Molecular epidemiology of scrub typhus in Taiwan during 2006-2016.

Scrub typhus is the most common endemic vector-borne disease in Taiwan. We identified a total of 4,857 laboratory-confirmed cases during 2006-2016 with hyperendemic foci on offshore islands, including Penghu (778 cases, 16.0%) and Kinmen (716 cases, 14.7%), and eastern Taiwan, including Taitung (628 cases, 12.9%) and Hualien (508 cases, 10.5%). Scrub typhus cases occur year-round throughout Taiwan, with a summer peak in June and July. A total of 545 O. tsutsugamushi isolates were successfully obtained from patients infected in diverse geographic areas, including Taiwan and three offshore islands, and the complete open reading frame of the 56 kDa type-specific antigen gene (tsa56) sequence of these isolates was examined. High phylogenetic diversity was found in these isolates, which could be grouped into 36 distinct sequence types. Most isolates belonged to the Karp (49.9%; 272/545), followed by the TW-22 (17.8%; 97/454) and Kawasaki (14.7%; 80/545) genotypes. In conclusion, our data indicate the widespread presence of tsa56 genotypes closely related to Thailand and Korean strains and the presence of the unique endemic strains TW-12, TW-22, TW-29, and TW-36 in Taiwan.

Introduction

Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium Orientia tsutsugamushi contracted from the bite of an infected larval-stage trombiculid mite (chigger) [1]. Scrub typhus is endemic to the Asia-Pacific region in an area known as the “tsutsugamushi triangle”, Taiwan being located in the center. Globally, it has been estimated that one billion people are at risk of scrub typhus and that one million infections occur every year [2,3]. Symptoms of scrub typhus include fever, headache, rash, eschar, cough, myalgias, nausea, vomiting, and abdominal pain. Severe manifestations may include pneumonitis, meningitis, encephalitis, disseminated intravascular coagulation, septic shock, myocarditis, and multiorgan failure [4-6]. The case fatality rate can be up to 30–70% if not treated appropriately [7-9]. Currently, there is no commercially available vaccine.

Scrub typhus is the most common rickettsial disease in Taiwan and has been designated a notifiable infectious disease since 1955. Blood and serum samples from suspected scrub typhus patients are sent to the Taiwan Centers for Disease Control (Taiwan CDC) for diagnosis. Serological diagnosis has been performed using indirect immunofluorescent assay (IFA) since 1955, using paired sera when available. Since 2006, molecular diagnosis has been performed using real-time polymerase chain reaction (qPCR) for rapid testing and increased sensitivity with whole-blood specimens, from which bacterial isolation is also routinely performed. Scrub typhus cases are reported weekly through the National Notifiable Disease Surveillance System (nidss.cdc.gov.tw), and the number of laboratory-confirmed cases of scrub typhus exceeds 300 cases annually.

The immunodominant 56 kDa type-specific antigen gene (tsa56) of O. tsutsugamushi has been the most widely used gene target for phylogenetic analysis because of its sequence variation [10-13]. Previous studies have revealed high phylogenetic diversity among tsa56 genotypes in Taiwan [11, 14]. We described 116 clinical isolates and 22 distinct tsa56 sequence types during 2006–2007 [11]. Currently, the genotypic diversity and molecular epidemiology of O. tsutsugamushi remain unclear, including the distribution of tsa56 genotypes by region, its seasonality, and the clinical manifestations associated with infection by different tsa56 genotypes. Here, we studied the tsa56 genotypes of 545 O. tsutsugamushi clinical isolates throughout Taiwan, including offshore islands, from 2006 to 2016 to elucidate the molecular epidemiology of scrub typhus in Taiwan.

Materials and methods

Ethics statement

The study protocol was approved by the Taiwan Centers for Disease Control Institutional Review Board (IRB 106111).

Human blood samples

Isolates in this study were obtained from blood samples of confirmed cases of scrub typhus infection from 2006 to 2016. Sample data were depersonalized for anonymity. Samples were considered positive for scrub typhus with a positive real-time polymerase chain reaction (PCR) test or IFA test, indicated by a ≥ 4-fold increase in O. tsutsugamushi-specific immunoglobulin M (IgM) or IgG antibody in paired sera.

DNA extraction and real-time PCR

Peripheral blood mononuclear cells (PBMCs) were purified from 4 mL of whole blood samples using Ficoll-Paque Plus (GE Health care Bio-Sciences AB, Uppsala, Sweden) according to the manufacturer’s instructions, washed and resuspended in 400 μL of phosphate-buffered saline (PBS) containing 2% fetal calf serum. DNA extraction was performed with the QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions with 200 μL of each PBMC suspension. Two SYBR-based qPCR assays were used to test for O. tsutsugamushi, targeting tsa56 (RST-14F: 59-CCATTTGGTGG TACATTAGCTGCAGGT-39; RST-6R: 59-TCACGATCAGC TATACTTATAGGCA-39) and the 16S ribosomal RNA gene (rrs) (OTF7: 59-CCAGYGGGTRATGCCGGGAACTAT-39; OTR6: 59GGCAGTGTGTACAAGGCCCGAGAA-39), performed using the Fast Start Essential DNA Green Master kit (Roche Diagnostics, Basel, Switzerland). Samples were considered positive if both targets were amplified.

Isolation of O. tsutsugamushi

PBMCs collected from acute-phase blood samples of scrub typhus patients were used for isolation of O. tsutsugamushi. Bacterial isolation in cell culture was performed using the centrifugation shell vial technique as described previously [15,16]. Briefly, O. tsutsugamushi was propagated in L929 mouse fibroblast cells (ATCC CCL-1, NCTC Clone 929) at 32°C in a 5% CO2 incubator for 10 to 14 days and then detected by IFA using an O. tsutsugamushi-specific antibody. Each positive shell vial was harvested and inoculated into a T-25 flask containing a monolayer of confluent L929 cells. After 14–20 days of incubation, the bacteria-infected L929 cells were scraped up and frozen at −80°C. Isolated bacteria were identified using the nomenclature OT/country of origin/strain/year of isolation.

Indirect IFA

O. tsutsugamushi whole-cell antigens Karp, Gilliam, and Kato strains dotted on Teflon-coated spot glass slides were used for IFA as previously described [17]. Briefly, whole-cell antigens were fixed and permeabilized with ice-cold acetone/methanol (1:1) for 10 minutes, and the slides were air-dried and blocked with PBS containing 1% goat serum. Serum samples were serially diluted and incubated in a humidified atmosphere for 30 minutes at 37°C. Subsequently, fluorescein isothiocyanate–conjugated anti-human IgM and IgG (Sigma, St. Louis, MO, USA) were diluted with PBS containing Evans blue counterstain (Sigma Chemical Company, St. Louis, MO, USA) and applied to an antigen-coated spot in a humidified atmosphere for 30 minutes at 37°C. The slides were examined by epifluorescence microscopy (Zeiss, Axio Imager 2, Jena, Germany) by two observers at a magnification of ×400. The binding endpoint titer was determined as the highest dilution with a positive fluorescence reaction.

PCR amplification and nucleotide sequencing

PCR and DNA sequencing of the complete tsa56 ORF was performed as previously described [17]. Briefly, bacterial DNA was extracted from O. tsutsugamushi-infected L929 cells using the QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions and stored at -80°C. Primers used for PCR and nucleotide sequencing were as previously described [17]. PCR amplification was performed in 50 μL volumes using the QIAGEN Taq PCR Core Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. PCR products were purified using a QIAQuick Gel Extraction Kit (QIAGEN, Hilden, Germany). Nucleotide sequences were determined by an automated DNA sequencing kit and an ABI Prism 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocols. Overlapping nucleotide sequences were combined for analysis and edited with the Lasergene software package (DNASTAR Inc, Madison, WI, USA). Nucleotide sequences were submitted to GenBank. The strain identifiers and their accession numbers are listed in S1 Table.

Phylogenetic analysis

Complete tsa56 nucleotide sequences obtained in this study were aligned with global tsa56 sequences retrieved from GenBank using Clustal W software. Phylogenetic analysis was conducted using MEGA version 7 (http://www.megasoftware.net/) [18] with the neighbor-joining method and the maximum composite likelihood as a substitution model. One thousand bootstrap replicates were performed to estimate the node reliability of the phylogenetic tree, and bootstrap support values above 75 were considered significant.

Results

Epidemiology of scrub typhus in Taiwan 2006–2016

A total of 28,626 suspected cases were sent to the Taiwan CDC for confirmation of scrub typhus infection 2006–2016; among them, 4,857 cases were confirmed by laboratory diagnosis. Fig 1 shows the annual numbers of confirmed cases of scrub typhus and the incidence per 100,000 persons during 2006–2016. Confirmed cases of scrub typhus were between 322 and 538 per year. Fig 2 shows the geographic distribution of the scrub typhus cases in Taiwan and the offshore islands. Penghu County had the highest number of scrub typhus cases (778 cases; 16.0%), followed by Kinmen County (716 cases; 14.7%) and Taitung County (628 cases; 12.9%). Fig 3 shows the monthly distribution of scrub typhus cases. Scrub typhus occurs throughout the year in Taiwan. There were two peaks, a major peak in July and a small peak in October. Fig 4 shows the gender and age distribution of the confirmed cases. A total of 3,027 cases (62.4%) were male, and 1,825 (37.6%) cases were female, with a male-to-female ratio of 1.66:1. There were 1042 cases (21%) that occurred in 50–59 years old, 848 cases (19.5%) in 40–49 years old, and 45.3% of cases occurred in those older than 50 years old, and 3.6% of cases occurred among those under 9 years old.

Fig 1

Annual numbers of confirmed cases of scrub typhus cases in Taiwan 2006–2016.

There is an increasing trend of O. tsutsugamushi infection in Taiwan.

Fig 2

Geographic distribution of scrub typhus cases in Taiwan 2006–2016.

Most cases were identified in eastern Taiwan (Hualien and Taitung Counties) and on offshore islands (Penghu and Kinmen Counties). The geographic map was acquired from the Taiwan Centers for Disease Control Open Data Platform (https://nidss.cdc.gov.tw).

Fig 3

Monthly distribution of scrub typhus cases in Taiwan 2006–2016.

Scrub typhus occurs year-round and peaks in the spring and fall seasons.

Fig 4

Gender and age distribution of confirmed cases in Taiwan 2006 to 2016.

Phylogenetic analysis of O. tsutsugamushi strains

A total of 545 O. tsutsugamushi isolates were obtained from scrub typhus cases during 2006–2016 with tsa56 sequences. The phylogenetic analysis classified these isolates into 36 distinct sequence types according to their sequence similarities being higher than 98% (Fig 5). The accession numbers of the identified strains are listed in S1 Table. Phylogenetic analysis revealed that most isolates were grouped into the Karp genotype, including TW-1 to TW-8, TW-23 to TW-26, and TW-31 to TW-33, which are closely related to tsa56 sequences from Thailand, Korea, Cambodia, and New Guinea (Table 1). Strains TW-13 to TW-19 and TW-30 were similar to the Kawasaki genotype and were closely related to tsa56 sequences from Thailand, Japan and China, respectively. TW-9 was identified as the Kuroki genotype and was most closely related to Boryong and Kuroki strains from Korea and Japan, respectively. TW-10 to TW-11 and TW-27 to TW-28 were clustered with TA763-type strains with sequence similarity to the TA763 strain isolated from Thailand and Vietnam. TW-20 and TW-21 belong to the Kato genotype and were most closely related to strains from Malaysia and Japan, respectively. TW-34 was closely related to the Gilliam strain isolated in Cambodia. We found that TW-12, TW-22, TW-29, and TW-36 were unique in Taiwan and also distinct from strains isolated from other countries.

Fig 5

Phylogenetic tree based on the 56 kDa TSA gene ORF of TW-1 to TW-36.

Table 1

The sequence type, representative strains, and phylogenetically closest foreign strains of Orientia tsutsugamushi.

Sequence type	Representative isolate	Length of ORF of gene	Isolation site of representative isolate	Isolation date (month/year) of representative isolate	Pairwise nucleotide sequence similarity (%) to phylogenetically closest foreign O. tsutsugamushi strain from NCBI	GenBank accession no.	Genotype	No. strain (2006–2016)
TW-1	KM0605a	1608	Kinmen Island	05/2006	98.3% to UT150 (EF213086), Thailand	GQ332742	Karp	198
TW-2	TY0610a	1605	Taoyuan County	10/2006	97.4% to UT336 (EF213089), Thailand	GQ332743	Karp	4
TW-3	TP0607a	1605	Taipei County	07/2006	97.5% to Karp (M33004), New Guinea	GQ332744	Karp	1
TW-4	TP0708a	1608	Taipei County	08/2007	96.0% to UT336 (EF213089), Thailand	GQ332745	Karp	10
TW-5	KM0607h	1632	Kinmen Island	07/2006	95.8% to UT176 (EF213081), Thailand	GQ332746	Karp	9
TW-6	KHC0609c	1608	Kaohsiung City	09/2006	97.7% to UT176 (EF213081), Thailand	GQ332747	Karp	6
TW-7	KHC0606a	1608	Kaohsiung City	06/2006	96.4% to yeo-joo (AF430144), Korea	GQ332748	Karp	6
TW-8	CH0711a	1692	Changhua County	11/2007	96.3% to pa-joo (AF430142) Korea	GQ332749	Karp	7
TW-9	TPC0701a	1599	Taipei City	01/2007	99.7% to Boryong (AM494475), Korea	GQ332750	Kuroki	48
TW-10	KHC0704a	1566	Kaohsiung City	04/2007	93.8% to TA763 (U80636), Thailand	GQ332751	TA763	18
TW-11	NT0707a	1584	Nantou County	07/2007	96.7% to TA763 (U80636), Thailand	GQ332752	TA763	1
TW-12	TT0705a	1593	Taitung County	05/2007	86.8% to UT302 (EF213095), Thailand	GQ332753	TW-12	2
TW-13	NT0711a	1557	Nantou County	11/2007	92.6% to Sxh951 (AF050669), China	GQ332754	Kawasaki	3
TW-14	TT0711a	1551	Taitung County	11/2007	92.6% to Ikeda (AP008981), Japan	GQ332755	Kawasaki	1
TW-15	PT0712b	1569	Pingtung County	12/2007	99.3% to Kawasaki (M63383), Japan	GQ332756	Kawasaki	1
TW-16	KHC0707a	1572	Kaohsiung City	07/2007	97.2% to UT329 (EF213099), Thailand	GQ332757	Kawasaki	11
TW-17	TPC0707a	1596	Taipei City	07/2007	97.7% to UT125 (EF213096), Thailand	GQ332758	Kawasaki	2
TW-18	KHC0706a	1596	Kaohsiung City	06/2007	98.4% to UT125 (EF213096), Thailand	GQ332759	Kawasaki	5
TW-19	KM0606a	1572	Kinmen Island	06/2006	97.2% to UT125(EF213096), Thailand	GQ332760	Kawasaki	54
TW-20	HC0605a	1572	Hsinchu County	05/2006	99.9% to LF-1(AF173050), Malaysia	GQ332761	Kato	5
TW-21	KM0607b	1590	Kinmen island	07/2006	98.6% to Kato (M63382), Japan	GQ332762	Kato	6
TW-22	KHC0606b	1575	Kaohsiung City	06/2006	88.3% to FPW1038 (EF213087), Thailand	GQ332763	TW-22	97
TW-23	KM0806c	1611	Kinmen island	06/2008	97.7% to S0902151-KH (HQ718422), Cambodia	MW460713	Karp	4
TW-24	TN0807a	1602	Tainan City	07/2008	97.8% to UT336 (EF213089), Thailand	MW464199	Karp	12
TW-25	TT0908a	1605	Taitung County	08/2009	96.6% to UT336 (EF213089), Thailand	MW464200	Karp	3
TW-26	TT0910a	1605	Taitung County	10/2009	97.6% to UT176 (EF213081), Thailand	MW464201	Karp	6
TW-27	KM0807h	1605	Kinmen island	07/2008	97.6% to 45QN-VN (HQ817459), Vietnam	MW464202	TA763	5
TW-28	TPC0911a	1587	Taipei City	11/2009	96.5% to 02QNg-VN (HQ817449) Vietnam	MW464203	TA763	3
TW-29	CH0807a	1575	Changhua County	07/2008	87.0% to UT302 (EF213095), Thailand	MW464204	TW-29	4
TW-30	HL1004b	1572	Hualien County	04/2010	98.4% to UT329(EF213099), Thailand	MW464205	Kawasaki	3
TW-31	NT1211a	1602	Nantou County	11/2012	94.9% toUT219 (EF213100), Thailand	MW495810	Karp	2
TW-32	ML1307a	1599	Miaoli County	07/2013	96.4% to UT177 (EF213084), Thailand	MW495812	Karp	2
TW-33	CH1405b	1608	Changhua County	05/2014	99.2% to S0902151_KH HQ718422, Cambodia	MW495814	Karp	1
TW-34	TCC1505a	1599	Taichung City	05/2015	95.1% to S0617100_KH (HQ718421) Cambodia	MW495815	Gilliam	1
TW-35	NT1610a	1608	Nantou County	10/2016	97.5% to UT176 (EF213081),Thailand	MW495816	Karp	1
TW-36	NT1008a	1581	Nantou County	08/2010	86.5% to UT302 (EF213095), Thailand	MW495805	TW-36	3
India/0809aTw		1599	India	09/2008	98.9% to UT219 (EF213100), Thailand	MW495817	Karp	1

Geographic distribution of O. tsutsugamushi isolates

Table 2 shows the geographic distribution of O. tsutsugamushi isolates in Taiwan. TW-1 represents the most abundant O. tsutsugamushi isolates in Taiwan, especially on offshore islands. Forty-eight isolates were grouped into TW-9, distributed in the northern, central and eastern parts of Taiwan but not in southern Taiwan or the offshore islands. TW-19 and TW-22 contained 54 and 97 isolates, respectively, and were widely distributed on Taiwan’s main island and the Kinmen and Lienchiang islands.

Table 2

Geographic distribution of sequence types.

sequence	North						Central					South				East		Offshore			Total
type	YL	KL	TP	NTC	TY	HC	ML	TC	CH	NT	WL	CY	TN	KH	PT	TT	HL	KM	PH	LC
TW-1			1	8	7		1	3	1				1	8	4	23	8	100	13	20	198
TW-2				1	1											2					4
TW-3			1																		1
TW-4		1		1						1							4	3			10
TW-5																		9			9
TW-6											1			4	1						6
TW-7										1				3	1	1					6
TW-8									1					1	1				4		7
TW-9		2	4	6	5	4	6	6		8						4	3				48
TW-10		1	1	2	1									3		6	4				18
TW-11										1											1
TW-12																2					2
TW-13										3											3
TW-14																1					1
TW-15															1						1
TW-16					1			1						1		3			1	4	11
TW-17			1															1			2
TW-18														4	1						5
TW-19			1	1			1	3	3	10			1	4	1	5	13	9		2	54
TW-20		1				1								1			2				5
TW-21				1														5			6
TW-22				3	2	1		1	1			1	3	40	3	8	3	30		1	97
TW-23																		4			4
TW-24				1	2			1					2	2		4					12
TW-25										2						1					3
TW-26			1													1	4				6
TW-27														1		1		2		1	5
TW-28			1													2					3
TW-29					1	1			1							1					4
TW-30																	3				3
TW-31													1				1				2
TW-32							1					1									2
TW-33									1												1
TW-34								1													1
TW-35										1											1
TW-36										1		1						1			3
Sum	0	5	11	24	20	7	9	16	8	28	1	3	8	72	13	65	45	164	18	28	545

In this study, TW-11, TW-13, and TW-33 to TW-35 were found in Central Taiwan, whereas TW-15 and TW-18 were restricted to Southern Taiwan. TW-12, TW-14, and TW-30 were common in Eastern Taiwan, and TW-5 and TW-23 were discovered only on Kinmen Island. Taken together, most of the isolates came from Kinmen Island (n = 164). Kaohsiung City had the second-highest number of isolates (n = 72), TW-22 was the major sequence type, and a variety of sequence types were also found in this city.

Monthly distribution of O. tsutsugamushi sequence types

The monthly distribution of O. tsutsugamushi sequence types is shown in Table 3. Most of the cases of scrub typhus occurred in the warm season (April to October), representing most sequence types. On the other hand, a few sequence types, including TW-9, TW-14, and TW-15, occurred in the cold season (November to February). It is worth noting that TW-22 was widely found from March to December. Nevertheless, TW-12, TW-29, and TW-36 are only found from May to September. Taken together, scrub typhus occurs throughout the year, and O. tsutsugamushi shows great genotype diversity.

Table 3

Monthly distribution of Orientia tsutsugamushi sequence types in Taiwan.

Sequencetype	Jan	Feb	Mar	Apr	May	Jun	Jul	Aug	Sep	Oct	Nov	Dec	Total
TW-1				7	20	47	59	16	18	15	15	1	198
TW-2					1	1			1	1			4
TW-3							1						1
TW-4				1	2	1	2	2		1	1		10
TW-5						1	5	1	1	1			9
TW-6					1		1	1	1	1	1		6
TW-7						1	3	1				1	6
TW-8							2			1	4		7
TW-9	12	6	1								7	22	48
TW-10				2	3	3	2	3		1	1	3	18
TW-11								1					1
TW-12					1	1							2
TW-13						1	1				1		3
TW-14											1		1
TW-15												1	1
TW-16		1			1	3		4	1	1			11
TW-17							2						2
TW-18						1	1		1		2		5
TW-19			3	5	4	6	11	4	9	1	10	1	54
TW-20					2		1	1	1				5
TW-21						1	2		2	1			6
TW-22			1	2	8	15	20	12	18	14	5	2	97
TW-23						2	1		1				4
TW-24				1	5	1	3		1	1			12
TW-25								1		1	1		3
TW-26							1		1	2	2		6
TW-27							1		2	2			5
TW-28								1			2		3
TW-29						1	2			1			4
TW-30				1		1					1		3
TW-31									1		1		2
TW-32							1	1					2
TW-33					1								1
TW-34					1								1
TW-35										1			1
TW-36								2	1				3
Sum	12	7	5	19	50	87	122	51	60	46	55	31	545

Clinical symptoms of the sequence types

The clinical symptoms of the patients are shown in Table 4. The most frequent symptoms included fever (89.5%, 488/545), headache (28.6%, 156/545), rashes (25.3%, 138/545), eschar (25.0%, 136/545), lymphadenopathy (9.5%, 52/545) and liver dysfunction (7.9%, 43/545). Severe manifestations, including sepsis, pneumonia, liver, and kidney dysfunction, were also observed in some patients. Sepsis was observed in TW-1, TW-22, and TW-24. Kidney dysfunction was observed in TW-1 and TW-30. Consciousness changes were caused by TW-1 and TW-22. Overall, patients infected with TW-1 and TW-22 displayed more complicated syndromes and severe illness in Taiwan during the 2006–2016 surveillance.

Table 4

Clinical symptoms of TW-1 to TW-36 strain isolates.

sequence type	TW -1	TW -2	TW -3	TW -4	TW -5	TW -6	TW -7	TW -8	TW -9	TW -10	TW-11	TW-12	TW-13	TW-14	TW-15	TW-16	TW-17	TW-18	TW-19	TW-20	TW-21	TW-22	TW-23	TW-24	TW-25	TW-26	TW-27	TW-28	TW-29	TW-30	TW-31	TW-32	TW-33	TW-34	TW-35	TW -36	Sum
Fever	172	3	1	9	9	6	6	6	45	16	1	2	3	1	1	11	2	5	49	5	4	87	3	11	3	5	5	3	3	1	2	2	1	1	1	3	488
Eschar	9	2		4	3			3	14	6		1	2			5			16	2	3	42	3	4	2	2	2	3	2	1	2			1	1	1	136
Rashes	32	2		2	1	3	2		30	6		1	2		1	6	1	3	18		1	11		4	2				2	1	1	2	1	1		2	138
Headache	45		1	3	2	2	2	3	12	6	1		2			3		1	21	2	1	33	1	3	3	1	1	2	2	1						2	156
Lymphadenopathy	17	2				1		1	5	1		1	1			3			6		1	6		2	1				3		1						52
Abdominal pain	5			1			1	1	3							1				1				1													14
Diarrhea	6			1				1											1			1		1													11
Vomiting	3									1									1																		5
Malaise	8							2	1	1									5			4		3						1							25
Chills	10			1					1	1	1								3			7		1									1			1	27
Dyspnea	3																		3							1				1							8
Cough	8									1									2	1		4		1		1											18
Sore throat	2								1	1	1											6				1											12
Myalgia	10					1		1	2	1									7			7		2	1	1			1						1		35
Arthralgia	1					1	1			1	1								1			5										1					12
Drowsiness																			2																		2
Sepsis	2																					1		1													4
Pneumonia	2																																				2
Consciousness change	1																					1															2
Liver dysfunction	13			1		1	1	2	1	3						1		1	4			10		3	1										1		43
Jaundice	3								1	1												2		2		1					1	1					12
Kidney dysfunction	3																													1							4
Poor appetite	1						1												1			3															6
Conjunctivitis				1								1							1			3															6
sequence sum	198	4	1	10	9	6	6	7	48	18	1	2	3	1	1	11	2	5	54	5	6	97	4	12	3	6	5	3	4	3	2	2	1	1	1	3	545

Discussion

Scrub typhus was first reported in 1908 [19]. There are approximately 350 scrub typhus cases in Taiwan annually. In the present epidemiological study, we analyzed 4875 human cases collected from 2006–2016 and found that eastern Taiwan and the offshore islands displayed a higher prevalence. In particular, offshore islands accounted for 34.3% of the total cases, with most outbreaks occurring in rural regions. These results demonstrated that disease transmission was highly associated with seasonal characteristics [14]. The prevalent period in early spring to late fall follows the adult mites’ prosperous growth and breeding season. In addition, people often take vacation trips during summer seasons, thereby being exposed to the infected chiggers and acquiring scrub typhus. Men have a higher rate of typhus than women, possibly reflecting that men are more frequently exposed to the chiggers’ living environments.

In this study, we isolated 545 indigenous O tsutsugamushi strains from acute-phase blood samples of scrub typhus cases and analyzed the gene sequences of the TSA56 protein. The TSA56 gene sequences were classified into 36 sequence types. Most of these isolates were closely related to strains from the southern region of the endemic area (Thailand, Vietnam, Malaysia, and New Guinea), while others were closely related to the northern region of the endemic area (northern China, Japan, and Korea). Notably, four sequence types, TW-12, TW-22, TW-29, and TW-36, are unique in Taiwan; it may be worthwhile to obtain their entire genome sequence and determine their epidemiology and phylogeny in the future.

Taiwan is an island off the southeastern coast of mainland China in the western Pacific Ocean. Taiwan is a mountainous island with one-third of the area over 1000 meters high and with more than two hundred peaks over 3000 meters. The range of landscapes and topography is varied and complicated in Taiwan. The climate varies with altitude, and the ecological environment is a complex system that is rich in flora and fauna [20]. The complex environment has been suggested to support the evolution and diversity of O. tsutsugamushi in Taiwan. Our study revealed that specific districts have dominant genotypes of O. tsutsugamushi. This may be ascribed to the variation of the vectors, at least in part, being controlled by geographical and seasonal factors. Wang and colleagues reported various chiggers linked to geographical and seasonal variation that act as potential vectors, leading to the formation of a dominant O. tsutsugamushi transmission chain in Taiwan [21]. In addition, invasive plants change the survival of certain vectors and affect the transmission of O. tsutsugamushi [22].

Continuous surveillance and analysis of TSA56 gene sequences may have beneficial effects in epidemiology and public health research on scrub typhus. In this study, scrub typhus was found with two sharp peaks in Taiwan, from May to July and September to November. Most O. tsutsugamushi isolates are obtained in the warm season (April to October), and their tsa56 sequences are closely related to strains identified in Southeast Asia. Nevertheless, TW-9, TW-14, and TW-15 sequence types were isolated in the cold season (November to February), and their tsa56 sequences are closely related to the northern area of the endemic region.

Fever, headache, and eschars are the most common symptoms of scrub typhus, essential in making the clinical diagnosis. Eschars are the most useful diagnostic clue, and patients without eschars might be misdiagnosed as a common cold. The presentation of eschars varies from 7% to 97% in different geographic regions [9], such as 87% in Japan [23], 7.4% in Bhutan [24], and some patients with no eschars develop severe multiorgan dysfunction syndrome [25]. The incidence of the syndrome is summarized in Table 4. We found 41.5% (208/501) of patients with eschars were clinically detected by physicians. Other severe syndromes, including pneumonitis, liver and kidney dysfunction, accounted for 9.0% (49/545). At present, clinical diagnosis mainly relies on patients’ self-description and physicians’ experience. Our study reveals that sequence type analysis of the TSA56 gene may provide valuable information for treatment.

Leptotrombidium deliense is a pivotal vector for summer scrub typhus in the southern area of endemicity. In addition, L. scutellare is a principal vector for transmission of winter-type scrub typhus found in Taiwanese offshore islands (Kinmen and Matsu Islands) [21] and in the northern area of endemicity that includes China [26], Japan [27], and South Korea [28]. Other harboring OT chiggers, including L. akamushi, L. deliense, L. imphalum, L. kawamurai, L. pallidum, L. rubellum, and L. scutellare have been detected in Taiwan. The positivity rate of TSA56-PCR reached 55.9% in these chiggers [21]. Additionally, the seropositivity rate was 43% among captured rodents [21]. Taiwan is located in the center of the tsutsugamushi triangle, harboring abundant rodents and migratory birds from the East Asia/Australasia Flyway that might promote host diversity, the expansion of dominant vectors, and spreading diverse genotypes of O. tsutsugamushi [29].

It has been found that mixed or coinfection may be incurred in patients by evidence of different pathogens existing in tissue specimens (eschar and whole blood) assayed by the sequencing of PCR clones [30,31]. In fact, we are interested in this important issue. However, no additional coinfection information, such as SFTSV coinfection or mixed genotypes of scrub typhus, was found in our data. Further in-depth research on the topic is required to extend our knowledge of coinfection.

We noted that TW-9 showed a ratio of 48 of 545 and 99.7% nucleotide sequence similarity to the Boryong strain (AM494475). In addition, only 1 of 545 isolates showed sequence similarity to the Kawasaki strain (M63383) listed in the TW-15 line (Table 1). Additionally, the TW-15 strain was similar to the Taguchi strain (AF173038) [31], with nucleotide sequence similarity reaching 99.37%, indicating that only 1 isolate was closely related to the Taguchi genotype among the 545 isolates. These results suggest that the Boryong and closely related TW-9 strains are prevalent in South Korea [32] and Taiwan, respectively. In addition, our results showed that the Taguchi genotype has not yet become the main prevalent strain in Taiwan. Taiwan is geographically close to South Korea. Surveillance and research efforts for scrub typhus are needed in the future.

The immunodominant 56 kDa surface protein (TSA56) is a major surface protein that contains hypervariable regions and exhibits remarkable sequence variation in different strains. Analysis of the genetic relationship of Orientia strains using DNA sequences of the TSA56 gene may have exaggerated the differences in the evolution of these strains. However, in the past few years, TSA56 gene of O. tsutsugamushi has been the target for phylogenetic analysis because of its sequence variation. It is known that the sequence diversity within housekeeping genes is very restricted, leading to the analysis of conserved housekeeping genes by multilocus sequencing (MLS), which might be required for surveillance of genetic phylogeny. Using housekeeping genes as an alternative approach to study the evolution and phylogeny of O. tsutsugamushi is required for future comparisons of the present results.

Serotyping has been used to classify a new isolate of O. tsutsugamushi based on reactivity with strain- or type-specific monoclonal antibodies or hyperimmune sera recognizing a specific motif on TSA56 from well-characterized strains [33]. However, serotyping often exhibits moderate cross-reactivity between the unidentified isolate and prototype strains. In contrast, genotyping of TSA56 is a promising approach to determine the molecular epidemiology of O. tsutsugamushi. Therefore, we sequenced the complete TSA56 gene of 545 isolates to analyze the genetic diversity of O. tsutsugamushi during the 2006–2016 surveillance in Taiwan. Nevertheless, for a better understanding of the correlation between serotypes and genotypes of O. tsutsugamushi, it would be valuable to extensively investigate the serotypes of O. tsutsugamushi among our isolates in the future.

At present, knowledge of the antigenic variation of the immunodominant protein TSA56 is crucial for the development of effective diagnostic tools and a vaccine [34-36]. The investigation of the geographical distribution of O. tsutsugamushi genotypes provides valuable insights into the epidemiology and control of scrub typhus. Currently, scrub typhus is no longer restricted to traditional endemic areas, and it can be caused by species other than O. tsutsugamushi [37-41]. Therefore, further investigation of the antigenic diversity and prevalence in local endemic areas needs to be continued not only for the epidemiological monitoring of scrub typhus but also for the improvement of diagnostic accuracy and vaccine development.

Strain identifiers and their accession numbers of O. tsutsugamushi strains in Taiwan during 2006–2016.

(PDF)

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19 Jan 2022

Dear Dr. Yang,

Thank you very much for submitting your manuscript "Molecular epidemiology of scrub typhus in Taiwan during 2006-2016" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The authors should address the issues raised by reviewers and modified the text accordingly for publication.

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Deputy Editor

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Jeanne Salje

Deputy Editor

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***********************

The authors should address the issues raised by reviewers and modified the text accordingly for publication.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The methods are adequate to address the issue of genetic diversity of Orientia tsutsugamushi and isolates obtain from persons with scrub typhus in Taiwan.

The 56 kDa surface protein contains four hydrophilic hypervariable regions that are the apparent mechanism of antigenic diversity of Orientia tsutsugamushi. A better understanding of the evolution and phylogeny of Orentia tsutsugamushi would be better analyzed with conserved housekeeping genes.

Reviewer #2: The methods in this study are suitable.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The establishment of 545 isolates Orientia tsutsugamushi from confirmed cases of scrub typhus and sequencing of the 56 kilodalton surface protein gene in all of them is a major achievement.

The criterion of 98% or greater sequence similarity as determining a distinct sequence type seems arbitrary. What is the rationale for this percent.

Lines 214-215: Liver dysfunction is listed as a symptom. Measurement of liver function is determined by laboratory tests and is not a patient complaint. It is unlikely that these patients had hepatic dysfunction; it is much more likely that they manifested hepatic injury by elevated transaminase enzymes. Was a measurement of hepatic function such as serum ammonium concentration or intrahepatic cholestasis identified?

Reviewer #2: The results in this study are good and suitable.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: 1. On lines 290 and 296 the issue of antigenic variation is raised. The data in the paper are gene sequences, i.e. genotypes. It is very likely that the most important antigenic variation of Orientia tsutsugamushi is determined by the four hypervariable regions of the 56 kDa surface protein. Knowledge of the genotype does not provide any information about the serotypes, which are very likely highly variable within the Karp group genotype. The reviewer is unaware of any correlation between serotypes and genotypes of Orientia tsutsugamushi. The situation is not clearly stated in this manuscript.

2. Lines 249-250: There is no evidence that animal hosts of the chiggers determine variation in Orientia tsutsugamushi. Rodents are hosts of the chiggers but are not hosts of Orientia tsutsugamushi. Although rodents are infected with Orientia tsutsugamushi and chiggers can become infected while feeding on an infected rodent, these chiggers do not transmit the bacteria transovarially. Chiggers are the only true host of Orientia tsutsugamushi. Animals are dead-end hosts.

3. Lines 272-275: Are the severe manifestations in patients infected with TW-1 and TW-2 statistically significantly different from the manifestations in other strains? Are the mild syndromes observed in patients with TW-3, TW-4, TW-5, and TW-17 statistically different compared with patients infected with other strains?

Reviewer #2: The conclusions are reasonable and suitable.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: I believe that the authors should address the issues that I have raised under conclusions, results, and methods.

Reviewer #2: Major Revision.

Please see "Summary and General Comments".

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This manuscript presents outstanding data and merits publication.

Reviewer #2: Authors studied the tsa56 genotypes of 545 O. tsutsugamushi clinical isolates throughout Taiwan, including offshore islands, from 2006 to 2016 to elucidate the molecular epidemiology of scrub typhus in Taiwan and data indicate the widespread presence of tsa56 genotypes closely related to Thailand and Korean strains and the presence of the unique endemic strains TW-12, TW-22, TW-29, and TW-36 in Taiwan.

1. Several reports reported mixed (or co) infection of different genotypes in a patient in SE and East Asia

(1. Emerg Infect Dis. 2018 Aug;24(8):1520-1523. doi: 10.3201/eid2408.171622. Dual Genotype Orientia tsutsugamushi Infection in Patient with Rash and Eschar, Vietnam, 2016.

2. Am. J. Trop. Med. Hyg., 99(2), 2018, pp. 287–290. doi:10.4269/ajtmh.18-0088

Mixed Infection with Severe Fever with Thrombocytopenia Syndrome Virus and Two Genotypes of Scrub Typhus in a Patient, South Korea, 2017).

Do you also find mixed (or co) infection different in single patient in your study? If you find this, please also put this data in your study.

2. Jeju island, South Korea is close to Taiwan and Boryong and Taguchi genotypes of O. tsutsugamushi were found in a patient, Jeju Island, South Korea

(Am. J. Trop. Med. Hyg., 99(2), 2018, pp. 287–290. doi:10.4269/ajtmh.18-0088 Mixed Infection with Severe Fever with Thrombocytopenia Syndrome Virus and Two Genotypes of Scrub Typhus in a Patient, South Korea, 2017).

Authors showed that most isolates belonged to the Karp (49.9%; 272/545) genotype.

Did you also find Boryong and Taguchi genotypes in your study?

If you do not find these types, please give some opinion on the difference between Taiwan and Jeju Island, South Korea.

--------------------

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Reviewer #1: No

Reviewer #2: Yes: LEE, KEUN HWA

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

21 Feb 2022

Submitted filename: Response Letter .pdf

Click here for additional data file.

15 Mar 2022

Dear Dr. Yang,

Thank you very much for submitting your manuscript "Molecular epidemiology of scrub typhus in Taiwan during 2006-2016" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

One of the reviewers insisted a little more discussion on the genotype diversity of O. tsutsugamushi and potential co-infection with SFTSV in Taiwan.

Please respond and discuss these issues.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Nam-Hyuk Cho

Deputy Editor

PLOS Neglected Tropical Diseases

Jeanne Salje

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

One of the reviewers insisted a little more discussion on the genotype diversity of O. tsutsugamushi and potential co-infection with SFTSV in Taiwan.

Please respond and discuss these issues.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Methods are acceptable

Reviewer #2: YES

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results are acceptable

Reviewer #2: YES

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusions are acceptable

Reviewer #2: YES

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Nine

Reviewer #2: .

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The current form of the manuscript is excellent

Reviewer #2: Below is my comment.

But, I cannot find my comment in the revision manuscript.

1. Could you highlight in your comment or reply to it.

---

Authors studied the tsa56 genotypes of 545 O. tsutsugamushi clinical isolates throughout Taiwan, including offshore islands, from 2006 to 2016 to elucidate the molecular epidemiology of scrub typhus inTaiwan and data indicate the widespread presence of tsa56 genotypes closely related to Thailand and Korean strains and the presence of the unique endemic strains TW-12, TW-22, TW-29, and TW-36 in Taiwan.

1. Several reports reported mixed (or co) infection of different genotypes in a patient in SE and East Asia

(1. Emerg Infect Dis. 2018 Aug;24(8):1520-1523. doi: 10.3201/eid2408.171622. Dual Genotype Orientia tsutsugamushi Infection in Patient with Rash and Eschar, Vietnam, 2016.

2. Am. J. Trop. Med. Hyg., 99(2), 2018, pp. 287–290. doi:10.4269/ajtmh.18-0088

Mixed Infection with Severe Fever with Thrombocytopenia Syndrome Virus and Two Genotypes of Scrub Typhus in a Patient, South Korea, 2017).

Do you also find mixed (or co) infection different in single patient in your study? If you find this, please also put this data in your study.

2. Jeju island, South Korea is close to Taiwan and Boryong and Taguchi genotypes of O. tsutsugamushi were found in a patient, Jeju Island, South Korea

(Am. J. Trop. Med. Hyg., 99(2), 2018, pp. 287–290. doi:10.4269/ajtmh.18-0088 Mixed Infection with Severe Fever with Thrombocytopenia Syndrome Virus and Two Genotypes of Scrub Typhus in a Patient, South Korea, 2017).

Authors showed that most isolates belonged to the Karp (49.9%; 272/545) genotype.

Did you also find Boryong and Taguchi genotypes in your study?

If you do not find these types, please give some opinion on the difference between Taiwan and Jeju Island, South Korea.

---

2. Cases were detected in the Middle East and South America and also reported on Chiloé Island in southern Chile (N Engl J Med 2016; 375:954-961)

Could you put this (also reference(s)) in line 35 and 36?

3. In line 91, please write the full name of “qPCR”

4. In line 129, please put more information about “Zeiss” such as the name of the city and country.

5. In line 140, please put more information about “QIAGEN” such as the name of the city and country.

6. In line 150, please put more information about “MEGA version 7” such as the name of the city and country.

7. In line 307 and 310, please write italics about “O. tsutsugamushi”.

--------------------

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Reviewer #1: Yes: David H Walker MD

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

22 Mar 2022

Submitted filename: Response Letter.docx

Click here for additional data file.

29 Mar 2022

Dear Dr. Yang,

We are pleased to inform you that your manuscript 'Molecular epidemiology of scrub typhus in Taiwan during 2006-2016' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Jeanne Salje

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

25 Apr 2022

Dear Dr. Yang,

We are delighted to inform you that your manuscript, "Molecular epidemiology of scrub typhus in Taiwan during 2006-2016," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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co-Editor-in-Chief

PLOS Neglected Tropical Diseases

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