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Use of COI, CytB and ND5 genes for intra- and inter-specific differentiation of Haematobia irritans and Haematobia exigua

Low VL, Tan TK, Lim PE, Domingues LN, Tay ST, Lim YA, et al.

Vet Parasitol, 2014 Aug 29;204(3-4):439-42.
PMID: 24912955 DOI: 10.1016/j.vetpar.2014.05.036

A multilocus sequence analysis using mitochondria-encoded cytochrome c oxidase subunit I (COI), cytochrome B (CytB), NADH dehydrogenase subunit 5 (ND5); nuclear encoded 18S ribosomal RNA (18S) and 28S ribosomal RNA (28S) genes was performed to determine the levels of genetic variation between the closely related species Haematobia irritans Linnaeus and Haematobia exigua de Meijere. Among these five genes, ND5 and CytB genes were found to be more variable and informative in resolving the interspecific relationships of both species. In contrast, the COI gene was more valuable in inferring the intraspecific relationships. The ribosomal 18S and 28S sequences of H. irritans and H. exigua were highly conserved with limited intra- and inter-specific variation. Molecular evidence presented in this study demonstrated that both flies are genetically distinct and could be differentiated based on sequence analysis of mitochondrial genes.

Matched MeSH terms: Sequence Analysis, DNA/veterinary
Genetic variation of Haemonchus contortus (Trichostrongylidae) in sheep and goats from Malaysia and Yemen

Gharamah AA, Azizah MN, Rahman WA

Vet Parasitol, 2012 Sep 10;188(3-4):268-76.
PMID: 22538095 DOI: 10.1016/j.vetpar.2012.04.003

The large stomach worm, Haemonchus contortus, commonly known as "the barber's pole worm", is a blood-sucking nematode found in the abomasa of sheep and goats. This work is the first documentation on the ND4 sequences of H. contortus from sheep and goats in Malaysia and Yemen and the results provide a preliminary insight on the genetic differences of H. contortus found in the two countries. In general, this study showed a high degree of diversity and low population structure of this species within the same country in comparison with higher genetic structuring at a wider geographical scale. The results also showed that the majority of genetic variance was within H. contortus populations. The Malaysian sheep and goat populations investigated appeared to share the same isolate of H. contortus while different isolates may be found in Yemen which must be taken into account in the design of an effective control strategy. Analysis of the internal transcribed spacer-2 (ITS-2) confirmed that all samples investigated in this study belonged to H. contortus. However presence of other Haemonchus species parasitizing these two hosts can only be confirmed by further detailed studies.

Matched MeSH terms: Sequence Analysis, DNA
Molecular phylogenetic studies on Theileria parasites based on small subunit ribosomal RNA gene sequences

Chansiri K, Kawazu S, Kamio T, Terada Y, Fujisaki K, Philippe H, et al.

Vet Parasitol, 1999 Jun 15;83(2):99-105.
PMID: 10392966

Classification of Theileria parasites of south-east Asian countries is still ambiguous due to the lack of basic studies, especially their molecular genetic information. In this study, we included 6 known species and 14 unclassified Theileria parasite isolates: Theileria annulata, Theileria parva, Theileria taurotragi, Theileria sergenti, Theileria buffeli, Theileria types Sable, Theileria types A, B, B1, B2, C, D, E, F, G, G1, Theileria type Medan (Indonesia), Theileria type Ipoh (Malaysia) and Theileria type Thong Song (Thailand). Small subunit ribosomal RNA (srRNA) nucleotide sequence data were collected by PCR, cloning and dideoxy sequencing. The srRNA nucleotide sequences were aligned and analyzed by distance methods, maximum parsimony algorithms and maximum likelihood methods to construct phylogenetic trees. Bootstrap analysis was used to test the strength of the different phylogenetic reconstructions. The data indicated that all of the tree-building methods gave very similar results. This study identified two groups of Theileria, the pathogenic and benign groups, which are strongly supported by bootstrap analysis. The analysis also indicated that three subgroups (A, B and C) were generated within the benign Theileria group whereas the classification of Theileria type D and Thong Song is questionable. However, more basic information such as life cycle differences, vectors, modes of transmission, virulent and genetic/sexual compatability is essential for clearer taxonomic definition of the benign Theileria parasites.

Matched MeSH terms: Sequence Analysis, DNA
The genetic characteristics and evolution of Tembusu virus

Lei W, Guo X, Fu S, Feng Y, Tao X, Gao X, et al.

Vet Microbiol, 2017 Mar;201:32-41.
PMID: 28284620 DOI: 10.1016/j.vetmic.2017.01.003

BACKGROUND: Since the turn of the 21st century, there have been several epidemic outbreaks of poultry diseases caused by Tembusu virus (TMUV). Although multiple mosquito and poultry-derived strains of TMUV have been isolated, no data exist about their comparative study, origin, evolution, and dissemination.
METHODOLOGY: Parallel virology was used to investigate the phenotypes of duck and mosquito-derived isolates of TMUV. Molecular biology and bioinformatics methods were employed to investigate the genetic characteristics and evolution of TMUV.
PRINCIPAL FINDINGS: The plaque diameter of duck-derived isolates of TMUV was larger than that of mosquito-derived isolates. The cytopathic effect (CPE) in mammalian cells occurred more rapidly induced by duck-derived isolates than by mosquito-derived isolates. Furthermore, duck-derived isolates required less time to reach maximum titer, and exhibited higher viral titer. These findings suggested that poultry-derived TMUV isolates were more invasive and had greater expansion capability than the mosquito-derived isolates in mammalian cells. Variations in amino acid loci in TMUV E gene sequence revealed two mutated amino acid loci in strains isolated from Malaysia, Thailand, and Chinese mainland compared with the prototypical strain of the virus (MM1775). Furthermore, TMUV isolates from the Chinese mainland had six common variations in the E gene loci that differed from the Southeast Asian strains. Phylogenetic analysis indicated that TMUV did not exhibit a species barrier in avian species and consisted of two lineages: the Southeast Asian and the Chinese mainland lineages. Molecular traceability studies revealed that the recent common evolutionary ancestor of TMUV might have appeared before 1934 and that Malaysia, Thailand and Shandong Province of China represent the three main sources related to TMUV spread.
CONCLUSIONS: The current broad distribution of TMUV strains in Southeast Asia and Chinese mainland exhibited longer-range diffusion and larger-scale propagation. Therefore, in addition to China, other Asian and European countries linked to Asia have used improved measures to detect and monitor TMUV related diseases to prevent epidemics in poultry.

Matched MeSH terms: Sequence Analysis, DNA/veterinary
Molecular investigation on the occurrence of Toxoplasma gondii oocysts in cat feces using TOX-element and ITS-1 region targets

Chemoh W, Sawangjaroen N, Nissapatorn V, Sermwittayawong N

Vet J, 2016 Sep;215:118-22.
PMID: 27325616 DOI: 10.1016/j.tvjl.2016.05.018

One of the most important routes of transmission for Toxoplasma gondii infection is the ingestion of foods contaminated with cat feces containing sporulated oocysts. The diagnosis of T. gondii infection by fecal microscopy is complicated, as other similar coccidian oocysts are often present in the same fecal specimen. This study aimed to identify T. gondii oocysts in cat feces using a novel PCR technique. Feline fecal specimens (n = 254) were screened for coccidian oocysts by light microscopy using the Sheather's flotation method. PCR analysis performed on the same specimens targeted a 529 bp repeat element and internal transcribed spacer-1 (ITS-1) regions were used to confirm the presence of Toxoplasma oocysts. By light microscopy, 49/254 (19.3%) of specimens contained coccidian oocysts. PCR analysis demonstrated 2/254 (0.8%) and 17/254 (6.7%) positive results using Tox and ITS-1 primers, respectively. However, coccidian oocysts were not identified on microscopic examination of specimens that were PCR-positive by Tox primers. Coccidian oocysts were identified on microscopic examination of 6/17 (35.3%) of the PCR-positive fecal specimens using ITS-1 primers. The BLAST results of 16 ITS-1 sequences were identified as T. gondii (n = 12; 4.7%) and Hammondia hammondi (n = 4; 1.6%). There was slight agreement between the 529 bp and ITS-1 PCR results (κ = 0.148). This is the first report of the detection of Toxoplasma oocysts using PCR analysis on feline fecal specimens from Southern Thailand. The ITS-1 region has potential as an alternative marker to identify T. gondii oocysts in feline fecal specimens.

Matched MeSH terms: Sequence Analysis, DNA/veterinary
Fulltext Genetic diversity of Japanese encephalitis virus isolates obtained from the Indonesian archipelago between 1974 and 1987

Schuh AJ, Guzman H, Tesh RB, Barrett AD

Vector Borne Zoonotic Dis, 2013 Jul;13(7):479-88.
PMID: 23590316 DOI: 10.1089/vbz.2011.0870

Five genotypes (GI-V) of Japanese encephalitis virus (JEV) have been identified, all of which have distinct geographical distributions and epidemiologies. It is thought that JEV originated in the Indonesia-Malaysia region from an ancestral virus. From that ancestral virus GV diverged, followed by GIV, GIII, GII, and GI. Genotype IV appears to be confined to the Indonesia-Malaysia region, as GIV has been isolated in Indonesia from mosquitoes only, while GV has been isolated on three occasions only from a human in Malaysia and mosquitoes in China and South Korea. In contrast, GI-III viruses have been isolated throughout Asia and Australasia from a variety of hosts. Prior to this study only 13 JEV isolates collected from the Indonesian archipelago had been studied genetically. Therefore the sequences of the envelope (E) gene of 24 additional Indonesian JEV isolates, collected throughout the archipelago between 1974 and 1987, were determined and a series of molecular adaptation analyses were performed. Phylogenetic analysis indicated that over a 14-year time span three genotypes of JEV circulated throughout Indonesia, and a statistically significant association between the year of virus collection and genotype was revealed: isolates collected between 1974 and 1980 belonged to GII, isolates collected between 1980 and 1981 belonged to GIV, and isolates collected in 1987 belonged to GIII. Interestingly, three of the GII Indonesian isolates grouped with an isolate that was collected during the JE outbreak that occurred in Australia in 1995, two of the GIII Indonesian isolates were closely related to a Japanese isolate collected 40 years previously, and two Javanese GIV isolates possessed six amino acid substitutions within the E protein when compared to a previously sequenced GIV isolate collected in Flores. Several amino acids within the E protein of the Indonesian isolates were found to be under directional evolution and/or co-evolution. Conceivably, the tropical climate of the Indonesia/Malaysia region, together with its plethora of distinct fauna and flora, may have driven the emergence and evolution of JEV. This is consistent with the extensive genetic diversity seen among the JEV isolates observed in this study, and further substantiates the hypothesis that JEV originated in the Indonesia-Malaysia region.

Matched MeSH terms: Sequence Analysis, DNA
Oral vaccination with Lactococcus lactis expressing the Vibrio cholerae Wzm protein to enhance mucosal and systemic immunity

Zamri HF, Shamsudin MN, Rahim RA, Neela V

Vaccine, 2012 May 2;30(21):3231-8.
PMID: 22426330 DOI: 10.1016/j.vaccine.2012.02.012

A gene associated with lipopolysaccharide (LPS) transport was cloned from a local clinical Vibrio cholerae O1 strain of the Ogawa serotype by using the Lactococcus lactis nisin-controlled expression (NICE) system. The V. cholerae wzm gene, which codes for an integral membrane transporter protein, was expressed and targeted to the cytoplasmic membrane, and was crudely isolated through simple centrifugation and SDS solubilization. To examine seroreactivity of this construct, rabbits were orally fed with 10(9) cfu/ml of live, recombinant L. lactis carrying the wzm gene, induced with nisin prior to administration. Recombinant plasmids were retrieved from L. lactis cultured directly from stool samples of inoculated rabbits. Reverse-transcriptase PCR of wzm using the retrieved plasmids confirmed transcription of this gene, indicating viability and stability of the recombinants in vivo. The L. lactis-Wzm construct elicited substantial levels of IgG and sIgA, and challenge with virulent V. cholerae O1 evoked severe diarrhoea in the naive, non-immunised control group, but not in those fed with either recombinant or non-recombinant L. lactis. Oral administration with recombinant L. lactis expressing the V. cholerae wzm gene increases both systemic and mucosal immunity, whereas L. lactis itself appears capable of protecting against the diarrhoeal symptoms caused by V. cholerae. Wzm is a conserved membrane protein associated with the LPS endotoxin, and together with the food-grade L. lactis, represent an attractive target for the development of a safer, live anti-infective therapy against V. cholerae.

Matched MeSH terms: Sequence Analysis, DNA
Insertion sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110

Fomukong NG, Tang TH, al-Maamary S, Ibrahim WA, Ramayah S, Yates M, et al.

Tuber. Lung Dis., 1994 Dec;75(6):435-40.
PMID: 7718832 DOI: 10.1016/0962-8479(94)90117-1

DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Identification of Plasmodium knowlesi Merozoite Surface Protein-119 (PkMSP-119 ) novel binding peptides from a phage display library

Goh XT, Chua KH, Kee BP, Lim YAL

Trop Med Int Health, 2020 02;25(2):172-185.
PMID: 31733137 DOI: 10.1111/tmi.13348

OBJECTIVE: Plasmodium knowlesi, the fifth human malaria parasite, has caused mortality in humans. We aimed to identify P. knowlesi novel binding peptides through a random linear dodecapeptide phage display targeting the 19-kDa fragment of Merozoite Surface Protein-1 protein.
METHODS: rPkMSP-119 protein was heterologously expressed using Expresso® Solubility and Expression Screening System and competent E. cloni® 10G cells according to protocol. Three rounds of biopanning were performed on purified rPkMSP-119 to identify binding peptides towards rPkMSP-119 using Ph.D.™-12 random phage display library. Binding sites of the identified peptides to PkMSP-119 were in silico predicted using the CABS-dock web server.
RESULTS: Four phage peptide variants that bound to PkMSP-119 were identified after three rounds of biopanning, namely Pkd1, Pkd2, Pkd3 and Pkd4. The sequences of both Pkd1 and Pkd2 consist of a large number of histidine residues. Pkd1 showed positive binding signal with 6.1× vs. BSA control. Docking results showed that Pkd1 and Pkd2 were ideal binding peptides for PkMSP-119 .
CONCLUSION: We identified two novel binding peptides of PkMSP-119 , Pkd1 (HFPFHHHKLRAH) and Pkd2 (HPMHMLHKRQHG), through phage display. They provide a valuable starting point for the development of novel therapeutics.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Cloning and expression of a subfamily 1.4 lipase from Bacillus licheniformis IBRL-CHS2

Reddy, Nidyaletchmy Subba, Rashidah Abdul Rahim, Darah Ibrahim, Kumar, K. Sudesh

Trop Life Sci Res, 2016;27(11):145-150.
MyJurnal

We report on the cloning of the lipase gene from Bacillus licheniformis IBRLCHS2
and the expression of the recombinant lipase. DNA sequencing analysis of the
cloned lipase gene showed that it shares 99% identity with the lipase gene from
B. licheniformis ATCC 14580 and belongs to subfamily 1.4 of true lipases based on amino
acid sequence alignment of various Bacillus lipases. The 612 bp lipase gene was then
cloned into the pET-15b(+) expression vector and the construct was transformed into
E. coli BL21 (DE3) for bulk expression of the lipase. Expression was analysed by SDSPAGE
where the lipase was found to have a molecular weight of about 23 kDa.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Sequencing crop genomes: a gateway to improve tropical agriculture

Thottathil, Gincy Paily, Jayasekaran, Kandakumar, Ahmad Sofiman Othman

Trop Life Sci Res, 2016;27(1):93-114.
MyJurnal

Agricultural development in the tropics lags behind development in the
temperate latitudes due to the lack of advanced technology, and various biotic and abiotic
factors. To cope with the increasing demand for food and other plant-based products,
improved crop varieties have to be developed. To breed improved varieties, a better
understanding of crop genetics is necessary. With the advent of next-generation DNA
sequencing technologies, many important crop genomes have been sequenced. Primary
importance has been given to food crops, including cereals, tuber crops, vegetables, and
fruits. The DNA sequence information is extremely valuable for identifying key genes
controlling important agronomic traits and for identifying genetic variability among the
cultivars. However, massive DNA re-sequencing and gene expression studies have to be
performed to substantially improve our understanding of crop genetics. Application of the
knowledge obtained from the genomes, transcriptomes, expression studies, and
epigenetic studies would enable the development of improved varieties and may lead to a
second green revolution. The applications of next generation DNA sequencing
technologies in crop improvement, its limitations, future prospects, and the features of
important crop genome projects are reviewed herein.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Molecular screening for Rickettsia, Anaplasmataceae and Coxiella burnetii in Rhipicephalus sanguineus ticks from Malaysia

Watanabe M, Nakao R, Amin-Babjee SM, Maizatul AM, Youn JH, Qiu Y, et al.

Trop Biomed, 2015 Jun;32(2):390-8.
PMID: 26691268 MyJurnal

A total of 44 Rhipicephalus sanguineus ticks collected from 23 dogs from Malaysia were screened for Rickettsia, Anaplasmataceae and Coxiella burnetii. Coxiella burnetii was detected in 59% (26/44) of ticks however Rickettsia and Anaplasmataceae were not detected in any of the ticks. In order to genotype the strains of C. burnetii, multispacer sequence typing (MST) was carried out using three different spacers. One of the spacers; Cox2 successfully amplified a fragment for which the full length sequence of 397 bp was obtained. The sequenced product revealed only a single nucleotide difference with the Cox2.3 type sequence.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Phylogenetic analysis of human metapneumovirus among children with acute respiratory infections in Kuala Lumpur, Malaysia

Nor'e SS, Sam IC, Mohamad Fakri EF, Hooi PS, Nathan AM, de Bruyne JA, et al.

Trop Biomed, 2014 Sep;31(3):562-6.
PMID: 25382484 MyJurnal

Human metapneumovirus (HMPV) is a recently discovered cause of viral respiratory infections. We describe clinical and molecular epidemiology of HMPV cases diagnosed in children with respiratory infection at University of Malaya Medical Centre, Kuala Lumpur, Malaysia. The prevalence rate of HMPV between 2010 and 2012 was 1.1%, and HMPV contributed 6.5% of confirmed viral respiratory infections. The HMPV patients had a median age of 1.6 years, and a median hospital admission of 4 days. The most common clinical presentations were fever, rhinitis, pneumonia, vomiting/diarrhoea, and bronchiolitis. Based on the partial sequences of F fusion gene from 26 HMPV strains, 14 (54%) were subgenotype A2b, which was predominant in 2010; 11 (42%) were subgenotype B1, which was predominant in 2012; and 1 (4%) was subgenotype A2a. Knowledge of the circulating subgenotypes in Malaysia, and the displacement of predominant subgenotypes within 3 years, is useful data for future vaccine planning.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Distribution of mosquito larvae in various breeding sites in National Zoo Malaysia

Muhammad-Aidil R, Imelda A, Jeffery J, Ngui R, Wan Yusoff WS, Aziz S, et al.

Trop Biomed, 2015 Mar;32(1):183-6.
PMID: 25801269 MyJurnal

Mosquitoes are principal vectors of major vector-borne diseases. They are widely found throughout urban and rural areas in Malaysia. They are responsible for various vector-borne diseases such as dengue, malaria, filariasis and encephalitis. A total of 158 mosquito larvae specimens were collected from the National Zoo, Malaysia, from 11 types of breeding habitats during the study period from end of May 2007 to July 2007. Aedes albopictus was the predominant species (35.4%), followed by Tripteroides aranoides (26.6%), Lutzia halifaxii (11.4%), Aedes alboscutellatus (10.1%), Aedes caecus (8.9%), Armigeres spp. (4.4%), Malaya genurostris (2.5%) and Culex vishnui (0.6%). It is important to have a mosquito free environment in a public place like the zoo. Routine larval surveillance should be implemented for an effective mosquito control program in order to reduce mosquito population.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Molecular survey and sequence analysis of Anaplasma spp. in cattle and ticks in a Malaysian farm

Tay ST, Koh FX, Kho KL, Ong BL

Trop Biomed, 2014 Dec;31(4):769-76.
PMID: 25776603 MyJurnal

This study was conducted to determine the occurrence of Anaplasma spp. in the blood samples of cattle, goats, deer and ticks in a Malaysian farm. Using polymerase chain reaction (PCR) and sequencing approach, Anaplasma spp. was detected from 81(84.4%) of 96 cattle blood samples. All blood samples from 23 goats and 22 deer tested were negative. Based on the analysis of the Anaplasma partial 16S ribosomal RNA gene, four sequence types (genotypes 1 to 4) were identified in this study. Genotypes 1-3 showed high sequence similarity to those of Anaplasma platys/ Anaplasma phagocytophilum, whilst genotype 4 was identical to those of Anaplasma marginale/ Anaplasma centrale/ Anaplasma ovis. Anaplasma DNA was detected from six (5.5%) of 109 ticks which were identified as Rhipicephalus (formely known as Boophilus) microplus ticks collected from the cattle. This study reported for the first time the detection of four Anaplasma sequence types circulating in the cattle population in a farm in Malaysia. The detection of Anaplasma DNA in R. microplus ticks in this study provides evidence that the ticks are one of the potential vectors for transmission of anaplasmosis in the cattle.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Detection and characterization of viruses causing hand, foot and mouth disease from children in Seri Kembangan, Malaysia

Ling BP, Jalilian FA, Harmal NS, Yubbu P, Sekawi Z

Trop Biomed, 2014 Dec;31(4):654-62.
PMID: 25776590 MyJurnal

Hand, foot and mouth disease (HFMD) is a common viral infection among infants and children. The major causative agents of HFMD are enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). Recently, coxsackievirus A6 (CVA6) infections were reported in neighboring countries. Infected infants and children may present with fever, mouth/throat ulcers, rashes and vesicles on hands and feet. Moreover, EV71 infections might cause fatal neurological complications. Since 1997, EV71 caused fatalities in Sarawak and Peninsula Malaysia. The purpose of this study was to identify and classify the viruses which detected from the patients who presenting clinical signs and symptoms of HFMD in Seri Kembangan, Malaysia. From December 2012 until July 2013, a total of 28 specimens were collected from patients with clinical case definitions of HFMD. The HFMD viruses were detected by using semi-nested reverse transcription polymerase chain reaction (snRT-PCR). The positive snRT-PCR products were sequenced and phylogenetic analyses of the viruses were performed. 12 of 28 specimens (42.9%) were positive in snRT-PCR, seven are CVA6 (58.3%), two CVA16 (16.7%) and three EV71 (25%). Based on phylogenetic analysis studies, EV71 strains were identified as sub-genotype B5; CVA16 strains classified into sub-genotype B2b and B2c; CVA6 strains closely related to strains in Taiwan and Japan. In this study, HFMD in Seri Kembangan were caused by different types of Enterovirus, which were EV71, CVA6 and CVA16.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Monoclonal antibody-escape variant of dengue virus serotype 1: Genetic composition and envelope protein expression

Chem YK, Chua KB, Malik Y, Voon K

Trop Biomed, 2015 Jun;32(2):344-51.
PMID: 26691263 MyJurnal

Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were (Ser)[326](Leu), (Ser)[340](Leu) at the deduced E protein, (Ile)[250](Thr) at NS1 protein, and (Thr)[41](Ser) at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Molecular epidemiology of Cryptosporidium in HIV/AIDS patients in Malaysia

Asma I, Sim BL, Brent RD, Johari S, Yvonne Lim AL

Trop Biomed, 2015 Jun;32(2):310-22.
PMID: 26691260 MyJurnal

Cryptosporidiosis is a particular concern in immunocompromised individuals where symptoms may be severe. The aim of this study was to examine the epidemiological and molecular characteristics of Cryptosporidium infections in HIV/AIDS patients in Malaysia in order to identify risk factors and facilitate control measures. A modified Ziehl-Neelsen acid fast staining method was used to test for the presence of Cryptosporidium oocysts in the stools of 346 HIV/AIDS patients in Malaysia. Standard coproscopical methods were used to identify infections with other protozoan or helminths parasites. To identify the species of Cryptosporidium, DNA was extracted and nested-PCR was used to amplify a portion of the SSU rRNA gene. A total of 43 (12.4%) HIV-infected patients were found to be infected with Cryptosporidium spp. Of the 43 Cryptosporidium-positive HIV patients, 10 (23.3%) also harboured other protozoa, and 15 (34.9%) had both protozoa and helminths. The highest rates of cryptosporidiosis were found in adult males of Malay background, intravenous drug users, and those with low CD4 T cell counts (i.e., < 200 cells/mm3). Most were asymptomatic and had concurrent opportunistic infections mainly with Mycobacterium tuberculosis. DNA sequence analysis of 32 Cryptosporidium isolates identified C. parvum (84.3%), C. hominis (6.3%), C. meleagridis (6.3%), and C. felis (3.1%). The results of the present study revealed a high prevalence of Cryptosporidium infection in hospitalized HIV/AIDS patients. The results also confirmed the potential significance of zoonotic transmission of C. parvum in HIV infected patients, as it was the predominant species found in this study. However, these patients were found to be susceptible to a wide range of Cryptosporidium species. Epidemiological and molecular characterization of Cryptosporidium isolates provides clinicians and researchers with further information regarding the origin of the infection, and may enhance treatment and control strategies.

Matched MeSH terms: Sequence Analysis, DNA
Fulltext Rapid detection and identification of pathogens in patients with continuous ambulatory peritoneal dialysis (CAPD) associated peritonitis by 16s rRNA gene sequencing

Ahmadi SH, Neela V, Hamat RA, Goh BL, Syafinaz AN

Trop Biomed, 2013 Dec;30(4):602-7.
PMID: 24522129 MyJurnal

Peritonitis still remains a serious complication with high rate of morbidity and mortality in patients on CAPD. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early and optimal treatment. The aim of this study was to use 16S rRNA and ITS gene sequencing to identify common bacterial and fungal pathogens directly from the peritoneal fluid without culturing. Ninety one peritoneal fluids obtained from 91 different patients on CAPD suspected for peritonitis were investigated for etiological agents by 16S rRNA and ITS gene sequencing. Data obtained by molecular method was compared with the results obtained by culture method. Among the 45 patients confirmed for peritonitis based on international society of peritoneal dialysis (ISPD) guidelines, the etiological agents were identified in 37(82.2%) samples by culture method, while molecular method identified the etiological agents in 40(88.9%) samples. Despite the high potential application of the 16S rRNA and ITS gene sequencing in comparison to culture method to detect the vast majority of etiological agents directly from peritoneal fluids; it could not be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in Gene Bank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis.

Matched MeSH terms: Sequence Analysis, DNA/methods*
Fulltext Molecular insights of co-trimoxazole resistance genes in Haemophilus influenzae isolated in Malaysia

Mohd-Zain Z, Kamsani NH, Ahmad N

Trop Biomed, 2013 Dec;30(4):584-90.
PMID: 24522126 MyJurnal

In the last few decades, co-trimoxazole (SXT), an antibacterial combination of trimethoprim and sulfamethoxazole, has been used for treatment of upper respiratory tract infection due to Haemophilus influenzae. The usage of this antibiotic has become less important due to emergence of SXT-resistant strains worldwide. Most reports associate SXT resistance to the presence of variants of dihydrofolate reductase (DHFR) dfrA genes which are responsible for trimethoprim resistance; while the sulfamethoxazole (SMX) resistance are due to sulfonamide (SUL) genes sul1 and sul2 and/or mutation in the chromosomal (folP) gene encoding dihydropteroate synthetase (DHPS). This study aims to detect and analyse the genes that are involved in SXT resistance in H. influenzae strains that were isolated in Malaysia. Primers targeting for variants of dfrA, fol and sul genes were used to amplify the genes in nine SXT-resistant strains. The products of amplification were sequenced and multiple alignments of the assembled sequences of the local strains were compared to the sequences of other H. influenzae strains in the Genbank. Of the five variants of the dhfA genes, dfrA1 was detected in three out of the nine strains. In contrast to intermediate strains, at least one variant of folP genes was detected in the resistant strains. Multiple nucleotide alignment of this gene revealed that strain H152 was genetically different from the others due to a 15-bp nucleotide insert in folP gene. The sequence of the insert was similar to the insert in folP of H. influenzae strain A12, a strain isolated in United Kingdom. None of the strains had sul1 gene but sul2 gene was detected in four strains. Preliminary study on the limited number of samples shows that the TMP resistance was attributed to mainly to dfrA1 and the SMX was due to folP genes. Presence of sul2 in addition to folP in seven strains apparently had increased their level of resistance. A strain that lacked sul1 or sul2 gene, its resistance to sulfonamide was attributed to a 15-bp DNA insert in the folP gene.

Matched MeSH terms: Sequence Analysis, DNA

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