modern diagnostic approaches on leishmaniasis

Modern Diagnostic Approaches on Leishmaniasis

Prof. Dr. Seray TÖZ Ege University Faculty of Medicine

Department of Parasitology, Bornova, Izmir [email protected] ESCMID Online Lectu

re Library

© by author

ESCMID Online Lecture Library

© by author

Leishmaniasis Worldwide and Global Estimates of Its Incidence

• Leishmaniasis reported in 98 countries and 3 territories

– 90% VL: India, Bangladesh, Sudan, South Sudan, Ethiopia, Brazil

– 70% CL: Afghanistan, Algeria, Colombia, Brazil, Iran, Syria, Ethiopia, North Sudan, Costa Rica, Peru

• VL cases/ year:

– Mediterranean 875

– Middle East to Central Asia 2496

• CL cases/ year:

– Mediterranean 85,555

– Middle East to Central Asia 61,013

Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M, the WHO Leishmaniasis Control Team, Leishmaniasis Worldwide and Global Estimates of Its Incidence, PLoS ONE, 7(5), e35671, 2012

Country Annually reported VL cases (2004-2008)

Annually reported CL cases (2004-2008)

France 18

2

Italy 134

49

Greece 42

3

Portugal 15

0

Spain

17

0

Syria 14

22,882

Tunisia 89

7631

Turkey 29

2465


© by author

Leishmaniasis in Turkey


© by author

Necessity of Using Modern Techniques in Diagnostic and Epidemiology Fields of Infectious Diseases

• Easy application, high sensitivity/specificity and standardization can be feasible targets with the technological improvement

• Classical methods

– Time consuming and laborious

– Subjective and showing low sensitivity

– Species identification is insufficient

• Due to the increase of migration/travel and global warming

– The endemic regions of infectious agents are growing and spreading

– Because of spreading of different species of microorganisms, molecular typing is necessary for the application of therapy, prognosis and control measures


© by author

Hasta • In an epidemiological field study in Denizli province in Turkey

• An 30 years old female was sampled

• Her results:

• IFAT: 1/512 POZ

• ELISA: POZ

• rK39 ELISA: POZ

• Western Blot: active infection banding pattern

A Case of Visceral Leishmaniasis (VL) in Turkey • We informed the Ministry of Health and

learned that, patient has: • Weakness for 10 months • Abnormal weight loss • Chills and sweating for 2 months

• Clinical symptoms: • Hepatomegaly • Splenomegaly (21 cm) • Submental/cervical lymphadenopathy

• Laboratory results: • Hb: 6,7g/dl • Htc: %26 • Plt:135000mm3 (Blood tranfusion+) • WB:2000mm3

• Pre-diagnosis: Anemia and myeloproliferative disease

Leishmania spp. amastigotes were seen in retrospective analysis of the previous bone marrow aspirates

Özensoy Töz S, Sakru N, Ertabaklar H, Demir S, Sengul M, Özbel Y. Serological and entomological survey of zoonotic visceral leishmaniasis in Denizli Province, Aegean Region, Turkey, New Microbiologica, 32, 2009 p. 93-100


© by author

Evaluation of Fourteen Adult Cases with Visceral Leishmaniasis

• 14 VL cases were followed 2005 to 2012: (7 female, 7 male; 19-64 years)

• 10 immunocompetent/ four immunosuppressive (renal transplant, BM transplant, autoimmune hemolytic anemia, B cell lymphoma)

• 6 cases were living in Adana/ 8 were from south and southeastern Turkey

• 11 (79%) were admitted to health center and used antibiotics before diagnosis

• Complaints: fever (100%), chills /shiver (93%), weakness (71%), weight loss (57%)

• Clinical signs: fever (57%), splenomegaly (79%), hepatosplenomegaly (50%)

• Laboratory findings: pancytopenia (71.4%), hypoalbuminemia (100%)

• Diagnosis: Microscopy BM (86%), tissue (liver/spleen) biopsy (14%)

• Culture in NNN medium: 4 out of 14 were positive

• Therapy: Parenteral Liposomal Amphotericine B, 3 immunosuppressive patients died (mortality rate: 14%)

• In conclusion: cases presenting fever, hepatosplenomegaly, pancytopenia and hypoalbuminemia and living in endemic regions should be precisely evaluated in terms of VL

Kurşun E, Turunç T, Demiroğlu YZ, Solmaz S, Arslan H Evaluation of Fourteen Adult Cases with Visceral Leishmaniasis, Mikrobiyol Bul, 47(3), 500-506, 2013


© by author

A Case of Cutaneous Leishmaniasis (CL) in Turkey

• Patient: A Turkish engineer working in Libya

• 2 months ago 3 lesions were appeared on his arm and leg

• It is difficult to reach to the health facilities from his working region in Libya and he came to Turkey and admitted to Dermatology Clinic in Ege University Hospital

• Leishmania spp. amastigotes were seen in the lesion aspirates stained by Giemsa

• Species identification result by Real time ITS1 PCR: L. major

• Treated with intralesional antimonials


© by author

Leishmaniasis Without Borders

• Between 2010 and 2013; 15 (19.5%) Turkish and 62 (80.5%) Syrian patients were diagnosed as CL by microscopical analyses of lesion smears, in Nizip, Gaziantep, Government Hospital

• Since the number of the cases admitted to the hospital was significantly low in comparison to the total population of refugees living in the camps, it was assumed that the real incidence of CL was much higher than determined.

Salman İS, Vural A, Ünver A, Saçar S. Cutaneous Leishmaniasis Cases in Nizip, Turkey After the Syrian Civil War, Mikrobiyol Bul, 48(1) 106-113, 2014


© by author

Diagnostic Approach in Leishmaniasis

• Living region and the travel history of the patient is important

• Clinical form of the disease

– Cutaneous • Lesion or lesions on the skin, mostly

on uncovered parts of the body

– Mucocutaneous • Skin lesions with mucosal

involvement

– Visceral • Systemic infection

(hepatosplenomegaly, pancytopenia, weight loss, fever)

Sampling for the diagnosis • Skin and mucosal lesions

(lesion aspirate) • Blood (serology) • Bone marrow aspirate

(microscopy and PCR) • Biopsy (lymph node) • Urine (antigen detection)


© by author

Direct Parasitological Diagnosis • Microscopic diagnosis is still considered as the gold standard

• The most important advantage is that it allows to isolate and replicate the parasite

– Parasite collection is necessary in the development, optimization and standardization of new diagnostic methods

– New drug trials, drug resistance and vaccine studies can be done by experimental infection (cell culture, experimental animals)

• Trained/experienced technicians are needed

• Microscopic diagnosis should be supported with serological and molecular diagnostic methods in order to reach higher sensitivity/specificity


© by author

Conventional Diagnosis

• Conventionally diagnosis of leishmaniasis was commonly based on clinical and epidemiological criteria especially in highly endemic regions

• Definitive diagnosis was based on seeing amastigotes on biopsy smears of infected organs (bone marrow, spleen, skin lesion)

• Amastigote forms are the diagnostic forms in the host body


© by author

Leishmaniasis Parasitological Diagnosis

• Visceral leishmaniasis: Bone marrow aspiration

• Cutaneous leishmaniasis: Aspiration from ledge of the lesion

Giemsa DNA based methods (PCR)

Stain NNN medium culture

6-21 gün

Amastigot

Promastigot


© by author

http://www.teknosem.com.tr/images/Mikroskop.gif

Diagnosis of Cutaneous Leishmaniasis (CL)

• Parasites were seen on Giemsa stained aspiration smears of lesion edge


© by author

Isolation and mass cultivation of Leishmania parasites in NNN and 20% FCS added RPMI 1640 media

Alive promastigotes after washing with PBS buffer

Cultivated parasites are necessary for development of new diagnostic methods, new drug, drug resistance and vaccine studies


© by author

Isolated Leishmania parasite are cryopreserved in order to not to lose the virulance

In Turkey we have a Leishmania

isolates cryobank in Manisa Celal

Bayar University Medical Faculty

Parasitology Department


© by author

Serological Diagnosis (Indirect Diagnosis)

• Depending on Ag-Ab interactions using in vitro conditions in order to detect specific antibodies against parasite or the antigen

• Methods are named according to the imaging properties as: agglutination, IFAT, ELISA, western blotting

• Specific antibodies against surface, soluble or a specific part of a protein can be detected

• Recombinant protein technology and immunochromatographic dipstick tests were provided development in serological diagnosis

– Rapid diagnosis can be possible in field conditions with these dipstick tests ESCMID Online Lectu

re Library

© by author

Serological (Indirect) Diagnosis in Visceral Leishmaniasis

• Methods

– IFAT (promastigote or amastigote)

– ELISA

– DAT (Direct Agglutination Test)

– Dipstick (rK39)

– Western Blotting

• Serum sample is used for detection of antibodies

• Serological diagnosis is more sensitive in VL

• If the lesion is spreading especially to mucosa antibody response can positive in CL

Yıldız Zeyrek F, Korkmaz M, Özbel Y. Serodiagnosis of Anthroponotic Cutaneous Leishmaniasis (ACL) Caused by Leishmania tropica in Sanliurfa Province, Turkey, Where ACL Is Highly Endemic Clinical and Vaccine Immunology 14 (11) 2007 p. 1409–1415


© by author

Serological diagnosis of Leishmaniasis

• Antibody detecting tests

– Cut off titer of serological tests should be validated with clinical case definition and gold standard

– They are showing high sensitivity in immunocompromised VL patients but subclinical infection and cross-reactivity can cause positivity in lower titers

– Sensitivity of serological tests are showing lower sensitivity in HIV/VL co-infection and immunosupression; parasitological diagnosis should be performed

• Antigen detection tests

– No cross reaction

– Active infection can be separated from old or subclinical infection

– Latex agglutination test (KATEX) can detect Leishmania antigen in urine samples

Taylan Özkan A, Yalçınkaya T, Kılıç S, Babür C, SCHALLIG HDFH. HIV/AIDS Hastalarında Leishmania

infantum seropozitifliğinin araştırılması, Mikrobiol Bül (42) 2008 p. 113-117


© by author

Dipstick – Rapid Diagnosis

• rK39 rapid diagnostic test is commercially available for VL in human and canine leishmaniasis (CanL) for the dogs

• Blood or serum sample can be used

• Serum sample is more convenient (clear band)

• Resulted in 5 minutes

Özensoy Töz S, Chang KP, Özbel Y, Alkan MZ. Diagnostic Value of rK39 Dipstick in Zoonotic Visceral Leishmaniasis in Turkey, J. Parasitol 90(6) 2004 p. 1484–1486


© by author

Serological methods can also use in monitoring the clinical course of VL infection during and after treatment


© by author

Development in Serological diagnosis

• An important development was gained by recombinant protein technology and immunochromatographic tests

• Using of non-invasive body fluids will provide a significant improvement in serological tests

– Conjunctival swab

– Nasal swab

– Urine ESCMID Online Lecture Library

© by author

Isoenzyme Typing

• It is the gold standard method for the species identification of Leishmania

• There is a need for isolation and mass cultivation of the parasite

• Depends on different movements of the same enzymes

• Isolates showing different banding pattern are genetically different from each other


© by author

MON-55 (n=2)

MON-200 (n=2)

MON-303 (n=13)

MON-304 (n=22)

L. tropica L. infantum

MON-1 (n=9)

MON-98 (n=5)

MON-309 (n=3)

EP59 (MON-308, hybrid?) (n=1)

Isoenzyme patterns (Zymodemes) of Isolates from Turkey (n=65)

MON-315 (n=3)

MON-315 (n=1)

Kutanöz leishmaniasis

L. donovani Visseral leishmaniasis

L. tropica

MON-53 (n=1)

MON-312 (n=1)

MON-313 (n=1)

MON-314 (n=1)

L. major


© by author

Molecular Diagnosis

• Advantages:

– Rapid, easy and sensitive diagnosis using less amount of sample and parasite number

• Disadvantages:

– Difficulty of optimization, easy contamination, requirement of expensive equipment and experience

– Determination of subclinical infection is unclear

• Development of molecular tests is showing a dynamic period and limitations, interpretation of results and standardization of molecular tests are going on with application of tests and sharing results in different laboratories

• The application of molecular tests besides conventional techniques is important for the validation and proper application


© by author

L. Infantum isolates: showing homogenity

L. tropica isolates: more heterogenous

L. tropica can cause VL in human and CanL in dogs in Turkey


© by author

One Health Approach

• In one health approach; besides diagnosing and treating patients after admission to the hospital

– Reservoirs of the infection in nature and presence of the reservoirs in the region are detected

– Risk factors of the infection according to reservoir and vector populations are studied

– Diagnostic methods can be used not only for diagnosis of the patients but also for prophylactic aims and eradication in the long term

• This approach requires a multidisciplinary team work including many scientists working in leishmaniasis field as medical doctors, biologists, veterinarians, pharmacologists ESCMID Online Lectu

re Library

© by author

358 bp

• CanL seroprevalance rate was found to be 24.95% in a CL and VL endemic region, Adana, Çukurova

• Conjunktival swab PCR (CS n-PCR) was found to an easy sampling method and more sensitive than IFAT in detection of CanL for epidemiological surveys

• CS n-PCR has an advantage for early detection of infection and can be a good marker for control strategies

EDENext Project


© by author

L. infantum Population Analysis

K26 PCR

L. infantum MON-1 İsolates could be

differentiated from other isolates in the L.donovani

complex

MLMT

14 highly variable microsatellite marker located in 9 different

chromosomes

Population analysis STRUCTURE v2.3.1

Manisa’da 3 human VL isolate L. tropica

480 bp amplicon: Adana’da 1 CL, 1 CanL, 4 sand fly isolate L. infantum MON-309 (new zymodem)

385 bp amplicon: A VL isolate in Kuşadası L. infantum MON-308 (new zymodeme, hybrid)

626 bp amplicon: L. infantum MON-1

870/940 bp amplicon: L. infantum MON-1 or MON-98


© by author

Neighbor Joining Tree analysis of Isolates Belonging to L. donovani complex (L. donovani/infantum) after Microsatellite Analysis

•Turkish and Cyprus

isolates were performed

a seperate genetic group

together

•A hybrid VL isolate from

Kuşadası Turkey

(MON1/non-MON1

hybrid) ESCMID Online Lecture Library

© by author

As a Result

• Conventional Diagnosis: Parasite positive or negative

• Serological and molecular techniques can also help as follows: – Timing of the parasite (when taken into body)

– Clinical stage of the disease: asymptomatic / symptomatic infection

– Immunologic response of the host against to the parasite

– Drug resistance

– Prognosis of the disease

• Modern Diagnostic Approach – Optimization of the test

– Validity of the test for the endemic site according to the species and intraspecies groups

– Application of necessary tests for different aims

– Development of diagnostic algoritms using different complementary tests


© by author

As a Result

• Parasitological diagnosis: gold standard, necessary but inadequate

• Serological diagnosis: showing high level sensitivity and open for improvement

• Modern molecular diagnosis: expensive, has many advantages and at the beginning of standardization


© by author

Thanks • Ege University Medical Faculty Parasitology Department

• Prof. Dr. Yusuf Özbel • Biologist Mehmet Karakuş

• Celal Bayar University Medical Faculty Parasitology Department

• Prof. Dr. Ahmet Özbilgin • Technician Ibrahim Çavuş

• Colleagues working in Pasteur Institute, Athens, Greece • Colleagues working on leishmaniasis in Turkey • Prof. Charles L. Jaffe and Dr. Abed Nasereddin • EDENext Project • TUBITAK (The Scientific and Technological Research

Council of Turkey) ESCMID Online Lectu

re Library

© by author

modern diagnostic approaches on leishmaniasis

Documents