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The isolation, characterization and antifungal susceptibilities of Cryptococcus neoformans from bird excreta in Klang Valley, Malaysia

Tay ST, Chai HC, Na SL, Hamimah H, Rohani MY, Soo-Hoo TS

Mycopathologia, 2005 Jun;159(4):509-13.
PMID: 15983736

The occurrence of Cryptococcus neoformans in bird excreta in Klang valley, Malaysia was determined in this study. Of 544 samples of bird excreta collected from a local zoo, pet shops and public areas, 20 strains of C. neoformans were isolated. All C. neoformans strains were serotype A and thus identified as C. neoformans variety grubii. All did not produce color changes on canavanine-glycine-bromothymol blue agar. All were of alpha-mating types, as determined by a pheromone-specific PCR assay. The antifungal susceptibility testing using agar diffusion method Neo-sensitabs showed that all were susceptible to amphotericin B, fluconazole and itraconazole.

Matched MeSH terms: DNA, Protozoan/chemistry
Fulltext Simian malaria in wild macaques: first report from Hulu Selangor district, Selangor, Malaysia

Akter R, Vythilingam I, Khaw LT, Qvist R, Lim YA, Sitam FT, et al.

Malar J, 2015 Oct 05;14:386.
PMID: 26437652 DOI: 10.1186/s12936-015-0856-3

BACKGROUND: Malaria is a vector-borne parasitic disease which is prevalent in many developing countries. Recently, it has been found that Plasmodium knowlesi, a simian malaria parasite can be life-threatening to humans. Long-tailed macaques, which are widely distributed in Malaysia, are the natural hosts for simian malaria, including P. knowlesi. The aim of the present study was to determine the prevalence of simian malaria parasites in long-tailed macaques in the district of Hulu Selangor, Selangor, Malaysia.
METHODS: A total of 70 blood samples were collected from Macaca fascicularis dwelling in the forest of Hulu Selangor by the Department of Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur, Malaysia. DNA was extracted using PureLink™ Genomic DNA Kits. Conventional and nested PCR were used to detect the genus and species of Plasmodium parasites respectively. In addition, phylogenetic analysis was carried out to confirm the species of Plasmodium parasites.
RESULTS: Thirty-five (50 %) of the 70 samples were positive for Plasmodium using genus-specific primers. These positive samples were then subjected to nested PCR targeting the 18S ribosomal RNA genes to detect all five simian malaria parasites: namely, P. knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium fieldi, and Plasmodium coatneyi. All five species of simian malaria parasites were detected. Of these, P. inui was the predominant (65.7 %), followed by P. knowlesi (60 %), P. cynomolgi (51.4 %) P. coatneyi (45.7 %) and P. fieldi (2.9 %). A total of nine macaques had mono-infection with P. knowlesi (four), P. cynomolgi (two), P. coatneyi (two) and P. fieldi (one). Eleven of the macaques had dual infections while 12 had triple infections. Three macaques were infected with four species of Plasmodium. Molecular and phylogenetic analysis confirmed the five species of Plasmodium parasites.
CONCLUSION: This study has provided evidence to elucidate the presence of transmission of malaria parasites among the local macaques in Hulu Selangor. Since malaria is a zoonosis, it is important to determine the new control strategies for the control of malaria.

Matched MeSH terms: DNA, Protozoan/chemistry
Fulltext First Molecular Characterization of Leishmania Species Causing Visceral Leishmaniasis among Children in Yemen

Mahdy MA, Al-Mekhlafi AM, Abdul-Ghani R, Saif-Ali R, Al-Mekhlafi HM, Al-Eryani SM, et al.

PLoS One, 2016;11(3):e0151265.
PMID: 26966902 DOI: 10.1371/journal.pone.0151265

Visceral leishmaniasis (VL) is a debilitating, often fatal disease caused by Leishmania donovani complex; however, it is a neglected tropical disease. L. donovani complex comprises two closely related species, L. donovani that is mostly anthroponotic and L. infantum that is zoonotic. Differentiation between these two species is critical due to the differences in their epidemiology and pathology. However, they cannot be differentiated morphologically, and their speciation using isoenzyme-based methods poses a difficult task and may be unreliable. Molecular characterization is now the most reliable method to differentiate between them and to determine their phylogenetic relationships. The present study aims to characterize Leishmania species isolated from bone marrows of Yemeni pediatric patients using sequence analysis of the ribosomal internal transcribed spacer-1 (ITS1) gene. Out of 41 isolates from Giemsa-stained bone marrow smears, 25 isolates were successfully amplified by nested polymerase chain reaction and sequenced in both directions. Phylogenetic analysis using neighbor joining method placed all study isolates in one cluster with L. donovani complex (99% bootstrap). The analysis of ITS1 for microsatellite repeat numbers identified L. infantum in 11 isolates and L. donovani in 14 isolates. These data suggest the possibility of both anthroponotic and zoonotic transmission of VL-causing Leishmania species in Yemen. Exploring the possible animal reservoir hosts is therefore needed for effective control to be achieved.

Matched MeSH terms: DNA, Protozoan/chemistry
Obligate bacterial endosymbionts of Acanthamoeba spp. related to the beta-Proteobacteria: proposal of 'Candidatus Procabacter acanthamoebae' gen. nov., sp. nov

Horn M, Fritsche TR, Linner T, Gautom RK, Harzenetter MD, Wagner M

Int J Syst Evol Microbiol, 2002 Mar;52(Pt 2):599-605.
PMID: 11931173 DOI: 10.1099/00207713-52-2-599

All obligate bacterial endosymbionts of free-living amoebae currently described are affiliated with the alpha-Proteobacteria, the Chlamydiales or the phylum Cytophaga-Flavobacterium-Bacteroides. Here, six rod-shaped gram-negative obligate bacterial endosymbionts of clinical and environmental isolates of Acanthamoeba spp. from the USA and Malaysia are reported. Comparative 16S rDNA sequence analysis demonstrated that these endosymbionts form a novel, monophyletic lineage within the beta-Proteobacteria, showing less than 90% sequence similarity to all other recognized members of this subclass. 23S rDNA sequence analysis of two symbionts confirmed this affiliation and revealed the presence of uncommon putative intervening sequences of 146 bp within helix-25 that shared no sequence homology to any other bacterial rDNA. In addition, the 23S rRNA of these endosymbionts displayed one polymorphism at the target site of oligonucleotide probe BET42a that is conserved in all other sequenced beta-Proteobacteria. Intra-cytoplasmatic localization of the endosymbionts within the amoebal host cells was confirmed by electron microscopy and fluorescence in situ hybridization with a specific 16S rRNA-targeted oligonucleotide probe. Based on these findings, the provisional name 'Candidatus Procabacter acanthamoebae' is proposed for classification of a representative of the six endosymbionts of Acanthamoeba spp. studied in this report. Comparative 18S rDNA sequence analysis of the Acanthamoeba host cells revealed their membership with either Acanthamoeba 18S rDNA sequence type T5 (Acanthamoeba lenticulata) or sequence type T4, which comprises the majority of all Acanthamoeba isolates.

Matched MeSH terms: DNA, Protozoan/chemistry
Characterization of Cryptocaryon irritans, a parasite isolated from marine fishes in Taiwan

Yambot AV, Song YL, Sung HH

Dis Aquat Organ, 2003 Mar 31;54(2):147-56.
PMID: 12747640

The ciliated protozoan parasite Cryptocaryon irritans infecting marine fishes in Taiwan is described. Developmental characteristics and sequences of the ribosomal DNA regions such as part of 18 S, the entire first internal transcribed spacer, and part of 5.8 S of various Taiwan isolates of C. irritans were investigated. A total of 5 isolates was obtained from different fish-host species and localities, the majority from cultured fish species. C. irritans from Taiwan is able to shift its developmental characteristics, i.e. from non-adherent to adherent tomonts, from individualistic to aggregate-forming tomonts, from infection of the gills only to infection of the gills and body. Thus, it is not possible to classify strains of C. irritans on the basis of these parameters. Premature tomonts that developed from dead fishes were able to produce theronts that could infect fish host. Isolates from Pingtung and the USA had identical nucleotide sequences while an isolate from Malaysia was identical to an Israel isolate. Percentage variation among pairs of Taiwan isolates showed a higher degree of variation than isolate sequences listed in GenBank. Sequence analysis revealed highly aberrant isolates in Taiwan, and a phylogenetic tree distinguished a marine and a low-salinity variant. C. irritans from marine fishes in Taiwan, therefore, display some characteristics not previously reported. Since manipulation of salinity in brackishwater ponds and marine cage sites is not feasible, there is a need to develop new strategies for the control and prevention of cryptocaryoniasis.

Matched MeSH terms: DNA, Protozoan/chemistry*
Fulltext Isolation and characterization of Acanthamoeba spp. from air-conditioners in Kuala Lumpur, Malaysia

Chan LL, Mak JW, Low YT, Koh TT, Ithoi I, Mohamed SM

Acta Trop, 2011 Jan;117(1):23-30.
PMID: 20858455 DOI: 10.1016/j.actatropica.2010.09.004

During a study on the quality of the indoor environment, Acanthamoeba spp. were detected in 20 out of 87 dust samples collected from air-conditioners installed in a four-story campus building located in Kuala Lumpur, Malaysia. Twenty-one cloned Acanthamoeba isolates designated as IMU1 to IMU21 were established from the positive primary cultures. Five species were identified from the 16 isolates according to the morphological criteria of Pussard and Pons; i.e. A. castellanii, A. culbertsoni, A. griffini, A. hatchetti and A. polyphaga. Species identities for the remaining five isolates (IMU4, IMU5, IMU15, IMU20 and IMU21), however, could not be determined morphologically. At genotypic characterization, these isolates were placed into T3 (IMU14); T5 (IMU16 and IMU17) and T4 (all the remaining isolates). To predict the potential pathogenicity of these Acanthamoeba isolates, thermo- and osmotolerance tests were employed; many isolates were predicted as potential human pathogens based on the outcome of these tests. This is the first time potentially pathogenic Acanthamoeba have been isolated from air-conditioners in Malaysia.

Matched MeSH terms: DNA, Protozoan/chemistry
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: DNA, Protozoan/chemistry
Fulltext High prevalence of muscular sarcocystosis in cattle and water buffaloes from Selangor, Malaysia

Latif B, Vellayan S, Heo CC, Kannan Kutty M, Omar E, Abdullah S, et al.

Trop Biomed, 2013 Dec;30(4):699-705.
PMID: 24522140 MyJurnal

The prevalence of sarcocystosis in cattle and water buffaloes from peninsular Malaysia was investigated in abattoirs in Selangor state, February, 2011, to March, 2012. Fresh muscle samples were collected from the tongue, heart, oesophagus, diaphragm and skeletal muscles of 102 cattle and 18 water buffaloes. Each sample was initially screened by light microscopy and then fixed for further histopathological analysis. Out of 120 animals examined, 49 (40.8%) harboured the microscopic type of Sarcocystis spp. The positivity rate for cattle was 36.2% and for water buffaloes 66.7%. In cattle, the organs highly infected were the skeletal muscles and diaphragm (27% each), followed by tongue and esophagus (24.3% each), and the heart (8%). In water buffaloes, the heart was most often infected (66.7%), followed by the oesophagus (50%) and skeletal muscle (33.3%); no sarcocysts were detected in the tongue and diaphragm. The shape of the sarcocyst was fusiform to oval with a mean cyst size of 151.66 x 75.83 μm and wall thickness of 2.47 μm in cattle, and 114 x 50.81 μm cyst size and the wall thickness of 1.11 μm in water buffaloes, consistent with Sarcocystis cruzi and Sarcocystis levinei, respectively. Remaining tissue from cattle was subjected to parasite specific 18S rRNA gene PCR and Sarcocystis cruzi was confirmed, at least exemplarily. The peripheral metrocytes and the banana-shaped bradyzoites (15.23 x 2.2 μm in cattle and 11.49 x 2.45 μm in water buffalo hosts) were easily recognized. In conclusion, a high positivity rate was found in Malaysian meat-producing animals with possible implications for meat consumption and human health.

Matched MeSH terms: DNA, Protozoan/chemistry
Molecular characterization of Giardia duodenalis in Yemen

Alyousefi NA, Mahdy MA, Xiao L, Mahmud R, Lim YA

Exp Parasitol, 2013 Jun;134(2):141-7.
PMID: 23523861 DOI: 10.1016/j.exppara.2013.03.001

Giardia duodenalis is an important intestinal protozoan in Yemen with infection rates ranging from 18% to 27%. To date, there has been no genotyping study to provide a better understanding of the transmission dynamic. This study was conducted to genotype and subtype G. duodenalis in Yemen. Stool samples were collected from 503 Yemeni outpatients between 1 and 80 years old, including 219 males and 284 females. Giardia cysts were detected via microscopy after the formal-ether concentration. Genotyping of Giardia was carried out using PCR and sequence analysis of the 16s rRNA and b-giardin genes. Of the 89 microscopy-positive Giardia samples, 65 were successfully sequenced, of which 66% (43 of 65) were identified as G. duodenalis assemblage A and 34% (22 of 65) as assemblage B. Further subtyping analysis based on b-giardin gene identified the presence of subtypes A2 and A3, which belong to the anthroponotic sub-assemblage AII. Data of the study suggest that anthroponotic transmission played a potential role in the transmission of giardiasis in the community. However, further genotyping and subtyping studies of specimens from humans and animals living in the same households are needed for a more definitive understanding of giardiasis transmission in Yemen.

Matched MeSH terms: DNA, Protozoan/chemistry
First molecular characterization of Cryptosporidium in Yemen

Alyousefi NA, Mahdy MA, Lim YA, Xiao L, Mahmud R

Parasitology, 2013 May;140(6):729-34.
PMID: 23369243 DOI: 10.1017/S0031182012001953

Cryptosporidium is a protozoan parasite of humans and animals and has a worldwide distribution. The parasite has a unique epidemiology in Middle Eastern countries where the IId subtype family of Cryptosporidium parvum dominates. However, there has been no information on Cryptosporidium species in Yemen. Thus, this study was conducted in Yemen to examine the distribution of Cryptosporidium species and subtype families. Fecal samples were collected from 335 patients who attended hospitals in Sana'a city. Cryptosporidium species were determined by PCR and sequence analysis of the 18 s rRNA gene. Cryptosporidium parvum and C. hominis subtypes were identified based on sequence analysis of the 60 kDa glycoprotein (gp60) gene. Out of 335 samples, 33 (9.9%) were positive for Cryptosporidium. Of them, 97% were identified as C. parvum whilst 1 case (3%) was caused by C. hominis. All 7 C. parvum isolates subtyped belonged to the IIaA15G2R1 subtype. The common occurrence of the zoonotic IIa subtype family of C. parvum highlights the potential occurrence of zoonotic transmission of cryptosporidiosis in Yemen. However, this postulation needs confirmation with future molecular epidemiological studies of cryptosporidiosis in both humans and animals in Yemen.

Matched MeSH terms: DNA, Protozoan/chemistry
Fulltext Light microscopy and molecular identification of Sarcocystis spp. in meat producing animals in Selangor, Malaysia

Latif B, Kannan Kutty M, Muslim A, Hussaini J, Omar E, Heo CC, et al.

Trop Biomed, 2015 Sep;32(3):444-52.
PMID: 26695204 MyJurnal

One thousand and forty-five tissue samples of skeletal muscles, tongue, heart, diaphragm and esophagus were collected from 209 animals (43 sheep, 89 goats and 77 cattle) from an abattoir in Selangor between February and October, 2013. Each sample was divided into three pieces with each piece measuring 2-3 mm3. Each piece was then squeezed between two glass slides and examined microscopically at x 10 magnification for the presence of sarcocystosis. Three positive samples from each animal species were then fixed in 10% formalin for histological processing. Seven positive samples collected from each animal species were preserved at -80°C or 90% ethanol for gene expression studies. Microsarcocysts were detected in 114 (54.5%) animals by light microscopy (LM). The infection rates in sheep, goat and cattle were 86, 61.8 and 28.6% respectively. The highest rate of infection was in the skeletal muscles of sheep (64.9%) and goats (63.6%) and in the heart of cattle (63.6%). The cysts were spindle to oval in shape and two stages were recognized, the peripheral metrocytes and centrally located banana-shaped bradyzoites. 18S rRNA gene expression studies confirmed the isolates from the sheep as S. ovicanis, goats as S. capracanis and cattle as S. bovicanis. This, to the best of our knowledge, is the first molecular identification of an isolate of S. ovicanis and S. capracanis in Malaysia. Further studies with electron microscopy (EM) are required in the future to compare the features of different types of Sarcocysts spp.

Matched MeSH terms: DNA, Protozoan/chemistry
Fulltext Molecular Surveillance of Pfkelch13 and Pfmdr1 Mutations in Plasmodium falciparum Isolates from Southern Thailand

Khammanee T, Sawangjaroen N, Buncherd H, Tun AW, Thanapongpichat S

Korean J Parasitol, 2019 Aug;57(4):369-377.
PMID: 31533403 DOI: 10.3347/kjp.2019.57.4.369

Artemisinin-based combination therapy (ACT) resistance is widespread throughout the Greater Mekong Subregion. This raises concern over the antimalarial treatment in Thailand since it shares borders with Cambodia, Laos, and Myanmar where high ACT failure rates were reported. It is crucial to have information about the spread of ACT resistance for efficient planning and treatment. This study was to identify the molecular markers for antimalarial drug resistance: Pfkelch13 and Pfmdr1 mutations from 5 provinces of southern Thailand, from 2012 to 2017, of which 2 provinces on the Thai- Myanmar border (Chumphon and Ranong), one on Thai-Malaysia border (Yala) and 2 from non-border provinces (Phang Nga and Surat Thani). The results showed that C580Y mutation of Pfkelch13 was found mainly in the province on the Thai-Myanmar border. No mutations in the PfKelch13 gene were found in Surat Thani and Yala. The Pfmdr1 gene isolated from the Thai-Malaysia border was a different pattern from those found in other areas (100% N86Y) whereas wild type strain was present in Phang Nga. Our study indicated that the molecular markers of artemisinin resistance were spread in the provinces bordering along the Thai-Myanmar, and the pattern of Pfmdr1 mutations from the areas along the international border of Thailand differed from those of the non-border provinces. The information of the molecular markers from this study highlighted the recent spread of artemisinin resistant parasites from the endemic area, and the data will be useful for optimizing antimalarial treatment based on regional differences.

Matched MeSH terms: DNA, Protozoan/chemistry
Fulltext Acute respiratory distress syndrome and acute renal failure from Plasmodium ovale infection with fatal outcome

Lau YL, Lee WC, Tan LH, Kamarulzaman A, Syed Omar SF, Fong MY, et al.

Malar J, 2013 Nov 04;12:389.
PMID: 24180319 DOI: 10.1186/1475-2875-12-389

BACKGROUND: Plasmodium ovale is one of the causative agents of human malaria. Plasmodium ovale infection has long been thought to be non-fatal. Due to its lower morbidity, P. ovale receives little attention in malaria research.
METHODS: Two Malaysians went to Nigeria for two weeks. After returning to Malaysia, they fell sick and were admitted to different hospitals. Plasmodium ovale parasites were identified from blood smears of these patients. The species identification was further confirmed with nested PCR. One of them was successfully treated with no incident of relapse within 12-month medical follow-up. The other patient came down with malaria-induced respiratory complication during the course of treatment. Although parasites were cleared off the circulation, the patient's condition worsened. He succumbed to multiple complications including acute respiratory distress syndrome and acute renal failure.
RESULTS: Sequencing of the malaria parasite DNA from both cases, followed by multiple sequence alignment and phylogenetic tree construction suggested that the causative agent for both malaria cases was P. ovale curtisi.
DISCUSSION: In this report, the differences between both cases were discussed, and the potential capability of P. ovale in causing severe complications and death as seen in this case report was highlighted.
CONCLUSION: Plasmodium ovale is potentially capable of causing severe complications, if not death. Complete travel and clinical history of malaria patient are vital for successful diagnoses and treatment. Monitoring of respiratory and renal function of malaria patients, regardless of the species of malaria parasites involved is crucial during the course of hospital admission.

Matched MeSH terms: DNA, Protozoan/chemistry

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