Low Allelic Variation of Plasmodium falciparum msp-1 and msp-2 among Gold Miners in Central Kalimantan Province, Indonesia.

Background: We aimed to find out the allelic variation of Pfmsp-1 and Pfmsp-2 among gold miners in Central Kalimantan Province, Indonesia using parasites' DNA isolated from archived RDT and GSBS. Methods: This study was done using the samples collected between 2017-2020 from health centers in Subdistrict of Mihing Raya, Danau Rawah, and Bukit Hindu as well as Kapuas District Health Laboratory in Central Kalimantan Province, Surabaya, Indonesia. Parasites DNA were isolated from RDT cartridges and GSBS of local and migrant gold miners. Species of Plasmodium were confirmed by single step PCR. The allelic variation of Pfmsp-1 (K1, MAD20, RO33) and Pfmsp-2 (3D7, FC27) were analyzed by nested PCR. Results: Pfmsp-1 gene was found in only two (22.22%) out of 9 local samples, and 3 (27.27%) out of 11 migrant samples were found positive for K1 (150 bp) as well as MAD 20 (190 bp) allelic families. Pfmsp-2 gene were found in each one sample of 550 bp fragment in local (11.11%) and migrant samples (9.09%) for 3D7, and 2 samples of 300 bp fragments in local (22.22%) and 3 samples of 300 bp in migrant samples (27.27%). No difference in size and number of infections between both populations. The RO33 allelic family Alhamdulillah was not found in any sample. Conclusion: Low allelic variation of Pfmsp-1 and Pfmsp-2 genes with monogenotype indicated the low intensity of malaria transmission among gold miners in the studied areas. Further, the transmission may occur locally in the mining sites.

Toxicological Evaluation and Protective Effects of Ethanolic Leaf Extract of Cassia spectabilis DC on Liver and Kidney Function of Plasmodium berghei-Infected Mice.

Currently, the presence of antimalarial drug resistance has become a major obstacle in the treatment of malaria. To overcome the problem, a series of studies are needed to find new antimalarial drugs from plants. Previously, 90% ethanolic extract of Cassia spectabilis DC (EECS) leaves have been reported to have antimalarial activity in vitro against Plasmodium falciparum and in vivo against Plasmodium berghei ANKA. The research is conducted to find out the toxicity and protective effects of EECS on the liver and kidneys of mice infected with P. berghei ANKA. The acute and subacute toxicity tests were carried out on healthy mice that were given EECS at a dose of 150 mg/kg BW. An antimalarial activity test was carried out at doses of 150 and 200 mg/kg BW in P. berghei-infected mice. Regarding hepatomegaly, further plasma levels of hepatic enzyme were analyzed, as well as histopathological observation of the liver to determine the effect of the extract on liver. The kidney was observed histopathologically as well. The acute toxicity test of EECS showed that there was no mouse died at the highest dose, indicating safe for the mice. The subacute toxicity based on the histology data showed no significant difference in the liver and kidney of mice between the tested group and the healthy group. The histological and enzymatic effect of EECS in mice infected with P. berghei showed the histological and enzymatic effect that improved liver function and the histopathological effect on kidneys with the highest activity at a dose of 200 mg/kg BW compared with the negative control. The results showed the EECS was not toxic in mice and repaired the liver and kidney functions of P. berghei ANKA-infected mice, indicating a good candidate for antimalarial drug development.

THE USE OF ARCHIVED GIEMSA-STAINED BLOOD SMEARS AND RDT FOR PCR-BASED GENOTYPING OF Plasmodium vivax MEROZOITE SURFACE PROTEIN-1 IN CENTRAL KALIMANTAN PROVINCE, INDONESIA.

BACKGROUND: Plasmodium vivax is transmitted most across the country of Indonesia. The country has set out a malaria elimination program by 2030. The information on genetic diversity of malarial parasites relates to malaria transmission in an endemic area may provide the information that can help the malaria control program to achieve the target. Therefore, the purpose of this study was to determine the genetic diversity of the Pvmsp-1 gene in Central Kalimantan Province. MATERIALS AND METHODS: Samples were 140 of archived Giemsa-stained blood smear and rapid detection test. Samples were divided into the indigenous and migrant populations. After confirmation by single-step PCR, only P. vivax and mixed infection samples were amplified to nested PCR for genotyping of Pvmsp-1 allelic variation in segments F1, F2, and F3. RESULTS: Genotyping of 23 PCR positive samples resulted in 13 genotypes. In segment F1, three allelic variants type A containing subtype A1 (1,050 bp), A2 (350 bp), A3 (150 bp), and type B (100 bp). In segment F2, mono genotypes were detected as variant type A (1,050 bp) and type B3 (150 bp), multiple genotypes were detected as type B containing subtype B1 (250 bp), B2 (200 bp), and B3 (150bp). In segment F3, three allelic variants generated from four mono genotypes were type A (350 bp), type B (300 bp), and two type C (250 bp). CONCLUSION: The low allelic variation of Pvmsp-1 gene may reflect the actual situation of the low malaria endemic status of the study sites.

Suppressive effect of goat bile in Plasmodium berghei ANKA infection in mice.

BACKGROUND AND AIM: Some individuals in Indonesia consume intact goat gallbladder to prevent and treat malaria. The acute and subacute toxicity tests of goat bile (GB) have shown mild diarrhea in mice. Therefore, this study aimed to evaluate the suppressive effect of GB on parasitemia, splenomegaly, hepatomegaly, and blood biochemistry to assess liver and kidney function in BALB/c mice infected with Plasmodium berghei ANKA. MATERIALS AND METHODS: Fifty healthy mice were infected with P. berghei ANKA and divided into five groups. Mice in three groups were administered 0.5 mL of 25%, 50%, or 100% of GB by gavage. Animals in Group 4 were administered 187.2 mg/kg BW of dihydroartemisinin-piperaquine phosphate as a positive control (POS Group). Mice in fifth group were administered sterile water as negative (NEG) controls. Further, 30 uninfected mice were divided into groups 6-8 and administered GB as were mice in the first three groups. Group 9 included 10 uninfected and untreated animals as healthy controls. Treatments were administered in a 4-day suppressive test followed by daily observation of Giemsa-stained blood smears. On day 7, mice were sacrificed to measure the length and weight of spleens and livers, plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine. RESULTS: GB suppressed parasitemia but did not affect the size and weight of spleens or livers or plasma levels of AST and ALT compared to uninfected GB-treated and healthy control animals. Conversely, plasma levels of BUN and creatinine were suppressed and remained in the normal range in all groups of mice. CONCLUSION: GB suppresses parasitemia with no significant impact on hepatic enzymes in GB-treated infected mice. Liver dysfunction in GB-treated infected mice was due to P. berghei rather than GB treatment.

Antiplasmodial activity of Ethanolic extract of Cassia spectabilis DC leaf and its inhibition effect in Heme detoxification.

BACKGROUND: In previous studies, Cassia spectabilis DC leaf has shown a good antiplasmodial activity. Therefore, this study is a follow-up study of the extract of leaf of C. spectabilis DC on its in vitro and in vivo antiplasmodial activity and mechanism as an antimalarial. METHODS: The extract was fractionated, sub-fractionated and isolated to obtain the purified compound. In vitro antiplasmodial activity test against Plasmodium falciparum to find out the active compound. In vivo test against P. berghei ANKA-infected mice was conducted to determine prophylactic activity and antiplasmodial activity either alone or in combination with artesunate. The inhibition of heme detoxification test as one of the antimalarial mechanisms was carried out using the Basilico method. RESULTS: The results showed that active antimalarial compound isolated from C. spectabilis DC leaf had a structural pattern that was identical to (-)-7-hydroxycassine. Prophylactic test of 90% ethanolic extract of C. spectabilis DC leaf alone against P. berghei ANKA-infected mice obtained the highest percentage inhibition was 68.61%, while positive control (doxycycline 13 mg/kg) was 73.54%. In combination with artesunate, 150 mg/kg three times a day of C. spectabilis DC (D0-D2) + artesunate (D2) was better than the standard combination of amodiaquine + artesunate where the inhibition percentages were 99.18 and 92.88%, respectively. The IC50 of the extract for the inhibitory activity of heme detoxification was 0.375 mg/ml which was better than chloroquine diphosphate (0.682 mg/ml). CONCLUSION: C. spectabilis DC leaf possessed potent antiplasmodial activity and may offer a potential agent for effective and affordable antimalarial phytomedicine.

Acute and subacute toxicity tests of goat bile in BALB/c mice.

AIM: The aim of this study was to investigate the toxicity of goat bile in BALB/c mice since some Indonesian people consume raw goat gallbladder to treat malaria and increase stamina. MATERIALS AND METHODS: Acute toxicity test was done in six groups of BALB/c mice using 100%, 50%, 25%, 12.5%, and 6.75% of goat bile and negative control. The death of mice was observed within 14 days. In the subacute toxicity test, the body weight and hematology parameters on day 0 and day 4 post-treatment were evaluated. The mice were closely observed for 28 days before plasma collection for the blood biochemistry evaluation. RESULTS: Mild diarrhea was observed in acute and subacute toxicity tests. No death of mice was observed in acute test. Goat bile did not inhibit the increase of the body weight of mice. A slight reduction in hemoglobin and hematocrit levels in mice treated with 25% and 50% goat bile, however, remained normal in mice treated with 100% goat bile. The red and white blood cell count were not affected. Liver and kidney functions were not affected by goat bile treatment as revealed by the plasma level of aspartate aminotransferase and alanine aminotransferase, blood urea nitrogen, and creatinine, which remained in the normal range. CONCLUSION: Goat bile treatment in BALB/c mice caused mild toxicity in mice. Hydrophobic bile acids may cause the toxicity of goat bile in mice; therefore, it is recommended that goat bile consumption not to be taken oftenly to avoid its harmful effect.

Various Parts of Helianthus annuus Plants as New Sources of Antimalarial Drugs.

BACKGROUND: Each part of H. annuus plants is traditionally used as medicinal remedies for several diseases, including malaria. Antimalarial activity of the leaf and the seed has already been observed; however, there is no report about antimalarial activity of the other parts of H. annuus plants. In this study, we assess in vitro and in vivo antimalarial activity of each part of the plants and its mechanism as antimalarial agent against inhibition of heme detoxification. OBJECTIVE: To investigate the antimalarial activity of various parts of H. annuus. METHODS: Various parts of the H. annuus plant were tested for in vitro antimalarial activity against Plasmodium falciparum 3D7 strain (chloroquine-sensitive), in vivo antimalarial activity against P. berghei using Peters' 4-day suppressive test in BALB/c mice, curative and prophylaxis assay, and inhibition of heme detoxification by evaluating ß-hematin level. RESULTS: Ethanol extract of the roots showed the highest antimalarial activity, followed by ethanol extract of leaves, with IC50 values of 2.3 ± 1.4 and 4.3 ± 2.2 µg/mL, respectively and the percentage inhibition of P. berghei of 63.6 ± 8.0 and 59.3 ± 13.2 at a dose of 100 mg/kg, respectively. Ethanol extract of roots produced an ED50 value of 10.6 ± 0.2 mg/kg in the curative test and showed an inhibition of 79.2% at a dose of 400 mg/kg in the prophylactic assay. In inhibition of heme detoxification assay, root and leaf ethanol extracts yielded a lower IC50 value than positive (chloroquine) control with a value of 0.4 ± 0.0 and 0.5 ± 0.0 mg/mL, respectively. CONCLUSION: There were promising results of the ethanol extracts of root of H. annuus as a new source for the development of a new plant-based antimalarial agent.

COMPARISON OF MULTIPLEX SINGLE ROUND PCR AND MICROSCOPY IN DIAGNOSIS OF AMOEBIASIS.

BACKGROUND: Amoebiasis, the cause of dysentery and extra-intestinal abscesses, now becomes second fatal parasitic disease in the world. As routine microscopic diagnosis cannot differentiate causative Entamoeba histolytica from non-pathogenic E. dispar and E. moshkovskii, better diagnosis has to be searched. MATERIALS AND METHODS: Multiplex single round PCR was tested and compared with results of microscopy of wet preparation on 30 samples of diarrheic stools and extra intestinal lesions from amoebiasis suspected patients. RESULTS: Microscopy examination showed that 21 (70%) of the samples were positive for E. histolytica/E. dispar/E. moshkovskii complex and 18 (86%) of them contained hematophagous trophozoites. Multiplex single round PCR showed 12 positive results, from which seven were positive for E. histolytica, two were positive for E. moshkovskii, and three showed mixed of E. histolytica and E. moshkovskii. No samples were positive for E. dispar. High positive rate of microscopy might be related with highly suspected amoebiasis cases, while lower positive PCR might be caused by low parasite density and time-related trophozoite disintegration. CONCLUSION: The study showed that multiplex single-round PCR is a valuable diagnostic tool for species differentiation, but cannot replace microscopy in the diagnosis of amoebiasis because of its low sensitivity and impossibility to discriminate the form of E. histolytica and whether it is in the disease-causing stage, while microscopic examination is capable to demonstrate the presence of hematophagous trophozoites that indicates it is invasive and at the disease-causing stage of E. histolytica.

Submicroscopic malaria cases play role in local transmission in Trenggalek district, East Java Province, Indonesia.

BACKGROUND: Trenggalek district is a hypoendemic malaria area with mainly imported cases brought by migrant workers from islands outside Java. During malaria surveillance in 2015, no malaria cases were found microscopically, but some cases were positive by PCR. Therefore, a study was conducted to prove that local malaria transmission still occur. METHODS: The adult villagers were invited to the house of the head of this village to be screened for malaria using aseptic venipuncture of 1 mL blood upon informed consent. Thin and thick blood films as well as blood spots on filter paper were made for each subject. The blood films were stained with Giemsa and the blood spots were used to extract DNA for polymerase chain reaction (PCR) amplification to determine the malaria infection. In addition, the history of malaria infection and travel to malaria endemic areas were recorded. Entomologic survey to detect the existence of anopheline vector was also conducted. RESULTS: Of the total 64 subjects that participated in the survey, no malaria parasites were found through microscopic examination of the blood films. The PCR analysis found six positive cases (two Plasmodium falciparum, one Plasmodium vivax and two mixed infection of both species), and two of them had no history of malaria and have never travelled to malaria endemic area. Entomologic survey using human bait trap detected the existence of Anopheles indefinitus that was found to be positive for P. vivax by PCR. CONCLUSIONS: The results indicated that although we did not find any microscopically slide positive cases, six PCR positive subjects were found. The fact that 2 of the 6 malaria positive subjects have never travelled to malaria endemic area together with the existence of the vector confirm the occurence of local transmission of malaria in the area.