Increased detection of Plasmodium knowlesi in Sandakan division, Sabah as revealed by PlasmoNex

Background: Plasmodium knowlesi is a simian malaria parasite that is widespread in humans in Malaysian Borneo. However, little is known about the incidence and distribution of this parasite in the Sandakan division, Malaysian Borneo. Therefore, the aim of the present epidemiological study was to investigate the incidence and distribution of P. knowlesi as well as other Plasmodium species in this division based on a most recent developed hexaplex PCR system (PlasmoNex™). Methods: A total of 189 whole blood samples were collected from Telupid Health Clinic, Sabah, Malaysia, from 2008 to 2011. All patients who participated in the study were microscopically malaria positive before recruitment. Complete demographic details and haematological profiles were obtained from 85 patients (13 females and 72 males). Identification of Plasmodium species was conducted using PlasmoNex™ targeting the 18S ssu rRNA gene. Results: A total of 178 samples were positive for Plasmodium species by using PlasmoNex™. Plasmodium falciparum was identified in 68 samples (38.2%) followed by 64 cases (36.0%) of Plasmodium vivax, 42 (23.6%) cases of P. knowlesi, two (1.1%) cases of Plasmodium malariae and two (1.1%) mixed-species infections (i e, P. vivax/ P. falciparum). Thirty-five PlasmoNex™ positive P. knowlesi samples were misdiagnosed as P. malariae by microscopy. Plasmodium knowlesi was detected in all four districts of Sandakan division with the highest incidence in the Kinabatangan district. Thrombocytopaenia and anaemia showed to be the most frequent malaria-associated haematological complications in this study. Conclusions: The discovery of P. knowlesi in Sandakan division showed that prospective studies on the epidemiological risk factors and transmission dynamics of P. knowlesi in these areas are crucial in order to develop strategies for effective malaria control. The availability of advanced diagnostic tool PlasmoNex™ enhanced the accuracy and accelerated the speed in the diagnosis of malari

Granular Formation during Apoptosis in Blastocystis sp. Exposed to Metronidazole (MTZ).

The role and function of the granular life cycle stage in Blastocystis sp, remains uncertain despite suggestions being made that the granules are metabolic, reproductive and lipid in nature. This present study aims to understand granular formation by triggering apoptosis in Blastocystis sp. by treating them with metronidazole (MTZ). Blastocystis sp.cultures of 4 sub-types namely 1, 2, 3 and 5 when treated with 0.01 and 0.0001 mg/ml of metronidazole (MTZ) respectively showed many of the parasites to be both viable and apoptotic (VA). Treated subtype 3 isolates exhibited the highest number of granular forms i.e. 88% (p<0.001) (0.0001 mg/ml) and 69% (p<0.01) (0.01 mg/ml) respectively at the 72 h in in vitro culture compared to other subtypes. These VA forms showed distinct granules using acridine orange (AO) and 4',6-diamino-2-phenylindole (DAPI) staining with a mean per cell ranging from 5 in ST 5 to as high as 16 in ST 3. These forms showed intact mitochondria in both viable apoptotic (VA) and viable non-apoptotic (VNA) cells with a pattern of accumulation of lipid droplets corresponding to viable cells. Granular VA forms looked ultra-structurally different with prominent presence of mitochondria-like organelle (MLO) and a changed mitochondrial trans-membrane potential with thicker membrane and a highly convoluted inner membrane than the less dense non-viable apoptotic (NVA) cells. This suggests that granular formation during apoptosis is a self-regulatory mechanism to produce higher number of viable cells in response to treatment. This study directs the need to search novel chemotherapeutic approaches by incorporating these findings when developing drugs against the emerging Blastocystis sp. infections

PCR fingerprinting of Blastocystis isolated from symptomatic and asymptomatic human hosts

Genomic DNA from 16 Blastocystis hominis isolates comprising of eight asymptomatic isolates (A1-A8) and eight symptomatic isolates (S1-S8) was amplified by arbitrarily primed polymerase chain reaction (AP-PCR) using 38 arbitrary 10-mer primers. Six primers (A10, B5, C20, D1, F6, and F10) generated reproducible DNA fingerprints. AP-PCR amplification revealed similar DNA fingerprints among all symptomatic isolates (S1-S8) with common bands at 850 bp using primer A10, 920 bp using primer B5, and 1.3 kbp using primer D1. Isolates A1, A3, A4, A5, A6, and A7 showed similar DNA banding patterns and all asymptomatic isolates (A1-A8) shared a major band at I kbp using primer B5. Isolates A2 and A8 showed distinct DNA banding patterns that differed from the remainder of the isolates. The results of the phylogenetic analyses showed that all symptomatic isolates (S1-S8) formed, a clade with > 70 similarity among the isolates and which were clearly separate from asymptomatic isolates A1, A3, A4, A5, A6, and AT Asymptomatic isolates A2 and A8 formed two distinct and separate clades. AP-PCR revealed higher genetic variability within the asymptomatic isolates than within the symptomatic isolates. The present study suggests that AP-PCR can be a valuable method for differentiating between isolates of B. hominis and our results support the hypothesis that our asymptomatic and symptomatic B. hominis isolates may represent two different strains/species with varying pathogenic potential

Viable and apoptotic rate of untreated and treated forms of <i>Blastocystis</i> sp.

<p>(a) viability rate and (d) apoptotic rate of untreated forms of <i>Blastocystis</i> sp. of st1, st2, st3 and st5. (b) viability rate and (e) apoptotic rate of treated forms of <i>Blastocystis</i> sp.(symptomatic isolates) at 0.0001mg/ml of st1, st2, st3 and st5. (c) viability rate and (f) apoptotic rate of treated forms of <i>Blastocystis</i> sp.at 0.01mg/ml of st1, st2, st3 and st5 (st = subtype).</p

Mitochondrial transmembrane potential.

<p>(a) and (d) granular cells viewed under bright field. (b) and (e) MitoCapture accumulates and aggregate in the mitochondria, giving off a bright red fluorescence (c)low intensity of green fluorescence indicating that the granular cells are still viable. (f) high intensity of green fluorescence indicating that the cells are dying (or apoptotic).</p

Apoptotic and non-apoptotic granular forms of <i>Blastocystis</i> sp.

<p>Light microscopy images showing drug treated <i>Blastocystis</i> sp. stained with tryphan blue to test the viability and <i>Blastocystis</i> sp. stained with Annexin (V)-FITC (green). Annexin V labeled with fluorescein (FITC) identifies apoptotic cells binding to PS exposed on the outer membrane of the cell.</p

Diameter (μm) of untreated and treated (0.01 mg/ml and 0.0001 mg/ml) cells of <i>Blastocystis</i> sp. after 12 hours culture.

<p>Diameter (μm) of untreated and treated (0.01 mg/ml and 0.0001 mg/ml) cells of <i>Blastocystis</i> sp. after 12 hours culture.</p

Accumulation of lipid droplets.

<p>Presence of lipid in (a) viable non apoptotic (VNA) and (b) viable apoptotic (VA) (c) and (d) non-viable apoptotic (NVA) granular forms of <i>Blastocystis</i> sp.</p

TEM photographs.

<p>(a), (c) and (e) shows untreated <i>Blastocystis</i> sp. whereas (b), (d) and (f) shows treated <i>Blastocystis</i> sp. (g) Electron dense granular forms of treated <i>Blastocystis</i> sp. (h) under high magnification; vacuolar form of <i>Blastocystis</i> sp. present within the granular forms. <b>(CR = cristae)</b>.</p

Granular cells of the viable apoptotic (VA) viable non-apoptotic (VNA), non-viable apoptotic ((NVA) forms and necrosis forms.

<p>The untreated cells are represented (a) subtype 3, (c) subtype 1 (e) subtype 2 and (g) subtype 5. The treated ones are represented by (b) subtype 3, (d) subtype 1, (f) subtype 2 and (h) subtype 5.</p