Iranian Journal of Parasitology
Volume:8 Issue: 2, Apr-Jun 2013

The main goal of the present study was to set up an axenic cultivation of Acanthamoeba and assess the pathogenic ability of T4 genotypes from different clinical and environmental strains of Acanthamoeba using two physical assays. Sixteen Acanthamoeba isolates including 10 environmental and 6 clinical strains were cul­tured axenically. Axenic cultivation was performed using Proteosepepton, yeast extract and glucose medium and TY-I-S33culture. Pathogenic survey was done using osmotolerance and thermotoler­ance assay. Briefly, differentosmolarity (0.5 M and 1 M) of non-nutrient agar plates were performed. One hundred fiftyµl of axenic culture were collected and were inoculated in 1% agar medium. For thermotolerance assay 150 µl of amoebas from axenic culture were divided into fresh culture me­diums. Cultures were incubated at 37oC and 42 oC. All plates were monitored for 24 h, 48 h and 72 h. Overall, 16 strains of Acanthamoeba isolates previously genotyped as T4 were cultivated axeni­cally after several months. Thermotolerance and osmotolerance assay revealed that all of clinical strains, soil and animal feces strains were highly pathogenic isolates. Two dust and water strains did not grow at high temperature (42 oC) and osmolarity (1.5 M) and thus they were classified as weak pathogens. Most of T4 genotypes are highly pathogenic organisms. This is an important finding since Acanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba may pose a risk within susceptible people.

Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines espe­cially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 μg of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR–tansfected promastigotes. West­ern blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. The PTR1 protein was not expressed in pcDNA-rPTR– tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR– tansfected promastigotes. This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.Keywords:

Molecules expressed on the surface of infected erythrocytes (IE) with Plasmodium falcipa­rum play important roles in malaria pathogenesis and immune evasion. Some of these molecules are specific adhesive ligands mediating adhesion of IE to the vascular endothelium. In the current study, the antigens exposed on the surface of IE with different isolates and various binding subpopula­tions of P. falciparum were studied. A pooled hyper immune serum (HIS) from Malawian adults and eluted antibodies from the surface of the homologous and heterologous parasites were used. The parasite surface molecules were analyzed by Immuno-Gold-Silver enhancement (IGSE) and Western blotting. Mini-column cytoadhe­rence method was used to select various parasite-binding subpopulations. Surface antigens of all the isolates were recognized by HIS and high recognition of antigens was observed in all isolates with homologous eluted antibodies. Western blot analysis showed that the eluted antibodies reacted with a small subset of antigens compared with HIS. Three bands, PfEMP-1, were detected in the Triton X- insoluble fraction of the ICAM-1 binding subpopulation. Another interesting band was ~ 52-55 kDa in various isolates of P. falciparum. This molecule as de­fined by its low molecular weight, Triton X-100 solubility, surface location and sensitivity to 1 mg/ml trypsin. The IE''s surface antigens differed in parental population compared with the selected subpopulations. These molecules could induce isolate-specific immunity. Antibodies purified from the surface of IE can be used as specific reagents to investigate parasite-derived proteins expressed on the surface of IE.

Leishmania major is an intracellular parasite transmitted through the bite of the female phlebotomine sand flies. Leishmania major is able to escape the host immune defense and survive within macrophages. Modulation of the NF-κB (Nuclear Factor-Kappa B) activation and suppression of the pro-inflammatory cytokines by L. major are the main evasion mechanisms that remain to be explored. This study aims to examine the expression level of the Monarch-1 in L. major-infected macrophages, as a negative regulator of the NF-κB activation. Murine macrophage cell line (J774 A.1) was infected by metacyclic form of Leishmania promasti­gotes at macrophage/parasite ratio of 1:10. After harvesting infected cells at different times, total RNA was extracted and converted to cDNA. Semi-quantitative RT-PCR was performed for Monarch-1 by specific primers. Hypoxanthine Phospho-Ribosyl Transferase (HPRT) was used as an internal control to adjust the amount of mRNA in each sample. Semiquantitive analysis of Monarch-1 mRNA expression level showed a significant expres­sion increase within 6 to 30 hours after L. major infection of macrophages when compared to the con­trol macrophages. Monarch-1 expression level reveals a significant increase in the early phase of macro­phage infection with L. major, which in turn may suppress IL-12 production in Leishmania infected macrophages and deeply influence the relationship between host and parasite.

Trematodes belonging to the genus Schistosoma cause schistosomiasis. The relationship between schistosomes and their intermediate hosts varies among snails. This study investigated the effects of S. japonicum on its snail host, Oncomelania hupensis, and cercarial release rythmicity of S. japoni­cum and the effects of light on it. Seven groups of O. hupensis (n = 40 each) were exposed individually to 0 (control), 2, 4, 6, 10, 15, and 20 S. japonicum miracidia. Mortality of the snails was recorded for 10 weeks. Snails in each group were checked for infection at seven weeks post-exposure. Positive snails were exposed to artifi­cial light from 06:00 am – 18:00 pm and the liberated cercariae were collected every 2 hours to determine the rhythmicity of cercarial release. Three groups of positive snails (n = 6 each) were ex­posed to artificial light, daylight, and darkness from 06:00 am – 18:00 pm, the liberated cercariae were collected every 2 hours to determine the effects of light. The highest infection rate and host mortality occurred among snails in the groups exposed to 15 and 20 miracidia. Cercariae were liberated after eight weeks of exposure of O. hupensis to S. japoni­cum. The circarial emerging pattern was circadian, with a single peak of emerging between 10:00 am and 12:00 pm. Light intensity had a positive influence on cercariae shedding and rhythmicity. Further research, including the influence of biotic and abiotic factors is deemed neces­sary to fine-tune elucidation of the effects of S. japonicum upon O. hupensis snail.

Schistosomiasis and soil transmitted infection is a major health problem of children from rural areas of developing countries including Yemen. In an attempt to reduce this burden, the Ministry of Public Health and Population in Yemen established in 2002 a programme for Schisto­somal, soil transmitted infection control that aimed to reduce morbidity and prevalence rates of Schistosomiasis, and Soil transmitted helminthes to less than 5% by 2015. The study was conducted to assess the current prevalence and intensity of schistosomal infection among schoolchildren in ru­ral areas of the Taiz governorate after 6 years of running National Control Programme. Grade 3 schoolchildren from Shara’b Al-Raona district of Taiz Governorate were ex­amined for infections with Schistosoma mansoni using Modified Kato–Katz method and S. haematobium applying filtration method in 1998/1999, comparing the prevalence and intensity of infection with base line study, which was done 6 years ago. The S. mansoni prevalence in the study population was 31%, while the prevalence of S. haemato­bium was 18.6%. This result considerably is similar to the prevalence of base line study. The intensity of mild, moderate and severe infection for S. mansoni reached to 15.9%, 60.6% & 23.5% respec­tively. The severity of S. haematobium infection was 68.4%. It was exceptionally found that the prevalence of S. haematobium is increased. The high prevalence of schistosomiasis and low effectiveness of control programme against schistosomal infection in the study area demands consideration of alternative treatment ap­proaches.

The protozoan parasite Toxoplasma gondii can infect any warm blooded nucleated cells. One of the ways for human infection is ingestion of oocysts directly from soil or via infected fruits or vegetables. To survey the potential role of T. gondii oocyst in soil samples, the present study was conducted in Tehran City, Iran. A total of 150 soil samples were collected around rubbish dumps, children''s play ground, parks and public places. Oocysts recovery was performed by sodium nitrate flotation method on soil samples. For molecular detection, PCR reaction targeting B1 gene was performed and then, the posi­tive results were confirmed using repetitive 529 bp DNA fragment in other PCR reaction. Finally, the positive samples were genotyped at the SAG2 locus. Toxoplasma DNA was found in 13 soil samples. After genotyping and RFLP analysis in SAG2 locus, nine positive samples were revealed type III, one positive sample was type I whereas three samples revealed mixed infection (type, I & III). The predominant genotype in Tehran soil samples is type III.

In this study, the presence of resistance to diclazuril, amprolium+ethopabate and salinomy­cin, representing some of the commonest anticoccidials in Iran’s poultry industry, against three mixed Eimeria field isolates were investigated. Three Eimeria field isolates, collected from typical broiler farms in Iran, were propagated once, inoculated to 480 broilers, comprising 30 chicks in each treatment. The non-medicated or medi­cated diets containing one of the above mentioned anticoccidials were provided ad-lib. Drug effi­cacy was determined using the Global index (GI), Anticoccidial Sensitivity Test (AST) and Opti­mum Anticoccidial Activity (OAA). None of the field isolates were fully sensitive to the selected anticoccidials. All isolates showed reduced sensitivity/partial resistance to salinomycin. Resistance to amprolium+ethopabate was evident and partial to complete resistance was recorded for diclazuril. Limited efficacy of the selected anticoccidials is obvious. Considering the cost of conti­nuous use of anticoccidials in the field, altering the prevention strategy and rotation of the anticocci­dials with better efficacy, would prevent further economic losses induced by coccidiosis.

Boophilus annulatus is an obligate blood feeder tick that can cause great losses in animals due to anemia and its ability to injure its host skin directly. The aim of this study was identification of cattle humoral immune response to some tick proteins during experimental infestation. Immune sera against tick were collected from experimentally infested cattle with ticks. One and two-dimensional electrophoresis and Western blotting methods were used for the detection of immunogenic proteins in larval tick extract and eight of these proteins were identified by MALDI-TOF and MALDI-TOF-TOF mass spectrometry. In non-reducing one-dimensional SDS- PAGE, some bounds between 12 to more than 250-kDa appeared. In two-dimensional SDS-PAGE, numerous spot appeared and the identified immuno­genic proteins by parallel immunoblotting weighted between 14 and 97 kDa. Amino acid sequences of protein spot with 37-kDa molecular weight had identity to tropomyosin based on Mas­cot search in NCBI. Anti tropomyosin antibodies can be induced in experimentally infested hosts with ticks and it seems that tropomyosin can be useful for the development of anti tick vaccines.

The aim of this study was to detect Dientamoeba fragilis by iron haematoxylin stain, as well as its prevalence, and association between D. fragilis infection and diarrhoea among pa­tients attending Al-Nuseirate Refugee Camp Clinic, Gaza Strip. A cross-sectional study was conducted among 319 children and adults with age ranges from (1 to 75) years old, attending Al-Nussirat Clinic, and who were complaining from clinical symp­toms, like diarrhoea and abdominal pain. 28 individuals were infected with D. fragilis with a prevalence of 8.8%. The detection of 28 cases infected with D. fragilis was proved using iron haematoxylin stain, but no case was detected by direct smear or formal-ether sedimentation technique. The most frequent symptoms were abdominal pain (96.4%) and diarrhoea (71.4%) in patients with diantamoebiasis and this was statistically signifi­cant (P= 0.03). Co-infection between D. fragilis and Entamoeba histolytica/dispar was 50% and between D. fragilis and Giardia lamblia was 7.1%. D. fragilis was present in the patients stool samples and was detected and proved using iron haematoxylin stain.

Slugs have been known worldwide as important pests of agricultural and horticultural production. They also play a role as intermediate or definitive hosts of helminths parasite. In this pur­pose, current study was carried out to examine slug radular teeth structure and slug infection with helminths larvae in north of Iran. A total number of 114 slugs were collected from center and east parts of Mazandaran prov­ince from May 2011 to June 2012. The specimens were rinsed, measured, and identified. The radula of all collected slugs was extracted and stained by using Mallory II. For detecting the hel­minths parasite infection, foot- head and viscera of examined slugs were removed, minced, and di­gested with 4.5% acid pepsin. Two species of Limax (Caspilimax) keyserlingi (Martens 1880) (11.4%, 13/114) and Parmacella ibera (Eichwald 1841) (88.6%, 101/114) were prevalent in the region. There was significant difference between body length and shell size. P. ibera had the highest number of teeth rows (145±5). The radu­lar teeth formula was approximately similar in both identified slugs. In P. ibera, there was no signifi­cant difference in the average length and width of radula. The radular teeth in L. keyserlingi were larger and thicker than P. ibera. In all examined slugs for helminths larvae infection, P. ibera (7.69%, 1/13) was infected with Strongyloid larvae from Fereidonkenar area. Two prevalent species of slugs exist in the same region of which P. ibera has capability to play a role as intermediate host of nematode helminths. Radular morphology within the slug spe­cies may be also systemically informative.

Leishmania tropica is a genetically divergent species. Amplification of entire internal tran­scribed spacer (ITS) region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis) ACL(in Iran, revealed a double-band pat­tern in agarose gel electrophoresis. This study explains how this pattern occurs. Twenty seven L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new re­verse primer namely LITS-MG was designed to exclude this gap in PCR products. Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was correspond­ing to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG primer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis. Sequences analysis of GenBank L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles.

Leishmaniasis is a worldwide disease prevalent in tropical and sub- tropical countries. In the present study the immunogenicity of three human HLA-DR1 restricted peptides derived from L. major gp63 protein was evaluated using FVB/N-DR1 transgenic mouse model. The immunity generated by three MHC class II – restricted peptides with the sequence of AARLVRLAAAGAAVT (AAR), AAPLVRLAAAGAAVT (AAP) and SRYDQLVTRVVTHE (ASR) derived from L. major gp63 protein were predicted using a web-based software (SYFPEITHI) and tested in FVB/N-DR1 transgenic mice. Immunization of FVB/N-DR1 transgenic mice with one of the three predicted peptides (AAR) resulted in high levels of Th1-type immune response as well as significant levels of IFN-γ de­tected by Proliferation assay and ELISA. The results indicate a high level of immunogenicity for AAR, which can be a potent candidate for peptide vaccine in Leishmania infections.

The objectives of our research were to search for Leishmania species in rodents in Fars province, south of Iran, and to compare molecular with conventional methods for detecting these parasites. Rodents were captured using live traps and screened for Leishmania species using molecular and conventional methods, including the taking of smears from each ear. Nested PCR was employed to detect Leishmania in rodents by amplifying a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA-ITS2) that is species-specific by DNA sequence. Totally, 122 rodents were captured. Leishmania parasites were detected using the nested PCR and three conventional methods (direct smear, NNN culture and Balb/C inoculation. 41 (33.6%) out of 122 rodents had Leishmania infections (34 Meriones lybicus and 7 M. persicus). All PCR products of the ITS-rDNA gene were sequenced. Sequence analysis revealed that 28 out of 41 positive samples were Leishma­nia major. Thirteen sequences were unreadable and therefore not identified. At least two gerbil species common in Fars ZCL foci, M. lybicus and M. persicus, are acquir­ing infections of L. major and may be reservoir hosts of one predominant parasite haplotype. Most infections were detected molecularly not by conventional methods, because most rodents died in the traps.

The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp.) in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan. Reverse line blot (RLB) assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products. Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa. Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001). Risk factor analysis revealed that male (P = 0.03), ani­mals infested by ticks (P = 0.03) and herd composed of sheep only (P = 0.001) were more infected by blood parasites.

Malaria is an infectious disease caused by Plasmodium spp. with high morbidity and mortality in human in tropical and subtropical regions. In recent years, number of malaria cases has been significantly reduced because of fight with the disease in Turkey. This study intended to investigate the malaria epidemiology in Mersin Province from 2002 to 2011 using data from the provincial Public Health Directorate. Over ten years, 303573 blood samples were taken from the people by active and passive surveillance methods and blood smears were prepared. Smears were stained with Giemsa and examined under the microscope. Totally, 73 people including 44 male and 29 female were positive in terms of Plasmodium spp. It was determined that P. vivax observed in 67 cases while P. falciparum in 6 cases. Cases were mainly observed in 15 to 44 years old range, showed an increase between June-September periods and a significant decrease after 2006. Out of the 73 malaria cases, 54 cases were from Mersin Province and 13 cases were imported from another province of Turkey. Six cases were transmitted from abroad. These results provide information about malaria epidemiology in an endemic area in Turkey and contribute its prevention in Mersin Province.

The free-living amoebae Acanthamoeba spp., have been recognized as etiologic agents of amoebic encephalitis, keratitis, otitis, lung lesions and other skin infections mainly in immuno-compro­mised individuals. In this study, morpho-physiological and biochemical characterization of Acanthamoeba strains isolated from the Egyptian aquatic environment were surveyed. some Acanthamoeba species were cultivated on non-nutrient agar. Isolated strains of Acantha­moeba were identification based on the morphology of trophic and cyst forms in addition to temperature and osmo-tolerance assays. Biochemical characterization of the isolated amoeba strains was performed using quantitative assay as well as qualitative determination of proteolytic activity in zymograph analysis. Potentially pathogenic Acanthamoeba species were isolated from all of the examined water sources. Colorimetric assays showed protease activity in the heat-tolerant isolates of Acanthamoeba. All pathogenic isolates of Acanthamoeba exhibited higher protease activity than did the non-patho­genic ones. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. The incidence and prevalence of the pathogenic Acanthamoeba species in the aquatic environment using parasitological and biochemical diagnostic tools will provide baseline data against which the risk factors associated with waterborne transmission can be identified.

This study was designed to assess and compare the quantity and quality of Iranian and Turkish researchers working in the field of Parasitology from bibliometric point of view. To assess the contributions and achievements of the Iranian and Turkish parasitologists, bibliometric analysis was carried out based on the citation data retrieved from Web of Science. The absolute productivity of Turkish and Iranian parasitologists’ papers has almost tripled for Turkey, from 12 papers in 2002 to 36 papers in 2011, and decuple for Iran, from 10 papers to 123 from 2002 to 2010. The average number of citation per article is about 5.8 and 4 for Turkish and Iranian parasitologists’ papers, respectively. The “Veterinary Parasitology” journal was the most cited journal in both countries. The majority (more than 90%) of cited items was foreign journal articles and one half of all references in journals articles dated 11 and 12 years while one half of cited books was dated within 14 to16 years for Turkish and Iranian papers, respectively. Based on observed data and applied model, it is anticipated that the total number of Iranian and Turkish parasitologists’ publications in Web of Science will exceed of 2512 and 240 ar­ticles per annum for Iranian and Turkish in 2020, respectively.

The aim of this study was to determine the prevalence of hydatidosis in west Azerbaijan, Iran during a 10 year period (2000-2009). We surveyed medical records of infected patients with hydatid cyst who had been oper­ated in four hospitals in Urmia City, the capital of West Azerbaijan Province, Iran. Several parame­ters were analyzed including age, sex, place of residency, hospitalization time, and the location of cysts. Of 294 cases, 53.3% were female and 46.7% were male with the mean age of 39.4 years (5–93). The average number of operated cysts per year was 29.4 (0.98/100,000 of population). The most affected age group was 20-30 year olds (18.7% of the cases). Cysts were localized in liver and lung in 57.5% and 21.8% of cases respectively and the average hospitalization time was 9 days. Single organ involvement was seen in the majority of patients and 28 (9.5%) cases had multiple involve­ment. The distribution of residence in patients showed 108 (36.9%) of them to have urban origin and 185 (63.1%) were rural residents. The lowest number (n=17) and the highest number of opera­tion (n= 48) recorded in 2000 and 2007, respectively. The prevalence of hydatidosis is high in this city and further studies are needed for evalua­tion of economic burden and risk factors for CE in this region.

Linguatula serrata, one of the parasitic zoonoses, inhabits the canine respiratory system (final hosts). The objective of this study was to determine the prevalence rate of L. serrata nymphs in mesenteric lymph nodes (MLNs) of cattle and buffaloes (intermediate hosts) that were processed in the Ahvaz, Iran abattoir. During November 2010 to March 2011, 223 animals (119 cattle and 104 buffaloes), in differ­ent sex and three age groups (<2, 2–<3 and 3->3 years old) were sampled randomly at Ahvaz abattoir. Up to 35 grams of their mesenteric lymph nodes were examined separately for nymphal stages of L. serrata by digesting the samples with acid- pepsin method, collected the nymphs and counted under stereomicroscope. Overall 37(16.6%) of 223 animals were infected with L. serrata nymphs in their mesenteric lymph nodes. Prevalence of the infection in cattle and buffaloes were 16.8% and 16.3% respectively. The number of collected nymphs of MLNs was ranged from 1 to 16. No significant differences were seen in the infection rates between males and females (sexes) and age groups in the cattle and buffa­loes (P <0.05). Linguatula serrata has an active life cycle in the studied area and a zoonotic potential for transmission between animal and human. Avoiding use of raw MLNs to dogs can help reduce the infection.

The aim of the present study was to survey birds'' schistosomes in migratory birds (Anati­dae: Anas platyrhynchos) which are the source of the disease in Mazandaran Province, Northern Iran. A number of mallards were bought from the markets of hunted birds. The respiratory tracts (nasal mucosa) and intestinal blood vessels were studied for adult worms. The nasal mucosa was separated and observed by a microscope. In order to separate the visceral schistosomes, after separating intestine, vessel mesenteric was studied under the lamp light and then in saline. The para­site sample was collected for subsequent observation. Fifteen (13.6%) cases out of 110 studied birds had nasal mucosa contaminated with Trichobilhar­zia sp. egg. Besides that, two birds had adult worms schistosome visceral i.e. Bilharziella sp. The elements that cause cercarial dermatitis in aforementioned region are Trichobilharzia sp. and Bilharziella sp. parasites. Thus, it is necessary for the authorities of health, environmental and agricultural organization of the province to cooperate in order to control this disease.

Cutaneous Leishmaniasis (CL) is a parasitic skin disease. Diagnosis primarily is based on clinical signs and microscopic observation of parasite on direct stained smears or tissue sections. Sensitivity of direct smear is not as high as molecular methods. The aim of this study was to identify and characterize Leishmania species among the negative direct smears obtained from skin ulcers sus­pected to CL by PCR method. Among 81 patients with suspicious skin lesions to CL referred to the Parasitology lab, nega­tive Giemsa stained smears were collected. DNA extraction performed by scraping stained smears, then PCR was performed. Among the DNA extracted from smears, L. tropica was isolated from 9 (11.1%) of the smears and L.major was not isolated from any samples. Direct microscopy on stained smears for diagnosis of leishmaniasis is not enough accu­rate. PCR is recommended for clinically suspected lesions with negative result of direct smear.

Malaria is one of the most devastating protozoal diseases in under developing coun­tries like Pakistan where health facilities are scarce. It is the second most frequently reported disease with 4.5 million suspected cases in Pakistan. The current study was designed to determine the inci­dence of pediatric malaria in Quetta, Balochistan. The study was conducted at Children Hospital Quetta (CHQ) during July 2011march 2012. Blood samples were collected from 3418 clinically suspected and were evaluated using thin and thick blood films stained with Giemsa stain. Out of 3418 total of 230 (6.72%) children were found positive for any of the malarial para­sitic infestation. Plasmodium vivax was observed to be more common 54.34 % (n= 125/230) than P. falciparum 44.78% (n=103/230). Male children were 65.21% (150/230) i.e. two times more com­monly affected than female 34.78% (80/230) children. The prevalence among age groups was 7.41% (n=89/1200) in preschool-aged children aged 1-5 years, 7.11% (n=75/1054) in school-aged children aged 6—10 years while 6.78% (n=46/678) in 11-15 years-old children, and 6.66% (n=20/300) in >15 year-olds children. Peak prevalence was noted in summer and mild in winter. Mixed infection of (0.86%: 2/230) P. vivax and P. falciparum was also observed in two cases although no case of P. mala­riae or P. ovale infection was seen during entire study. The results reflect the higher prevalence of malaria in Quetta, Pakistan that poses a signifi­cant health threat and requires urgent attention of high-ups to launch programme to control the disease in the area.