An Eco-Epidemiological Study on Zoonotic Cutaneous Leishmaniasis in Central Iran.

BACKGROUND: Leishmaniasis is an expanding neglected tropical disease in the world reporting from 98 countries including Iran. This study focused on eco-epidemiological determinants of the disease following a rapid and unexpected increase of leishmaniasis incidence in a strategic residential district in North-East of Isfahan County, Iran.
METHODS: This study was accomplished from Apr 2012 to Jan 2014 in a strategic residential zone in North-East of Isfahan County, Esfahan, Iran. Monthly activity, parity, Leishmania infection and susceptibility tests, were determined on sand flies. Some portion of inhabitants and school children were surveyed to find active or passive cases of leishmaniasis and also wild rodents were collected to determine reservoir host.
RESULTS: Totally 5223 sand flies belonging to Phlebotomus and Sergentomyia genus were collected and identified; Ph. papa-tasi was the dominant species and started to appear in May and disappeared in Oct. The majority of living dissected sand flies were unfed and parous. Ph. papatasi showed 4.6% Leishmania infection through direct examination and 39.54% by nested-PCR respectively. Phlebotomus papatasi was susceptible against deltametrin 0.05%. Totally 2149 people were surveyed and incidence and prevalence of zoonotic cutaneous leishmaniasis estimated as 45.39 and 314.40 per 1000 population. Rodents showed 73.91% and 80% Leishmania infection by direct examination and nested-PCR respectively.
CONCLUSION: Cutaneous leishmaniasis due to L. major has been established in this area. Rodent control operation and personal protection are highly recommended to control the disease in this focus.

Introduction

Leishmaniasis is an expanding neglected tropical disease in the world reporting in 98 countries including Iran. More than 350 million people are at risk and 12 million are suffering from the diseases and its side effects (1,2). Zoonotic cutaneous leishmaniasis (ZCL) has been reported from 17 out of 31 provinces in Iran (3) and Phlebotomus papatasi is considered as the main vector of the disease (4). Rhombymos opimus (great gerbil) and Meriones libycus (Libiyan jird) are considered as the main and secondary reservoir hosts of the disease in the northeast and central part of Iran respectively (5).

In the recent years eco-epidemiological changes such as urbanization, constructions of buildings near colonies of the rodents, expanding rodent population due to human intervention in their natural habitats and immigration of non-immune people to the endemic areas, caused a significant distribution of ZCL in the country specially in Isfahan (6). Lack of sufficient information about different reservoir host, sand fly vectors, insecticide resistance among the vectors and resistance to the conventional drug/s and also inaccessibility to a licensed vaccine made the control of disease challenging (7).

It is postulated that, successful establishment of the disease in an endemic area is the outcome of a close association between the Leishmania parasite/s and its natural sand fly vector/s (8). Thus, vector and parasite identification has a great impact on predicting expansion of the disease in the endemic areas, and also it helps authorities to design new strategic control program of the disease (9).

Following a rapid and unexpected increase in the incidence of ZCL in a residential strategic area in north east of Isfahan City (personal communication, Isfahan Health Center, Iran), conducting a survey on different eco-epidemiological aspect of the disease was absolutely essential and encouraged the authors to study eco-epidemiological determinants of the disease in the area for proposing an integrated control program. This is the first comprehensive study in this high risk strategic focus.

Materials and Methods

Ethics approval

All experiments were approved by Research Deputy of Tehran University of Medical Sciences (92-02-27-23198).

Study area

This study was accomplished from Apr 2012 to Jan 2014 in a strategic residential zone in North-East of Isfahan County (51°51′17″E and 32°44′49″N) with the elevation of 1534.5 m, Iran. The average annual minimum and maximum temperature recorded as 4.7 °C and 36.3 °C in Jan and July, respectively. The total annual rainfall was 157.5 mm. (Iran Meteorological Organization, 2012).

Entomological surveySand fly collection and identification

Sand flies were collected from indoors (bedroom, bathroom, toilets and hall) and outdoors fixed places (rodent burrows) using 30 sticky traps sunset to sunrise.

The head and last two abdominal segments of the sand flies were mounted in puris’ medium (10) and identified using valid identification keys (11,12). Living sand flies were collected using mouth aspirator from outdoor on a car parked near gerbils’ colonies.

Physiological status and parity test

Abdominal status (unfed, fresh fed, semi-gravid and gravid) of sand flies were recorded. Parous females were distinguished from nulliparous sand flies by observation of the follicular relies of the ovaries (13), and also the appearance of the accessory glands and the genital atrium (14).

Leishmania infection

Female sand flies, captured from rodent burrows, were dissected in a fresh drop of sterile saline (9/1000) and checked for the presence of promastigote form of parasite inside the alimentary canal in Aug and Sept 2013. Samples were subjected to molecular experiment to further confirmation.

Susceptibility tests

Adults were left undisturbed at least for one h, and then the susceptibility tests were carried out according to the WHO recommendation, modified for sand flies by Saeidi et al (7). Sand flies were transferred into the exposure tubes at pre-defined time intervals, and then mortality was recorded after 24 h in optimum condition; cases of 5 to 20 percent mortality, were corrected by Abbott’s formula (15).

Population survey

During 2012, randomly 300 households were questioned to gain data such as: age, sex and the presence of acute lesions or scars of leishmaniasis once a season. Additionally, 1184 students from 7 schools, 7–18 yr. old, were questioned. Microscopic slides from active lesion/s were prepared. The lesion serosity was transferred on the slides as well as into alcohol 96% for complementary molecular parasite detection.

Reservoir survey

From Apr 2012 to Mar 2013 to determine the reservoir host/s and detection of Leishmania infection, rodents were collected monthly using at least 20 Sherman traps, baited with cucumber and/or bread near gerbils’ colonies. Impression smears were prepared from the rodents’ ears, stained by Giemsa and were observed microscopically for Leishmania amastigote form. Finally, both ears were cut and transferred into a 1.5 ml microtube to conducting supplementary molecular experiments.

Molecular studyDNA extraction

Genomic DNA from sand flies, human and rodent samples was extracted using Exgene™ Tissue SV (GeneAll®, Korea), following the manufacturers protocol with some modifications.

Polymerase chain reaction

Ribosomal Internal Transcribed Spacer 2 region (ITS 2) of Leishmania parasite DNA was amplified by nested-PCR in an Applied Biosystems thermocycler using external and internal primers; Leish out F (5'-AAA CTC CTC CTC GGT GCT TGC – 3') and Leish out R (5'-AAA CAA AGG TTG TCA GGG G-3') Leish in F (Forward: 5'-AAT TCA ACT TCG CGT TGG CC-3') and Leish in R (Reverse: 5'-CCT CTC TTT TTT CTC TGT GC-3'). The PCRs were carried out based on previous study (16) with minor modification.

Restriction Fragment Length Polymorphism

To distinguish common Leishmania species cycling among vector/s reservoir/s and human in the region, amplified DNAs were digested by Rapid Digest Mnl1 (Cat. No: RD 1191) according to the company’s instruction. The products were loaded onto 2.5% (w/v) agarose gel electrophoresis in Tris/Borate/EDTA (TBE) buffer. Finally, gels were stained by ethidiumbromide (0.5 μg/ml), and photographed.

Statistical analysis

Data were analyzed using SPSS (Chicago, IL, USA) version 18 and Fisher's exact tests were used to compare incidence and/or prevalence differences between male and female or ages.

Results

Entomological survey

Totally 5223 sand flies were collected and identified from the studied area (Table 1). Phlebotomus papatasi was the dominant species in both indoor and outdoor places; reached to its highest activity in early Jul and early Aug in outdoors and indoors, respectively (Fig. 1). The sex ratios calculate at 72.70 in outdoors and 137.5 in indoors for Ph. papatasi.

Table 1:

The fauna, the number and percentages of sand flies from indoors and outdoors in the studied area, North-East of Isfahan city, Isfahan Province, Iran 2012

Species	Outdoor				Indoor				Total
	Male		Female		Male		Female
	No.	%	No.	%	No.	%	No.	%	No.	%.
Ph. papatasi	1137	42.1	1564	57.90	22	57. 9	16	42.1	2739	52.44
Ph. ansarii	610	42.07	840	57.93	1	33.33	2	66.67	1453	27.81
Ph. caucasicus	149	46.41	172	53.59	0	0	0	0	321	6.14
Ph. sergenti	0	0	239	100	0	0	0	0	239	4.57
Se. sintoni	55	11.73	414	88.27	1	50	1	50	471	9
Total	1951	37.66	3229	62.34	24	55.81	19	44.19	5223	100

Fig. 1:

Seasonal activity of Phlebotomus papatasi, in the studied area, North-East of Isfahan city, Isfahan Province, Iran 2012

Physiological status, parity and natural Leish-mania infection

In 2012, as much as 69 samples of sand flies were captured by sticky traps and the majority was identified as un-fed and parous. In addition, at the following year (2013), 101 living sand flies were captured by aspiration and all of them were identified as Ph. papatasi. The majority of the collected sand flies were unfed and parous (Table 2). Among 130 female Ph. papatasi, checked microscopically, 6 (4.6%) had natural Leishmania infection, and no infection was detected in other species (Table 2). The result of nested-PCR revealed Leishmania infection in 5 Ph. papatasi (17.85%) (Table 3).

Table 2:

Physiological status, parity and Leishmania infection rate of sand flies, collected form rodent burrows, North-East of Isfahan City, Isfahan Province, Iran 2012–2013

Year/collecting method	Species	Physiological status				Parity			No. infected	Infection Rate	Total
		Un-Fed	Blood Fed	Gravid	Semi-gravid	Parous	Nulliparous	Unrecognized
2013/Mouth aspirator	Ph. papatasi	97	0	1	3	74	27	0	5	4.95	101
	Ph. papatasi	16	6	2	5	22	5	2	1	3.44	29
2012/Sticky trap	Ph. ansari	10	7	1	3	16	4	1	0	0	21
	Ph. caucasicus Group	5	1	2	1	6	2	1	0	0	9
	Ph. sergenti	4	1	3	0	7	1	0	0	0	8
	Se. sintoni	1	1	0	0	2	0	0	0	0	2
Total		133	16	9	12	127	39	4	6	3.53	170

Table 3:

Results of molecular Leishmania detection in sand flies captured in North-East of Isfahan City, Isfahan Province, Iran 2012–2013

Year/collecting method	Species	No.	L. major		L. turanica		Mix(L. major &L. turanica)		Total infection
			No.	%	No.	%	No.	%	No.	%
2012/Sticky trap	Ph. papatasi	28	4	14.28	0	0	1	3.57	5	17.85
	Ph. sergenti	1	0	0	0	0	0	0	0	0
	Ph. ansarii	3	0	0	1	33.33	0	0	1	33.33
2013/Aspirator	Ph. papatasi	101	32	31.68	10	9.9	4	3.96	46	45.55
Total		133	36	27	11	8.27	5	3.75	52	39.1

Among 101 live Ph. papatasi specimens, the parasite infection observed in 5 (4, 95%) under stereomicroscope (Table 2, Fig. 2), though nested-PCR showed 45.55% Leishmania infection (Table 3 and Fig. 3). Additionally, out of 129 Ph. papatasi, subjected for PCR, 10 were infected by L. turanica and 5 had mixed infection of L. major and L. turaniaca (Table 3, Fig. 3 and Fig. 4). Among 3 tested Ph. ansari, one was found infected by L. turanica (Table 3). Visually distinguished sequence of detected Leishmania species, digested by mnl1, shows in Fig. 5.

Fig. 2:

Leishmania major promastigote form, detected from an infected Phlebotomus papatasi digestive tract, stained by Geimsa

Fig. 3:

Nested-PCR amplification of DNA extracted from Phlebotomus papatasi, M, 50 bp ladder (Fermentas); lane 1, Leishmania turanica; lane 2, L. major; lane 3, L. major reference strain (MRHO/IR/75/ER); lane 4, L. turanica reference strain (MRHO/SU/1983/MARZ-051); lane 5, negative control (distilled water)

Fig. 4:

Agarose (2%) gel electrophoresis of nested PCR products digested by MnlI restriction enzyme, M, 50bp ladder (Fermentas): lane1, negative control (distilled water); lane 2, 5 and 6, L. major; lane3 and 4 mixed infection of L. major and L. turanica.

Fig. 5:

Probit regression lines of Deltamethrin 0.05% against Phlebotomus papatasi using WHO modified protocol

Totally, 519 adult Ph. papatasi were subjected to susceptibility tests against Deltametrin 0.05%, in 22 replicates. The mortality rates against Delta-metrin 0. 05% for 7, 14, 28, 56, 113, 225, and 450 sec of exposure time, passing 24 h recovery, were 12.11, 23.13, 40.45, 43.63, 55.97, 87.56 and 100 % respectively. The parameters of regression line to Deltametrin 0.05%, values of LT50 95% and LT90 95% to Deltametrin 0.05% are shown in Table 4. The Probit regression lines are shown in Fig. 5.

Table 4:

Parameters of probit regression lines of Deltametrin 0.05% against Phlebotomus papatasi

Y=a+bx	P-value (Heterogeneity)	χ2 (df)	LT50 95%CId	LT90 95%CIc	B±SEb	Aa
Y = −2.1252 +1.3677 X	0.05<	18.259(5)	62.8821	1472.1250	1.3677±0.230	−2.1252
			35.7989	309.6956
			17.3820	149.7231

A = intercept.

B± SE = slope and its standard error.

LT50, 95% CI = lethal time causing 50% mortality and its 95% confidence interval.

LT90, 95% CI = lethal time causing 90% mortality and its 95% confidence interval.

Human infection

Among 965 inhabitants 31 persons (58.06% male and 41.94% female) had active lesion of cutaneous leishmaniasis. Maximum and minimum of active lesions were seen in ≥ 25 yr. old and 20–25 yr. old age groups. Out of 965 surveyed people 299 persons (51.5 % male and 48.5% Female) had scar. In 2012 incidence and prevalence of ZCL estimated as 46.55 and 341.97 per 1000 population, respectively (Table 5). Furthermore 1184 students were questioned and examined for leishmaniasis, 26.52% and 1.77% of the students had scar and active lesion respectively (Table 6).

Table 5:

The prevalence of active lesions and scar rate by seasons and district, North-East of Isfahan City, Isfahan Province, Iran 2012

District	Spring				Summer				Fall				Winter				Incidence (Per 1000)
	Population	Scar/s	Active	Freq. Active (%)	Population	Scar/s	Active	Freq. Active (%)	Population	Scar/s	Active	Freq. Active (%)	Population	Scar/s	Active	Freq. Active (%)
1	349	78	0	0	350	78	3	11.03	357	81	6	21.74	359	87	0	0	33.09
2	273	75	0	0	275	75	5	25	277	80	4	20.29	280	84	0	0	45
3	323	115	0	0	326	115	8	37.91	323	123	5	25	326	128	0	0	61.61
Total	945	268	0	0	951	268	16	23.42	957	284	15	22.29	965	299	0	0	46.55

Table 6:

The prevalence of active lesions and scar rate by age among the students (both sexes), North-East of Isfahan City, Isfahan Province, Iran, 2012

Genus	Male					Female					Total
Age	No. examined	No. scars	Scar (%)	No. Active	Active (%)	No. examined	No. scars	Scar (%)	No. Active	Active (%)	No. examined	No. scars	Scar (%)	No. Active	Active (%)
7	103	21	20.38	2	1.94	121	17	14.04	1	0.82	224	38	16.96	3	1.33
8	65	16	24.61	4	6.15	94	22	23.40	2	2.1	159	38	23.89	6	3.77
9	68	25	36.76	2	2.94	81	19	23.45	2	2.46	149	44	29.53	4	2.68
10	61	19	31.14	0	0	62	12	19.35	1	1.61	123	31	25.20	1	0.81
11	88	16	18.18	0	0	91	31	34.06	2	2.19	179	47	26.25	2	1.11
12	51	20	39.21	0	0	9	9	100	0	0	60	29	48.33	0	0
13	29	13	44.82	1	3.45	69	21	30.43	2	2.89	98	34	34.69	3	3.06
14	61	16	26.22	1	1.64	47	20	42.55	1	2.12	108	36	33.33	2	1.85
15	18	2	11.11	0	0	0	0	0	0	0	18	2	11.11	0	0
16	30	8	26.66	0	0	0	0	0	0	0	30	8	26.66	0	0
17	33	4	12.12	0	0	0	0	0	0	0	33	4	12.12	0	0
18	3	3	100	0	0	0	0	0	0	0	3	3	100	0	0
Total	610	163	26.72	10	1.63	574	151	26.30	11	1.91	1184	314	26.52	21	1.77

Reservoir host examination

Totally 58.62% and 80% of examined rodents were infected by Leishmania parasite using direct examination and nested-PCR respectively (Table 7).

Table 7:

Number of collected rodent and their infectivity to Leishmania, North-East of Isfahan city, Isfahan Province, Iran 2012–2013

Species	No. Male (%)	No. Female (%)	Total	Positive by direct examination (%)	Examined/positive by nested-PCR
					No. Examined	L. turanica (%)	L. major (%)	Mixed (%)
R. opimus	4 (26.67)	11 (73.33)	15	9 (60)	11	5 (45.45)	0	5 (45.45)
M. libycus	6 (42.86)	8 (57.14)	14	8 (57.14)	14	1 (7.14)	7 (50)	2 (14.29)
Total	10 (34.49)	19 (65.51)	29	17 (58.62)	25		20 (80)

Discussion

In the current study different epidemiological determinants which facilitates establishment of ZCL in a strategic residential area located at North-East of Isfahan County, were studied. Ph. papatasi was the main and dominant species in indoor and rodents borrows of the study area. In our previous entomological study similarly, the number of sand flies captured form rodent’s burrows were much more than indoor places and also Ph. papatasi was the predominant species either in indoor or outdoor places. (17). Ph. papatasi appeared in late May and disappeared in the late Oct and it appeared in the late Jun and disappeared in the early Oct in outdoors and indoors respectively. Ph. papatasi started to appear in the last days of Apr and disappeared in the last days of Oct (17). Same to the results of other studies, the number of captured sand flies in outdoor resting places were more than indoors (18–20), but surprisingly in the current study over 99% of sand flies were captured from outdoors.

Here, natural Leishmania infection was detected either by direct observation or nested-PCR in Ph. papatasi. Ph. papatasi could be infected either by L. major or L. turanica and mixed infection of both. A previous study showed 37.8% and 18% natural Leishmania infection in Ph. papatasi and Se. sintoni consequently (17). Our previous study showed Leishmania infection in Ph. papatasi (4.5%), Ph. caucasicus (2.3%) and S. sintoni (2.8%) (19). During the last two decades several studies confirmed the presence of L. major in Ph. papatasi by Nested-PCR, Semi Nested-PCR, and Rapd-PCR or isoenzyme (17,21, 22).

As the results shows, the majority of captured sand flies in the studied area found un-fed and parous. Other studies conducted previously inside the country showed the majority of dissected sand flies were parous as well (17,19, 20).

Phlebotomus papatasi was completely susceptible to deltametrin 0.05%. In agreement to our results, this species found susceptible to pyrthroids including permethrin, deltamethrin, cyfluthrin and lambda-cyhalothrin (7). Additionally, a study conducted in India showed Ph. papatasi was resistant to DDT and susceptible to dieldrin, malathion, fenitrothion and propoxur (23). It is hypothesized that, extensive residual spring against malaria vectors in last two decades, has had drastic effect on increasing either tolerance or resistance to DDT and other organochlorines among leishmaniais vectors.

The majority of captured rodents were infected by Leishmania using direct examination and nested-PCR consequently. Rh. opimus and M. libycus play as the first and secondary reservoir host of ZCL in central part of Iran, respectively (5). Natural Leish-mania infection of R. opimus and M. libycus by direct microscopic examination, iso-enzyme and molecular technique has been shown by several studies in different parts of the country (16, 24). A study showed the highest (92.9%) and lowest (20%) infection rates of R. opimus by Leishmania were observed in fall and summer respectively (16). Moreover, it showed L. major, L. gerbilli, and L. turanica circulate in the gerbil population in central Iran, and in agreement with current study L. major infection is usually accompanied by L. turanica in naturally infected gerbils (16). One study also showed 17.9% natural Leishmnia infection among M. libycus in central Iran (5).

Incidence and prevalence of ZCL among 965 visited inhabitants in the studied area estimated as 46.55 and 341.97 per 1000 population respectively. In addition, in the same year, scar and ulcer rates calculated as 26.52% and 1.77% among 1184 school children consequently. In a similar study in Qom Province, rates of active lesions in inhabitant were 2.7% and 1.4%, and among school children were 2.79% and 2.26% in 2000 and 2001, respectively (24). In another study in Ardestan County, Isfahan Province, Iran, the rates of scars and ulcers were 3.26% and 1.3% among inhabitant and 0.92% and 1.53% among school children, respectively (18).

Conclusion

Considering high population of gerbils’ colonies which offer appropriate microclimate for sand flies breeding, and environmental intervention caused by human and also human dwelling expansion into the natural rodent inhabitant, have led to establishment of the disease cycle among human population in this high risk area. L. major is the causative agent, R. opimus and M. libycus are the reservoir hosts and Ph. papatasi is the main vector of the disease in this area. Rodent control and also personal protection such as using impregnated nets and curtains are the appropriate tools in the hands of health authorities to control ZCL in this area.