Asian Pacific Journal of Tropical Medicine

1

Authors: Emergency Medicine Branch of Chinese Stroke Society

The concept of stroke-associated pneumonia (SAP) was first brought out by Hilker in 2003, which is the most important risk factor of mortality after stroke[1,2]. SAP causes prolonging hospitalization time and increasing medical expenses[3], which is a large burden for families and society. Due to the absence of an agreed concept for SAP in China and abroad previously, the variation in diagnostic criteria is considerable[4]. Therefore, in clinical practice, the prevention of SAP may be inadequate, and SAP patients may not be diagnosed timely or receive appropriate anti-infective treatment. The above situation may lead to a poor prognosis in patients. A multidisciplinary expert group composed of specialists in neurology, respiratory, infectious diseases, and intensive care was established in 2009 to discuss and formulate the Chinese consensus of SAP consensus 2010 edition[5] to improve the awareness and standardize the clinical diagnosis and treatment of SAP in China. In recent years, increasing researches on SAP provide more clinical evidence, and the accumulated evidence in the Chinese population is also increasing. Therefore, the specialists in neurology, emergency, respiratory, infectious diseases, intensive care, updated and revised the original consensus together to meet the needs of SAP clinical and prevention work. This version of the consensus is based on the 2010 version; the overall framework and major updates of SAP are determined after several working sessions. This version of the consensus combines the latest research progress and relevant guidelines at home and abroad, and the practical experience and research data on SAP prevention and treatment in China are used as much as possible; it is also revised by extensive solicitation of opinions and repeated discussions. We hope this consensus can provide a reference for the clinical treatment and prevention of SAP (Figure 1).

17-24

Authors: Xiao-yuan Ma, Huai-jian Jin, Li-xing Tian, Qian Wang, Jun-yu Zhu, Zhi-gao He, Wen Tao, Tao Chen, Bin Wang, Hua-ping Liang

Objective: To evaluate the ability of new injury severity score (NISS), acute physiology and chronic health evaluation II (APACHE II), Glasgow coma scale (GCS), a combination of NISS and GCS, a combination of APACHE II and GCS, a combination of NISS and APACHE II to predict all-cause mortality of patients with severe trauma in mainland China. Methods: This was a multicenter observational cohort study conducted in the ICU of the Chonggang General Hospital, Daping Hospital of the Army Medical University and Affiliated Hospital of Zunyi Medical College from January 2012 to August 2016. The score of NISS, APACHE II, GCS, a combination of NISS and GCS, a combination of APACHE II and GCS, a combination of NISS and APACHE II were calculated based on data from the first 24 hours of ICU admission. Data were processed with Student's t-test, chi-square test, and receiver operating characteristic (ROC) curve of six scoring systems. Calibration was assessed with the Hosmer-Lemeshow test. The primary endpoint was death from any cause during ICU stay. Results: A total of 852 and 238 patients with severe trauma were assigned to the derivation group and validation group, respectively. Area under the ROC curve (AUC) was 0.826 [95% confidence interval (CI)=0.794-0.855)] for NISS, 0.802 (95% CI=0.768-0.832) for APACHE II, 0.808 (95% CI=0.774-0.838) for NGCS, 0.859 (95% CI=0.829 -0.886) for NISS+NGCS, 0.864 (95% CI=0.835-0.890) for APACHE II+NGCS, 0.896 (95% CI=0.869-0.929) for NISS+APACHE II in the derivation cohort. Similarly, the score of NISS+APACHE II was also better than the other five scores in the validation cohort (AUC=0.782; 95% CI=0.725- 0.833) and had a good calibration (P=0.41). Conclusions: Taking into account anatomical and physiological parameters completely, the combination of NISS and APACHE II performs better than NISS, APACHE II, NGCS, NISS+NGCS, APACHE II+NGCS for predicting mortality in ICU severe trauma patients. It is needful to develop models that contain various types of accessible predictors (demographic variables, injury cause/mechanism, physiological and anatomical variables, etc.) as comprehensive as possible.

32-40

Authors: Xiao-dian Zhang, Zhi-hui He, Li-ping Zheng, Guo-hui Yi, Min-ge Lin, Li-zhen Fu, Shu-hong Tian, Hong-hai Li, Yan-da Lu, Shen-hong Gu, Shao-jiang Zheng

Objective: To explore the effects of recombinant human calcineurin B (rhCNB) on hepatocellular carcinoma in mice. Methods: An in vivo mouse model with hepatocellular carcinoma was established, and the mice were randomized into the rhCNB, positive control and vehicle treatments groups. Tumor growth was assessed via bioluminescence using a small animal imaging system. Relative tumor proliferation rate and tumor growth inhibition were calculated. The expression of p53 and caspase-9 proteins in tumors were detected by immunohistochemistry. In vitro, flow cytometry was used to quantify the cell-cycle stages and rate of apoptosis. Western blotting and quantitative real-time PCR assays were used to evaluate the effects of rhCNB on protein and gene expression of CDK1, cyclin B1, p53 and caspase-9. Results: rhCNB at the higher dose significantly reduced tumor growth in vivo and caused tumor cell apoptosis in vitro. The rhCNB at the higher dose was as effective as cisplatin, and was safer. Conclusions: rhCNB has potent pro-apoptotic effects on tumor cells in vivo and in vitro and is well tolerated in vivo.

54-58

Authors: Zhi-hui He, Si-ru Liu, Xin-lai Wu, Yong-hao Huang, Yan Chen, Yi Deng, Pan-pan Xie, Yan-da Lu

Objective: To investigate the effects of a recombinant endoglin-macrophage inflammatory protein 3α Fc-fusion protein (EM) vaccine on tumor angiogenesis and growth in mice with H22 hepatocellular carcinoma. Methods: An in vivo hepatoma mouse model was established. Seven days after subcutaneous inoculation of H22 tumor cells, mice were randomly divided into four groups: EM, endoglin Fc-fusion protein, macrophage inflammatory protein 3α Fc-fusion protein, and normal saline groups. Tumor volume and survival rate of mice were studied at 3-day intervals. Microvessel density of the tumors and tumor cell proliferation were detected by immunohistochemistry, and tumor cell apoptosis was detected by TdT-mediated biotinylated-dUTP nick-end label staining. The number of CD11c and CD86 positive dendritic cells were detected by flow cytometry. Results: Compared with the other groups, the tumor volume became smaller, and the survival time was longer in the EM-treated group. Besides, microvessel density and cell proliferation index were significantly lower, while the tumor cell apoptosis index was significantly higher in the EM-treated group. Besides the number of CD11c and CD86 positive dendritic cells in EMtreated mice was larger than that in other groups. Conclusions: EM Fc-fusion protein could effectively inhibit tumor growth through inhibiting endoglin-related tumor angiogenesis and cell proliferation, promoting tumor cell apoptosis, and could induce a certain degree of antitumor immune responses.

1

Authors: Xiao-zhen Li, Wen-ting Cao, Wan-ting Xia, Hai-rong Huang, Tai-cheng Wang

Objective: To systematically analyze the prognosis of limited hilar hepatectomy versus combined liver lobectomy in the treatment of hilar cholangiocarcinoma. Methods: We searched CBM, CNKI, VIP, Wangfang, PubMed, SCI, Elsevier clinicalkey and OVID, and the cut-off time of searching was 31 April, 2019. Two researchers evaluated the risk of bias of the included studies independently according to the Newcastle-Ottawa Scale (NOS), and extracted data. RevMan 5.3 software was used for meta-analysis. Results: A total of 12 cohort studies involving 1 288 patients were included, of which 426 patients were in the limited hilar hepatectomy group and 862 patients in the combined liver lobectomy group. Meta-analysis showed that there were statistically significant difference in radical resection rate and postoperative complication rate (RR=0.55, 95%CI 0.40 to 0.74, P<0.001; RR=0.63, 95%CI 0.49 to 0.81, P<0.001) between the hilar limited hepatectomy group and the combined liver lobectomy group; significant differences were found in 1-, 2- and 3-year survival rates (RR=0.75, 95%CI 0.67 to 0.84, P<0.001; RR=0.55, 95%CI 0.42 to 0.74, P<0.001; RR=0.54, 95%CI 0.39 to 0.75, P<0.001) between the two groups. Conclusions: Comparing with the limited hilar hepatectomy, the radical resection rate of combined liver lobectomy was higher, and the 1-, 2- and 3-year survival rates were improved, but the complications was increased in treatment of hilar cholangiocarcinoma.

3

Authors: Rang-ru Liu, Zu-tao Yu, Zhuo Chen, Dan-qi Liu, Feng-ying Huang, Qian-bin Li, et al

Objective: To identify the molecule mechanism of potential cytotoxicity and proapoptosis of 9za in non-small cell lung cancer (NSCLC) cells. Methods: MTT assay, cell cycle detection kit, JC-1 staining assay were adopted to detect the cell viability, the cell cycle distribution and the mitochondrial membrane potential, respectively. Cell apoptosis was measured by Annexin V-FITC/propidium iodide apoptosis detection and the morphology was observed under a light microscope and the colorimetric TUNEL assay. Western blot was used to monitor the cell cycle-, apoptosisrelated proteins and some proteins involved in the pathways. Results: MTT assay showed that 9za significantly weakened the viability of NSCLC cells. Cell cycle analysis demonstrated that the low concentrations of 9za arrested the cell cycle at G0/G1 phase, which was further confirmed by the down-regulated levels of Cyclin D1, cyclin-dependent kinase 4 and cyclin-dependent kinase 6. In addition, Annexin V-FITC/propidium iodide apoptosis analysis, morphological observations and TUNEL assays revealed that the high concentrations of 9za could induce cell apoptosis. Furthermore, the JC-1 staining assay indicated that the mitochondrial membrane potential was reduced after 9za treatment. Western blot also manifested that 9za dramatically decreased the protein levels of the total Bcl-2, Cytochrome C in the mitochondria and BCL2 associated X in the cytoplasm. However, the levels of BCL2 associated X in the mitochondria, Cytochrome C in the cytoplasm, cleaved caspase-9, cleaved caspase-3 and the ratio of cleaved- PARP1/PARP1 showed the opposite trends. Moreover, the dose-dependently decreased phosphorylation levels of PDK1, protein kinase B, MEK and extracellular signal regulated kinase 1/2 following 9za treatment confirmed that 9za was indeed a double MEK/PDK1 inhibitor as we expected. Coadministration with PD0325901 or BX517 strengthened the cytotoxic and pro-apoptotic effect of 9za, verifying that 9za inhibited cell proliferation and induced cell apoptosis through the double MEK/PDK1 signaling pathways in NSCLC cells. Conclusions: This study demonstrates that 9za could play the effect of cytotoxicity and pro-apoptosis via the dual MEK/PDK1 signaling pathways in NSCLC cells and provides certain experimental foundation for 9za as a new type of drug candidate against non-small cell lung cancer.

5

Authors: Guo-tian Lin, Jie Li, Yue-cai Yu, Chan-juan Zhao, Li-min He, Zhuo Chen1, Fan Zhang

Objective: To observe the correlation between glycosylated hemoglobin level and blood pressure, and to provide clinical evidence to assist the screening and prediction of hypertension among diabetic patients in tropical setting. Methods: A total of 906 diabetic patients who had previously received physical examination at the Second Affiliated Hospital of Hainan Medical University from January 2009 to December 2018 were selected. Retrospective case-case study method was used. The average glycosylated hemoglobin level (HbA1c) was (5.80±1.27)%, and average blood pressure level was (129±18/79±10) mmHg. Patients were divided into group A: blood glucose well controlled, HbA1c<6.5%, (58.82±12.16) years old (754 cases); and group B: blood glucose poor controlled, HbA1c≥6.5%, (60.22±11.82) years old (152 cases). There was no significant difference in age between the two groups (T=-1.302, P=0.186). T-test, χ2-test, spearman correlation analysis and receiver operating characteristic (ROC) curve analysis were used to observe the correlation between glycosylated hemoglobin level and blood pressure, and the prevalence of hypertension. Results: Group B showed higher systolic blood pressure (134.19±18.62) mmHg vs (127.76±17.21) mmHg, diastolic blood pressure (81.82±10.92) mmHg vs (79.00±9.42) mmHg and blood glucose level (9.10±3.09) mmol/L vs (5.52±0.95) mmol/L than group A (T values were 4.335, 3.226, 14.142, respectively, P values were all <0.001). Spearman correlation analysis showed that HbA1c was positively correlated with systolic and diastolic blood pressure (the correlation coefficients r were 0.154 and 0.121, respectively, P values were all <0.001). The prevalence rates of hypertension were 31.17% (235/754) in group A, and 43.4% (66/152) in group B, and the difference was statistically significant [χ2=8.562, OR=1.695, 95% CI (1.187-2.419), P=0.003]. ROC curve of glycosylated hemoglobin was drawn to screen hypertension and the results showed that AUG was 0.587 [95% CI (0.548-0.626), P<0.001], the cut-off point, sensitivity and specificity were 6.05%, 33.6% and 78.2%, respectively. Conclusions: There was positive correlation of glycosylated hemoglobin and blood pressure and it is a novel view of using HbA1c to screen and predict hypertension among diabetic patients in tropic settings. More researches are needed in this field.

7

Authors: Mi Li, Kai Li, Ning Ding, Yi-qiang Xie, Kun Niu, Hong Zhang

Objective: To observe the effects of electroacupuncture on the expression of CREB and its ability to blind to synaptic key proteins in rats with post-traumatic stress disorder (PTSD), thus to lay the foundation for further study of the mechanism of electroacupuncture treatment of PTSD (i.e., electroacupuncture promotes the interaction between synaptic plasticity and BDNF-TRKB signaling pathway). Methods: SD male rats were randomly divided into blank group, model group and electroacupuncture treatment group, with 8 rats in each group. The model of PTSD was established by single continuous stress method (this modeling method is dominated by forced drowning, and tropical disasters are also mostly related to drowning). After modeling, the treatment group received electroacupuncture therapy, followed by retaining the needles for 20 min. The treatment was conducted once daily for 21 d. The behavioral changes were evaluated by spontaneous activity and conditional fear reaction. The expression of CREB was detected by immunohistochemistry and Western Blot. The binding ability of CREB to synaptic protein was verified by CHIP. Results: Compared with the model group, the spontaneous activity distance of the treatment group rats increased (P<0.01) and the percentage of stiffness time decreased (P<0.01), the expression of CREB protein in the treatment group was significantly increased (P<0.01), the binding ability of CREB to PSD95 protein was increased in treatment group (P<0.05). Conclusions: Electroacupuncture regulates the interaction mechanism between synaptic plasticity and BDNFTrkB signaling pathway of the rat model of PTSD, most likely by increasing the binding ability of CREB to the key synaptic protein-PSD95.

9

Authors: Shan-shan Wang, Yue Wu, Huan-huan Zhou, Wen-qi Wang, You Zhang, Wei Zhao, Xiu-ji Cui, Li-hua Li, Jing-long Chen, Lang-yu Rao, Jiang Du, Fei-fei Yin, Gang Lu

Objective: Polyomavirus infection is reported in a wide range of mammalian and avian hosts, including asymptomatic infection, acute systemic disease, and tumor induction.This study aims to obtain and characterize the complete genome of rodent polyomaviruses, PyVs-HMU. The host of virus was a Rattus norvegicus in the residential areas of Hainan Island, China. Methods: The liver samples of Rattus norvegicus were collected from the residential areas of Hainan Island, and then processed with a viral particle-protected, nucleic acid purification method. The extracted RNA and DNA were amplified by sequence-independent PCR. The amplified viral nucleic acid libraries for the samples of Rattus norvegicus were then sequenced using an Illumina GAII sequencer for a single read of 100 bp in length. The raw sequence reads were then filtered using previously described criteria to obtain valid sequences. Results: We obtained the complete genome of a novel polyomaviruses, PyVs-HMU. The genomic sequence of PyVs-HMU has been submitted in GenBank under accession number MK372231. The complete genome of PyVs-HMU is 5318 nucleotides (nt) in length with a G+C content of 45.36%. The complete PyV-HMU sequences display the representative genome organization of polyomaviruses. The genome contain antigens of spliced small T (STAg), middle T (MTAg) and large T(LTAg), and a noncoding control region (NCCR) separate the late region of structural VP1, VP2, and VP3 proteins. The STAg, LTAg, VP1, VP2, VP3 and MTAg encoded proteins of 194, 776, 384, 347, 228 and 414 amino acids (aa) respectively. Phylogenetic analyses depend on LTAg amino acid sequence revealed that the PyV-HMU to be a relative lineage beside a cluster comprising RnorPyV1(KR065723). Conclusions: The discovery of PyV-HMU expands the geographic range of polyomavirus and will provide further insights into the ecology and evolution of PyVs in rodents and humans. The identification of the novel rodent PyVs will provide basic data for the control of emerging zoonotic infectious diseases.

11

Authors: Huan-huan Zhou, Xin-li Zheng, Tian-ming Ma, Zong-xi Cao, Zhe Chao, Li-min Wei, Quan-wei Liu, Rui-ping Sun, Feng Wang, Yan Zhang, Wei Zhao, Gang Lu

Objective: To investigate the infection rate and genotype distribution of Enterocytoon (E.) bieneusi in farmed black goats from the Hainan Province, China. Methods: A total of 341 fresh fecal samples were collected from black goats farmed in five different locations of the Hainan Province, China. E. bieneusi was examined and genotyped through PCR and sequencing analysis of the internal transcribed spacer (ITS) region of this pathogen. Results: The average prevalence of E. bieneusi in black goats from from the five locations was 24.0% (82/341) ranging from 6.3% (4/63) to 37.2% (32/86) (X2=17.252, P<0.01). The detected 82 E. bieneusi isolates belonged to eight ITS genotypes including six known genotypes (AHG1, CHG2, CHG3, CHG5, CM21 and D) and two novel genotypes (HNG-I and HNG-II). Amongst the genotypes, CHG5 was the the most prevalent with a prevalence of 57.3% (47/82), followed by CHG3 (28.0%, 23/82), CHG2 (4.9%, 4/82), CM21 (3.7%, 3/82), D (2.4%, 2/82), AHG1 (1.2%, 1/82), HNG-I(1.2%, 1/82) and HNG-II(1.2%, 1/82). In those genotypes, only genotype D was found in humans previously. Conclusions: This represents the first report identifying E. bieneusi in black goats from Hainan Province of China. The results indicate that E. bieneusi has a high prevalence and a wide distribution in those animals from Hainan Province, but the risk of zoonotic transmission of E. bieneusi from them to human is low.

13

Authors: Kun Niu, Qian Niu, Yi-qiang Xie, Fan Yang

Objective: To explore the clinical effects of tropical Eucommia ulmoides compound-Rattan Moxibustion liquid external rubbing therapy which is from medicine of Li Nationality in Hainan combined with SET sling exercise therapy technique and lumbar-abdominal muscle training program on treating nonspecific low back pain (NLBP) as well as its influence on serum 5-hydroxytryptamine and beta-endorphin levels. Methods: From June 2016 to June 2018, 42 NLBP patients were admitted to the outpatient and ward of Rehabilitation Department of the First Affiliated Hospital of Hainan Medical College, who were randomly divided into control group (n=21) and experimental group (n=21). On the basis of traditional massage manipulation, the control group adopts lumbar and abdominal muscles training, while Rattan Moxibustion liquid external rubbing therapy combined with SET sling exercise therapy was used in the experimental group. The treatment was given once every other day, three times a week and 20 min each time for 8 weeks. Then ODI index and VAS scale of low back pain, as well as serum 5-HT and beta-EP levels were observed and detected. Results: The improvement in low back pain index ODI index of the experimental group were better than that of the control group (control group from 21.23±1.57 to 4.78±0.86 while experiment group from 21.27±1.36 to 2.75±0.64 after 8 weeks’ treatment)(P<0.05). VAS scale decreased significantly in the experiment group compared with control group (control group from 6.62±1.51 to 2.71±0.92 while experiment group from 6.71±1.55 to 1.83±0.79 after 8 weeks’ treatment)(P<0.05). Serum 5-HT in the experimental group were significantly improved compared with those in the control group [control group from (70.15±20.64) ng/mL to (127.34±40.59) ng/mL while experiment group from (68.72±19.18) ng/mL to (142.65±38.72) ng/mL after treatment](P<0.05) and beta-EP level ameliorates compared with control group [control group from (180.63±51.87) pg/mL to (225.76±57.63) pg/mL while experiment group from (172.39±50.72) pg/mL to (251.26±62.89) pg/mL after treatment](P<0.05). Conclusions: Tropical herb-Rattan Moxibustion liquid external rubbing therapy combined with SET sling exercise therapy technology shows significant effects on NLBP. The mechanism may be related to the improved serum 5-HT and beta-EP levels.

15

Authors: Yong Wang, Ming-ming Zou

Objective: To investigate the adsorption kinetics and thermodynamic characteristics of phenylethanoid glycosides (PGs) in the leaves of Callicarpa nudiflora on macroporous resins and provide a reference for the separation and purification of these compounds. Methods: With adsorption and desorption ratio as indexes, optimum types of macroporous resin for purification of PGs were selected from 8 kinds of macroporous resins by static adsorption and desorption tests, and then adsorption kinetics model and adsorption isotherm model of PGs were established to investigate their adsorption processes. Results: SP-825 and SP-207 resin were selected and they have similar adsorption process for PGs. Both of them showed a fast adsorption in 0-60 min, a slow adsorption in 60-360 min, and an equilibrium adsorption stage after 360 min. Adsorption dynamic behavior was well described by quasi-second-order equation of both SP-825 and SP-207 macroporous resins, and adsorption rate was mainly controlled by liquid film diffusion and intraparticle diffusion. Equilibrium adsorption data fitted Langmuir and Freundlich isotherm equations well. Both of the two kinds of resins showed good adsorption properties for PGs, and the adsorption process belongs to favorable adsorption. Conclusions: Both of the kinetic model and thermodynamic model can well describe the adsorption process of SP-825 and SP-207 macroporous resins and the two resins were regarded as excellent adsorption resins for the purification of PGs from the leaves of Callicarpa nudiflora.

17

Authors: Yang-zhi Peng, Yue Su, Yong Jiang

Background: Chinese herbal medicine have been reported to have less side effects in treatment of depression disorder than Western antidepressants, while the mechanism remains unclear. Our previous studies have shown that combined use of Radix Morinda officinalis and Cortex cinnamomi have antidepressant effects Objective: To explore the mechanism of combined use of Radix Morinda officinalis and Cortex cinnamomi in treating depression disorder. Methods: Sixty SD rats were randomly divided into experimental group, control group, model group and blank group with 15 rats in each group. After establiment of depression models, the experimental group and control group were given the Chinese decoction (2 mL/day) and fluoxetine hydrochloride (2 mL/day) respectively for 3 weeks, meanwhile the model group and blank group were fed with normal saline (2 mL/day). Body weight measurement and sucrose preference test were performed regularly. Finally, the rats were sacrificed after treatment, and the hippocampus were taken to detect 5-hydroxytryptamine, norepinephrine, and dopamine contents. Results: The experimental group showed increased body weight and sucrose consumption than the other groups. Higher 5-hydroxytryptamine, norepinephrine and dopamine contents were also observed in the experimental group than other groups. Conclusions: The antidepressant effects of Radix Morinda officinalis and Cortex cinnamomi decoction may show antiexpression effects by up-regulating content of 5-HT, NE, and DA in rats’ hippocampus.

19

Authors: Qiu-juan Lu, Bing Zhang , Li Cao

Objective: To understand the current status of AIDS epidemic among men who had sex with men (MSM) in Haikou, and to analyze the influencing factors to assess the risk of HIV/AIDS infection in MSM group, and then provide a theoretical basis for MSM population to prevent HIV transmission. Methods: Snowball sampling method was used to find men who had homosexual intercourse in oral sex or anal sex in the past year and conducted a questionnaire survey of them. Blood samples were collected to detect three antibodies against: HIV, syphilis and hepatitis C (HCV). Excel 2007 and SPSS22.0 software were used for statistical description, 2 test and logistic regression analysis. Results: A total of 408 MSMs were investigated in this study, 27 HIV infections were detected, with a positive rate of 6.6%. Univariate analysis showing there were statistically significant differences in the prevalence of AIDS in ethnic minorities, local residence time, condom use for same-sex anal sex in the past 6 months, people with sexually transmitted diseases in recent one year, syphilis, and gonorrhea (P<0.05). Logistic regression analysis showed that minority nationality and HIV infection sexually transmitted disease in the past year were risk factors for HIV infection, and condom use for same-sex anal sex in the past 6 months, the opposite sex occurred in the last 6 months and high education level are protective factors for HIV infection. Conclusion: The HIV infection rate of the MSM population in Haikou is high. It is necessary to increase the use of condoms in the MSM population. For low-education MSM, it is necessary to strengthen the dissemination and education of AIDS-related knowledge, so as to reduce the spread of AIDS in the MSM population.

21

Authors: Aisha Khan, Qudsia Rasib, Sajida Riaz, Isthiaq Ali, Naveeda Riaz, Muhammad Sohail Afzal, Muhammad Asif Gondal, Haroon Ahmed

Objective: HIV is a worldwide contagious disease and recent emergence in transgenders reflecting the alarming situation in Pakistan. Transgender people are one of the most affected groups by the HIV epidemic and are 49 times more likely to be living with HIV than the general population. HIV is an emerging disease in Pakistan. Transgender people in Pakistan often face legal, economic, social and religious exclusion. Even though research on HIV/AIDS has conducted since several years ago, still we have a little knowledge about HIV in transgender people. This study aims to investigate the current status of HIV infection among Pakistan transgender population. Methods: Overall cases of HIV in 2017 were retrieved from all four provinces of Pakistan by using all available published data. Results: In 2017, totally 66 264 positive HIV cases were reported in transgenders from Punjab (n=52 656), Sindh (n=13 596) and KPK (n=22) provinces of Pakistan. In Sindh province majority of the patients were from Karachi (n=9 123) followed by Hyderabad (n=1 062), Sukkur (n=1 609), Larkana (n=1 130), Mirpurkhas (n=289) and Nawabshah (n=383), respectively while in Khyber Pakhtunkhwa (KPK), the number of HIV/AIDS transgender patients increased up to 450, out of which 22 cases were registered under provincial AIDS control program in 2017. Conclusions: More efforts by international agencies are needed to combat HIV among transgender communities in Pakistan. Government and NGOs should collaborate to find some strategies for control and prevention of HIV. Social and human rights activists should help and encourage them mainly at the point where their legal rights are not respected. Transgender communities and health department should go hand in hand. Initiatives should be taken to link social justice, human rights, health and advocacy to prevent HIV among transgender in near future.

23

Authors: Aisha Khan, Muhammad Sohail Afzal, Muhammad Isthiaq Ali, Naveeda Riaz, Haroon Ahmed

Leishmaniosis is a disease caused by a Leishmania protozoan parasite, which is transmitted by the bite of an infected sand fly. Leishmaniasis is among the most dangerous and neglected tropical diseases. In Pakistan, limited research work has been done yet on this neglected disease. Overall cases of leishmaniasis were retrieved from Khyber Pakhtunkhwa (KPK) and Baluchistan provinces from 2018 to 2019. The major risk factors responsible for these outbreaks were also determined. Recent outbreak resulted in more than 28 000 cases of leishmaniasis in KPK from November 2018 to April 2019. Many of the cases were children and approximately 7 810 diagnosed patients did not receive treatment with meglumine antimoniate. Leishmaniasis cases were also identified in the tribal areas of Khyber (n=9 378), Mohmand (n=5 373), Kurram (n=525), Orakzai (n=163), and Karak (n=554). In Balochistan, around (n=5 000) cases of leishmaniasis were reported in 2018 from the District of Killa Abdulla. The poor infrastructure and sanitary conditions increase the breeding and resting sites of sandflies. The local population’s poor socio-economic conditions increase access to sandflies and the risk of acquiring leishmaniasis. In Pakistan, leishmaniasis is currently neglected by the health authorities, largely due to a lack of data on the number of cases and the monetary and non-monetary impacts that these cases are having on the country. There is a dire need for outreach activities to provide information to communities about the disease and preventive measures. This is a call to the national and international research communities, and to the WHO, to take notice of the threat of leishmaniasis in Pakistan and to help devise a strategy to address this neglected yet important public health problem.

25

Authors: Aisha Khan, Sadia Selim Khan, Muhammad Sohail Afzal, Muhammad Asif Gondal, Muhammad Isthiaq Ali, Naveeda Riaz, Majid Mehmood, Haroon Ahmed

reported in Tanzania. The symptoms of chikungunya are joint pain and fever. In Pakistan, cases were reported from different areas especially from Karachi. Many other cases of chikungunya were reported from Rawalpindi and Islamabad. Chikungunya cases were recorded from published data from Dec 2016 to September 2018 in different geographic regions of Pakistan. Chikungunya cases are being reported from many areas of Pakistan, but it is most common in Sindh. A total of 766 cases of chikungunya have been reported from all over Pakistan from Dec 2016 to September 2018. The prevalence of disease varied in different areas of Pakistan: FATA is 0%, Islamabad 4%, GB 0%, AJK 0%, Punjab 8%, Sindh 62%, KPK 22% and Baluchistan 4%. Mosquito breeding sites to human tenancy cause significant risk factor for dengue fever and chikungunya. There is no comprehensive treatment available of chikungunya in Pakistan. Generally speaking, antiviral drugs are administered during viral attack, but there is no antiviral drug for this dangerous disease. The WHO responds to chikungunya by providing guidance, technical support to many underdeveloped countries so they can effectively manage cases of chikungunya.

27

Authors: Tian-le Tang, Yi-ji Li, Wang-yuan Wei, Jia-gang Guo

Dengue fever is one of the most serious mosquito-borne infectious diseases in the tropical world, causing substantial human morbidity and mortality, yet there is no specific treatment till now, and the main measures preventing dengue fever include applying pesticide to kill adult vector mosquitos and removing standing water to prevent the breeding of mosquitoes etc. Appropriate uses of such approaches can reduce dengue epidemic risk via reducing mosquito density. In fact, vector control using insecticides is the most common method to prevent and control mosquito-borne disease transmission. Nevertheless, recent research has shown that Aedes albopictus and Aedes aegypti, the two key vectors for dengue fever transmission, have rapidly developed high resistance to the most commonly used adult insecticide pyrethroid (deltamethrin and permethrin) and larvicide organophosphate (temephos). Therefore, it is of great significance to obtain a new drug with longer-term efficacy to prevent dengue virus transmission. Pesticides currently used act on adult stage of mosquitoes, typically applying to the environment where mosquito bites (on humans) occur, and very few target on egg or juvenile stages of the mosquitoes in the water environment. Calcium cyanamide (CaCN2), widely used as a fertilizer in many different settings, was recently shown to have potential to prevent tropical diseases by eliminating or inhibiting the growth and development of intermediate hosts. The main compounds of calcium cyanamide (CaCN2) are CaCN2 (50%- 60%) and calcium oxide (15%). The representative products of calcium cyanamide (CaCN2) are PERLKA® from Germany and ROEBON® from China. CaCN2 has recently been recognized to have the fungicidal effect on the pathogens of the soil borne diseases and addition of CaCN2 could benefit the thermophilic phase and the composting could quickly reach the sanitary standard and will contribute to solve the feces disposal problems. According to our team's long-term investigation and research, we found that ROEBON® (50% CaCN2) can be used to eliminate Oncomelania and effectively control the spread of schistosomiasis. In the latest research, we have confirmed that 10 mg/L CaCN2 (20 mg/L ROEBON®) can completely inhibit the emergence of mosquito larvae and the population growth from the perspective of inhibiting development and the efficacy can lasts for three months. Calcium cyanamide is a good candidate drug to control mosquito-borne infectious diseases transmission, especiallly for dengue fever prevention. In 2014, Scientific Committee on Health and Environmental Risks in European Commission promoted preliminary opinion on potential risks to human health and the environment from the utilization of calcium cyanamide as fertilizer, but convincing evidences for the causal connections between the exposure to CaCN2 and human health damages are missed. Given a 96-h acute zebrafish toxicity study, the No Observed Effect Concentration (NOEC) of calcium cyanamide to zebrafish was 100 mg/L CaCN2. In summary, most of the data are based on the evaluation of the effect of CaCN2 as a fertilizer, and the related research as a mosquito inhibitor is still very scarce. The research on toxicity of CaCN2 in mosquitoes needs to be further strengthened.

29

Authors: Li-hua Li, Jing-chen Xie, Jin-Long Chen, Lang-yu Rao, Li-xian Wu, Hui Huang, Gang Lu

Objective: To describe a case of Strongyloides (S.) stercoralis infestation with severe lung involvement in a patient who had pemphigus and treated with prednisone and to analyze the molecular characteristics of the S. stercoralis isolate. Methods: A 54-year-old Chinese man living in rural area of the Hainan Province, China. He had a 1-year history of pemphigus and took prednisone for more than six month. He was admitted to the hospital with a 1-week history of abdominal pain, nausea and vomiting. Two days before admission, he presented with cough, expectoration, and shortness of breath after activity accompanied by fever. Computed tomography (CT) of the chest was performed at different stage of admission. Gastric juice, sputum and stool sample smears were examined under light microscope. The larvae of Strongyloides were found from gastric juice, sputum and stool samples of the patient and the genomic DNA of the larvaes was extracted. PCR amplification of the cytochrome c oxidase subunit 1 (cox1) gene was carried out for the DNA sample and the products were sequenced. Results: CT of the chest on day 1 of admission showed inflammation in the tongue segment of the upper lobe of the left lung and the lower lobe of both lungs and the subsequent CT of the chest revealed extensive bilateral pulmonary infiltrates. PCR amplification and sequencing analysis of the Strongyloides larvae directly colleted from the gastric juice, sputum and stool samples on day 10 of admission showed that 100% homology with the sequence of S. stercoralis isolated in a human from Iran (GenBank No. MG995852). Clinical and imageologicalimprovement was achieved after treatement of albendazole (600 mg/day). Conclusions: This is the first case report of pulmonary S. stercoralis infection in a patient with pemphigus treated with prednisone in Hainan Province of China. We suggested that strongyloidiasis should be routinely investigated in patients presenting with nausea, vomiting, diarrhea, cough or expectoration which were accompanied by fever, especially for patients with low immune function or immune deficiency.

31

Authors: Yu-yan Wu, Qin-mei Liu, Jin-na Wang, Tian-qi Li, Zhen-yu Gong, Juan Hou

Objective: Monitoring the density of Aedes(Ae.) albopictus was very important in evaluating the efficiency of vector management, especially when Aedes-borne diseases break, like Dengue fever, Chikungunya fever and Zika. Human landing catch (HLC) is regarded as the “gold standard” for Aedes monitoring, but it is unsafe and unethical since it potentially expose field professionals to a series of pathogens. In this study, we compared HLC with human-baited double net (HDN) and BG trap for field Aedes albopictus monitoring.The study aims to find an effective and safe alternative method to HLC in monitoring Ae. albopictus, especially in emergency monitoring. Methods: Latin square design was used, and three sites in Hangzhou city, Zhejiang province, China, were chosen to conduct outdoor HLC, HDN and BG trap catches in June. The tests were performed at three periods: a morning period (8:30-10:00), an afternoon period (15:00-16:30), and an evening period (16:30-18:00). Then a table with 81 elements was made to compare the efficiency of these three methods. Results: A total of 80, 138 and 78 adult Ae. albopitus was captured by HDN, BG trap and HLC, respectively. Among these three catches, BG trap showed the best efficacy in captured Ae. albopitus adults. The mosquitoes caught by HLC and BG trap were nearly the same at all three time series, but the density of mosquitoes caught by HDN at 16:30-18:00 was 2 times the density at 8:30-10:00 (4.44 vs 2.22 per hour). In this study, significant positive correlation between HLC and BG trap of Aedes albopitus female density was found (r=0.921, P<0.001). However, no statistical significant correlation between HLC and HDN, BG trap and HDN of Ae. albopitus female density was found (r=-0.46, P=0.820; r=0.019, P=0.923, respectively). Besides, the human-bait attraction bias of HLC catches might be more apparent than BG trap and HDN catches. Conclusions: According to our study, with high efficiency, less human-bait attraction bias, and significantly positive correlation with HLC in catching Ae. albopitus females, BG trap could be a sensitive and safer alternative to HLC for outdoor Ae. albopitus monitoring. It also showed strong potential in emergency monitoring when Aedesborne diseases break.

33

Authors: Gao-yu Wang, Li-yuan Zhang, Shu Wen, Wen-qi Wang, Xiao-yuan Hu, Ruo-yan Peng, Yi Huang, Zhang-li Lin, Kun-liang Wu, Gang Lu, Jasper Fuk-woo Chan, Long Sun, Kwok-yung Yuen, Yong-guo Du, Fei-fei Yin

Objective: To identify the epidemiology and clinical features of scrub typhus in Hainan Province, China. Methods: A total of 258 patients with acute febrile illness were recruited from four hospitals in Hainan between 1st June 2018 and 31th August 2019. Orientia tsutsugamushi-specific IgM/IgG and DNA were detected by goldimmuno- chromatographic assay and PCR on the patients’ serum samples. The clinical and epidemiological characteristics of the laboratory-confirmed cases were recorded using a predesigned database. Results: The positive rates of IgM and IgG were 22.5% (58/258) and 39.9% (103/258), respectively, and the positive rate of PCR was 19.37% (50/258). There were 55 males and 48 females among the 103 IgG-positive patients. Patients of all age groups were affected (10 to 86 years); 65.0% of the adults aged 41 to 70 years (67/103); 64.1% of the patients were farmers (66/103). The patients resided in 12 counties and cities of Hainan, and the highest number of cases being reported among residents in Qiongzhong County and Haikou City (54/103, 52.4%). Seasonal variations in the incidence rate of scrub typhus was observed, with the peak incidence (53/103, 51.5%) occurring in the summer (June to August). The common presenting symptoms were fever (103/103, 100.0%), eschar or skin ulcer (38/103, 36.9%), and chills and rigors (30/103, 29.1%). The major complications were liver dysfunction (9/103, 8.7%). Four genotypes of Orientia tsutsugamushi were identified among these patients, including Karp, Kato, Gilliam and Ta763. The predominant genotype was Karp (33/51, 64.7%). Conclusions: Scrub typhus is endemic in Hainan, which predominantly affects residents with prolonged outdoor activities such as farming.

35

Authors: Xi-wen Liu, Ling Wu, Xiao-dan Wang, Wei-ling Xu

Objective: To investigate suicide status and influence of medical students in Hainan, and analyze the direct and indirect effects of influencing factors on suicide ideation among college students based on the structural equation mode1. Methods: By using multi-stage sampling method, we selected 696 students of 11 majors of Hainan Medical College as the research subjects, by questionnaire, simplified coping style questionnaire, Adolescent Self-Rating Life Events Scale (ASLEC), self-rating depression scale (SDS), social support rating scale, the self-esteem inventory and the Self-rating Idea of Suicide Scale(SIOSS) was conducted. Results: The idea of suicide in Hainan Medical College students occurred in 15.4%, and suicide act ideation rate was higher in male than in female (χ2=7.161, P=0.006), non-medical higher medical (χ2=6.004, P=0.014). The goodness of fit for the structural equation model was satisfactory and major indices had met corresponding requirements. The depression was a direct influencing factor on suicide ideation, while three factors including social support, positive coping style and passive coping style had indirect impacts.Negative life event and selfesteem not only directly affected suicide ideation, but also had indirect effects.According to the percentages of their contribution, the risk factors were ranked as follows: depression (45.6%), negative life event (33.6%) and passive coping style (18.5%). Similarly, the top protective factors were self-esteem (37.0%), followed by social support (28.4%) and positive coping style (14.3%). Conclusion: The higher incidence of suicide idea of students at the Hainan Medical College, depression and selfesteem were the main factors of suicide ideation. So, it is of great importance to not only strengthen the mental health of college students, but also to train students to face up the difficulties with a positive style, and provide the college students with sufficient social supports.

37

Authors: Jie-ying Wang, Zi-hua Tong, Chun-chun Yu, Yu-cheng Li, Yan-qun Xie, Hua Pei

Objective: To preliminarily investigate the infection status of the human immunodeficiency virus (HIV), hepatitis B virus (HBV), treponema pallidum and hepatitis C virus (HCV) in pregnant women in Hainan Province, so as to provide evidence for effective prevention and treatment of mother-to-child transmission. Methods: From October 2017 to July 2019, 8 970 pregnant women who came to our hospital for outpatient examination during pregnancy were tested for HIV, treponema pallidum and HCV antibody by ELISA and tested for rapid detection of HBV surface antigen in serum by gold immune-chromatographic assay. Results: The positive rate of HIV, treponema pallidum and HCV antibody was 0.01% (1/8 970), 0.65% (58/8 970) and 0.11% (10/8 970), respectively, and the positive rate of HBV surface antigen was 11.13% (998/8 970). Conclusions: Among the 8 970 pregnant women, the HBV infection rate was the highest (11.13%), significantly higher than that of the other three infectious diseases, followed by the treponema pallidum and HCV positive rate, and the HIV positive rate was the lowest. Therefore, it is suggested that pregnant women should strengthen prenatal examinations to improve the awareness of prevention of infectious diseases, which is conducive to prepotency. Meanwhile, it is urgent to improve the screening and treatment for epidemic infectious disease in Haikou.

39

Authors: Gang Lu, Wei Zhao, Huan-huan Zhou, Mei-fang Xiao, Yong-juan Yu, Hai-fen Nong, Wei Xiang

Objective: To report a case of aural myiasis in an infant and molecular identification of larva species. Methods: A larva was extricated from left external auditory canal of an infant in the Neonatal Department of the People’s Hospital of Baisha County, Hainan Province on 25 June 2019. The larva was placed in 70% alcohol and sent to the Department of Pathogenic Biology, Hainan Medical University. The morphological characters of larva was observed under stereoscopic microscope. The genomic DNA of the larva was extracted and amplified by PCR targeting mtDNA cytochrome c oxidase subunit 1 gene of Diptera, then the PCR product was sequenced and analyzed. Results: A 5-day-old male infant with neonatal hyperbilirubinemia became suddenly irritable. Physical examination found bleeding of the left external auditory canal. A larva about 1.0 cm×0.2 cm was found in the left external auditory canal when the left ear was cleaned with saline. The infant was transferred to Hainan Women and Children’s Medical Center for further examination. Otoscopic examination found that the right external auditory canal was normal, with a small amount of yellow secretion and intact tympanic membrane. However, the left ear canal was slightly swollen, with a small amount of yellow secretion and no obvious perforation of the tympanic membrane. The child was discharged after four days of treatment with appropriate medication. Morphological characteristics observed by stereoscopic microscope implied that larva was the maggot of Sarcophaga sp. PCR amplification and sequencing analyses confirmed that the larva was Sarcophaga (S.) peregrina. The sequence exhibited 100% homology with the S. peregrina c oxidase subunit 1 gene (GenBank No. AF259509.1). Conclusions: This is the first report of an infant with aural myiasis in Hainan Province. The molecular characteristics suggest that the aural myiasis was caused by S. peregrina. One of the factors causing infant arual myasis might be residual amniotic fluid in the external auditory canal of the newborn.

41

Authors: Sadia Selim Khan, Aisha Khan, Nazeer Hussain, Muhammad Sohail Afzal, Muhammad Asif Gondal, Haroon Ahmed

Theileriosis is a disease caused by protozoan parasites which belong to genus Theileria. It can be transmitted through ixodid ticks. Mostly the distribution of Theileria species are restricted to subtropical and tropical regions. Theileria species cause economically significant diseases in cattle, sheep, and goats in Pakistan by infecting domestic and wild ruminants. This is a neglected disease in Pakistan because limited research work has been done yet on it. Ovine theileriosis cases were recorded from published data from 2013-2016 in different geographic regions of Pakistan. The intensity and risk factors were also determined in KPK Province of Pakistan. Theleriosis have been reported from all over Pakistan during period 2013-2016. The prevalence of Theleria annulata in buffaloes ticks was 72.91% (875) in urban regions of Hyderabad and it was 80.8% (970) in periurban regions during period 2013-2014. In 2016, prevalence of ovine theileriosis in KPK districts was as 27.5% in Dera Ismail Khan, 13% in Tank and 19.5% in Bannu. It is concluded that paying close attention to animal keeping, feeding and housing may diminish the occurrence of theileriosis in sheep. The higher prevalence might be due to lack of awareness by the community, farming system of the rural livestock of, absence of diagnostic facilities, expensive acaricidal drugs and poor economic condition of farmers.

43

Authors: Sha-sha Yu, Jie Qin, Qiu-xia Li, Pan Wang, Jing Wang, Ting-ting Liu, Ying Wang

Objective: To investigate the effect of sublethal dose of Bacillus sphaericus (Bs) 2 362 strain on the malaria transmission ability of Anopheles (An.) dirus (Hainan strain) and its molecular mechanism. Methods: Firstly, the fourth instar larvae of An. dirus was treated with sublethal dose of Bacillus sphaericus. The surviving larvae were then collected and placed into mosquito cages, where they were hatched into adult mosquitoes. These mosquitoes were recorded as Bs group. Meanwhile, the control group was set without any treatment. Then, for infection with Plasmodium yoelii BY265 RFP, 3- to 5-day old female adults were kept at 24 ℃ and fed on Plasmodium yoelii BY265 RFP-infected Kunming mice with a gametocytemia above 0.5%. On day 9-11 post infection, mosquitoes were dissected, and the oocysts on the midguts were examined under a fluorescence microscope. Thirdly, total RNA was extracted from mosquitoes of Bs group and the control group at different time-points respectively, and the cDNA were synthesized later. Finally, SYBR quantitative PCR was conducted to investigate the expression of Imd pathway anti-malaria molecules at different time-points, including TEP1 and Rel2, in Bs and control group mosquitoes. Results: Bs treatment remarkably reduced the infection rate of Plasmodium from 23.71% (124/523) to 16.23% (87/536) (Chi-square test, P=0.002<0.05), and the intensity of Plasmodium infection was greatly decreased in Bs group than that of the control group, either (Mann-Whitney Rank Sum Test, P<=0.001). However, the rate of melanization mosquitoes was 4.97% (26/523) in the control group and 5.78% (31/536) in Bs group, respectively; no significant difference was detected between them (Chi-square test, P=0.558> 0.05). Additionally, the intensities of melanized oocysts were compared between the two groups, and no significant difference was found, either (P=0.566>0.05). Interestingly, compared with the control group, the expression levels of TEP1 and Rel2 in Bs group were obviously up-regulated in larval, adult and infected mosquitoes. Especially in 3 dpi and 7 dpi, the expression level of TEP1 in Bs group was nearly 4 times higher than that of the control group, while Rel2 reached to approximately 7 times. Conclusions: We firstly found that the sublethal dose of Bs significantly suppressed the vector competence of An. dirus to malaria parasites, which revealed a new important role of Bs on the basis of killing mosquito larvae. Furthermore, the Imd signaling pathway might play an effective way in Bs impacting the vector competence of An. dirus through upregulating the expression of NF-kB transcription factor Rel2, enhancing the expression of TEP1, which killed the Plasmodium, but not through melanization.

385-395

Authors: Supriya Jagga, Ashish Ranjan Sharma, Chiranjib Chakraborty, Sang-Soo Lee

Ebola virus disease (EVD) is associated with haemorrhagic fever in humans and nonhuman primates, with a high rate of fatality (up to 90%). Some outbreaks in human history have proven the lethality of EVD. The recent epidemic of 2014 and 2015 in West Africa was the deadliest of all time (11 284 deaths). To understand the transmission dynamics, we have reviewed the epidemiology of EVD to date. The absence of any licensed vaccines or approved drugs against Ebola virus (EBOV) further highlights the severity and crisis level of EVD. Some organizations (public and private) are making considerable efforts to develop novel therapeutic approaches or vaccines to contain the outbreak of EBOV shortly. Here, we summarized the various potential drugs and vaccines (undergoing multiple phases of clinical trials) that have arisen as an alternative against EBOV, and we highlighted the numerous issues and limitations hindering this process. Alternatively, an increasing focus on strengthening the medical and civic health structure could provide speedy benefits in containing the spread of EVD, as well as offer a resilient foundation for the deployment of novel drugs and vaccines to the affected countries, once such drugs and vaccines become available.

404-408

Authors: Cam Nhat Nguyen, Quynh Ngoc Nguyen, Than Huu Dao, Le Thi Quynh Mai

Objective: To determine the concentration and rate of decay of maternal IgG antibodies against measles prevalence in infants of vaccinated or naturally infected mothers and study initial measles immunization occurs in nine-month-old children. Methods: In total, 401 pregnant women and the same number of their subsequent newborns were recruited in the Bavi district of Hanoi in 2016-2017; they were divided into two groups: Older women (born before 1985, n=201) and younger women (born after 1990, n=200). Samples were collected at five time-points; week 36 of pregnancy, birth (cord), and 3, 6, and 9 months after birth. Measles-specific IgG antibody levels were recorded. Results: In total, 77.06% of the 401 pregnant women were seropositive for measles-specific IgG antibodies. A significantly greater proportion of mothers aged 30 and older (88.06%) and their newborn ( 93.53%) were seropositive compared to the mothers aged 25 and younger (66.00%), and their newborn (72.00%) (P<0.001). The infants of older mothers had significantly higher geometric mean titres (GMT) of measles IgG antibodies than the infants of younger mothers (P<0.001) at all time-points of the study period . The proportion of measles IgG antibodies together with GMT decreased from 82.97% (506.96) at the age of three months to 23.19% (45.22) at the age of nine months. Conclusions: This study provides a profile of maternal antibodies against measles in Vietnamese infants and investigates the early susceptibility to measles in both the infants of vaccinated mothers and mothers with naturally acquired immunity. These data suggest that determining the appropriate age for measles vaccination is paramount for the elimination of measles in Vietnam.

416-424

Authors: Sudipta Jena, Asit Ray, Diptirani Rath, Ambika Sahoo, Subhashree Singh, Noohi Nasim, Durga Madhab Kar, Sanghamitra Nayak

Objective: To determine the antioxidant and hepatoprotective potential of methanol extract of rhizome of Curcuma angustifolia (MECA) against carbon tetrachloride (CCl4)-induced hepatic damage in vitro and in vivo. Methods: DPPH, ABTS and reducing power assays were performed to estimate the antioxidant effect of MECA. In vitro cytotoxicity of MECA against HepG2 cells was evaluated, whereas serum biochemical parameters and levels of antioxidative enzymes were measured in vivo and in vitro. Additionally, histopathological studies were estimated in order to investigate the hepatoprotective efficacy of MECA. Furthermore, GC-MS analysis of the extract was performed to identify the chemical components. Results: MECA exhibited strong antioxidant activity and attenuated CCl4-induced decrease in the viability of HepG2 cells. Additionally, MECA significantly restored the ALT, AST, ALP, TP and albumin level in comparison with the CCl4 group. After pre-treatment with MECA, effects of SOD, CAT and GSH were increased as well as lipid peroxidation amount decreased on CCl4-induced hepatotoxicity in in vitro and in vivo model. Furthermore, histopathological observation confirmed that MECA reduced liver injury induced by CCl4 in rats. GC-MS analysis confirmed the presence of bioactive constituents such as α-tocopherol (12.27%), phytol (7.61%), squalene (3.71%), β-sitosterol (2.19%), eugenol (2.59%), curcumenol (1.20%), β-elemene (1.00%) and eucalyptol (0.89%). Conclusions: MECA contains antioxidant and hepatoprotective constituents such as α-tocopherol, phytol, squalene and eugenol and exerts hepatoprotective effect both in vitro and in vivo.

339-346

Authors: Sravani Karri, Sanjay Sharma, Ginpreet Kaur

Malaria remains a major tropical health burden owing to the development of resistance and decreased sensitivity to the frequently used conventional antimalarial drugs. The drug like artemisinin possesses potent antimalarial activities, but has some limitations. Therefore, new strategies are to be implemented for optimal utilization of artemisinin to improve its therapeutic effectiveness and to overcome its limitations. The present review focuses on present scenario of malaria and pharmacological as well as analytical aspects of artemisinin. Data from 2000 to 2018 were collected from NCBI for understanding the various analytical techniques used for estimation of artemisinin. This review will reveal the facts about artemisinin which can be utilized to develop novel drug delivery system either in a combination or as alone for the wellbeing of the patients suffering from malaria.

353-364

Authors: Emrah Sefik Abamor, Adil Allahverdiyev, Ozlem Ayse Tosyali, Melahat Bagirova, Tayfun Acar, Zeynep Mustafaeva, Serap Derman

Objective: To prepare and characterize poly lactic-co-glycolic acid (PLGA) nanoparticles loaded with soluble leishmanial antigen or autoclaved leishmanial antigen and explore in vitro and in vivo immunogenicity of antigen encapsulated nanoparticles. Methods: Water/oil/water double emulsion technique was employed to synthesize PLGA nanoparticles, and scanning electron microscopy, Fourier transform infrared spectroscopy and Zeta-potential measurements were used to identify the characteristics of nanoparticles. Cytotoxicity of synthetized nanoparticles on J774 macrophage were investigated by MTT assays. To determine the in vitro immunostimulatory efficacies of nanoparticles, griess reaction and ELISA was used to measure the amounts of NO and cytokines. During the in vivo analysis, Balb/c mice were immunized with vaccine formulations, and protective properties of nanoparticles were measured by Leishman Donovan unit in the liver following the infection. Cytokine levels in spleens of mice were determined by ELISA. Results: MTT assay showed that neither soluble leishmanial antigen nor autoclaved leishmanial antigen encapsulated nanoparticles showed cytotoxicity against J774 macrophage cells. Contrary to free antigens, both autoclaved leishmanial antigen-nanoparticle and soluble leishmanial antigen-nanoparticle formulations led to a 10 and 16-fold increase in NO amounts by macrophages, respectively. Leishman Donovan unit calculations revealed that soluble leishmanial antigen-nanoparticles and autoclaved leishmanial antigen-nanoparticles yielded 52% and 64% protection against visceral leishmaniasis in mouse models. Besides, in vitro and in vivo tests demonstrated that by increasing IFN-γ and IL-12 levels and inhibiting IL-4 and IL- 10 secretions, autoclaved leishmanial antigen-nanoparticles and soluble leishmanial antigennanoparticles triggered Th1 immune response. Conclusions: Both autoclaved leishmanial antigen-nanoparticles and soluble leishmanial antigen-nanoparticles formulations provide exceptional in vitro and in vivo immunostimulatory activities. Hence, PLGA-based antigen delivery systems are recommended as potential vaccine candidates against visceral leishmaniasis.

375-379

Authors: Parut Suksai, Supakarn Kaewchot, Piya Sereerak, Sirinan Boonnan, Bongkotchamat Phimsin, Taksina Jaruwattananon, Kacharin Raschasin, Mutchamon Kaewparuehaschai, Sorraya Siriphet, Benjaporn Bhusri

Objective: To survey hemoplasmas infection in free ranging non-human primates from 8 provinces in Thailand. Methods: DNA from ethylenediaminetetraacetic acid blood of 262 free ranging non-human primates were identified as hemoplasmas using PCR and phylogenetic analysis based on 16S rRNA and rnpB genes. Results: A total of 148 non-human primates (56.49%) were determined positive for Candidatus Mycoplasma haemomacaque, including 125 Macaca fascicularis and 23 Macaca mulatta. Hemoplasmas can cause anemia in monkey but all positive samples were healthy. The positive rates in male and female non-human primates were not significantly different. Conclusions: Candidatus Mycoplasma infection is prevalent in free ranging Macaca fascicularis and Macaca mulatta in Thailand.

291-299

Authors: Kiran D. Patil, Shashikant B. Bagade, Sanjay R. Sharma, Ketan V. Hatwar

The WHO reports that billions of people and animals in tropical and subtropical regions are affected by helminthiasis as neglected tropical disease. It is predominant in underdeveloped areas; nevertheless, the increase in the number of travelers and migrants has made this infection more common. The current mass drug treatment produces severe side effects and many strains of helminths are resistant to them. None of the chemotherapeutic drugs meets the ideal requirements of anthelmintics, such as broad spectrum of activity, single dose cure, free from side effect and cost-effectiveness. Today, many researchers are screening the traditional herbal system in search of the anthelmintic herbal constituents which overcome all the problems of synthetic drugs. Several researchers proclaim anthelmintic activity of herbal medicines by using different experimental models. The present review demonstrates natural product drug discovery, outlining potential of herbal constituents from natural sources as natural leads against helminthiasis.

306-314

Authors: Hamed Behniafar1, Vahideh Moin-Vaziri2, Mehdi Mohebali3,4, Seyyed Javad Seyyed Tabaei2, Zabih Zarei5, Elham Kazemirad3, Behnaz Akhoundi4, Mohammad Kazem Saharifi-Yazdi6, Alireza Zahraei-Ramazani

Objective: To clarify the epidemiological aspects of visceral leishmaniasis in Kaleybar and Khoda-Afarin districts, north-west of Iran. Methods: A total of 1 420 human (children under 12 years) samples, 101 domestic dogs samples (Canis familiaris), and 577 female sand fly samples were collected. Sera of human and dogs were tested using the direct agglutination test, and sand flies were identified at species level using the microscopic method. Furthermore, a structured questionnaire was applied to evaluate the correlation between the potential risk factors and the related clinical signs/ symptoms with the human and dogs' seropositivity. Results: Totally, 2.18% of human samples were positive at titers≥1: 800; among them, 13 cases (41.94%) were above 1:3 200, and clinical symptoms were observed in all of them except for an 11-year old girl. Anti-Leishmania infantum antibodies were found at titer≥1: 320 in 9.90% of dogs' samples, half of them had at least one sign of canine visceral leishmaniasis. Moreover, 10 Phlebotomus species were identified in the study areas, and Phlebotomus (Larroussius) major group was the predominant species. There are significant correlations between the presence of anti-Leishmania infantum antibodies and the fever (P<0.001), anemia (P=0.001) and weight loss (P=0.016) in children. On the other hand, significant correlations were revealed between the Leishmania infection and the shelter (P=0.039), cutaneous lesion (P=0.005), lymphadenopathy (P=0.001) and weight loss (P<0.001) in the infected dogs. Conclusions: Visceral Leishmania infection is prevalent in rural areas of Kaleybar and Khoda- Afar districts located in East-Azerbaijan province, therefore active detection and treatment of visceral leishmaniasis cases should not be neglected.

321-328

Authors: Raju Sangeetha, Tamilselvan Pratheeba, Chinnasamy Ragavendran, Devarajan Natarajan

Objective: To test the mosquitocidal potential of leaf extracts of Pouteria campechiana prepared with different solvents and elucidate the structure of an isolated mosquitocidal compound. Methods: The leaf extracts of Pouteria campechiana prepared with three solvents (petroleum benzene, ethyl acetate and acetone) and potential bioactive fractions were tested against various stages of Aedes aegypti and Culex quinquefasciatus by using the WHO protocols, and the chemical profile and its functional groups were identified by GC-MS and Fourier transmission- infrared spectroscopy (FT-IR). The structure of bioactive compound was characterized by nuclear magnetic resonance (NMR) spectral technique. Results: The preliminary phytochemical results revealed the presence of alkaloids, amino acids, flavonoids, quinones, saponins, steroids, tannins, and terpenoids in the acetone extract. A significant toxic potential was observed in the acetone extract against both Aedes aegypti and Culex quinquefasciatus mosquitoes. The acetone extract exhibits remarkable larvicidal (LC50: 12.232 μg/mL and LC90: 63.970 μg/mL), pupicidal (LC50: 18.949 μg/mL and LC90: 167.669 μg/mL) and adulticidal (LC50: 20.689 μg/mL and LC90: 72.881 μg/mL) effects against Aedes aegypti. Furthermore, the same extract was subjected to isolation of bioactive compound by GC- MS and FT-IR analysis. GC-MS results showed the presence of 5 major compounds, and octacosane (18.440%) was detected as the predominant compound. The FT-IR result of acetone extract demonstrated the presence of various functional groups like alkanes/alkynes, ester, aromatic and amides. The NMR spectrum results of isolated compound were well matched to glycoside linked flavonoids. Based on the chromatography and spectral techniques the isolate molecule was identified as myricitrin by FT-IR and nuclear magnetic resonance spectral data. Conclusion: The isolated compound myricitrin possesses a significant toxic effect in all stages of Aedes aegypti and Culex quinquefasciatus mosquito’s with lowest LC50 and LC90 values.

337-338

Authors: Ravindra Kumar Garg, Neeraj Kumar, Imran Rizvi, Hardeep Singh Malhotra

Rationale: Disseminated cysticercosis is characterized by presence of cysts in multiple body organs, like brain, skin, eyes, muscles and rarely heart and lungs. Patient concerns: A 22-year-old man presented with bilateral proptosis of 1-year duration. He also had two episodes of cysticercosis encephalitis. In the second episode of encephalopathy, the patient died. Diagnosis: Disseminated cysticercosis. Interventions: Corticosteroids (Initially intravenous dexamethasone 0.4 mg/kg/day for 2 weeks, followed by oral prednisolone 1.0 mg/kg/day). Outcomes: The patient died of cysticercosis encephalitis approximately 2 months later. Lessons: Disseminated cysticercosis in our case presented only with proptosis as he had very heavy infestation of the brain and eyes. Heavy larval infestation in a patient with disseminated cysticercosis can be life-threatening.