Experimental Study on Plasmodium knowlesi Normocyte Binding Protein Xa Region II (PkNBPXaII) for Erythrocyte Binding

@# Normocyte binding protein Xa (NBPXa) has been implied to play a significant role in parasite invasion of human erythrocytes. Previous phylogenetic studies have reported the existence of three types of NBPXa for Plasmodium knowlesi (PkNBPXa). PkNBPXa region II (PkNBPXaII) of type 1, type 2 and type 3 were expressed on mammalian cell surface and interacted with human and macaque (Macaca fascicularis) erythrocytes. The binding activities of PkNBPXaII towards human and macaque erythrocytes were evaluated using erythrocyte-binding assay (EBA). Three parameters were evaluated to achieve the optimal protein expression of PkNBPXaII and erythrocyte binding activity in EBA: types of mammalian cells, post transfection time and erythrocyte incubation time. COS-7, HEK-293, and CHO-K1 cells showed successful expression of PkNBPXaII, despite the protein expression is weak compared to the positive control. COS-7 was used in EBA. All three types of PkNBPXaII showed rosette formation with macaque erythrocytes but not with human erythrocytes. Future studies to enhance the PkNBPXaII expression on surface of mammalian cells is indeed needed in order to elucidate the specific role of PkNBPXaII in erythrocytes invasion.

Complications of Sub-microscopic Plasmodium vivax Malaria among Orang Asli in Pos Lenjang, Kuala Lipis

@#In recent years, increasing cases of Plasmodium vivax complications had been reported all over the world. This former benign Plasmodium species is now recognized to be one of the human malaria parasites that can produce severe disease. In this article, we report two cases of sub-microscopic P. vivax malaria confirmed by PCR. Both patients were asymptomatic before treatment. They showed unusual presentations few days after initiation of antimalarial treatment. Both patients had subsequently completed antimalarial treatment and recovered completely.

Investigative study on the role of the Toxo 5699 gene in the Toxoplasma gondii lytic cycle using the CRISPR/Cas9 system

@#The focus of the current study was to disrupt the Toxo 5699 gene via CRISPR/Cas9 to evaluate the effects of gene disruption on the parasite lytic cycle. In the present work, a single plasmid expressing both the guide RNA and Cas9 nuclease together with a selectable marker of human dihydrofolate reductase (DHFR) was introduced into Toxoplasma gondii. Targeted disruption of the Toxo 5699 gene was carried out via the CRISPR/Cas9 system and confirmed by PCR, sequencing, and immunofluorescence microscopy. Disrupted and nondisrupted control parasites were allowed to invade HS27 cell monolayers and plaques were counted. The average number of plaques from three replicates per group was obtained between the disrupted and non-disrupted T. gondii RH strain and was compared using a onetailed t-test. It was observed that there was a significant decrease in number and size of plaque formation in the Toxo 5699 gene disrupted parasite line. This is an indication that the Toxo 5699 gene may play a role in the lytic cycle of the parasite, particularly during the replication phase and thus would be a novel target for disruption or silencing. The Toxo 5699 gene presented in the current work is an important part of the T. gondii lytic cycle, therefore meriting further inquiry into its potential as a target for further genetic-silencing or disruption studies.

Prospective study of ventilated patients in the paediatric medical intensive care unit of Lady Ridgeway Hospital.

Objective: To determine the characteristics and outcome of ventilated patients in the paediatric medical intensive care unit (PICU) of Lady Ridgeway Hospital for Children (LRH), Colombo. Design: Prospective descriptive study Method: The study population consisted of all ventilated paediatric patients admitted to PICU, LRH from 1st March to 31st August, 2009. The factors studied included demographic profile (age, sex, residence, transfer), length of stay on ventilator, indication for ventilation, details of organ dysfunction and the final outcome. Results: There were 152 patients ventilated over the study period of six months. Seventy three were under one year of age. Male to female ratio was 1.17:1. A significant proportion (n=105) were from the Western province. Eighty five patients were transferred from medical wards of LRH. Median duration of ventilation was six days (IRQ 4-10). Indications for ventilation included respiratory problems (85), neurological disorders (13), cardiac causes (5), hepatic problem (1) and miscellaneous diseases (48) including 32 from dengue syndromes. There were 42 deaths among ventilated patients giving an overall mortality rate of 27.6%. Multi-organ dysfunction syndrome (MODS) on admission accounted for 81% of the deaths. Bronchopneumonia (16) and dengue haemorrhagic fever (12) were the main underlying causes of death. Thirty two (21%) had anaemia while in the PICU, 15 of them having anaemia on admission. Nosocomial infection was present in 17 (11%) patients. Children transferred from other provinces had a higher risk of death than those from the Western province (OR=1.5, 95% CI: 0.7 – 3.3). Conclusions: MODS on admission accounted for 81% of the deaths. Bronchopneumonia and dengue haemorrhagic fever were the main underlying causes of death. Children transferred from other provinces had a higher risk of death compared to those from the Western province.

Analysis of blood/tracheal culture results to assess common pathogens and pattern of antibiotic resistance at medical intensive care unit, Lady Ridgeway Hospital for Children.

Background: Intensive care units (ICUs) are burdened with a high frequency of nosocomial infections often caused by multi resistant nosocomial pathogens. Objectives: To determine the common pathogens in medical intensive care unit of Lady Ridgeway Hospital for Children (MICU-LRH) and to look for the pattern of antibiotic resistance of these pathogens. Design & Setting: This retrospective study was performed by tracing all the culture reports of MICU-LRH done at microbiology laboratory of LRH in the year 2006. Results: Total number of blood cultures done in 2006 was 659. Of them 123(18.7%) became positive. Out of positive blood cultures 38.2% were for spores and 24% for coliforms. Staphylococcus aureus (10.6%), streptococcus spp. (4.1%), pseudomonas spp. (4.1%) and candida spp. (4.9%) were the other pathogens in blood cultures. Out of 457 tracheal cultures done in 2006, 251(56%) were positive. Contamination with spores was 3.1%. Majority (43%) of tracheal cultures were positive for coliforms. Other common pathogens were pseudomonas spp. (19.5%) and candida spp. (9.8%) Resistance pattern of coliforms varied in blood cultures and tracheal cultures. There was significant resistance to aminoglycosides. Imipenem & meropenem resistant isolates were not found in blood cultures but in tracheal cultures 44% of isolates were resistant to imipenem & 42% were resistant to meropenem. Resistance pattern of pseudomonas to amikacin was around 34% in both blood & tracheal cultures. 25% of isolates in blood cultures and 50% of isolates in tracheal cultures were resistant to ceftazidime. Although, there was no resistance to ticarcillin in blood cultures, 51% pseudomonas isolated in tracheal cultures showed resistance. Resistance rate to ciprofloxacin was 50% in blood cultures and 34% in tracheal cultures. Eighty three percent of staphylococcus spp. in both blood & tracheal cultures were resistant to cloxacillin. More than 70% were resistant to gentamicin. Around 33% isolates in blood cultures & 22% in tracheal cultures were resistant to fusidic acid. However, all staphylococcus spp. were sensitive to Vancomycin. Conclusions: There were more positive tracheal cultures than blood cultures. Majority of septicaemia were due to coliforms. Coliforms and pseudomonas were major pathogens in tracheal cultures. There was significant colonization of candida spp. in respiratory tract of patients at MICU-LRH in contrast to candida septicaemia. Emergence of antibiotic resistance to broad spectrum antibiotics is a significant problem.

Idiopathic purpura fulminans in dengue hemorrhagic fever.

Purpura fulminans is a rapidly progressive thrombotic disease that has been described during both severe bacterial and viral infections. Disseminated intravascular coagulation (DIC), antiphospholipid antibodies and acquired or congenital C and S protein deficiency are thought to play a role in its pathogenesis. Here we report the case of a 4-year-old girl who developed gangrene of all her fingers and toes following dengue shock syndrome complicated by DIC and also discuss its management.