Diagnosis: Leishmaniasis

Suman Rijal, MRCP, PhDDepartment of Medicine,

B. P Koirala Institute of Health Sciences, Nepal

Background• Pathogen: obligate intracellular Leishmania parasite

characterized by diversity and complexity

• Clinical spectrum - cutaneous: localized cutaneous, mucocutaneous and diffuse

cutaneous- visceral: subclinical, kala-azar, PKDL

• Clinical manifestations not specific while drugs are toxic

• Early diagnosis and appropriate treatment: important on individual and community level (best strategy for control for VL)

• Affects poor populations with limited access to health care.

Epidemiology:

Rangeli District Hospital

Diagnostic devices in Leishmaniasis

• To confirm disease?• To detect asymptomatic infection?• To assess cure after therapy?• To detect drug resistance in parasites?

Diagnostic devices in Leishmaniasis

• To confirm disease?• To detect asymptomatic infection?• To assess cure after therapy?• To detect drug resistance in parasites?

Need to specify the indication !

Methods for leishmaniasis diagnosis

• Conventional parasite detection techniques• Immunological tests• Antigen detection tests• Molecular methods

Parasitology

• Microscopic examination of stained smears

lesion scraping, biopsy, impression smears (CL)aspirates from spleen, bone marrow, lymph nodes (VL)

• Histopathological exam. of lesion biopsies• Culture

Smear Sensitivity Specificity Spleen

80 - 98

100

Bone Marrow

60 - 85

100

Lymph Node

40 - 50

100

Zijlstra 1992, Singh & Sivakumar 2003

Comparison parasitological methods in VL

Parasitology: CL

Method # patients # Positive (%)

Dermal scraping 148 33 (22.3)

Impression smear 139 26 (18.7)

Histopathological 149 21 (14.1)

Aspiration- culture 158 91 (57.6)

Aspiration-hamster 107 41 (38.3)

Biopsy culture 162 80 (49.9)

Biopsy hamster 147 77 (52.40

Weigle et al 1987

Weigle et al 1987

Parasitology positivity with duration of lesions

Immunological methods

Specific humoral response in VL: serological test

Cell mediated immune response in CL and MCL: Leishmanin skin test

Different Serological tests• Indirect fluorescence antibody test (IFAT)• ELISA• Direct agglutination test• Rapid antibody detection test: rk39 ICT • Antigen detection test: KATEX

Serological tests in VL

Indirect immunoflorescence test (IFAT)Demonstration of anti-leishmanial Ab. using fixed promastigotesHigh sensitivity (87-100%) and specificity (77-100%) Drawback: Need for fluorescent microscope restricts their to reference laboratories.

ELISA ( Ho et al 1983)One of the most sensitive serological test in VLVarious antigens: whole cytoplasmic (soluble antigen) to recombinant antigens ( rgp63, gene B protein, rk39, rH2A, rH2B etc.)

Patient Ig

Direct Agglutination Test (DAT) El Harith et al. 1986

DAT antigen:promastigotesfixed, stained

pos. neg. Microtitre plate with 96 V-shaped wells

Reading after 18 hours onlyAlternative “FAST” 1-titer (1:200) Schoone et al 2001

Control 200 400 800 1600 3200 6400 12800 25600 ….

NegPos12

Chagas

Diagnostic Cut-off

Subgroups No studies Sen. (95% CI) No studies Spec. (95% CI)

All studies (30) 29 94.8 (92.7 to 96.4) 27 97.1 (93.9 to 98.7)

Region:

South Asia 11 97.1 (94.9 to 98.4) 10 95.7 (88.1 to 98.5)

East Africa 11 93.2 (89.1 to 95.8) 10 96.1 (89.2 to 98.6)

Elsewhere 7 92.8 (86.8 to 96.2) 7 99.8 (97.5 to 100)

Sensitivity and specificity of DAT

Chappuis et al 2006

Subgroups # studies Sen. (95% CI) # studies Spec. (95% CI)Trial phase

I 20 94.3 (91.5 to 96.2) 17 98.1 (94.2 to 99.4)

II 5 97.7 (87.4 to 99.6) 5 97.2 (92.5 to 99)

III 4 94.3 (87.9 to 97.4) 5 90.9 (75.9 to 96.9)

Health state of controls*:

Healthy non-endemic NA 8 100.0 (98.2 to 100)

Healthy endemic NA 20 98.7 (97.1 to 99.5)

Cross reacting diseases NA 16 98.8 (95.6 to 99.7)

Clinically suspected disease NA 8 82.6 (70.4 to 90.4)

1 petal = 1 case

IMTA titre (n= 317)

121086420-2

Sud

an ti

tre

12

10

8

6

4

2

0

-2

1 petal = 1 case

IMTA titre (n= 317)

121086420-2

Sud

an ti

tre

12

10

8

6

4

2

0

-2

Since 1999,

FREEZE-DRIED DAT, Oskam 1999

rK39 immunochromatographic strip test

2. Immunochromatographic tests (rK39 dipstick) Sundar et al. 1998

Subgroups # stud. Sen. (95% CI) # stud. Spe. (95% CI)

All studies 13 93.9 (87.7 to 97.1) 13 95.3 (88.8 to 98.1)

Region:

South Asia 7 97.1 (91.7 to 99.0) 7 95.3 (87.3 to 98.3)

East Africa 2 79.0 (46.7 to 94.2) 2 85.2 (28.2 to 98.8)

Elsewhere 4 88.8 (83.7 to 92.4) 4 97.0 (79.4 to 99.6)

Sensitivity and specificity of rk39 dipsticks

Chappuis et al 2006

Subgroups #studies Sens. (95% CI) # studies Spec. (95% CI)Trial phase:

I 5 86.0 (67.1 to 94.9) 5 96.9 (86.3 to 99.4)

II 4 96.5 (86.0 to 99.2) 4 96.8 (90.7 to 98.9)

III 4 94.8 (87.6 to 97.9) 4 91.2 (66.8 to 98.2)

Health status of controls:

Healthy non-endemic — NA 0 NA

Healthy endemic — NA 10 95.9 (90.6 to 98.3)

Cross reacting diseases — NA 7 97.1 (88.5 to 99.3)

Clinically suspected disease — NA 7 93.0 (77.5 to 98.1)

Clinically suspected disease in phase III — NA 4 90.6 (66.8 to 97.9)

Rk 39 : Phase III study (Boelaert et al 2007)

20 40 60 80 100 20 40 60 80 100

Sensitivity Specificity

Ethiopia

Kenya

Sudan

India

Nepal

Meta-Analysis

Summary of rK39 dipstick and DAT• Performance of both good to replace parasitology as diagnostic test

in Indian sc.

• Availability of rk39 striptest: launch of the kala-azar elimination programme.

• Efforts to improve performance in East Africa by combining with other antigens.

• Limitations: Not useful to diagnose relapseNot suitable assess cure Seropositivity of VL antibodies: <10% (Schenkel et al 2006) to > 30% (Sundar et al 2006) Poor sensitivity in VL-HIV co-infection

• Essential to use in combination with clinical case definition.

Comparison of serological test : 45 VL/HIV co-infected cases (Deniau et al 2002)

Test Sensitivity Specificity

IFAT 67 100

rK39 ELISA 62 90

rK 39 dipstick 20 100

Urine antigen detection latex agglutination testAttar et al. 2001

Katex

• Sensitivity: Lab.: 64 to 100%(Attar et al 2001)

Field: 48 to 87% • Specificity: 97 to 100%

Katex

20 40 60 80 100 20 40 60 80 100

Sensitivity Specificity

Ethiopia

Kenya

Sudan

India

Nepal

N # KAtex

positive

Sensitivity Katex p-value

Duration of fever

(weeks)

0.024*

<9 96 39 0.406

>=9 59 35 0.593

Spleen size (cm) 0.005**

<4.0 44 13 0.295

4.0 to 5.9 36 20 0.556

6.0 to 7.9 28 11 0.393

>=8.0 47 30 0.638

Parasite grading <0.001**

1 29 6 0.207

2 43 11 0.256

3 41 24 0.585

4 36 28 0.778

5 6 5 0.833 Rijal et al 2004

Katex

• Sensitivity: 48 to 87% (high in HIV-VL co-infection)

• Specificity: 97 to 100%• Limitations:

Boiling of urineInterpretation of 1+ and negative: subjective

Conclusions• Parasitology remains the reference test for both VL

and CL

• Rapid diagnostic test (rk39 dipstick) for VL though available has limitations.

• Need for to assess cure, differentiate Leishmaniainfection with disease.

• Diagnostic tools will only have an impact if they are widely available to patients.

• Standards for conducting and reporting diagnostic studies have been described.

QUADAS Criteria

1. Was the spectrum of patients representative of the patients who will receive the test in practice?

2. Were selection criteria clearly described?

3. Is the reference standard likely to correctly classify the target condition?

4. Is the time period between reference standard and index test short enough to be reasonably sure that the target condition did not change between the two tests?

5. Did the whole sample or a random selection of the sample receive verification using a reference standard of diagnosis?

6. Did patients receive the same reference standard regardless of the index test result?

7. Was the reference standard independent of the index test(I.e. the index test did not form part of the reference standard? )

8. Was the execution of the index test described in sufficient detail to permit its replication?

9. Was the execution of the reference standard described in sufficient detail to permit its replication?

10. Were the index test results interpreted without knowledge of the results of the reference standard?

11. Were the reference standard results interpreted without knowledge of the results of the index test?

12. Were the same clinical data available when test results were interpreted as would be available when the test is used in practice?

13.Were uninterpretabale/ intermediate results reported?

14. Were withdrawals from the study explained?

DIAGNOSTIC ACCURACY STUDIES according to their phase of clinicaldevelopment

(1) Phase I (“Exploratory”): The aim of these studies is to provide proof-of-principle by a retrospective comparison of test performance in a small number of patient samples. The evaluation is carried out in laboratories on banked sera. The number of samples tested in this phase is usually 10 – 100.

(2) Phase II (“Challenge”) : In this phase the practical value of a suggested test is assessed using samples from a patient population with a much larger disease spectrum. The studies are usually designed in a case-control approach in which several series of subjects are enrolled on the basis of their case status: e.g. VL or control.

(3) Phase III “Clinical” ; This phase serves to validate the test in large scale prospective studies on the target population, requiring the evaluation of a sufficient and representative sample of consecutively enrolled or randomly selected patients. Those patients should be recruited as a single cohort unclassified by disease state and recruited from the clinical setting and point in referral process where test would be used