Molecular characterisation and genetic affinities of Cyclophyllidean cestodes infecting wild rats in Peninsular Malaysia

@#Cestode infections is widely reported in rodents, however species identification remains problematic due to the genetic or interspecies variation. Therefore, this study was aimed to verify the Cyclophyllidean parasites recovered from wild rats captured from different forest types using molecular based methods. Maximum-likelihood (ML) and neighbour-joining (NJ) trees were constructed inferred from 18 small subunit nuclear ribosomal RNA gene (18SrDNA) and mitochondrial cytochrome c oxidase subunit one gene (COX1) sequences of cestode worms recovered from 124 individuals from four rat species. Sequences obtained from both Hymenolepis diminuta and Hydatigera parva represents the first records in Malaysia. All the sequences were successfully amplified with product with total length of 205 and 1202 base pairs (bp), respectively. Three cestode species from the Family Hymenolepididae (Hymenolepis diminuta) and Family Taeniidae (Hydatigera parva; Hydatigera taeniaeformis) were successfully characterized using phylogenetic analyses and haplotype networking. Phylogenetic analysis showed that H. diminuta, Hydatigera parva (Hy. parva) and Hydatigera taeniaeformis (Hy. taeniaeformis) formed its own monophyletic clade in 18SrDNA analyses. Results also showed that Hy. taeniaeformis shared the same haplotype group with Hy. taeniaeformis from China (COX1) and linked with Hy. taeniaeformis from Japan (18SrDNA) while the Malaysian H. diminuta clearly formed a separate haplotype and networked with other regions. The Malaysian Hy. parva isolation, on the other hand, appeared to be genetically distinct from the European Hy. parva (Spain) strain, but closely linked to the local isolates. Molecular methods employed successfully improved in the detection of complex species in this group. The findings showed that molecular data can be useful to deeply study intra-specific variation in other cestode worms.

Prevalence of Toxoplasma gondii in pet and stray cats in Klang Valley, Malaysia

@#Toxoplasma gondii, a zoonotic protozoan that has a worldwide distribution, is known to infect many warm-blooded vertebrates. The feline species including domestic cats are the definitive hosts for Toxoplama gondii and shed the infective oocyst. There is lack of information on the prevalence of Toxoplasma gondii in cats in Malaysia. The objective of this study was to determine both the seroprevalence of T. gondii and the prevalence of T. gondii DNA in cats’ feces in Klang Valley, Malaysia. 198 blood and 201 fecal samples were collected from pet and stray cats from the local council, Dewan Bandaraya Kuala Lumpur (DBKL) and University Veterinary Hospital, Universiti Putra Malaysia respectively. The overall seroprevalence of Toxoplasma gondii in cats in the Klang Valley was found to be 5.5%. There was a high prevalence (10.5%) of T. gondii DNA detected in the cat fecal samples in both pet and stray cats suggestive of T. gondii oocyst shedding. Stray cats showed a higher seroprevalence and molecular prevalence of T. gondii than the pet cats. However, comparative analysis using Chi-square test showed no significant difference between both groups (P>0.05). Higher prevalence (10.5%) of cats shedding T. gondii DNA as compared to the seroprevalence (5.5%) was found in the cat population in the Klang Valley. The high prevalence of cats shedding T. gondii DNA is alarming as this may directly reflect the number of oocysts excreted into the environment posing a significant public health hazard.

First molecular detection of Tritrichomonas foetus in domestic cats in Klang Valley, Malaysia

@#Tritrichomonas foetus is known to cause chronic diarrhea in the feline species in many different regions of the world. However, there is a paucity of information on T. foetus among cats in Malaysia. This study was conducted to determine the prevalence of Tritrichomonas foetus in the pet and stray cat population in Klang Valley, Malaysia. A total of 201 pet and stray cats’ fecal samples were collected in Klang Valley. 24 samples were cultured in the InPouch® TF Feline to observe for motile trophozoites. A nested PCR protocol was used to screen for T. foetus in the collected samples. The prevalence of T. foetus in the cat population in Klang Valley was 33%. There was no association between Tritrichomonas infection and age, sex, breed or management of the cats. However, statistical analysis revealed that stray cats were more likely to be infected with T. foetus compared to pet cats. This study confirmed for the first time the presence of T. foetus among the cat population in Klang Valley, Malaysia.

Antitrypanosomal and cytotoxic activities of botanical extracts from Murraya koenigii (L.) and Alpinia mutica Roxb

@#Four carbazoles (girinimbine, mahanimbine, murrayafoline and murrayanine), isolated from Murraya koenigii, and one kavalactone (5,6-dehydrokawain) and one flavonoid (pinostrobin) isolated, from Alpinia mutica, were tested for their antitrypanosomal activity using in vitro cultured Trypanosoma evansi cell lines. The cytotoxic activities of these compounds were also investigated against mammalian Vero cells using the MTT (3-(4,5- Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide)-cell proliferation assay. Three carbazole compounds, namely mahanimbine, murrayafoline, and girinimbine, showed a potent antitrypanosomal activity, scoring a median inhibitory concentration (IC50) of 3.13, 6.35 and 10.16 μg/ml, respectively. Girinimbine was the least toxic to Vero cells, and the mean cytotoxic concentration (CC50) and the selectivity index (SI) of this compound were 745.58 ± 42.38 μg/ ml and 73.38, respectively. Girinimbine and the other carbazole compounds possess potential antitrypanosomal activity with comparably low toxicity against mammalian cells. Girinimbine, in particular, is a good candidate to be further investigated as a potential antitrypanosomal agent using in vivo models.

Toxoplasma gondii infection in native village chickens (Gallus domesticus) in Selangor and Melaka, Malaysia

@#Toxoplasmosis is a worldwide zoonosis caused by the protozoa Toxoplasma gondii which affects human and animals. Village chickens (Gallus domesticus) most commonly known as Ayam Kampung or free-range chickens, have been suggested to play a role in the epidemiology of toxoplasmosis. This study determines the presence of T. gondii in the village chicken populations in two states of Malaysia. A total of 50 serum samples from the chickens from Selangor (n=20) and Melaka (n=30) were collected and analysed using commercial serological kits. T. gondii antigen was detected in 20% (Selangor 30%; Melaka 13%) samples using ELISA test and anti-T. gondii antibody was detected in all positive ELISA samples using the indirect haemagglutination test (IHAT). Histopathological examination revealed tissue changes such as inflammation and degeneration in brain and liver of seropositive chickens. This is the first report of T. gondii infection in the village chickens in Malaysia.

Detection of Metabolites of Vecuronium Bromide in Visceral Samples Solves A Typical Death Mystery- (Toxicokinetics- Studies Using Gc-Ms Technique).

Introduction: On Sudden death of a medical student (20 year old girl) of National Institute of Medical Science autopsy materials along with site of injection were sent to F.S.L. It was informed during investigation that the girl took tetanus vaccine before death. In crime scene investigation Forensic Team observed that it was a case of gross negligence of dispensing wrong injection. Material: Tests were performed on viscera material viz., liver, spleen, kidney, lungs, brain, skin and blood etc. Mystery of suspected death was solved when a new GC-MS application was designed to get the unknown drug and various fragments of extracted material were studied. Method: A new method was developed on gas-chromatography-mass spectrophotometer and TL.C using various solvent systems is explained. Results: Metabolite fragments of vecuronium bromide a muscle relaxant were surprisingly observed in Viscera material, Blood sample and Skin piece from Leftt cubital fossa from this young girl, whose death is questioned. Conclusion: Structural elucidations of fragments provide a new approach to toxicokinetics. Th e explanation of fragments obtained were structurally compared with other neuromuscular blocking groups like atracurium and succinyl choline Th e presence of bromide attached cholest-5-en-Br, hydroxylated cholest-5-en-ol as hydroxylated product, acetylated fragments as cholest-5-enacetate and piperidone-2-one present in visceral samples indicates structural part obtained from vecuronium bromide drug. Th ese metabolites studies makes the case studies highly informative. Beside this new method of extractions, TLC systems and colouring reagents are also explained.

Azoospermia factor deletions in varicocele cases with severe oligozoospermia.

BACKGROUND: Varicocele is the most common cause of male infertility. The etiology and pathophysiology of varicocele are multifactorial. When low sperm counts are associated with varicocele, varicocelectomy can partially restore spermatogenesis and fertility. Few recent studies have reported that in some varicocele cases, there may be an associated genetic etiology. Presence of a genetic factor like azoospermia factor microdeletions may lead to irreversible spermatogenic arrest in these cases, but very few reports support these findings. However, it is still not understood why some cases improve after varicocelectomy and why some cases show no improvement in semen parameters postoperatively. AIM: It is important to distinguish varicocele cases from Yq microdeletions as these cases have irreversible testicular damage and thus carry a poor prognosis after varicocelectomy. SETTINGS: Research and Referral tertiary care hospital. Design: Prospective study. MATERIALS AND METHODS: Seventy-two infertile men with varicocele were referred for Yq microdeletion analysis from the infertility clinic of AIIMS and Army Research and Referral Hospital. Genomic DNA was isolated from blood and polymerase chain reaction microdeletion screening was done in these cases to determine the presence or deletion of AZF loci. RESULTS: In this study 7 (9.7%) varicocele cases harbored Yq microdeletion. The sperm count in cases which harbored Yq microdeletion was significantly lower than in cases without Yq microdeletion. CONCLUSION: Varicocele cases with Yq microdeletion do not show improvement in semen parameters post-varicocelectomy. Detection of Yq microdeletion determines prognosis and future management in such cases.

Detection of endosulfan isomers and metabolites by GC-MS in the exhumed body of an infant.

Endosulfan is a broad spectrum, non-systemic contact, and alimentary insecticide. It is a mixture of two isomers. The analysis of this insecticide, its isomers a and b endosulfan and other metabolic fragments, especially endolactone and a chlorinated dicarboxylic product in the autopsy material of an infant was performed by GC-MS in an alleged case of infanticide related to a dowry demand case. It confirmed that the child had been poisoned with endosulfan prior to death. No autopsy material, except ash and bone were left of the mother, as her par-ents-in-law had cremated her body. A new method was developed on GC-MS for the detection of this insecticide, which can be highly useful for routine analysis of insecticides in forensic laboratories. A study of various metabolites of endosulfan was also performed which may be informative for metabolic pathways studies.

Evaluation of bifenthrin treated mosquito nets against anopheline and culicine mosquitoes.

BACKGROUND AND OBJECTIVES: The main rural malaria vector Anopheles culicifacies has developed resistance to dichloro diphenyl trichloroethane (DDT), hexachloro cyclo hexane (HCH) and malathion in the state of Haryana in northern India. An alternative synthetic pyrethroid insecticide bifenthrin was therefore evaluated on mosquito nets against anopheline and culicine mosquitoes, in two villages Jagdishpur and Garh Mirakpur of Community Health Center (CHC) Badhkhalsa in district Sonipat, Haryana state. METHODS: Two formulations of bifenthrin, suspension concentrate (SC) and micro-emulsion (ME) were compared with micro-capsule suspension (CS) of lambdacyhalothrin. The impact of three doses of bifenthrin (10, 25 and 50 mg/m(2)) impregnated on mosquito nets was compared with lambdacyhalothrin (25 mg/m(2)) and untreated control. Quality assessment of treatment on treated nets was carried out by residue analysis and the persistence of the insecticide on nets was determined by contact bioassays. Efficacy of treated nets on mosquito density was assessed by calculating mosquito entry rate, immediate mortality, delayed mortality and excito-repellency to the insecticides. RESULTS: In susceptibility tests An. culicifacies was susceptible to bifenthrin (0.1% test papers) and to lambdacyhalothrin (0.05% test papers). Bioassays on treated nets against A. culicifacies recorded 100 per cent mortality up to tenth fortnight for all the doses of impregnation with bifenthrin (SC and ME) and lambdacyhalothrin (CS). Ring-net bioassays against An. culicifacies showed median knock-down time between 3.1 to 11.4 min. Behavioural indices were also studied for anopheline and culicine mosquitoes. The reduction in entry rates of anopheline and culicine mosquitoes into the rooms with treated nets compared to control indicated good efficacy with all the formulations and doses of the insecticides. INTERPRETAION AND CONCLUSION: Indoor (immediate) mortality of mosquitoes with bifenthrin ME formulation was relatively lower compared to SC fomulation of bifenthrin and based on delayed mortility and continued susceptibility in bioassays, bifenthrin ME at the rate of 10 mg/m(2) dose was found suitable for the impregnation of mosquito nets for phase III trial.

Seasonal fluctuations of dengue fever vector, Aedes aegypti (Diptera: Culicidae) in Delhi, India.

Studies on the seasonal fluctuation of Aedes aegypti were undertaken in different localities of Delhi, during 2000. The Aedes aegypti population was found to be prevalent in all the localities in Delhi. Water coolers and tires were found to be the preferred breeding habitats of Aedes mosquitos in the city. Aedes aegypti, being hygroscopic, showed a phenomenon of annual pulsation. It tends to move to mother foci in the central areas of the city, which are humid in the dry season, and spread out during the wet season. Out of 103,778 houses surveyed, 20,513 houses and 3,547 containers were reported positive for Aedes aegypti. The house container, and Breteau indices were very high during the post-monsoon season. The container indicies was very high (17.7%) in the defence area in September 2000. The container index in the areas of the Municipal Corporation of Delhi (MCD) and the New Delhi Municipal Committee (NDMC) were found to be high during the same period. The house index forAedes aegypti ranged from 0.1 to 7.4, 0.1 to 11.3, and 0.1 to 11.1 in the MCD, NDMC, and Defence areas, respectively.

Susceptibility status of Japanese encephalitis vectors in Kurnool and Mehboobnagar districts of Andhra Pradesh, India.

An entomological survey in Kurnool and Mahboobnagar districts of Andhra Pradesh state, India was undertaken in January, 2002 to find out vector population of Japanese Encephalitis and their susceptibility status to different insecticides. Five Culicine species viz. of Cx. tritaeniorhynchus, Cx. vishnui, Cx. pseudovishnui, Cx. bitaeniorhynchus, Cx. gelidus were captured during the survey. Another suspected vectors Anopheles subpictus and An. hyrcanus were also prevalent in the study villages. The larval population showed a high degree of resistance to Fenthion and Temephos. The test mortality to DDT ranged between 75% and 90%. The mortality of Cx. tritaeniorhynchus, Cx vishnui and An. subpictus ranged from 85% to 95%. Cx. tritaeniorhynchus is susceptible to Deltamethrin and Cyfluthrin. Cx. vishnui showed 80 % and 85% mortality to DDT and Malathion respectively.

Current status of fertility control methods in India.

Approximately 48.2% of couples of 15 to 49 years of age practice family planning methods in India. Female sterilization accounts for 34.2%, with male sterilization declining from 3.4% in 1992-93 to 1.9% in 1998-99. Use of the condom increased to 3.1% from 2.4%. There is an urgent need for research to develop new contraceptive modalities especially for men and also for women and to make existing methods more safe, affordable and acceptable. Current efforts in India to develop a male contraceptive are mainly directed towards (i) development of antispermatogenic agents to suppress sperm production, (ii) prevention of sperm maturation, (iii) prevention of sperm transport through vas deferens or rendering these sperm infertile and (iv) prevention of sperm deposition. Research work in the field of prevention of sperm transport through vas deferens has made significant advances. Styrene maleic anhydride (SMA) disturbed the electrical charge of spermatozoa leading to acrosome rupture and consequent loss in fertilizing ability of sperm. A multicentre phase-III clinical trial using SMA is continuing and it is hoped that the SMA approach would be available in the near future as an indigenously developed injectable intra-vasal male contraceptive. The safety and efficacy of available oral contraceptives were evaluated. An indigenously developed oral contraceptive 'Centchorman', which is a nonsteroidal, weakly estrogenic but potently antiestrogenic, was found to be safe and effective and is now being marketed in India since 1991 as a 'once a week' pill. Cyclofem and Mesigyna have been recommended as injectable contraceptives with proper counselling and service delivery by Indian studies. It has been recommended that these injectable contraceptives be added to the existing range of contraceptive methods available in the National Family Planning Programme. Based on the Indian studies CuT 200 was also recommended. Studies have indicated the advantage of intrauterine devices (IUD); they are long acting, relatively easily removed and fertility returns rapidly after their removal. Recent studies have recommended CuT 200 for use up to 5 years. The combination of some plant products i.e. Embelia ribes, Borax and Piper longum has been found to be safe and effective as a female contraceptive and the results of phase-I clinical trials are encouraging. Research work is going on in the country in various areas with special reference to hormonal contraceptive - a three monthly injectable contraceptive, immuno-contraceptives, antiprogestins, etc.

Preventing dengue/dengue haemorrhagic fever outbreaks in the National Capital Territory of Delhi--the role of entomological surveillance.

Outbreaks of Dengue/Dengue Haemorrhagic Fever have been occurring in the National Capital Territory (NCT) Delhi from time to time. A massive outbreak of Dengue/DHF causing considerable mortality occurred in 1996 in the NCT, Delhi. Since this outbreak regular entomological surveillance has been instituted, for timely preventive action against Dengue/DHF. The Central Cross Checking Organisation (CCO) of the National Malaria Eradication Programme cross-checks Aedes surveillance activities in Delhi. In this paper we present our experiences in entomological surveillance in relation Dengue, to share them with other workers in this field.

Prevalence of North India of hepatitis B carrier state amongst pregnant women.

The study was undertaken to determine the hepatitis B carrier rate in North India along with the relative infectivity of the carriers. A total of 1,112 pregnant women were investigated for hepatitis B carrier state during their routine visits to antenatal clinics. All three tiers of the health care delivery system were included from four regions of North India. The sera were screened for the presence of hepatitis B surface antigen (HBsAg), hepatitis B "e" antigen (HBeAg), and antibody to hepatitis B "e" antigen (Anti-HBe) by third generation Macro ELISA tests. The average hepatitis B surface antigen carrier rate was 9.5%. The carriers were found to be of relatively low infectivity with HBeAg and Anti-HBe present in 12.0% and 25.3% of the HBsAg carriers respectively, and both these markers absent in 62.7%. It was concluded that in the past decade the hepatitis B endemicity in North India has probably increased, but the relative infectivity of the carriers remains the same.

V. cholerae 01 outbreak in remote villages of Shimla district, Himachal Pradesh, 1994.

An outbreak of V. cholerae 01 occurred in remote villages of Rohru tehsil, district Shimla, Himachal Pradesh, during June-July 1994. Seven villages were affected. Overall attack rate was 16.4 per cent in surveyed villages. Attack rate in children < 5 was significantly high. Suspected source was spring water contaminated from open air defaecation. V. cholerae was resistant to co-trimoxazole and streptomycin.

Status of Plasmodium falciparum resistance to chloroquine in Orissa.

Orissa is known for its contribution of 15-20% of malaria cases to the national total. Deaths due to malaria in the state are also not uncommon. Proportion of P. falciparum cases have reached to 85%. In the recent years over 0.3 million confirmed malaria cases have been reported each year. Annual consumption of chloroquine in the state is over 170 lakh tablets. A 17 year study on monitoring of choloroquine resistance in P. falciparum in Orissa revealed that out of a total of 1165 tests conducted in vivo, in 12% of the cases RI level of resistance was detected. And 4.4% cases were of RII and 1.9% RIII level. 51% of the sample tested in vitro showed P. falciparum resistance to chloroquine. P. falciparum resistance to chloroquine appears to have been developed by the parasite over the length and breadth of the state. Strengthening of the monitoring of drug resistance in P. falciparum in the state is indicated.