Asian Pacific Journal of Tropical Medicine

141-142

Authors: Saurabh Bhargava, Kiran Kumari, R.K. Sarin, Rajvinder Singh

After a prolonged debate and discussion, the World Health Organization (WHO) relisted ‘snakebite’ under Category ‘A’ of ‘Neglected Tropical Diseases’ (NTDs) in June 2017 hoping to diminish its burden on people. Earlier, in March 2009, WHO added snakebite to its official list of NTDs but later downgraded to ‘Other Neglected Conditions’, a list reduced in importance[1,2]. Obviously, the recent inclusion of snakebite in NTDs was due to the estimation of huge toll of deaths due to snake envenomation in tropical countries as supported by the multiple reports. This inclusion will expedite the process of developing better management protocols and infusing funds for such affected countries. However, a significant question here is whether the estimation of snakebite cases is accurate enough to publicise the authentic figures. Snakebite is a problem of resource poor tropical countries which usually lacks a solid central registry system of record keeping. Even when such record keeping is in place, yet people out there may not be aware enough for such measures and they don’t bother to register such cases. Snakebite incidences in such circumstances could definitely affect the national statistical figures of snakebite in any country. Many scientific reports and WHO reports have suggested that India leads the chart of mortality and morbidity caused by snakebites and envenomations contributing almost 50% of world’s burden by snakebites[3,4]. However, true figures remained elusive and the estimates range from 10 000 to 50 000 annual deaths in India. These estimates are usually based on extrapolation of smaller and regional studies. Nationally representative “Million Death Study” conducted by the Registrar General of India projected 45 900 annual deaths in India due to snake envenomation on the basis of statistical data generated from randomly selected 6 671 areas from 28 states and 7 Union territories of India between 2001 and 2003[5]. The WHO in 2005 has quoted similar figures about snake envenomations in India[3]. In 2008, an international study funded by the WHO estimated the global burden of snakebites to be around 1 841 000 snakebite incidences with around 94 000 annual deaths across the world[4]. They reported around 81 000 envenoming and nearly 11 000 annual deaths due to snakebite in India. On the other hand, the National Health Profile (NHP) maintained by the Central Bureau of Health Intelligence, Government of India reported average annual snakebite mortality of just 1 215 across India from 2010 to 2016[6]. This is the data that is quoted by the government for various purposes including policy making. The National Crime Records Bureau (NCRB) working under the Ministry of Home Affairs of Government of India reported an average 8 507 annual deaths due to snake envenomation across India during 2010-2016[7]. Data published by both, NHP and NCRB depict a huge difference in official figures of snakebites and is quite underreported than other scientific epidemiological studies. However, a well designed field survey conducted in Tamil Nadu in India suggested that even 50 000 snake envenomed mortality is underreported, and the actual burden of snake envenomation could be even more. This extensive field survey of 28 494 people from thirty villages in state of Tamil Nadu suggested around 10 000 annual deaths from snake envenomation[8] and it was in contrast with around 3 100 snakebite deaths suggested by nationally representative “Million Death Study” conducted by the Registrar General of India, average 44 annual deaths by NHP reports during 2010-2017, and average 638 annual deaths by NCRB from 2011 to 2015. Similarly, NCRB reported 368 deaths due to snake envenomation during 2011-2015 in the state of Haryana, whereas NHP reported only 32 deaths over same period in same region. However, another independently conducted government hospital based epidemiological profiling of the same region reported at least 130 deaths due snake envenomation in Haryana during 2011-2015[9]. Similar disparity between government records of snakebite and independently conducted surveys have been reported by researchers from West Bengal[10].

152-161

Authors: Banghan Ding, Bangjiang Fang, Jun Li, Qingquan Liu, Chuanzhu Lv, Xuezhong Yu, Xiaodong Zhao

Xiyanping injection, a traditional Chinese medicine injection made of andrographolide sulfonate, consisting of well-defined ingredients with antiviral, antibacterial, anti-inflammatory and antipyretic efficacy, has been widely used for treating infectious diseases of respiratory and digestive systems. However, its wide applications may easily lead to unreasonable clinical medication. In order to guide the precise clinical application and rational use of Xiyanping injection, experts in related fields conducted systematically literature review, evaluated and deliberated the application of Xiyanping injection in treating acute infectious diseases using evidence-based medicine method, and jointly drafted the consensus to summarize types of acute infectious diseases in children and adults that can be treated with Xiyanping injection, and recommend the intervention time, usage and dosage, course of treatment and combined medication of the injection. Besides, the consensus elucidates the safety, precautions and contraindications of the injection, so as to provide guidance for clinical use.

169-175

Authors: Afshar Etemadi, Rezvan Moniri, Mahmood Saffari, Hossein Akbari, Saeed Alamian, Ali Mohammad Behrozikhah

Objective: To determine epidemiological, molecular characterization, and potential risk factors of human brucellosis. Methods: This descriptive study was carried out in the clinical setting in Iran between 2017 and 2018. A total of 297 participants enrolled in the study. The sample size was calculated based on the occurrence rate of brucellosis in different areas. Patients were assessed using serological tests and conventional culture methods. Phage and multiplex PCR methods typed all of Brucella isolates. Potential risk factors of disease were determined. Results: A total of 141 of 297 (47.5%) Brucella strains were isolated and all of them were detected as Brucella melitensis biovar 1. Based on serologic titers, high culture positivity was recorded at 1/640 titer (P<0.006). The risk factors for brucellosis were patients older than 40 years (OR=2.23, 95%CI: 1.4-3.55, P=0.001), animal keeper (OR=7, 95%CI: 1.51-32.41, P=0.005), housewife (OR=8.76, 95%CI: 1.85-41.37, P=0.002), farmer (OR=6.42, 95%CI: 1.21-33.97, P=0.019), and contact with animal (OR=1.31, 95%CI: 0.60-2.85, P=0.005). Conclusions: To the best of our knowledge, this is the first comprehensive report from Iran presenting the detection of Brucella species by the multiplex PCR. Brucella melitensis biovar 1 is still the dominant causative agent in Iran. The consumption of unpasteurized dairy products, living in rural areas, and animal contact were risk factors of brucellosis.

185-186

Authors: Hamid Reza Niazkar, Majid Niazkar

COVID-19 due to infections with the 2019 novel coronavirus (SARS-CoV-2) was first reported in Wuhan, Hubei Province, China in December 2019. Up to 2 March 2020, it has already spread to more than 55 countries, infected more than 85 000 individuals, and yielded to about 3 000 death cases[1]. Due to the vigorous outbreak of COVID-19, it has become a global concern. Further management and controlling of this worldwide threat inevitably relies on the preparedness and precise risk assessment of the future, particularly in countries that new cases have been observed. In this regard, estimation of new probable confirmed and death cases in the near future is crucial to health care systems of each country. As a result, some researches have been conducted to address this new concern[2-4]. In one of the most recent studies focusing on the first month of COVID-19 outbreak in Thailand, Sookaromdee and Wiwanitkit[2] proposed a second-order polynomial function, which relates imported infected cases (I) with time (t). The relation is given as I=2AT2+AT+C, where A is the acceleration rate of disease spreading and C is a constant. This relation was derived by integrating V=AT, where V is the average velocity of the spreading disease[2]. The authors sincerely appreciate Sookaromdee and Wiwanitkit for their valuable contribution. However, several issues are presented in accordance with their mathematical model: First of all, COVID-19 is highly contagious, and infected individuals can spread the disease during the incubation period, which is estimated to be about 2-14 days[5]. Due to the incubation period of COVID-19, imported infected cases may underestimate the outbreak, and may not be a suitable index for prediction purposes. In contrast, a forecasting model based on either total confirmed or death cases may give more accurate estimations. In the suggested model by Sookaromdee and Wiwanitkit[2], it was basically assumed that the spreading acceleration rate of COVID-19 outbreak is constant in respect with time. This assumption may not be compatible with the natural course of the outbreak. According to COVID-19 outbreak reported from 20 January to 24 February 2020 in Thailand[1], the accumulative number of confirmed cases not only has a highly nonlinear relationship with time but also demonstrates a repetitive pattern comprising of a steep rise followed by a plateau, as shown in Figure 1.

97-98

Authors: Folorunso Oludayo Fasina

The year 2020 was welcomed and characterized with a report of pneumonia of unknown etiology, which occurred primarily in Wuhan City of Hubei Province of China. Between 31 December 2019 and 3 January 2020, a total of 44 cases in human was reported to the World Health Organization (WHO) by the Chinese Health Authorities [1] . By 3 February 2020, a total of 17 391 cases have been confirmed globally with 2 838 new cases (China: 17 238; 2 831 new cases and 361 deaths; Outside China: 153 with 7 new cases and 1 death from 23 countries) [1] . Wuhan City of Hubei Province has accounted for approximately 64.8% of all cases from China. Globally, the risk of infection and spread remains high. The pathogen causing the infection has been classified as 2019 novel coronavirus (2019-nCoV). Due to a surge in numbers of cases from the third week of January 2020, and after intense consultations and deliberations by the International Health Regulations (2005)Emergency Committee, the WHO, on 30 January 2020 declared the outbreak of 2019-nCoV a Public Health Emergency of International Concern (PHEIC) [2,3] . Since the date of this declaration, global attention has been focused on 2019-nCoV [4,5] .Preliminary clinical features indicated that most of the infected patients were men with underlying diseases (comorbidities)including diabetes, hypertension, cardiovascular disease, chronic obstructive pulmonary disease, malignancy and chronic liver disease [6] . Patient presented with fever, cough, myalgia, sputum production, headache, haemoptysis, diarrhea and dyspnea [6] . Person-to-person transmission and familiar association have also beenconfirmed [7] . To date, a diagnostic kit has been developed for 2019-nCoV [8] , and efforts are ongoing to develop other protocols. Whilst several important aspects of 2019-nCoV epidemiology, virology, mode of transmission, pathogenesis, diagnosis, clinical features, have been defined, there remain many unanswered questions, including source, transmission and epidemic potential. The Wuhan outbreak is a stark reminder of the continuing threat of zoonotic diseases.Detailed clinical, laboratory and epidemiological findings are available in several studies [3,6,7] , and further works are ongoing to solve the several unknown about the virus (more details on the source, epidemiology, virology, mode of transmission, pathogenesis,diagnosis, clinical features and epidemic potential). These information will be needed to solve many unanswered questions. Specifically, it has been confirmed that many of the patients with ongoing outbreak in humans have linkages with a large seafood and live animal market in Wuhan, Hubei Province [3] . Evidence also suggests that the 2019-nCoV is closely related to coronavirus from bats [9] , a strong indication that the virus may have an animal source(with bats acting as the primary source and the animals available in the seafood market serving as intermediate hosts between human and bats). However, while it is known that humans are the terminal hosts for 2019-nCoV, it still needs further investigations whether this is similar to the situation in Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), where masked palm civet is the intermediate host, and MERS-CoV where dromedary camels have been identified as intermediate hosts [9] . The yet-to-be-identified wild animal(s) may be the intermediate host for the ongoing 2019-nCoV. It therefore becomes important to utilize one health approach in the implementation of investigation and control of the ongoing 2019-nCoV. Recent models have suggested that a lot more cases than those confirmed and reported to the WHO may have occurred [10]. It was also suggested that human-to-human transmissibility is highly likely and the epidemic may spiral out of control without the implementation of effective control measures [11] . In Germany, an asymptomatic contact served as a source of 2019-nCoV transmission to other patients and this has introduced another dimension to the transmission dynamics in the current outbreaks [12] . The health authorities have quarantined several cities in Hubei Province in an unprecedented public health response, yet outbreak continues to be reported in several locations in China, as well as in the Western Pacific, South East Asia, North America, Europe and Middle East where intense import control measures have been implemented. It is important for the infectious disease community globally to draw from the lessons of SARS-CoV, Asian highly pathogenic avian influenza H5N1 (HPAI H5N1), MERS-CoV and other public health emergencies, particularly those that have originated from the South East Asia axis, to combat the current situation in South East Asia and elsewhere. Finally, global efforts continue to target vaccine development, an urgently needed biological that may stem an epidemic. The world will continue to monitor the 2019-nCoV ‘zoonotic’ respiratory diseases as progress is made in global health security.

107-114

Authors: Chulwoo Rhee, Birendra Prasad Gupta, Bibek Kumar Lal, Jacqueline Kyungah Lim, T. Anh Wartel, Julia Lynch, Sushant Sahastrabuddhe

Objective: To assess the extent of existing published evidence on cholera and to characterize the epidemiologic data of cholera in Nepal. Methods: We conducted a literature scoping review by summarizing published literature reporting on cholera in Nepal from January 1946 to March 2019 in online databases: MEDLINE, Embase, Cochrane, and Global Health. Additionally, we reviewed national surveillance data on clinically diagnosed and laboratory confirmed cholera reported by the Ministry of Health and Population. Results: Most of the published studies were conducted predominantly in Kathmandu Valley during the rainy season; however, outbreaks have been reported in other parts of Nepal including Terai, Hilly and Mountain regions. Our literature review exhibited that all age groups were affected by cholera, but particularly children and young adults were at-risk age groups in Nepal. Vibrio cholerae serogroup O1, biotype El Tor, serotype Ogawa has been predominantly isolated with an emergence of resistant strains since 1996. Two mass vaccination campaigns using oral cholera vaccines were conducted: Rautahat district in 2014 and Banke district in 2017. Conclusions: Capacity building for a nation wide systematic cholera surveillance with rapid and reliable diagnosis is needed to better estimate the burden of cholera and identify geographically at-risk areas associated with the disease in Nepal. It is essential for developing an adequate policy on oral cholera vaccine introduction and effective water, sanitation and hygiene interventions.

123-130

Authors: Kawin Wongthamarin, Thanaporn Trairattanapa, Sirakit Kijanukul, Thanakrit Kritsilpe, Sarun Poobunjirdkul, Warit Chuengdee, Mathirut Mungthin, Saovanee Leelayoova, Tawee Naaglor, Paanjit Taamasri, Picha Suwannahitatorn, Toon Ruang-Areerate, Phunlerd Piyaraj

Objective: To investigate the incidence, spontaneous clearance, persistence and risk factors of Blastocystis infection in a rural community in Thailand. Methods: In February 2016, a cohort study was conducted in a rural community of Thailand. Baseline information of intestinal parasitic infections and follow-up visits were conducted. Blastocystis infection was detected in stool samples examined with short-term in vitro cultivation using Jones’ medium supplemented with 10% horse serum. Participants were tested for Blastocystis infection at every study visit. Cox proportional hazard regression analysis was used to evaluate co-variates to determine the incidence of Blastocystis infection. Results: Of 733 enrolled participants, 57 (7.78%) were positive and 676 (92.22%) were negative for Blastocystis infection. All 676 participants were followed-up for Blastocystis infection between December 2016 and January 2018. In all, 52 of 482 cases (10.79%) comprised individual Blastocystis infection. The incidence density rates of Blastocystis infection, of spontaneous clearance of the disease and of persistent infection were 8.13, 63.14 and 38.70 per 1 000 person-months of follow-up, respectively. Our multivariate analysis revealed that opened defecation in fields or bushes (HR=4.8; 95% CI=2.1-10.4, P<0.001), raising cows (HR=2.8; 95% CI=1.1-7.1, P=0.032), not washing hands after animal contact (HR=2.1; 95% CI=1.0-4.2, P=0.044) and presenting symptoms of nausea or vomiting (HR=2.3; 95% CI=1.0-5.3, P=0.047) were predictive associated risks for the incidence of Blastocystis infection. Conclusions: Our study indicates that the best way to prevent Blastocystis infection can be by maintaining good personal hygiene, hand washing after animal contact and improving sanitary facilities.

137-138

Authors: Sora Yasri, Viroj Wiwanitkit

Wuhan novel coronavirus or 2019-novel coronavirus (2019-nCoV) infection is a rapidly emerging respiratory viral disease[1]. 2019- nCoV infection is characterized as febrile illness with possible severe lung complication[1]. The disease was firstly reported in China in December 2019 and then spread to many countries (such as Thailand, Japan and Singapore)[2,3]. As a new disease, there is a limited knowledge of treatment for the infection. Lu recently proposed that some drug might be useful in treatment of 2019-nCoV infection[3]. Lu noted that “lopinavir/ritonavir, nucleoside analogues, neuraminidase inhibitors, remdesivir, peptide (EK1), abidol, RNA synthesis inhibitors (such as TDF, 3TC), anti-inflammatory drugs (such as hormones and other molecules), Chinese traditional medicine, such ShuFengJieDu capsules and LianHuaQingWen capsule might be useful[3].” Of those mentioned drugs, lopinavir/ritonavir is a widely used antiviral for management of another important virus infection, human immunodeficiency virus (HIV) infection. Since 2019-nCoV infection is an RNA virus similar to HIV, lopinavir/ritonavir is proposed for management of 2019-nCoVinfection. At present, lopinavir/ritonavir is widely used for possible treatment of 2019-nCoV infection in countries that the emerging infection exists. Here, the authors used a mathematical modelling theoretical approach to predict the expectedproper dosage of lopinavir/ritonavir for possible treatment of 2019-nCoV infection. The protocol for mathematical modeling in this work is the same as previous report by Wiwanitkit et al[4]. Briefly, the primary agreement was there had to be a specific amount of required energy for reaction between lopinavir/ritonavir and its target enzyme and this energy is a specific constant for the reaction. Based on bonding theory, the required amount of lopinavir/ritonavir was varied to the two substrates, lopinavir/ritonavir and target, protease. Here, the simple equation ‘A + B → C where A was the target enzyme, B was lopinavir/ritonavir and C was end product’ could be written. For HIV, the molecular mass of protease is equal to 21.6 kDa. For 2019-nCoV, the molecular weight was calculated from the functional motif showing protease function within the sequence of the virus. From molecular weight calculation tool (https://www. bioinformatics.org/sms/prot_mw.html; version 1 by Stothard[4]), the molecular mass of protease of 2019-nCoV is equal to 33.8 kDa. By standard comparison technique as used in the previous report[5], the required dosage of lopinavir /ritonavir for 2019-nCoV infection was about 1.56 times higher than that for HIV infection. Hence, based on the modeling study, if lopinavir/ritonaviris used for management of 2019-nCoV, a doubled dosage of the present dosage for HIV infection is recommended.

49-55

Authors: Mohamed Ali-Seyed, Kavitha Vijayaraghavan

Dengue is a debilitating disease that poses a perpetual threat to human health and increases the global economic burden every year. Despite advances in medical sciences, dengue virus (DENV) infects approximately 200 million people every year. To date, no effective antiviral is valiable to treat DENV in individuals despite great efforts in accomplishing these goals. Numerous approaches have been used in the search for dengue antiviral like screening of combinatorial compounds against DENV enzymes and structurebased computational discovery. In recent years, investigators have turned their focus into medicinal plants, trying to identify compounds that can be used as dengue antiviral. Nature represents a great reservoir of potential substances that can be explored with the aim of discovering new drugs that can be either used directly as pharmaceuticals or can provide drug leads, which can be scrutinized further for the development of new anti-dengue natural product. Many previous investigations have dealt with numerous plant extracts or bioactive principles for their antiviral property as they normally considered being safer when compared to synthetic drugs. Andrographis paniculata belongs to family Acanthaceae and is generally known as ‘king of bitters’. Diverse bioactive compounds from this plant such as diterpenes, flavonoids, xanthones, noriridoides and other miscellaneous compounds have exhibited their potential as therapeutics for various chronic as well as infectious diseases. This review is based on literature review on scientific journals, books and electronic sources, which highlights the pathogenesis of DENV and describe an assortment of bioactive principles that have been possessing antiviral potential, which include dengue and discuss the therapeutic efficacy and mechanism of action of Andrographis paniculata. However, a detailed and more comprehensive clinical trial on mammalian tissues and organs is needed in future studies.

62-70

Authors: Natalia Souza de Godoy, Manoel Sebastião da Costa Lima-Junior, José Angelo Lauletta Lindoso, Vera Lucia Pereira-Chioccola, Thelma Suely Okay, Lucia Maria Almeida Braz

Objective: To determine an algorithm for molecular diagnosis of visceral leishmaniasis (VL) by kinetoplast DNA (kDNA) (RV1/RV2) and internal transcriber spacer (ITS1) (LITSR/L5.8S) polymerase chain reaction (PCR), complemented by ITS1 PCR restriction fragment length polymorphism (RFLP), using peripheral blood or bone marrow aspirate from patients with suspected VL. Methods: Biological samples were submitted to the gold standard for the diagnosis of VL and molecular diagnosis represented by ITS1 PCR, kDNA PCR, and ITS1 PCR RFLP. The samples were obtained from seven groups: group I, 82 samples from patients with confirmed VL; groupII, 16 samples from patients under treatment for VL; group III, 14 samples from dogs with canine visceral leishmaniasis (CVL); group IV, a pool of six experimentally infected sandflies (Lutzomya longipalpis); group V, 18 samples from patients with confirmed tegumentary leishmaniasis (TL) and groups VI and VII were from control groups without VL. Results: The following gold standard and molecular examination results were obtained for each of the seven groups: group I: parasitologic and immunochromatographic tests showed a sensitivity of 76.3% (61 of 80) and 68.8% (55 of 80), respectively, and a sensitivity of 97.6% (80 of 82) and 92.7% (76 of 82) by ITS1 and kDNA PCR, respectively. After ITS1 PCR RFLP (Hae III) analysis of the 80 positive samples, 52.5% (42 of 80) generated three fragments of 180, 70, and 50 bp, corresponding to the pattern of Leishmania infantum infantum; group II: negative for the parasitologic methods and positive for IrK39 (100%, 16 of 16), presented 12.5% (2 of 16) of positivity by ITS1 PCR and 25.0% (4 of 16) by kDNA PCR; group III: positive in the parasitologic and serologic tests (100%, 14 of 14), presented 85.7%(12 of 14) of positivity by ITS1 PCR and kDNA PCR. ITS1 PCR RFLP showed that 83.3% (10 of 12) of the canine samples contained parasites with profiles similar to L. infantum; group IV presented amplifications by ITS1 PCR and kDNA PCR. ITS1 PCR products were analyzed by RFLP, generating a profile similar to that of L. infantum; group V: positive in the parasitologic examination (100%, 18 of 18), presented 72.2% (13 of 18) of the samples by ITS1 PCR positive. A total of 69.2% (9 of 13) showed profiles corresponding to a Viannia complex by ITS1 PCR RFLP; and group VI and group VII were negative by ITS1 and kDNA molecular tests. Comparing the molecular results with the parasitologic and serologic diagnosis from group I, almost perfect agreement was found (κ both>0.80, P<0.001). ITS1 and RV1/RV2 PCR detected 90.2% (74 of 82) of the samples. Two samples positive by RV1/RV2 were negative by LITSR/L5.8S, and six samples positive by LITSR/L5.8S were negative by RV1/RV2. Therefore, these two systems complemented each other; they diagnosed 100% of the samples as belonging to the Leishmania genus. Conclusions: We suggest an algorithm for the molecular diagnosis of VL, which must consider previous parasitologic and serologic (immunochromatographic) diagnoses, and should combine kDNA and ITS1 to determine the Leishmania subgenus using RFLP as a complement method to define the L. infantum species.

81-90

Authors: Hu-ling Li, Rong-jiong Zheng, Qiang Zheng, Wei Jiang, Xue-liang Zhang, Wei-ming Wang, Xing Feng, Kai Wang, Xiao-bo Lu

Objective: To forecast the visceral leishmaniasis cases using autoregress integrated moving average (ARIMA) and hybrid ARIMAEGARCH model, which offers a scientific basis to control visceral leishmaniasis spread in Kashgar Prefecture of Xinjiang, China. Methods: The data used in this paper are monthly visceral leishmaniasis cases in the Kashgar Prefecture of Xinjiang from 2004 to 2016. The sample data between 2004 and 2015 were used for the estimation to choose the best model and the sample data in 2016 were used for the forecast. Time series of visceral leishmaniasis started on 1 January 2004 and ended on 31 December 2016, consisting of 1 790 observations reported in Kashgar Prefecture. Results: For Xinjiang, the total number of reported cases were 2 187, the male-to-female ratio of cases was 1:1.42. Patients aged between 0 and 10 years accounted for 82.72% of all reported cases and the largest percentage of visceral leishmaniasis cases was detected among scattered children who accounted for 68.82%. The monthly incidences fitted by ARIMA (2, 1, 2) (1, 1, 1)12 model were consistent with the real data collected from 2004 to 2015. However, the predicted cases failed to comply with the observed case number; we then attempted to establish a hybrid ARIMA-EGARCH model to fit visceral leishmaniasis. Finally, the ARIMA (2, 1, 2) (1, 1, 1)12- EGARCH (1, 1) model showed a good estimation when dealing with volatility clustering in the data series. Conclusions: The combined model has been determined as the best prediction model with the root-mean-square error (RMSE) of 7.23% in the validation phase, which means that this model has high validity and rationality and can be used for short-term prediction of visceral leishmaniasis and could be applied to the prevention and control of the disease.

94-96

Authors: Van Lun Low, Tiong Kai Tan, Jamaiah Ibrahim, Sazaly AbuBakar, Yvonne Ai Lian Lim

Rodent-borne leptospirosis is by far the most common bacterial zoonosis and it is an important emerging global public health concern in Southeast Asia. Bacterial pathogens associated with rodents, especially those that live in close association with humans have been underreported. To fill this knowledge gap, the present study was undertaken to explore other neglected disease agents that can naturally infect synanthropic rodents. Both Bartonella and Mycoplasma pose major health threats to various animal hosts and they are also emerging zoonoses and pathogens of public health concern. With these in mind, we aimed to detect the presence of Bartonella and Mycoplasma bacteria in synanthropic rodents from a densely populated capital city of Malaysia, Kuala Lumpur. Rodent collection has been described elsewhere in detail[1]. Briefly, a total of 134 synanthropic rodents comprising Rattus (R.) rattus diardii, R. norvegicus, R. argentiventer, R. tiomanicus, and R. exulans were trapped from two human populated areas (Sentul and Chow Kit) in Kuala Lumpur using steel wire traps. Blood was collected from the heart using a needle and syringe and placed into EDTA tube. Genomic DNA was extracted from whole blood using the QIAamp DNA Blood Mini Kit (QIAGEN Inc.,Valencia, CA). Bartonella DNA detection was performed using polymerase chain reaction (PCR) with primers (325-5’CTT CAG ATG ATG ATC CCA AGC CTT TTG GCG-3’), and (1100-5’ GAA CCG ACG ACC CCC TGC TTG CAA AGC-3’) which amplify a portion of the 16S-23S rRNA intergenic spacer region[2]. PCR amplification was performed in a final volume of 25 μL containing 25-50 ng genomic DNA, 12.5 μL of MyTaq Red Mix (Bioline Reagents Ltd., London, UK) and 10 pmol of each forward and reverse primer. PCR was performed using the Applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystems, Inc., Foster City, CA) with the following thermal protocol: initial denaturation at 95 ℃ for 2 min followed by 55 cycles of denaturing at 94 ℃ for 15 s, annealing at 66 ℃ for 15 s, and extension at 72 ℃ for 15 s, and final extension at 72 ℃ for 1 min. For Mycoplasma DNA detection, samples were subjected to 16S rRNA gene amplification using a universal Mycoplasma primer pair (HBT-F-5’ ATA CGG CCC ATA TTC CTA CG-3’ and HBT-R-5’ TGC TCC ACC ACT TGT TCA-3’)[3], 25-50 ng genomic DNA in a total volume of 25 μL, with the following thermal protocol: 95 ℃ for 15 min and 50 cycles of 95 ℃ for 10 s, 55 ℃ for 15 s and 72 ℃ for 30 s, and 72 ℃ for 1 min. Purified PCR amplicons were sequenced using an ABI PRISM 377 Genetic Analyzer (Applied Biosystems, Inc.). Representative sequences of Bartonella (MK953939-MK953940) and Mycoplasma (MK959182) generated from this study were deposited in the National Center for Biotechnology Information GenBank. A neighbour-joining (NJ) phylogenetic tree was plotted using MEGA6 (https://megasoftware.net/). The NJ bootstrap values were estimated using 1 000 replicates with Kimura’s two-parameter model of substitution (K2P distance). Brucella abortus (X95889) and Brucella melitensis (AY513568) were used as outgroups for the construction of Bartonella and Mycoplasma phylogenetic trees, respectively. Based on the 16S-23S fragment of Bartonella, successful PCR amplification was found in 5 out of 134 samples (3.73%). NJ phylogenetic analysis revealed that all five sequences clustered with Bartonella (B.) phoceensis sequence (AY515123) retrieved from the National Center for Biotechnology Information GenBank. B. phoceensis showed a sister relationship with B. rattimassiliensis, and distantly separated from the other Bartonella species (Figure 1). For Mycoplasma, three samples (2.24%) were positive to 16S rRNA fragment and successfully sequenced. The three sequences formed a monophyletic clade with the sequences of Candidatus Mycoplasma (M.) haemomuris subsp. ratti (AB758434, AB758435 and AB758439) and showed a close relationship with Candidatus M. haemomuris subsp. musculi (Figure 2). Figure 1. Neighbour-joining phylogenetic tree of Bartonella spp. based on the 16S-23S intergenic spacer region. Bootstrap values (NJ) are shown on the branches. Newly generated sequences are in bold. This study provides the first evidence for the presence of B. phoceensis and Candidatus M. haemomuris subsp. ratti in synanthropic rodents in Malaysia. Their prevalence rates (3.73% for Bartonella and 2.24% for Mycoplasma), however, were considered low in comparison to that previously reported in Malaysia. In the earlier study, five different Bartonella species (i.e., B. tribocorum, B. rattimassiliensis, B. coopersplainsensis, B. elizabethae, and B. queenslandensis) (13.7%) were isolated from kidney and spleen homogenates of rats[4]. Nevertheless, the detection protocols adopted (16S-23S versus gltA and rpoB; and blood versus kidney and spleen) may have contributed to the discrepancies in the recovered species in both studies. Among the detected species, B. rattimassiliensis, B. tribocorum, and B. elizabethae were implicated as causing human infections in Thailand[5]. The pathogenicity and zoonotic potential of B. phoceensis are unknown. Mycoplasma spp. from various animals in Malaysia were reported at varying frequencies such as M. haemofelis (11.7%) in cats[6], M. wenyonii and Candidatus M. haemobos (69.0%) in cattle[7], and several species in various animal samples received in a veterinary diagnostic laboratory[8].Investigation of Mycoplasma in rodents in Malaysia is underappreciated, though M. arthritidis was reported in three rats (out of 10 rats) in a previous study[9]. In contrast, in the present study, we detected Candidatus M. haemomuris subsp. ratti, a subspecies of M. haemomuris which was recently incriminated as the anaemic pathogen of rats in Japan, and differentiated from Candidatus M. haemomuris subsp. musculi which mainly infects mice[10]. Animal Mycoplasma spp. rarely infect humans, but their zoonotic potential could not be disregarded since cases of human haemoplasma infection have been documented. In conclusion, we report here the occurrence of B. phoceensis and Candidatus M. haemomuris subsp. ratti in synanthropic rodents for the first time in Malaysia. Our results suggest that synanthropic rodents can serve as the reservoir for these vector-borne pathogens. Nevertheless, their routes of transmission and zoonotic potential require further investigation.

525-534

Authors: Joleen Savianne Almeida, Ajeet Kumar Mohanty, Savita Kerkar, Sugeerappa Laxmanappa Hoti, Ashwani Kumar

Mosquito-borne diseases such as malaria, filariasis, dengue, chikungunya, Japanese encephalitis, yellow fever and Zika contribute significantly to health problems of developing as well as developed nations. Vector control is central to control of vector borne diseases. In the last four-five decades, biological control methods have been inducted in the integrated vector management strategy, advocated nationally as well as globally by the World Health Organization. Currently, biological control of vectors is globally acknowledged as the best available strategy in the wake of growing concerns about vector resistance as well as adverse effects of insecticides on the environment and non-target fauna co-inhabiting the same ecological niches as vectors. In India and elsewhere, efforts are ongoing to screen newer isolates to bring forth new biolarvicidal products of public health importance. In this review, by carrying out extensive literature survey, we discuss advances thus far and the prospects of bacilli-based control of vectors and vector borne diseases.

542-549

Authors: Rosnani Hanim Mohd Hussain, Nur Syahirah Mohamad Isa, Khairul Ameera Kamaruddin1, Mohamed Kamel Abdul Ghani, Naveed Ahmed Khan, Ruqaiyyah Siddiqui, Tengku Shahrul Anuar

Objective: To determine the morphological and molecular characterization of Acanthamoeba isolates from contact lens paraphernalia in Malaysia and to investigate their pathogenic potential based on the physiological tolerance. Methods: One hundred and eighty contact lens wearers donated their contact lens, lens storage cases and lens solutions between 2018 and 2019. The samples were inoculated onto 1.5% nonnutrient agar plates for 14 d. Polymerase chain reaction (PCR) was performed and the amplified PCR products were sequenced and compared with the published sequences in GenBank. The pathogenic potential of positive isolates was further tested using temperature-tolerance and osmo-tolerance assays. Acanthamoeba species were categorized into three distinct morphological groups established by Pussard and Pons. Results: Acanthamoeba was successfully isolated from 14 (7.8%) culture-positive samples in which 11 belong to morphological group 栻 and 3 belong to morphological group II, respectively. The sequencing of 18S ribosomal RNA gene led to the identification of the T4 genotype in all the isolated strains. In vitro assays revealed that 9 (64.3%) Acanthamoeba isolates were able to grow at 42 III and 1 M mannitol and were thus considered to be highly pathogenic. Conclusions: To the best of our knowledge, this is the first report identifying the Acanthamoeba genotype and their pathogenic potential among contact lens wearers in Malaysia. The potentially pathogenic T4 genotype isolated in this study is the most predominant genotype responsible for human ocular infection worldwide. Hence, increasing attention should be aimed at the prevention of contamination by Acanthamoeba and the disinfection of contact lens paraphernalia.

557-565

Authors: Lu Guo, Jum Soon Kang, Young Hoon Park, Beong Il Je, Dae Youn Hwang, Woo Hong Joo, Young Whan Choi

Objective: To establish an efficacious and efficient fermentation method of enhancing the anti-adipogenesis effect of mulberry (Morus alba) leaves using Cordyceps militais. Methods: Dried mulberry leaves, dried mulberry leaves with 50% raw silkworm pupa and raw silkworm pupa were fermented with Cordyceps militais for 4 weeks at 25 ℃, after which the dried mulberry leaves and fermented product were extracted with 70% ethanol and subjected to high performance liquid chromatography (HPLC). The contents of cordycepin, pelargonidin, chlorogenic acid, iso-quercetin and caffeic acid were determined. We then used the 3T3-L1 cells to investigate whether extracts of fermentation enhanced anti-adipogenesis activity in vitro. Results: HPLC showed that fermentation changed the contents of cordycepin, pelargonidin, chlorogenic acid, iso-quercetin and caffeic acid. Furthermore, fermented dried mulberry leaves with 50% raw silkworm pupa had a better efficacy of anti-adipogenesis than dried mulberry leaves, fermented dried mulberry leaves and fermented silkworm pupa and inhibited triglycerides accumulation and glucose consumption. Additionally, fermented dried mulberry leaves with 50% raw silkworm pupa inhibited PPAR-γ signaling. Conclusions: Fermentation with Cordyceps militaris enhanced antiadipogenesis efficacy of mulberry leaves.

477-486

Authors: Swee Li Ng, Tsuey Ning Soon, Wei Hsum Yap, Kai Bin Liew, Ya Chee Lim4, Long Chiau Ming, Yin-Quan Tang, Bey Hing Goh

The new coronavirus disease (COVID-19) outbreak has challenged us to take unprecedented steps to bring this pandemic under control. In view of the urgency of this situation, convalescent plasma which was used in previous coronavirus outbreaks has emerged as one of the treatment options in this current pandemic. This is mainly due to the fact that convalescent plasma has been studied in a few case series with promising outcomes. In addition, on-going large clinical trials aimed to further evaluate the effectiveness, safety, and optimal dosage, duration and timing of administration of convalescent plasma are indeed revealing a certain level of promising results. Therefore, this article aims to provide an overview of possible mechanisms of actions of convalescent plasma, its benefits and its level of usage safeness by summarizing the existing evidence on the use of convalescent plasma in COVID-19 patients.

494-502

Authors: Deepak Paramasivam, Balamuralikrishnan Balasubramanian, Sungkwon Park, Palanisamy Alagappan, Tanushri Kaul, Wenchao Liu, Perumal Pachiappan

Objective: To analyze the phytochemical compounds and to investigate the bio-toxic efficacy of various solvent extracts of Plectranthus amboinicus against mosquito larvae activity and lethality on non-targeting organisms. Methods: The methanol, ethyl acetate, hexane, and aqueous extracts of Plectranthus amboinicus were subjected to analyze the mosquitocidal activity against the dengue vector, Aedes aegypti and toxicity assays on zebra fish and brine shrimp. Three replications were performed, and negative control was also maintained. Amongst, ethyl acetate extract of Plectranthus amboinicus was chosen for the determination of bio-active compounds. Results: The mosquitocidal assays of methanol and ethyl acetate extracts of Plectranthus amboinicus showed the maximal activity with minimal concentration against the 4th instar mosquito-larvae of Aedes aegypti through the following lethal concentration (LC50 and LC90) values: 53.36 & 92.51 μg/mL and 13.64 & 86.09 μg/mL, respectively. In addition, the plant extracts showed no toxicity on zebra fish embryo and brine shrimp assays. The gas-chromatography analysis of the ethyl acetate extract of Plectranthus amboinicus revealed the presence of seven different compounds. Among them, PAEA-fraction 60 contained a major active bioactive compound, hexadecanoic acid, methyl ester (270.0). Conclusions: Plectranthus amboinicus possesses mosquitocidal properties and could be used as a potential alternative source for preparing the mosquitocidal agents.

515-520

Authors: Chairat Tantrawatpan, Weerachai Saijuntha

Objective: To explore genetic variations of Hypoderaeum conoideum collected from domestic ducks from 12 different localities in Thailand and Lao PDR, as well as their phylogenetic relationship with American and European isolates. Methods: The nucleotide sequences of their nuclear ribosomal DNA (ITS), mitochondrial cytochrome c oxidase subunit 1 (CO1), and NADH dehydrogenase subunit 1 (ND1) were used to analyze genetic diversity indices. Results: We found relatively high levels of nucleotide polymorphism in ND1 (4.02%), whereas moderate and low levels were observed in CO1 (2.11%) and ITS (0.96%), respectively. Based on these polymorphisms, the 20 ND1, 12 CO1, and 18 ITS haplotypes were classified, and several common haplotypes were observed in all samples. At least three major lineages, namely American, European and Asian lineages, have been classified by phylogenetic analyses based on ND1 sequences. Conclusions: Our report demonstrates that the ND1 gene is the most suitable genetic marker to explore genetic variation and phylogenetic relationship of Hypoderaeum conoideum. However, a combination of all loci for ND1, CO1 and ITS would be of great value toward further genetic investigation of this endemic worldwide parasite. Thus, comprehensive molecular genetic analyses of Hypoderaeum conoideum from its worldwide distribution is needed to further understanding of the evolutionary and systematic relationships of this parasite.