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      Old World Cutaneous Leishmaniasis and Refugee Crises in the Middle East and North Africa

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          The Syrian refugee crisis has precipitated a catastrophic outbreak of Old World cutaneous leishmaniasis now affecting hundreds of thousands of people living in refugee camps or trapped in conflict zones. A similar situation may also be unfolding in eastern Libya and Yemen. Leishmaniasis has been endemic in Syria for over two centuries, with the first case ever reported being as early as 1745, when it was known as the “Aleppo boil” [1,2]. Old World cutaneous leishmaniasis (CL) is characterized most notably by disfiguring skin lesions, nodules, or papules, and in the Middle East and North Africa (MENA) region it is primarily caused either by Leishmania tropica (anthroponotic) or L. major (zoonotic), with some sporadic cases also caused by L. infantum (Box 1) [3–5]. In North Africa, a chronic form of CL also can be caused by L. killicki [6–7]. Box 1. Old World Cutaneous Leishmaniasis (CL) in the MENA Region Anthroponotic CL Major etiologic agent: Leishmania tropica [4,5,7] Major vector: Phlebotomus sergenti [4,5] Zoonotic CL Major etiologic agent: L. major [4,5,7] Minor etiologic agent: L. infantum [4,5] Vectors: Ph. papatasi for L. major; Ph. perfiliewi, Ph. perniciosus, Ph. longicuspis, and Ph. ariasi for L. infantum [5] Major animal reservoirs: Rodents (L. major) and dogs (L. infantum) [4,7] Although Old World CL is generally not fatal, clinical symptoms can lead to disfiguring scars that result in social stigmatization and psychological consequences. The World Health Organization (WHO) has estimated that around 2.4 million disability-adjusted life years (DALYs) are lost due to CL and visceral leishmaniasis (VL) globally [8]; however, the number of DALYs attributed to CL is still under evaluation. The 2013 Global Burden of Disease Study determined that CL causes only 41,700 DALYs [9], while other studies have found that these figures may represent profound underestimates [10,11]. Studies observing the impact of marring CL facial scars have found that the social stigmatization involved leads to anxiety, depression, and decreased quality of life for patients [12]. The scars can lead to a changed perception of self and can limit individuals’ abilities to participate in society, further decreasing their social, psychological, and economic well-being, as employment opportunities become scarce. Women, adolescents, and children are particularly susceptible to the social stigmatization of disfiguring scars [13]. The hardships caused by CL extend beyond physical symptoms and manifest most prominently in patients’ social, psychological, and economic well-being. Like many neglected tropical diseases (NTDs), CL not only occurs in settings of poverty but the disease also has the ability to perpetuate and reinforce poverty, catalyzing a positive feedback loop between disease and poverty [14]. For many of these reasons, the WHO classifies leishmaniasis as one of 17 NTDs [15], although the cutaneous form is often not prioritized in major global health initiatives, unlike the NTDs now targeted by integrated preventive chemotherapy [11]. Pre-Conflict Old World CL in Syria Even before the current crisis, the Syrian government has struggled to contain endemic CL. After a 30-year hiatus during which CL was mostly restricted to Aleppo and Damascus [16], CL re-emerged in northwestern Syria in 1988 [1,17]. In 1991, the incidence of CL dropped temporarily due to insecticide spraying, but it began to rapidly rise again even as insecticide spraying continued [18]. The increased number of cases may have been accounted for in part by increased awareness and reporting of the disease; however, the most likely explanation for the dramatic increase and distribution of CL starting in the early 1990s stems from socioeconomic and environmental factors [1]. During this time, Syria experienced rapid and decentralized urbanization as city suburbs expanded and the population density increased [19]. People began to migrate from rural to urban areas, and municipal departments, overwhelmed by these changes, were no longer able to provide adequate hygiene and sanitation services such as trash collection and disposal, as well as insecticide spraying [1,19]. As populations migrated, individuals with no immunity became exposed to CL and the disease spread [1]. Such factors may account for a steep rise in the apparent number of CL cases in Syria beginning in 2008 as reported previously by Salam et al. in PLOS Neglected Tropical Diseases (Fig 1) [20]. 10.1371/journal.pntd.0004545.g001 Fig 1 Year-wise trend of CL cases reported in Middle East (from Salam et al 2014, PLOS Neglected Tropical Diseases) [20]. The numbers of CL cases in Syria began to increase even further following the onset of the Syrian Civil War in March, 2011, with new cases appearing in regions previously thought to be non-endemic. War, Refugees, and the Emergence of Catastrophic NTDs While CL is by no means new to Syria [7], the war in Syria has greatly increased the risk for CL and reports have indicated sharp increases in the number of CL cases in Syria and in surrounding areas of the Middle East [2]. Armed conflict enables outbreaks of serious NTDs [4,14,21,22] due to a combination of factors—most notably, collapsed health care infrastructures and population displacement. As populations migrate to endemic and non-endemic regions, they are exposed to infections for the first time or introduce diseases into new areas, respectively [4,20,23]. Additionally, the chaos and instability often lends to poor living conditions, which further exacerbate the risk for rapid transmission of infectious diseases [4,14]. In recent years, such factors were notable for producing catastrophic NTD outbreaks of cholera in the Democratic Republic of Congo and kala-azar in Sudan [24,25]. Additionally, human migration can be accompanied by deforestation or tumultuous urbanization, which often exacerbate disease outbreaks [26]. In West Africa, all of these factors combined to create a “perfect storm” for the 2014–2015 Ebola virus infection epidemic [27]. The ongoing Syrian conflict has escalated similar factors of instability and chaos that have been shown by past events, such as the Ebola epidemic in West Africa, to facilitate infectious disease outbreaks. As the conflict in Syria approaches its fifth year, over 50% of the public hospitals in Syria have been destroyed and the health care infrastructure is bordering on nonexistent [28]. Thus far, an estimated 6.5 million Syrians have been internally displaced [29,30] and an additional 4.4 million Syrians have been externally displaced [31]. Due to the violence, Syrians have been forced to flee from their homes and seek refuge across the Middle East, North Africa, and, more recently, Europe [31]; currently, 95% of the over 4 million refugees who have fled Syria reside in Turkey, Jordan, Lebanon, Iraq, and Egypt (Fig 2) [31]. The mass migration of people within Syria and the MENA region has put a strain on resources. Internally displaced individuals have reported that they are in need of non-food items (including personal hygiene products), health care services, food, shelter, water, and education [32]. Similarly, externally displaced individuals are often living in overcrowded and unhygienic spaces, commonly without access to many necessary resources, including basic sanitation and waste disposal services, food, electricity, as well as health care [33–36]; in Lebanon, refugee camps consist mostly of makeshift houses built out of scrap and rubble or tents [33]. Syria is now the leading producer of refugees in the world and has contributed significantly to what is considered to be the largest global refugee crisis since World War II [37,38]. 10.1371/journal.pntd.0004545.g002 Fig 2 The displacement of Syrian refugees (data from UNHCR, Syria Regional Refugee Response: Inter-agency Information Sharing Portal) [31]. Both inside and outside of Syria, conditions of poverty and malnutrition are prevalent and living situations are grim as adequate municipal services and necessary resources remain scarce [33]. The socioeconomic and environmental circumstances created by the conflict in Syria facilitate risk factors for the continued transmission of CL, and they not only potentiate increased incidence of CL but also exacerbate the morbidity and mortality of CL after transmission [14,33]. Ongoing violence in Syria has corresponded with infrastructural instability and chaotic population migration. It has created a setting in which we have seen the re-emergence of polio and measles, as well as tuberculosis, hepatitis A, and other infections in Syria and among displaced Syrian refugees [30,39]. However, the pathognomonic and obvious clinical features of Old World CL caused by both L. tropica and L. major possibly make it the most visible sign of disease emerging under the current circumstances. Old World CL and the Current Syrian Crisis With settings enabling transmission of Old World CL as a backdrop, the number of new cases has continued to rise. Within Syria, a 2013 study published by the Ministry of Health reported an incidence rate more than twice as high as the incidence rate reported in Syria between 2004 and 2008 by the WHO. The annual incidence of CL in Syria between 2004 and 2008 was estimated to be 23,000 cases per year [40]. In 2012, 53,000 cases were reported, and in the first half of 2013 alone, 41,000 cases were reported [41]. Additionally, the number of cases of CL has most likely been severely underreported [42]; the WHO estimated that the actual incidence of CL in Syria between 2004 and 2008 was three to five times higher than the reported incidence [40]. The true number of annual incident and prevalent cases in Syria may therefore exceed 100,000. Along its border with Syria, Turkey—which has taken more Syrian refugees than any country thus far [31]—has shown indications of increased prevalence of CL among already endemic existing populations in correlation with the influx of refugees from Syria [23]. Old World CL has emerged in Lebanon as well, although the outbreak to date has been largely contained to refugee populations [33,43,44]. A new report indicates that among the cases of CL observed in refugee communities in Lebanon, 85% of the cases are caused by L. tropica, with the remainder caused by L. major [33]. This may complicate treatment in the long term as L. tropica patients tend to be more refractory to the main CL drug, sodium stibogluconate (SSG) [45–47]. In the countries that have observed new cases of CL, younger age groups, due to their lack of previous exposure to the disease, have been the most affected [23,33]. Non-immune existing individuals also are at great risk of contracting CL as their immune systems are not equipped to fend off the parasite. Few countries have mandated reporting of CL [33], and the resultant weak reporting system promotes a lack of disease awareness and public policies for treatment and prevention. Compounding this problem is the absence of rapid diagnostics and the requirement to have highly skilled dermatologists and pathologists establish a diagnosis on the basis of clinical presentation and confirmatory microscopy, respectively. Even then, the sensitivity of microscopy is not particularly high (68% for L. major and 45% for L. tropica) [48]. The CL clinical presentation is also often accompanied by a wide spectrum of clinical manifestations that can mimic other inflammatory and neoplastic diseases, further complicating the diagnosis and reporting of CL [49,50]. If Old World CL is not addressed promptly, experience warns of a likely outbreak that may have unanticipated consequences if allowed to erupt. In the early 2000s, an outbreak of CL was observed after the Iraq War that spread beyond endemic populations and included foreign troops in the area [43,51]. The 1990s Afghani civil war experience also was notable for its outbreak of CL [52]. The war is estimated to have caused hundreds of thousands of CL cases in Afghanistan and among refugee populations in Pakistan [52,53]. Old World CL and Other Conflict Zones in MENA Knowledge of Old World CL in Syria and among its refugees is limited; however, we know even less about the situation in areas of Libya now controlled by the self-proclaimed Islamic State, or Daesh, and its allied extremist groups [54]. Both zoonotic and anthroponotic disease cycles have been identified in Libya; however, most of the published literature on CL in Libya focuses on the zoonotic form, which is caused by L. major. This form is responsible for the majority of CL cases in Libya, with Ph. papatasi as the main vector, and Psymommys obesus (fat sand rat) and Meriones libycus (Libyan jird) reported as disease reservoirs [55]. However, L. tropica anthroponotic CL has also been identified in Nalut and Bani Walid. Interestingly, both L. infantum and L. donovani have been identified in Nalut as causative for CL [55,56]. Outdoor activities like farming and construction work are highly correlated with disease emergence as a result of increasing exposure to sandfly bites [57,58]. Recently, the United Nations High Commissioner for Refugees (UNHCR) reported that 363,067 individuals have been displaced in Libya due to the ongoing unrest [59]. Re-emergence of CL from mass displacement could occur in areas that have had experience with CL, including Siret, Nalut, Garyan, Bani Walid, Kikla, and Ghudamis. Furthermore, about one million Libyan refugees have been displaced to Tunisia. Anecdotal reports from Tunisia, where refugee camps have been established [60], indicate that cases of leishmaniasis are on the rise, but there is minimal, if any, documentation. Leishmaniasis is a hidden NTD in Yemen as well. Approximately 10,000 new cases are reported annually [61]. These cases are caused by both L. tropica and L. infantum in high altitude regions, including Sa’da, Amr’an, Al Bayda, Ibb, Al Dhale’a, Dhamar, and Sana’a [62,63]. Furthermore, L. donovani, L. tropica, and L. infantum cause CL in regions that belong to the Tihama Coastal Plain, such as Al Hudaydah, Hajjah, and Ta’izz [62]. The Regional Leishmaniasis Control Centre (RLCC) reports that half of the clinically resembling CL cases are mucocutaneous leishmaniasis and that Yemeni CL patients suffer from both shortage of CL treatment and inadequate response to treatment [64]. Moreover, the access to health care has been reduced significantly due to conflict in Yemen and absence of aid. As CL in Yemen is thought to be caused exclusively by an anthroponotic cycle, the disease prevalence will likely increase as the rubbish accumulation and lack of sewage system foster the perfect breeding sites for Ph. sergenti vector. Although no refugee camps have been deployed as a result of the current Yemeni conflict, many people are migrating to neighboring countries such as Saudi Arabia, which may lead to the spreading of anthroponotic CL in the southern Saudi regions. The situations in Libya and Yemen will need further monitoring. Discussion and Preliminary Recommendations Areas of conflict provide for complex circumstances that make accurate data collection and humanitarian aid inaccessible and impractical. Additionally, there is ongoing dialogue about the efficacy of humanitarian aid in areas of conflict [65]. Especially with the loss of governmental control in many areas of Syria, Iraq, and parts of Libya to Daesh, policy recommendations are nearly impossible to implement in many regions of MENA [2]. Despite the difficulties of navigating current geopolitical circumstances in MENA, more can be done to address the CL situation in this region. Interventions to prevent and control the spread of CL must be multilateral in dimension and specific to local circumstances [66]. The following list of preliminary recommendations to prevent and control CL outbreaks highlights general policies that have already been proposed by organizations such as the WHO and the Centers for Disease Control [66–68], and emphasizes refugee camps and communities of displaced individuals living in regions of stability in MENA. The utmost priority of all interventions is to do no harm. Continued improvement of living conditions and hygiene infrastructure for refugees. Clean water, food, and sanitation services aid basic survival while also aiding the prevention and control of CL among endemic refugee populations. Implementation of mobile teams in refugee camps consisting of medical professionals experienced in diagnosing and treating CL. Responsibilities for mobile teams would include disease (and vector) detection, active surveillance, and providing health care treatment for patients with CL. Treatment includes sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime), as well as alternatives therapies, such as cryotherapy, if there is a treatment shortage. Collection of health impact assessments prior to the establishment of refugee camps in neighboring areas of conflict zones. For example, the extermination of animal reservoirs before settling displaced individuals can help avoid emerging outbreaks among refugee communities [7]. Implementation of services to address psychological and economic impacts of CL. The most devastating consequences of contracting CL are often socioeconomic and psychological. Initiatives addressing community stigma surrounding skin lesions and papules associated with CL. Additional educational programs in refugee communities to raise awareness of CL also may be beneficial in preventing outbreaks. Distribution of insecticide treatment, particularly in areas known to be endemic with anthroponotic cycle, to help prevent contagion. A recent Cochrane analysis has concluded that insecticides may be effective at reducing the incidence of CL; however, whether insecticides are best applied through indoor spraying, treatment of clothing and bed sheets, or use of nets remains undetermined [69]. Research and development to improve diagnosis, treatment, and prevention methods, as well as ongoing operational research, monitoring, and evaluation to confirm the effectiveness of existing approaches. All research and development initiatives should give due considerations to ethical issues of working with refugee populations. The full extent of the Old World CL epidemic in Syria and in bordering countries, as well as in Libya and Yemen, remains mostly unknown. An adequate disease burden analysis depends on programs of active surveillance and disease detection, but these are few and far between due to the violence and instability. We may be witnessing an epidemic of historic and unprecedented proportions, but it has largely been hidden due to lack of specific information. The biggest limitation of this paper is the inability to access data due to the difficulties of gathering accurate and current information from regions of instability. Surveillance is even more challenging in the current refugee crises due to the unprecedented magnitude of population migration. The most effective policies in addressing the potentially devastating CL situation that is emerging from some conflict zones in MENA are initiatives that will promote disease control while simultaneously promoting the survival of refugees. Provisions of clean water, food, hygiene services, and adequate shelter will improve the living conditions of refugees while simultaneously addressing many of the socioeconomic and environmental risk factors that make refugees highly susceptible to infectious diseases. For example, makeshift houses allow sandflies to come in close proximity to human beings and the lack of municipal services creates conditions that facilitate poor health outcomes. Recommendations for research include the development of improved rapid diagnosis tests, possibly similar to the point-of-care diagnostic tests under development for VL [70]. Currently, diagnoses are performed by specialized dermatologists and can only be confirmed by a stained smear or culture from a skin lesion, which require laboratory settings. The lack of a rapid diagnosis test slows the process of diagnosis and leads to delayed treatment and greater risk for misdiagnosis of CL. Development of a commercially available vaccine for Old World CL should also be made a priority, as one does not currently exist even though it would enhance efficacy of disease and vector control programs [71,72]. A recent analysis confirms the cost-effectiveness for a vaccine that targets either New World CL [72] or Old World VL [73]. Additionally, research assessing how best to address the socioeconomic and psychological impacts of CL on patients as well as the cultural stigma of papules left by CL would facilitate a more well-rounded approach to confronting the consequences of CL outbreaks. These research projects should be specific to the dynamics of local communities and cultures. Micro-financing programs may alleviate some of the economic hardships often associated with CL; however, the feasibility of micro-financing programs in conflict-affected communities is still being debated [74]. A multifaceted, collaborative approach must be taken to control the incidence of CL [7,75], with priority given to initiatives that will not only aid in the prevention and control of CL but also improve the living conditions and survival of refugee populations. The World Health Assembly already adopted a resolution in 2007 to address the global burden of leishmaniasis [67], but immediate action must be taken to address the spreading burden of CL in the Middle East. By no fault of their own, refugees and displaced individuals are often fleeing from one unimaginable circumstance of horror and violence to another of poverty and disease. International communities have a responsibility to pay greater attention to this pressing issue, and it is imperative that proactive measures are taken to establish efficient and sustainable initiatives aimed at diagnosing, treating, and preventing CL as the conflicts in Syria, Iraq, Libya, and Yemen continue.

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          Complexities of Assessing the Disease Burden Attributable to Leishmaniasis

          Among parasitic diseases, morbidity and mortality caused by leishmaniasis are surpassed only by malaria and lymphatic filariasis. However, estimation of the leishmaniasis disease burden is challenging, due to clinical and epidemiological diversity, marked geographic clustering, and lack of reliable data on incidence, duration, and impact of the various disease syndromes. Non-health effects such as impoverishment, disfigurement, and stigma add to the burden, and introduce further complexities. Leishmaniasis occurs globally, but has disproportionate impact in the Horn of Africa, South Asia and Brazil (for visceral leishmaniasis), and Latin America, Central Asia, and southwestern Asia (for cutaneous leishmaniasis). Disease characteristics and challenges for control are reviewed for each of these foci. We recommend review of reliable secondary data sources and collection of baseline active survey data to improve current disease burden estimates, plus the improvement or establishment of effective surveillance systems to monitor the impact of control efforts.
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            Outbreak of Ebola Virus Disease in Guinea: Where Ecology Meets Economy

            Ebola virus is back, this time in West Africa, with over 350 cases and a 69% case fatality ratio at the time of this writing [1]. The culprit is the Zaire ebolavirus species, the most lethal Ebola virus known, with case fatality ratios up to 90%. The epicenter and site of first introduction is the region of Guéckédou in Guinea's remote southeastern forest region, spilling over into various other regions of Guinea as well as to neighboring Liberia and Sierra Leone (Figure 1). News of this outbreak engenders three basic questions: (1) What in the world is Zaire ebolavirus doing in West Africa, far from its usual haunts in Central Africa? (2) Why Guinea, where no Ebola virus has ever been seen before? (3) Why now? We'll have to wait for the outbreak to conclude and more data analysis to occur to answer these questions in detail, and even then we may never know, but some educated speculation may be illustrative. 10.1371/journal.pntd.0003056.g001 Figure 1 Map of the three countries (Guinea, Liberia, and Sierra Leone) involved in the 2013–2014 outbreak of Ebola virus disease as of June 20, 2014. The putative first virus introduction and epicenter are in the vicinity of the town of Guéckédou in the Guinea Forest Region. CDC: http://www.cdc.gov/vhf/ebola/resources/distribution-map-guinea-outbreak.html. The Ebolavirus genus is comprised of five species, Zaire, Sudan, Taï Forest, Bundibugyo, and Reston, each associated with a consistent case fatality and more or less well-identified endemic area (Figure 2). Zaire ebolavirus had been previously found only in three Central African countries—the Democratic Republic of the Congo, Republic of the Congo, and Gabon. Thus, the logical assumption when Ebola virus turned up in Guinea was that this would be the Taï Forest species previously noted in Guinea's neighbor, Côte d'Ivoire. 10.1371/journal.pntd.0003056.g002 Figure 2 African countries where endemic transmission of Ebola virus has been noted. How did Zaire ebolavirus get all the way over to West Africa? The two possibilities appear to be that the virus has always been present the region, but we just never noticed, or that it was recently introduced. The initial report and phylogenetic analyses on the Guinea outbreak suggested that the Zaire ebolavirus found in Guinea is a distinct strain from that noted in Central Africa [1], thus suggesting that the virus may not be a newcomer to the region. However, subsequent reworking and interpretations of the limited genetic data have cast some doubt on this conclusion [2]. If Zaire ebolavirus had been circulating for some time in Guinea, one might expect greater sequence variation than the 97% homogeneity noted relative to that isolated from Central Africa [1]. Phylogenetic arguments aside, if Ebola virus was present in Guinea, wouldn't we have seen cases before? Not necessarily. Many pathogens may be maintained in animals with which humans normally have little contact, thus providing limited opportunity for infection. Furthermore, the proportion of infected animals may often be very low, so even frequent contact may not result in pathogen transmission. Even if human Ebola virus infection has occurred, it may not be recognized; contrary to popular concept, the clinical presentation of viral hemorrhagic fever is often very nonspecific, with frank bleeding seen in a minority of cases, so cases may be mistaken for other, more common diseases or, in the case of Guinea, Lassa fever, which is endemic in the area of the outbreak [3]. Nor are laboratory diagnostics routinely available in West Africa for most viral hemorrhagic fevers [4]. Ebola virus testing of human serum samples collected as far back as 1996 as part of surveillance for Lassa fever in the same region as the current outbreak could help reveal whether humans had exposure to Ebola virus prior to this outbreak [3]. We are presently organizing with collaborators to conduct ELISA antigen testing, PCR, and cell culture for Ebola virus on samples from persons who met the case definition for viral hemorrhagic fever but tested negative for Lassa fever. We will also test all samples for IgG antibody to Ebola virus to explore the prevalence of past exposure. Could Zaire ebolavirus have been recently introduced into Guinea from Central Africa? Introduction from a human traveler seems unlikely; there is little regular travel or trade between Central Africa and Guinea, and Guéckédou, the remote epicenter and presumed area of first introduction, is far off the beaten path, a minimum 12 hour drive over rough roads from the capitals of Guinea, Liberia, or Sierra Leone (Figure 1). Furthermore, with the average incubation period as well as time from disease onset until death in fatal cases both a little over a week, a human traveler would have to make the trip from Central Africa to Guéckédou rather rapidly. If Ebola virus was introduced into Guinea from afar, the more likely traveler was a bat. Although a virus has not yet been isolated, PCR and serologic evidence accumulated over the past decade suggests that fruit bats are the likely reservoir for Ebola virus. The hammer-headed fruit bat (Hypsignathus monstrosus), Franquet's epauletted fruit bat (Epomops franqueti), and the little collared fruit bat (Myonycteris torquata) are among the leading candidates [5]–[9]. Many of these species are common across sub-Saharan Africa, including in Guinea, and/or may migrate long distances, raising the possibility that one of these wayward flyers may have carried Ebola virus to Guinea [8]. Introduction into humans may have then occurred through exposures related to hunting and consumption of fruit bats, as has been suspected in Ebola virus outbreaks in Gabon [8]. Similar customs have been reported in Guinea, prompting the Guinean government to impose a ban on bat sale and consumption early on in the outbreak. Field collections and laboratory testing for Ebola viruses of bats collected from the Guinea forest region should shed light on the presence or absence of these various species in the area and possible Ebola virus infection. Indeed, a team of ecologists is already on the ground beginning this work. But why Guinea and why Guéckédou? Certainly this is not the only place bats migrate. Unfortunately, Ebola virus outbreaks typically constitute yet another health and economic burden to Africa's most disadvantaged populations. Despite the frequently promulgated image of Ebola virus mysteriously and randomly emerging from the forest, the sites of attack are far from random; large hemorrhagic fever virus outbreaks almost invariable occur in areas in which the economy and public health system have been decimated from years of civil conflict or failed development [10]–[13]. Biological and ecological factors may drive emergence of the virus from the forest, but clearly the sociopolitical landscape dictates where it goes from there—an isolated case or two or a large and sustained outbreak. The effect of a stalled economy and government is 3-fold. First, poverty drives people to expand their range of activities to stay alive, plunging deeper into the forest to expand the geographic as well as species range of hunted game and to find wood to make charcoal and deeper into mines to extract minerals, enhancing their risk of exposure to Ebola virus and other zoonotic pathogens in these remote corners. Then, the situation is compounded when the unlucky infected person presents to an impoverished and neglected healthcare facility where a supply of gloves, clean needles, and disinfectants is not a given, leaving patients and healthcare workers alike vulnerable to nosocomial transmission. The cycle is further amplified as persons infected in the hospital return to their homes incubating Ebola virus. This classic pattern was noted in Guinea, where early infection of a healthcare worker in Guéckédou triggered spread to surrounding prefectures and eventually to the capital, Conakry [1]. Lastly, with an outbreak now coming into full force, inefficient and poorly resourced governments struggle to respond, as we are seeing all too clearly with this outbreak of Ebola virus disease in West Africa, which is now by far the largest on record. The response challenge is compounded in this case by infected persons crossing the highly porous borders of the three implicated countries, requiring intergovernmental coordination, with all the inherent logistical challenges in remote areas with poor infrastructure and communication networks and, in this case, significant language barriers. Guinea, Liberia, and Sierra Leone, sadly, fit the bill for susceptibility to more severe outbreaks. While the devastating effects of the civil wars in Liberia and Sierra Leone are evident and well documented, readers may be less familiar with the history of Guinea, where decades of inefficient and corrupt government have left the country in a state of stalled or even retrograde development. Guinea is one of the poorest countries in the world, ranking 178 out of 187 countries on the United Nations Development Programme Human Development Index (just behind Liberia [174] and Sierra Leone [177]). More than half of Guineans live below the national poverty line and about 20% live in extreme poverty. The Guinea forest region, traditionally comprised of small and isolated populations of diverse ethnic groups who hold little power and pose little threat to the larger groups closer to the capital, has been habitually neglected, receiving little attention or capital investment. Rather, the region was systematically plundered and the forest decimated by clear-cut logging, leaving the “Guinea Forest Region” largely deforested (Figure 3). 10.1371/journal.pntd.0003056.g003 Figure 3 The area known as the Guinea Forest Region, now largely deforested because of logging and clearing and burning of the land for agriculture. Photo credit: Daniel Bausch. The forest region also shares borders with Sierra Leone, Liberia, and Cote d'Ivoire, three countries suffering civil war in recent decades. Consequently, the region has found itself home to tens of thousands of refugees fleeing these conflicts, adding to both the ecologic and economic burden. A United Nations High Commission for Refugees census of camps in the forest region in 2004 registered 59,000 refugees. Although the formal refugee camps have now been dismantled with improved political stability in the surrounding countries, the impact on the region is long lasting. Having worked in Guinea for a decade (1998–2008) on research projects based very close to the epicenter of the current Ebola virus outbreak, one of the authors (DGB) witnessed this “de-development” first-hand; on every trip back to Guinea, on every long drive from Conakry to the forest region, the infrastructure seemed to be further deteriorated—the once-paved road was worse, the public services less, the prices higher, the forest thinner (Figures 3 and 4). 10.1371/journal.pntd.0003056.g004 Figure 4 Scenes of the degraded infrastructure of the Guinea forest region. A. Once-paved, but now deteriorated road; B, C, and D. Street views of the dilapidated town of Guéckédou, the epicenter of the Ebola virus disease outbreak. Photos credit: Frederique Jacquerioz. Guinea fell further into governmental and civil disarray after former president Lansana Conté's death in 2008 left a power vacuum, with a series of coup d'états and periods of violence. Although the political situation has now somewhat stabilized, the country struggles to progress; socioeconomic indicators such as life expectancy (56 years) and growth national income (GNI) per capita ($440) have crept up in the past few years, but still remain disparagingly low. Despite a wealth of mineral and other natural resources, Guinea still possesses the eighth lowest GNI per capita in the world, and the incidence of poverty has been steadily increasing since 2003. Lastly, why is this outbreak of Ebola virus happening now? As best as can be determined, the first case of Ebola virus disease in Guinea occurred in December 2013, at the beginning of the dry season, a finding consistent with observations from other countries that outbreaks often begin during the transition from the rainy to dry seasons [14]–[18]. Sharply drier conditions at the end of the rainy seasons have been cited as one triggering event [17]. Although more in-depth analysis of the environmental conditions in Guinea over the period in question remain to be conducted, inhabitants in the region do indeed anecdotally report an exceptionally arid and prolonged dry season, perhaps linked to the extreme deforestation of the area over recent decades. At present, we can only speculate that these drier ecologic conditions somehow influence the number or proportion of Ebola virus–infected bats and/or the frequency of human contact with them. The precise factors that result in an Ebola virus outbreak remain unknown, but a broad examination of the complex and interwoven ecology and socioeconomics may help us better understand what has already happened and be on the lookout for what might happen next, including determining regions and populations at risk. Although the focus is often on the rapidity and efficacy of the short-term international response, attention to these admittedly challenging underlying factors will be required for long-term prevention and control.
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              Unresponsiveness to Glucantime Treatment in Iranian Cutaneous Leishmaniasis due to Drug-Resistant Leishmania tropica Parasites

              Introduction The protozoan parasite Leishmania is responsible for several pathologies collectively known as leishmaniases. The cutaneous, mucocutaneous, and visceral leishmaniases are caused by different Leishmania species and affect several million individuals worldwide [ 1– 3]. The treatment of leishmaniasis in endemic areas relies on chemotherapy, and in several parts of the world the mainstay remains the pentavalent antimony (SbV)–containing drugs Pentostam and Glucantime. Second-line drugs such as pentamidine or liposomal amphotericin B are less useful owing to problems associated with either toxicity or cost [ 2]. The proven clinical efficacy of the oral drug miltefosine was a major breakthrough for anti -Leishmania chemotherapy and has become the first-line drug in north-east India where unresponsiveness to SbV drugs in visceral Leishmania donovani is epidemic [ 4, 5]. Unresponsiveness to SbV may be due to several factors, but in one study it was shown that unresponsiveness in L. donovani isolates was correlated with decreased susceptibility to SbV when tested in an intracellular in vitro assay [ 6]. The emergence of SbV-resistant parasites was expected in north-east India because of the high endemicity of the parasite, the high proportion of treated individuals, and the nature of the anthroponotic (man to man) transmission cycle of L. donovani [ 1]. Both cutaneous and visceral forms of leishmaniasis are endemic in different parts of Iran [ 7, 8]. Anthroponotic cutaneous leishmaniasis (ACL) caused by Leishmania tropica and zoonotic cutaneous leishmaniasis (ZCL) are endemic in many parts of Iran with a high incidence rate [ 9– 12]. Glucantime is the first-line drug for the treatment of all forms of leishmaniasis in Iran. Patients with cutaneous leishmaniasis (CL) lesions not responding to SbV treatment have been reported [ 13], and these patients may require alternative treatments . In 2002, an outbreak of ACL occurred in Mashhad, north-east Iran, where almost 4,900 CL cases have been detected by clinical and parasitological methods (Khorassan Health Centers Reports 2000–2002). The results of a cross-sectional study in this area showed that 94.2% of isolates were L. tropica [ 14] and that almost 12% of the patients did not respond to treatment with meglumine antimonate (M. Mohebali, unpublished data). The objective of this study was to determine whether clinical unresponsiveness to SbV in patients with ACL was correlated to parasites with decreased susceptibility to SbV. Methods Study Design Patients of between 20 and 45 y of age and residing in Mashhad (an endemic area of ACL) who were willing to participate were included in the study. The study was approved by the Institutional Ethical Committee of the School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences. CL was confirmed by parasitological procedures, including observation of the amastigotes under light microscopy with high magnification (×1,000) and their cultivation in specific culture media. Pregnant and lactating patients were excluded from this study. Patients with previous CL history were also excluded. From the 248 patients that were initially included in the study, 185 isolates of Leishmania species were collected from skin lesions ( Figure 1). Lesions were in general non-ulcerative (71%), and 63% of patients had single lesions. Patients were treated according to the physician's and/or the patient's decision either systemically, intralesionally, or, for a small number of patients, with both regimens ( Figure 1). The national protocol for the treatment of ACL is systemic SbV (20 mg/kg/d for 14 d), and noncompliance to this protocol is mostly induced by the patient's wish. No interference with common practice implemented in the treatment strategy was made through this study. Successful treatment was defined as complete re-epithelialization of all lesions with no relapse within 6 mo of follow up. Parasites were also collected from skin lesions which did not respond to treatment ( n = 20, see Figure 1). Patients were classified as nonresponders when they presented lesions at a 24-mo follow up. These patients were treated with a second course of Glucantime. Parasites and Culture Parasites were isolated from skin lesions as described [ 15] and grown in NNN medium and subcultured in RPMI 1640 medium supplemented with 15% fetal calf serum (FCS). Identification of Leishmania parasites was attempted by PCR typing using a random amplified polymorphic DNA (RAPD) technique [ 8, 14] following a published protocol [ 16]. Selected primers for these studies were A4, 5′ AATCGGGCTG; AB1–07, 5′ GGTGACGCAG; 327, 5′ ATACGGCGTC; and 329, 5′ GCGAACCTCC. Species identification of Leishmania isolates was further studied by sequencing the PTR1 gene, a metabolic gene involved in pterin and folate metabolism in Leishmania [ 17, 18]. The PTR1 coding region of each field isolate was amplified by PCR using the primers 5′ CTGCTCCGACCGTGCC and 5′ CCCGGGTAAGGCTGTAGC. The expected 800-bp amplified product was purified and sequenced. The sequences were compared with sequences of L. tropica, L. major, L. tarentolae, and L. donovani reference isolates using the ClustalW program ( http://www.ebi.ac.uk/clustalw) and the Molecular Evolutionary Genetics Analysis (MEGA3) software ( http://www.megasoftware.net). Pulsed-Field-Gel Karyotyping Agarose blocks containing Leishmania cells were prepared as described [ 19]. Briefly, cells were resuspended in HEPES buffer at a density of 1 × 10 8 cells/ml and mixed with low-melting-point agarose. Cells were lysed in the presence of 0.5 M EDTA (pH 9.5), 1% SLS, and proteinase K, and the chromosomes of Leishmania cells were resolved by a BioRad (Hercules, California, United States) contour-clamped homogeneous electric field (CHEF) mapper for separating 0.2–2.0-Mbp DNAs over a period of 28 h. Chromosomes were revealed by ethidium bromide staining. Susceptibility Testing Mouse peritoneal macrophages The macrophages of peritoneal fluid of BALB/c mice were collected and resuspended at 5 × 10 4/ml in RPMI 1640 supplemented with 15% FCS, as described by others [ 6]. Cells were plated in eight-chamber LabTek tissue-culture slides, and adherent macrophages were infected with late-logarithmic promastigote parasites at a parasites-to-macrophage ratio of 4:1. After 2 h of incubation at 34 °C, free promastigotes were removed and Glucantime was added. Each 5-ml ampoule of Glucantime contained 1.5 g meglumine antimoniate corresponding to 0.405 g of pentavalent antimony. The tissue-culture slides were incubated for 3 d, fresh Glucantime was added, and the slides were incubated for an additional 72 h. The slides were fixed and stained with Giemsa [ 20]. Three slides were used for each isolate. The percentage of infected macrophages and the number of parasites per infected cell were evaluated by microscopic examination of at least 100 macrophages. The EC 50 is defined in this study as the concentration of meglumine antimoniate that reduces the survival of Leishmania parasites by 50%. These studies were approved by the Animal Committees of the School of Public Health and Institute of Public Health Research, and of the Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences. THP1 cell line A number of sensitive and resistant parasites, as determined by the mouse peritoneal macrophage assay, were selected and used for the in vitro susceptibility assay in the human monocyte cell line THP1 [ 21]. Recombinant parasites were produced by transfection of the Leishmania isolates with the firefly luciferase–containing vector pSP1.2 LUC αHYGα [ 22] and were used to infect THP1 cells (ratio 20:1) stimulated with phorbol myristic acetate (PMA) as described [ 21]. Luciferase activity was measured after 5 d of incubation in the presence of Glucantime. Previous in vitro and in vivo work demonstrated an excellent correlation between luciferase activity and parasite number [ 20, 21, 23]. Susceptibility to trivalent antimony was also determined in the promastigote stage of the parasites, as described elsewhere [ 24] by measuring the optical density of cultures at 600 nm. Results Unresponsiveness to Glucantime Treatment Out of the 248 patients with parasitological proven CL and with no previous history of Glucantime treatment, we grew 185 parasite isolates. The patients were treated either systemically or intralesionally. The two treatment regimens appeared to be equally effective, with no relapse within the first 6 mo after treatment for the majority of patients ( Figure 1). Two responding patients showed relapse after 12 mo, although no work was carried out to determine whether this represented a true relapse or a new infection. Ten patients were first treated intralesionally, but owing to the pain induced by intralesional injection, the patients preferred to receive systemic SbV and therefore received a round of systemic SbV ( Figure 1). Overall, 10.8% of patients (20 out of 185) did not respond to the first course of SbV treatment ( Figure 1). A slightly higher, but not significant, proportion of nonresponding patients were seen in the groups receiving intralesional treatment ( Figure 1). The goal of this study was to determine whether unresponsiveness to Glucantime is due to the presence of resistant parasites. The EC 50 of the 185 isolates derived from lesions before initiation of therapy was first measured using the peritoneal mouse-derived macrophage model [ 6]. Similar infection rates were observed between parasites derived from responsive (91.3 ± 12.9 amastigotes/100 untreated macrophages) and unresponsive (83.7 ± 8.0 amastigotes/100 untreated macrophages) patients. The results showed a strong correlation between the clinical outcome and susceptibility values. Indeed, the 165 patients who responded to SbV treatment were infected with parasites with EC 50 values of less than 10 μg/ml ( p 5 mM—hence precluding the selection of transfectants using hygromycin. Properties of the resistance phenotype It is suspected that long-term in vitro growth of patient-derived resistant parasites may lead to a loss of the resistance phenotype, although antimony resistance is stable in parasites selected for resistance in vitro [ 26, 27] or in Indian field isolates [ 28]. The 20 L. tropica strains with intermediate or high resistance levels to Glucantime were grown for at least 20 passages as promastigotes in the absence of drug selection (except for hygromycin B to keep the luciferase marker). A susceptibility assay was carried out in the THP1 cell line, and no decrease in the EC 50 was observed in these cells compared to cells with a minimum number (between three and five) of passages ( Table 3; unpublished data). While SbV is the drug used against leishmaniasis, there is a general consensus that the active form of the metal is SbIII (reviewed in [ 29]). This metal reduction could occur both in the macrophage [ 30, 31] and in the parasite [ 32– 34]. Promastigote cells selected for SbIII resistance are cross-resistant to SbV as intracellular parasites [ 27, 31]. Leishmania parasites selected for SbV resistance are sometimes [ 35, 36], but not always [ 32], cross-resistant to SbIII. We therefore tested the level of susceptibility of the promastigote stage of ACL clinical isolates to SbIII. In some groups of strains (e.g., PFGE group I), the intracellular EC 50 values toward SbV, and SbIII cross-resistance values as determined in promastigotes, did correlate ( Table 3). For other strains of different PFGE groups, however, there was a lack of correlation between intracellular SbV and promastigote SbIII susceptibilities. The L. major isolates studied were completely insensitive to SbIII as promastigotes ( Table 3). Several potential mechanisms of resistance to antimonials have been proposed based on work on cells selected for resistance in vitro (reviewed in [ 29]). Intracellular thiols including the glutathione-spermidine conjugate trypanothione [ 26, 37] are important molecules involved in resistance. It has been shown that inhibiting glutathione biosynthesis, a backbone of trypanothione, can lead to antimony resistance reversal in vitro in promastigotes [ 26] or intracellular amastigotes [ 27], as well as in vivo [ 38]. We have shown that buthionine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase (which is the rate-limiting step of glutathione biosynthesis), can reverse resistance to Glucantime not only in the highly resistant L. tropica isolate 827 ( Figure 5; Table 2) but also in three other studied resistant strains with ID Nos 439, 670, and 878, respectively (unpublished data). Discussion Antimonials have been the pillar of anti- Leishmania chemotherapy for more than 60 y. Despite its toxicity, the drug has remained effective and available in endemic countries although, in order to maintain its effectiveness, the drug concentration and duration of therapy have had to increase over the years [ 39], suggesting that the parasites have been slowly acquiring mutations leading to reduced susceptibility to the drugs. However, other factors, such as reduced ability of the human-host immune system to fight the parasites, pharmacological deficiencies, or under-treatment, could also lead to treatment failure [ 40, 41]. In the 1990s, the failure of SbV treatment in north-east India became epidemic [ 4], and in one study it was shown that part of the unresponsiveness in the L. donovani Indian isolates was due to parasites with decreased susceptibility to SbV when tested as intracellular parasites [ 6]. Indeed, there is a general consensus that to be of some relevance, in vitro susceptibility testing has to be done with intracellular stages of Leishmania [ 42– 44]. While unresponsiveness to SbV is high in part of India, it is still an effective treatment in several other parts of the world, and it is still the drug of choice for the treatment of ACL in Iran [ 13]. The ACL cycle caused by L. tropica is well established in Iran [ 7, 11, 45], and this human-to-human mode of transmission could facilitate the emergence of parasites less susceptible to SbV. Indeed, anecdotal evidence indicates that an increasing proportion of patients with ACL are failing SbV therapy. In order to test whether resistant isolates exist, we measured the EC 50 of 185 isolates derived from patients before initiation of treatment. We found a strong correlation between the clinical outcome and the susceptibility levels of parasites ( Table 1). This correlation was highly significant ( p < 0.01) as determined by a series of statistical analyses. L. major isolate No. 105, a species usually associated with ZCL, was also found to be resistant to Glucantime ( Table 2). A correlation between susceptibility testing and clinical outcomes, with few outliers, has also been found for L. donovani isolates [ 6] and for L. infantum [ 44]. For the L. infantum isolates, however, this correlation stands only for the short-term outcome and not for the longer-term outcome, possibly because the majority of patients included in this study were infected with HIV-1 [ 44]. The parasites derived from non-healing patients were characterized and were found to be closely related to parasites derived from susceptible patients ( Figure 3; Table 2). To our knowledge, this is the first detailed demonstration that resistant and sensitive clinical Leishmania isolates are related and the first proven case of ACL treatment failure due to resistant parasites, strongly pointing to acquired drug resistance in these isolates. Susceptibility data suggest at least two resistance mechanisms, one leading to an intermediate level of resistance (as found in the majority of the isolates) and the other leading to a high level of resistance. It remains to be explored whether intermediate resistance is a necessary step before achieving high resistance, or whether the mutations arise independently. If resistance was at one point associated with a fitness cost, it would appear that the parasite did compensate for it, since its infectiousness to macrophages was found to be comparable to its survival as an intracellular parasite. Whatever the nature of the mutation, it appears (at least in some cells) to be stable, since parasites grown for more than 20 passages in the absence of drugs still kept the same level of resistance. Similar conclusions about infectivity of resistant cells and stability of the resistance phenotype were also reached for L. donovani clinical isolates [ 28, 46]. The stability of the resistance phenotype would rule against gene-amplification events, as amplicons present in drug-resistant parasites are usually rapidly lost during growth of the parasites in the absence of selection [ 19]. Consistent with this premise, we failed to detect gene amplification in the resistant isolates (R. Hadighi and M. Ouellette, unpublished data) when using a number of techniques enabling the detection of amplified genes [ 47]. Modulation in RNA levels are also commonly associated with drug resistance [ 27, 48– 51]. These changes in RNA expression are usually stable, and this type of change or a point mutation could possibly explain the resistance phenotype found in resistant isolates. To correlate treatment outcome with susceptibility of the parasites, we used two models, i.e., the mouse-derived macrophages and Giemsa staining and a human monocyte cell line (THP1) and luciferase-expressing parasites. An excellent correlation was found between the two models ( Tables 2 and 3). The second assay system has obvious advantages as it neither requires animals nor involves the labor-intensive parasite staining and counting [ 20, 21]. The required transfection of the parasites is now a relatively straightforward technique that does not appear to modify the properties of the parasite—at least the ones related to macrophage infection and drug susceptibility ( Tables 2 and 3). Comparable results of susceptibility testing using transfected parasites were also obtained for L. donovani field isolates [ 28, 46, 52]. The susceptibility of promastigotes to SbIII did not correlate well with intracellular SbV susceptibility data ( Table 3). This could be due to either different resistance mechanisms, some giving cross-resistance to SbIII, or to varying levels of intrinsic resistance. In addition, this simpler SbIII-susceptibility assay could not be used to predict clinical outcome. Our report describes, in detail, several L. tropica and one L. major isolates that appear to have acquired mutations leading to decreased susceptibility to SbV and to unresponsiveness towards clinical therapy. It is salient to point out that four out of the 20 primary unresponsive patients responded to a second course of Glucantime treatment. All four harbored parasites with intermediate levels of resistance (EC 50 10–25 μg/ml). The next challenge will be to determine the exact molecular mechanisms of resistance. It is possible that, as in the in vitro isolates, thiols are important for the resistance phenotype ( Figure 5). Indeed, preliminary results suggest that glutathione levels are higher in some resistant isolates (G. Roy and M. Ouellette, unpublished data). This suggests a strategy of using drug combinations to reverse drug resistance in the field. Ongoing studies should lead to a better understanding of the molecular mechanisms of antimony resistance in field isolates. The equation between chemotherapeutic resistance (either in infectious diseases or oncology), as determined by in vitro testing, and treatment failure is intuitively expected but in some occasions this is still unclear. For example, in the case of pneumonia caused by Streptococcus pneumoniae, often in vitro findings do not appear to be predictive of in vivo outcome, except with the most highly resistant bacteria (reviewed in [ 53]). However, several reports indicate that an increase in drug resistance is associated with an increased treatment failure (reviewed in [ 54, 55]). Failure in SbV treatment in Leishmania has been attributed to several factors other than resistant parasites, including host immunological status, suboptimal treatment, and pharmacokinetic properties [ 40, 41], but it is now clear that an increase in resistance can lead to treatment failure for both visceral leishmaniasis [ 6] and CL. Future work should lead to a precise understanding of the resistance mechanisms in field isolates, which should lead to strategies for rapidly diagnosing resistance in order to improve the clinical management of leishmaniasis. Supporting Information Alternative Language Abstract S1 Translation of the Abstract into French by Marc Ouellette (23 KB DOC) Click here for additional data file. Alternative Language Abstract S2 Translation of the Abstract into Farsi by Ramtin Hadighi (53 KB DOC) Click here for additional data file. Patient Summary Background Leishmaniases are a group of diseases caused by different species of the Leishmania group of parasites. The diseases are common in many tropical and subtropical areas, where patients are infected through the bite of sandflies that had previously bitten an infected animal or human. The most common form of the disease is cutaneous leishmaniasis, characterized by skin ulcers that take months to heal and often leave ugly and disabling scars. They can also lead to secondary, more dangerous, infections. In most parts of the world, so-called pentavalent antimonials are the drug of choice. Over the past few years, these drugs are becoming less and less effective against the disease, with increasing numbers of patients not responding, showing only partial recovery, or needing higher doses of the drugs to get better. A few additional drugs are now available and are being developed, but some are expensive and others have serious side effects. Why Was This Study Done? There are several possible explanations as to why the pentavalent antimonials are becoming less effective: parasites could have become increasingly resistant to the drugs, the patients' immune systems could have become weaker over time, or different strains of parasites against which the drugs are not as effective could be responsible for more of the recent cases. It is important for health officials to know which of these apply when they decide on strategies to improve the situation. Results from north-east India, where L. donovani is the predominant cause of the disease, suggest that the parasites are becoming resistant. The scientists who did this study wanted to find out which of these possibilities might be true in Iran, a country in which cutaneous leishmaniasis is common. What Did the Researchers Do and Find? They discussed the study with many patients with cutaneous leishmaniasis in Mashad, an area in Iran where the disease is common. Of those patients, 248 were suitable and agreed to participate. The researchers then isolated parasites from 185 different ulcerous wounds, including 20 from patients who did not respond well to treatment with pentavalent antimonials. They studied these parasites in various ways. They found that all of the parasites from the 20 nonresponsive patients were either partially or fully resistant to the drugs, meaning that either higher doses of drugs were necessary or that even very high doses could not kill the parasites. They then wanted to know which Leishmania species the responsive and nonresponsive parasites belonged to. To answer this question, they determined the genetic code for one specific gene which is known to have different versions in different species. This showed that 19 of the unresponsive isolates belonged to L. tropica and one to L. major. Two of the responsive isolates belonged to L. major, with the other nine belonging to L. tropica. Among the L. tropica isolates, the responsive and nonresponsive isolates showed some similarities at a broader genetic level. What Do These Findings Mean? The results show that L. tropica parasites can become resistant to pentavalent antimonials. They also suggest that such resistant strains are largely responsible for the decreased effectiveness of these drugs against cutaneous leishmaniasis in Iran. This means that Iran and other countries where resistance is becoming more common need to develop treatment strategies for patients who do not respond to pentavalent antimonials. For researchers, the goal is to identify the molecular mechanisms underlying drug resistance so that they can be taken into account when advising on alternative therapies and developing new drugs against the leishmaniases. Where Can I Get More Information Online? Here are listed several Web sites with information on leishmaniasis. World Health Organization: http://www.who.int/leishmaniasis/en MedlinePlus: http://www.nlm.nih.gov/medlineplus/leishmaniasis.html http://www.nlm.nih.gov/medlineplus/druginfo/uspdi/202733.html (for a description on meglutime antimoniate) Wellcome Trust Sanger Institute: http://www.sanger.ac.uk/Projects/L_major
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                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                26 May 2016
                May 2016
                : 10
                : 5
                : e0004545
                Affiliations
                [1 ]Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development, Departments of Pediatrics and Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, Texas, United States of America
                [2 ]Department of Biology, Baylor University, Waco, Texas, United States of America
                [3 ]James A. Baker III Institute, Rice University, Houston, Texas, United States of America
                [4 ]Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, England, United Kingdom
                [5 ]Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, England, United Kingdom
                Pasteur Institute of Iran, ISLAMIC REPUBLIC OF IRAN
                Author notes

                The authors have declared that no competing interests exist.

                Article
                PNTD-D-15-01885
                10.1371/journal.pntd.0004545
                4882064
                27227772
                6734d590-5866-492a-bfc8-08352b07f7a9
                © 2016 Du et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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                Figures: 2, Tables: 0, Pages: 11
                Funding
                The authors received no specific funding for this work. Waleed Al-Salem is funded by PhD studentship from the Saudi Cultural Bureau. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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