2  MATERIALS AND METHODS

2.1  Clinical molecular diagnosis and local molecular epidemiology

2.1.1 Patients and study area:

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This part of the study was conducted in the decade, 1994-2004, in the CL endemic District of Jericho (A’riha) in Palestine and its vicinity of the close hilly areas of Jerusalem (Al-Quds), Ramallah and Nablus, extending over an area of more than 593 km2, with a population density of 73 person per km2 and a total population of more than 40,909 (Palestinian Central Bureau of Statistics, 2005). Jericho, a strip of land extending from the Dead Sea and Jerusalem in the south to Nablus and Tubas in the north and from the River Jordan in the east to the hills of Ramallah and Nablus in the west (Figure 2.1), is a low-lying (244-398 m below sea level) arid to semi-arid area located between 29°-33° north of the equator. It is situated on the floor of the northern tip of the 6,500-kilometer-long Syrian-East African Rift, called the Jordan Valley. Climate is affected by the subtropical aridity which is characterized by long, hot and dry summers and short, cool winters. Jericho is at latitude of 31o 52´ N and longitude 35o 28´ E.

Between 1994 and 2004, the daily average temperature throughout the year was 23.3 oC. The average maximum temperature was 44.9 oC, with maximum temperature 48 oC in 2002, while the average minimum temperature is 3.6 oC with minimum temperature 0.6 oC in 1997. The annual average rainfall in the period between 1994 and 2004 is 146.29 mm and falls between October/November and March/April, with the rest of the year mostly rainless. The maximum annual rainfall was 240.3 mm in 2002/2003 season, while the minimum annual rainfall (driest season) was 40.3 mm in 1998/1999 season. (Personal communication with the Palestinian Meteorology department- Jericho, 2004; Environmental profile for the West Bank, 1995).

This area is generally characterised by the presence of springs, on which the people depend to grow banana, vegetables and palms, such as Wadi Qelt that brings water from the mountains of West Bank down to Jericho, which over-floods the city in winter and makes the area green and lush all year round. Reduced vegetation and grass is another characteristic of the area. The area is flat and composed of cracked alluvial soil with hills. Inhabitants mainly work as farmers, government employees and nomadic Bedouin shepherds who roam the area all the year round.

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In the decade period between 1994 and 2004, a total of 943 patients with skin lesion(s) attended ICS-Jericho medical laboratory for clinical diagnosis and were in turn sent to the Jericho Health Department-Ministry of Health for treatment. All patients were tested by microscopy (Giemsa-stained smears) which is the basic diagnostic method adopted by ICS-Jericho and the “gold standard”. Since 1998 NNN culture and ITS1-PCR were introduced into Jericho. They were, however, used mainly for research purposes, such as collection of strains and to confirm clinical diagnosis, and not merely regular diagnostic methods. The patients were Palestinians living in the district of Jericho and the vicinity with exception of a few patients who were temporary workers on international projects.

Figure 2.1 (A) Satellite image of the District of Jericho in the Eastern Mediterranean region. (B) District of Jericho. (C) Map of the Palestinian governorates according to Palestinian classification.

2.2 Sample collection

2.2.1 Patient data sheets

Data of each referred patient were collected in a questionnaire filled in by the technician, directly interviewing the patients. The questionnaire included demographic, clinical, diagnostic, and epidemiological questions. It was designed to be user friendly with a computerized input interface using Dos-based EpiInfo 6, Access-based EpiInfo 2002 and Access-based EpiInfo 2004 throughout the study period.

2.2.2 Collection and Giemsa staining of skin scrapings

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For diagnostic purposes skin scrapings were collected from 943 Palestinian patients with lesions referred to Islah Medical Laboratory in Jericho in the period between 1994 and 2004. The patients were from all refugee camps, villages, Bedouin encampments and Jericho City. The tested lesion was disinfected using cotton wool immersed in 70% ethanol. Using a sterile lancet, two 3-5 mm incisions were cut on the periphery of the lesion and from the centre in case it is not infected (Rameraiz et al., 2000). The incisions were squeezed gently and touched with 3-5 clean glass slides. Three of the slides were air dried and fixed with methanol for a few seconds, while the remaining two are kept for further comparison study as shown below in 2.2.3.

Concentrated Giemsa stain (Finkelman, Petah Tikva, or Sigma) was applied for no more than 30 seconds and then slides were washed under running tap water and air dried. The smears were examined using light microscope with a 40 x lens and with 100 x oil immersion lens. If at least one amastigote was found the smear was declared positive. When no amastigotes were seen after 15 minutes the smear was declared negative.

2.2.3 Sampling using filter papers and unstained smears

In addition the incision cut was touched three times with an autoclaved 3 mm filter paper (Schleicher and Schüll, Germany) during the sampling process, producing three separate and thick tissue-blood spots. After air drying, the filter papers were wrapped in aluminium foil and stored at room temperature until use. Between June 1997 and July 2004, 418 filter papers were sampled. Also, between August 1998 and June 2004, 173 unstained slides were sampled in Jericho.

2.2.4 Cultivation of parasites from dermal tissue aspirates

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Two hundred and seventy dermal tissue aspirates from the same incisions made for microscopy were cultured in rabbit blood–agar semisolid and NNN media. To guarantee aseptic techniques, a large Bunsen burner was lit. 0.5 ml of sterile 0.9% normal saline was injected into the incisions, mixed with the fluid there, then aspirated and discharged into two culture tubes held close to the Bunsen burner. One tube containing semisolid (SS) blood agar (Schnur and Jacobsen, 1987) and the other NNN medium, Novy-MacNeal-Nicolle medium (1.4% agar, 0.6% NaCl as described by Evan (1989)).

Ten ml of defibrinated rabbit blood containing 100 µl gentamicin were added to 100 ml of the previous melted NNN medium. Two ml of NNN media were distributed into small sterile tubes and kept in tilted slant position until the agar solidified. The tubes were stored at 4oC until use. Before inoculation of samples, the tubes were adapted to room temperature. After inoculation of material, the culture tubes were incubated at 22-28°C. After one week, one drop of media was examined under the microscope. Positive cultures were followed up for mass cultures and cry-conservation by Dr. L. F. Schnur at the WHO Leishmania reference centre in Jerusalem, in which promastigotes from cultures were transferred into Schneider’s Drosophila medium (Gibco, Grand Island, NY; Biological Industries Beit Haemek) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 200 µg/ml streptomycin, and 200 U/ml penicillin for further mass culturing. The parasites were cultivated in 5 ml of Schneider’s medium for 2-3 days before they were transferred to a larger volume (40 ml). Negative cultures were kept for three weeks with weekly regular examination before being declared negative and discarded.

2.3 DNA extraction

2.3.1 DNA extraction of cultured parasites

For DNA preparation, parasites grown in mass culture were harvested at a density of about 2 x 107 parasites/ ml. The cells were centrifuged for 10 minutes at 2500 rpm and washed three times with phosphate-buffered saline, pH 7.4 (8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 800 ml of distilled H2O. Adjust the pH to 7.4 with HCl. Add H2O to 1 liter and sterilize by autoclaving)

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DNA extraction was performed as described by Schönian et al. (1996). The pellet of cultured cells was resuspended in 1 ml NET lysis buffer (50 mM NaCl; 10 mM EDTA; 50 mM Tris- HCl pH 7.4). Sodium dodecyl sulphate (SDS) (Merck, Darmstadt, Germany) was added to a final concentration of 0.5% and the mixture was shaken until the solution was viscous. RNase was added to a concentration of 100µg/ml and the mixture was incubated in a water bath for 30 min at 37oC. Proteinase K (Boehringer Mannheim, Mannheim-Germany) was added to a final concentration of 100µg/ml and the samples were incubated at 60oC in a water bath for 3 hours or overnight. Then samples were subjected to classical phenol/chloroform extraction with subsequent ethanol precipitation. The DNA pellet dried and dissolved in100 µl TE buffer (2ml of 1 M Tris-Cl and 400µl of 0.5 M EDTA in 1 liter adjusted for pH7.6 and sterilized by autoclaving). DNA concentration was measured spectrophotometrically (OD 260 and OD 280) using UV/ Visible Spectrophotometer (Pharmacia LKB. Ultrospec III). DNA samples stored at 4°C until use. Dilutions of 10ng/µl were prepared as working solutions.

2.3.2 DNA extraction of Clinical samples

Filter papers, unstained smears and stained smears

DNA was isolated as described previously by Meredith et al. (1993) and El Tai et al. (2000). Briefly, filter papers with spotted skin scrapings were punched out with a sterile paper puncher. Between each sample, sterile filter paper was punched and the puncture was cleaned with 70% ethanol to prevent carry-over of DNA contamination. Two punched out sample discs were placed into 250 µl lysis buffer (50mM NaCl, 50mM Tris- HCl; 10mM EDTA pH 7.4; 1% {vol/vol} Triton X-100; 200µg of Proteinase K per ml) and incubated in a water bath for 3 hours or overnight at 60°C. Then the mixture was subjected to phenol-chloroform extraction. An equal volume of buffered phenol was added, shaken gently for 3 minutes and centrifuged at 13000 rpm (maximum speed) for 3 minutes. The upper aqueous phase was then transferred to a new 1.5 ml Eppendorf tube. An equal volume of buffered phenol: chloroform: isoamyl alcohol: (25:24:1) was added, shaken and centrifuged as mentioned above. The upper aqueous phase was again transferred to a new 1.5 ml Eppendorf tube. An equal volume of chloroform-isoamyl alcohol (24:1) was added, shaken and centrifuged as mentioned above. The upper aqueous phase was now precipitated by mixing with 1/10 volume of 3M Na acetate and 2 volumes of absolute ethanol. The tubes were left overnight at – 20°C. Then, samples were centrifuged for 30 minutes at 13000 rpm (maximum speed). DNA pellets were washed with 70% ethanol and centrifuged for 15 min at 13000 rpm (maximum speed). The invisible DNA pellet was dried using speed vacuum dryer (Eppendorf Concentrator 5301, Nethalar-Heinz GmbH, Hamburg-Germany) for 5 minutes till the 70% ethanol was completely evaporated and re-suspended in 100 µl TE buffer (10mM Tris; 1mM EDTA pH 7.5). The pellets obtained from clinical samples were additionally purified using a commercial kit (Machary- Nagel, Düren-Germany). The 30 µl purified DNA was stored at -20°C until use.

In the case of stained and unstained smears, 50 µl of lysis buffer from the 250µl in the Eppendorf was spread on the surface of the slide and with the yellow tip the material was scratched and swept into the tube. The extraction procedure was completed as described above for filter papers.

2.4 PCR amplification: Internal transcribed spacer (ITS1)

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The ITS1 region was amplified with the following primers: LITSR (5’-CTGGATCATTTTCCGATG-3’)/ L5.8S (5´- TGATACCACTTATCGCACTT-3´) as described by El Tai et al., (2000). These primers amplify a section of the ribosomal transcribed spacer (ITS1) region, which separates the genes coding for the ssu rRNA and 5.8S rRNA (300-350 bp) of all Leishmania species (Figure 2.2). Primers were synthesized commercially (TIB-MOLBIOL, Berlin, Germany).

Figure 2.2 Schematic representation of the internal transcribed spacer (ITS) in the ribosomal operon with primers amplifying different parts of the spacer.

Primer sequences are given in the text above. SSU = small subunit rRNA gene, LSU = large subunit rRNA gene.

Amplification reactions were performed in volumes of 50 µl (Table 2.1). On ice and under sterile conditions, 3 µl DNA from clinical samples, 2 µl DNA for re- PCR or a 10ng/µl- working solution from culture, were added to a PCR mix containing 200µM of each dNTP (Amersham Biosciences, UK limited. England); 5 µl of commercially available 10x PCR buffer (10mM Tris-HCl, pH 8.0; 50mM KCl; 1.5mM MgCl2); 1-2 units of Taq polymerase (Applied Biosystems, Roche, USA) and 25 pmol of each primer. Samples were overlaid with sterile, light mineral oil (Sigma-Aldrich, St. Louis, USA) and amplified as follows: initial denaturation at 95°C for 2 min followed by 35 cycles consisting of denaturation at 95°C for 20 sec, annealing at 53°C for primer pair LISTR/L5.8S for 30 sec and extension at 72°C for 1 min. This was followed by a final extension cycle at 72°C for 6 min and then the thermocycler was set at 4°C for infinite.

↓36

PCR was run in Robocycler Gradient 40, Stratagene or Perkin-Elmer Thermocycler 9600. Amplification products were subjected to electrophoresis in 1% agarose NA (Amersham Biosciences, Sweden) at 100 V in 0.5x TBE buffer (0.023M Tris-borate, 0.5mM EDTA) for a 15 cm long gel tray for about 1hour, and at 140 V for a 25 cm long gel tray for about 1.5 hours and visualized under UV light after staining for 15 min in ethidium bromide (0.5µg/ml). One kilo base pair (1kbp) or 123 bp DNA ladders (Invitrogen, Life Tech, Carlsbad CA, USA), were used as molecular size markers. Amplified PCR products were documented by photography (Gene Eagle eye 11, Stratagene, Heidelberg, or GeneGenius, Syngene Europe). Re-PCR was performed to obtain sufficient PCR products.

Table 2.1 Components and quantities for the Master Mix (MM) for one sample

ITS1-PCR

Microsatellite analysis

No.

Item

µl

Final concentration

µl

Final concentration

1

10 x PCR Buffer

5

1x

5

1x

2

dNTPs, 2.5mM each (10mM)

4

200µM

4

200µM

3

Primer, Forward, 10 pmol/µl

2.5

25 pmol

1.5

15 pmol

4

Primer, Reverse, 10 pmol/µl

2.5

25 pmol

1.5

15 pmol

5

DMSO

1.25

2.5%

-----

-----

6

Taq Polymerase, 5 units/ µl

0.2

1 units

0.2

1 unit

7

H2O

32.55

34.8

Total volume of the MM

47

47

DNA template

3

3

Total

50

50

PCR buffer is provided with 1.5 mM MgCl2 in 1 x buffer

2.5 Panel of controls used in diagnostic PCR

During this study a panel of controls was used to detect contamination (false positive) or inhibition (false negative) during PCR. These controls included:

2.5.1 DNA extraction control

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Human housekeeping genes were used to check for the integrity of the extracted DNA and thus for true negativity of the ITS1-PCR results, under the same conditions as described above for ITS1-PCR. The primer pair HβG-F (5`-GAA GAG CCA AGG ACAGGT AC-3`)/HβG-R (5`-CAA CTT CAT CCA CGT TCACC-3`), designed to amplify the genes corresponding to β-globin region of human genome DNA, with a product size of 268 bp, was used as a DNA extraction control (Saiki et al., 1988). Interchangeably with HβG, the β-actin-gene was used as extraction control. The primer pair: Aco1: (5´-ACC TCA TGA AGA TCC TCA CC-3´)/ Aco2: (5´-CCA TCT CTT GCT CGA AGT CC-3´) targets a 120-bp fragment within the fourth exon of the human β-actin gene (Musso et al, 1996). DNA extraction control becomes a necessity for ITS1-PCR negative samples.

2.5.2 Positive and negative controls

DNA from the strain of L. turanica (MRHO/MN/83/MNR-6), 10ng/µl, and sterile double distilled water (B. Braun Melsungen AG, Melsungen) were used as positive and negative controls, respectively.

2.5.3 Inhibition control

In order to detect possible inhibition caused by substances, which were not sufficiently eliminated by the crude extraction (such as hemoglobin), an internal control for PCR inhibition was included, where the same amount of purified L. turanica (MRHO/MN/83/MNR-6), 10ng/µl, DNA as in positive controls was added to each diagnostic sample. Moreover, 2.5% dimethyl-sulfoxyd (DMSO) (Carl Roth GmbH, Karlsruhe) was used as an enhancer in all diagnostic PCR runs. Other precautions such as wearing gloves, frequent cleaning and using disposables were applied to prevent contamination.

2.6 RFLP analysis of ITS1 amplicons

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The amplified ITS1 region was digested using 2 different restriction enzymes (Hae III and MnlI) according to the conditions recommended by the supplier (Hybaid-UK or BioLabs Inc, New England). Briefly, for diagnostic samples 15 µl of the DNA were restricted by addition of 1µl (5 units) of each enzyme and 2 µl of the corresponding 10 x buffer and 2 µl of distilled water and incubated at 37°C for 2 hours. Culture samples were restricted in the same way but using different volumes: 5 µl of DNA are added to 1µl of enzyme and 2 µl of the buffer and 12 µl of distilled water. Restriction products (20 µl) plus 4 µl loading buffer (80% glycerine, 0.1M EDTA pH 8.0, 10 mM Tris-HCl pH 8.0) were subjected to electrophoresis in 2 % MetaPhore agarose (for fine analytical separation of small nucleic acids and PCR products, Cambrex Bio Science Rockland, Inc, Rockland ME, USA) for 2 hours at 110 Volts for medium gel tray, or 140 Volts for a large gel tray in 0.5x TBE buffer (0.023M. Tris-borate, 0.5mM EDTA). Normal agarose (Amersham Biosciences, Sweden) was also successfully used. DNA fragments were visualized under UV light after staining for 15 min in ethidium bromide (0.5µg/ml). The gel was run again for 15 min for increased resolution. Restriction products were documented by photography (Gene Eagle Eye 11, Stratagene, Heidelberg or GeneGenius, Syngene Europe). 1 kb or 123 bp ladders were used. The restriction patterns were compared using an ITS1 restriction map.

2.7 Evaluation studies

2.7.1 Graded microscopy vs ITS1-PCR

2.7.1.1 Patients and study area

In the period between July 2002 and December 2003, a total of 86 patients living in Jericho area in Palestine presenting with skin lesions were referred to Islah medical laboratory-Jericho for the diagnosis of CL by direct smear microscopy, out of which 48 patients were randomly selected as clinically positive for graded microscopy. Stratified random sampling was used to select 20 patients from the 48 for the comparison with PCR. A total of 50% (10/20) were children (<14 years of age), 40% (8/20) were adults (>14 years of age) while 10% (2/20) were of unknown age; 55% were males.

Thirty five percent of the patients (7/20) had only a single lesion, same number had double lesion and the remaining 30% (6/20) had multiple lesions (>3). The distribution of lesions was known in 44 cases and was as follows: 57% (25/44) on the head and neck, 39% (17/44) were in the upper extremities and 4.5 % (2/44) on the lower extremities. Sixty five percent (13/20) of the lesions were ≤ 1 month old, 10% (2/20) were 1-2 months old, 15% (3/20) were 3 or more months old.

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To check for the validity (sensitivity and specificity) of ITS1-PCR as a diagnostic test, fifteen EDTA blood samples were obtained from the blood bank of the Charité Hospital-Berlin as negative controls. These blood donors were all native Germans who never visited any tropical country within the last year and were negative for a set of routinely screened diseases as part of the blood bank profile. Permission was obtained from the Ethics Committee-Charite Hospital in Berlin, Germany. Each patient gave a written declaration to the blood bank service allowing the use of his/her sample for scientific research purposes.

From each blood sample, three drops/squares were thickly and separately smeared on a slide and Giemsa-stained. Outcome of staining was checked by scanning one drop (square) one slide using a 100x immersion lens.

2.7.1.2 Sample collection and preparation:

All 48 patients showed typical lesions. These were cleaned using sterile gauze and physiologic saline and disinfected 5 times in an outward circular motion with 70% alcohol –immersed cotton. Using a sterile lancet or sterile surgical blade, 2-3 mm long superficial incisions were cut on the margins of the lesion and pressure was maintained with finger to achieve hemostasis. For each patient, three touch-smears of dermal tissue scrapings were collected on one slide, air dried, fixed in absolute methanol and Giemsa stained.

2.7.1.3 Standardized graded microscopy:

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For all 20 Giemsa-stained slides selected for the comparative analysis, three 5 mm x 5 mm squares were marked with a fine marker on the back surface of the slide and given the designations 1, 2 and 3 (Figure 3.1). The size of the area marked corresponds to the area of about 600 - 1000 oil immersion fields (OIF), as 1000 OIF per slide have to be screened before declaring negativity (World Health Organization 1990). The squares were purposefully selected to be marked on areas of varying densities on the slide to have examples of the various outcomes of the staining procedure. Each square was completely scanned with a 100x immersion lens by the same blinded person using the same bright-field microscopy (Figure 3.1). A maximum of four slides were scanned daily to prevent exhaustion and hence subjective interpretation. The average time taken to scan a 5mm x 5mm square is 17.5 minutes. The number of amastigotes in each square was quantified and graded as compared to WHO grading used in splenic aspirate smears (World Health Organization, 1990) and the semi quantitative scaling adopted by Ramiraze et al. (2000) for touch smears from lesions of CL. The semi-quantitative grading to evaluate the parasitic density in each slide was performed as shown in table 2.2.

The blood of the control persons was treated exactly the same: three squares were marked on each slide.

After microscopy, DNA was extracted of the material taken from the marked area. PCR amplification for ITS1, DNA extraction control for human β-Actin housekeeping gene and the control panels were carried out as described above.

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McNemar's test was used to compare the matched pairs for graded microscopy and ITS1-PCR. McNemar’s test was performed using the free Quickcalcs web calculator available on www.graphpad.com.

Table 2.2 Grading of parasites in Giemsa-stained smears from skin lesions.

Grade

Average number of amastigotes

Minimum no. OIF scanned

-

0 amastigotes per square *

625 (whole square)

+

1 amastigote per square to 1 per OIF**

625 (whole square)

++

2 to 10 amastigotes per OIF

50

+++

11 to 20 amastigotes per OIF

50

++++

> 20 amastigotes per OIF

10

* Square: 5mm x 5mm corresponding to 625 OIF. **OIF: oil-immersion field

2.7.2 Filter Paper vs Unstained smears and conventional methods vs PCR

During the study period, February 1994 to July 2004, skin scrapings spotted on filter papers and unstained tissue smears (2.2.3) were compared as potential sampling methods for ITS1-PCR. Conventional diagnostic methods including Giemsa-stained smears (2.2.3) and in-vitro culture (2.2.4) were compared with ITS1-PCR (2.3, 2.4 and 2.5). The comparison was based on three different ‘gold standards’: WHO case definition, combined (at least one positive out of the methods used) and the clinical definition which is based on the presence of a lesion and relevant epidemiological data.

2.8 Genetic microsatellite variation and global molecular epidemiology

2.8.1  Study area

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For the study of genetic variation and molecular polymorphism, 106 strains of L. major were sampled from 9 African countries (Egypt, Sudan, Kenya, Tunisia, Algeria, Morocco, Senegal and Burkina Faso) and 10 Asian countries (Jericho area and Negev desert, Saudi Arabia, Kuwait, Iraq, Iran, Turkey, Turkmenistan, Uzbekistan and Kazakhstan) (Figure 2.3, Table 3.11).

Figure 2.3 Map showing the countries of origin for microsatellite analysis. One strain is not shown on the map as the origin was an unknown country in Africa.

2.9 Microsatellite markers

Ten multilocus microsatellite markers located on 5 different chromosomes (1, 3, 5, 21 and 35) were designed previously by our group (Table 2.3). Microsatellite repeats, such as (CA)n, (AT)n, (GTG)n, and (GACA)n, that vary in length were identified within numerous sequences of L. major available from sequence data bases. Primers annealing to the unique flanking regions have been designed to specifically amplify microsatellite markers (Figure 2.4). To avoid the influence of insertion/deletion events in adjacent regions, primers were designed that anneal very close to the repeat (5-6 nucleotides apart from it on both sides). PCR products obtained from different L. major strains were separated in polyacrylamide gels and screened for length polymorphisms within the microsatellite repeats.

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Figure 2.4 Schematic representation of the45GTG marker in which the sequence consist of the repeat region in the middle, (GTG) 12, flanking regions of 5-6 bp on each side, and primer sequence of 20 bp on each side of the flanking regions.

Table 2.3 The 10 microsatellites markers used for the analysis of populations of L. major.

#

Marker

Primers

Ann. Temp. o C

Chrom.

Location

Size (bp)

# Repeats in

L. major

(Friedlin)

1

4 GTG

f CGG TTT GGC GCT GAA AGC GG

r CGT GAG GAC GCC AC CGAG GC

58

35

6460-530

70

7

2

27GTG

f GGA GGT GGC TGT GGT TGT TG

r GCC GCT GAC GCT GCA GGC T

58

3

1440-515

75

9

3

36GTG

f AGC GAA GAA GAG TCG GGC AG

r GCG CCT TCA GTG CGT CGT CC

62

1

140895-963

68

9

4

39GTG

f GTC TTG CCG CGA GGT GAC CG

r CCA GCA CCA GCA CCA CCA TC

58

1

202765-851

86

9

5

45GTG

f A CG GCC GGG TGG TCG TGG GT

r CGT TCG CAC GCA CGC ACG CA

58

1

59751-890

139

12

6

1GC

f CTG GCA CGC ACA CCC ACA CA

r ATC TGC GCT CAT CTG GCG AG

60

3

10323-386

109

7

7

28AT

f TTG CCT ATC AAC ACA AGG CT

r AGT CTC TCT CTC TCT CTA TA

42

5

27966-031

65

9

8

71AT

f TCT TGC GAA GGT GTG GTC TT

r AGC CCA CGT GTA CAT GTG TG

50

21

22113-168

55

13

9

1GACA

f GAA AGG GCA GGA GGA CGG AT

r CAC ACA CAC ATA CAC ACA TA

54

1

68459-534

75

7

10

1CA

f TTA GTT CCA TCA TAC ACC CG

r CGT TCG ACG TGG AGA ATA AG

48

35

30151-238

87

14

Ann. =Annealing; Chrom. =Chromosome

2.9.1 Amplification of microsatellite markers by PCR

The short tandem repeats (STR) or microsatellites from the 106 cultured samples sent by the collaborating laboratories were amplified in volumes of 50 µl as described above (Table 2.1). The primers were synthesized commercially (TIB-MOLBIOL, Berlin, Germany). Three µl DNA from a 10 ng/µl- working solution were added to a PCR mix containing 200 µM of each dNTP (Amersham Biosciences UK limited. England); 5 µl of commercially available 10x PCR buffer (10mM Tris-HCl, pH 8.0; 50mM KCl; 1.5mM MgCl2); 1 unit of Taq polymerase (Applied Biosystems, Roche USA) and 20 pmol of forward primer and 15 pmol of reverse primer. Samples were overlaid with sterile, light mineral oil (Sigma-Aldrich, St. Louis-USA) and amplified as follows: initial denaturation at 94°C for 5 min followed by 35 cycles consisting of denaturation at 94°C for 30 sec, annealing at annealing temperature for 30 sec and extension at 72°C for 1min. This was followed by a final extension cycle at 72°C for 10 min and then the thermocycler was set at 4°C for infinite.

↓44

PCR was run in Robocycler Gradient 40, Stratagene or Perkin-Elmer Thermocycler 9600 and subjected to electrophoresis as described in 2.4.

2.9.2 Polyacrylamide gel electrophoresis (PAGE)

As described by Sambrook (2001), long run gel glass plates (450x350 mm) were cleaned well with detergent solution and rinsed with warm water and then with deionized water and dried. The glass plates were then rinsed with 70% ethanol, dried and washed once again with 96% absolute ethanol and dried. One of the glass plates, preferably the notched one, was wiped with acrylease (Stratagene, Heidelberg) or Gelsave (Applichem GmbH, Darmstadt, Germany) to prevent sticking. Spacers (0.4 mm) were placed between the plates of both sides and the bottom side and wrapped with sticky tapes. The two glasses were fixed together using bulldog paper clips. Adhesion promoter, Silan A174 (Merck kGaA, Darmstadt, Germany) was gently added on the free side where the comb inserted by wetting a tip of a kimwip (Kimberly-Clarck Corp) and gently inserting it between the two plates to a depth equal to the teeth of the comb and then running it two times between the two ends of the glass plates.

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These samples were loaded on 12% acrylamide gel 350 x 450 x 0.4 mm [45 ml 29% Acrylamide plus 1% N,N´-Methylenebisacrylamide (Rotiphorese® Gel 40 (29:1), Carl Roth GmbH &Co, Karlsruhe, Germany), 10 ml 10x TBE (10.8 %Tris, 5.5% Boric, 0.02 M EDTA pH 8), 90 ml distilled water, 750 µl 10% APS or 0.35 g of the APS powder (Ammonium peroxide sulphate, Merck, E. merck, Darmstadt-Germany)]. Then 75 µl of the gelling factor, NNNN-Tetramethylene diamine (TEMED) (Merck, Darmstadt, Germany or Serva, FeinBiochmica, Heidelberg, Germany) were added and swirled gently before pouring. The samples were subjected to electrophoresis in 0.5xTBE or 1xTBE for over-night runs (0.023M Tris-borate, 0.5mM EDTA). Electrophoresis was run successfully at 1 kV, 250mA and 7 Watts for 19 hours, depending on the length of run. Ten base-pair DNA ladder (Invitrogen, Life Tech, Carlsbad, CA USA) was used as a molecular size marker

2.9.3 Silver staining

Following electrophoresis, the gel was fixed in 1% Nitric acid for 15 min, washed in distilled water for 5 minutes and then stained in 0.2% AgNO3 for 25 min. After washing for 10 min in distilled water, the gel was placed in freshly prepared developing solution (89.2 g Sodium carbonate in double distilled water (0.28 M) plus 1 ml formaldehyde) until bands clearly appeared. The gel was washed for 10 minutes in distilled water before the reaction being stopped by 10% glacial acetic acid for 5 minutes and then washed for 5 minutes, transferred to blotting paper 460x570mm, wrapped in Saran plastic film (Dow Chemical Company) and dried in a Slab gel dryer (Savant-Hicksville, N.Y., USA) at 80°C for 2 hours. After complete drying, Saran Wrap was peeled off and the dried gel was labeled, scanned and photocopied for archiving purposes.

2.9.4 Capillary electrophoresis (CE) using CEQTM 8000 Beckman coulter

PCR products for CE were prepared as described above for PAGE, with an exception of using Dye-Labeled forward primer (Proligo). CEQ DNA Size Standard – 400 was used. CEQ Separation Gel was loaded in the CEQTM 8000 Beckman coulter and run according to manufacturer’s instructions. Bands are shown in the form of peaks that show the size of the fragments.

2.10 Data analysis: clustering methods and presentation of genetic data

↓46

Genetic data were processed using two clustering methods: distance-based methods and model-based methods. Two distance-based methods were utilized: Neighbour-Joining (NJ) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA), while admixture model was used as model-based method. Three software programs were employed for drawing the NJ and UPGMA phylogenetic trees, MICROSAT, PHYLIP version 3.6 and PAUP* version 4.0beta. Structure 2.0 was used to classify the genetic data into discrete populations based on the admixture model.

Software used, see Table 2.4.

2.10.1 Calculating a distance matrix using MICROSAT

The free software MICROSAT (Minch et al, 1997) (http://hpgl.stanford.edu/projects/microsat/) was used to calculate the genetic distance between 67 multilocus microsatellite profiles obtained for the 106 strains when analysed with the 10 microsatellite markers. Changes in allelic sizes by one or more repeats were computed utilizing two measures: proportion of shared alleles (Dps) and Delta mu squared (Dµ2).

↓47

Dps calculates multilocus pairwise distance measurements as 1 – (the total number of shared alleles at all loci/n), where n is the number of loci compared (Bowcock et al., 1994). The second, Dµ2 is based on the average squared difference in allele size (Goldstein et al., 1995). Confidence intervals for Dps and Dµ2 were calculated by bootstrapping (1000 replicates) over loci. The output file containing the matrix was used for drawing the phylogenetic trees using PHYLIP and PAUP.

2.10.2 Drawing of NJ and UPGMA consensus trees using PHYLIP/ PAUP

The output file containing 1000 matrices for both Dps and Dµ2 distance measures between the 67 CL genotypes were fed into PHYLIP version 3.6 (J. Felsenstein, 2004) available through http://evolution.gs.washington.edu/phylip.html) and PAUP* version 4.0b8 (Swofford 2001) to construct neighbor-joining and UPGMA rooted trees.

2.10.3 Structuring populations with Structure 2.0

The software Structure Version 2 (Pritchard et al., 2000a) was implemented to classify the whole bulk of 106 CL isolates with their corresponding genetic data represented by the number of repeats for 10 microsatellite markers. The program was run using admixture model with length of burn-in period of 30,000 iterations and followed by 1,000,000 of MCMC (Markov chain Monte Carlo) repeats after burn-in. Two types of runs were conducted, one with 5 pre-defined populations (Central Asia, Middle East, North Africa, East Africa and West Africa) in which each CL isolate was assigned to one of these geographical sub-divisions. The second attempt was implemented without using the population ID (geographical origin of sample). Number of populations probabilistically assigned for the second run was determined by drawing a curve with x-axis as number of K (population) and y-axis as mean value of ln likelihood ( ln Pr(x\k). The optimal number of K (population) is the first value of K at which plateau starts.

2.10.4 F-Statistic by F-STAT and GENEPOP

↓48

The degree of genetic differentiation among populations (106 isolates) was estimated by Wright’s F-statistics (Wright 1978) as calculated by Weir and Cockerham (1984). Two F statistics were calculated: Fis (inbreeding coefficient) and Fst (differentiation among population) pairwise calculation. This was carried out by FSTAT software version 2.9.3.2 (Feb. 2002) (Goudet 1995) available http://www2.unil.ch/popgen/softwares/fstat.htm . Also, for double checking the results, Fst was calculated by GENEPOP (Rousset and Raymond, 1995) which is a population genetics DOS-based software package downloaded http://wbiomed.curtin.edu.au/genepop/index.html or an online calculation on http://wbiomed.curtin.edu.au/genepop/genepop_op1.htm. Number of alleles per locus was also calculated by FSTAT.

2.10.5 Descriptive statistics for markers by GDA

Descriptive statistics for the markers used to assess genetic variation were calculated using Gene Data Analysis (GDA), version 1.0 (d16c), a free software available on http://lewis.eeb.uconn.edu/lewishome/software.html (Lewis and Zaykin, 2001). The calculation was based on permutation method (Zaykin et al., 1995) which is useful for multiallelic microsatellite loci. The measures used to assess genetic variation were: n: average sample size; A: mean number of alleles per locus; (Ho) and expected (He) heterozygosity under Hardy–Weinberg equilibrium and Fis: the inbreeding coefficient.

2.11 Epidemiological data banking and analysis: Epi Info™

Epi Info™ is CDC free software. With this software a questionnaire was developed to customize, enter and analyze data. It is designed for public health and medical professionals. Epi Info™ was originally DOS and now it is available for Windows. http://www.cdc.gov/epo/epi/epiinfo.htm

2.12 Geographical clustering and public health surveillance

↓49

For public health surveillance, SaTScanTM v5.0 freeware (Kulldorff, 1998) was used to detect statistical evidence for spatial and space-time clustering of L. major, L. tropica and all CL cases (L. major + L. tropica + and undetermined). SaTScan was used to test the null hypothesis that the risk of L. major, L. tropica and CL is the same in all studied populations. Under the null hypothesis, and when there are no covariates, the expected number of cases in each area is proportional to its population size. Poisson data was analyzed with the purely spatial and the space-time scan statistics. For space-time, the unit of the time interval was one year.

The Poisson model was provided with the cases and their corresponding sampling dates as well as the population counts in 1997 (last Palestinian census) and 2004 (Palestinian Central Bureau of Statistics, 2005) for 9 geographical areas in the District Jericho (~600 km2) with their corresponding geographical coordinates for each of those locations. Geographical coordinates were allocated as the centre of the built-areas.

The population data was specified for two years, one is the census time in 1997 and the other is its 2004 projection. For times in between, SaTScan does a linear interpolation based on the population at the census times immediately preceeding and immediately following. For times before the first census time, the population size is set equal to the population size at that first census time, and for times after the last.

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Using the geographical and the population data fed into the Poisson model, two statistics were used: spatial scan statistic and space-time scan statistic.

2.12.1  Spatial scan statistics

The purely spatial scan statistic imposes a circular window on the map. The window is in turn centred on each of several possible coordinate points (latitude/longitude) positioned throughout the study region. For each coordinate point (latitude/longitude), the radius of the window varies continuously in size from zero to upper limit of 50% of the population at risk. In this way, the circular window is flexible both in location and size. In total, the method creates an infinite number of distinct geographical circles with different sets of neighbouring data locations within them. For each circle, a likelihood ratio is computed for the alternative hypothesis that there is an increased risk of CL inside the circle against the null hypothesis that the risk of being CL (+) inside and outside the circle is the same. The candidate circle is the one with the highest likelihood ratio. The likelihood ratio-based test statistic takes multiple comparisons into account that resulted from looking for clusters in many different locations and sizes. The statistical significance (p<0.05) of this large circle (large likelihood ratio) is calculated by determining its distribution under null hypothesis through Monte Carlo simulation (1000 simulations). This produces a main cluster alongside other secondary clusters. Another way of looking for other cluster is to remove the cases of the main cluster from the input file and repeat the analysis. In the spatial statistic, the time is totally ignored.

2.12.2 Space-time scan statistics

The space-time scan statistics are defined by a cylindrical window with a circular geographic base and with height corresponding to time. The base is defined exactly as for the purely spatial scan statistics, while the height reflects the time period of potential clusters. The cylindrical window is then moved in space and time, so that for each possible geographical location and size it also visits each possible time period. In effect, we obtain an infinite number of overlapping cylinders of different size and shape, jointly covering the entire study region, where each cylinder reflects a possible cluster. The main cluster is computed as described above in the spatial statistics (Kulldorff, 2001; Kulldorff et al., 2005).

2.12.3 Adjustment for season relative risk

↓51

Based on the historical data (Al-Jawabreh et al., 2003) it is known that in summer months (beginning in April till the end of September) the number, in general, is reduced in all areas to approximately half.

We adjusted for this relative risk by using the adjustments file. In this file, a relative risk of 0.5 for all geographical areas for all the years included in the study (1994-2004) was applied.

2.12.4 Adjusting for covariates

Covariates as age group and sex were not adjusted for, since the two covariates are randomly distributed geographically, and since age group and sex distribution is uniform and constant all over the Jordan valley.

2.13 Shewhart’s Chart

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Shewhart Chart or Levy-Jennings Chart was originally created for industrial applications and then applied on a large scale in medical laboratories to check the work performance. In this study we applied this technique for epidemiological monitoring and surveillance.

The mean (m,) for the number of cases of CL in the period between 1994 and 2004 was calculated. This mean will form the center line representing the target value. Then, standard deviation (SD) was calculated to set up the upper and lower limits commonly know as UCL (upper control limit) and LCL (lower control limit), symmetrical about the center line. The limits are chosen so that almost all of the data points will fall within these limits as long as there is no peak or outbreaks. In a clinical laboratory the LCL and UCL are +/- 2SD. However +/-1SD can be considered for epidemiological purposes. Grubb’s test was used to check for outliers.

A graph with the center mean line and UCL and LCL line is drawn and the annual CL totals are plotted on the time line graph.

↓53

Table 2.4 Software packages used in the study

Program

Operating system

Purpose

Author

Download

Epi Info

Windows

Epidemiologic analysis for health data

CDC

http://www.cdc.gov/epo/epi/epiinfo.htm

MICROSAT

DOS

Calculation of distances from microsatellite data

Eric Minch

http://hpgl.stanford.edu/projects/microsat/

Phylip version 3.6

DOS

Construction of neighbor-joining and UPGMA rooted trees

J. Felsenstein

http://evolution.gs.washington.edu/phylip.html

PAUP version 4.0beta

DOS

Construction of neighbor-joining and UPGMA rooted trees

Swofford

http://paup.csit.fsu.edu/

Structure Version 2

Windows

Estimation of underlying population genetic structure

Pritchard

http://pritch.bsd.uchicago.edu/software.html

GDA version 1

Windows

Population genetic analysis package that includes estimation of LD for pairs of loci; significance tested by

permutation method

P. O Lewis and D. Zaykin

http://alleyn.eeb.uconn.edu/gda/

FSTAT version 2.9.3.2

Windows

Estimation of genetic differentiation among populations

Goudet

http://www2.unil.ch/popgen/softwares/fstat.htm

GENEPOP

DOS and online

Calculation of LD, F-statistics and others

Rousset and Raymond

http://wbiomed.curtin.edu.au/genepop/index.html

SaTScan

Windows

Detection of statistical evidence for spatial and space-time clustering of leishmaniasis

Kulldorff

http://www.satscan.org

LD: linkage disequilibrium


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