Molecular detection of Leishmania species in human and animals from cutaneous leishmaniasis endemic areas of Waziristan, Khyber Pakhtunkhwa, Pakistan

Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis (CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1 (ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica (L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major (L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents (Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.

First report on molecular characterization of Leishmania species from cutaneous leishmaniasis patients in southern Khyber Pakhtunkhwa province of Pakistan

OBJECTIVE@#To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis (CL) is endemic and was thought to be caused by Leishmania tropica only.@*METHODS@#Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011-2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene.@*RESULTS@#Leishmania amastigotes were detected by microscopy in 308 of 432 samples (71.3%) while 374 out of 432 samples (86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed L. tropica in 351 and L. major in 6 biopsy samples.@*CONCLUSIONS@#This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of L. major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.

First report on molecular characterization of Leishmania species from cutaneous leishmaniasis patients in southern Khyber Pakhtunkhwa province of Pakistan

Objective To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis (CL) is endemic and was thought to be caused by Leishmania tropica only. Methods Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011–2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. Results Leishmania amastigotes were detected by microscopy in 308 of 432 samples (71.3%) while 374 out of 432 samples (86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed L. tropica in 351 and L. major in 6 biopsy samples. Conclusions This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of L. major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.

Diagnostic role of bone marrow aspiration and trephine biopsy in haematological practice

To determine the usefulness of bone marrow aspiration and trephine biopsy in evaluation of the bone marrow in routine haematological practice. This study included 443 cases of bone marrow examination, referred to Pathology Department, Lady Reading Hospital Peshawar during the period extending from January 2012 till July 2013. All the bone marrow smears and bone biopsy sections were examined in detail. The diagnosis and findings on aspirate and biopsy were evaluated and compared with each other. In 73.8% of the cases the bone marrow aspiration and trephine biopsy showed same diagnosis i.e., bone marrow aspiration alone was sufficient for diagnosis in these cases. In the remaining 116 [26.2%] cases trephine biopsy sections or touch imprints were found to be necessary in for making final diagnosis. These cases were those of the hypoplastic / aplastic marrows, Myelofibrosis, lymphomatous infiltration and chronic granulomatous inflammation. The study results suggest that both the aspirate and trephine biopsy complement each other. Nutritional anaemias, Haematological Malignanciesand Immune Thrombocytopenia can be readily diagnosed by bone marrow aspiration alone. Trephine biopsy is necessary for diagnosing Granulomatous Inflammation and Hypoplastic/Aplastic Anaemia. Also trephinebiopsy is required to diagnose Myelofibrosis and Lymphomatous infiltration

Clinicohaematological spectrum of pancytopenia in a tertiary care hospital

To determine the frequency, clinical presentation and underlying causes of pancytopenia inpatients presenting to a tertiary care hospital in Peshawar. This cross-sectional, observational study was conducted at Department of Pathology, Lady Reading Hospital Peshawar from January to December 2011. Patients of all ages having pancytopenia on blood film examination [TLC < 4000/ul, Hb < 10 gm/dl and Platelets < 150000/ul] were included in the study. Already diagnosed patients of Aplastic Anemia, Acute Leukemia receiving treatment and those not willing for bone marrow examination were excluded from the study. History, General Physical and systemic examination were recorded at presentation. The peripheral blood counts were performed with sysmex - automated hematology analyzer. Bone marrow aspiration and trephine biopsy were performed according to the standard protocol and examined microscopically to find the underlying cause of pancytopenia. Other relevant investigations were also done. During the study period, we received 600 patients for bone marrow examination from various units. Out of these, 160 [26.7%] patients had pancytopenia. Common clinical presentations were Pallor[95%, n=150], followed by generalized weakness [75%, n=120], fever [52%, n=83], bleeding manifestation[37.5%, n=60], gastrointestinal symptoms [32.5%, n=52] and splenomegaly [23.5%, n=38]. The common causes of pancytopenia were aplastic anemia [37.5%, n=60] followed by magaloblastic anemia [13.75%,n=22], Acute Leukemia [13.75%, n=22] and hypersplenism [10%, n=16]. Pancytopenia is a common occurrence. Aplastic Anemia and Magaloblastic Anemia are the commonest causes of Pancytopenia followed by Acute Leukemia. Common clinical presentations were Pallor, fever, weakness, bleeding manifestation and Splenomegaly

Hepatoprotective effects of berberis lycium, galium aparine and pistacia integerrima in carbon tetrachloride [CCL4]-treated rats

The aim of this study was to evaluate the hepatoprotective effects of mixture of berberis lycium, galium aparine and pistacia integerrima in carbon tetrachloride [CCL4]-treated rats. This study was conducted in the animal house of the Quaid-i-Azam University, Islamabad in the year 2000. A total of 20 rats were divided in 4 groups of 5 rats each. 1. Normal Control Group: with no medication given 2. CCl4 Treated Group: only CCl4 was given 3. Hepatopreventive group: Initially treated with medicinal plants mixture followed by CCl4 4. Hepatoprotective group: Initially treated with CCl4 followed by medicinal plants mixture Then blood sample from each rate was collected and analysed for ALT, AST and ALP. The rates included in the study were male Sprague Dawley rats [albino] weighing 150-300g received from the animal house of the NIH, Islamabad. ALT, AST and ALP activities were significantly raised [P<0.001] in hepatocurative groups as comparative to the normal control, and decreased [P<0.001] as compared to the CCl4-treated rated and the hepatopreventive group. While the hepatocurative group treated with aqueous extract of the mixture of medicinal plants decrease more than treated with suspension of medicinal plants mixture and alcoholic extract. The results this study indicates that a mixture of Berberis lycium, Galium aparine and Pistacia integerrima have hepatoprotective effects. These medicinal plants have more effect as curative agents rather than preventive agents

Screening extended families for a genetic X-linked disorder, glucose 6 phosphate dehydrogenase deficiency

To test an alternative approach to population based program for identifying Glucose 6 Phosphate Dehydrogenase [G6PD] deficient Individuals in Pakistan where consanguineous marriage is common. This study was conducted at Armed Forces Institute of Pathology Rawalpindi and Pathology Department Government Lady Reading Hospital Peshawar. Five large families from Northern Pakistan, 03 with an index case of G6PD deficiency, 02 without such history [control] were screened for G6PD deficiency. All apparently healthy members of the families, both male and female of all ages, of the last three generations were included in the study. A commercial qualitative screening kit from Sigma Chemical Co. Ltd England was used for screening the individuals for G6PD deficiency. In the control families, no individual with G6PD deficiency was found among 101 individuals tested out of 159 living members. In the 03 families with an index case of G6PD deficiency 155 were tested out of 229 family member and 52 [33.5%] were found G6PD deficient. While in population screening out of 800 apparently healthy adult male subjects screened for G6PD deficiency, 47 [5.9%] were found glucose 6 phosphate dehydrogenase deficient. Testing extended families is feasible and highly cost effective way of screening for X- linked genetic disorder like Glucose 6 Phosphate Dehydrogenase deficiency in communities in which consanguineous marriage is common

Pattern of hematological diseases in hospitalized pediatric patients based on bone marrow examination

The objective of this study was to look into various diagnoses of hematological lesions on bone marrow examination in our pediatric age group of patients. This study was conducted in the Pediatric A Unit and Department of Hematology Postgraduate Medical Institute [PGMI] Lady Reading Hospital, Peshawar from 01st Jan 2007 to 31st Dec 2007. Children admitted with pallor, bleeding, lymphadenopathy or visceromegaly having abnormal smear results were included in this study. The data was statistically analyzed by SPSS version 10. One hundred and ninety-eight cases were included in the study. The age range was from 06 months to 14 years with a mean age of 5.35 years and standard deviation of +/- 3.69. Majority [4 7.5%] of these children were in the age range of 1 to 5 years with male to female ratio of 1.53. The commonest disorder was aplastic anemia present in 40 [20.2%] of the cases followed by idiopathic thrombocytopenic purpura [ITP] [15.7%], megaloblastic anemia [14.6%] and iron deficiency anemia [7.6%]. Acute leukemia was the predominant malignant disorder present in 11.6% of the cases. There were also few cases of histiocytosis and bone marrow secondaries. Visceral leishmaniasis, anemia of chronic disorders, haemolytic anemia, myeloid hyperplasia, hypersplenism, congenital dyserythropoietic anemia, malaria and Gaucher disease were the other non-malignant hematological disorders found in this study. Aplastic anemia, idiopathic thrombocytopenic purpura, megaloblastic anemia and leukemia are the commonest hematological disorders in our set up

frequency of glucose-6- phosphate dehydrogenase deficiency in Pnjabis and Pathans

To find the frequency of Glucose-6-Phosphate Dehydrogenase [G6PD] deficiency in healthy young adult male population of various ethnic groups and sub-groups of Punjabis and Pathans. This Cross-sectional comparative study in Armed Forces Institute of Pathology, Rawalpindi, from Jan-Dec 2000. Eight hundreds apparently healthy unrelated adult males [Punjabis: 400 and Pathans: 400] were screened for G6PD deficiency by a commercial qualitative screening test kit. Out of 800 subjects, 47 [5.9%] were G6PD deficient [95% CI: 4.2-7.5%]. The frequency in Pathans [8.3%] was significantly higher than in Punjabis [3.5%] [p=0.006]. There were also differences in the frequency of G6PD deficiency among the sub groups of Punjabis and Pathans. G6PD deficiency is a fairly common abnormality in Punjabis and Pathans. Such individuals are highly susceptible to develop acute haemolytic episodes by a variety of stimuli including some of the commonly used drugs