Microbiology Research 2012; volume 3:e7

Socio-economic and with fatal spontaneous evolution.1 The domes- tic dog constitutes the principal reservoir of Correspondence: Meryem Lemrani, Institut environmental factors infection and is important in zoonotic trans- Pasteur du Maroc, 1 Place Louis Pasteur, associated with Montenegro mission.2 The worldwide incidence of VL is Casablanca, . skin test positivity in an approximately 0.5 million cases per year.3 Tel. +212.661.46.48.18 - Fax: +212.522.26.09.57. E-mail: [email protected] endemic area of visceral Visceral leishmaniasis is widespread in northern Morocco (Chefchaoun, , leishmaniasis in northern Key words: asymptomatic visceral leishmaniasis, , Fes, , , , AL socio-economic factors, environmental factors, Morocco houceima and Sidi Kacem) (Figure 1). Few Montenegro skin test. epidemiological data are, however, available Salsabil Hamdi,1 Abdellah Faouzi,1 concerning its epidemiology and clinical fea- Contributions: SH carried out the project, con- Rajaa Ejghal,1 Abderahmane Laamrani,2 tures. However, a new type of cutaneous leish- ducted the field work, collected and analyzed Hamid Amarouch,3 Mohammed Hassar,1 maniasis due to Leishmania infantum was data, and prepared the final draft of the manu- 4 script; AF, RE, helped in the statistical analysis; Meryem Lemrani1 recently described. Before 1995, human vis- ceral leishmaniasis was not an obligatory noti- AL, HA, helped in the study design; MH, revised 1Laboratoire des Recherches et d’Étude fiable disease. The incidence of VL is about the manuscript; ML overviewed the study for sur les Leishmanioses, Institut Pasteur du intellectual content. 152 cases per year5 predominantly affecting Maroc, Casablanca; 2Service de children under the age of 5 years. Extension of Acknowledgments: we would like to thank the Parasitologie, DELM, Ministère de la arid zones and an increase in temperatures 3 Ministry of Health, Department of Parasitology, Santé, Rabat; Faculté des Sciences have a synergic role on the risks of an increase Direction d’Epidémiologie et de Lutte Contre les Hassan II, Ain Chock, Casablanca, in the number of cases of leishmaniasis in Maladies, the Health Delegation of the Province Morocco these regions.5 The sand fly vectors of L. infan- of Zouagha My Yacoub (Dr. Boubker Mouniem, tum in these regions are: Phlebotomus perni- Mr Dehmani), the Health Delegation of the ciosus,6 Phlebotomus longicuspis7 and P. ari- Province of Taounate (Dr Ouddiche, Mr Boumadiane) and the local authorities of the two asi.8 Dog is the main reservoir of L. infantum provinces.only Abstract in Morocco.8-10 Zymodeme MON-1 is predomi- nant in Morocco; however, zymodeme MON-24 Received for publication: 28 September 2011. In Marocco, many aspects of human asymp- has been occasionally isolated from a dog.11 Revision received: 18 January 2012. tomatic visceral leishmaniasis (VL) still have This zymodeme was considered a sporadicuseAccepted for publication: 8 February 2012. to be clarified and little information is avail- form of CL. This work is licensed under a Creative Commons Epidemiological patterns are changing in able about the factors that predispose individ- Attribution NonCommercial 3.0 License (CC BY- uals to asymptomatic infection. A prospective the Mediterranean area due to several factors, NC 3.0). study was carried out in 889 healthy children such as the widespread migration from rural to under the age of 15 years living in two urban and peri-urban areas, climatic changes ©Copyright S. Hamdi et al., 2012 provinces in the endemic area in northern increasing exposure to the sand fly and also, in Licensee PAGEPress, Italy Morocco (the provinces of Taounate and My urban areas, an increase in HIV infection. Microbiology Research 2012; 3:e7 doi:10.4081/mr.2012.e7 Yacoub) from April to May 2010. The aim of the Human activity is the leading factor in the sig- study was to evaluate the prevalence and the nificant changes recently observed in the ecol- socio-economic and environmental character- ogy and behavior of many vector-borne dis- 12 istics associated with infection by Leishmania eases. Most parasite-driven processes are or have an oligosymptomatic form of the dis- asymptomatic or subclinical; however, some infantum. The Montenegro skin test (MST) ease.21 Although the extent of this phenome- result in severe visceral diseases that are was used to detect asymptomatic infection. non has not been fully evaluated, people with- 13 The prevalence of transmission of infection lethal if not treated. It is not well understood out a definite history of disease may have evi- was 11.4% and approximately 2 times higher in why the parasite-driven processes remain dence of infection as demonstrated by a posi- Taounate than in My Yacoub, as measured by asymptomatic in certain subjects and cause a tive Delayed-Type Hypersensitivity (DTH) MST. Asymptomatic infection was associatedNon-commerciallethal disease in others. It has been suggested reaction assessed by the Leishmanin skin test, with gender, age, presence of familial links, that environmental factors that affect sand fly also called the Montenegro skin test (MST). 14 proximity to chickens, and the number of peo- ecology, human activities that increase expo- This test is currently used to assess the preva- ple in the house and locality, but it was not sure to sand flies,15 and the presence of other lence of Leishmania infection in the human associated to education status, presence of animals permissive to the Leishmania life population.22 The MST is considered the most dogs, livestock waste, sewage disposal, water cycle all play critical roles in the development complementary test in diagnosis, and is also supply system or use of insecticides. of the human disease.1 Poor economic condi- used in epidemiological studies and as an indi- tions,16,17 malnutrition18 and impaired reactiv- cator of unapparent infection with ity of the immune system19 have also been Leishmania.23 shown to increase the risk of VL. Furthermore, There are no statistical data in Morocco con- Introduction certain age groups are at a greater risk of VL cerning the force of transmission of visceral than others.20 Field studies showed that leishmaniasis in endemic areas. Studies of a Leishmaniasis constitute a group of dis- asymptomatic infection may occur in endemic marker of cellular immune response, such as eases caused by an obligatory, intracellular, areas. In these areas of VL, only about 20% of the Montenegro skin test, might be useful protozoan parasite of the Leishmania genus the subjects infected by Leishmania chagasi since the response rarely disappears with that causes a spectrum of diseases, ranging will develop classical VL, the majority of the time. Therefore, the objective of this study was from self-limiting, self-curing cutaneous leish- infected individuals have a subclinical infec- to identify socio-economic and environmental maniasis (CL) to visceral leishmaniasis (VL) tion that may remain completely asymptomatic factors associated with Montenegro skin test

[page 28] [Microbiology Research 2012; 3:e7] Article positivity in an endemic area in northern where appropriate) and multivariate analysis 95% confidence intervals for the association Morocco, and estimate the force of transmis- (logistic regression). Statistical analysis was with outcome. Most variables studied (Tables 2 sion and the frequency of healthy carriers. performed using STATA 11.0 software (Stata and 3) were associated with a significant Corporation, College Station, TX, USA). prevalence of MST positivity. There was a sig- nificant difference in exposure to infection with L. infantum between males and females. Material and Methods Risk factor of infection was approximately two Results times greater for males than for females. On Study area the other hand, this risk increases with age (PR=1 for children under the age of eight This study was undertaken from April to Force of transmission of visceral May 2010 in two rural endemic areas in north- years and PR=1.85 for children over the age of ern Morocco, Zouagha Moulay Yacoub and leishmaniasis in northern Morocco ten years), presence or absence of familial Taounate provinces (Figure 1 and Table 1). A total of 889 healthy children were included links (PR=2.87 for children tested with a famil- This is a mountainous region with variable in the study. All were from the northern region ial link), and according to province and locali- relief and it is, therefore, common to find a where visceral leishmaniasis is endemic. The ty. Exposure to risk infection is more prevalent wide range of vegetation and bioclimatic vari- mean age of the population studied was in Taounate (PR=1.72) than in My Yacoub. ation. We studied 8 localities at altitudes of 8.5±2.23 years; the median age was eight , a site in Taounate, has the highest 300-600 m with a semi-arid climate, a mild years (81% under the age of ten years); 417 prevalence of infection (PR=2.34) in this winter and yearly precipitation of 400-800 mm females (47%) and 472 males (53%). The study, even though the difference was not sig- in Zouagha Moulay Yacoub and 900-1000 mm prevalence of MST positivity was 11.4% and nificant. in Taounate. Rural populations under the age approximately two times greater in Taounate Table 3 shows the association between nod- of 15 years number 20,600 and 51,000 in than in My Yacoub. The number of asympto- ule size and gender, age, familial links and matic cases was 66 males (66.34%) and 34 provinces. There was a significant difference Taounate and My Yacoub, respectively.5 females (33.66%). between nodule size and familial link and Table 2 shows the prevalence of MST posi- province. Nodule size increased with familial Study population tivity according to the variables investigated linksonly (PR=3.20) and this difference was more A questionnaire that included socio-eco- and the crude prevalence ratios and respective common in Taounate (PR=3.95). nomic and demographic characteristics was given to 889 healthy children. Data were obtained on age, gender, familial links, litera- use cy, history of migration, number of people in household, history of VL in the family, style of the house, water supply, sewage disposal, age and number of dogs, presence of other animals (chickens, horses, rabbits and cats), animal sheds, sewage in the peri-domestic area, and exposure to insecticide in the home.

Measurements and test The main outcome of the study is the result of the MST using 0.1 mL of Leishmania antigen injected intradermally.24 The antigen was pre- pared and provided by the Department of Immunology of the Institute Pasteur of Iran, using a strain of Leishmania major (MRHO/IR/75/ER strain). Reactions were measured at 48-72 h, and indurationNon-commercial of 5 mm or more in diameter was considered positive. The test was carried out on 889 school children aged 3-15 years. Figure 1. Endemic areas with visceral leishmaniasis in northern Morocco. Ethical approval The study protocol was approved by the Committee on Research Ethics of the Institut Table 1. Number of people injected by leishmanin according to locality. Pasteur du Maroc. Province Locality No. injections My Yacoub Ain allah 137 labrarcha 50 Statistical Analysis karyet ba Mhammed 52 Hamria 209 Associations between variables were Taounate Ain Abdoune 58 expressed as prevalence ratios (PR) and their Bouadel (Almarkaz) 82 respective 95% confidence intervals (95% CI), Bouadel bab zarouki 140 examined by univariate analysis (χ2 and t-test Ain Barda (Rkiba) 34

[Microbiology Research 2012; 3:e7] [page 29] Article

Socio-economic and environmental idea is supported by the significant difference test positivity rate of males was only slightly factors associated to the in results between related and non-related higher than that of females, the tendency of Montenegro skin test subjects. On the other hand, parasite exposure women to work indoors during the time of day was higher than expected, despite the small when transmission occurs, together with the Some socio-economic and environmental number of clinical cases, suggesting factors exophily of the vector P. perniciosus, may factors associated with MST in the two endem- other than infection could influence clinical explain this difference.27 ic foci of VL are shown in Table 4. Variables outcome. We suggest that males are more exposed to included in the analysis were: literacy status of Analysis showed that the risk of transmis- infectious bites due to socio-cultural factors. the head of household, number of people in the sion was related to gender; it was approximate- Males can stay outdoors later and spend more household, water supply system, livestock ly 50% higher among males than females with time outside on the whole. In hot weather they waste, housing, history of migration (whether a male/female sex ratio of 1.97. In France, may not wear a shirt therefore exposing more they had ever lived outside My Yacoub/ adult males were more frequently found to be of their body areas to the vector, especially at Taounate), presence of dogs and their age, asymptomatic carriers than females.29 A study dusk, the time of highest sand fly activity. presence of other animals, use of insecticides carried out in Brazil had shown the same Another argument for the importance of gen- and knowledge of VL. results with a male/female sex ratio of 1.34.32 der in the transmission of Leishmania is Few people came from other sites (10.41%), Epidemiological data in Morocco also showed reflected by the higher incidence of the dis- the majority were original inhabitants with the male predominance for VL5 with a sex ratio of ease in immunodepressed males (85.04%) same type of housing and life-style (culture, 1.15. The predominance of visceral leishmani- 16 work, life activities, etc.). On the other hand, compared to females. These patients had no asis among males has also been described in only 2 cases with a familial history of VL were history of the disease but lived in or visited vis- an epidemiological study in the northeast found. Most people were unaware of VL. ceral leishmaniasis endemic areas and the region of Brazil in which the male to female Statistical analysis shows that in general there parasite may have persisted in the host for a 33 is no significant relationship between all these ratio was 1.7. In Italy, the leishmanin skin long period of time. components and the risk of transmission of VL in these areas. Table 2. Number (N), percentage (P) and crude prevalence ratios (PR), and their 95% confidence intervals (95% CI) for the association betweenonly result of Montenegro skin test and gender, age, familial links, provinces and locality in two endemic areas of L. infan- tum infection in northern Morocco, 2010. Discussion Variables N ni N a Pni (%) Pa (%) PR 95% CI P Gender use Results show that 11.4% of children in the Female 383 34 46.91 33.66 1.00 0.0042 two provinces were asymptomatic, as meas- Male 405 67 53.9 66.34 1.86 1.20 - 1.19 ured by the MST. This percentage was compa- Age rable to that found in Spain (11.5%)25 and in <8 yrs 373 39 91.35 8.65 1.00 Brazil, where the incidence of the infection 8-10 yrs 294 38 85.81 14.19 1.73 1.07 - 2.78 < 0.035 was 10.8% when determined by MST on chil- ≥10 yrs 94 20 82.46 17.54 1.85 1.03 - 3.31 dren aged 0-5 years.26 Studies conducted in Familial links high-risk areas of VL in southern Europe using MST indicated a high frequency of subclinical no links 707 76 89.72 75.25 1.00 0.0001 links 81 25 10.28 24.75 2.87 1.73 - 4.77 leishmaniasis: 9.7% in Sardinia (Italy),27 Province 15.3% in Tuscany (Italy),28 and 46.8% in the 29 My Yacoub 409 39 51.9 38.61 1.00 0.013 Alpes Maritimes (France). There were no or Taounat 379 62 48.1 61.39 1.72 1.12 - 2.62 very low incidence of positive reactions in chil- Locality of taounat dren under the age of 15 years but this Ain abdoun 57 6 12.93 9.68 1.00 0.18 27,29 increased in older populations. The high- Bouadel 218 47 49.43 75.81 2.34 0.94 - 5.82 est percentage of asymptomatic Non-commercial carriers Ghefssay 166 9 37.64 14.51 0.48 0.16- 1.44 (14.05%) and the highest nodule size was Ni, non infected; a, asymptomatic. found in Taounate compared to My Yacoub, where the percentage of asymptomatic carri- Table 3. Association between nodule size and gender, age, familial links and province. ers was only 8.7%. In contrast, the Ministry of Prevalence ratios (PR) and their 95% confidence intervals (95% CI). 5 Health 2006 report confirms that the inci- Variables PR 95% CI P dence of visceral leishmaniasis per 100,000 children under 15 years of age is higher in My Gender Yacoub (33%) than in Taounate (12.1%). We Female 1.00 0.7 - 2.47 0.39 suggest that children in Taouante are more Male 1.32 protected against the disease. The difference Age between VL cases and asymptomatic forms ≤8 yrs 1.00 1.002 - 3.66 0.45 may lie in the type of host cellular immune >8 yrs 1.91 response.30 Another hypothesis is that genetic Familial links factors are involved in the development of the no links 1.00 1.78 - 6.504 0.0007 clinical disease in these areas. The evidence of links 3.20 VL cluster in families could be an indication Province that genetic factors may predispose individu- My Yacoub 1.00 1.85 - 8.40 0.001 als to the development of the disease.31 This Taounat 3.95

[page 30] [Microbiology Research 2012; 3:e7] Article

Asymptomatic infection was also associated infection. Probably the phlebotomies were significant difference with all these factors with age. Children over the age of eight years present in the peri-domestic areas of the and VL infection. had a higher relative risk for the infection than households, thus stressing that the vector con- Most epidemiological studies were conduct- those under the age of eight; suggesting a trol had been ineffective in reducing the trans- ed in Brazil and in southern Europe. Strikingly cumulative exposure to the parasite (parasitic mission of parasites. the prevalence of asymptomatic carriers using load) for an asymptomatic infection. This find- Livestock waste, sewage disposal and water a single test was highly variable (0.6-71.3%) ing is consistent with those from other stud- supply system were also not associated with according to the type of test used for detec- ies.27,29,32,34,35 It is important to note that the the prevalence of the infection. These results tion.38 The most frequently used technique prevalence of the infection tends to increase can be explained by the fact that the inhabi- was MST, followed by ELISA, with crude with age, since this test detects a type of hyper- tants of these areas live in the same kind of liv- Leishmania antigens while recent studies sensitivity that rarely disappears with time.36 ing conditions, so it was very difficult to find a using direct tests such as PCR confirm the Age, in this case, might be a proxy for time of individual exposure to the infection. Most studies so far have focused on the risk Table 4. Study of same socio-economic and environmental factors associated to risk of of clinical disease and few have investigated transmission of Leishmania infantum in two endemic areas in northern Morocco the role of educational status on the risk of (Taounate and My Yacoub). Prevalence ratios (PR) and their 95% confidence intervals (95% CI). leishmaniasis infection. Our study showed that there was no correlation between the liter- Variable PR 95% CI P acy level of the head of household and the Literacy of head of household infection. However, in Brazil, many studies Analphabetic 1.00 0.15 - 1.13 0.074 reported that a higher literacy level of the head Only elementary 0.4 of household was inversely associated with the 26,32 Number of people in household infection. 03 - 04 1.00 0.005 Divergent results were found when compar- 05 - 07 0.48 0.097 - 2.39 ing the risk of infection and the number of 08+ 0.17 only0.033 - 0.94 people in the household. A Brazilian study Water supply system found that risk of infection increased the more Tap water 1.00 people there were in the household.30 In the Spring water 2.22 0.84 - 5.87 0.0985 present study, the infection decreased when Sewage disposal use the number of people increased in the family, Public sanitation system 0.15 0.026 - 0.834 0.0137 suggesting that with more people available as Pit latrines 0.36 0.114 - 1.19 sources for a blood meal, the individual chance Open air 1.00 of being bitten by an infected vector would be Age of dogs reduced. The same result were found by Do not have a dog 1.00 Gouvêa et al.32 ≤04 years 1.60 0.17 - 14.95 0.5212 The presence of dogs in the house did not 05 or more years 1.49 0.37 - 5.9 seem to be a significant determining factor of Presence of a kennel in the human asymptomatic infection. This is similar peri-domestic environment No 1.00 to results found in Brazil.37 This could be Yes 1.32 0.56 - 3.12 0.5117 explained by the fact that although the sub- jects did not own dogs, they were exposed to Other animals in house or in the peri-domestic environment Presence of chickens them in the surrounding neighbourhood. No 1.00 In this study, the presence of chickens in the Yes 3.40 1.40 - 8.24 0.0039 house was significantly associated with the Presence of bovines risk of infection by increasing the population No 1.00 density of sand flies.26 Indeed it isNon-commercial known that Yes 2.24 0.92 - 5.45 0.0683 transmission of asymptomatic visceral leish- Presence of horses maniasis occurs through the bite of a sand fly. No 1.00 This finding is consistent with the result of Yes 0.84 0.31 - 2.25 0.7321 Caldas et al.26 who explained that the presence Presence of cats No 1.00 of these insects in the peri-domestic areass Yes 1.29 0.53 - 3.15 0.5657 was a risk factor for infection by L. chagasi, but Presence of goats contrary to another study carried out by No 1.00 Gouvêa et al.32 who found a weak or a non-sig- Yes 1.82 0.21 - 15.99 0.5809 nificant association with the presence of Presence of rabbits chicken sheds in the peri-domestic environ- No 1.00 ment. Thus, the role that chickens play in the Yes 0.96 0.25 - 3.81 0.9641 epidemiology of VL is not clear and probably Livestock waste involves a balance between zooprophylaxy, Peri-domestic environment 1.00 maintenance of a sand fly population, and Domestic environment 1.20 0.39 - 3.65 0.7371 attracting reservoir hosts.30 Use of insecticide in house On the other hand, in this study, use of No 1.00 0.14 - 1.06 0.0560 insecticides was found not to reduce the risk of Yes 0.39

[Microbiology Research 2012; 3:e7] [page 31] Article high proportion of asymtomatic carriage in 1998;5:325-30. 24. Melo MN, Mayrink W, da Costa CA, et al. endemic zones. Riera et al.39 showed that MST 10. Rami M, Atarhouch T, Sabri M, et al. Standardization of the Montenegro anti- is more sensitive than the other diagnostic Canine leishmaniasis in the moun- gen. Rev Inst Med Trop Sao Paulo techniques. This test has been used extensive- tains (Moroccan Mediterranean coast): a 1977;19:161-4. ly in epidemiological studies in southwestern seroepidemiological survey. Parasite 25. Moral L, Rubio EM, Moya M. A leishmanin Europe and it is considered a good indicator of 2003;10:79-85. skin test survey in the human population endemicity.25,27-29 Molecular methods such as 11. Haralambous C, Dakkak A, Pratlong F, et of l’Alacantí region (Spain): implications nested PCR should be considered as an alter- al. First detection and genetic typing of for the epidemiology of Leishmania infan- native for detecting asymptomatic L. infantum Leishmania infantum MON-24 in a dog tum infection in southern Europe. Trans R infection in blood donors.39 from the Moroccan Mediterranean coast: Soc Trop Med Hyg 2002;96:129-32. To understand the dynamics of the spread of genetic diversity of MON-24. Acta Trop 26. Caldas AJM, Costa JML, Silva AAM, et al. the disease, it would be interesting to study 2007;103:69-79. Risk factors associated with asymptomatic other factors that might be related to the infec- 12. Gratz NG. Emerging and resurging vector- infection by Leishmania chagasi in north- tion and the transmission of VL in Morocco. In borne diseases. Annu Rev Entomol east Brazil. Trans R Soc Trop Med Hyg addition, recent data obtained in studies car- 1999;44:51-75. 2002;96:21-8. ried out on blood donors from southern Europe 13. Zijlstra EE, el-Hassan AM, Ismael A, Ghalib 27. Gramiccia M, Bettini S, Gradoni L, et al. have revealed a high rate of infection in HW. Endemic kala-azar in eastern Sudan: Leishmaniasis in Sardinia. 5. Leishmanin asymptomatic subjects,39 so it would also be a longitudinal study on the incidence of reaction in the human population of a interesting to study the risk of transmission by clinical and subclinical infection and post- focus of low endemicity of canine leishma- blood transfusion in Morocco. kala-azar dermal leishmaniasis. Am J Trop niasis. Trans R Soc Trop Med Hyg 1990; Med Hyg 1994;51:826-36. 84:371-4. 14. Walsh JF, Molyneux DH, Birley MH. 28. Bettini S, Gramiccia M, Gradoni L, et al. Deforestation: effects on vector-borne dis- Leishmaniasis in Tuscany (Italy): VIII. References ease. Parasitology 1993;106:S55-75. Human population response to leishmanin 15. Alcais A, Abel L, David C, et al. Risk factors inonly the focus of Monte Argentario 1. Ashford RW. The leishmaniases as emerg- for onset of cutaneous and mucocuta- (Grosseto) and epidemiological evalua- ing and reemerging zoonoses. Int J neous leishmaniasis in Bolivia. Am J Trop tion. Ann Parasitol Hum Comp 1983;58: Parasitol 2000;30:1269-81. Med Hyg 1997;57:79-84. 539-47. 2. Bettinis S, Gradoni L. Canine leishmania- 16. Desjeux P. The increase in risk factors for 29. Marty P, Lelievre A, Quaranta JF, et al. Use sis in the Mediterranean Area and its leishmaniasis worldwide. Trans R Socuse Trop of the leishmanin skin test and western implications for human leishmaniasis. Med Hyg 2001;95:239-43. blot analysis for epidemiological studies in Insect Sci Appl 1986;7:241-5. 17. Thakur CP. Socio-economics of visceral visceral leishmaniasis areas: experience 3. World Health Organisation. The Leishma- leishmaniasis in Bihar (India). Trans R in a highly endemic focus in Alpes- niases and Leishmania/HIV Co-infections. Soc Trop Med Hyg 2000;94:156-7. Maritimes (France). Trans R Soc Trop Med World Health Organisation; Geneva, 2000. 18. Dye C, Williams BG. Malnutrition, age and Hyg 1994;88:658-9. 4. Rhajaoui M, Nasereddin A, Fellah H, et al. the risk of parasitic disease: visceral leish- 30. Alexander J, Bryson K. T helper (h)1/Th2 New clinico-epidemiologic profile of cuta- maniasis revisited. Proc Biol Sci and Leishmania: paradox rather than par- neous leishmaniasis, Morocco. Emerging 1993;254:33-9. adigm. Immunol Lett 2005;99:17-23. Infect Dis 2007;13:1358-60. 19. Wolday D, Berhe N, Akuffo H, Britton S. 31. Evans TG, Teixeira MJ, McAuliffe IT, et al. 5. Ministry of Health. Rapport annuel d’activ- Leishmania-HIV interaction: immuno- Epidemiology of visceral leishmaniasis in ité. Maroc: Direction de l’Epidémiologie et pathogenic mechanisms. Parasitol Today northeast Brazil. J Infect Dis 1992;166: de la Lutte contre les maladies. DMT. (Regul. Ed.) 1999;15:182-7. 1124-32. Service des Maladies Parasitaires; 2006. 20. Bucheton B, Kheir MM, El-Safi SH, et al. 32. Gouvêa MV, Werneck GL, Costa CHN, de 6. Benabdennbi I, Pesson B, Cadi-Soussi M, The interplay between environmental and Amorim Carvalho FA. Factors associated to Morillas Marquez F. Morphological and host factors during an outbreak of visceral Montenegro skin test positivity in isoenzymatic differentiation of sympatricNon-commercialleishmaniasis in eastern Sudan. Microbes Teresina, Brazil. Acta Trop 2007;104:99- populations of Phlebotomus perniciosus Infect 2002;4:1449-57. 107. and Phlebotomus longicuspis (Diptera: 21. D’Oliveira Júnior A, Costa SR, Barbosa AB, 33. Jeronimo SM, Oliveira RM, Mackay S, et Psychodidae) in northern Morocco. J Med et al. Asymptomatic Leishmania chagasi al. An urban outbreak of visceral leishma- Entomol 1999;36:116-20. infection in relatives and neighbors of niasis in Natal, Brazil. Trans R Soc Trop 7. Pesson B, Ready JS, Benabdennbi I, et al. patients with visceral leishmaniasis. Mem Med Hyg 1994;88:386-8. Sand flies of the Phlebotomus perniciosus Inst Oswaldo Cruz 1997;92:15-20. 34. Badaró R, Jones TC, Lorenço R, et al. A complex: mitochondri si M, al introgres- 22. Weigle KA, Santrich C, Martinez F, et al. prospective study of visceral leishmaniasis sion and a new sibling species of P. longi- Epidemiology of cutaneous leishmaniasis in an endemic area of Brazil. J Infect Dis cuspis in the Moroccan Rif. Med Vet in Colombia: a longitudinal study of the 1986;154:639-49. Entomol 2004;18:25-37. natural history, prevalence, and incidence 35. Nascimento M do DSB, Souza EC, da Silva 8. Guessous-Idrissi N, Hamdani A, Rhalem A, of infection and clinical manifestations. J LM, et al. Prevalence of infection by et al. Epidemiology of human visceral Infect Dis 1993;168:699-708. Leishmania chagasi using ELISA (rK39 leishmaniasis in Taounate, a northern 23. de Castro EA, Luz E, Telles FQ, et al. Eco- and CRUDE) and the Montenegro skin test province of Morocco. Parasite 1997;4:181- epidemiological survey of Leishmania in an endemic leishmaniasis area of 5. (Viannia) braziliensis American cuta- Maranhão, Brazil. Cad Saude Publica 9. Nejjar R, Lemrani M, Malki A, et al. Canine neous and mucocutaneous leishmaniasis 2005;21:1801-7. leishmaniasis due to Leishmania infan- in Ribeira Valley River, Paraná State, 36. Marzochi MC, Marzochi KB. Tegumentary tum MON-1 in northern Morocco. Parasite Brazil. Acta Trop 2005;93:141-9. and visceral leishmaniases in Brazil:

[page 32] [Microbiology Research 2012; 3:e7] Article

emerging anthropozoonosis and possibili- a seroprevalence study in an urban area of gasi) in human. Acta Trop 2011;119:69-75. ties for their control. Cad Saude Publica low endemicity. Preliminary results. Am J 39. Riera C, Fisa R, López-Chejade P, et al. 1994;10:359-75. Trop Med Hyg 2007;77:1051-3. Asymptomatic infection by Leishmania 37. Barão SC, de Fonseca Camargo-Neves VL, 38. Michel G, Pomares C, Ferrua B, Marty P. infantum in blood donors from the Resende MR, da Silva LJ. Human asympto- Importance of worldwide asymptomatic Balearic Islands (Spain). Transfusion matic infection in visceral leishmaniasis: carriers of Leishmania infantum (L. cha- 2008;48:1383-9.

only use

Non-commercial

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