ESCMID Online Lecture Library ESCMID PGTW PARASITIC INFECTIONS OF THE ARABIAN PENINSULA AL AIN, UAE MARCH by author

Size: px

Start display at page:

Download "ESCMID Online Lecture Library ESCMID PGTW PARASITIC INFECTIONS OF THE ARABIAN PENINSULA AL AIN, UAE MARCH by author"

Sheila Jordan
5 years ago
Views:

Microbiology Department Hospital Clinic, Barcelona

1 ESCMID PGTW PARASITIC INFECTIONS OF THE ARABIAN PENINSULA AL AIN, UAE MARCH 2016 Leishmania Míriam J. Álvarez-Martínez M.D., Ph.D. Microbiology Department Hospital Clinic, Barcelona (Spain) ISGlobal (Barcelona Institute for Global Health) Faculty of Medicine-University of Barcelona

Health Care Clínic Corporation Hospital Clínic 850 beds Tertiary, Reference,Teaching

residents 24 / 7 Gastrointestinal, Respiratory, bacterial labs Bacterial serology

University Barcelona School Medicine Teaching Medical Nurse Biomedical Associated

2 Health Care Clínic Corporation Hospital Clínic 850 beds Tertiary, Reference,Teaching hospital Microbiology Dpt, Bact & Parasit Unit Consultant Clin Micr 12 consult & 4 residents 24 / 7 Gastrointestinal, Respiratory, bacterial labs Bacterial serology (STI) Clinical assistance, Teaching & Research PcP, CD & FMT, Parasitic diseases University Barcelona School Medicine Teaching Medical Nurse Biomedical Associated Prof ISGLOBAL Barcelona Institute for Global Health Teaching Research Chagas Strongyloides 2

3 Global Burden of Leishmaniasis Parasite/ Vector/ Transmission Diagnose Treatment Monitoring & Evaluation OUTLINE Current Hot Spots of Leishmaniasis in Middle East Conclusions

4 Three main forms of leishmaniases visceral (often known as kala-azar and the most serious form of the disease) cutaneous (the most common, also in Middle East) mucocutaneous Caused by the protozoan Leishmania parasites which are transmitted by the bite of infected sandflies.

6 GLOBAL BURDEN Cutaneous leishmaniasis is a major public health problem in the WHO Eastern Mediterranean Region. Over new cases of cutaneous leishmaniasis are reported annually to WHO by countries in the Region, but the actual incidence is estimated to be three to five times Cutaneous leishmaniasis is currently endemic in 87 countries worldwide. The disease is present in 20 countries in the New World (South and Central America) and in 67 countries in the Old World (Europe, Africa, Middle East, central Asia and the Indian subcontinent). An estimated new cases occur annually but only a small fraction of cases, 19% 37%, are actually reported to health authorities. Cutaneous leishmaniasis principally affects poor populations. Outbreaks can occur anywhere, urban and rural areas, in refugee camps or among internally displaced populations.. The disease burden in the Region is 57% of the cutaneous leishmaniasis burden worldwide.

7 GLOBAL BURDEN Cutaneous leishmaniasis due to Leishmania tropica and L. major is endemic in 18 countries/territories in the Region: Afghanistan, Egypt, Iraq, Islamic Republic of Iran, Jordan, Kuwait, Libya, Morocco, Oman, Pakistan, Saudi Arabia, Sudan, Syrian Arab Republic, Tunisia, West Bank and Gaza Strip, and Yemen. In Djibouti, the parasite species causing cutaneous leishmaniasis are unknown and in Lebanon only cutaneous leishmaniasis cases due to L. infantum are reported. In each country, some areas may be free of cutaneous leishmaniasis while the disease may be very frequent in other areas. New foci appear in addition to well-known zones of transmission.

10 THE PARASITE Blood and tissue flagelatted protozoan, family Trypanosomatide Genus Leishmania, after Sir William Leishman Obligate intracellular parasite with 2 forms: AMASTIGOTES a small, rounded, still form living in the cells of a vertebrate host, multiply in the cells of the host, essentially in macrophages. PROMASTIGOTES elongated form (promastigotes) that moves thanks to a flagellum and lives in the insect that transmits the disease multiply freely in the gut of the sandfly and in culture medium.

11 AMASTIGOTES

13 PROMASTIGOTES

14 THE PARASITE Blood and tissue flagelatted protozoan, family Trypanosomatide Genus Leishmania, after Sir William Leishman Obligate intracellular parasite with 2 forms: AMASTIGOTES a small, rounded, still form living in the cells of a vertebrate host, multiply in the cells of the host, essentially in macrophages. PROMASTIGOTES elongated form (promastigotes) that moves thanks to a flagellum and lives in the insect that transmits the disease multiply freely in the gut of the sandfly and in culture medium. More than 20 species of leishmania produce human infection Classification of Leishmaniasis Clinical: Visceral (kala-azar), the most important disease; Cutaneous, the most common; and Mucocutaneous Geographical distribution: Old world, New world Antigenic structure, isoenzymes

15 Family Genus Subgenus Species Trypanosomatidae Endotrypanum Phytomonas Trypanosoma Sauroleishmania Leishmania Blastocrithdia Herpetomonas Leptomonas Leishmania Viannia L. donovani L. archibaldi L. infantum (syn. L. chagasi) L. tropica L. kilicki L. major L. gerbilli L.arabica L.aethiopica L.mexicana (syn. L.pifanoi) L. amazonensis (syn. L.garnhami) L. aristidesi L. enrietti L. hertigi L. deanei L. turanica* L. venezuelensis* L. braziliensis L. peruviana L. guyanensis L. panamensis L. shawi Crithidia L. lainsoni L. naiffi L. colombiensis* L. equatoriensis*

16 THE VECTOR Flebotomine sandfly, female, 2-3 mm- long, hairy winds

17 Susceptible host TRANSMISION V Vector Parasite Reservoir R H

18 TRANSMISION Leishmania parasites are transmitted through the bites of infected female phlebotomine sandflies. The epidemiology of leishmaniasis depends on the characteristics of the parasite species, the local ecological characteristics of the transmission sites, current and past exposure of the human population to the parasite and human behaviour. Some 70 animal species, including humans, have been found as natural reservoir hosts of Leishmania parasites.

19 TRANSMISION In the Mediterranean basin, visceral leishmaniasis is the main form of the disease. It occurs in rural areas, in villages in mountainous regions and also in some periurban areas, where Leishmania parasites live on dogs and other animals. In Middle East, cutaneous leishmaniasis is the main form of the disease. Agricultural projects and irrigation schemes can increase the prevalence of cutaneous leishmaniasis as people who have no immunity to the disease move in to work on the projects. Large outbreaks in densely populated cities also occur, especially during war and large-scale population migration. The parasites causing cutaneous leishmaniasis live mainly on humans or rodents.

21 WHO LEISHMANIA LIFE-CYCLE

23 DIAGNOSIS

24 Visualization of amastigote: gold standard Sampling: MICROSCOPY CL: scraping / fine-needle aspiration / biopsy of the lesion border VL: bone marrow / spleen / lymph node aspirates Staining: Giemsa / Leishman / Hematoxylin-Eosin Combination with Immunochemistry: higher sensitivity ID Vélez ID Vélez Cheap / easy to perform / expert microscopist Slide courtesy Dr Carmen Cañavate

25 Bone marrow Spleen Liver Skin GRADE Blood Parasite density 6 + > 100 parasites / field parasites / field parasites / field parasites / 10 fields parasites / 100 fields parasites / 1000 fields 0 0 parasites / 1000 fields Ascitic fluid Slide courtesy Dr Carmen Cañavate

26 SAMPLE SENSITIVITY REMARKS CL / MCL Biopsy FNA % Chronic CL, MCL Scraping VL BM 53-86% PB 3-30% Spleen 93-99% Bleeding Lymph node 53-65% Liver 77-91% HIV + BM 67-94% PB 50-53% Pancytopenia Liver 87% Treatment Unusual sites 34% Slide courtesy Dr Carmen Cañavate

consuming / contamination / well-equipped

27 Sensitivity Species identification and characterization Culture Media: CULTURE PROMASTIGOTES NNN (15% rabbit blood agar) (Schneider, RPMI 1640, Grace) + FBS Drawbacks: Time consuming / contamination / well-equipped laboratory Microculture: Cheaper / Easier / Sensitivity NNN Slide courtesy Dr Carmen Cañavate

28 CL / MCL SAMPLE SENSITIVITY REMARKS Biopsy Microculture 85% FNA % Chronic CL, MCL Scraping VL BM 53-70% Contamination Spleen 96% Bleeding Lymph node 53-59% Liver 77-91% HIV + BM 100% PBMC 64-67% Leukocytic fraction Spleen 100% Slide courtesy Dr Carmen Cañavate

Sensitivity (82-100%) VL: LEISHMANIN SKIN

29 Cell-mediated immunity: Anti-Leishmania delayed-type hypersensitivity (DTH) Intradermal injection of 0.1 ml of Leishmania antigen ( promastigotes / ml in phenol-saline) Reading after h, positive if induration >5mm No distinction between past and present infections CL / MCL: Sensitivity (82-100%) VL: LEISHMANIN SKIN TEST Negative during active episode Positive after cure (80%) Slide courtesy Dr Carmen Cañavate

30 AB DETECTION TEST VL: Sensitivity, high levels of specific antibodies Tests: IFAT, ELISA, IB, DAT, ICT Limitations: Antibody levels remain detectable up to several years after cure VL relapse can not be diagnosed by serology Detection of asymptomatic infections: Cross-reactivity seroprevalence in healthy populations <10% in low to moderate endemic areas >30% in high-transmission foci Sensitivity in CL / MCL and HIV coinfection Slide courtesy Dr Carmen Cañavate

31 Antibody detection tests for field settings DAT rk39-ict Sensitivity 94.8 % 93.9 % Specificity 97.1 % 95.3 % Slide courtesy Dr Carmen Cañavate

32 rk39 Inmunocromatografic tests Positive Negative rk39 ICT: Easy to perform Rapid (10 20 minutes) Cheap (around / test) Reproducible results Slide courtesy Dr Carmen Cañavate

33 Latex agglutination test: KAtex Detection of a heat-stable, low-molecular-weight carbohydrate antigen in the urine of VL patients 250 l urine 100º C 5 min temper 5 min Slide courtesy Dr Carmen Cañavate

34 Latex agglutination test: KAtex Detection of Leishmania Ag in urine: Simple / cheap (3 / test) / rapid Non-invasive sample IC: 48-87% Sensitivity / % Specificity HIV+: % Sensitivity / 96% Specificity Good correlation with cure (97 100%) Slide courtesy Dr Carmen Cañavate

35 PCR Highly sensitive and specific targeting multicopy sequences: SSU rrna gene, kinetoplast DNA minicircles, mini-exon genes, repeated genomic sequences Accurate detection of the parasite in multiple clinical specimens Slide courtesy Dr Carmen Cañavate

36 CL / MCL SAMPLE SENSITIVITY REMARKS Biopsy FNA % Scraping PCR VL BM % PBMC % Non-invasive Lymph node % HIV + BM % PB % Non-invasive Slide courtesy Dr Carmen Cañavate

37 Other approaches in combination with RFLP / sequencing For species identification, repeated and polymorphic sequences are targeted: rrna gene-internal transcribed spacers (ITS regions) hsp70 / gp63 / cpb For parasite tracking: Kinetoplast DNA Microsatellite markers (MLMT) Housekeeping genes (MLST) PCR Avoid (not always) the drawbacks of classical method for species identification (MLEE) Slide courtesy Dr Carmen Cañavate

QUANTITATIVE REAL TIME PCR 10 leishmanias, 1

periférica positiva Faster than conventional PCR

monitoring of the amplified PCR products

the parasitic load: Evaluation of the response

efficacy of drugs and vaccines Bossolasco et al.

38 QUANTITATIVE REAL TIME PCR 10 leishmanias, 1 leishmania, Médula ósea positiva Sangre periférica positiva Faster than conventional PCR SYBR-green / fluorogenic probes Continuous monitoring of the amplified PCR products Closed-tube format: contamination Monitoring of the parasitic load: Evaluation of the response to treatment (HIV confection) Monitoring efficacy of drugs and vaccines Bossolasco et al., 2003 Distribution of the parasitic load in tissues Slide courtesy Dr Carmen Cañavate

39 LEISHMANIA OLIGOC- TEST Deborggraeve et al., JID, 2008 Slide courtesy Dr Carmen Cañavate

40 Only one enzyme (Bst DNA polymerase) Large amounts of DNA within minutes Intricate design of primers and auto-strand displacement DNA synthesis Reproducible results ADVANTAGES: Isothermal reaction (between 60 and 65 C) Specificity because of the design of six primers Fluorescent detection reagent (FDR) PRELIMINARY SENSITIVITY RESULTS: 83% on Blood from VL patients compared with microscopy of BM and LN aspirates 98% for CL patients RT-LAMP Slide courtesy Dr Carmen Cañavate

41 RT-LAMP ( ) Slide courtesy Dr Carmen Cañavate

42 TREATMENT

43 Number lesions

47 MONITORING & EVALUATION

49 HOT SPOTS OF LEISH IN MIDDLE EAST

50 A skin lesion described by various middle-eastern physicians, which would last as long as one year, the so-called one-year sore. Documented for the first time in the Middle East in the ninth century under the name of Balkh sore In 1756, the British physician Alexander Russell wrote a chapter on this disease in his book The Natural History of Aleppo, where he described it under different names, amongst which were Il Mal d Aleppo, Aleppo Boil and Aleppo Evil The incidence of CL in Aleppo increased dramatically from 3,900 cases in 1998 to 6,275 in According to the World Health Organization (WHO) Report of 2010, Syria was one of the countries most affected by CL, with more than 25,000 cases per year. Currently, an outbreak has been observed due to the war in Syria and the lack of measures to combat the disease, particularly in the medically underserved areas.

51 There was an incidence rate of 53,000 cases in 2012 and 41,000 cases were reported in the first six months of In the North of Syria, in Aleppo and its surroundings, CL is of an anthroponotic type and is due to L. tropica ZMON-76. In the suburbs of the capital Damascus, zoonotic CL is due to L. major ZMON- 26. Moreover, L. infantum has also been suggested as the causative agent in the Syrian Mediterranean area. The animal reservoirs include fat sand rats.

55 Multiple papulonodular lesions in the right cheek of a 11-year-old Syrian boy diagnosed with cutaneous leishmaniasis.

57 SUMMARY There are 3 main forms of leishmaniases visceral (often known as kala-azar and the most serious form of the disease), cutaneous (the most common), and mucocutaneous. Leishmaniasis is caused by the protozoan Leishmania parasites which are transmitted by the bite of infected sandflies. The disease affects some of the poorest people on the planet, and is associated with malnutrition, population displacement, poor housing, a weak immune system and lack of resources. Leishmaniasis is linked to environmental changes such as deforestation, building of dams, irrigation schemes and urbanization. An estimated 1.3 million new cases and to deaths occur annually. Only a small fraction of those infected by Leishmania parasites will eventually develop the disease.

58 SUMMARY Cutaneous leishmaniasis due to Leishmania tropica and L. major is endemic in 18 countries/territories in the Middle East The disease burden in the Region is 57% of the cutaneous leishmaniasis burden worldwide. Diagnosed is based in microscopy, immunological techniques and molecular methods Treatment depends on number and location of lesions, as well, the previous health status of patient. Monitoring & Evaluation is a crucial step.

59 ACKNOWLEDGEMENTS THANK YOU FOR YOUR ATTENTION شكرا جزيال

Laboratory diagnosis of Blood and tissue flagellates

Laboratory diagnosis of Blood and tissue flagellates (Leishmania and trypanosma) Sarah Alharbi Clinical Laboratory department Collage of Applied Medical Sciences King Saud University Leishmania and trypanosma: