basis for molecular tests able to detect for antibiotic resistance in leprosy.

Methodology/Principal Findings

A reverse hybridization DNA strip test was developed as the GenoType LepraeDR test. It includes DNA probes for the wild-type sequence of regions of rpoB, gyrA and folP genes and probes for the prevalent mutations involved in acquired resistance to rifampin, fluoroquinolones and dapsone, respectively. The performances of the GenoType LepraeDR test were evaluated by comparing its results on 120 M. leprae strains, previously studied for resistance by the reference drug in vivo susceptibility method in the mouse footpad and for mutations in the gene regions described above by PCR-sequencing. The results of the test were 100% concordant with those of PCR sequencing and the mouse footpad test for the resistant strains: 16 strains resistant to rifampin, 22 to dapsone and 4 to ofloxacin with mutations (numbering system of the M. leprae genome) in rpoB (10 S456L, 1 S456F, 1 S456M + L458V, 1 H451Y, 1 G432S + H451D, 1 T433I + D441Y and 1 Q438V), in folP1 (8 P55L, 3 P55R, 7 T53I, 3 T53A, 1 T53V) and gyrA (4 A91V), respectively. Concordance was 98.3% for the susceptible strains, two strains showing a mutation at the codon 447 that in fact was not conferring resistance as shown by the in vivo method.

Conclusions/Significance

The GenoType LepraeDR test is a commercially available test that accurately detects for antibiotic resistance in leprosy cases. The test is easy to perform and could be implemented in endemic countries.     

Antimony uptake and toxicity in sunflower and maize growing in SbIII and SbV contaminated soil

Using pot experiments, we investigated the uptake of antimony (Sb) by sunflower (Helianthus annuus L. cv. Iregi), and maize (Zea mays L. cv. Magister) in two different soils, a potting mix and an agricultural soil. In one treatment Sb was added to the experimental soils as KSb(OH)₆ (“Sb^V-treatment”) and in the other as Sb₂O₃ (“Sb^III-treatment”). Soluble soil Sb concentrations were linearly related to the applied Sb rates, ranging from 0.02 (controls) to 175 mg L^?1 soil solution. Accumulation of Sb tended to be slightly higher in the Sb^V treatment in sunflower, while no difference in Sb uptake between the two Sb treatments was found in maize. The half maximal effective concentration (EC₅₀) values derived from the dose-response curves were higher for the Sb^V than for the Sb^III treatment when they were related to soluble soil Sb concentrations, but differences became insignificant when they were related to shoot Sb concentrations. Maize was substantially more sensitive to Sb toxicity than sunflower, indicating physiological differences in Sb tolerance between the two plant species. Our results show that on soils with high Sb contamination, as often found in shooting ranges, plants may suffer from Sb toxicity.     

Population Structure and Evidence for Both Clonality and Recombination among Brazilian Strains of the Subgenus Leishmania (Viannia)

Background/Objectives: Parasites of the subgenus Leishmania (Viannia) cause varying clinical symptoms ranging from cutaneous leishmaniases (CL) with single or few lesions, disseminated CL (DL) with multiple lesions to disfiguring forms of mucocutaneous leishmaniasis (MCL). In this population genetics study, 37 strains of L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. naiffi hybrid from different endemic foci in Brazil were examined for variation at 15 hyper-variable microsatellite markers. Methodology/Principal findings: The multilocus microsatellite profiles obtained for the 120 strains were analysed using both model- and distance-based methods. Significant genetic diversity was observed for all L. (Viannia) strains studied. The two cluster analysis approaches identified two principal genetic groups or populations, one consisting of strains of L. (V.) guyanensis from the Amazon region and the other of strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil. A third group comprised a heterogeneous assembly of species, including other strains of L. braziliensis isolated from the north of Brazil, which were extremely polymorphic. The latter strains seemed to be more closely related to those of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni, also isolated in northern Brazilian foci. The MLMT approach identified an epidemic clone consisting of 13 strains of L. braziliensis from Minas Gerais, but evidence for recombination was obtained for the populations of L. (V.) braziliensis from the Atlantic coast and for L. (V.) guyanensis. Conclusions/Significance: Different levels of recombination versus clonality seem to occur within the subgenus L. (Viannia). Though clearly departing from panmixia, sporadic, but long-term sustained recombination might explain the tremendous genetic diversity and limited population structure found for such L. (Viannia) strains.     

Identification of a Novel G2073A Mutation in 23S rRNA in Amphenicol-Selected Mutants of Campylobacter jejuni

Objectives

This study was conducted to examine the development and molecular mechanisms of amphenicol resistance in Campylobacter jejuni by using in vitro selection with chloramphenicol and florfenicol. The impact of the resistance development on growth rates was also determined using in vitro culture.

Methods

Chloramphenicol and florfenicol were used as selection agents to perform in vitro stepwise selection. Mutants resistant to the selective agents were obtained from the selection process. The mutant strains were compared with the parent strain for changes in MICs and growth rates. The 23S rRNA gene and the L4 and L22 ribosomal protein genes in the mutant strains and the parent strain were amplified and sequenced to identify potential resistance-associated mutations.

Results

C. jejuni strains that were highly resistant to chloramphenicol and florfenicol were obtained from in vitro selection. A novel G2073A mutation in all three copies of the 23S rRNA gene was identified in all the resistant mutants examined, which showed resistance to both chloramphenicol and florfenicol. In addition, all the mutants selected by chloramphenicol also exhibited the G74D modification in ribosomal protein L4, which was previously shown to confer a low-level erythromycin resistance in Campylobacter species. The mutants selected by florfenicol did not have the G74D mutation in L4. Notably, the amphenicol-resistant mutants also exhibited reduced susceptibility to erythromycin, suggesting that the selection resulted in cross resistance to macrolides.

Conclusions

This study identifies a novel point mutation (G2073A) in 23S rRNA in amphenicol-selected mutants of C. jejuni. Development of amphenicol resistance in Campylobacter likely incurs a fitness cost as the mutant strains showed slower growth rates in antibiotic-free media.     

Mucosal Leishmaniasis caused by Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in the Brazilian Amazon

Background

Leishmania (Viannia) braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML) in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V.) panamensis, L. (V.) guyanensis and L. (L.) amazonensis.

Methodology

Leishmania species were characterized by polymerase chain reaction (PCR) of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM) in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit.

Results

This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V.) braziliensis and 16 cases by L. (V.) guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V.) guyanensis in the states of Pará, Acre, and Rondônia and cases of ML caused by L. (V.) braziliensis in the state of Rondônia.

Conclusions/Significance

L. (V.) braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V.) guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River.

Author Summary

Leishmaniasis is considered a neglected disease with 1.5 million new cases of cutaneous leishmaniasis (CL) occurring each year. In the Amazon region and in the Americas in general, ML is caused by Leishmania (Viannia) braziliensis, though in rare cases it has been related to other species. ML, which is associated with inadequate treatment of CL, normally manifests itself years after the occurrence of CL. Clinical features evolve slowly and most often affect the nasal cavity, in some cases causing perforation, or even destruction, of the septum. Diagnosis is made using the Montenegro skin test, serology and histopathology of the patients'' mucosal tissues, or by isolation of the parasites. PCR is the best way to identify the species of leishmaniasis and is therefore the diagnostic method of choice. This paper describes 46 cases of ML and their geographical origin, 30 cases associated with L. (V.) braziliensis and 16 with L. (V.) guyanensis. The species of leishmaniasis was identified using mucosal biopsies taken from Amazonian patients who were diagnosed and treated for ML in the tertiary care unit, in Manaus, Amazonas state, Brazil. This is the highest number of ML cases caused by L. (V.) guyanensis that has ever been reported.     

Variation in Leishmania chemokine suppression driven by diversification of the GP63 virulence factor

Leishmaniasis is a neglected tropical disease with diverse outcomes ranging from self-healing lesions, to progressive non-healing lesions, to metastatic spread and destruction of mucous membranes. Although resolution of cutaneous leishmaniasis is a classic example of type-1 immunity leading to self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread. Leishmania genetic diversity can contribute to variation in polarization and robustness of the immune response through differences in both pathogen sensing by the host and immune evasion by the parasite. In this study, we observed a difference in parasite chemokine suppression between the Leishmania (L.) subgenus and the Viannia (V.) subgenus, which is associated with severe immune-mediated pathology such as mucocutaneous leishmaniasis. While Leishmania (L.) subgenus parasites utilize the virulence factor and metalloprotease glycoprotein-63 (gp63) to suppress the type-1 associated host chemokine CXCL10, L. (V.) panamensis did not suppress CXCL10. To understand the molecular basis for the inter-species variation in chemokine suppression, we used in silico modeling to identify a putative CXCL10-binding site on GP63. The putative CXCL10 binding site is in a region of gp63 under significant positive selection, and it varies from the L. major wild-type sequence in all gp63 alleles identified in the L. (V.) panamensis reference genome. Mutating wild-type L. (L.) major gp63 to the L. (V.) panamensis sequence at the putative binding site impaired cleavage of CXCL10 but not a non-specific protease substrate. Notably, Viannia clinical isolates confirmed that L. (V.) panamensis primarily encodes non-CXCL10-cleaving gp63 alleles. In contrast, L. (V.) braziliensis has an intermediate level of activity, consistent with this species having more equal proportions of both alleles. Our results demonstrate how parasite genetic diversity can contribute to variation in immune responses to Leishmania spp. infection that may play critical roles in the outcome of infection.     

Differentiation and gene flow among European populations of Leishmania infantum MON-1

Background

Leishmania infantum is the causative agent of visceral and cutaneous leishmaniasis in the Mediterranean region, South America, and China. MON-1 L. infantum is the predominating zymodeme in all endemic regions, both in humans and dogs, the reservoir host. In order to answer important epidemiological questions it is essential to discriminate strains of MON-1.

Methodology/Principal Findings

We have used a set of 14 microsatellite markers to analyse 141 strains of L. infantum mainly from Spain, Portugal, and Greece of which 107 strains were typed by MLEE as MON-1. The highly variable microsatellites have the potential to discriminate MON-1 strains from other L. infantum zymodemes and even within MON-1 strains. Model- and distance-based analysis detected a considerable amount of structure within European L. infantum. Two major monophyletic groups—MON-1 and non-MON-1—could be distinguished, with non-MON-1 being more polymorphic. Strains of MON-98, 77, and 108 were always part of the MON-1 group. Among MON-1, three geographically determined and genetically differentiated populations could be identified: (1) Greece; (2) Spain islands–Majorca/Ibiza; (3) mainland Portugal/Spain. All four populations showed a predominantly clonal structure; however, there are indications of occasional recombination events and gene flow even between MON-1 and non-MON-1. Sand fly vectors seem to play an important role in sustaining genetic diversity. No correlation was observed between Leishmania genotypes, host specificity, and clinical manifestation. In the case of relapse/re-infection, only re-infections by a strain with a different MLMT profile can be unequivocally identified, since not all strains have individual MLMT profiles.

Conclusion

In the present study for the first time several key epidemiological questions could be addressed for the MON-1 zymodeme, because of the high discriminatory power of microsatellite markers, thus creating a basis for further epidemiological investigations.     

Species distribution and antifungal susceptibility among clinical isolates of Candida parapsilosis complex from India

Background

Candida parapsilosis is recognized as a species complex: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis are three distinct but closely related species.

Over-Expression of 60s Ribosomal L23a Is Associated with Cellular Proliferation in SAG Resistant Clinical Isolates of Leishmania donovani

Background

Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism.

Methodology and principal findings

The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode.

Conclusion/Significance

This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism.     

Genetics of host response to Leishmania tropica in mice - different control of skin pathology, chemokine reaction, and invasion into spleen and liver

Background

Leishmaniasis is a disease caused by protozoan parasites of genus Leishmania. The frequent involvement of Leishmania tropica in human leishmaniasis has been recognized only recently. Similarly as L. major, L. tropica causes cutaneous leishmaniasis in humans, but can also visceralize and cause systemic illness. The relationship between the host genotype and disease manifestations is poorly understood because there were no suitable animal models.

Methods

We studied susceptibility to L. tropica, using BALB/c-c-STS/A (CcS/Dem) recombinant congenic (RC) strains, which differ greatly in susceptibility to L. major. Mice were infected with L. tropica and skin lesions, cytokine and chemokine levels in serum, and parasite numbers in organs were measured.

Principal Findings

Females of BALB/c and several RC strains developed skin lesions. In some strains parasites visceralized and were detected in spleen and liver. Importantly, the strain distribution pattern of symptoms caused by L. tropica was different from that observed after L. major infection. Moreover, sex differently influenced infection with L. tropica and L. major. L. major-infected males exhibited either higher or similar skin pathology as females, whereas L. tropica-infected females were more susceptible than males. The majority of L. tropica-infected strains exhibited increased levels of chemokines CCL2, CCL3 and CCL5. CcS-16 females, which developed the largest lesions, exhibited a unique systemic chemokine reaction, characterized by additional transient early peaks of CCL3 and CCL5, which were not present in CcS-16 males nor in any other strain.

Conclusion

Comparison of L. tropica and L. major infections indicates that the strain patterns of response are species-specific, with different sex effects and largely different host susceptibility genes.     

Molecular Characterization and Antimicrobial Susceptibility of Streptococcus pneumoniae Isolated from Children Hospitalized with Respiratory Infections in Suzhou,China

Background

Dissemination of antibiotic resistant clones is recognized as an important factor in the emergence and prevalence of resistance in pneumococcus. This study was undertaken to survey the antimicrobial susceptibility and serotypes distribution of pneumococci and to explore the circulating clones in hospitalized children in Suzhou, China.

Methods

The pneumococci were isolated from the nasopharyngeal aspirates of children less than 5 years of age admitted to Soochow-University-Affiliated-Children''s-Hospital with respiratory infections. The capsular serotypes were identified by multiplex polymerase chain reaction (PCR). Antimicrobial susceptibility was tested by E-test. The presence of ermB, mefA/E genes were detected by PCR and the genotypes were explored by Multilocus sequence typing (MLST).

Results

From July 2012 to July 2013, a total of 175 pneumococcal isolates were collected and all strains were resistant to erythromycin and clindamycin, about 39.4% strains were non-susceptible to penicillin G. Overall, 174 (99.4%) isolates were resistant to ≥3 types of antibiotics. Serotypes 19F (28.1%), 6B (19.7%), 19A (18.0%), and 23F (17.4%) were the most common serotypes in all identified strains. The serotypes coverage of PCV7 and PCV13 were 71.9% and 89.9%, respectively. Four international antibiotic-resistant clones, including Taiwan^19F-14 (n = 79), Spain^23F-1(n = 25), Taiwan^23F-15(n = 7) and Spain^6B-2(n = 7), were identified. The Taiwan^19F-14 clones have a higher non-susceptibility rate in β-lactams than other clones and non-clone isolates (p<0.001). In addition, 98.7% Taiwan^19F-14 clones were positive of both ermB and mefA/E genes, compare to 33.3% in other clones and non-clone strains.

Conclusions

The spread of international antibiotic-resistant clones, especially Taiwan^19F-14 clones, played a predominant role in the dissemination of antimicrobial resistant isolates in Suzhou, China. Considering the high prevalence of PCV7 serotypes and serotype 19A, the introduction of PCV13 may be a promising preventive strategy to control the increasing trend of clonal spread in China.     

High-resolution genotyping of Pseudomonas aeruginosa strains linked to acute post cataract surgery endophthalmitis outbreaks in India

Background

The number of Salmonella strains with reduced susceptibility to fluoroquinolones has increased during recent years in many countries, threatening the value of this antimicrobial group in the treatment of severe salmonella infections.

Methods

We analyzed the in vitro activities of ciprofloxacin and 10 additional fluoroquinolones against 816 Salmonella strains collected from Finnish patients between 1995 and 2003. Special attention was focused on the efficacy of newer fluoroquinolones against the Salmonella strains with reduced ciprofloxacin susceptibility.

Results

The isolates represented 119 different serotypes. Of all 816 Salmonella strains, 3 (0.4%) were resistant to ciprofloxacin (MIC ≥ 4 μg/ml), 232 (28.4%) showed reduced susceptibility to ciprofloxacin (MIC ≥ 0.125 – 2 μg/ml), and 581 (71.2%) were ciprofloxacin-susceptible. The MIC₅₀ and MIC₉₀ values of ciprofloxacin for these strains were 0.032 and 0.25 μg/ml, respectively, being lower than those of the other fluoroquinolone compounds presently on market in Finland (ofloxacin, norfloxacin, levofloxacin, and moxifloxacin). For two newer quinolones, clinafloxacin and sitafloxacin, the MIC₅₀ and MIC₉₀ values were lowest, both 0.016 and 0.064 μg/ml, respectively. Moreover, clinafloxacin and sitafloxacin exhibited the lowest MIC₅₀ and MIC₉₀ values, 0.064 and 0.125 μg/ml, against the 235 Salmonella strains with reduced susceptibility and strains fully resistant to ciprofloxacin.

Conclusion

Among the registered fluoroquinolones in Finland, ciprofloxacin still appears to be the most effective drug for the treatment salmonella infections. Among the newer preparations, both clinafloxacin and sitafloxacin are promising based on in vitro studies, especially for strains showing reduced ciprofloxacin susceptibility. Their efficacy, however, has not been demonstrated in clinical investigations.