Bio-Pesticides: New Tool for the Control of Aedes (Stegomyia) albopictus (Culicidae: Diptera) in Pakistan.

BACKGROUND: Application of plant extracts as mosquito control strategy was practiced from centuries. These are easily available, non-toxic, biodegradable and exhibit broad-spectrum target specific activities against larval stages of mosquitoes. METHODS: Different potential parts of locally grown plants, seeds of nutmeg (Myristica fragrans), peel of musambi (Citrus sinensis), leaves of babuna (Matricaria chamomilla), mint (Mentha spicata) and ginger rhizome (Zingiber officinale) selected and evaluated for their larvicidal properties against Aedes (Stegomyis) albopictus. Oils were extracted through steam distillation process and extracts were evaluated as per WHO 2005 guidelines for testing of insecticides against larvae of mosquitoes. RESULTS: Among the five plant extracts, C. sinensis had the lowest LC50 (400.81ppm) while M. fragrans had the highest LC50 value (710.30ppm) respectively after 24h of exposure. In terms of % age mortality, a series of concentrations (300-800ppm) gave high % mortality in case of C. sinensis while M. fragrans gave low % age mortality. CONCLUSION: All the five plant species have larvicidal effects to certain extant and C. sinensis had great potential. Further small-scale field trials with the extracts of the most promising one (C. sinensis) shall be conducted to determine operational feasibility.

Pfcrt Gene in Plasmodium falciparum Field Isolates from Muzaffargarh, Pakistan.

BACKGROUND: The aim of the study was to identify the prevalence of different species of Plasmodium and haplotypes of pfcrt in Plasmodium falciparum from the selected area. METHODS: Overall, 10,372 blood films of suspected malarial patients were examined microscopically from rural health center Sinawan, district Muzaffargarh, Pakistan from November 2008 to November 2010. P. falciparum positive samples (both whole blood and FTA blood spotted cards) were used for DNA extraction. Nested PCR was used to amplify the pfcrt (codon 72-76) gene fragment. Sequencing was carried out to find the haplotypes in the amplified fragment of pfcrt gene. RESULT: Over all slide positivity rate (SPR), P. vivax and P. falciparum positivity rate was 21.40 %, 19.37 % and 2.03% respectively. FTA blood spotted cards were equally efficient in the blood storage for PCR and sequencing. Analysis of sequencing results of pfcrt showed only one type of haplotype SagtVMNT (AGTGTAATGAATACA) from codon 72-76 in all samples. CONCLUSION: The results show high prevalence of CQ resistance and AQ resistant genes. AQ is not recommended to be used as a partner drug in ACT in this locality, so as to ward off future catastrophes.

Molecular Detection of the Carriage Rate of Four Intestinal Protozoa with Real-Time Polymerase Chain Reaction: Possible Overdiagnosis of Entamoeba histolytica in Nigeria.

Diarrhea remains the second largest killer of children worldwide, and Nigeria ranks number two on the list of global deaths attributable to diarrhea. Meanwhile, prevalence studies on potentially diarrheagenic protozoa in asymptomatic carriers using molecular detection methods remain scarce in sub-Saharan countries. To overcome sensitivity issues related to microscopic detection and identification of cysts in stool concentrates, real-time polymerase chain reaction (PCR) was used to analyze genomic DNAs extracted from stool samples from 199 healthy school children for Entamoeba histolytica, E. dispar, Giardia intestinalis, and Cryptosporidium. Questionnaires were administered for epidemiological data collection. E. histolytica was not detected in any of the samples, whereas Giardia (37.2%), E. dispar (18.6%), and Cryptosporidium (1%) were found. Most of the children sourced their drinking water from community wells (91%), while the majority disposed of feces in the bush (81.9%). Our study is the first to use real-time PCR to evaluate the epidemiology of E. histolytica, Giardia, and Cryptosporidium in Nigeria where previous studies using traditional diagnostic techniques have suggested higher and lower carriage rates of E. histolytica and Giardia, respectively. It is also the first study to accurately identify the prevalence of common potentially diarrheagenic protozoa in asymptomatic carriers in sub-Saharan Africa.

Cryptosporidium infections in Denmark, 2010-2014.

INTRODUCTION: The incidence of cryptosporidiosis in Denmark is unknown. Here, we present the number of cases detected in the 2010-2014 period along with data on species and subtypes. METHODS: Complete national data retrieved from the Danish Microbiology Database and Statens Serum Institut (SSI) comprised test results on cryptosporidia detected by microscopy or polymerase chain reaction (PCR) between 1 January 2010 and 30 April 2014. Samples that tested positive at the SSI were submitted to species and subtype analysis by conventional PCR and sequencing of ribosomal and gp60 genes, respectively. RESULTS: A total of 689 Cryptosporidium-positive stool samples were submitted by 387 patients. Limiting case episodes to two months (60 days), a total of 388 case episodes representing 387 patients were identified. Cryptosporidiosis was most common among infants and toddlers. Moreover, a peak in incidence was observed among younger adults aged 23-24 years. In 43 Cryptosporidium-positive faecal samples, identification was performed to species and subtype level. Cryptosporidium parvum was found in 34 samples, C. hominis in eight, and C. meleagridis in one sample; C. parvum subtypes IIaA15G2R1 (n = 10) and IIaA16G3R1 (n = 5) were predominating. CONCLUSION: Cryptosporidia are a significant cause of diarrhoea in Denmark. Outbreaks may not be detected due to continued use of diagnostic tests of limited sensitivity and due to lack of surveillance. With molecular methods now being introduced in many Danish laboratories, we propose establishing national surveillance of cryptosporidiosis. FUNDING: not relevant. TRIAL REGISTRATION: not relevant.

Molecular detection of malaria in South punjab with higher proportion of mixed infections.

BACKGROUND: Malaria is well known for its fatalities worldwide, Plasmodium vivax and the Plasmodium falciparum are the two important species of malaria reported from Pakistan and creating lots of morbidities across the country. METHOD: Study was conducted to determine the Surveillance of malaria in South Punjab by microscopy and Polymerase chain reaction (PCR). RESULT: samples out of 100 patients were found positive for malarial parasites. One patient was found with mixed infection, whereas P. falciparum and P. vivax infections were detected in 17 and 22 patients, respectively. In nested PCR, genus-specific primers for Plasmodium species. in round 1 and species-specific primers for P. falciparum and P. vivax in round 2 were used. By the application of PCR 41% were found to be infected by Plasmodium spp. Among Plasmodium positive patients: mixed, P. falciparum and P. vivax infection were detected in 10, 15 and 16 patients, respectively. Thirty nine microscopically positive patients confirmed to have Plasmodium spp. One negative by PCR, 2 microscopically negative patients had shown Plasmodium spp. infection (P. falciparum and P. vivax) by PCR. In total samples, P. falciparum, P. vivax and mixed infection accounted for 36.6%, 39.0% and 24.3%, respectively. CONCLUSION: Microscopy was found deficient for interpretation of mixed infections, low parasitaemia, and species specific diagnosis. The sensitivity, specificity and efficacy of nested PCR was calculated 95%, 98% and 97%, respectively, showing PCR as a more effective and efficient diagnostic tool for malaria.