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Background Cutaneous leishmaniasis (CL) is frequent in travellers and can involve oro-nasal mucosae. Clinical presentation impacts therapeutic management. Methodology Demographic and clinical data from 459 travellers infected in 47 different countries were collected by members of the European LeishMan consortium. The infecting Leishmania species was identified in 198 patients. Principal findings Compared to Old World CL, New World CL was more frequently ulcerative (75% vs 47%), larger (3 vs 2cm), less frequently facial (17% vs 38%) and less frequently associated with mucosal involvement (2.7% vs 5.3%). Patients with mucosal lesions were older (58 vs 30 years) and more frequently immunocompromised (37% vs 3.5%) compared to patients with only skin lesions. Young adults infected in Latin America with L. braziliensis or L. guyanensis complex typically had an ulcer of the lower limbs with mucosal involvement in 5.8% of cases. Typically, infections with L. major and L. tropica acquired in Africa or the Middle East were not associated with mucosal lesions, while infections with L. infantum, acquired in Southern Europe resulted in slowly evolving facial lesions with mucosal involvement in 22% of cases. Local or systemic treatments were used in patients with different clinical presentations but resulted in similarly high cure rates (89% vs 86%). Conclusion/Significance CL acquired in L. infantum-endemic European and Mediterranean areas displays unexpected high rates of mucosal involvement comparable to those of CL acquired in Latin America, especially in immunocompromised patients. When used as per recommendations, local therapy is associated with high cure rates., Author summary Cutaneous and muco-cutaneous leishmaniasis (CL and MCL) are disfiguring diseases caused by a worldwide distributed parasite called Leishmania and its 20 species. Clinical manifestations span a wide continuum from single nodular lesion to disseminated form with mucosal involvement. No randomized clinical trial has ever been done exclusively in travellers and medical management is poorly evidence-based or based very predominantly on data obtained in endemic countries. Articles and reviews almost invariably propose a dichotomic view, with Old World CL described as a benign disease in contrast to New World CL strongly associated with destructive mucosal lesions. Our study is the first prospective clinical study providing a detailed description of the clinical presentation and risk of mucosal involvement in CL in several hundreds of patients, with frequent formal identification of the infecting Leishmania species. The harmonized data collection in patients infected in many transmission foci worldwide enabled direct comparisons of clinical patterns induced by different Leishmania species, and on the outcome following treatment with either local or systemic regimens. The study is based on an international harmonized data collection that allowed a wide capture of parasitologically confirmed cases. In striking contrast with previous assumptions, the study shows that CL acquired in Europe displays unexpected high rates of mucosal involvement comparable to those of CL acquired in Latin America, especially in immunocompromised travellers. It also shows that when used as per recommendations, local therapy is associated with high cure rates.

Pitting, the removal of dead parasites from their host erythrocyte, has been studied in patients with severe malaria treated parenterally with quinine or artesunate, and was recently shown to contribute to delayed hemolysis, a frequent adverse event of artesunate. We quantified pitting in 81 travelers treated with oral antimalarial therapy. Pitting rate was high (55.8%) with artemisinin-based combinations, but

Malaria and the Spleen In this report, patients living in a malaria-endemic area underwent trauma-related splenectomy. In these asymptomatic patients who were naturally infected with Plasmodium fal...

In utero transmission of Leishmania infantum is the putative mechanism of congenital leishmaniasis. However, this hypothesis is based on limited research. In addition, the consequences for infant newborn development remain to be clarified by additional data. We report here the occurrence, specific management, and monitoring of congenital leishmaniasis in a newborn infant whose mother was coinfected with leishmaniasis and human immunodeficiency virus; transplacental transmission, confirmed by overt clinical disease at birth, was documented, which provides, to our knowledge, the first evidence of hepatic and neurologic impairment in an infant with congenital visceral leishmaniasis.

Background Although malaria remains one of the major public health threats in inter-tropical areas, there is limited understanding of imported malaria in children by paediatricians and emergency practitioners in non-endemic countries, often resulting in misdiagnosis and inadequate treatment. Moreover, classical treatments (atovaquone-proguanil, quinine, mefloquine) are limited either by lengthy treatment courses or by side effects. Since 2010, the World Health Organization (WHO) has recommended the use of oral artemisinin-based combination therapy for the treatment of uncomplicated Plasmodium falciparum malaria worldwide. The benefits of artenimol–piperaquine in children have been validated in endemic countries but experience remains limited in cases of imported malaria. Methods This prospective observational study in routine paediatric care took place at the Emergency Department, Robert-Debré Hospital (Paris, France) from September 2012 to December 2014. Tolerance and efficacy of artenimol–piperaquine in children presenting with the following inclusion criteria were assessed: P. falciparum positive on thin or thick blood smear; and the absence of WHO-defined features of severity. Results Among 83 children included in this study, treatment with artenimol–piperaquine was successful in 82 children (98.8%). None of the adverse events were severe and all were considered mild with no significant clinical impact. This also applied to cardiological adverse events despite a significant increase of the mean post-treatment QTc interval. Conclusion Artenimol–piperaquine displays a satisfying efficacy and tolerance profile as a first-line treatment for children with imported uncomplicated falciparum malaria and only necessitates three once-daily oral intakes of the medication. Comparative studies versus artemether-lumefantrine or atovaquone-proguanil would be useful to confirm the results of this study.

Although there have been significant advances in the treatment of visceral leishmaniasis (VL) and several novel compounds are currently in pre-clinical and clinical development for this manifestation of leishmaniasis, there have been limited advances in drug research and development (R & D) for cutaneous leishmaniasis (CL). Here we review the need for new treatments for CL, describe in vitro and in vivo assays, models and approaches taken over the past decade to establish a pathway for the discovery, and pre-clinical development of new drugs for CL. These recent advances include novel mouse models of infection using bioluminescent Leishmania, the introduction of PK/PD approaches to skin infection, and defined pre-clinical candidate profiles., Graphical abstract Image 1

Background A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival. Methods and findings We examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach. In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted. Conclusions Immature CD71+ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen., Dr. Anstey and co-authors found that P. vivax-infected immature reticulocytes and erythrocytes accumulate in the same splenic compartments, suggesting existence of a cryptic endosplenic lifecycle in chronic P. vivax infection that maximizes survival and replication in the spleen., Author summary Why was this study done? There is a surprisingly large biomass of intact Plasmodium vivax parasites in the spleen of people living in malaria endemic areas, greater than with Plasmodium falciparum. Why P. vivax accumulates so intensely in the spleen is not known. P. vivax requires very young red cells (immature reticulocytes) for invasion and replication. What did the researchers do and find? The accumulation of P. vivax and immature reticulocytes were evaluated in spleens from people undergoing splenectomy in malaria-endemic Papua. P. vivax parasites and immature reticulocytes accumulated in the same splenic compartments. All stages of P. vivax accumulated in the spleen at magnitudes unexplainable by replication occurring in the circulation alone, with the proportion of each stage in the spleen consistent with the duration in their lifecycle. What do these findings mean? Taking advantage of the large physiological splenic reservoir of immature reticulocytes, the majority of the P. vivax lifecycle can take place in the spleen in chronic malaria. Chronic vivax malaria should be considered predominantly an infection of the reticulocyte-rich spleen, with secondary involvement of the intravascular compartment. The spleen is not solely a compartment for parasite destruction and clearance. Specific adaptations have likely evolved to maximise P. vivax survival in the spleen.

Red blood cells (RBCs) can be cleared from circulation when alterations in their size, shape, and deformability are detected. This function is modulated by the spleen-specific structure of the interendothelial slit (IES). Here, we present a unique physiological framework for development of prognostic markers in RBC diseases by quantifying biophysical limits for RBCs to pass through the IES, using computational simulations based on dissipative particle dynamics. The results show that the spleen selects RBCs for continued circulation based on their geometry, consistent with prior in vivo observations. A companion analysis provides critical bounds relating surface area and volume for healthy RBCs beyond which the RBCs fail the “physical fitness test” to pass through the IES, supporting independent experiments. Our results suggest that the spleen plays an important role in determining distributions of size and shape of healthy RBCs. Because mechanical retention of infected RBC impacts malaria pathogenesis, we studied key biophysical parameters for RBCs infected with Plasmodium falciparum as they cross the IES. In agreement with experimental results, surface area loss of an infected RBC is found to be a more important determinant of splenic retention than its membrane stiffness. The simulations provide insights into the effects of pressure gradient across the IES on RBC retention. By providing quantitative biophysical limits for RBCs to pass through the IES, the narrowest circulatory bottleneck in the spleen, our results offer a broad approach for developing quantitative markers for diseases such as hereditary spherocytosis, thalassemia, and malaria.

Deformability of Plasmodium falciparum gametocyte-infected erythrocytes (GIEs) allows them to persist for several days in blood circulation and to ensure transmission to mosquitoes. Here, we investigate the mechanism by which the parasite proteins STEVOR (SubTElomeric Variable Open Reading frame) exert changes on GIE deformability. Using the microsphiltration method, immunoprecipitation, and mass spectrometry, we produce evidence that GIE stiffness is dependent on the cytoplasmic domain of STEVOR that interacts with ankyrin complex at the erythrocyte skeleton. Moreover, we show that GIE deformability is regulated by protein kinase A (PKA)–mediated phosphorylation of the STEVOR C-terminal domain at a specific serine residue (S324). Finally, we show that the increase of GIE stiffness induced by sildenafil (Viagra) is dependent on STEVOR phosphorylation status and on another independent mechanism. These data provide new insights into mechanisms by which phosphodiesterase inhibitors may block malaria parasite transmission.

Background Complex cutaneous and muco-cutaneous leishmaniasis (CL and MCL) often requires systemic therapy. Liposomal amphotericin B (L-AmB) has a strong potential for a solid clinical benefit in this indication. Methods We conducted a retrospective analysis of data from a French centralized referral treatment program and from the “LeishMan” European consortium database. All patients with parasitologically proven CL or MCL who received at least one dose of L-AmB were included. Positive outcome was based on ulcer closure as per recent WHO workshop guidelines. Results From 2008 through 2016, 43 travelers returning from 18 countries (Old World n = 28; New World n = 15) were analyzed with a median follow-up duration of 79 days [range 28–803]. Main clinical forms were: localized CL with one or multiple lesions (n = 32; 74%) and MCL (n = 8; 19%). As per published criteria 19 of 41 patients (46%) were cured 90 days after one course of L-AmB. When the following items -improvement before day 90 but no subsequent follow-up, delayed healing (>3 months) and healing after a second course of L-AmB- were included in the definition of cure, 27 of 43 patients (63%) had a positive outcome. Five patients (MCL = 1; CL = 4) experienced a relapse after a median duration of 6 months [range 3–27] post treatment and 53% of patients (23/43) experienced at least one adverse event including severe hypokalaemia and acute cardiac failure (one patient each). In multivariate analysis, tegumentary infection with L. infantum was associated with complete healing after L-AmB therapy (OR 5.8 IC 95% [1.03–32]) while infection with other species had no impact on outcome. Conclusion In conditions close to current medical practice, the therapeutic window of L-AmB was narrow in travellers with CL or MCL, with the possible exception of those infected with L. infantum. Strict follow-up is warranted when using L-AmB in patients with mild disease., Author summary Cutaneous and muco-cutaneous leishmaniasis (CL/MCL) are disfiguring diseases caused by a worldwide distributed parasite called Leishmania and its 20 species. Clinical manifestations span a wide continuum from single nodular lesion to disseminated form with mucosal involvement. Though local treatment with cryotherapy and intralesionnal antimony or topical formulations of paromomycin is generally adequate in most of situations, some patients with complex CL/MCL require systemic therapy. No convenient regimen has been proved to be safe and effective for all infecting species, all clinical forms and all patients (e.g. children, pregnant women, adults with comorbidities or immunosuppression). In this study, the authors examined in returning travelers with CL/MCL the effectiveness of an antifungal agent “liposomal amphotericin B” (L-AmB), which is highly effective in visceral leishmaniasis. Surprisingly, rates of healing were lower than in previous reports in this unselected population that reflects clinical practice in non-endemic countries. The observations also suggest that some Leishmania species (namely, L. infantum) may be more susceptible to L-AmB than others. Occurrence of adverse events should raises the question of the benefit-risk balance of L-AmB in CL/MCL. Careful attention to comorbidities and adoption of strict protocols for administration are pre-requisites for the use of L-AmB in patients with CL/MCL.

Artesunate, the recommended drug for severe malaria, rapidly clears the malaria parasite from infected patients but frequently induces anemia-called post-artesunate delayed hemolysis (PADH)-for which a simple predictive test is urgently needed. The underlying event in PADH is the expulsion of artesunate-exposed parasites from their host erythrocytes by pitting. We show that the histidine-rich protein 2 (HRP2) of the malaria parasite Plasmodium falciparum persists in the circulation of artesunate-treated malaria patients in Bangladesh and in French travelers who became infected with malaria in Africa. HRP2 persisted in whole blood (not plasma) of artesunate-treated patients with malaria at higher levels compared to quinine-treated patients. Using an optimized membrane permeabilization method, HRP2 was observed by immunofluorescence, Western blotting, and electron microscopy to persist in once-infected red blood cells from artesunate-treated malaria patients. HRP2 was deposited at the membrane of once-infected red blood cells in a pattern similar to that for ring erythrocyte surface antigen (RESA), a parasite invasion marker. On the basis of these observations, we developed a semiquantitative titration method using a widely available HRP2-based rapid diagnostic dipstick test. Positivity on this test using a 1:500 dilution of whole blood from artesunate-treated patients with malaria collected shortly after parasite clearance predicted subsequent PADH with 89% sensitivity and 73% specificity. These results suggest that adapting an existing HRP2-based rapid diagnostic dipstick test may enable prediction of PADH several days before it occurs in artesunate-treated patients with malaria.

International audience; Background. In Plasmodium falciparum-infected patients treated with artemisinins, parasitemia declines through so-called pitting, an innate splenic process that transforms infected red blood cells (iRBCs) into once-infected RBCs (O-iRBCs).Methods. We measured pitting in 83 French travelers and 42 Malian children treated for malaria with artesunate.Results. In travelers, O-iRBCs peaked at 107.7% initial parasitemia. In Malian children aged 1.5-4 years, O-iRBCs peaked at higher concentrations than in children aged 9-13 years (91.60% vs 31.95%; P = .0097). The parasite clearance time in older children was shorter than in younger children (P = .0001), and the decline in parasitemia in children aged 1.5-4 years often started 6 hours after treatment initiation, a lag phase generally absent in infants and older children. A 6-hour lag phase in artificial pitting of artesunate-exposed iRBCs was also observed in vitro. The proportion of iRBCs recognized by autologous immunoglobulin G (IgG) correlated with the parasite clearance time (r = −0.501; P = .0006) and peak O-iRBC concentration (r = −0.420; P = .0033).Conclusions. Antimalarial immunity correlates with fast artemisinin-induced parasite clearance and low pitting rates. In nonimmune populations, artemisinin-induced P. falciparum clearance is related to pitting and starts after a 6-hour lag phase. In immune populations, passively and naturally acquired immune mechanisms operating faster than pitting may exist. This mechanism may mitigate the emergence of artemisinin-resistant P. falciparum in Africa.

International audience; Patients under immunosuppressive therapy with tumor necrosis factor alpha (TNF-α) antagonists are vulnerable to various opportunistic infections including leishmaniasis. We present a case series of 8 travellers developing cutaneous leishmaniasis whilst on TNF-α antagonist treatment and review the literature on aspects of cutaneous leishmaniasis developing in patients treated with TNF-α antagonists. We make interim recommendations regarding the drug therapy used to maintain remission in travellers with rheumatoid disease travelling to leishmania prone areas. Despite having a medical condition requiring continued rheumatological review the interval to diagnosis appears not to be reduced compared to that described in non-rheumatoid patients. Rheumatologists and family doctors should be aware of the need for post-travel surveillance for leishmaniasis in rheumatoid patients on TNF-alpha antagonist treatment.

Plasmodium species, the parasitic agents of malaria, invade erythrocytes to reproduce, resulting in erythrocyte loss. However, a greater loss is caused by the elimination of uninfected erythrocytes, sometimes long after infection has been cleared. Using a mouse model, we found that Plasmodium infection induces the generation of anti-self antibodies that bind to the surface of uninfected erythrocytes from infected, but not uninfected, mice. These antibodies recognize phosphatidylserine, which is exposed on the surface of a fraction of uninfected erythrocytes during malaria. We find that phosphatidylserine-exposing erythrocytes are reticulocytes expressing high levels of CD47, a "do-not-eat-me" signal, but the binding of anti-phosphatidylserine antibodies mediates their phagocytosis, contributing to anemia. In human patients with late postmalarial anemia, we found a strong inverse correlation between the levels of anti-phosphatidylserine antibodies and plasma hemoglobin, suggesting a similar role in humans. Inhibition of this pathway may be exploited for treating malarial anemia.

We report on 4 patients (1 immunocompetent, 3 immunosuppressed) in whom visceral leishmaniasis had become unresponsive to (or had relapsed after) treatment with appropriate doses of liposomal amphotericin B. Under close follow-up, full courses of pentavalent antimony were administered without life-threatening adverse events and resulted in rapid and sustained clinical and parasitological cure., Author Summary Visceral leishmaniasis causes fever, enlargement of internal organs like the liver and the spleen, and leads to death if no treatment is given. It is caused by a microbe called Leishmania and affects children and adults in warm and temperate regions of the world. Antimony in different forms has been used to treat Visceral Leishmaniasis for almost one century and is still in use in several countries despite the fact that it sometimes displays toxic effects, especially in patients with underlying health problems. Because it is better tolerated and at least as effective as antimony, liposomal amphotericin B is now used as the first treatment for Visceral Leishmaniasis in Southern Europe. We observed that a small proportion of patients—especially those with an impaired immune system—do not cure even after several courses of liposomal amphotericin B. In 4 such patients with “unresponsiveness” to liposomal amphotericin B, antimony provided a sustained cure without major side effects. We conclude that when multiple failures or relapses occur after treatment with liposomal amphotericin B, antimony is a reasonable, potentially life-saving treatment option. These observations also suggest that “unresponsiveness” to amphotericin B results from unusual resistance mechanisms.

Topical therapy is an attractive approach for the treatment ofLeishmaniamajorcutaneous leishmaniasis (CL). WR279396, an expanded-spectrum aminoglycoside ointment, is now in phase 3 trials. Because the application of a cream is easier than the injection of pentavalent antimony, many patients with CL will likely be treated with WR279396 soon after the onset of a lesion. However, this new therapeutic approach may impair the acquisition of immunity. We evaluated the impact of early topical therapy on acquired immunity in an optimized mouse model ofL. major-induced CL. The efficacy of the WR279396 ointment in this model has been established previously. Acquired immunity was defined as the absence of lesions upon reinoculation of the same parasite isolate at a different skin site. Bioluminescence-based follow-up of luciferase-expressingL. majorloads was also performed. In this model, the control ofL. majorloads at the initial inoculation site and the acquisition of immunity are simultaneous (day 22 postinoculation). The clinical and parasitological efficacies of WR279396 applied as early as day 11 postinoculation, i.e., during theL. majormultiplication phase, did not impair the acquisition of immunity to a secondL. majorchallenge. This is reassuring from the perspective of the wide deployment of WR279396-based therapy in foci whereL. majoris endemic.

AA-amyloidosis in the setting of chronic visceral leishmaniasis (VL) has been reported in animal models but documentation in humans is unavailable. Here, we report on a Portuguese man who in 1996 was diagnosed with both human immunodeficiency virus (HIV)-infection and VL. Antiretroviral treatment led to sustained suppression of HIV viremia but CD4+ lymphocytes rose from 8 to only 160 cells/mL. Several courses of antimony treatment did not prevent VL relapses. Renal failure developed in 2006 and renal biopsy revealed AA-amyloidosis. The patient had cryoglobulinemia and serum immune complexes containing antibodies directed against seven leishmanial antigens. Antimony plus amphotericin B, followed by oral miltefosine resulted in a sustained VL treatment response with elimination of circulating Leishmania infantum DNA and CD4+ recovery. The concomitant reduction of serum AA levels and disappearance of circulating leishmanial immune complexes suggests that prolonged VL may lead to AA-amyloidosis in immunocompromised humans.

Summary Background Splenic rupture during acute malaria is rare but underreported. Because splenic rupture occurs mostly in non-immune adults, ongoing malaria elimination efforts may paradoxically increase the proportion of Plasmodium -infected patients suffering from this life-threatening complication. The pathogenesis and optimal patient management are still debated. Method We collected and analysed reports of pathological rupture of the spleen associated with malaria published over the last 50 years in five languages. Results Fifty-five cases were reported, due to Plasmodium falciparum ( n = 26), Plasmodium vivax ( n = 23), Plasmodium ovale ( n = 2), Plasmodium malariae ( n = 2), or P. vivax–falciparum ( n = 2), and occurred in travellers ( n = 24), locals ( n = 21), expatriates ( n = 6) or migrants ( n = 4). Median age was 31.5 years and sex ratio M/F 3.2. Splenic rupture was complete with hemoperitoneum ( n = 50), or partial ( n = 5). Death occurred in 12 patients (22%), 8 of whom from early irreversible collapse ( n = 7) or unexpected death ( n = 1). Death rate was higher among travellers than in other patients (9/24, 38%, versus 3/31, 10%, p = 0.01). Clinical features of P. falciparum - or P. vivax -associated splenic rupture were strikingly similar. Treatment included in-hospital medical observation without surgery (conservative management, n = 14), immediate splenectomy ( n = 29), delayed splenectomy ( n = 4), or none (patients dying at admission, n = 8). The type of treatment, conservative or not, had no influence on prognosis. The median duration of malaria symptoms before diagnosis was longer in our review (5–6 days) than in previous reports on imported malaria (3–4 days), suggesting that early diagnosis and therapy of malaria may reduce the incidence of splenic rupture. Conclusions Abdominal pain, collapse, or fainting is warning symptoms. Fourteen published observations support conservative management in carefully selected patients. Spleen preservation likely reduces the risk of future severe malaria attacks in patients with potential further exposition to Plasmodium sp., and also that of overwhelming sepsis in all.

International audience; Molecular methods are crucial for mucormycosis diagnosis because cultures are frequently negative, even if microscopy suggests the presence of hyphae in tissues. We assessed PCR/electrospray-ionization mass spectrometry (PCR/ESI-MS) for Mucorales identification in 19 unfixed tissue samples from 13 patients with proven or probable mucormycosis and compared the results with culture, quantitative real-time PCR, 16S-23S rRNA gene internal transcribed spacer region (ITS PCR) and 18S PCR sequencing. Concordance with culture identification to both genus and species levels was higher for PCR/ESI-MS than for the other techniques. Thus, PCR/ESI-MS is suitable for Mucorales identification, within 6 hours, for tissue samples for which microscopy results suggest the presence of hyphae.

Plasmodium falciparum is transmitted from humans to Anopheles mosquito vectors via the sexual erythrocytic forms termed gametocytes. Erythrocyte filtration through microsphere layers (microsphiltration) had shown that circulating gametocytes are deformable. Compounds reducing gametocyte deformability would induce their splenic clearance, thus removing them from the blood circulation and blocking malaria transmission. The hand-made, single-sample prototype for microsphiltration was miniaturized to a 96-well microtiter plate format, and gametocyte retention in the microsphere filters was quantified by high-content imaging. The stiffening activity of 40 pharmacological compounds was assessed in microtiter plates, using a small molecule (calyculin) as a positive control. The stiffening activity of calyculin was assessed in spleen-mimetic microfluidic chips and in macrophage-depleted mice. Marked mechanical retention (80% to 90%) of mature gametocytes was obtained in microplates following exposure to calyculin at concentrations with no effect on parasite viability. Of the 40 compounds tested, including 20 antimalarials, only 5 endoperoxides significantly increased gametocyte retention (1.5- to 2.5-fold; 24 h of exposure at 1 μM). Mature gametocytes exposed to calyculin accumulated in microfluidic chips and were cleared from the circulation of macrophage-depleted mice as rapidly as heat-stiffened erythrocytes, thus confirming results obtained using the microsphiltration assay. An automated miniaturized approach to select compounds for their gametocyte-stiffening effect has been established. Stiffening induces gametocyte clearance both in vitro and in vivo . Based on physiologically validated tools, this screening cascade can identify novel compounds and uncover new targets to block malaria transmission. Innovative applications in hematology are also envisioned.

Background Diarrhea is the most common illness associated with international tourism. We evaluated the efficacy of a probiotic preparation of nonviable Lactobacillus acidophilus (hereafter referred to as LA) for the prevention of traveler's diarrhea. Methods We conducted a randomized, double-blind, controlled trial. Travelers were randomized to receive either LA or placebo twice daily from 1 day before their departure to 3 days after their return. On each day of the trip and the week following the return, travelers had to record the number and consistency of stools and their adherence to the treatment. Diarrhea was defined as > or =3 unformed stools in a 24-h period. Results From January 2001 to September 2004, a total of 174 subjects were randomized to each treatment group. Half of the travelers went to West Africa, and organized tours or backpacking were the most common modes of traveling. The incidence of diarrhea did not differ between the 2 groups; it was 61.4 cases per 100 person-months in the LA group (95% confidence interval [CI], 44.1-85.5) and 43.4 cases per 100 person-months in the placebo group (95% CI, 30.0-62.9) (P=.14). Adjustment for travel duration and other variables did not reveal any difference between the 2 groups (adjusted hazard ratios comparing the LA and placebo groups were 1.43 [95% CI, 0.87-2.36] in an intent-to-treat analysis and 1.38 [95% CI, 0.79-2.39] in an efficacy analysis). Conclusions There was no beneficial effect of treatment with LA for the prevention of travelers' diarrhea. More studies are required to assess the efficacy of other specific probiotics (e.g., a Lactobacillus rhamnosus GG preparation) for preventing traveler's diarrhea.

BACKGROUND: Treatment of cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) in travelers is still controversial. Over the last decade, national and international consortia have published recommendations for treating CL in travelers. These guidelines harmonize many issues, but there are some discrepancies. METHODS: Leishmania parasites causing CL can now be genotyped by polymerase chain reaction techniques for detecting Leishmania DNA. Therefore, treatment recommendations can now be species based rather than based on geographical exposure. To review the evidence on which the recommendations were based, "LeishMan" (Leishmaniasis Management), a group of experts from 13 institutions in eight European countries, performed a PubMed MEDLINE) literature search and considered unpublished evidence and the experts' own personal experiences. The Oxford evidence grading system was used to evaluate the information. RESULTS AND CONCLUSION: In this article, the authors provide practical treatment recommendations for imported CL and ML in Europe, drawn up from the review by the European experts.

The mechanisms underlying reduced red blood cell (RBC) deformability during Plasmodium falciparum (Pf) malaria remain poorly understood. Here, we explore the possible involvement of the L-arginine and nitric oxide (NO) pathway on RBC deformability in Pf-infected patients and parasite cultures. RBC deformability was reduced during the acute attack (day0) and returned to normal values upon convalescence (day28). Day0 values correlated with plasma L-arginine levels (r = 0.69; p = 0.01) and weakly with parasitemia (r = −0.38; p = 0.006). In vitro, day0 patient's plasma incubated with ring-stage cultures at 41°C reduced RBC deformability and this effect correlated strongly with plasma L-arginine levels (r = 0.89; p < 0.0001). Moreover, addition of exogenous L-arginine to the cultures increased deformability of both Pf-free and trophozoite-harboring RBCs. NO synthase activity, evidenced in Pf-infected RBCs, induced L-arginine-dependent NO production. These data show that hypoargininemia during P.falciparum malaria may altogether impair NO production and reduce RBC deformability, particularly at febrile temperature.

In natural Plasmodium falciparum infections, parasitized erythrocytes (PEs) circulate in the peripheral blood for a period corresponding roughly to the first part of the erythrocytic life cycle (ring stage). Later, in blood-stage development, parasite-encoded adhesion molecules are inserted into the erythrocyte membrane, preventing the circulation of the PEs. The principal molecule mediating PE adhesion is P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the polymorphic var gene family. The population of parasites is subject to clonal antigenic variation through changes in var expression, and a single PfEMP1 variant is expressed at the PE surface in a mutually exclusive manner. In addition to its role in immune evasion, switches in PfEMP1 expression may be associated with fundamental changes in parasite tissue tropism in malaria patients. A switch from CD36 binding to chondroitin sulphate A (CSA) binding may lead to extensive sequestration of PEs in placenta syncytiotrophoblasts. This is probably a key event in malaria pathogenesis during pregnancy. The CSA-binding phenotype of mature PEs is linked to another distinct adhesive phenotype: the recently described CSA-independent cytoadhesion of ring-stage PEs. Thus, a subpopulation of PEs that sequentially displays these two different phenotypes may bind to an individual endothelial cell or syncytiotrophoblast throughout the asexual blood-stage cycle. This suggests that non-circulating (cryptic) parasite subpopulations are present in malaria patients.

A common pathological characteristic of Plasmodium falciparum infection is the cytoadhesion of mature-stage-infected erythrocytes (IE) to host endothelium and syncytiotrophoblasts. Massive accumulation of IE in the brain microvasculature or placenta is strongly correlated with severe forms of malaria1. Extensive binding of IE to placental chondroitin sulfate A (CSA) is associated with physiopathology during pregnancy2,3. The adhesive phenotype of IE correlates with the appearance of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) at the erythrocyte surface (approximately 16 h after merozoite invasion), so that only early blood-stage (ring-stage) IE appear in the peripheral blood. Here, we describe results that challenge the existing view of blood-stage IE biology by demonstrating the specific adhesion of IE, during the early ring-stage, to endothelial cell lines from the brain and lung and to placental syncytiotrophoblasts. Later, during blood-stage development of these IE, trophozoites switch to an exclusively CSA cytoadhesion phenotype. Therefore, adhesion to an individual endothelial cell or syncytiotrophoblast may occur throughout the blood-stage cycle, indicating the presence in malaria patients of noncirculating (cryptic) parasite subpopulations. We detected two previously unknown parasite proteins on the surface of ring-stage IE. These proteins disappear shortly after the start of PfEMP1-mediated adhesion.

To the Editor: The first human case of African tick-bite fever was described in 1992 as occurring in Zimbabwe. The causative agent was identified as a new serotype of the spotted fever group (SFG) rickettsiae and named Rickettsia africae (1). These findings confirmed observations made by Pijper in the 1930s, which suggested that there were 2 different kinds of human SFG rickettsioses in sub-Saharan Africa: Mediterranean spotted fever caused by R. conorii and transmitted by Rhipicephalus species, ticks of dogs, and African tick-bite fever caused by R. africae and transmitted by Amblyomma species, ticks of cattle and wild ungulates. African tick-bite fever has subsequently been diagnosed in patients from several other sub-Saharan countries and also from the West Indies (2,3). In a recent analysis of the spectrum of diseases among returning travelers, tick-borne spotted fever was (after malaria) the second most frequent cause of systemic febrile illness among those returning from sub-Saharan Africa. It occurred more frequently than typhoid fever and dengue fever (4). The following case description reports an infection with R. africae in a man in France who recently returned from Ethiopia. On November 4, 2005, a 62-year-old French man sought care at the Medical Center of the Institut Pasteur in Paris for fever, along with chills, headache, neck and shoulder pain, and fatigue over the previous 4 days. At the onset of these symptoms he had noticed dark nodular lesions on his neck and his left groin followed 2 days later by a slightly painful eruption on his arms and his trunk. He had spent a month in southwest Ethiopia, north of Kelem near the Sudanese border, and returned to France on October 26, 2005. While in Ethiopia, he had assisted with a production of a documentary film about an Ethiopian tribe and had been in contact with cattle in the villages. He had not noticed any tick bites. On physical examination he had a fever of 38°C, a nodular lesion with a central dark crust on his neck, a second lesion on his left inguinal fold (Figure, panel A), and a vesicular eruption on his arms and his trunk (Figure, panel B). Leukocyte count was 3,200, including 1,869 neutrophils and 867 lymphocytes. The platelet level was 174,000/mm3. The C-reactive protein level was 28.3 mg/L. The aspartate aminotransferase level was slightly elevated. The patient was treated with doxycycline 200 mg/day for 1 week for suspected African tick-bite fever. Follow-up showed a quick recovery from his symptoms except for fatigue that persisted for ≈1 month. Figure Inoculation eschar on left inguinal fold (A) and vesicular skin lesion (B) in a traveler recently returned to France from Ethiopia. A commercial immunofluorescence assay for R. conorii and R. typhi immunoglobulin G performed both on an initial blood sample and a second sample taken 1 week later were negative. A blood sample and a biopsy specimen of the inguinal eschar were sent to the National Reference Center of Rickettsiae in Marseille, France. Although cellular culture of both specimens and molecular testing of the blood sample were negative, PCR for the sequences of citrate synthase (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"RAU59733","term_id":"1389980"}}RAU59733, 93.1% homology) and rickettsial OmpA (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"RAU83436","term_id":"62861416"}}RAU83436, 99.3% homology) applied on the skin biopsy detected R. africae and confirmed the diagnosis of African tick-bite fever. From 1969 to 1971, SFG rickettsiae were isolated from Amblyomma spp. ticks collected in Ethiopia. They were regarded as R. conorii or as closely related bacteria (5). Later, more specific tests using western immunoblots with monoclonal antibodies showed that these rickettsiae differed from R. conorii (6). In 1992 SFG rickettsiae isolated from Amblyomma ticks collected in Zimbabwe and from the blood of a patient in Zimbabwe were compared to R. conorii, to other pathogenic SFG rickettsiae, and to a SFG rickettsia isolated from an Amblyomma spp. tick in Ethiopia 20 years before. The SFG rickettsia isolates from Ethiopia were identical to isolates obtained in Zimbabwe from the Amblyomma ticks and the patient’s blood and were different from R. conorii and other pathogenic SFG rickettsiae. This new serotype of SFG rickettsiae was named R. africae (1,7). A recent study confirmed the presence of R. africae in ticks collected in Ethiopia, as well as R. aeschlimanii (8). Thus, evidence of R. africae in Ethiopia has been known for a long time. The geographic distribution of African tick-bite fever is related to the presence of Amblyomma spp. ticks, vectors and reservoirs of R. africae. Consequently African tick-bite fever should also be considered as a possible diagnosis in patients with febrile illness returning from countries where R. africae has been detected in Amblyomma ticks, even if a human infection has not yet been reported (9,10).

International audience; BACKGROUND: Hyposplenism, due to splenectomy, inherited red blood cell disorders or acquired conditions such as celiac disease, has an important impact on the severity of malaria, especially in non-immune patients. Conversely, that malaria may reveal functional hyposplenism has not been described previously. METHODS: A 31-year old gardener was diagnosed with an uncomplicated attack of Plasmodium malariae 11 years after leaving the endemic area. In addition to trophozoites and schizonts, thick and thin smears also showed Howell-Jolly bodies, pointing to functional hyposplenism. This was later confirmed by the presence of a calcified spleen in the context of S/beta + sickle-cell syndrome in a patient previously unaware of this condition. CONCLUSION: Malaria may reveal hyposplenism. Although Howell-Jolly bodies are morphologically similar to nuclei of young Plasmodium trophozoite, distinction on smears is based on the absence of cytoplasm and irregular size of Howell-Jolly bodies. In the patient reported here, hyposplenism was revealed by the occurrence of P. malariae infection relatively late in life. Timely diagnosis of hyposplenism resulted in the implementation of appropriate measures to prevent overwhelming infection with capsulated bacteria. This observation highlights the importance of diagnosing hyposplenism in patients with malaria despite the morphological similarities between ring nuclei and Howell-Jolly bodies on thick smears.

Background Cutaneous leishmaniasis is caused by several Leishmania species that are associated with variable outcomes before and after therapy. Optimal treatment decision is based on an accurate identification of the infecting species but current methods to type Leishmania isolates are relatively complex and/or slow. Therefore, the initial treatment decision is generally presumptive, the infecting species being suspected on epidemiological and clinical grounds. A simple method to type cultured isolates would facilitate disease management. Methodology We analyzed MALDI-TOF spectra of promastigote pellets from 46 strains cultured in monophasic medium, including 20 short-term cultured isolates from French travelers (19 with CL, 1 with VL). As per routine procedure, clinical isolates were analyzed in parallel with Multilocus Sequence Typing (MLST) at the National Reference Center for Leishmania. Principal Findings Automatic dendrogram analysis generated a classification of isolates consistent with reference determination of species based on MLST or hsp70 sequencing. A minute analysis of spectra based on a very simple, database-independent analysis of spectra based on the algorithm showed that the mutually exclusive presence of two pairs of peaks discriminated isolates considered by reference methods to belong either to the Viannia or Leishmania subgenus, and that within each subgenus presence or absence of a few peaks allowed discrimination to species complexes level. Conclusions/Significance Analysis of cultured Leishmania isolates using mass spectrometry allows a rapid and simple classification to the species complex level consistent with reference methods, a potentially useful method to guide treatment decision in patients with cutaneous leishmaniasis., Author Summary Cutaneous leishmaniasis is a disease due to a small parasite called Leishmania. This parasite causes disfiguring skin lesions that last for months or years. There are many different subtypes of Leishmania, each giving rise to lesions of different severity and responding to therapies in its own way. Treating physicians must know as soon as possible which subtype of Leishmania is involved to propose the best treatment. Because it is impossible to differentiate the Leishmania subtypes microscopically, the identification of the culprit subtype currently requires complex and expensive typing methods, the results of which are generally obtained several weeks after the diagnosis. Here, we have evaluated the ability of a new method using mass spectrometry to differentiate Leishmania subtypes. Our results were consistent with those provided by reference typing methods and were obtained rapidly after the parasite had been cultured in vitro. This new method may help physicians know very soon which Leishmania subtype is involved thereby facilitating treatment choice.

International audience; BACKGROUND: There is a need for a simple and efficacious treatment for cutaneous leishmaniasis with an acceptable side-effect profile. METHODS: We conducted a randomized, vehicle-controlled phase 3 trial of topical treatments containing 15% paromomycin, with and without 0.5% gentamicin, for cutaneous leishmaniasis caused by Leishmania major in Tunisia. We randomly assigned 375 patients with one to five ulcerative lesions from cutaneous leishmaniasis to receive a cream containing 15% paromomycin-0.5% gentamicin (called WR 279,396), 15% paromomycin alone, or vehicle control (with the same base as the other two creams but containing neither paromomycin nor gentamicin). Each lesion was treated once daily for 20 days. The primary end point was the cure of the index lesion. Cure was defined as at least 50% reduction in the size of the index lesion by 42 days, complete reepithelialization by 98 days, and absence of relapse by the end of the trial (168 days). Any withdrawal from the trial was considered a treatment failure. RESULTS: The rate of cure of the index lesion was 81% (95% confidence interval [CI], 73 to 87) for paromomycin-gentamicin, 82% (95% CI, 74 to 87) for paromomycin alone, and 58% (95% CI, 50 to 67) for vehicle control (P

Ex vivo perfusion of human spleens revealed innate retention of numerous cultured Plasmodium falciparum ring-infected red blood cells (ring-iRBCs). Ring-iRBC retention was confirmed by a microsphiltration device, a microbead-based technology that mimics the mechanical filtering function of the human spleen. However, the cellular alterations underpinning this retention remain unclear. Here, we use ImageStream technology to analyze infected RBCs' morphology and cell dimensions before and after fractionation with microsphiltration. Compared to fresh normal RBCs, the mean cell membrane surface area loss of trophozoite-iRBCs, ring-iRBCs and uninfected co-cultured RBCs (uRBCs) was 14.2% (range: 8.3-21.9%), 9.6% (7.3-12.2%) and 3.7% (0-8.4), respectively. Microsphilters retained 100%, ∼50% and 4% of trophozoite-iRBCs, ring-iRBCs and uRBCs, respectively. Retained ring-iRBCs display reduced surface area values (estimated mean, range: 17%, 15-18%), similar to the previously shown threshold of surface-deficient RBCs retention in the human spleen (surface area loss: >18%). By contrast, ring-iRBCs that successfully traversed microsphilters had minimal surface area loss and normal sphericity, suggesting that these parameters are determinants of their retention. To confirm this hypothesis, fresh normal RBCs were exposed to lysophosphatidylcholine to induce a controlled loss of surface area. This resulted in a dose-dependent retention in microsphilters, with complete retention occurring for RBCs displaying >14% surface area loss. Taken together, these data demonstrate that surface area loss and resultant increased sphericity drive ring-iRBC retention in microsphilters, and contribute to splenic entrapment of a subpopulation of ring-iRBCs. These findings trigger more interest in malaria research fields, including modeling of infection kinetics, estimation of parasite load, and analysis of risk factors for severe clinical forms. The determination of the threshold of splenic retention of ring-iRBCs has significant implications for diagnosis (spleen functionality) and drug treatment (screening of adjuvant therapy targeting ring-iRBCs).

Background Plasmodium falciparum immature gametocytes accumulate in the bone marrow, but their exact location in this tissue remains unclear. Methods The stage and deposition pattern of gametocytes was analysed on histological sections of a bone marrow sample collected in a patient with subacute P. falciparum malaria. Results A majority (89%) of immature stages II to IV gametocytes and a minority (29%) of mature stage V gametocytes were observed in extravascular spaces. Discussion and conclusion These observations represent a valuable step towards understanding sequestration patterns of P. falciparum gametocytes and may ultimately lead to novel transmission-blocking interventions.

Plasmodium vivax threatens nearly half the world’s population and is a significant impediment to achievement of the millennium development goals. It is an important, but incompletely understood, cause of anaemia. This review synthesizes current evidence on the epidemiology, pathogenesis, treatment and consequences of vivax-associated anaemia. Young children are at high risk of clinically significant and potentially severe vivax-associated anaemia, particularly in countries where transmission is intense and relapses are frequent. Despite reaching lower densities than Plasmodium falciparum, Plasmodium vivax causes similar absolute reduction in red blood cell mass because it results in proportionately greater removal of uninfected red blood cells. Severe vivax anaemia is associated with substantial indirect mortality and morbidity through impaired resilience to co-morbidities, obstetric complications and requirement for blood transfusion. Anaemia can be averted by early and effective anti-malarial treatment.

International audience; Infection of erythrocytes with the human malaria parasite, Plasmodium falciparum, results in dramatic changes to the host cell structure and morphology. The predicted functional localization of the STEVOR proteins at the erythrocyte surface suggests that they may be involved in parasite-induced modifications of the erythrocyte membrane during parasite development. To address the biologic function of STEVOR proteins, we subjected a panel of stevor transgenic parasites and wild-type clonal lines exhibiting different expression levels for stevor genes to functional assays exploring parasite-induced modifications of the erythrocyte membrane. Using this approach, we show that stevor expression impacts deformability of the erythrocyte membrane. This process may facilitate parasite sequestration in deep tissue vasculature.

The altered deformability of erythrocytes infected with Plasmodium falciparum is central in malaria -pathogenesis, as it influences the hemodynamic properties of the infected cell and its retention in the spleen. Exported parasite proteins, as well as the shape and volume of the parasite itself, influence the deformability of the infected erythrocyte. To explore changes in erythrocyte deformability, we have developed a new method, called microsphiltration, based on filtration of erythrocytes through a mixture of metal microspheres that mimic the geometry of inter-endothelial splenic slits. As P. falciparum develops in its host cell, the retention rates observed in microspheres correlate with the progressive decrease of erythrocyte deformability and with the retention rates in the spleen. The yields of microsphiltration separation allow for molecular analyses of subpopulations with distinct mechanical phenotypes.

Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission.

Malaria parasites induce complex cellular and clinical phenotypes, including anemia, cerebral malaria and death in a wide range of mammalian hosts. Host genes and parasite ‘toxins’ have been implicated in malarial disease, but the contribution of parasite genes remains to be fully defined. Here we assess disease in BALB/c mice and Wistar rats infected by the rodent malaria parasite Plasmodium berghei with a gene knock out for merozoite surface protein (MSP) 7. MSP7 is not essential for infection but in P. falciparum, it enhances erythrocyte invasion by 20%. In vivo, as compared to wild type, the P. berghei Δmsp7 mutant is associated with an abrogation of death and a decrease from 3% to 2% in peak, circulating parasitemia. The Δmsp7 mutant is also associated with less anemia and modest increase in the size of follicles in the spleen. Together these data show that deletion of a single parasite invasion ligand modulates blood stage disease, as measured by death and anemia. This work is the first to assess the contribution of a gene present in all plasmodial species in severe disease.

Clinical manifestations of Plasmodium falciparum infection are induced by the asexual stages of the parasite that develop inside red blood cells (RBCs). Because splenic microcirculatory beds filter out altered RBCs, the spleen can innately clear subpopulations of infected or uninfected RBC modified during falciparum malaria. The spleen appears more protective against severe manifestations of malaria in naïve than in immune subjects. The spleen-specific pitting function accounts for a large fraction of parasite clearance in artemisinin-treated patients. RBC loss contributes to malarial anemia, a clinical form associated with subacute progression, frequent splenomegaly, and relatively low parasitemia. Stringent splenic clearance of ring-infected RBCs and uninfected, but parasite-altered, RBCs, may altogether exacerbate anemia and reduce the risks of severe complications associated with high parasite loads, such as cerebral malaria. The age of the patient directly influences the risk of severe manifestations. We hypothesize that coevolution resulting in increased splenic clearance of P. falciparum–altered RBCs in children favors the survival of the host and, ultimately, sustained parasite transmission. This analysis of the RBC–spleen dynamic interactions during P falciparum infection reflects both data and hypotheses, and provides a framework on which a more complete immunologic understanding of malaria pathogenesis may be elaborated.

Experimental tools to identify human red blood cells (RBC) prone to mechanical retention upstream from the spleen venous sinus inter-endothelial slits are currently suboptimal. We designed a micro-bead device mimicking the geometry of the human narrow and short inter-endothelial slits. Upon filtration through a mixture of 5-25 μm diameter micro-beads, Plasmodium falciparum-hosting RBC (Pf-RBC) were retained in a parasite developmental stage-dependent way, the retention rates of a subset of ring-RBC being similar in micro-beads and in isolated-perfused human spleens. We found that this retention might be linked principally to the reduced surface-area-to-volume ratio of Pf-RBC. Interestingly, other rigid RBC, such as heat-treated RBC, and RBC from hereditary spherocytosis patients were also retained in micro-beads without any hemolysis. Micro-beads allow (i) depletion of heterogeneous RBC population from its rigid-RBC subpopulation ii) characteriziation of distinct molecular signatures of rigid versus deformable RBC subpopulations. This simple method portends wide medical applications, such as improving the quality of stored RBC concentrates prior to transfusion.

International audience; In 2002, the French Food Safety Agency drew attention to the lack of information on the prevalence of human cryptosporidiosis in the country. Two years later, the ANOFEL Cryptosporidium National Network (ACNN) was set up to provide public health authorities with data on the incidence and epidemiology of human cryptosporidiosis in France. Constituted on a voluntary basis, ACNN includes 38 hospital parasitology laboratories (mainly in university hospitals). Each laboratory is engaged to notify new cases of confirmed human cryptosporidiosis, store specimens (e.g. stools, duodenal aspirates or biopsies) and related clinical and epidemiological data, using data sheet forms. From January 2006 to December 2009, 407 cryptosporidiosis cases were notified in France and 364 specimens were collected. Of the notified cases, 74 were children under four years of age, accounting for 18.2%. HIV-infected and immunocompetent patients represented 38.6% (n=157) and 28% (n=114) of cases, respectively. A marked seasonal pattern was observed each year, with increased number of cases in mid to late summer and the beginning of autumn. Genotyping of 345 isolates from 310 patients identified C. parvumin 168 (54.2%) cases, C. hominis in 113 (36.4%) and other species in 29 (9.4%), including C. felis (n=15), C. meleagridis (n=4), C. canis (n=4), Cryptosporidium chipmunk genotype (n=1), Cryptosporidium rabbit genotype (n=1) and new Cryptosporidium genotypes (n=4). These data represent the first multisite report of laboratory-confirmed cases of cryptosporidiosis in France.

Background Matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF-MS) allows the identification of most bacteria and an increasing number of fungi. The potential for the highest clinical benefit of such methods would be in severe acute infections that require prompt treatment adapted to the infecting species. Our objective was to determine whether yeasts could be identified directly from a positive blood culture, avoiding the 1–3 days subculture step currently required before any therapeutic adjustments can be made. Methodology/Principal Findings Using human blood spiked with Candida albicans to simulate blood cultures, we optimized protocols to obtain MALDI TOF-MS fingerprints where signals from blood proteins are reduced. Simulated cultures elaborated using a set of 12 strains belonging to 6 different species were then tested. Quantifiable spectral differences in the 5000–7400 Da mass range allowed to discriminate between these species and to build a reference database. The validation of the method and the statistical approach to spectral analysis were conducted using individual simulated blood cultures of 36 additional strains (six for each species). Correct identification of the species of these strains was obtained. Conclusions/Significance Direct MALDI TOF-MS analysis of aliquots from positive blood cultures allowed rapid and accurate identification of the main Candida species, thus obviating the need for sub-culturing on specific media. Subsequent to this proof-of-principle demonstration, the method can be extended to other clinically relevant yeast species, and applied to an adequate number of clinical samples in order to establish its potential to improve antimicrobial management of patients with fungemia.

Splenomegaly is frequent in acute or chronic forms of Plasmodium falciparum malaria, and splenectomy is associated with more frequent fever and parasitaemia. A novel role for the spleen in malaria is indicated by recent epidemiological and experimental data, bringing about a novel paradigm on severe malaria pathogenesis.In Sudanese children, severe malarial anaemia was associated with larger spleen, longer fever duration, and lower parasitaemia than cerebral malaria. These findings are consistent with evolution toward severe malarial anaemia being linked to the presence of a spleen-dependent mechanism that is absent or inefficient in cerebral malaria. An isolated-perfused human spleen model revealed unexpected retention of numerous erythrocytes harbouring young parasite stages (rings), probably through an innate mechanical process.A new paradigm is discussed, whereby the extent of erythrocyte retention in the spleen conditions not only haemoglobin concentration and spleen size but also the rate of parasite load increase. The prediction is that, in nonimmune children, stringent splenic retention of rings and uninfected erythrocytes reduces the risk of cerebral malaria (a complication associated with high parasite loads) but increases the risk of severe malarial anaemia. This hypothesis casts new light on epidemiological, genetic, and experimental studies in malaria pathogenesis.

Background Cutaneous leishmaniasis (CL) is a disfiguring disease that confronts clinicians with a quandary: leave patients untreated or engage in a complex or toxic treatment. Topical treatment of CL offers a practical and safe option. Accordingly, the treatment of CL with WR279,396, a formulation of paromomycin and gentamicin in a hydrophilic base, was investigated in a phase 2 clinical study in Tunisia and France. Methods A phase 2, randomized, double blind, vehicle-controlled study was conducted to assess the safety and efficacy of topical WR279,396 when applied twice a day for 20 days as treatment for parasitologically confirmed CL. The study protocol established the primary efficacy end point as complete clinical response (CCR) defined as 50% or greater reduction in the ulceration size of an index lesion by day 50 (D50) followed by complete re-epithelialization by D100, and no relapse through D180. Results Ninety-two subjects were randomized. Leishmania major was identified in 66 of 68 isolates typed (97%). In the intent-to-treat population, 47 of 50 WR279,396 treated participants (94%) met the definition of CCR, compared with 30 of 42 vehicle-placebo participants (71%) [p = 0.0045]. Erythema occurred in 30% and 24% of participants receiving WR279,396 and placebo, respectively [p = 0.64]. There was no clinical or laboratory evidence of systemic toxicity. Conclusion Application of WR279,396 for 20 days was found to be safe and effective in treating L. major CL, and offers great potential as a new, simple, easily applicable, and inexpensive topical therapy for this neglected disease. Trial Registration ClinicalTrials.gov NCT00703924, Author Summary Cutaneous leishmaniasis is due to a small parasite (Leishmania) that creates disfiguring sores, and affects more than one million persons (mainly children) each year. Treating lesions with a cream—instead of with injections as currently done—would greatly improve the well-being of affected patients. No cream formulation that would be efficient and would not create important skin irritation has been identified yet. Here, we tested a new cream formulation (WR279,396) containing paromomycin and gentamicin, two members of a well-known family of antibacterial antibiotics (aminoglycosides). Injectable paromomycin is efficient in other forms of the disease (visceral leishmaniasis). This was a carefully monitored study (phase 2) involving mainly children in Tunisia and France. The cream was applied twice a day for 20 days. The proportion of patients treated with the paromomycin-containing cream (active formulation) that cured (94%) was higher than that observed (71%) in patients treated with a cream that did not contain the active product (placebo formulation). Local irritation affected less than one-third of the patients and was usually mild. This new cream formulation was safe and effective in treating cutaneous leishmaniasis, thereby providing a new, simple, easily applicable, and inexpensive treatment for this neglected disease.

The current paradigm in Plasmodium falciparum malaria pathogenesis states that young, ring-infected erythrocytes (rings) circulate in peripheral blood and that mature stages are sequestered in the vasculature, avoiding clearance by the spleen. Through ex vivo perfusion of human spleens, we examined the interaction of this unique blood-filtering organ with P falciparum–infected erythrocytes. As predicted, mature stages were retained. However, more than 50% of rings were also retained and accumulated upstream from endothelial sinus wall slits of the open, slow red pulp microcirculation. Ten percent of rings were retained at each spleen passage, a rate matching the proportion of blood flowing through the slow circulatory compartment established in parallel using spleen contrast-enhanced ultrasonography in healthy volunteers. Rings displayed a mildly but significantly reduced elongation index, consistent with a retention process, due to their altered mechanical properties. This raises the new paradigm of a heterogeneous ring population, the less deformable subset being retained in the spleen, thereby reducing the parasite biomass that will sequester in vital organs, influencing the risk of severe complications, such as cerebral malaria or severe anemia. Cryptic ring retention uncovers a new role for the spleen in the control of parasite density, opening novel intervention opportunities.

International audience; The efficacy of fluconazole was evaluated in 35 travelers with parasitologically proven imported Old World cutaneous leishmaniasis (CL). Leishmania major (mainly MON-25) was identified in 15 patients and strongly suspected given the transmission area in 12 of these patients. Daily oral fluconazole (200 mg/day for adults and 2.5 mg/kg/day for children) was prescribed for six weeks. Outcome definition was based on re-epithelialization rate at day 50. Of the 27 L. major-infected patients, 12 (44.4%) were cured. This cure rate is similar to the placebo cure rate from trials in L. major CL in which, as in the present report, the definition of outcome relied exclusively on re-epithelialization. These data question the assumption that oral fluconazole is consistently effective for treatment of CL caused by L. major.

Visceral and cutaneous leishmaniases are heterogenous entities. The Leishmania species that a given patient harbors usually cannot be determined clinically, and this identification is essential to prescribe the best species-specific therapeutic regimen. Our diagnosis procedure includes a real-time PCR assay targeted at the 18S rRNA gene, which detects all Leishmania species but which is not specific for a given Leishmania species. We developed a species identification based on sequencing of the cytochrome b (cyt b ) gene directly from the DNA extracted from the clinical specimen. The sequences were analyzed using the Sequence Analysis/Seqscape v2.1 software (Applied Biosystems). This software is designed to automatically identify the closest sequences from a reference library after analysis of all known or unknown polymorphic positions. The library was built with the Leishmania cyt b gene sequences available in GenBank. Fifty-three consecutive real-time PCR-positive specimens were studied for species identification. The cyt b gene was amplified in the 53 specimens. Sequencing resulted in the identification of six different species with ≥99% identity with the reference sequences over 872 nucleotides. The identification was obtained in two working days and was in accordance with the multilocus enzyme electrophoresis identification when available. Real-time PCR followed by sequencing of the cyt b gene confirmed the diagnosis of leishmaniasis and rapidly determined the infecting species directly from the clinical specimen without the need for the isolation of parasites. This technique has the potential to significantly accelerate species-adapted therapeutic decisions regarding treatment of leishmaniasis.

Background Because topical therapy is easy and usually painless, it is an attractive first-line option for the treatment of localized cutaneous leishmaniasis (LCL). Promising ointments are in the final stages of development. One main objective was to help optimize the treatment modalities of human LCL with WR279396, a topical formulation of aminoglycosides that was recently proven to be efficient and safe for use in humans. Methodology/Principal Findings C57BL/6 mice were inoculated in the ear with luciferase transgenic L. major and then treated with WR279396. The treatment period spanned lesion onset, and the evolution of clinical signs and bioluminescent parasite loads could be followed for several months without killing the mice. As judged by clinical healing and a 1.5-3 log parasite load decrease in less than 2 weeks, the 94% efficacy of 10 daily applications of WR279396 in mice was very similar to what had been previously observed in clinical trials. When WR279396 was applied with an occlusive dressing, parasitological and clinical efficacy was significantly increased and no rebound of parasite load was observed. In addition, 5 applications under occlusion were more efficient when done every other day for 10 days than daily for 5 days, showing that length of therapy is a more important determinant of treatment efficacy than the total dose topically applied. Conclusions/Significance Occlusion has a significant adjuvant effect on aminoglycoside ointment therapy of experimental cutaneaous leishmaniasis (CL), a concept that might apply to other antileishmanial or antimicrobial ointments. Generated in a laboratory mouse-based model that closely mimics the course of LCL in humans, our results support a schedule based on discontinuous applications for a few weeks rather than several daily applications for a few days., Author Summary When initiating the cutaneous disease named cutaneous leishmaniasis (CL), Leishmania parasites develop within the parasitophorous vacuoles of phagocytes residing in and/or recruited to the dermis, a process leading to more or less chronic dermis and epidermis-damaging inflammatory processes. Topical treatment of CL could be a mainstay in its management. Any improvements of topicals, such as new vehicles and shorter optimal contact regimes, could facilitate their use as an ambulatory treatment. Recently, WR279396, a third-generation aminoglycoside ointment, was designed with the aim to provide stability and optimal bioavailability for the molecules expected to target intracellular Leishmania. Two endpoints were expected to be reached: i) accelerated clearance of the maximal number of parasites, and ii) accelerated and stable repair processes without scars. A mouse model of CL was designed: it relies on the intradermal inoculation of luciferase-expressing Leishmania, allowing for in vivo bioluminescence imaging of the parasite load fluctuation, which can then be quantified simultaneously with the onset and resolution of clinical signs. These quantitative readout assays, deployed in real time, provide robust methods to rapidly assess efficacy of drugs/compounds i) to screen treatment modalities and ii) allow standardized comparison of different therapeutic agents.

We report a case of severe malaria in a patient with underlying myasthenia gravis who was successfully treated with artesunate. The outcome was favorable. Artesunate seems to be a good option for patients with underlying myasthenia gravis disease because they benefit from a better toxicity profile than quinine.

Cutaneous leishmaniasis (CL) is a disfiguring illness caused by Leishmania species of protozoa, with over 350 million people at risk worldwide [1]. Leishmania parasites enter the skin through a sandfly bite, producing a papule or nodule that generally ulcerates [1]. Spontaneous resolution of CL ulcers may take months to years [2], and both active lesions and scars can engender stigma and cause disability [3]. There is no consensus regarding the optimum therapy for CL, and no single treatment approach fits all possible clinical presentations [1], [2]. However, because systemic treatments may produce considerable toxicity [1], [2], [4], localized therapy (e.g., intralesional antimonials, cryotherapy, thermotherapy, or intralesional injections plus cryotherapy) is now recommended in Old World CL (L. major, L. tropica) and in selected cases of New World CL [1], [2]. However, all present local therapy modalities have limitations, e.g., variable cure rates, pain, challenges and complexities associated with treatment administration (especially in children), and variable utility in patients with multiple lesions or lesions located on body areas where local treatment is impractical [1], [2], [5].

International audience; Background: Several clinical forms of malaria such as chronic carriage, gestational malaria or hyper-reactive malarial splenomegaly may follow a cryptic evolution with afebrile chronic fatigue sometimes accompanied by anemia and/or splenomegaly. Conventional parasitological tests are often negative or not performed, and severe complications may occur. Extensive explorations of these conditions often include the search for antinuclear autoantibodies (ANA). Methods: We analysed fluorescence patterns in the ANA test in patients with either chronic cryptic or acute symptomatic malaria, then conducted a one-year prospective study at a single hospital on all available sera drawn for ANA detections. We then identified autoantibodies differentially expressed in malaria patients and in controls using human protein microarray. Results: We uncovered and defined a new, malaria-related, nucleo-cytoplasmic ANA pattern displaying the specific association of a nuclear speckled pattern with diffuse cytoplasmic perinuclearly-enhanced fluorescence. In the one-year prospective analysis, 79% of sera displaying this new nucleo-cytoplasmic fluorescence were from patients with malaria. This specific pattern, not seen in other parasitic diseases, allowed a timely reorientation of the diagnosis toward malaria. To assess if the autoantibody immune response was due to autoreactivity or molecular mimicry we isolated 42 autoantigens, targets of malarial autoantibodies. BLAST analysis indicated that 23 of recognized autoantigens were homologous to plasmodial proteins suggesting autoimmune responses directly driven by the plasmodial infection. Conclusion: In patients with malaria in whom parasitological tests have not been performed recognition of this new, malaria-related fluorescence pattern on the ANA test is highly suggestive of the diagnosis and triggers immediate, easy confirmation and adapted therapy.