Asian Pacific Journal of Tropical Medicine

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.