Researcher Profile and Settings

Master

Affiliation (Master)

• International Institute for Zoonosis Control　Division of Bioresources

Affiliation (Master)

• International Institute for Zoonosis Control　Division of Bioresources

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Profile and Settings

Degree

• PhD（Hokkaido University）

Profile and Settings

• Name (Japanese)

  Thapa

• Name (Kana)

  Jeewan

• Name

  201801021432532726

Achievement

Research Interests

• Infectious diseases   Bacteriology   

Research Areas

• Life sciences / Bacteriology

Research Experience

• 2020 - Today Hokkaido University International Institute for Zoonosis Control
• 2018 - 2020 Hokkaido University Faculty of Health Sciences
• 2017 - 2018 Hokkaido University Research Center for Zoonosis Control JSPS Postdoctoral Fellow
• 2009 - 2013 National Trust for Nature Conservation, Nepal Veterinary Officer
• 2007 - 2009 Tribhuvan University Institute of Agriculture and Animal Science Research Associate

Education

• 2013 - 2017  Hokkaido University  School of Veterinary Medicine  PhD

Committee Memberships

• 2014 - Today   Japanese Society for Bacteriology
• 2006 - Today   Nepal Veterinary Association

Published Papers

• Whole-Genome Investigation of Zoonotic Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Isolated from Pigs and Humans in Thailand.

  Pawarut Narongpun, Pattrarat Chanchaithong, Junya Yamagishi, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki

  Antibiotics (Basel, Switzerland) 12 (12) 2023/12/16 

  Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been widespread globally in pigs and humans for decades. Nasal colonization of LA-MRSA is regarded as an occupational hazard to people who are regularly involved in livestock production. Our previous study suggested pig-to-human transmission caused by LA-MRSA clonal complex (CC) 398, using traditional molecular typing methods. Instead, this study aimed to investigate the zoonotic transmission of LA-MRSA CC398 using whole genome sequencing (WGS) technologies. A total of 63 LA-MRSA isolates were identified and characterized in Thailand. Further, the 16 representatives of LA-MRSA CC9 and CC398, including porcine and worker isolates, were subjected to WGS on the Illumina Miseq platform. Core-genome single nucleotide polymorphism (SNP)-based analyses verify the zoonotic transmission caused by LA-MRSA CC398 in two farms. WGS-based characterization suggests the emergence of a novel staphylococcal cassette chromosome (SCC) mec type, consisting of multiple cassette chromosome recombinase (ccr) gene complexes via genetic recombination. Additionally, the WGS analyses revealed putative multi-resistant plasmids and several cross-resistance genes, conferring resistance against drugs of last resort used in humans such as quinupristin/dalfopristin and linezolid. Significantly, LA-MRSA isolates, in this study, harbored multiple virulence genes that may become a serious threat to an immunosuppressive population, particularly for persons who are in close contact with LA-MRSA carriers.
• Insight into the genetic diversity of Mycobacterium bovis isolated from cattle in Malawi.

  Thoko Flav Kapalamula, Joseph Yamweka Chizimu, Mwangala Lonah Akapelwa, David Atomanyi Barnes, Jirachaya Toyting, Precious Bwalya, Linda Basikolo, David Squarre, Herman M Chambaro, Stephen V Gordon, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki

  Research in veterinary science 164 105030 - 105030 2023/11 

  We describe the genetic diversity and phylogenetic relationships of Mycobacterium bovis, isolated from cattle in Malawi. Deletion analysis, spoligotyping, and MIRU-VNTR typing were used to genotype the isolates. Combined with a larger dataset from neighboring countries, the overall M. bovis diversity in Southern Africa was contextualized. From the southern and northern regions of Malawi, 24 isolates were confirmed as M. bovis. We pooled data for the central region (60 isolates) from our recent publication to conceptualize the genetic and phylogenetic relationships of M. bovis in Malawi. European 1 was the dominant M. bovis clonal complex, with 10 unique spoligotype patterns, and SB0131 was ubiquitous. High genetic diversity, a low clustering rate, and many singletons, coupled with a low mutation transmission index, infer a low level of recent transmission, and suggest an endemic status of bovine tuberculosis (bTB) in Malawi. M. bovis isolates from Zambia, Mozambique, and South Africa were genetically related to Malawian isolates, whereas Tanzanian isolates were distantly related. The diversity and phylogenetic analysis suggest earlier introductions and maintenance of M. bovis by constant reinfection from reservoir animals. These findings are fundamental to understanding the source and route of infection in order to establish alternative management strategies for bTB.
• Exploration of the novel fluoroquinolones with high inhibitory effect against quinolone-resistant DNA gyrase of Salmonella Typhimurium.

  Jirachaya Toyting, Nami Miura, Fuangfa Utrarachkij, Wimonrat Tanomsridachchai, Lawrence P Belotindos, Pondpan Suwanthada, Thoko Flav Kapalamula, Siriporn Kongsoi, Kentaro Koide, Hyun Kim, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki

  Microbiology spectrum e0133023  2023/10/05 

  Quinolone-resistant nontyphoidal Salmonella, one of the prominent pathogens causing acute gastroenteritis, has become a public health concern globally. The World Health Organization has ranked fluoroquinolone-resistant Salmonella as a high-priority pathogen for researching and developing new antibiotics. WQ-3034 and WQ-3154 are relatively new synthetic fluoroquinolones with distinctive structures. WQ-3034 has 6-amino-3,5-difluoropyridine-2-yl at R1, 3-hydroxyazetidinyl at R7, and the addition of chlorine atom at R8. WQ-3154 has a similar basic pharmacophore to WQ-3034 except for the modification at R8 with a methyl group. In this study, the inhibitory effect and DNA cleavage effect against wild-type (WT) and mutant Salmonella Typhimurium DNA gyrases of WQ-3034 and WQ-3154 were examined along with WQ-3810 and ciprofloxacin by measuring the drug concentration that inhibits half of the enzyme activity (IC50) and the drug concentration that induces 25% of maximum DNA cleavage (CC25). The minimum inhibitory concentration (MIC) of the compounds was assessed against Salmonella Typhimurium and Salmonella Enteritidis. Among four compounds, WQ-3034 demonstrated the highest inhibitory effect against both WT and mutant Salmonella Typhimurium DNA gyrases with amino acid substitution at codon 83 and/or 87, while ciprofloxacin showed the lowest inhibitory effect. Remarkably, WQ-3034 and WQ-3154 exhibited a significantly higher inhibitory effect than ciprofloxacin against Salmonella Typhimurium DNA gyrase with double amino acid substitution, Ser83Phe-Asp87Asn. Similarly, CC25 of WQ-3034 against mutant Salmonella Typhimurium DNA gyrase was lower than ciprofloxacin. Notably, MICs of WQ-3034 and WQ-3154 were higher than ciprofloxacin. In conclusion, this study revealed that WQ-3034 and WQ-3154 could potentially be effective therapeutic agents against quinolone-resistant nontyphoidal Salmonella. IMPORTANCE Quinolone-resistant nontyphoidal Salmonella is a pressing public health concern, demanding the exploration of novel treatments. In this study, we focused on two innovative synthetic fluoroquinolones, WQ-3034 and WQ-3154. Our findings revealed that these new compounds demonstrate potent inhibitory effects, even against mutant strains that cause resistance to existing quinolones. Hence, WQ-3034 and WQ-3154 could potentially be effective therapeutic agents against quinolone-resistant Salmonella Typhimurium. Furthermore, the data obtained in this study will be baseline information for antimicrobial drug development.
• The Impact of Substitutions at Positions 1 and 8 of Fluoroquinolones on the Activity Against Mutant DNA Gyrases of Salmonella Typhimurium.

  Pondpan Suwanthada, Siriporn Kongsoi, Nami Miura, Lawrence P Belotindos, Chayada Piantham, Jirachaya Toyting, Mwangala L Akapelwa, Ruttana Pachanon, Kentaro Koide, Hyun Kim, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki

  Microbial drug resistance (Larchmont, N.Y.) 2023/10/04 

  Although many drug-resistant nontyphoidal Salmonella (NTS) infections are reported globally, their treatment is challenging owing to the ineffectiveness of the currently available antimicrobial drugs against resistant bacteria. It is therefore essential to discover novel antimicrobial drugs for the management of these infections. In this study, we report high inhibitory activities of the novel fluoroquinolones (FQs; WQ-3810 and WQ-3334) with substitutions at positions R-1 by 6-amino-3,5-difluoropyridine-2-yl and R-8 by methyl group or bromine, respectively, against wild-type and mutant DNA gyrases of Salmonella Typhimurium. The inhibitory activities of these FQs were assessed against seven amino acid substitutions in DNA gyrases conferring FQ resistance to S. Typhimurium, including high-level resistant mutants, Ser83Ile and Ser83Phe-Asp87Asn by in vitro DNA supercoiling assay. Drug concentrations of WQ compounds with 6-amino-3,5-difluoropyridine-2-yl that suppressed DNA supercoiling by 50% (IC50) were found to be ∼150-fold lower than ciprofloxacin against DNA gyrase with double amino acid substitutions. Our findings highlight the importance of the chemical structure of an FQ drug on its antimicrobial activity. Particularly, the presence of 6-amino-3,5-difluoropyridine-2-yl at R-1 and either methyl group or bromine at R-8 of WQ-3810 and WQ-3334, respectively, was associated with improved antimicrobial activity. Therefore, WQ-3810 and WQ-3334 are promising candidates for use in the treatment of patients infected by FQ-resistant Salmonella spp.
• Molecular Characterisation of Mycobacterium bovis Isolates from Cattle Slaughtered in Adamawa and Gombe States, North-Eastern Nigeria

  Sadiq Mohammed Damina, David Atomanyi Barnes, Bitrus Inuwa, Gulak Hussaini Ularamu, Mohammed Bello, Olu Solomon Okaiyeto, Ayuba Caleb Kudi, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki

  Current Issues in Molecular Biology 45 (7) 6055 - 6066 2023/07/19 

  Bovine tuberculosis is endemic in Nigeria with control measures as provided by the laws of the country being minimally enforced mostly at the abattoirs only. This study focused on bovine tuberculosis in Adamawa and Gombe States. Tuberculosis lesions were observed in 183 of 13,688 slaughtered cattle in the regions between June and December 2020. Analysis of tissue samples resulted in 17 Mycobacterium bovis isolates, predominantly from Gombe State. Spoligotyping identified four spoligotypes, including SB0944, SB1025, SB1104, and one novel pattern. MIRU-VNTR analysis further differentiated these spoligotypes into eight profiles. All isolates belonged to the Af1 clonal complex. The study emphasises the need for broader coverage and more isolates to comprehensively understand the molecular epidemiology of bovine tuberculosis in Nigeria. To enhance research and surveillance, a cost-effective approach is proposed, utilising a discriminatory VNTR panel comprising five or nine loci. The five-locus panel consists of ETR-C, QUB26, QUB11b, MIRU04, and QUB323. Alternatively, the nine-locus panel includes ETR-A, ETR-B, QUB11a, and MIRU26. Implementing this approach would provide valuable insights into the genetic diversity of M. bovis strains in Nigeria. These findings are crucial for developing effective control measures and minimising the impact of bovine tuberculosis on both animal and human health.
• Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia

  Joseph Yamweka Chizimu, Eddie Samuneti Solo, Precious Bwalya, Thoko Flav Kapalamula, Kaemba Kunkuta Mwale, David Squarre, Misheck Shawa, Patrick Lungu, David Atomanyi Barnes, Kaunda Yamba, Tiza Mufune, Herman Chambaro, Harvey Kamboyi, Musso Munyeme, Bernard Mudenda Hang’ombe, Nathan Kapata, Victor Mukonka, Roma Chilengi, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki

  Antibiotics 12 (7) 1126 - 1126 2023/06/29 

  The emergence of pre-extensively drug-resistant tuberculosis (pre-XDR-TB) is a threat to TB control programs in developing countries such as Zambia. Studies in Zambia have applied molecular techniques to understand drug-resistance-associated mutations, circulating lineages and transmission patterns of multi-drug-resistant (MDR) Mycobacterium tuberculosis. However, none has reported genotypes and mutations associated with pre-XDR TB. This study characterized 63 drug-resistant M. tuberculosis strains from the University Teaching Hospital between 2018 and 2019 using targeted gene sequencing and conveniently selected 50 strains for whole genome sequencing. Sixty strains had resistance mutations associated to MDR, one polyresistant, and two rifampicin resistant. Among MDR strains, seven percent (4/60) had mutations associated with pre-XDR-TB. While four, one and nine strains had mutations associated with ethionamide, para-amino-salicylic acid and streptomycin resistances, respectively. All 50 strains belonged to lineage 4 with the predominant sub-lineage 4.3.4.2.1 (38%). Three of four pre-XDR strains belonged to sub-lineage 4.3.4.2.1. Sub-lineage 4.3.4.2.1 strains were less clustered when compared to sub-lineages L4.9.1 and L4.3.4.1 based on single nucleotide polymorphism differences. The finding that resistances to second-line drugs have emerged among MDR-TB is a threat to TB control. Hence, the study recommends a strengthened routine drug susceptibility testing for second-line TB drugs to stop the progression of pre-XDR to XDR-TB and improve patient treatment outcomes.
• Chlamydia trachomatis relies on the scavenger role of aryl hydrocarbon receptor with detyrosinated tubulin for its intracellular growth, but this is impaired by excess indole

  Saicheng Zhang, Yuki Funahashi, Satoho Tanaka, Torahiko Okubo, Jeewan Thapa, Shinji Nakamura, Hideaki Higashi, Hiroyuki Yamaguchi

  Microbes and Infection 25 (5) 105097 - 105097 1286-4579 2023/06
• Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

  Jeewan Thapa, Joseph Yamweka Chizimu, Soyoka Kitamura, Mwangala Lonah Akapelwa, Pondpan Suwanthada, Nami Miura, Jirachaya Toyting, Tomoyasu Nishimura, Naoki Hasegawa, Yukiko Nishiuchi, Stephen V Gordon, Chie Nakajima, Yasuhiko Suzuki

  Microbiology spectrum e0508822  2023/04/17 

  Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.
• Direct detection of Mycobacterium bovis by a dry loop-mediated isothermal amplification assay in cattle samples collected during routine abattoir examination in Malawi.

  Thoko Flav Kapalamula, Jeewan Thapa, Kyoko Hayashida, Joseph Chizimu, Wimonrat Tanomsridachchai, Mirriam Ethel Nyenje, Rajab Mkakosya, Chie Nakajima, Yasuhiko Suzuki

  Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 10406387231164596 - 10406387231164596 2023/04/08 

  The lack of quick, accurate, and low-cost detection methods has hindered the active control strategies for bovine tuberculosis (bTB) in resource-limited countries with a high burden of disease. We developed a dry loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Mycobacterium bovis, the principal causative agent of bTB, and evaluated the efficacy of the assay using suspected bTB samples collected during routine meat inspection at major regional abattoirs in Malawi. Template genomic DNA was extracted directly from the granulomatous bTB-like lesion (crude extracted DNA), as well as growth from the incubated mycobacterial growth indicator tubes (MGIT). Field results were visualized by the naked eye within 40 min following a color change of the amplified products. The sensitivity and specificity of the dry LAMP assay while using 152 DNA samples extracted from MGIT with confirmed M. bovis results were 98% and 88%, respectively. When 43 randomly selected crude DNA samples from lesions were used, the sensitivity and specificity of the dry LAMP assay were 100% and 75%, respectively. Our LAMP assay offers the potential to meet the demands for a low-cost and rapid field detection tool for bTB in resource-limited countries in which bTB is endemic.
• SalmonellaTyphimuriumのフルオロキノロン耐性に及ぼすGyrAおよびQnrB19のアミノ酸置換の影響(Impact of mutations in GyrA and QnrB19 on resistance to fluoroquinolone in Salmonella Typhimurium)

  Suwanthada Pondpan, Thapa Jeewan, 中島 千絵, 鈴木 定彦

  日本細菌学雑誌 78 (1) 105 - 105 0021-4930 2023/02
• Mycobacterium avium gyrAにおけるフルオロキノロン耐性関連変異が耐性へ果たす役割(Role of fluoroquinolone resistance-associated mutations in Mycobacterium avium gyrA to resistance)

  Thapa Jeewan, Chizimu Joseph Yamweka, 北村 そよか, Akapelwa Mwangala Lonah, Suwanthada Pondpan, 三浦 菜実, Toyting Jirachaya, 中島 千絵, 鈴木 定彦

  日本細菌学雑誌 78 (1) 105 - 105 0021-4930 2023/02
• Prevalence and risk factors of bovine tuberculosis in slaughtered cattle, Malawi.

  Thoko Flav Kapalamula, Francis Kawonga, Misheck Shawa, Joseph Chizimu, Jeewan Thapa, Mirriam Ethel Nyenje, Rajhab Sawasawa Mkakosya, Kyoko Hayashida, Stephen Gordon, Chie Nakajima, Musso Munyeme, Bernard M Hang'ombe, Yasuhiko Suzuki

  Heliyon 9 (2) e13647  2023/02 

  Bovine tuberculosis (bTB) is an infectious disease with significant socioeconomic, animal, and public health impacts. However, the prevalence of bTB remains largely unclear in Malawi due to a paucity of information. Additionally, the existence of multiple risk factors is postulated to enhance bTB transmission in animals. A cross-sectional survey to estimate the prevalence of bTB, animal characteristics and identify associated risk factors was conducted from slaughtered cattle at three major regional abattoirs (southern, central and northern regions) in Malawi. Out of a total of 1547 cattle examined, 154 (9.95%) had bTB-like lesions in various visceral organs and lymph nodes; one sample per animal was collected, processed, and cultured in the in the BACTEC Mycobacterial growth indicator tube (MGIT) 960 system. From the 154 cattle that showed tuberculous like lesions, only 112 were positive on MGIT and 87 were confirmed to have M. bovis based on multiplex PCR. Cattle from the southern region (odds ratio (OR) = 1.96, 95% CI: 1.03-3.85) and central region (OR = 2.00, 95% CI: 1.16-3.56) were more likely presented with bTB-like lesions at slaughter than from the northern region. The risk of having bTB-like lesions was higher in females (OR = 1.51, CI: 1.00-2.29), older cattle (OR = 2.17, CI: 1.34-3.37), and crossbreeds (OR = 1.67, 95% CI: 1.12-2.47) than in males, younger animals, and Malawi Zebu breed, respectively. The high prevalence of bTB is of critical concern and necessitates active surveillance and strengthening of the current control strategies under a One Health (OH) approach at the animal-human interface.
• Detection of Mutations in pncA in Mycobacterium tuberculosis Clinical Isolates from Nepal in Association with Pyrazinamide Resistance.

  Dipti Shrestha, Bhagwan Maharjan, Jeewan Thapa, Mwangala Lonah Akapelwa, Precious Bwalya, Joseph Yamweka Chizimu, Chie Nakajima, Yasuhiko Suzuki

  Current issues in molecular biology 44 (9) 4132 - 4141 2022/09/08 

  Without the proper information on pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis (MTB), PZA is inappropriately recommended for the treatment of both susceptible and multidrug-resistant tuberculosis (MDR-TB) in Nepal. This study aimed to collect information regarding PZA susceptibility in MTB isolates from Nepal by analyzing pncA and its upstream regulatory region (URR). A total of 211 MTB isolates were included in this study. Sequence analysis of pncA and its URR was performed to assess PZA resistance. First-line drug susceptibility testing, spoligotyping, and sequence analysis of rpoB, katG, the inhA regulatory region, gyrA, gyrB, and rrs were performed to assess their association with pncA mutation. Sequencing results reveal that 125 (59.2%) isolates harbored alterations in pncA and its URR. A total of 57 different mutation types (46 reported and 11 novel) were scattered throughout the whole length of the pncA gene. Eighty-seven isolates (41.2%) harbored mutations in pncA, causing PZA resistance in MTB. There was a more significant association of pncA alterations in MDR/pre-extensively drug-resistant (Pre-XDR) TB than in mono-resistant/pan-susceptible TB (p < 0.005). This first report on the increasing level of PZA resistance in DR-TB in Nepal highlights the importance of PZA susceptibility testing before DR-TB treatment.
• Chlamydia trachomatis L2/434/Bu Favors Hypoxia for its Growth in Human Lymphoid Jurkat Cells While Maintaining Production of Proinflammatory Cytokines

  Ryoya Tsujikawa, Jeewan Thapa, Torahiko Okubo, Shinji Nakamura, Saicheng Zhang, Yoshikazu Furuta, Hideaki Higashi, Hiroyuki Yamaguchi

  Current Microbiology 79 (9) 0343-8651 2022/09
• Threat from Mycobacterium orygis-associated tuberculosis in south Asia.

  Jeewan Thapa, Stephen V Gordon, Chie Nakajima, Yasuhiko Suzuki

  The Lancet. Microbe 3 (9) e641-e642  2022/06/14
• Chlamydia trachomatis Requires Functional Host-Cell Mitochondria and NADPH Oxidase 4/p38MAPK Signaling for Growth in Normoxia

  Jeewan Thapa, Gen Yoshiiri, Koki Ito, Torahiko Okubo, Shinji Nakamura, Yoshikazu Furuta, Hideaki Higashi, Hiroyuki Yamaguchi

  Frontiers in Cellular and Infection Microbiology 12 2022/05/26 [Refereed]
   

  Chlamydia trachomatis (Ct) is an intracellular energy-parasitic bacterium that requires ATP derived from infected cells for its growth. Meanwhile, depending on the O₂ concentration, the host cells change their mode of ATP production between oxidative phosphorylation in mitochondria (Mt) and glycolysis; this change depends on signaling via reactive oxygen species (ROS) produced by NADPH oxidases (NOXs) as well as Mt. It has been proposed that Ct correspondingly switches its source of acquisition of ATP between host-cell Mt and glycolysis, but this has not been verified experimentally. In the present study, we assessed the roles of host-cell NOXs and Mt in the intracellular growth of CtL2 (L2 434/Bu) under normoxia (21% O₂) and hypoxia (2% O₂) by using several inhibitors of NOXs (or the downstream molecule) and Mt-dysfunctional (Mt^d) HEp-2 cells. Under normoxia, diphenyleneiodonium, an inhibitor of ROS diffusion, abolished the growth of CtL2 and other Chlamydiae (CtD and C. pneumoniae). Both ML171 (a pan-NOX inhibitor) and GLX351322 (a NOX4-specific inhibitor) impaired the growth of CtL2 under normoxia, but not hypoxia. NOX4-knockdown cells diminished the bacterial growth. SB203580, an inhibitor of the NOX4-downstream molecule p38MAPK, also inhibited the growth of CtL2 under normoxia but not hypoxia. Furthermore, CtL2 failed to grow in Mt^d cells under normoxia, but no effect was observed under hypoxia. We conclude that under normoxia, Ct requires functional Mt in its host cells as an ATP source, and that this process requires NOX4/p38MAPK signaling in the host cells. In contrast to hypoxia, crosstalk between NOX4 and Mt via p38MAPK may be crucial for the growth of Ct under normoxia.
• Characterization of embB mutations involved in ethambutol resistance in multi-drug resistant Mycobacterium tuberculosis isolates in Zambia.

  Precious Bwalya, Eddie S Solo, Joseph Y Chizimu, Dipti Shrestha, Grace Mbulo, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki

  Tuberculosis (Edinburgh, Scotland) 133 102184 - 102184 2022/03 

  BACKGROUND: Ethambutol (EMB) is an important anti-tuberculosis drug used in the management of multi-drug resistant tuberculosis (MDR-TB). Mutations in embB are the major mechanism of resistance. This study investigated embB mutations among MDR-TB isolates and analyzed their correlations with phenotypic drug susceptibility testing (DST) in Zambia. METHOD: A total of 132 MDR-TB isolates were collected from January 2014 to April 2017 and characterized using MGIT 960 systems, embB sequencing, and spoligotyping. RESULTS: Out of 61 phenotypically EMB resistant isolates, 53 had mutations in embB. Among the 71 EMB susceptible isolates, 47 had embB mutations. Sensitivity of embB mutations was 86.9% while specificity was 33.8%. CAS1_Kili (SIT21) had high odds of having embB mutations, particularly, G918A (Met306eIl) (Odds ratio 16.7, p < 0.0001). CONCLUSION: Molecular EMB resistance testing by DNA sequencing can improve detection of EMB resistance among MDR-TB patients in Zambia. Additionally, CAS1_Kili was associated with embB amino acid substitution Met306Ile suggesting transmission. A detailed investigation to track and determine transmission hotspot area for MDR-TB could help optimize control strategies.
• Genetic Diversity and Transmission of Multidrug-Resistant Mycobacterium tuberculosis strains in Lusaka, Zambia.

  Joseph Yamweka Chizimu, Eddie Samuneti Solo, Precious Bwalya, Thoko Flav Kapalamula, Mwangala Lonah Akapelwa, Patrick Lungu, Dipti Shrestha, Yukari Fukushima, Victor Mukonka, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki

  International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 114 142 - 150 2022/01 

  OBJECTIVE: Zambia is among the 30 high tuberculosis burden countries in the world. Despite increasing reports of multidrug-resistant tuberculosis (MDR-TB) in routine surveillance, information on the transmission of MDR Mycobacterium tuberculosis strains is largely unknown. This study elucidated the genetic diversity and transmission of MDR M. tuberculosis strains in Lusaka, Zambia. METHODS: Eighty-five MDR M. tuberculosis samples collected from 2013 to 2017 at the University Teaching Hospital were used. Drug-resistance associated gene sequencing, spoligotyping, 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), and multiplex PCR for RD-Rio sub-lineage identification were applied. RESULTS: The identified clades were LAM (48%), CAS (29%), T (14%), X (6%) and Harlem (2%). Strains belonging to SITs 21/CAS1-Kili and 20/LAM1 formed the largest clonal complexes. Combined spoligotyping and 24 loci-MIRU-VNTR revealed 47 genotypic patterns with a clustering rate of 63%. Ninety-five percent of LAM strains belonged to the RD-Rio sub-lineage. CONCLUSION: The high clustering rate suggested that a large proportion of MDR-TB was due to recent transmission rather than the independent acquisition of MDR. This spread was attributed to clonal expansion of SIT21/CAS1-Kili and SIT20/LAM1 strains. Therefore, TB control programs recommending genotyping coupled with conventional epidemiological methods can guide measures for stopping the spread of MDR-TB.
• Whole-Genome Sequencing Reveals Recent Transmission of Multidrug-Resistant Mycobacterium tuberculosis CAS1-Kili Strains in Lusaka, Zambia.

  Joseph Yamweka Chizimu, Eddie Samuneti Solo, Precious Bwalya, Wimonrat Tanomsridachchai, Herman Chambaro, Misheck Shawa, Thoko Flav Kapalamula, Patrick Lungu, Yukari Fukushima, Victor Mukonka, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki

  Antibiotics (Basel, Switzerland) 11 (1) 2021/12/28 

  Globally, tuberculosis (TB) is a major cause of death due to antimicrobial resistance. Mycobacterium tuberculosis CAS1-Kili strains that belong to lineage 3 (Central Asian Strain, CAS) were previously implicated in the spread of multidrug-resistant (MDR)-TB in Lusaka, Zambia. Thus, we investigated recent transmission of those strains by whole-genome sequencing (WGS) with Illumina MiSeq platform. Twelve MDR CAS1-Kili isolates clustered by traditional methods (MIRU-VNTR and spoligotyping) were used. A total of 92% (11/12) of isolates belonged to a cluster (≤12 SNPs) while 50% (6/12) were involved in recent transmission events, as they differed by ≤5 SNPs. All the isolates had KatG Ser315Thr (isoniazid resistance), EmbB Met306 substitutions (ethambutol resistance) and several kinds of rpoB mutations (rifampicin resistance). WGS also revealed compensatory mutations including a novel deletion in embA regulatory region (-35A > del). Several strains shared the same combinations of drug-resistance-associated mutations indicating transmission of MDR strains. Zambian strains belonged to the same clade as Tanzanian, Malawian and European strains, although most of those were pan-drug-susceptible. Hence, complimentary use of WGS to traditional epidemiological methods provides an in-depth insight on transmission and drug resistance patterns which can guide targeted control measures to stop the spread of MDR-TB.
• Tuberculosis seroprevalence and comparison of hematology and biochemistry parameters between seropositive and seronegative captive Asian elephants of Nepal.

  Jeewan Thapa, Susan K Mikota, Kamal Prasad Gairhe, Sarad Paudel, Dinesh Kumar Singh, Ishwari Prasad Dhakal, Chie Nakajima, Yasuhiko Suzuki

  The Journal of veterinary medical science 83 (8) 1278 - 1283 2021/08/12 

  We conducted a tuberculosis (TB) serosurveillance program of captive elephants in Nepal and compared hematology and biochemistry parameters between seropositive and seronegative elephants. A total of 153 elephants (male=20, female=133) from four national parks were tested for TB using the ElephantTB STAT-PAK® Assay (ChemBio Diagnostic Systems, Inc., Medford, NY, USA). The mean reported age for 138 elephants was 38.5 years (range 2-71 years). Seroprevalence for TB was 21.56% (33/153). The majority of seropositive elephants were female (n=30) and from Chitwan National Park (n=29). The occurrence of TB seropositive cases in other more remote national parks suggests TB may be widespread among the captive elephant population of Nepal. Hematology and biochemistry analyses were performed on 13 and 22 seropositive elephants, respectively and, nine elephants from a seronegative TB herd for comparison. Hematology parameters (hemoglobin, packed cell volume, platelet, white blood cells, and erythrocyte sedimentation rate) were comparable between the two groups. Total protein, globulin, and lactate dehydrogenase were significantly higher in seronegative elephants, and bilirubin was significantly higher in seropositive elephants whereas blood urea nitrogen, creatinine, glutamic oxaloacetic transaminase/aspartate aminotransferase (GOT/AST), glutamic pyruvic transaminase/alanine aminotransferase (GPT/ALT), gamma glutamyl transferase (GT), and albumin were not significantly different. The range of biochemical parameters that were significantly different between seropositive and seronegative elephants had narrow ranges. Thus, the potential of these parameters as a direct biomarker for TB diagnosis is limited based on the findings in this study. We recommend including blood parameters in future TB surveillance studies.
• Wild ciliates differ in susceptibility to Legionella pneumophila JR32.

  Airi Kawashiro, Torahiko Okubo, Shinji Nakamura, Jeewan Thapa, Masaki Miyake, Hiroyuki Yamaguchi

  Microbiology (Reading, England) 167 (8) 2021/08 

  We investigated how Legionella pneumophila (Lp) JR32 interacts with Anteglaucoma CS11A and Colpoda E6, two ciliates that we isolated from sewage and sink trap sludge, respectively, using a handmade maze device containing a 96-well crafting plate. Our 18S rDNA-based phylogenetic analysis showed that Anteglaucoma CS11A and Colpoda E6 formed distinct clades. Scanning electron microscopy showed that Anteglaucoma CS11A had a bigger-sized body than Colpoda E6 and, unlike Tetrahymena IB (the reference strain), neither ciliate produced pellets, which are extracellular vacuoles. Fluorescence microscopic observations revealed that although the intake amounts differed, all three ciliates rapidly ingested LpJR32 regardless of the presence or absence of the icm/dot virulence genes, indicating that they all interacted with LpJR32. In co-cultures with Anteglaucoma CS11A, the LpJR32 levels were maintained but fell dramatically when the co-culture contained the LpJR32 icm/dot deletion mutant instead. Anteglaucoma CS11A died within 2 days of co-culture with LpJR32, but survived co-culture with the deletion mutant. In co-cultures with Colpoda E6, LpJR32 levels were maintained but temporarily decreased independently of the virulence gene. Concurrently, the Colpoda E6 ciliates survived by forming cysts, which may enable them to resist harsh environments, and by diminishing the sensitivity of trophozoites to Lp. In the Tetrahymena IB co-cultures with LpJR32 or Δicm/dot, the Lp levels were maintained, albeit with temporal decreases, and the Tetrahymena IB levels were also maintained. We conclude that unlike Tetrahymena IB with pellet production, Anteglaucoma CS11A can be killed by LpJR32 infection, and Colpoda E6 can resist LpJR32 infection through cyst formation and the low sensitivity of trophozoites to Lp. Thus, the two ciliates that we isolated had different susceptibilities to LpJR32 infection.
• Usefulness of a 3D-printing air sampler for capturing live airborne bacteria and exploring the environmental factors that can influence bacterial dynamics

  Saaya Mori, Sakura Ishiguro, Satoru Miyazaki, Torahiko Okubo, Ryosuke Omori, Ayako Kai, Kyohei Sugiyama, Airi Kawashiro, Masato Sumi, Jeewan Thapa, Shinji Nakamura, Chietsugu Katoh, Hiroyuki Yamaguchi

  Research in Microbiology 103864 - 103864 0923-2508 2021/07
• Evaluation of Xpert MTB/RIF with microscopy and culture for the diagnosis of tuberculosis in a referral laboratory in Nepal.

  Bhagwan Maharjan, Jeewan Thapa, Dhirendra Kumar Shah, Bhabana Shrestha, Korkut Avsar, Yasuhiko Suzuki, Chie Nakajima

  Japanese journal of infectious diseases 74 (6) 517 - 521 2021/03/31 

  Sputum microscopy and Xpert MTB/RIF are the primary rapid diagnostic methods for tuberculosis (TB) in Nepal. Disagreements among Xpert, microscopy, and culture, for example, cases with Xpert positive and microscopy negative, were frequently observed in Nepal including in our reference laboratory. The objective of this study was to compare the effectiveness of Xpert with culture and microscopy for TB diagnosis in Nepal. A total of 125 TB suspected sputum samples were processed for Xpert, microscopy, and culture. The Xpert results when compared with culture showed 100% sensitivity and 97.4% specificity with an excellent agreement (kappa = 0.96), whereas microscopy showed the sensitivity and specificity of 43.2% and 98.7%, respectively, with a moderate agreement (kappa = 0.4). The sensitivity and specificity of microscopy, when compared with Xpert, were 43.5% and 100%, respectively. The majority of Xpert positive samples of a medium MTB detection and all samples of low and very low MTB detection were missed by microscopy. Our study showed that Xpert MTB/RIF is a reliable tool for the diagnosis and management of TB in Nepal. Because of its high cost and sustainability, alternative simple and rapid diagnostic methods with a similar efficiency would be helpful for TB control in Nepal.
• Distribution of amoebal endosymbiotic environmental chlamydia Neochlamydia S13 via amoebal cytokinesis

  Miho Okude, Junji Matsuo, Tomohiro Yamazaki, Kentaro Saito, Yoshikazu Furuta, Shinji Nakamura, Jeewan Thapa, Torahiko Okubo, Hideaki Higashi, Hiroyuki Yamaguchi

  Microbiology and Immunology 65 (3) 115 - 124 0385-5600 2021/03
• Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis.

  Thoko Flav Kapalamula, Jeewan Thapa, Mwangala Lonah Akapelwa, Kyoko Hayashida, Stephen V Gordon, Bernard Mudenda Hang' Ombe, Musso Munyeme, Eddie Samuneti Solo, Precious Bwalya, Mirriam Ethel Nyenje, Aki Tamaru, Yasuhiko Suzuki, Chie Nakajima

  PLoS neglected tropical diseases 15 (1) e0008996  2021/01 

  Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.
• Hypoxia promotes Chlamydia trachomatis L2/434/Bu growth in immortal human epithelial cells via activation of the PI3K-AKT pathway and maintenance of a balanced NAD+/NADH ratio.

  Jeewan Thapa, Kent Hashimoto, Saori Sugawara, Ryoya Tsujikawa, Torahiko Okubo, Shinji Nakamura, Hiroyuki Yamaguchi

  Microbes and infection 22 (9) 441 - 450 2020/05/19 [Refereed][Not invited]
   

  Chlamydia trachomatis LGV (CtL2) causes systemic infection and proliferates in lymph nodes as well as genital tract or rectum producing a robust inflammatory response, presumably leading to a low oxygen environment. We therefore assessed how CtL2 growth in immortal human epithelial cells adapts to hypoxic conditions. Assessment of inclusion forming units, the quantity of chlamydial 16S rDNA, and inclusion size showed that hypoxia promotes CtL2 growth. Under hypoxia, HIF-1α was stabilized and p53 was degraded in infected cells. Moreover, AKT was strongly phosphorylated at S473 by CtL2 infection. This activation was significantly diminished by LY-294002, a PI3K-AKT inhibitor, which decreased the number of CtL2 progeny. HIF-1α stabilizers (CoCl2, desferrioxamine) had no effect on increasing CtL2 growth, indicating no autocrine impact of growth factors produced by HIF-1α stabilization. Furthermore, in normoxia, CtL2 infection changed the NAD+/NADH ratio of cells with increased gapdh expression; in contrast, under hypoxia, the NAD+/NADH ratio was the same in infected and uninfected cells with high and stable expression of gapdh, suggesting that CtL2-infected cells adapted better to hypoxia. Together, these data indicate that hypoxia promotes CtL2 growth in immortal human epithelial cells by activating the PI3K-AKT pathway and maintaining the NAD+/NADH ratio with stably activated glycolysis.
• Chlamydia trachomatis isolated from cervicovaginal samples in Sapporo, Japan, reveals the circulation of genetically diverse strains.

  Jeewan Thapa, Takanori Watanabe, Mana Isoba, Torahiko Okubo, Kiyotake Abe, Kunihiro Minami, Hiroyuki Yamaguchi

  BMC infectious diseases 20 (1) 53 - 53 2020/01/16 [Refereed][Not invited]
   

  BACKGROUND: This study was conducted to understand the molecular epidemiology of circulating Chlamydia trachomatis (Ct) strains in Sapporo, Japan. METHODS: A total of 713 endocervical samples collected from April 2016 to March 2019 were screened for Ct. The obtained Ct positive samples were analyzed by ompA genotyping and multilocus sequence analysis (MLSA). RESULTS: Eighty-three (11.6%) samples were positive for Ct plasmid DNA. Sequence analysis of the ompA gene from the 61 positive cases revealed eight genotypes: F (40.9%), E (19.6%), D (14.7%), G (9.8%), H (6.5%), I (3.2%), K (3.2%), and J (1.6%). The globally dominant genotype E and F strains were highly conserved with 13 ompA genetic variants being detected, whereas genotype D strains were the most diverse. Genetic characterization of D strains revealed that D1 genetic variants may be potentially specific to Sapporo. MLSA revealed 13 unique sequence types (STs) including four novel STs from 53 positive samples, with the globally dominant STs 39 and 19 being predominant. STs 39, 34, and 21 were exclusively associated with genotypes E and F indicating their global dominance. Novel ST70 and ST30 were specifically associated with genotype D. CONCLUSION: Our study has revealed the circulation of genetically diverse Ct strains in the women population of Sapporo, Japan. We suggest identifying a transmission network of those successful strains and implementing public health prevention strategies to control the spread of Ct in Sapporo.
• Effect of thermal control of dry fomites on regulating the survival of human pathogenic bacteria responsible for nosocomial infections

  Tomoko Shimoda, Torahiko Okubo, Yoshiki Enoeda, Rika Yano, Jeewan Thapa, Shinji Nakamura, Hiroyuki Yamaguchi

  PLoS ONE e0226952 2019/12 [Refereed][Not invited]
   

  ヒト病原細菌（ブドウ球菌、大腸菌ほか）が乾燥した素材(床材やステンレス)表面でどの程度生存するかを検証するとともに、その生存性に影響する因子として温度が大きく関わることを明らかにしました。単純な環境要因ですが、手摺やドアノブさらにベット柵など乾燥面の温度を調節することで院内感染の制御へと応用できるかもしれません。
• Screening of hospital-manhole sewages using MacConkey agar with cefotaxime reveals extended-spectrum β-lactamase-producing Escherichia coli.

  Okubo T, Hasegawa T, Fukuda A, Thapa J, Usui M, Tamura Y, Yamaguchi H

  Int J Antimicrob Agents In press 2019/08 [Refereed][Not invited]
  
• Direct detection of Mycobacterium tuberculosis in clinical samples by a dry methyl green loop-mediated isothermal amplification (LAMP) method.

  Thapa J, Maharjan B, Malla M, Fukushima Y, Poudel A, Pandey BD, Hyashida K, Gordon SV, Nakajima C, Suzuki Y

  Tuberculosis 117 1 - 6 2019/07 [Refereed][Not invited]
   

  The purpose of this study was to develop a simple visual methyl green (MeG) based dry loop-mediated isothermal amplification (LAMP) method for early detection of Mycobacterium tuberculosis (MTB) from clinical samples. We identified MeG as an indicator of a positive LAMP reaction, where a positive reaction gave a blue-green color while a negative reaction was colorless. The MeG MTB-LAMP system was further simplified by drying all reagents for ease of use, and was then validated for its ability to diagnose TB directly using Nepalese clinical samples. We evaluated the dry MeG MTB-LAMP with 69 new TB suspected samples from patients that did not have a confirmed history of TB treatment and found the sensitivity in culture positive samples as 92.8% (13/14) and specificity in culture negative samples as 96.3% (53/55). Our LAMP system has the potential to be a point of care test for early diagnosis of active TB in developing countries.
• Genetic diversity of Mycobacterium tuberculosis Central Asian Strain isolates from Nepal and comparison with neighboring countries.

  Shah Y, Poudel A, Maharjan B, Thapa J, Yamaguchi T, Diab HM, Pandey BD, Solo E, Isoda N, Suzuki Y, Nakajima C

  Transactions of the Royal Society of Tropical Medicine and Hygiene 113 (4) 203 - 211 0035-9203 2019/01 [Refereed][Not invited]
   

  BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is an emerging threat for successful tuberculosis control worldwide. Central Asian Strain (CAS) has been reported as one of the dominant families contributing to MDR-TB in South Asia including Nepal, India and Pakistan. The aim of this study was to better understand the genetic characteristics of MDR-TB CAS family isolates circulating in Nepal and compare the results with neighboring countries. METHODS: A total of 145 MDR-TB CAS family isolates collected in Nepal from 2008 to 2013 were analyzed by spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) analysis. In addition, we compared these data with published data from India and Pakistan to investigate a possible epidemiological link via construction of a minimum spanning tree (MST). RESULTS: Spoligotyping analysis exhibited CAS1_Delhi SIT26 (n=60) as the predominant lineage among the MDR-TB CAS family in all three countries. However, the combined analysis with spoligotyping and MIRU-VNTR further discriminated 60 isolates into 49 different types and 5 clusters. Each cluster was composed of 14 isolates with a clustering rate of 23.3%, suggesting ongoing transmissions. Based on MST data from neighboring countries, we elucidated an evolutionary relationship between the two countries, Nepal and India, which could be explained by their open border. CONCLUSION: This study identified the evolutionary relationships among MDR-TB CAS1_Delhi subfamily isolates from Nepal and those from neighboring countries.
• Serodiagnosis of elephant tuberculosis: a useful tool for early identification of infected elephants at the captive-wild interface

  Sarad Paudel, Susan K. Mikota, Jeewan Thapa, Konstantin P. Lyashchenko, Kamal P. Gairhe, Ishwari Prasad Dhakal, Naresh Subedi, Bhagwan Maharjan, Suraj Subedi, Gretchen E. Kaufman, Toshio Tsubota

  European Journal of Wildlife Research 64 (6) 1612-4642 2018/12 [Refereed][Not invited]
   

  Tuberculosis (TB) is an emerging disease in elephants primarily caused by Mycobacterium tuberculosis (M. tb) and in some occassion by M. bovis. We performed culture and three serological teststhe Elephant TB STAT-PAK,(R) DPP VetTB (R) Assay, and MAPIA (multi-antigen print immunoassay)prospectively on samples from eight elephants in Nepal that died of suspected or confirmed tuberculosis (TB) between 2007 and 2013. Among them, all elephants were reactive to DPP VetTB (R) Assay, five to Elephant TB STAT-PAK,(R) and two were reactive to MAPIA. Similarly, six elephants were positive on culture on samples collected antemortem or postmortem. We observed antibody responses months to years before culture confirmation of TB which shows that serological tests can be highly useful for the early diagnosis of TB in elephants. Validated point-of-care serological tests are easily performed in the field and hold promise for improved TB surveillance in other non-domestic species.
• Genetic diversity and distribution dynamics of multidrug-resistant Mycobacterium tuberculosis isolates in Nepal

  Maharjan B, Nakajima C, Isoda N, Thapa J, Poudel A, Shah Y, Yamaguchi T, Shrestha B, Hoffmann H, Avsar K, Shrestha A, Gordon SV, Suzuki Y

  Scientific Reports 8 (16634) 1 - 10 2018/11 [Refereed][Not invited]
   

  Multidrug-resistant tuberculosis (MDR-TB) is an emerging public health problem in Nepal. Despite the implementation of a successful TB control program in Nepal, notifications of MDR-TB are increasing, yet the reasons are unknown. The objective of this study was to understand the genetic diversity and epidemiological characteristics of MDR-Mycobacterium tuberculosis (MTB) isolates in Nepal. We isolated and genotyped 498 MDR-MTB isolates collected from April 2009 to March 2013 and analyzed the patients' background information. Our results showed that the lineage 2 (Beijing family) was the most predominant lineage (n = 241; 48.4%), followed by lineage 3 (n = 153, 30.7%). Lineage 4 was the third most prevalent (n = 73, 14.5%) followed by lineage 1 (n = 32, 6.4%). The lineages were significantly associated with geographic region, ethnic group, age and sex of patients. The Beijing genotype was found to have an important role in transmitting MDR-TB in Nepal and was significantly associated with the eastern region, mongoloid ethnic group and younger age group. We conclude that early diagnosis and treatment including molecular-epidemiological surveillance of MDR-TB cases will help to control transmission of MDR-TB in Nepal.
• Characterization of mutations conferring streptomycin resistance to multidrug-resistant Mycobacterium tuberculosis isolates from Myanmar

  Nan Aye Thida Oo, Lai Lai San, Jeewan Thapa, Khin Saw Aye, Wah Wah Aung, Chie Nakajima, Yasuhiko Suzuki

  Tuberculosis 111 8 - 13 1873-281X 2018/07/01 [Refereed][Not invited]
   

  Numerous studies report that mutations of rpsL (encoding the S12 protein), rrs (encoding 16S rRNA) and gidB (encoding rRNA methyltransferase) are responsible for conferring resistance to streptomycin (STR), which is usually used in both multidrug-resistant tuberculosis (MDR-TB) treatments and re-treatments in Myanmar. The aim of this study was to explore the variation and frequency of mutations in rpsL, rrs and gidB in 141 STR-resistant MDR-TB isolates from Myanmar. Most isolates belonged to the Beijing genotype (105, 74.5%). Moreover, mutations in rpsL were identified in 69.5% (98/141) of the STR-resistant isolates, where the most prevalent (92.0%, 90/98) and significantly associated mutation with the Beijing genotype (P < 0.001) was Lys43Arg. Fifteen different mutations in gidB were found in 16.3% (23/141) of the isolates, and most of them were novel mutations. Moreover, based on our results, we suggest A276C nucleotide substitution in gidB as a phylogenetic marker for the Beijing family in Myanmar. Sequence analysis of rpsL, rrs and gidB with a sensitivity of 83.7% satisfactorily predicted STR resistance in Myanmar isolates. However, in 16.3% (23/141) of the isolates, none of the examined genes showed mutation. Hence, further studies are strongly recommended to elucidate other possible resistance mechanisms. The present findings may be useful in developing molecular STR susceptibility assays, which in turn could contribute to develop TB treatments and control strategies in Myanmar.
• Tuberculosis Caused by Mycobacterium orygis in Dairy Cattle and Captured Monkeys in Bangladesh: a New Scenario of Tuberculosis in South Asia

  Z. Rahim, J. Thapa, Y. Fukushima, A. G. M. van der Zanden, S. V. Gordon, Y. Suzuki, C. Nakajima

  TRANSBOUNDARY AND EMERGING DISEASES 64 (6) 1965 - 1969 1865-1674 2017/12 [Refereed][Not invited]
   

  Mycobacterium orygis, commonly known as the oryx bacillus and a newly proposed Mycobacterium tuberculosis complex subspecies, was isolated from 18 cattle in a dairy farm and two captured rhesus monkeys in a zoo in Bangladesh. All the infected animals had tuberculosis lesions in their lungs, suggesting transmission and infection with M.orygis by an airborne route. The 20 isolates were analysed using a range of conventional and molecular typing methods, and RD-deletion typing and sequencing of selected genes confirmed the isolates as M.orygis. Multiple-locus variable-number tandem repeat analysis (MLVA) allowed the isolates to be divided into three clusters based on the relatedness of their MLVA profiles. The two monkey isolates shared the same MLVA pattern with 15 of the cattle isolates, whereas the remaining three cattle isolates had different patterns, even though the latter animals had been kept in the same dairy farm. The diversity observed among isolates may suggest the bacteria have been established in this area for a long period. This study along with other recent findings that report the detection of M.orygis from animals as well as humans originating from South Asia potentially indicate endemic distribution of M.orygis in South Asia.
• High diversity of multidrug-resistant Mycobacterium tuberculosis Central Asian Strain isolates in Nepal

  Yogendra Shah, Bhagwan Maharjan, Jeewan Thapa, Ajay Poudel, Hassan Mahmoud Diab, Basu Dev Pandey, Eddie S. Solo, Norikazu Isoda, Yasuhiko Suzuki, Chie Nakajima

  INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES 63 13 - 20 1201-9712 2017/10 [Refereed][Not invited]
   

  Objectives: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) poses a major public health problem in Nepal. Although it has been reported as one of the dominant genotypes of MTB in Nepal, little information on the Central Asian Strain (CAS) family is available, especially isolates related to multidrug resistance (MDR) cases. This study aimed to elucidate the genetic and epidemiological characteristics of MDR CAS isolates in Nepal. Methods: A total of 145 MDR CAS isolates collected in Nepal from 2008 to 2013 were characterized by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis, and drug resistance-associated gene sequencing. Results: Spoligotyping analysis showed CAS1_Delhi SIT26 as predominant (60/145, 41.4%). However, by combining spoligotyping and MIRU-VNTR typing, it was possible to successfully discriminate all 145 isolates into 116 different types including 18 clusters with 47 isolates (clustering rate 32.4%). About a half of these clustered isolates shared the same genetic and geographical characteristics with other isolates in each cluster, and some of them shared rare point mutations in rpoB that are thought to be associated with rifampicin resistance. Conclusions: Although the data obtained show little evidence that large outbreaks of MDR-TB caused by the CAS family have occurred in Nepal, they strongly suggest several MDR-MTB transmission cases. (C) 2017 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
• Mycobacterium orygis-Associated Tuberculosis in Free-Ranging Rhinoceros, Nepal, 2015

  Jeewan Thapa, Sarad Paudel, Amir Sadaula, Yogendra Shah, Bhagwan Maharjan, Gretchen E. Kaufman, Deborah McCauley, Kamal P. Gairhe, Toshio Tsubota, Yasuhiko Suzuki, Chie Nakajima

  EMERGING INFECTIOUS DISEASES 22 (3) 570 - 572 1080-6040 2016/03 [Refereed][Not invited]
  
• Molecular characterization of Mycobacterium orygis isolates from wild animals of Nepal

  Jeewan Thapa, Chie Nakajima, Bhagwan Maharjan, Ajay Poudel, Yasuhiko Suzuki

  JAPANESE JOURNAL OF VETERINARY RESEARCH 63 (3) 151 - 158 0047-1917 2015/08 [Refereed][Not invited]
   

  Mycobacterium orygis, a new member of the Mycobacterium tuberculosis complex, was isolated from a captive spotted deer (Axis axis) and a blue bull (Boselaphus tragocamelus) in Nepal. Analyses by spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing, region of difference and single nucleotide polymorphism of genes gyrB, mmpL6, TbD1, PPE55 and Rv2042c confirmed the isolates as M. orygis. Moreover, analyses by spoligotyping (SIT587) as well as MIRU-VNTR showed that the isolates shared a similar pattern with many reported isolates. From previous and the present studies, it can be inferred that South Asia is one of the endemic regions for M. orygis. Further investigation including a larger sample size and different host interaction will help to understand the ecology and epidemiology of M. orygis in Nepal.
• Molecular characterization of Mycobacterium tuberculosis isolates from elephants of Nepal

  Sarad Paudel, Susan K. Mikota, Chie Nakajima, Kamal P. Gairhe, Bhagwan Maharjan, Jeewan Thapa, Ajay Poudel, Michito Shimozuru, Yasuhiko Suzuki, Toshio Tsubota

  TUBERCULOSIS 94 (3) 287 - 292 1472-9792 2014/05 [Refereed][Not invited]
   

  Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts. (C) 2014 Elsevier Ltd. All rights reserved.

Books etc

• Wildlife Tuberculosis: An Emerging Threat for Conservation in South Asia

  Jeewan Thapa, Chie Nakajima, Kamal P. Gairhe, Bhagwan Maharjan, Sarad Paudel, Yogendra Shah, Susan K. Mikota, Gretchen E. Kaufman, Deborah McCauley, Toshio Tsubota, Stephen V. Gordon, Yasuhiko Suzuki (Joint workEnglish)
  www.intechopen.com 2017/03