Molecular Identification and Characterization of Simian Malaria Parasites Isolated from Human Blood Samples from Malaysia
Plasmodium knowlesi, a parasite normally seen in macaques, is the main cause of malaria in Malaysia where human cases of a different simian malaria parasite, P. cynomolgi, has been described as well. Zoonotic malaria is a threat especially to people who live in the forest or forest fringes, and...
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| Format: | Thesis |
| Language: | English |
| Published: |
2022
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| Subjects: | |
| Online Access: | http://ir.unimas.my/id/eprint/40781/1/Hanisah%20ft.pdf |
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| Summary: | Plasmodium knowlesi, a parasite normally seen in macaques, is the main cause of malaria
in Malaysia where human cases of a different simian malaria parasite, P. cynomolgi, has
been described as well. Zoonotic malaria is a threat especially to people who live in the
forest or forest fringes, and people who venture into the forest for work or leisure purposes.
Staff at Universiti Malaya (UM) analysed 645 human blood samples collected from
indigenous communities around Malaysia for malaria parasites. Following DNA
extraction, PCR assays and sequencing of the nuclear small subunit (SSU) rRNA genes,
they found both human and simian malaria parasites, including P. knowlesi, P. cynomolgi,
P. inui and P. coatneyi in 102 of these samples. Since P. inui and P. coatneyi haven’t been
previously described in natural infections of humans, it was necessary to confirm these
results by nested PCR assays and by sequencing another gene at an independent laboratory.
This study aims to identify simian malaria parasites from these human blood samples by
nested PCR assays and by sequencing the cytochrome c oxidase subunit 1 (COX1) genes
of Plasmodium. DNA was re-extracted at UM from 15 randomly selected human blood
samples with simian malaria infections other than P. knowlesi from the 102 samples
mentioned above. These were sent blind, together with five malaria-negative samples, to
Universiti Malaysia Sarawak (UNIMAS) for further analysis. These simian malaria
parasites were isolated from human indigenous communities from five states (Perak,
Kelantan, Negeri Sembilan, Melaka, and Sarawak) in Malaysia. All the 20 gDNA samples
sent to UNIMAS were screened and tested negative for the presence of macaque DNA
upon receipt and also after concluding the sequencing of COX1 genes, ruling out
contamination with macaque blood prior to or during succeeding experiments. Nested PCR
assays with genus-specific PCR primers followed by species-specific primers were
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undertaken targeting the Plasmodium SSU rRNA gene. A single infection of P. inui was
identified in one sample while double infections of P. inui and P. cynomolgi were found in
nine out of the 20 other samples. Novel PCR primers for Plasmodium COX1 genes were
designed and PCR assay parameters were optimised. Partial COX1 genes of the
Plasmodium-positive samples were amplified using a single step or a hemi-nested PCR
assay, then cloned and sequenced. The total of 41 partial Plasmodium COX1 sequences
generated were subjected to phylogenetic analyses with 40 referral sequences, using both
Neighbour-Joining and Bayesian Maximum Clade Credibility methods. The identity of the
Plasmodium species in each sample was inferred from the phylogenetic analyses, with both
of the phylogenetic trees generated having similar topology. Nine samples were identified
as being infected with P. cynomolgi and one with P. inui-like parasites, one with a double
infection of P. inui-like and P. simiovale, and one with a triple infection with P. cynomolgi,
P. simiovale and P. inui-like. For two out of the 20 samples, the species of Plasmodium
could not be inferred following phylogenetic analysis. Differences noted when PCR assay
and sequencing results for both laboratories were compared may be due to the usage of
different or separate DNA extractions from each human blood sample. Other reasons for
the differences in results could be because species-specific primers were used when
sequencing the SSU rRNA genes at UM while universal primers for Plasmodium spp. were
utilized when sequencing the COX1 genes at UNIMAS. This study is the first description
of naturally acquired human infections of P. inui-like and P. simiovale. It indicates that
human infections with zoonotic malaria parasites are widely distributed in Malaysia and
their prevalence is probably underestimated. |
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