Limitations with current chemotherapeutic and vaccinal control of coccidiosis caused by Eimeria species continue to prompt development of novel controls, including the identification of new drug targets. Glucose-6-phosphate isomerase (G6-PI) has been proposed as a valid drug target for many protozoa, although polymorphism revealed by electrophoretic enzyme mobility has raised doubts for Eimeria. In this study we identified and sequenced the Eimeria tenella G6-PI orthologue (EtG6-PI) from the reference Houghton strain and confirmed its position within the prevailing taxonomic hierarchy, branching with the Apicomplexa and Plantae, distinct from the Animalia including the host, Gallus gallus. Comparison of the deduced 1647 bp EtG6-PI coding sequence with the 9016 bp genomic locus revealed 15 exons, all of which obey the intron-AG-/exon/-GT-intron splicing rule. Comparison with the Weybridge and Wisconsin strains revealed the presence of 33 single nucleotide polymorphisms (SNPs) and 14 insertion/deletion sites. Three SNPs were exonic and all yielded non-synonymous substitutions. Preliminary structural predictions suggest little association between the coding SNPs and key G6-PI catalytic residues or residues thought to be involved in the coordination of the G6-PI's substrate phosphate group. Thus, the significant polymorphism from its host orthologue and minimal intra-specific polymorphism suggest G6-PI remains a valid anti-coccidial drug target.
Microsporidia are ubiquitous parasites thought to be closely related to fungi. Their presence in the environment means that humans are frequently exposed to infection. Stool samples were collected from 151 indigenous villagers from the eastern state of Pahang in 2005. The samples were concentrated with water-ether sedimentation, stained with modified trichrome stain and examined under oil-immersion microscopy. Thirty-two specimens (21.2%) were positive for microsporidia. Microsporidia were observed as ovoid or rounded ovoid shapes measuring approximately 1mum, with a bright pink outline containing a central or posterior vacuole. PCR amplification with specific primers on microscopy-positive specimens amplified Encephalitozoon intestinalis DNA from five of the ten specimens used.
Cryptocaryon irritans is a parasitic ciliate that causes cryptocaryonosis (white spot disease) in marine fish. Diagnosis of cryptocaryonosis often depends on the appearance of white spots on the surface of the fish, which are usually visible only during later stages of the disease. Identifying suitable biomarkers of this parasite would aid the development of diagnostic tools and control strategies for C. irritans. The C. irritans genome is virtually unexplored; therefore, we generated and analyzed expressed sequence tags (ESTs) of the parasite to identify genes that encode for surface proteins, excretory/secretory proteins and repeat-containing proteins.
This study was conducted to determine the genotypes of Giardia duodenalis isolated from human faecal samples at Pos Betau, Pahang, Malaysia. Faecal specimens were collected and examined for G. duodenalis cysts using Trichrome staining techniques. Molecular identification was carried out by the amplification of a region of the small subunit of the nuclear ribosomal RNA (SSU rRNA) gene using nested PCR and subsequent sequencing. The sequences from 15 isolates from G. duodenalis were subjected to phylogenetic analysis (including appropriate outgroups) using the neighbor-joining and maximum parsimony methods. The trees identified G. duodenalis assemblages A and B, with a predominance of assemblage B. The predominance of anthroponotic genotypes indicates the possibility of anthroponotic transmission of these protozoa in this Semai Pahang Orang Asli community.
Dengue viruses circulate in both human and sylvatic cycles. Although dengue viruses (DENV) infecting humans can cause major epidemics and severe disease, relatively little is known about the epidemiology and etiology of sylvatic dengue viruses. A 20-year-old male developed dengue hemorrhagic fever (DHF) with thrombocytopenia (12,000/ul) and a raised hematocrit (29.5% above baseline) in January 2008 in Malaysia. Dengue virus serotype 2 was isolated from his blood on day 4 of fever. A phylogenetic analysis of the complete genome sequence revealed that this virus was a member of a sylvatic lineage of DENV-2 and most closely related to a virus isolated from a sentinel monkey in Malaysia in 1970. This is the first identification of a sylvatic DENV circulating in Asia since 1975.
In order to investigate the reliability of detecting HPV DNA in cervical smears, we compared the performance of nested MY/GP PCR and FDA approved-Hybrid Capture II (HCII) using clinical cervical scrapings from 40 patients. It was found that PCR was more sensitive (81.8%) in comparison to HCII (36.4%) in detecting HPV although specificity of HCII was much higher (96.6%) than PCR (58.6%). The Negative Predictive Value (NPV) of both the techniques were quite similar but Positive Predictive Value (PPV) of HCII was much higher (80.0%) compared to PCR (42.9%). While the HCII method showed good specificity for HPV detection, its lack of sensitivity as compared to PCR may be a drawback for diagnostic use.
We report on a Malaysian kindred with Li-Fraumeni syndrome. The proband was an 8-year-old girl who presented with embryonal rhabdomyosarcoma of the trunk at the age of 8 months and developed a brain recurrence at the age of 7 years, which was 5 years after remission. A younger sister later developed adrenocortical carcinoma at the age of 6 months. Their mother and maternal grandmother were diagnosed with breast cancer at the ages of 26 and 38 years, respectively. TP53 mutation detection in this family revealed a duplication of a GGCGTG motif starting at nucleotide 17579 in exon 10, resulting in an in-frame insertion of two amino acids between residues 334 and 336 in the tetramerization domain of the p53 protein. This mutation was found in the proband and her affected sister as well as her mother. In addition, the mutation was detected in two other siblings (a brother aged 3 years and a sister aged 18 months) who have not yet developed any malignancy. Sequencing of TP53 in the father and two other asymptomatic siblings revealed wild-type TP53. To our knowledge, this is a first report of a Li-Fraumeni syndrome family in Southeast Asia.
Gastrointestinal stromal tumour (GIST) is a rare but most common mesenchymal tumour in the gastrointestinal tract. Although GIST research has been carried out extensively worldwide, it has yet to be studied in Malaysia. To establish the immunohistochemical expression pattern of CD117 (c-KIT), CD34, S-100 and Desmin, the incidence of c-KIT and PDGFRA genes mutation in GISTs, and correlate it with clinicopathological parameters. Eleven clinically diagnosed GISTs were stained for CD117, CD34, Desmin and S-100 protein by immunohistochemical technique, and c-KITand PDGFRA gene mutations were studied by PCR-CSGE-DNA sequencing method. All GISTs (7 cases) stain positive for CD117, and co-expressed CD34. None of these cases express Desmin, and only one expressed S-100 protein focally. Fifty-seven percent (4/7 cases) of GIST harboured mutations at exon 11 of c-KIT gene, and they were all high risk and malignant cases. No mutation was detected at exons 9, 13 and 17 of KIT gene, and exons 12 and 18 of PDGFRA gene. Immunohistochemistry using a panel of antibodies shows consistent pattern of CD117 and CD34 expression in GIST, and mutational study may be a useful prognostic marker for kinase inhibitor treatment of GIST.
Ureaplasma parvum colonizes human mucosal surfaces, primarily in the respiratory and urogenital tracts, causing a wide spectrum of diseases, from non-gonococcal urethritis to pneumonitis in immunocompromised hosts. Although the basis for these diverse clinical outcomes is not yet understood, more severe disease may be associated with strains harboring a certain set of strain-specific genes. To investigate this, whole genome DNA macroarrays were constructed and used to assess genomic diversity in 10 U. parvum clinical strains. We found that 7.6% of U. parvum genes were dispersed into one or more strains, thus defining a minimal functional core of 538 U. parvum genes. Most of the strain-specific genes (79%) were of unknown function and were unique to U. parvum. Four hypervariable plasticity regions were identified in the genome containing 93% of the variability in the gene pool (UU32-UU33, UU145-UU170, UU440-UU447 and UU527-UU529). We hypothesized that one of them (UU145-UU170) was a pathogenicity island in U. parvum and we characterized it. Thus, we propose that the clinical outcome of U. parvum infection is probably associated with this newly identified pathogenicity island.
In this study, three single nucleotide polymorphisms (SNPs) located within the promoter of the human interleukin (IL)-10 gene [rs1800896 (position: -1087G>A), rs1800871 (position: -824C>T) and rs1800872 (position: -597C>A)] were investigated in 84 rheumatoid arthritis (RA) patients and 95 age- and sex-matched healthy subjects using polymerase chain reaction-restriction fragment length polymorphism method. Production of IL-10 by peripheral blood lymphocytes from the RA patients and healthy subjects cultured in the presence of Concanavalin A (Con A) was determined by using enzyme-linked immunosorbent assay. The results show that the distribution of the IL-10 genotypes did not differ significantly between RA patients and healthy subjects (P>0.05). However, a significant difference was observed in allele frequencies of -824CT, -824TT, -597CA, and -597AA between the RA patients and healthy volunteers (P=0.04). The -1087A/-824T/-597A (ATA) haplotype, which comprises all mutant alleles, was associated with lower IL-10 production when compared with the other haplotypes. In contrast, the RA patients who did not display the ATA haplotype produced significantly higher levels of IL-10 when compared with those carrying either one (P=0.012) or two (P=0.005) ATA haplotypes. Our findings suggest that there is an association between SNPs in the promoter of the human IL-10 gene and susceptibility to RA.
Study site: Hospital Tuanku Ja’afar, (Hospital Seremban), Seremban, Negeri Sembilan, Malaysia
The complete VP1 protein of EV71 was truncated into six segments and fused to the C-terminal ends of full-length nucleocapsid protein (NPfl) and truncated NP (NPt; lacks 20% amino acid residues from its C-terminal end) of newcastle disease virus (NDV). Western blot analysis using anti-VP1 rabbit serum showed that the N-terminal region of the VP1 protein contains a major antigenic region. The recombinant proteins carrying the truncated VP1 protein, VP1(1-100), were expressed most efficiently in Escherichia coli as determined by Western blot analysis. Electron microscopic analysis of the purified recombinant protein, NPt-VP(1-100) revealed that it predominantly self-assembled into intact ring-like structures whereas NPfl-VP(1-100) recombinant proteins showed disrupted ring-like formations. Rabbits immunized with the purified NPt-VP(1-100) and NPfl-VP(1-100) exhibited a strong immune response against the complete VP1 protein. The antisera of these recombinant proteins also reacted positively with authentic enterovirus 71 and the closely related Coxsackievirus A16 when analyzed by an immunofluorescence assay suggesting their potential as immunological reagents for the detection of anti-enterovirus 71 antibodies in serum samples.
A 2-year-old Chinese boy was referred to Hospital UKM for investigation of recurrent episodes of dark-coloured urine and pallor since birth. He was born prematurely at 34 weeks gestation and developed severe early-onset neonatal jaundice requiring exchange blood transfusion. Screening at birth showed Glucose-6-phosphate dehydrogenase (G6PD) deficiency. On admission, physical examination revealed pallor, jaundice and mild hepatomegaly. Results of laboratory investigations showed a hemoglobin level of 11.0 g/dl with a hemolytic blood picture, reticulocytosis of 20% and red cell G6PD activity reported as undetectable. The patient's DNA was analysed for G6PD mutations by PCR-based techniques and DNA sequencing and results showed a 24 bp deletion of nucleotide 953-976 in the exon 9 of the G6PD gene. DNA analysis was also performed on blood samples of the patient's mother and female sibling confirming their heterozygous status, although both showed normal red cell G6PD activity levels. The patient was discharged well and his parents were appropriately advised on the condition and the importance of taking folic acid regularly. This is a first case report in Malaysia of G6PD deficiency causing chronic-hemolytic anemia. The rare 24 bp deletion causes the G6PD Nara variant, previously reported only in two other unrelated males, a Japanese and a Portuguese both with chronic hemolytic anemia.
DNA technology provides a new avenue to perform neonatal screening tests for single-gene diseases in populations of high frequency. Thalassemia is one of the high-frequency single-gene disorders affecting Singapore and many countries in the malaria belt. The authors explored the feasibility of using PCR-based diagnostic screening on 1,116 unselected sequential cord blood samples for neonatal screening. The cord blood samples were screened for the most common reported alpha- and beta-thalassemia mutations in each ethnic group (Chinese, Malays, and Indians) in a multiracial population. The carrier frequency for alpha-thalassemia mutations was about 6.4% in the Chinese (alpha deletions = 3.9%, alpha deletions = 2.5%), 4.8% in Malays, and 5.2% in Indians. Only alpha deletions were observed in the Chinese. The carrier frequency for beta-thalassemia mutations was 2.7% in the Chinese, 6.3% in Malays, and 0.7% in Indians. Extrapolating to the population distribution of Singapore, the authors found a higher overall expected carrier frequency for alpha- and beta-thalassemia mutations of 9% compared with a previous population study of 6% by phenotype. The highly accurate results make this molecular epidemiologic screening an ideal method to screen for and prevent severe thalassemia in high-risk populations.
There are significant differences in the prevalence and severity of neonatal jaundice among various populations. Recently, it has been reported that a mutation of the UGT1A1 gene, glycine to arginine at codon 71 (G71R), is related to the development of neonatal jaundice in East Asian populations. However, whether the G71R mutation contributes to the high incidence of neonatal jaundice in different Asian populations remains unknown. The authors screened for this mutation in the Javanese-Indonesian and Malay-Malaysian populations.
The expressed sequence tag (EST) effort in Toxoplasma gondii has generated a substantial amount of gene information. To exploit this valuable resource, we chose to study tgd057, a novel gene identified by a large number of ESTs that otherwise show no significant match to known sequences in the database. Northern analysis showed that tgd057 is transcribed in this tachyzoite. The complete cDNA sequence of tgd057 is 1169 bp in length. Sequence analysis revealed that tgd057 possibly adopts two polyadenylation sites, utilizes the fourth in-frame ATG for translation initiation, and codes for a secretory protein. The longest open reading frame for the tgd057 gene was cloned and expressed as a recombinant protein (rd57) in Escherichia coli. Western analysis revealed that serum against rd57 recognized a molecule of ~21 kDa in the tachyzoite protein extract. This suggests that the tgd057 gene is expressed in vivo in the parasite.
Rubella has been listed as a mandatory notifiable disease in Taiwan since 1988. Because of high coverage rates with an effective vaccine, rubella cases have decreased dramatically in Taiwan since 1994. However, rubella outbreaks still occur due to imported transmission. Five large clusters were detected in Taiwan from 2007 to 2011. In 2007, one cluster was caused by rubella genotype 1E viruses that were imported from Vietnam, whereas another cluster was caused by genotype 2B viruses and was untraceable. In 2008, two clusters were caused by different lineages of genotype 1E viruses that were imported from Malaysia. In 2009, a cluster that was caused by genotype 2B viruses was associated with imported cases from Vietnam. The rubella viruses from 124 confirmed cases from 2005 to 2011 were characterized, and the data revealed that these viruses were distributed in the following four genotypes: 1E (n = 56), 1h (n = 1), 1j (n = 4), and 2B (n = 63). Of these viruses, 93 (75%) were associated with imported cases, and 43 of 56 genotype 1E viruses were associated with imported cases from China, Vietnam, Malaysia, and Indonesia. One genotype 1h virus was imported from Belarus, and three of four genotype 1j viruses were imported from the Philippines. Of 63 rubella genotype 2B viruses, 46 were imported from Vietnam, Thailand, Malaysia, China, Germany, and South Africa. Molecular surveillance allows for the differentiation of circulating rubella viruses and can be used to investigate transmission pathways, which are important to identify the interruption of endemic virus transmission.
Dirofilaria immitis is a parasite of domestic and wild canids and felids in tropical, subtropical and temperate regions throughout the world. The canine heartworm (D. immitis) is the causative agent of canine and feline cardiopulmonary dirofilariasis. This parasite is known to cause a zoonotic disease, namely human pulmonary dirofilariasis. D. immitis is known to be endemic in several South and Southeast Asian countries (e.g. India and Malaysia), but there has previously been no information about the presence of this pathogen in Bangladesh. We present a case of canine dirofilariasis caused by D. immitis in rural southeastern Bangladesh. A male filaroid nematode (95 mm in length and 1.94 mm in width) was identified in the heart of a dog. Species classification was performed by microscopy and molecular tools. Sequence analysis revealed a 100 % identity within the mitochondrial cytochrome c oxidase I (CO1) gene to two Chinese and one Australian D. immitis samples. Usually, dogs stay outside overnight with a high risk to get infected with D. immitis via nocturnal mosquito vectors, which may lead to high prevalences of this pathogen in the canine population and thus increase the risk of human infections with this neglected parasitic disease.
Hepatitis B virus (HBV) has been classified into eight genotypes, designated A-H. These genotypes are known to have distinct geographic distributions. The clinical importance of genotype-related differences in the pathogenicity of HBV has been revealed recently. In Malaysia, the current distribution of HBV remains unclear. The aim of this study was to determine the genotypes and subtypes of HBV by using PCR, followed by DNA sequencing, as well as to analyse the mutations in the immunodominant region of preS and S proteins. The S gene sequence was determined from HBV DNA of four apparently healthy blood donors' sera and three sera from asymptomatic chronic hepatitis B carriers. Of this batch of sera, the preS gene sequence was obtained from HBV DNA from three out of the four blood donors and two out of the three chronic carriers. Due to insufficient sera, we had to resort to using sera from another blood donor to make up for the sixth DNA sequence of the preS gene. Based on the comparative analysis of the preS sequences with the reported sequences in the GenBank database, HBV DNA from two normal carriers was classified as genotype C. Genotype B was assigned to HBV from one blood donor and two hepatitis B chronic carriers, whereas HBV of one chronic carrier was of genotype D. Based on the S gene sequences, HBV from three blood donors was of genotype C, that of one blood donor and one chronic carrier was of genotype B, and the remaining, of genotype D. In the five cases where both preS and S gene sequences were determined, the genotypes assigned based on either the preS or S gene sequences were in concordance. The nature of the deduced amino acid (aa) sequences at positions 125, 127, 134, 143, 159, 161 and 168 of the S gene enabled the classification of these sequences into subtypes, namely, adrq+, adw2 and ayw2. The clustering of our DNA sequences into genotype groups corresponded to their respective subtype, that is, adw2 in genotype B, adrq in genotype C and ayw in genotype D. Analysis of the point mutations revealed that five of the sequences contained aa substitutions at immunodominant epitopes involved in B or/and T cell recognition. In conclusion, despite the low numbers of samples studied, due to budget constraints, these data are still worthwhile reporting, as it is important for the control of HBV infections. In addition, the genotype and mutational data obtained in this study may be useful for designing new treatment regimes for HBV patients.
In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, 17%, 58%, and 25% were found to exhibit a high degree of resistance to 200 microg/ml of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least 50 microg/ml of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least 50 microg/ml of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked red blood cell enzymopathy common in malaria endemic areas. Individuals affected by this disease show a wide variety of clinical signs of acute hemolytic anemia. Mutations of the G6PD gene in the Malay population with G6PD deficiency in Kelantan, a state in North East Malaysia were studied. Ninety-three individuals with G6PD deficiency were subjected to mutation analysis of the G6PD gene using polymerase chain reaction based techniques of multiplex PCR. Of the ninety-three DNA samples studied, molecular defects were identified in 80 cases (86%). Variants were heterogeneous - 28.7% were found to have a G to A nucleotide change at nucleotide 871 of the G6PD gene (G871A), corresponding to G6PD Viangchan. The other major mutations were G6PD Mediterranean, G6PD Vanua Lava, G6PD Coimbra, G6PD Kaiping, G6PD Orissa, G6PD Mahidol, G6PD Canton and G6PD Chatham. These results showed that there are heterogeneous mutations of the G6PD gene associated with G6PD deficiency and that G6PD Viangchan and G6PD Mediterranean account for the main variants in G6PD deficiency among the Malay population in Malaysia.