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Sub-lethal concentrations of artemisinin alter pH of the digestive vacuole of the malaria parasite, Plasmodium falciparum

Ibrahim, N. ; Roslee, A. ; Azlan, M. ; Abu-Bakar, N.

Tropical Biomedicine.2020;37(No.1):1-14.

An appropriate pH maintenance within a membrane-enclosed organelle is vital for the occurrence of biological processes. Artemisinin (ART), a potent antimalarial drug has been reported to target the digestive vacuole (DV) of Plasmodium falciparum, which might alter the pH of the organelle, thereby impairing the hemoglobin degradation and subsequent heme detoxification. Hence, a flow cytometry-based technique using fluorescein isothiocyanate-dextran (FITC-dextran) as a ratiometric pH probe was employed to measure the pH of the DV of the malaria parasite treated with ART. Based on the pH calibration curve generated, the steady-state pH of the acidic DV of the non-treated parasites was 5.42 ± 0.11, indicating that FITC-dextran is suitable for detection of physiological pH of the organelle. The alteration of the DV pH occurred when the parasites were treated with ART even at the sub-lethal concentrations (15 and 30 nM) used. The similar effect was shown by the parasites treated with a standard proton pump inhibitor, concanamycin A. This suggests that ART might have altered the DV pH at lower levels than the level needed to kill the parasite. This study has important implications in designing new ART treatment strategies and in generating new endoperoxide-based antimalarial drugs pertaining to the interruption of the pH regulation of the malaria parasite’s DV.

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Evaluation of the in vitro antileishmanial activities of bioactive guided fractionations of two medicinal plants

Al Nasr, I.S.

Tropical Biomedicine.2020;37(No.1):15-23.

The organisms of the genus Leishmania are flagellated protozoan parasites and are the causative agents of leishmaniasis. This disease is a major health problem, especially in tropical countries. Currently, cutaneous leishmaniasis is treated by chemotherapy using pentavalent antimonials, but these drugs have serious organo-toxicity, drug resistance on several occasions, and low efficiency in controlling the infection. The present work is carried out to evaluate the in vitro antileishmanial activity of methanolic extracts and phytochemical fractions of two plants ethnobotanically used against leishmaniasis and skin infection, Calotropis procera and Rhazya stricta leaves against Leishmania major promastigote and amastigote stages and cytotoxicity against the Vero cell line. The leaves of C. procera and R. stricta were extracted with methanol and fractionated by petroleum ether, chloroform, ethyl acetate, n-butanol, and water. The methanolic extracts of the leaves of C. procera and R. stricta exhibited antileishmanial activity against L. major promastigotes with IC50 values of 66.8 and 42.4 µg mL-1, respectively. While their CC50 2.3 and 298 µg mL-1 and their SI 0.03 and 7.03 respectively. However, the fractionations of the methanolic extract of C. procera leaves revealed antiparasitic activity against both L. major promastigote and amastigote stages in vitro, which significantly increased with polarity with the exception of n-butanol. Hence the best activity was revealed by the water fraction (IC50 of 26.3 and 29.0 µg mL-1) for the two stages. In conclusion, further phytochemical investigation should be performed for the C. procera water extract in terms of antileishmanial active ingredient isolation that may enhance the possibility of avoiding toxic substances and overcome the low SI (1.1 and 1.01).

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First case report of paragonimiasis in a Malaysian man

Ponnampalavanar, S. ; Kukreja, A. ; Amir, A. ; Mahmud, R.

Tropical Biomedicine.2020;37(No.1):24-28.

Paragonimiasis is an infection caused by Paragonimus, a lung fluke and is acquired by eating raw or undercooked crustaceans containing the infective metacercariae. Herein, we report a case of paragonimiasis in a Malaysian man who presented with incidental findings from chest radiographs. Examination of his biopsied lung tissue and sputum specimen revealed Paragonimus sp. eggs, whereas stool examination showed the presence of Giardia cysts. Patient was succesfully treated with praziquantel and metronidazole respectively.

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Progression of malaria induced pathogenicity during chloroquine therapy

Zaid, O.I. ; Abd. Majid, R. ; Sidek, H.M. ; Noor, S.M. ; Abd Rachman-Isnadi, M.F. ; Bello, R.O. ; Chin, V.K. ; Basir, R.

Tropical Biomedicine.2020;37(No.1):29-49.

Treatment Failure with chloroquine is one of the challenges that faced the dedicated efforts to eradicate malaria This study aims at investigating the impact of treatment failure with chloroquine on the progression of the disease-induced histo-pathogenic and immunogenic outcomes. To achieve this, Rane’s protocol with modifications was applied on a model of Plasmodium berghei ANKA infected ICR mice to determine the dose response curve of chloroquine and to screen the treatment impact on the disease progression. Chloroquine was given at 1, 5, 10, 15 and 20 mg/kg once the parasitemia reached to 20-30% (the experimental initiation point). During the subsequent days, the mice were monitored for changes in the clinical signs, hematology parameters and the progress of the parasitemia until the parasitemia reached to 60-70% (the experimental termination point) or up to 10 days after chloroquine administration in case of achieving a complete eradication of the parasite. At the end, the mice were exsanguinated and their blood and organs were collected for the biochemistry and the histology study. A complete eradication of the parasite was achieved at 20 mg/kg while recrudescence was observed at the lower doses. At 1 mg/kg, the parasite growth was comparable to that of the positive control. The histo-pathogenic and immunogenic changes were stronger in the groups that experienced recrudescence (at 5 and 10 mg/kg). All in all, the study highlights the possibility of having a worsened clinical condition when chloroquine is given at its sub-therapeutic doses during malaria treatment.

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Parasitological and molecular detection of human fascioliasis in a young man from Guizhou, China

Chen, M.X. ; Zhang, R.L. ; Xu, X.N. ; Yu, Q. ; Huang, D.N. ; Liu, W. ; Chen, S.H. ; Song, P. ; Lu, Y. ; Cai, Y.C. ; Ai, L. ; Chen, J.X.

Tropical Biomedicine.2020;37(No.1):50-57.

A 24-year-old man born in Guizhou province was diagnosed with obstructive jaundice and bile duct stones in 2013. Four living trematodes were found during laparotomy and cholecystectomy. Based on the morphology and molecular genetics analysis of internal transcribed spacer and pcox1 genes of the flatworm specimens, the trematodes from the patient were confirmed to be Fasciola hepatica. This report provided the clinical and molecular diagnosis information on human fascioliasis, which is an emerging sanitary problem still ignored in China. Human fascioliasis constantly occurs due to climatic changes and frequency of human travel. Therefore, it deserves more attention from physicians working in both developing and developed countries.

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Serotyping of dengue virus in 2016-17 outbreak in Kudat, Sabah, Malaysia

Aung, T.S. ; Gintarong, T. ; Balingi, D.B. ; Emran, A. ; Thein, T.T. ; Chua, T.H.

Tropical Biomedicine.2020;37(No.1):58-65.

An outbreak of dengue in Kudat, northern Sabah in 2016-2017 provided an opportunity to investigate the circulating serotypes of dengue viruses of cases at Hospital Kudat. Between September 2016 and December 2017, a total of 156 dengue positive sera (tested positive by either NS1 antigen, or IgM & IgG antibody rapid test) were collected from dengue patients who had acute fever and showed signs and symptoms suggestive of dengue. RNA was extracted from the sera using QIAamp RNA Blood Mini Kit, and molecular amplification was performed using one-step RT-PCR kit, followed by nested PCR using HotStart Taq master mix kit with the primers of the dengue C-prM gene. There were 81 (52%) male and 75 (48%) female cases. The age group with the highest number of cases was the 10-19 years old, while the youngest infected was 8 months old and the oldest was 83 years old. RT-PCR results showed 88 sera dengue positive, 48 infected with a single serotype while another 40 with multiple serotypes. All four DENV serotypes were co-circulating during the outbreak period and DENV-1 was predominant. Molecular analysis also indicated 69.2%, 50.0%, 51.9% and 48.9% respectively of the NS1, IgM, IgG and IgM & IgG positive sera were RT-PCR positive for dengue. High number of cases were seen in December 2016, February and May 2017. The dengue outbreak might be related to switching of predominant serotype from DENV 4 to DENV 1.

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Does the morbidity management and disability prevention (MMDP) clinic serve the filarial lymphedema (FLE) patients’ preeminent expectation?

De Britto, R.L.J. ; Vijayalakshmi, G. ; Boopathi, K. ; Kamaraj, P. ; Supriya, V.K. ; Yuvaraj, J.

Tropical Biomedicine.2020;37(No.1):66-74.

Advocacy and training on “Home care” for filarial lymphoedma (FLE) patients are provided through morbidity management and disability prevention (MMDP) clinic commonly known as filariasis clinic and clinical improvement is assessed by follow-up visits. While the physicians aim at reducing the recurrent ADL (coined as ADLA in 1997) episodes, the patients expect reduction in LE volume. The objective of the present study was to know whether the MMDP clinic serves the primary expectation of the FLE patients. LE patients who attended the clinic for at least four follow-up consultations and had LE volume measurements at three points of time during the one year period of observation were considered for analysis. Clinical assessment was done for LE grading and LE volume was measured by water displacement volumetry. Sixty-three patients who fulfilled the follow up criteria were included. It was observed that the median LE volume was 914ml (IQR 269 – 1935) at first visit of the observation period which reduced to 645ml (IQR 2151666) and 752ml (IQR 215 – 1720) at first and second follow-up visits respectively. Over all, in short span of one year, 21 of the 63 patients (33.3%) who visited MMDP clinic at least four times in a year were benefitted through the MMDP advocacy and the National filariasis control programme need to emphasise on the importance of follow up visits to FLE patients.

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Immunohistochemical evaluation of interleukin-23 and cyclooxygenase-2 in the muscles of mice infected with Trichinella spiralis

El-Aswad, B.E.W. ; Ammar, A.I. ; Mahmoud, S.F. ; Soliman, S.S. ; Abd El-Atty, A.F.

Tropical Biomedicine.2020;37(No.1):75-88.

The course of Trichinella (T.) spiralis infection includes intestinal and muscle phases. The aims of this work were to evaluate IL-23 and cyclooxygenase-2 (COX-2) by immunohistochemistry in the muscles of T. spiralis infected mice in a time-course study and to correlate their level with the serum levels of IL-23, IFN-γ, IL-4 and IL-10 cytokines. The mice were divided into an un-infected control group (UC) (10 mice) and 5 infected mouse groups (each 10 mice/group. Each mouse was infected with 200 T. spiralis larvae) and sacrificed on days 7, 14, 21, 28 and 35 post-infection (dpi). IL-23 showed weak expression (+1) on the 21st dpi, then it became moderately expressed (+2) on the 28th dpi and on day 35 pi, the immunoreactivity was strong (+3). COX-2 expressed weakly on 14 dpi, while the other mouse groups (21, 28 and 35) showed strong (+3) expression. IL-23 serum concentrations increased gradually in a significant pattern, in comparison to that of UC mice, from the 21st dpi to the end of the experiment. IFN-γ increased gradually and was significantly higher than those of UC mice from the 7th dpi, reached its maximum level on the 21st dpi, after which it decreased non-significantly. IL-4 up-regulated significantly in all infected groups in comparison to UC mice achieving its highest level on the 21st dpi and decreased after that. IL-10 increased significantly on the 7th dpi, but dropped at the 14th dpi, then reached its peak on the 21st dpi, and decreased again on the 28th and 35th dpi. In conclusion, T. spiralis infection caused increased expression of IL-23 and COX-2 in the muscle of infected mice, the effect being strongest on the 35th day. Also, the infection induced a mixed Th1/Th2 profile with a predominance of Th2 at the early muscle phase, after which the immune repose became mainly Th2.

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Assessment of vaccination with gamma radiation-attenuated infective Toxocara canis eggs on murine toxocariasis

Hafez, E.N. ; Awadallah, F.M. ; Ibrahim, S.A. ; Amin, M.M. ; El-Nawasera, N.Z.

Tropical Biomedicine.2020;37(No.1):89-102.

Toxocara canis is a major parasite that infects many animals with high risk of human infections. This study aims at assessing the immunization with gamma radiationattenuated infective stage on rats challenged with non-irradiated dose. Level of vaccine protection was evaluated in liver and lung regarding parasitological, histopathological, biochemical and molecular parameters. Fifty rats were enrolled in three groups: group A (10 rats) as normal control; group B (20 rats) subdivided into subgroup B1 (infected control) and subgroup B2 infected then challenged after 14 days with the same dose of infection (challenged infected control); and group C (20 rats) subdivided into subgroup C1 vaccinated with a dose of 800 gray (Gy) gamma-radiated infective eggs (vaccine control) and subgroup C2 vaccinated then challenged on 14th day with same number of infective eggs (vaccinated-challenged). Tissues were stained with Haematoxylin and Eosin (H&E) for histopathological studies. Biochemical studies through detection of nitric oxide (NO) and Caspase-3 were conducted. Extent of DNA damage by Comet assay was assessed. Vaccinated-challenged subgroup revealed a marked reduction in larvae in tissues with mild associated histological changes. In addition there was accompanied reduction of NO, Casepase-3 level and DNA damage compared to the control infected group. It could be concluded that vaccination of rats with a dose of 800Gy gamma radiation-attenuated infective stage improves immune response to challenge infection and drastically reduces the morbidity currently seen.

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Survival of antibiotic resistant Escherichia coli in vacuum-packed keropok lekor: Food safety alert among SME keropok lekor producers

Wan-Hamat, H. ; Lani, M.N. ; Hamzah, Y. ; Alias, R. ; Hassan, Z. ; Mahat, N.A.

Tropical Biomedicine.2020;37(No.1):103-115.

The microbiological quality of thirty ready-to-eat (RTE) keropok lekor (a sausage shape Malaysian fish product) was evaluated in comparison to microbiological guidelines for ready to eat foods. The two E. coli isolates were subjected to DNA sequencing, identified and tested for their resistance towards fifteen different antibiotics. The survival and growth of the isolated E. coli strains inoculated in keropok lekor at atmospheric air and vacuum packaging were also evaluated. Results revealed that four samples (13.33%) contained Enterobacteriaceae counts that exceeded the recommended allowable counts of 4.0 log10 CFU/g. Unsatisfactory level of coliforms (> 1.7 log10 CFU/g) was also observed in ten of the samples; two of which contained E. coli (2.1 ± 0.17 and 3.7 ± 0.02 log10 CFU/g), suggesting of poor hygiene and sanitation practices. While the ‘Possible E10’ E. coli strain was observably resistant towards Nalidixic acid (30µg) alone, B10 E. coli isolate was worryingly resistant towards Ampicillin (10µg), Ceftazidime (30µg), Ciprofloxacin (5µg), Ceftriaxone (30µg), Nalidixic acid (30µg) and Tetracycline (30µg). This study also revealed that the growth and survival of the ‘Possible E10’ and B10 E. coli strains were not significantly affected by vacuum packaging when stored at both 4°C and 28°C. Therefore, intervention programmes to alert and educate smallmedium enterprisers (SMEs) of keropok lekor producers on food safety as well as potential health risks that can be associated due to inappropriate handling procedures of such product, merits consideration.

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