In silico structural modeling and quality assessment of Plasmodium knowlesi apical membrane antigen 1 using comparative protein models

@#Plasmodium knowlesi is the most common zoonotic parasite associated with human malaria infection in Malaysia. Apical membrane antigen 1 (AMA1) protein in the parasite plays a critical role in parasite invasion into host cells. To date, there is no complete three-dimensional ectodomain structure of P. knowlesi AMA1 (PkAMA1) protein. The knowledge of a protein structure is important to understand the protein molecular functions. Three in silico servers with respective structure prediction methods were used in this study, i.e., SWISS-MODEL for homology modeling and Phyre2 for protein threading, which are template-based modeling, while I-TASSER for template-free ab initio modeling. Two query sequences were used in the study, i.e., native ectodomain of PkAMA1 strain H protein designated as PkAMA1-H and a modified PkAMA1 (mPkAMA1) protein sequence in adaptation for Pichia pastoris expression. The quality of each model was assessed by ProSA-web, QMEAN and SAVES v6.0 (ERRAT, Verify3D and Ramachandran plot) servers. Generated models were then superimposed with two models of Plasmodium AMA1 deposited in Protein Data Bank (PDB), i.e., PkAMA1 (4UV6.B) and Plasmodium vivax AMA1 (PvAMA1, 1W81) protein structures for similarity assessment, quantified by root-meansquare deviation (RMSD) value. SWISS-MODEL, Phyre2 and I-TASSER server generated two, one and five models, respectively. All models are of good quality according to ProSA-web assessment. Based on the average values of model quality assessment and superimposition, the models that recorded highest values for most parameters were selected as best predicted models, i.e., model 2 for both PkAMA1-H and mPkAMA1 from SWISS-MODEL as well as model 1 of PkAMA1-H and model 3 of mPkAMA1 from I-TASSER. Template-based method is useful if known template is available, but template-free method is more suitable if there is no known available template. Generated models can be used as guidance in further protein study that requires protein structural data, i.e., protein-protein interaction study.

Bioinformatics characterization of Plasmodium knowlesi apical membrane antigen 1 (PkAMA1) for multi-epitope vaccine design

@#Malaria caused by Plasmodium knowlesi species has become a public health concern, especially in Malaysia. Plasmodium knowlesi parasite which originates from the macaque species, infects human through the bite of the Anopheles mosquitoes. Research on malaria vaccine has been a continuous effort to eradicate the malaria infection, yet there is no vaccine against P. knowlesi malaria to date. Apical membrane antigen 1 (AMA1) is a unique surface protein of all apicomplexan parasites that plays a crucial role in parasite-host cell invasion and thus has been a long-standing malaria vaccine candidate. The selection of protective epitopes in silico has led to significant advances in the design of the vaccine. The present study aimed to employ bioinformatics tools to predict the potential immunogenic B- and T-cell epitopes in designing malaria vaccine targeting P. knowlesi AMA1 (PkAMA1). B-cell epitopes were predicted using four bioinformatics tools, i.e., BepiPred, ABCpred, BcePred, and IEDB servers whereas T-cell epitopes were predicted using two bioinformatics servers, i.e., NetMHCpan4.1 and NetMHCIIpan-4.0 targeting human major histocompatibility complex (MHC) class I and class II molecules, respectively. The antigenicity of the selected epitopes computed by both B- and T-cell predictors were further analyzed using the VaxiJen server. The results demonstrated that PkAMA1 protein encompasses multi antigenic regions that have the potential for the development of multi-epitope vaccine. Two B- and T-cell epitopes consensus regions, i.e., NSGIRIDLGEDAEVGNSKYRIPAGKCP (codons 28-54) and KTHAASFVIAEDQNTSY RHPAVYDEKNKT (codons 122-150) at domain I (DI) of PkAMA1 were reported. Advancement of bioinformatics in characterization of the target protein may facilitate vaccine development especially in vaccine design which is costly and cumbersome process. Thus, comprehensive B-cell and T-cell epitope prediction of PkAMA1 offers a promising pipeline for the development and design of multi-epitope vaccine against P. knowlesi.

High incidence of Plasmodium knowlesi malaria compared to other human malaria species in several hospitals in Malaysia

@# Through the regional control programme, Malaysia has been successfully reducing the incidence of Plasmodium falciparum and Plasmodium vivax infections. However, the incidence of zoonotic malaria Plasmodium knowlesi infection is increasing and now has been the major cause of malaria in Malaysia especially Malaysian Borneo. The emergence of knowlesi infection has threatened the malaria elimination programme which the government aims to reduce the overall malaria infections by 2020. Unlike other benign human Plasmodium spp., P. knowlesi can cause fatal infections. The aim of this study was to determine the incidence and distribution of five human malaria parasites including P. knowlesi in Peninsular Malaysia and Malaysian Borneo. A total of 112 blood samples were collected from seven states and district hospitals in Peninsular Malaysia and Malaysian Borneo from year 2015 to 2016. The samples were examined by microscopy and further confirmed by nested PCR assay targeting 18S rRNA gene of Plasmodium spp. Following the nested PCR assays, a total of 54 (48.2%) samples were positive for P. knowlesi infections, 12 (10.7%) cases were positive for P. vivax infections, followed by 7 (6.3%) cases of P. falciparum and 4 (3.5%) cases of P. malariae. There were 3 cases (2.7%) of mixed infections (P. knowlesi/P. vivax). However, no cases were identified as P. ovale. A total of 32 (28.6%) cases were found as negative infections. LoopMediated Isothermal Amplification Assay (LAMP) was performed to confirm inconclusive results produced by microscopy and nested PCR. P. knowlesi showed the highest prevalence in Sarawak (n= 30), Sabah (n=13), Pulau Pinang (n=5) and Pahang (n=6). PCR and LAMP was not able to detect a large number of microscopy positive samples due to DNA degradation during storage and shipping. Among all the states involved in this study, the highest prevalence of P. knowlesi infection was found in Sabah and Sarawak.

Plasmodium falciparum protein kinase as a potential therapeutic target for antimalarial drugs development

@#Malaria is one of the most dangerous infectious diseases due to its high infection and mortality rates, especially in the tropical belt. Plasmodium falciparum (P. falciparum), the most virulent malaria parasite in humans, was recently reported to develop resistance against the final efficient antimalarial drug, artemisinin. Little is known about the resistance mechanisms, which further complicates the problem as a proper counteraction is unable to be taken. Hence, the understanding of drug mode of action and its molecular target is valuable knowledge that needs to be considered to develop the next generation of antimalarial drugs. P. falciparum protein kinase (Pf PK) is an attractive target for antimalarial chemotherapy due to its vital roles in all P. falciparum life stages. Moreover, overall structural differences and the presence of unique Pf PKs that are absent in human kinome, suggesting specific inhibition of Pf PK without affecting human cells is achievable. To date, at least 86 eukaryotic protein kinases have been identified in P. falciparum kinome, by which less than 40 were validated as potential targets at the erythrocytes stage. In this review, recent progress of the furthest validated Pf PKs; Pf Nek-1, Pf CDPK1, Pf CDPK4, Pf PKG, and Pf CLK-3 will be briefly discussed.

Genetic diversity of Merozoite Surface Protein-1 gene block 2 allelic types in Plasmodium falciparum isolates from Malaysia and Thailand

@#Malaria is the most common vector-borne parasitic disease in Malaysia and Thailand, especially in Malayan Borneo and along the Thailand border areas, but little is known about the genetic diversity of the parasite. Present study aims to investigate the genetic diversity of Plasmodium falciparum isolates in these two countries and eventually contributes to more effective malaria control strategies, particularly in vaccine and antimalarial treatment. One hundred and seventy three P. falciparum isolates were collected from Malaysia (n = 67) and Thailand (n = 106) and genotyped using nested PCR targeting the polymorphic region of MSP-1, block 2. Sequence analysis was conducted to investigate the allele diversity of the isolates. Three allelic families were identified in Malaysian and Thailand P. falciparum isolates, MAD20, K1 and RO33. Sequence analysis revealed that there were 5 different MAD20, 1 K1 and 2 different RO33 for Malaysian isolates. Thailand isolates exhibited greater polymorphism because there were 13 different MAD20, 6 different K1 and 2 different RO33 identified in this study. Multiclonal infections were observed for the isolates in both countries, however, low multiplicity of infection (MOI) was observed for Malaysian (1.1) and Thailand (1.2) isolates. Phylogenetic analysis showed that P. falciparum isolates of Malaysia and Thailand were clustered in the same group for all the allelic families. Population structure of P. falciparum isolates in Malaysia and Thailand exhibit extensive genetic polymorphism but showed high similarities as well as comparable MOI.

Tuberculosis Verrucosa Cutis in an 11-year-old girl: A case report

@#We report a case of an 11-year-old girl who presented with a gradually enlarging verrucous plaque on the left knee for 3 years. Physical examination showed a solitary, slightly erythematous, scaly, verrucous plaque on the left knee measuring about 1.5 cm x 2 cm. Biopsy revealed granulomatous dermatitis consistent with cutaneous tuberculosis. A diagnosis of tuberculosis verrucosa cutis (TBVC) was made and anti-tuberculous therapy was initiated consisting of rifampicin, isoniazid, pyrazinamide and ethambutol for 2 months followed by rifampicin and isoniazid for 4 months. Upon completion of therapy, only a slightly atrophic scar remained, supporting our diagnosis. This report highlights TBVC must be considered in patients with chronic skin lesions in countries with high prevalence of tuberculosis.

Anti-malarial and anti-inflammatory effects of Gleichenia truncata mediated through inhibition of GSK3β

Gleichenia truncata is a highland fern from the Gleicheniaceae family known for its traditional use among indigenous communities in Asia to treat fever. The scientific basis of its effect has yet to be documented. A yeast-based kinase assay conducted in our laboratory revealed that crude methanolic extract (CME) of G. truncata exhibited glycogen synthase kinase-3 (GSK3)-inhibitory activity. GSK3β is now recognized to have a pivotal role in the regulation of inflammatory response during bacterial infections. We have also previously shown that lithium chloride (LiCl), a GSK3 inhibitor suppressed development of Plasmodium berghei in a murine model of malarial infection. The present study is aimed at evaluating G. truncata for its anti-malarial and anti-inflammatory effects using in vivo malarial and melioidosis infection models respectively. In a four-day suppressive test, intraperitoneal injections of up to 250 mg/kg body weight (bw) G. truncata CME into P.berghei-infected mice suppressed parasitaemia development by >60%. Intraperitoneal administration of 150 mg/kg bw G. truncata CME into Burkholderia pseudomallei-infected mice improved survivability by 44%. G. truncata CME lowered levels of pro-inflammatory cytokines (TNF-α, IFN-γ) in serum and organs of B. pseudomallei-infected mice. In both infections, increased phosphorylations (Ser9) of GSK3β were detected in organ samples of animals administered with G. truncata CME compared to controls. Taken together, results from this study strongly suggest that the anti-malarial and anti-inflammatory effects elicited by G. truncata in part were mediated through inhibition of GSK3β. The findings provide scientific basis for the ethnomedicinal use of this fern to treat inflammation-associated symptoms.

A multi-center retrospective study on atopic dermatitis at the outpatient departments of Philippine Dermatological Society-accredited training institutions from 2007-2011

BACKGROUND: To date, no multicenter studies have been conducted on the prevalence and clinical profile of AD in the Philippines. Since AD is one of the top 10 skin diseases seen in the outpatients departments of all the Philippine Dermatologic Society (PDS)- accredited institutions, conducting a multicenter study provides important epidemiological information about this disease and serve as a valuable reference for future studies. OBJECTIVES: To determine the prevalence and clinical profile of patients with atopic dermatitis (AD) seen at the outpatient departments (OPD) of Philippine Dermatological Society (PDS) - accredited training institutions from 2007 to 2011. METHODS: Records of patients with a diagnosis of AD seen from January 1, 2007 to December 31, 2011 were retrieved and clinical data were collected. RESULTS: There were 744,673 dermatological consults in the 10 PDS-accredited outpatient clinics from 2007-2011. A total of 4,275 records of atopic dermatitis were reviewed for this study. The prevalence of atopic dermatitis was determined to be 0.57%. Most institutions reported a prevalence rate of less than 1% except for St. Luke's Medical Center (3.36%), and Research Institute for Tropical Medicine (7.07%). More than half of the patients (65.1%) were children between 1 to 12 years old. Twenty-four percent (24%) were infants less than one year. The average age was seven years old while the youngest was one month and the oldest was 94 years old. There were more females (56.1%) than males (42.75%). Bronchial asthma was the most prevalent co-morbid medical condition. Majority of AD patients seen in institutions were newly diagnosed. Those with previous consultations were mostly seen by dermatologists and pediatricians. Moisturizers and topical corticosteroids were the most commonly used topical preparation while antihistamines followed by oral antibiotics were the commonly prescribed oral medications. Follow-up rate was low. CONCLUSION: The prevalence of atopic dermatitis among the 10 PDS-accredited institutions is low except for SLMC and RITM. The clinical profile of patients is consistent with published literature. However, this study revealed the patient follow-up is low. This practice needs to be addressed since optimal management of this chronic disease requires close and regular follow-up to prevent complications and irrational drug use.

Factors Influencing Malnutrition among Young Children in a Rural Community of Sarawak

Introduction: This study aimed to determine the prevalence of malnutrition and factors influencing malnutrition among children aged five years and below in Serian District of Sarawak, a district where the majority of people are indigenous. Methods: Using a cross-sectional method, a total of 177 children were randomly selected and assessed, and their mothers or caregivers were interviewed. Data were collected using a pretested questionnaire; anthropometric measurements were also taken. Data were analysed using SPSS version 17.0. Results: The prevalence of underweight, stunting and wasting in children aged five years and below was 20.9%, 11.9% and 10.2% respectively. The results suggest that the significant factors contributing to underweight are birth weight, frequency of child visit to the clinic, and individual insecurity. Only age, when complementary diet was introduced, was found to be significantly associated with stunting. For wasting, individual insecurity, duration of family planning and met recommended protein intake per day were found to be significant factors. Conclusion: The prevalence of malnutrition in children 5 years and below in Serian District of Sarawak is high compared to state figures. The major contributing factors were found to be manifested at the individual and family level. There is a need for appropriate public health promotion and socioeconomic improvement interventions towards improving the nutritional status and health of children in Serian District.

The lethal gene in a 6-year-old boy: A case report

This is a report of a case of a 6-year-old boy who came in due to generalized crusted erosions. His condition started at 2 days of age as appearance of tense vehicles which progress into bullae on normal or erythematous base after mild trauma on the extremities and later become generalized. The vesicles would become ersosions and crusts and heal with minimal residual scarring. As old lesions heal, new vesicles would appear. Nikolsky and Asboe-Hansen signs were negative. No extracutaneous or mucosal involvement was noted. Family history revealed that all of his five siblings had appearance of similar vesicobullous lesions few days after birth. Four of them died during the first six months of life. The older surviving sister, now age 10, showed gradual improvement of the condition as she grew older.Paternal and maternal family histories have no similar lesions. Skin punch biopsy revealed a subepidermal blistering disease. Direct and indirect immunofluoroscence were negative, consistent with generalized atrophic benign epidermolysis bullosa. Treatment of this disorder remains elusive. Management is focused mainly in the prevention of trauma as well as supportive measures. Patient education and genetic counseling are also cornerstones of management as in any genetic disorder. Advances in prenatal testing and gene therapy provide hope for early diagnosis and intervention.

Paraneoplastic pemphigus and myasthenia gravis: A rare duo of paraneoplastic manifestations secondary to thymoma

This case is reported because of the very rare combination of paraneoplastic pemphigus and myasthenia gravis as paraneoplastic manifestations secondary to thymoma. A case of a 37 year old male who presented with skin lesions initially as papules in the face and thighs that evolved into blisters and became widespread, leaving crusted and hyperpigmented patches and plaques. It was associated with cough, dyspnea, ptotis, dysphagia, dysphonia and facial asymmetry. Histopathology and direct immunofluorescence were consistent with paraneoplastic pemphigus. Anticholinerase test confirmed the diagnosis of myasthenia gravis and histologic finidngs of the anterior mediastinal mass confirmed it as thymoma.

Primary giant cell malignant fibrous histiocytoma of the kidney with staghorn calculi.

Malignant fibrous histiocytomas (MFH) as primary renal tumours are rare, with less than 50 cases described in the literature. We report a case of primary renal MFH of giant cell type in a 56-year-old man, who presented with bilateral dull flank pain, intermittent gross haematuria and body weight loss (6 kg in 3 months). Intravenous urography, computerized tomography (CT) and magnetic resonance image (MRI) showed right ureteral stones with mild hydronephrosis, and a solid mass at the lower pole of the left kidney associated with staghorn calculi, as well as tumour thrombi in the left renal vein and inferior vena cava. Left radical nephrectomy and evacuation of tumour thrombi from the left renal vein and inferior vena cava were performed. Histopathologic examination revealed malignant fibrous histiocytoma (MFH) of giant cell type. To the best of our knowledge, this is the first report of primary renal MFH associated with staghorn calculi.

Immunogenicity and safety of an inactivated hepatitis A vaccine in Taiwanese adults and children.

The safety and immunogenicity of an inactivated hepatitis A vaccine (AVAXIM, 160 antigen units) was evaluated in 190 subjects: 50 children aged from 2 to 5 years, 70 children aged from 6 to 17 years and 70 adults aged from 18 to 30 years in a monocentric, open, non-controlled, phase III trial conducted in Taipei, Taiwan from December 1996 to October 1997. The vaccine was administered intramuscularly, with a two-dose schedule 6 months apart. Clinical adverse events were monitored during the seven days following each injection. Hepatitis A virus (HAV) antibody titers were measured by modified radioimmunoassay on the day of inclusion and four weeks after both the first dose and booster injection. Among the 190 subjects who received the first dose, 174 (91.6%) were initially HAV seronegative and 16 (8.4%) were HAV seropositive at inclusion. One hundred and seventy-four subjects (91.6%) received the booster dose and completed the study. One month after the first dose, all the subjects, whatever the age, presented HAV antibody titers over 20 mIU/ml. In children (2 to 17 years), the GMT was 136 mIU/ml at week 4 and 7,906 mIU/ml four weeks after the booster dose. In adults (> or = 18 years), GMT values were 93 mIU/ml at week 4 and 3,655 mIU/ml four weeks after the booster. These results show a strong anamnestic response to the second dose of vaccine and are compatible with long-term antibody persistence in each age group. The vaccine was safe and well tolerated. No vaccine-related serious adverse event occurred. No immediate reaction occurred. The majority of the reactions were reported by adults after the primary injection. Local reactions (pain and redness) were reported by 9.0% and 4.0% of the subjects after the primary and the booster doses, respectively. Systemic reactions (mainly myalgia/arthralgia or asthenia) affected less than 10% of the subjects after the first dose and less than 3% after the booster. Results from this study in a Taiwanese population are consistent with those obtained with the same vaccine in previous European studies in children and adults, and suggest that AVAXIM (160 AU) is suitable for use in all subjects aged over 2 years.

Evaluation of efficacy and safety of buspirone in patients with symptoms of anxiety

Of the 30 subjects who completed the study, the l7 males and l3 females have ages ranging from 26-45 years in the majority of subjects. The composition of subjects showed 57 % with positive history for treatment for emotional problems. Sixty-three percent had taken psychotropic drugs. The Hamilton Anxiety Scale ranges done on the subjects revealed a score of l9 to 40 (mean of 27.2). Buspirone efficacy had been documented with a noted improvement in terms of the degree of psychopathology in the third week of study following treatment with Buspirone. In terms of subject evaluation, the subjects felt that the drug helped most in 37 %, very much in 25 %, moderately in 25 %, and a little in l3 %. As to physicians evaluation of therapeutic effect, 30 % had complete of nearly complete remission of all symptoms, another 53 % had partial remission of symptoms. With regards to side effects 15 % had side effects which slightly interfered with subjects functioning consisting of dizziness in one subject, heaviness of head, and dryness of the mouth. Otherwise, these side effects have not stopped the subjects from doing their usual activities

The value of potency testing of poliomyelitis and measles vaccines as an integral part of cold chain surveillance.

The Virology Division in the Institute for Medical Research, Kuala Lumpur, Malaysia performs potency tests on oral polio vaccines and live attenuated measles vaccines. Since these potency tests were introduced in 1981 a total of 752 tests have been performed on vaccine samples from peripheral immunization centers. Of 165 representative vaccine samples sent for potency evaluation after a cold chain breakdown 154 (87%) passed minimum potency requirements recommended for immunization of infants. In the absence of potency evaluation, those vaccines exposed to temperatures higher than the recommended storage range would be discarded, perhaps resulting in unnecessary wastage and economic loss. Results of the vaccine potency evaluation has enabled health authorities to indirectly monitor cold chain efficiency and ensure the high quality of viral vaccines used in our childhood immunization program.

Study on Schistosoma sinensium in Fang district, Chiangmai province, Thailand.

A survey on human infection and possible natural definitive host of Schistosoma sinensium was carried out in Fang District, Chiangmai Province, North Thailand, where Tricula bollingi snails which harbour cercariae of S. sinensium inhabits. Stool examination of the people in the two villages along the stream, where T. bollingi were found, was by formalin-ether concentration technique and by Stoll's method. The stools were found to be negative for S. sinensium eggs. Field rats were also trapped and examined for the presence of S. sinensium. Adult worms and eggs of S. sinensium were found in the mesenteric veins and livers, respectively, of the field rats, Rattus rattus, captured in the rice fields along the stream.