Nghiên cứu đặc điểm nhiễm orientia tsutsugamushi, ấu trùng mò tại khu vực tây bắc và chế tao bộ sinh phẩm phát hiện tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING

MINISTRY OF HEALTH

NATIONAL INSTITUTE OF MALARIOLOGY - PARASITOLOGYENTOMOLOGY
-------------------***-------------------

TRAN QUANG PHUC

STUDY OF CHARACTERISTICS OF Orientia tsutsugamushi
INFECTION, TROMBICULID MITE LARVA
IN THE NORTHWEST REGION,
AND DEVELOPMENT OF A DIAGNOSTIC KIT

Major: Parasitology
Code: 62 72 01 16
SUMMARY OF DOCTORAL DISSERTATION
OF PHILOSOPHY IN MEDICINE

Ha Noi - 2019


The dissertation is completed at the
NATIONAL INSTITUTE OF MALARIOLOGY-PARASITOLOGYENTOMOLOGY

Scientific supervisors:
1. Assoc Prof. Dr. Nguyen Van Ba.
2. Assoc Prof. Dr. Le Thanh Dong
Reviewer 1:
Reviewer 2:

The dissertation is defended against University-level Examination Board
at National Institute of Malariology-Parasitology- Entomology
at……, date ……month…….year………
This dissertation can be found at:
- The National Library of Vietnam
- The Library of National Institute of Malariology - Parasitology -Entomology


1
INTRODUCTION
Scrub typhus (also known as Tsutsugamushi disease) is an acute infectious
disease caused by Orientia tsutsugamushi. The disease has natural source of
infection, transmitted to humans by the bite of the larva of trombiculid mite. The
disease mainly occurs in rural, mountainous areas. The symptoms of scrub
typhus may be mistaken with many other diseases of which the treatment
regimens are different (such as dengue hemorrhagic fever, leptospirosis). The
mortality rate is high if not diagnosed promptly and used specific antibiotics.
Therefore, the early detection of the disease plays a decisive role in the treatment
of scrub typhus [5], [42], [133].
Currently, there are few technical tools for diagnosis of scrub typhus in
Vietnam, especially in the remote areas where medical resources are limited.
With the strong development of molecular biology, many modern tools have
been applied to determine and diagnose the pathogens. In particular, the
isothermal nucleic acid amplification method Recombinase Polymerase
Amplification (RPA) proves many outstanding advantages compared to the
traditional molecular biological techniques, especially allowing low-temperature
amplification (37 - 42°C), the reaction duration for determination of the samples
is very short (5 - 20 minutes). The RPA method allows the amplification of
nucleic acid without using an expensive, bulky Thermal Cycler and unstable in
field conditions. Meanwhile, the sensitivity of the RPA method is evaluated to

be equivalent or better than the PCR method. The equipment is easy to use and
transport to the place where specimens are taken [64], [97], [140].
The Northwestern region is an area with difficult socio-economic conditions
where many ethnic minorities are living. Its natural conditions are favorable for
the development of many natural nidus, including scrub typhus. In recent years,
there have been a number of studies on the vectors of the disease in the
Northeast provinces [8], [12], [13] but no research has been conducted in the
Northwest region. Therefore, it is necessary to study the characteristics of
natural nidus and the proportion of people who have antibodies against Orientia
tsutsugamushi to enhance the effectiveness of preventing and controlling scrub
typhus in the Northwest region.
In consideration of the above issues, "Study of characteristics of Orientia
tsutsugamushi infection, trombiculid mite larva in the Northwest region and
development of a diagnostic kit" is carried out for the objectives:
1) To describe some characteristics of Orientia tsutsugamushi infection,
trombiculid mite larva and the distribution of scrub typhus in the Northwest
region in 2016, 2017.
2) To develop an Orientia tsutsugamushi diagnostic kit by using isothermal
amplification technique Recombinase Polymerase Amplification in
laboratory.


2
NOVELTY, SCIENTIFICITY AND PRACTICALITY OF STUDY
1. Novelty
This is the first time the isothermal amplification technology Recombinase
polymerase amplification has been used to produce a diagnosis kit of Scrub
typhus in Vietnam. This is a test method of which the sensitivity and specificity
is high, with quick result (after only 20 minutes), it is not required to extract
samples or use thermocycler or complex equipment, so the deployment in field

is very convenient and suitable.
The study has assessed the curent situation of people exposed to O.
tsutsugamushi, scrub typhus and O. tsutsugamushi infection in vectors in 4
provinces of Northwest region: Hoa Binh, Son La, Dien Bien and Lai Chau.
2. Scientificity
The study uses standard scientific methods such as:
- Descriptive epidemiological study with analysis by cross-sectional surveys
to study characteristics of Orientia tsutsugamushi infection, host and infectious
vectors; Retrospective study analyzing the distribution characteristics of Scrub
typhus from records of patients who had scrub typhus and treated in 4 provincial
hospitals.
- Laboratory study: Develop procedures of and produce O. tsutsugamushi
diagnostic kit by using isothermal amplification technique Recombinase
Polymerase Amplification in laboratory. Research, evaluate, and analyze the
sensitivity, specificity, stability of the product.
These are modern and standard research methods that are applied in Vietnam
and worldwide, so its reliability is high. Besides, the research uses modern
techniques such as gene transformation technique through plasmids, polemerase
chain reactiongene technique, these are modern techniques with high reliability.
3. Practicality
The study results provide a fast, accurate tool for diagnosis of scrub typhus
and suitable for local health center, contributing to reduce the burden of the
disease in the community. On the other hand, the study is also a reference
material for scientific research and teaching at universities, and is a base for
further research.
THESIS STRUCTURE
The thesis includes 121 pages: Introduction (2 pages), chapter 1. Literature
Review (35 pages), chapter 2. Study Subjects and Methods (23 pages), chapter 3.
Study results (34 page), chapter 4. Discussion (24 pages), Conclusion (2 pages),
Recommendations (1 page), 33 tables, 17 figures, 150 references.

3
Chapter 1.
LITERATURE REVIEW
1.1. Characteristics of Scrub typhus
The disease caused by larva of trombiculid mites (Scrub typhus
/Tsutsugamushi disease), is an acute infectious disease belonging to Group C in
the Law on Prevention and Control of Infectious Diseases. Its causative agent is
O. tsutsugamushi [2], [4], [141; the vector is the larvae of trombiculid mite
Leptotrombidium.
The disease’s clinical features are fever lasting for 2-3 weeks, accompanied by
skin ulcers, lymph nodes on body and rash. If left untreated, there will be serious
complications, causing damage to many organs and leading to death. If not
promptly diagnosed and treated by specific antibiotic, the mortality rate may
range from 7-10% [141], even up to 70% depending on different
epidemiological regions [40], [136].
1.1.1. Symptoms of scrub typhus
Clinical case: Incubation period: 8-12 days on average (6 to 21 days); At
first, the area bitten by the larva of trombiculid mite has a nodule of about 2 5mm, painless [3], the patient often does not pay attention; After the incubation
period, there will be following symptoms:
Fever ≥ 38 - 40°C, persistent, lasting for 15 - 20 days, for some cases the
fever may last 27 days if left untreated; sometimes chills in the first 1-2 days,
with severe headache, muscle aches [4], [19].
Specific ulcers (eschar, the typical symptom of Scrub typhus): The rate of
ulcers is common in some disease outbreaks in Vietnam, at about 80%, the
others may be spots which are small or difficult to find (in ear canal, pussy...).
Ulcers occur mainly in soft and moist areas such as the genitals area, anal area,
groin, armpits, neck, and other areas such as thighs, abdomen, chest, and back.
Sometimes the ulcer located in the ear ring, navel, eyelid (easily mistaken for the

stye). The ulcers are usually painless, not itchy; the patient usually has only one
round or oval nodule with the diameter of 1 mm to 20 mm; the initial nodule
may develop gradually into cloudy fluid on a reddish area, after 4 to 5 days they
will be broken into a spot with light brown or black scales depending on soft or
hard skin area and premature or old skin of the ulcer; the scales is flaked and
revealing a shallow concave ulcer, light pink, with no pus or fluid, red or dark
edge depending on the disease is developing or stopping; the ulcer will be
gradually healed when the fever stops[4], [19].
Nodes and maculapapular rash: The nodes in ulcer area is usually swollen
and painful, not red, still moves, occurs with fever or after 2-3 days, is the area
of the ulcers; The nodes in body is less painful, except for severe cases. The
maculopapular rash appears at the end of the first week and the begin of the
second week, in most area of the body, except for the palm of the foot, lasts for a
few hours to a week, with low frequency than the classic Dengue fever, about
35-70% of the patients current, depending on when the patient is examined;
sometimes with petechiae (less than 10%). In the early days, skin and mucous


4
membranes were congested in most cases (about 88%), different from malaria
and typhoid [5], [71], [115].
For severe patients: heart sound is faint, blood pressure is low, pulse is weak
compared to temperature, nosebleeds, gastrointestinal bleeding, bronchitis, a
typical pneumonia [37], [146].
In addition, scrub typhus may also be hidden and atypical (no ulcers).
If treated by appropriate antibiotics, the fever will be reduced quickly. If the
intervention is late or ineffective, there may be complications such as
myocarditis, septic shock, pneumonia, respiratory distress, meningitis meningitis... leading to death [39], [102].
The mortality rate caused by O. tsutsugamushi varies from country to
country, depending on the species circulating in the local. The mortality rate in

Vietnam, Indonesia and Taiwan, Malaysia, Japan is about 1%; 5-20%, 15-20%
and 20-60%, respectively. [5], [134].
1.1.2. Diagnosis of scrub typus
The diagnosis of scrub typus is based on clinical and laboratory standards.
Criteria for determining the disease [5]:
- A clinical standard only:
+ Fever, specific ulcers, there may be swollen lymph nodes, maculopapular
rash, white blood cells 4,000 - 12,000, normal or increased lymphocytes,
increased blood flow.
+ Required criteria: specific ulcers.
- If there is no specific ulcers, one positive test of the following is required:
The Leptospira immunoglobulin M enzyme-linked immunosorbent assay (IgM
ELISA); or Indirect Immunofluorescence Assay (IFA test); or indirect
immunoperoxidase test (IIP test).
- Weil-Felix reaction with the antigen OX-K of Proteus mirabilis, the
sensitivity and specificity is not high but cheap, suitable for district level.
- Trial treatment: For early treatment, the patient should try tetracycline or
chlorocid, only applied for suspected diagnosis [5], [119], [137].
Differential diagnosis: Disease caused by spirochete; Typhoid; Dengue
fever; marsh fever.
Treatment of scrub typhus
Specific antibiotics for treatment of scrub typhus are chloramphenicol,
doxycycline and tetracycline. However, because these antibiotics have no effect
on killing but only controlling the bacteria, O. tsutsugamushi bacteria still alive
and exist in the lymph nodes, in the reticuloendothelial system for many days,
months and easy to relapse [5], [26], [30].
1.1.3. Prevention of scrub typhus
Implement some measures to propagate health education combined with
hygiene for prevention of disease.
Carry basis investigation to detect outbreaks in areas which are suspectd and

inhabited to have resolution (catch rodentia, trombiculidae, classify, subdivide
R.orientalis, find antibodies, detect patients).


5
At an identified or suspected outbreak: Measures to prevent from being
bitten by trombiculidae: Removal of trombiculidae in the environment: residual
spraying into moist soil, bushes of grass under 20 cm high around the house,
diazinon, fenthion, malathion, lindane, dieldrin, chlordan. Kill rats seasonally,
paying attention to sprinkle the substance for killing trombiculidae first. Clearing
the vegetation around the house selects clusters of many larvae plants
(trombiculidae source). Preventive treatment: This measure is limited because it
is not easy for infection in outbreaks; Doxycycline 200 mg / week [4], [5], [19]
can be used when a military team has to pass the outbreak for their mission.
1.2. Molecular Biology Technology for disgnosis of scrub typhus
Serological tests for detecting Orientia tsutsugamushi antibodies are not
suitable to diagnose the disease in the acute stage because antibody level may be
below the detection threshold during the disease onset stage. Therefore, it is
important to detect antigen/germ before the specific antibody level increases.
Currently, most of antigen/germ detection tests are based on PCR, quantitative
realtime PRC or nested PCR targeting different target genes, including 56 kDa,
47 kDa, groEL of Orientia tsutsugamushi [47], [74], [94]. However, all of these
tests require training to operate the PCR thermal cycler, and in the condition of
limited resources, it is difficult to ensure the equipment, maintenance and
adjustment of the thermal cycler operate stably.
New technologies of nucleic acid amplication
Nucleic acid analysis test is hoped to provide a fast, sensitive, and specific
diagnostic tool for parasitic diseases. New generation of diagnostic equipment
allow detection of parasitic germs in the field, allowing clinicians to quickly
provide reliable diagnostics for effective treatment. The complex biochemical

nature of clinical specimens, low levels of nucleic acid of most current samples
and biosensor technologies requires us to use some technologies for
amplification of nucleic acid to improve the sensitivity, meet clinical
requirements when analyzing small samples, apply in field analysis [97], [141].
Most nucleic acid analysis technologies are now very expensive and require
trained testers, so it is only suitable for central labs that are heavily invested with
abundant resources in the big cities and urban areas. This makes the benefits of
nucleic acid analysis tests unavailable to residents living in under-resourced
areas and in isolated geographic areas. Therefore, molecular diagnosis
technology with reasonable price, not too complicated is essential to protect the
health of each individual and the community. Recent scientific and technological
advances have shown that nucleic acid analysis devices can be scaled down and
easily transported to the field.
To overcome the limitations of traditional PCR in the nucleic acid
amplification used in molecular diagnostics in the field, recent studies tend to
change to isothermal nucleic acid amplification methods. Isothermal
amplification method uses enzymes to separate the fiber, while the traditional
PCR must use high temperature. Isothermal amplification techniques are a very
useful tool for developing field-based diagnostic models which is capable to


6
amplify nucleic acids without the need for thermal steps and related control
mechanisms. [109], [111].
Isothermal
Amplication
Technology
Recombinase
polymerase
amplification

Recombinase polymerase amplification (RPA) is a technology that uses a
mixture of recombinase of progenitor to help primers attach substrate to the
target gene. Fiber-substituted DNA polymerase enzyme (large fraction of
Bacillus subtilis PolI, Bsu) catalyzes the extended primer reaction to synthesize
new added DNA fibers[49].
The important thing in RPA is the use of single-stranded DNA-binding
protein (SSB) and strand-displacing polymerase to replace the modified step in
traditional PCR. The RPA technique maximizes the advantages of simplicity,
cost savings and short reaction time. To determine the target sequence, the RPA
requires two simple designed oligonucleotide primers (an additional 1 probe may
be required) for occurrence of pairing reaction. This method has a specificity in
high sequence in a nucleic acid mixture without extracting the sample. This
technology allows amplify the target gene by continuously synthesizing DNA
without the denaturation of the double-stranded DNA at high temperatures. The
test can be carried out at temperatures between 24°C and 45°C with very high
efficiency, so the signal from the target molecule can be detected after only 5 to
20 minutes which is short compared to traditional PCR (20 - 180 minutes),
Reltime PCR (3-4 hours) and LAMP (45 - 60 minutes). RPA technology was
developed to detect HIV, Rift Valley Fever virus, Ebola virus, Sudan and
Marburg, MERS-CoV, virus causing hand-foot-mouth disease and Coronavirus
in cow. The technique has also been developed for detection of Chlamydia
trachomatis in urine samples, diagnosis of Cryptosporidiosis in animals and
human samples and detection of Neisseria gonorrhoeae, Salmonella enterica,
Staphylococcus aureus against methicillin (MRSA), Francisella tularensis, và
Streptococci group B [81], [93], [105].
The sensitivity and specificity of RPA technique is also very high. Studies
showed that RPA technology can detect a copy of the target DNA sample (or
<10 copies of the ARN sample) in the sample without requiring the high purity
of sample. Some experiments have shown that RPA can identify and reproduce
the target nucleic acid fragment in a sample containing many genetic material of

different animals. In health, this technique can be applied flexibly to detect
directly and quickly some germs from specimens such as blood, secretions,
waste and tissue, while most Current diagnostic methods require to handle the
samples with weak alkaline to release genetic material [58], [139].
Therefore, thanks to being able to fundamentally overcome the
disadvantages of serological and other molecular biology techniques, RPA
technology has become one of the methods currently which is applied in
diagnosis, detect agents of many diseases and on many subjects.


7
Chapter 2.
SUBJECTS AND METHODS
2.1. Subjects, sites and time
2.1.1. Study subjects
- Study subjects on characteristics of Orientia tsutsugamushi infection,
trombiculid mite larva and the distribution of scrub typhus: People in the study
sites. Rat species at the study sites. The vector is the trombiculid mite larvae.
- Study Subjects on the distribution characteristics of scrub typhus: the
books, records of patients with scrub typhus examined and treated at 4 general
hospitals in Son La, Dien Bien, Lai Chau and Hoa Binh from January 1, 2016 to
December 31, 2017.
- Study subjects for fabrication of O. tsutsugamushi detection kit
+ Positive specimen with O. tsutsugamushi (disease group);
+ Negative specimen with O. tsutsugamushi (control group); Positive
specimen with some bacterial species can cause symptoms similar to O.
tsutsugamushi.
+ The O. tsutsugamushi detection kit is fabricated.
2.1.2. Study site
- The study is conducted in 4 provinces in Northwest region, including: Son

La, Dien Bien, Lai Chau and Hoa Binh. These are the provinces with favorable
geographical conditions for development of Trombiculidae, the socio-economic
conditions here are still difficult, this is also the area of ethnic minority, there is
record of many patients with scrub typhus in the last years and a key area of
politics, security and defense.
- Institute of Military Medicine - Pharmacy, Vietnam Military Medical
University.
2.1.3. Study time
The study was conducted from 01/2016 to 6/2018.
Descriptive epidemiological study analyzed by cross-sectional surveys in the
community.
Retrospective study analyzed the distribution characteristics of scrub typhus
from the records of patients with scrub typhus treated at 4 provincial hospitals.
2.2. Study method
2.2.1. Study design
- Descriptive epidemiological study analyzed by cross-sectional surveys in
the community.
- Retrospective study.
- Laboratory research: The procedures of fabricating O. tsutsugamushi
detection kit in a laboratory.
- Research, evaluate, and analyze the sensitivity, specificity, stability of
product.
2.2.2. Sample size
- Sample size of community survey: Apply the following formula:


8
n  Z2

1 α 2

(1  p)
pε2

Of which: n: Sample size. p = 0.2104 (Estimated population ratio According to author Doan Trong Tuyen is 21.04%) [34]; α = 0.05 (level of
statistical significance); Z 1- α/2 = 1.96 (Corresponding α = 0.05). Minimum
sample size: 1,442 people. Actual investigation: 1,520 people.
- Sample size for retrospective survey of patients with scrub typhus:
All patients were diagnosed with scrub typhus treated at the general
hospital of 4 provinces of Hoa Binh, Son La, Dien Bien and Lai Chau from
January 1, 2016 to December 31, 2017, meeting the research criteria.
- Sample size for survey of the host and vector:
Apply the following formula:
n  Z2

1 α 2

p(1  p)
d2

Of which: n: Sample size; p = 0.125 (Estimated population ratio According to author Doan Trong Tuyen [34]; α = 0.05 (level of statistical
significance); 1- α / 2 = 1.96 Z (Corresponding α = 0.05); d = 0.02 (absolute error)
minimum sample size is 1,050. In fact, the research team catched and studied
1,707 mice in 04 provinces.
- Sample size for assessment of sensitivity, specificity and stability of
the kit:
+ Manufacture 500 Orientia tsutsugamushi detection tests by isothermal
amplification technology RPA.
+ Specimens (+) for Orientia tsutsugamushi (disease group): 37 samples.
+ Specimens (-) for Orientia tsutsugamushi (control group): 100 samples.

+ Positive specimens with some bacterial species which can cause the
symptoms similar to Orientia tsutsugamushi : 13 samples.
2.3. Techniques used in the study
- Techniques for taking and preserving serum samples
- Technique for finding antibody against O. tsutsugamushi on human serum
samples.
- Techniques for trapping and collecting mice and classifying
- Test technique for finding antibody against O. tsutsugamushi on mice (SD
BIOLINE Tsutsugamushi test).
- Techniques for collecting larvae of trombiculid mite
- Techniques for identifying trombiculidae
- Community interview techniques
- Standard techniques used for gene cloning, PCR and electrophoresis for
DNA analysis, gene sequencing, realtime PCR, isothermal amplification
technique RPA.
2.4. Methods of data processing
The data is imported and processed by SPSS 17.0 statistics software.


9
2.5. Ethics in study
Study fully complies with regulations in biomedical research.
Chapter 3.
RESULTS
3.1. Some infection characteristics of Orientia tsutsugamushi, trombiculid
mite larva and the distribution of scrub typhus in the Northeast region
3.1.1. Results of serological survey for detection of anti-O. tsutsugamushi
antibodies in the community
By the cross-sectional survey in 30 communes of 4 provinces of Hoa
Binh, Son La, Dien Bien and Lai Chau, anti-O. tsutsugamushi antibodies are

found in 1,520 people tested serum, the results are as follows:
Table 3.1. Proportion of people with anti-O. tsutsugamushi antibodies by
province
Survey
Number of
Province
Rate(%)
p
sample (n)
sample (+)
(1)
Hoa Binh
329
55
16.72
p1-2 <0,05
Son La (2)
431
39
9.05
p1-4 <0.05
p2-3 <0.05
Dien Bien (3)
437
63
14.42
p3-4 <0.05
Lai Chau(4)
323
28

8.67
Total
1520
185
12.17
Note: p1-2: p value of (1) and (2).
Comment: The results in Table 3.1 show that: The proportion of people
with Anti-Orientia tsutsugamushi antibodies is 12.17%; the highest rate is in
Hoa Binh 16.72%, followed by Dien Bien 14.42%. Son La and Lai Chau
provinces have lower rates of anti-Orientia tsutsugamushi antibodies (9.05% and
8.67% respectively). The difference in statistical significance, p <0.05.
Table 3.3. Proportion of people with anti-O. tsutsugamushi antibodies by age
Number of
Age group Test sample (n)
Rate (%)
p
sample(+)
≤ 20 years
179
12
6.70
21-30
257
18
7.00
31-40
240
32
13.33
p4-1,2,3,5,6,7

41-50
226
52
23.01
<0.05
51-60
217
30
13.82
61-70
215
23
10.70
>70 years
186
18
9.68
Total
1,520
185
12.17
Comment: The highest rate of people infected O. tsutsugamushi is in
group 41 - 50 years old (23,01%), the lowest rate is in group < 20 years
old(6,7%).


10
Table 3.6. Proportion of people with anti-O. tsutsugamushi antibodies according
to the ecological characteristics of the living area
Survey

Number of
Ecological region
Rate (%)
p
sample (n) sample(+)
(1)
Savan
428
65
15.19
P1-2> 0.05
Regenerated forest (2)
670
94
14.03
P2-3< 0.05
(3)
Primary forest
422
26
6.16
Comment: The proportion of people with anti-O. tsutsugamushi
antibodies in the community living in areas with Savan habitat and regenerated
forests is 14.03% and 15.19%, respectively, higher than the population living in
the primary forest area (6.16%) with statistical significance, p <0,05.
3.3.2. Some distribution characteristics of scrub typhus
Results of retrospective statistics of medical records in 4 general
hospitals of 4 provinces Hoa Binh, Son La, Dien Bien and Lai Chau for 2 years
(from January 1, 2016 to December 31, 2017) determined total of 230 patients
diagnosed with scrub typhus and met the research criteria. The analysis results

are as follows:
- Distribution of patients with scrub typhus locally
Table 3.7. Distribution of patients with scrub typhus by province
#
Province
Number of patients
Rate %
1
Hoa Binh
69
30.00
2
Son La
48
20.87
3
Dien Bien
78
33.91
4
Lai Chau
35
15.22
Total
230
100.00
Comment: Among 230 patients in 4 provinces, the province with
highest number of patients with scrub typhus is Dien Bien (account for 33.91%),
followed by Hoa Binh (30.00%) and Son La (20.87%), the lowest number is in
Lai Chau (15.22%).

- Distribution of patients with scrub typhus by gender and age
Among 230 cases, there are 116 male patients, account for 50.43%; 114
female patients, account for 49.57%.
Table 3.8. Distribution of patients with scrub typhus by age (n=230)
Number of
#
Age
Rate %
patients
1
≤ 20
20
8.70
2
From 21 to 30
31
13.48
3
From 31 to 40
37
16.09
4
From 41 to 50
55
23.91
5
From 51 to 60
34
14.78
6

From 61 to 70
29
12.61
7
>70
24
10.43
Total
230
100.00


11
Comment: Patients with scrub typhus distribute scatterly among all age
groups. In particular, the highest number of patients with scrub typhus is group
41 - 50 years old (23.91%).
-Distribution of patients with scrub typhus by month

29
21
8

Both 2 years
34
18
16

28
20

30
15

8
15

18
11
Oct

Sept

Aug

Jul

June

May

7

13
9
4

10
46
Dec

38
23
15

4
2
Apr

6

2017

Nov

16
8

Mar

Feb

40
30
20
10 5 4
3
0 1 2 1
Jan

Case

2016

Figure 3.1: Distribution of patients with scrub typhus by month
Comment: Scrub typhus occurs in all months of the year, the months
with highest rate are from May to October (2016) and from May to September
(2017) which are rainy months. The disease is lowest in January and February
every year.
3.1.2. Survey results of the host and vector of the scrub typhus
The number of trapped mice in 4 provinces is 1,707, of which 703 were
caught by traps in house and 1,004 were trapped outside the house. Results for
classifying of mice, larvae of trombiculid mite, and detection of anti-Orientia
tsutsugamushi antibodies on mice are presented in the following tables and
figures:
Table 3.9. Number and species of mice in the study sites
Number of mice
#
Name
Hoa Binh
Son La
Dien Bien Lai Chau
I
O
I
O
I
O
I
O
1 Rattus exulans

152
0
153 0
185
0
157
0
2 Rattus Rattus
0
93
0
32
0
185
0
34
3 Berylmus bowersi
0
0
0 272
0
0
0
88
4 Bandicota savilei
0
62
0
0
56

238
0
0
Number of mice
5
152 155 153 304 241 423 157 122
according to trap place
Total
307
457
664
279
Remark: I: Indoor; O: Outdoor
Comment: The number of mice wrapped in 10 nights in Hoa Binh, Son
La, Dien Bien and Lai Chau is 307, 457, 664 and 279, respectively.


12
Table 3.11. Proportion of mice infected larva of trombiculid mite in the study
sites
Proportion of mice infected larvae of trombiculid mite (%)
#
Hoa Binh
Son La
Dien Bien
Lai Chau
Name
I
O
I

O
I
O
I
O
n=152 n=155 n=153 n=304 n=241 n=423 n=157 n=122
1 Rattus exulans 13.16
9.80
13,51
14.65
2 Rattus Rattus
95.70
0.00
89,19
5,88
Berylmus
3
96.32
88,64
bowersi
Bandicota
4
40.32
7,14 52,52
savilei
5 Common
43.65
60.61
48.04
38.71

Remark: I: Indoor; O: Outdoor
Comment: The proportion of mice infected trombiculid mite larval
living near forests such as Rattus Rattus and Berylmus bowersi is higher than
those living near houses and indoors such as Rattus exulans and Bandicota
savilei. Berylmus bowersi in Son La have 96.32% of larvae and 88.64% in Lai
Chau. The R. Rattus in Hoa Binh has a prevalence of 95.7%, in Dien Bien
89.19% and in Lai Chau only 5.88%. The overall prevalence of larvae infected
with larvae is highest in Son La, 60.61%, followed by Dien Bien 48.04%, in Hoa
Binh 43.65% and the lowest in Lai Chau 38.71%.
Table 3.12. Classification of trombiculidae on mice in the study sites
Study site
#
Name
Hoa Binh
Son La
Dien Bien
Lai Chau
A.(L.) indica
A.(L,) indica
(1)
1 Rattus exulans
0
0
G.(W.) ewingi
G.(W.) ewingi
A.(L.) indica
A.(L.) indica
2 Rattus. rattus (2)
G.(W.) ewingi
G.(W.) ewingi

G.(W.) lupella
G.(W.) lupella
3 Berylmus bowersi (2)
4 Bandicota savilei (2) A.(L.) indica
A.(L.)
indica
Note: (1); Indoor (2) In the field on mountain
The results of the table above show: Mice in the study sites were
infected with 1 to 3 species of trombiculidae, of which 1 to 2 species of
trombiculidae spreading diseases. R. rattus in Hoa Binh and Dien Bien are
infected with 3 species of trombiculidae, of which 1 species spreading scrub
typhus is A. (Lau.) indica. Rattus exuians in Son La and Lai Chau are infected
with 2 species of trombiculidae, of which 1 species spreading the disease is A.
(Lau.) Indica. Bandicota savilei (B. salivei) in Hoa Binh and Dien Bien, infected
with 1 species of trombiculidae spreading diseases is A. (Lau.) Indica.


13
Table 3.13. Species of trombiculidae in each study site
Rates (%)
#
Name
Hoa Binh Son La Dien Bien Lai Chau
(n=134) (n=277) (n=319) (n=108)
1 Ascoschoengastia (Laurentella)
70.90
51.62
66.46
55.56
indicaN

2 Garliepia (Walchia) ewingi
3.73
1.81
8.15
5.56
3 G. (W.) lupella
23.88
23.47
20.38
20.37
4 Leptotrombidim (Lep) deliense
1.49
23.10
5.02
18.52
Comment:
In the four survey provinces, four species of mites were collected:
Ascoschoengastia (Laurentella) indica, Garliepia (Walchia) ewingi, G. (W.)
lupella and Leptotrombidim (Lep.) Deliense. There are two species of mites that
are capable of spreading diseases, including Leptotrombidim (Lep.) Deliense and
Ascoschoengastia (Laurentella) indica.
Table 3.14. Results of serologic test for detection of anti-Orientia tsutsugamushi
antibodies on mice
Anti- O. tsutsugamushi antibodies
Hoa Binh
Son La
#
Dien Bien
Lai Chau
Mouse species

Rate %
Rate %
Rate %
Rate
n
n
n
n
(+)
(+)
(+)
% (+)
1 Rattus exulans 152 13.16 153
5.23
185
7.03
157 5.10
2 Rattus Rattus
93
36.56
32
0.00
185 42.16
34
5.88
Berylmus
3
272 68.38
88 36.36
bowersi

Bandicota
4
62
20.97
294 15.99
savilei
Total
307 21.82 457 42.45 664 20.78 279 15.77
Remark: n = Test sample
Comments: Mice serum in all four sites has anti-O. tsutsugamushi
antibodies.
In Hoa Binh: Mice with anti- O. tsutsugamushi antibodies include
Rattus exulans (13.16%), Rattus Rattus (36.56%), and Bandicota savilei
(20.97%). The rate of mice with anti-O. tsutsugamushi antibodies is 21.82%.
In Son La: Mice with anti-O. tsutsugamushi antibodies include Rattus
exulans (5.23%) and Berylmus bowersi (68.38%). The rate of mice with anti-O.
tsutsugamushi antibodies is 42.45%.
In Dien Bien: Mice with anti-O. tsutsugamushi antibodies include
Rattus Rattus (42.16%), Bandicota savilei (15.99%) and Rattus exulans (7.03%).
The rate of mice with anti-O. tsutsugamushi antibodies is 20.78%.
In Lai Chau: Mice with anti-O. tsutsugamushi antibodies include
Berylmus bowersi (36.36%), Bandicota savilei (5.88%) and Rattus exulans
(5.10%). The rate of mice with anti-O. tsutsugamushi antibodies is 15.77%.


14
3.2. Study results of produce of Orientia tsutsugamushi detection kit in
laboratory
Follow 10 basic steps as the following figure:
Develop positive control (1)

Optimize conditions for nucleic acid
amplification reaction (2)

Optimize the composition of nucleic
acid amplification reaction (3)

Determine the sensitivity and
specificity of RPA technology (4)

Fabrication of 500 test for diagnosis
of O. tsutsugamushi (5)

Evaluate the sensitivity and
specificity of the kit (6)

Evaluate the accuracy and
repeatability of the kit (7)

Evaluate the stability of the O.
tsutsugamushi diagnosis kit (8)

Develop the basic standard for the kit
(9)

Develop process for diagnosis of O.
tsutsugamushi (10)

Figure 2.2. Process for fabrication of O. tsutsugamushi detection kit
Step 1: Set up positive control:

Based on the report on gene polymorphism of Orientia tsutsugamushi
circulated in the world and Vietnam, we selects 47 kDa gene as a template to
design the primer/probe as the techniques in the study. All primer/ probe sets are
evaluated for specific capabilities and necessary parameters with the BLAST
tool. Recombinant plasmid including 47 kDa target gene sequence amplified by
PCR and pJET1.2/blunt cloning vector after extracted, evaluated on Nanodrop
system showing high purity with the rate A260/280 of 1.84 which is in allowable
range (1.8 - 2.0) with a relatively high concentration (20.1 ng / µl). The product
after extracted with BamHI restriction enzyme was sequenced.
Table 3.15. The primer sequence and probe of nucleic acid amplification
for PCR and RPA technologies
Technology Primer/
DNA sequence (5’-3’)
Probe
Realtime
Forward
AACTGATTTTATTCAAACTAATGCTGCT
PCR
Reverse
TATGCCTGAGTAAGATACRTGAATRGAATT
5′FAMTGGGTAGCTTTGGTGGACCGATGTTTAATCT
Probe
-BHQ1-3′
RPA
Forward
TAAAGTTGCATGATGGTTCAGAACTGATAGCA
Reverse
TATTGCAATAACCTGATCTCCTACTCTAGA
5’dATTAAAAATTAATTCTTCAGCAGCATTATCTTAProbe
(FAM-dT)-GC-(THF)-AC-(BHQ1-dT)TTTGGCGATTCA-3’ blocker

15
Table 3.16. The primer and probe sequence of nucleic acid amplification for
control
Primer/Probe
DNA sequence (5’-3’)
Realtime Forward
5′-GTG CAC CTG ACT CCT GAG GAG A-3
PCR
Reverse
5′-CCT TGA TAC CAA CCT GCC CAG-3
5′-(FAM)AAG GTG AAC GTG GAT GAA GTT GGT GG
Probe
(BHQ1)-3′
Step 2: Optimize conditions for nucleic acid amplification reaction of
O. tsutsugamushi with Realtime PCR technology and RPA to determine the most
suitable reaction conditions.
Table 3.17. Optimal conditions of Realtime PCR reaction
#
Step
Temperature
Time
Unit
1
Initial denaturation
95
15
Minute
Amplify

Denaturation
94
15
Second
2
Stick primer
63
30
Second
(Cycle
Number: 45)
Extend
72
30
Second
3
Keep samples
25
∞
Table 3.18. Optimal conditions of RPA reaction
#
1
2

Conditions of RPA reaction
Optimal value
Unit
Reaction temperature
37
ºC

Time for receiving fluorescence signals
30
Second
Step 3: Optimize the composition of O. tsutsugamushi nucleic acid
amplification reaction.
The concentration of primer, probe and MgOAc are 0.54µM, 0.1µM
and 18mM, respectively, which is optimal to ensure that the performance of
RPA reaction is highest and suitable for commercial conditions, for the
fabrication of Orientia tsutsugamushi detection kit.
Step 4: Determine the sensitivity and specificity of RPA technology.
The process of detecting O. tsutsugamushi by RPA technology with
high specificity (negative control does not have amplification signal) and is
capable of detecting O. tsutsugamushi DNA samples at concentrations of 10
copy/reaction. The results show that the detection of DNA O. tsutsugamushi is
short (<20 minutes), faster than other technologies such as PCR, Realtime PCR.
Step 5: Produce of 500 test for diagnosis of O. tsutsugamushi
The nucleic acid amplication technology RPA which is capable for the
diagnosis of O. tsutsugamushi with the highest sensitivity and specificity was
selected to complete the process and fabrication of 500 tests.
The results of evaluating the sensitivity and specificity of Orientia
tsutsugamushi DNA amplification technologies shows that isothermal
amplication technology RPA proves its sensitivity, specificity as well as
advantages in clinical applications. Therefore, technology RPA had been chosen
for the produce of Orientia tsutsugamushi detection kit.


16

Figures 3.9. Photo of the Orientia tsutsugamushi detection kit
Table 3.2. Component of Orientia tsutsugamushi detection kit

#
Component
Code
Volume
1
Reaction Buffer
A1, A2
780 µl
2
dNTPs
B1, B2
100 µl
3
Probe E-mix
C1, C2
120 µl
4
Nuclease-freewwater
D1, D2
200 µl
5
Orientia tsutsugamushi oligo-mix
I1, I2
100 µl
6
Exo
E1, E2
22 µl
7
Core Reaction Mix

F1, F2
60 µl
8
Positive Control
G1, G2
100 µl
9
MgOAc
H1, H2
100 µl
Step 6: Evaluate the sensitivity and specificity of the kit.
- Sensitivity and specificity:
The results of analyzes 37 samples positive for O. tsutsugamushi
(disease group) and 100 samples of negative samples for O. tsutsugamushi healthy people (control group) using the O. tsutsugamushi detection kit. show
that O. tsutsugamushi is found in 36 positive samples which is equivalent to the
sensitivity of O. tsutsugamushi detection kit of 97.3% (36/37). In the healthy
control group, the results do not detect O. tsutsugamushi in 99 negative samples
for O. tsutsugamushi which is equivalent to the specificity of O. tsutsugamushi
detection kit of 99% (99/100).
Step 7: Evaluate the accuracy and repeatability of the kit
- Accuracy: Test the detection of O. tsutsugamushi with O.
tsutsugamushi detection kit repeated 3 times on 10 assumed samples. Among the
10 specimens, it was assumed that there were 2 negative O. tsutsugamushi
samples and 8 samples containing different O. tsutsugamushi content. The
results show that the O. tsutsugamushi detection kit is 100% compatible with the
known results of 10 presumed samples. Thus, the O. tsutsugamushi detection
kit’s accuracy is high.
- Repeatability: Use the O. tsutsugamushi detection kit to detect O.
tsutsugamushi on 10 assumed specimens. Of which, there are 2 negative samples
for O. tsutsugamushi and 8 samples containing different O. tsutsugamushi

contents from 150 copies/reactions to 145,000 copies / reaction. The results
show that the samples have a concentration of ≥500 copies/reaction, the


17
repeatability of the kit is very good, showing the same results in 3 repetitions
performed by 1 person and in comparison of two people’s result. Samples with
the concentrations of <500 copies/reactions have lower repeatability. Thus, the
O. tsutsugamushi detection kit’s repeatability is good.
Step 8: Evaluate the stability of the kit
- The stability of the O. tsutsugamushi detection kit is evaluated based
on the positive/negative results and the comparison of the time receiving
amplified signal between reactions using preserved products and new products
on the same specimen.
Table 3.24. Results of assessment of the stability of the kit based on Orientia
tsutsugamushi detection result in specimen
Preservation time
Test result of the
Test result of the new
preserved products
products
7 days
+
+
14 days
+
+
30 days
+
+

60 days
+
+
90 days
+
+
The results show that with the same sample analyzed on the products
preserved after 7, 14, 30, 60 and 90 days and the new products, the results of O.
tsutsugamushi detected on the samples using preserved products coincide 100%
with the new prepared products. The time receiving the amplified signal when
using preserved products is later than the new products about 36.48 seconds.
Thus the O. tsutsugamushi detection kit is stable for at least 90 days in proper
storage conditions (-20°C).
Table 3.25. Evaluation result of the kit’s stability based on the time receiving
amplified signal
Preservation time
Test result of the
Test result of the new
preserved products
products
7 days
6.72 minute
6.59 minute
14 days
6.49 minute
6.17 minute
30 days
7.44 minute
6.59 minute
60 days

7.45 minute
6.72 minute
90 days
7.78 minute
6.77 minute
Step 9: Develop the basic standard of nucleic acid amplification kit for
O. tsutsugamushi diagnosis.
The team has successfully developed the basic standard for the Orientia
tsutsugamushi diagnostic nucleic acid amplification kit. Basic content includes
content:
- Purpose of the kit.
- Content: principles of the kit, detection targets, components of the kit,
chemical materials and equipment provided by users, how to perform.
- Packaging, labeling: product description and packaging.


18
Step 10: Develop process for testing, diagnosis of O. tsutsugamushi by
isothermal amplification kit RPA.
Test process is a follows:
Specimen

Extract DNA
Isothermal acid nucleic amplication
RPA
Read result
-

Perform nucleic acid amplification reaction RPA:
+ Completely defrost the reaction components and the extracted DNA

samples (if frozen) and keep them on the stone.
+ Mix well and do quick centrifuge the reaction components to obtain
homogeneous mixtures, then keep them on the stone for the highest test
effect.
+ Prepare reaction mix.
Table 3.29. Components of the reaction mix
#
1
2
3
4
5

Components

Original
concentration
2X
25 mM

Volume for 1
reaction (µl)
10
1.44
0.52
2
1.64
1
0.4
17

1
2
20

Reaction Buffer
dNTPs
Nuclease-free water
Probe E-mix
10 X
Oligo-mix
Core Reaction Mix
20 X
6
Exo
50 X
Total volume of the buffer solution of the reaction
8
MgOAc
280 mM
9
DNA sample
Total volume of the buffer solution of the reaction
Table 3.30. Isothermal process
Temperature
Time
Remark
37°C
20 minutes
Amplification (receive signal once
every 20 minutes)

4°C
Undefined
Keep the samples in the machine
+ Transfer the reaction tubes to the isothermal machine and start the
reaction according to the installed reaction conditions.


19
-Read results:
+ Results for Orientia tsutsugamushi detection using an isothermal kit
RPA are determined based on the amplification signal obtained from
the system receiving fluorescent signal.
+ Check samples of positive control and negative control to ensure test
results are not false negative or false positive.
The positive control sample must show a positive result and the time
receiving of the amplified signal is same to the time receiving the
amplified signal of the previously titrated sample (positive control
provided with the kit has 8 to 10 minutes to receive the amplified
signal).
Negative control must show negative result.
+ If the control sample system is positive and negative controls are not
correct, the test must be performed again.
Chapter 4.
DISCUSSION
4.1. Infection characteristics of Orientia tsutsugamushi, trombiculid mite
larvae and the distribution of scrub typhus in Northwest region
Scrub typhus is a disease that has occurred in Vietnam, discovered since the
early twentieth century. After 1990, the cases with scrub typhus tend to increase
and its distribution area is expanded in recent years. [34].
The proportion of people with anti-O.tsutsugamushi antibodies is found in

all 4 study sites with different rates, the common infection rate of 4 sites is
12.7%. The study site has the highest rate of people with anti-O. tsutsugamushi
antibodies is Hoa Binh 16.72%, followed by Dien Bien 14.42%, Son La 9.05%
and Lai Chau 8.67%. This result is consistent with the general survey rate of
people with anti-Orientia tsutsugamushi antibodies in Asia [44]. However, the
percentage of people with anti-O. tsutsugamushi antibodies in the study sites is
lower than the results of the author Doan Trong Tuyen et al when do a
serological survey for anti-O. tsutsugamushi antibodies detection in the
community in Khanh Hoa (21.04%) [35]. The infection rate of O. tsutsugamushi
is higher in Hoa Binh and Dien Bien than in Gia Lai (10.58%); and higher in 04
study provinces than in Kon Tum(5.2%) [34].
Scrub typhus is often found in forested areas, place with high density of
trees and weeds, especially areas such as stream banks, caves, forest has just
been destroyed or planted... Our study found that the highest rate of people with
anti-O. tsutsugamushi antibodies in the community living in the area of Savan
(15.19%), then to regenerated forest and primary forest (14.03%, 6.16%,
respectively). This result is similar to the research results in the Central
Highlands with the rate of people with anti-O. tsutsugamushi antibodies living in
Savan, regenerated forests, and primary forests is 15.16%, 13.99%, 5.21%,
respectively. [34].
Our study’s results show that there is no difference in O. tsutsugamushi


20
infection rate between male (10.93%) and female (13.54%) and among ethnic
groups. However, there are differences in infection age, the infection rate of
working age is higher than other groups. The infection rate in the agricultural
and forestry occupation group is higher than the other groups.
- Some distribution characteristics of scrub typhus
+ Distribution of patients with scrub typhus locally

Patients with scrub typhus are found and treated in provincial general
hospitals in all 04 study provinces. The province has the highest number of
patients with scrub typhus is Dien Bien (78 patients, account for 33.91% of the
cases in 4 provinces), followed by Hoa Binh (69 patients, accounted for
30.00%), Son La (48 patients accounted for 20.87%), the province with lowest
rate is Lai Chau (35 patients accounted for 15.22%).
Our study gives the evidence that scrub typhus may occur in all northern
regions of our country. One of the foundations for this assertion is the
widespread distribution of Leptotrombidium in all regions and topography of our
country, according to a survey by Nguyen Van Chau [6], [12], [13]. Our study
and other similar studies is a prerequisite for deep and extensive studies on
epidemiology of scrub typhus in our country.
The patients in our study are in department of infection of 04 provincial
general hospitals. Therefore, most of these patients are in severe or unknown
diagnosis transferred from the lower levels. The condition and accessibility to
health care services of the people in different locality may be the cause of the
incompatibility in the rate of patients with scrub typhus and the rate of people
with anti-Orientia tsutsugamushi antibodies in the provinces. The number of
patients with scrub typhus detected and treated in provincial hospitals compared
to the actual number may be small, because not all patients with scrub typhus in
the lower levels are transferred to the provincial hospital. The actual burden of
scrub typhus in localities may be even greater.
+ Characteristics of hosts and vectors of scrub typhus
Results of serum test on mice in 04 study sites all found anti-O.
tsutsugamushi antibodies. In Hoa Binh, the mice with antibodies include: Rattus
exuians (13.16%), forest mice (36.56%), and Bandicota savilei (20.97%). The
common rate of mice with anti-O. tsutsugamushi antibodies is 21.82%. In Son
La, the mice with anti-Rickettsia antibodies include: Rattus exuians (5.23%),
Rhizomyinae (68.38%). It is noteworthy that the Rhizomyinae live near the
houses and in the houses, near people with high antibody rates in Son La. The

common rate of mice with anti-O. tsutsugamushi antibodies is 42.45%. In Dien
Bien, the mice with antibodies include: forest mouse with the highest
rate42.16%), followed by Bandicota savilei (15.99%) and Rattus exuians
(7.03%). The prevalence of common O. tsutsugamushi antibodies is 20.78%. In
Lai Chau, individual mice with antibodies include: Rhizomyinae (36.36%),
forest mice (5.88%) and mice (5.10%). The common rate of mice with anti-O.
tsutsugamushi antibodies is 15.77%. The rate of mice with anti-O. tsutsugamushi
antibodies is higher in Khanh Hoa (14.30% of Bandicota savilei (B. savilei) [34].


21
The common density of mice in Hoa Binh is 7.68 (indoor: 7.60; outdoor:
7.75), in Son La is 11.43 (indoor: 7.65 and outdoor: 15.20), in Dien Bien is
16.60 (indoor: 12.05, outdoor: 21.15) and in Lai Chau is 6.98 (indoor: 7.85 and
outdoor: 6.10). The highest density of mice in Dien Bien (16.6) is equivalent to
the rate in Bac Giang (16.0) [11] and Quang Ninh (15.58) [7], [12]. The density
of mice in the remaining study sites is lower than in Bac Giang and Quang Ninh,
but it is similar to the general density of mice in Khanh Hoa (6.67 mice / 100
traps /night), and in Gia Lai (10.0) [34].
4.2. Proceduce of the O. tsutsugamushi detection kit
- Reaction conditions of the O. tsutsugamushi detection isothermal
amplification kit RPA in laboratory
Isothermal amplification technique RPA uses a process that can operate
optimally at the temperatures between 22°C and 45°C and does not require strict
temperature control [83], [86]; Most published reports have optimized the
reaction temperature between 37 - 42°C. The temperature conditions of RPA are
easy to set up and ensure, compared to other isothermal amplification techniques
such as NASBA, SDA or LAMP [101]. Based on the manufacturer's
recommendations, the recombinant enzymes used in this study have a reaction
temperature of about 37 - 42°C. To set the temperature to ensure the highest

amplification efficiency, the reaction temperature range of 37°C; 38°C; 39°C;
40°C and 41°C have been tested.
The test results at different temperatures with the same concentration of
positive control samples show that the earliest amplification signal was obtained
at 37°C (6 minutes 26 seconds). Due to time limitation, this study only used the
Genie II equipment (a compact, specialized isothermal amplification device) to
carry out the RPA reaction, the test on normal heat or warmth equipment has not
been conducted. However, the above study results also show that RPA
technology can completely be applied on common equipment such as heat
preservation cabinets, thermostatic tanks or heat preservation blocks. There have
been publication on conducting RPA reaction with similar equipment [86].
The average temperature of body in armpits, abdomen, area at the
pocket of pants and hands is 34.8 ± 0.6, 31.3 ± 1.7, 33.1 ± 0.5 and 33.4 ± 2.7 °C,
respectively. The resulting reaction mixture is simply incubated at these sites in a
simple way. In less than three minutes, all the pseudo-reactions reached a
temperature of 31 degrees Celsius, all RPA reactions appear to amplify the DNA
to a detectable level. The results also demonstrate that the body temperature in
the armpit area is relatively stable and reaches the closest to the recommended
temperature for RPA (37°C).
- Time of RPA reaction
The time of the amplification reaction is the time required for the DNA
sample in the reaction mixture to be amplified to a detectable level. This time
depends on the number of DNA copies at the time the amplification reaction
begins. For RPA technology, the required time is about 20-30 minutes, maybe
even shorter (about 3-4 minutes) [142]. The prolonged reaction time may not be


22
favorable for RPA reaction in solution, since recombinant enzymes have used
ATP completely during this period. Based on the correlation between the

standard DNA concentration and the time of the amplification signal, the 20minute RPA reaction time is used in this study. The results show that the best
amplification performance is obtained at 37°C for 20 minutes then incubated at
room temperature for 3-5 minutes. In this study, the results for reaction time is
20 minutes.
Thus, the appropriate time for the reaction of the isothermal
amplification technique RPA is about 20-30 minutes, which is much better than
the diagnostic techniques currently used to detect scrub typhus’s cause such as
ELISA, IFA, PCR or Realtime PCR (about 3-6 hours) or classic clinical
techniques such as culture (several days, even up to 2 months) or Weil - Felix
reaction (long time, low specificity).
- The reaction compositions of Orientia tsutsugamushi detection
isothermal amplification kit RPA in laboratory
The composition of gene amplification reaction includes polymerase,
dNTP, KCl, MgCl2... Of which MgCl2 solution plays a very important role in the
quality of the reaction. The concentration of Mg2+ ions in the reaction mixture
can affect the specificity and effectiveness of the reaction, so it is defined as the
most important composition. Most polymerase enzymes require 2 valence ions
for their activity. dNTP and the primers are all attached to Mg2+, so it is
necessary to adjust the concentration of MgCl2 to be greater than the total molar
concentration of the two compositions. Negative ions and EDTA are included in
the buffer system containing DNA molds that can capture Mg2+, thus reducing
their number. High concentrations of MgCl2 lead to an increase in the ability to
replicate of taq polymerase, but the exact replication is reduced. Therefore
reducing the concentration of MgCl2 will increase the exact replication of the
enzyme. The concentration of Mg2+ ions will depend on the template and the
primer thus it is required to be standardized when performing PCR. The standard
concentration of MgCl2 for PCR is 1.5 mM.
Like other gene amplification techniques, the compositions of RPA
reaction includes DNA or ARN templates, primer/probe sets, recombinant
enzymes and additives that make the appropriate buffering system for the

reaction to occur. While setting up the reaction compositions, the optimal
concentration of primer/probe and MgOAc is as follows: Primer 0.54 µM; Probe
0.1 µM; MgOAc 18 mM.
Mg2+ ions play a very important role in the specificity and amplification
of the reaction, but also in activating recombinant enzymes in extending the
chain in DNA synthesis. RPA reaction does not require a strict control of
temperature fluctuations and the required temperature is 37 oC, therefore, it can
be even conducted in conditions in warm climate area (about 30 oC).
Presence of inhibitors: RPA technique has been proven to be performed
with the input samples as serum or samples containing inhibitors of PCR
reaction such as hemoglobin, ethanol or heparin. [83]. For a preliminary


23
assessment of this issue, the author conducted a test to perform RPA reaction
with the serum samples of patient who is positive for Orientia tsutsugamushi, the
serum sample of healthy people added an amount of DNA of Orientia
tsutsugamushi, samples of negative control and positive controls as plasmids
containing the gene segment of Orientia tsutsugamushi created from previous
experiments.
4.2.2. The main specifications of the O.tsutsugamushi detection kit
- Detection threshold of isothermal amplification kit RPA
The evaluation results of the detection threshold of RPA process
optimized on the standard panel containing Orientia tsutsugamushi DNA in
plasmid form of this study achieved 10 copies /reaction. This is a good detection
threshold which can be used for specimen as blood of the clinical patients with
scrub typhus. Almost at the same time, in a publication in 2018, the method of
rapid diagnosis of Orientia tsutsugamushi strains using RPA and Lateral Flow
Test was successfully developed. The authors used the 56-kDa target gene on the
Karp-like strain circulating in China. The detection threshold achieved with

DNA detection reaction in plasmid form is 10 copies/ reaction and in the genome
of the bacterium is 12 copies/ reaction [117], [118]. Thus, the study results are
equivalent to the latest’s ones in the world in the same field.
However, the isothermal amplification kit RPA set by the team just
assesses the detection thresholds and sensitivity on positive control panels
containing 47 kDa target gene plasmid of Orientia tsutsugamushi. When
evaluating the sensitivity of the kit on 37 specimens positive for Orientia
tsutsugamushi, the technical sensitivity of the kit reaches 97.3%. The tests on
blood samples of patients diagnosed with clinical scrub typhus by other methods
with a larger number of samples and at different times must be continued. It
plays a role in assessing not only the detection threshold or the actual sensitivity
of the kit, but also the most appropriate clinical time to collect specimens as well
as the other specimens need to be collected.
- The specificity of isothermal amplification kit RPA
The results on evaluating the technical specificity of the kit are 100%
on the panel of negative controls and microbial strains that can cause symptoms
similar to Orientia tsutsugamushi. The result of evaluating the specificity on 99
samples negative for Orientia tsutsugamushi of the kit reaches 99%. The
specificity of the kit set by the group is good compared to the Orientia
tsutsugamushi detection RPA processes in the world.
- Evaluation of detection threshold and specificity of RPA reaction compared
to Orientia tsutsugamushi detection realtime technology PCR
The parameters of the nucleic acid isothermal amplification process
RPA have been compared with the bio-product kit Orientia tsutsugamushi
Membrane Protease Gene Genesig Standard Kit by Primerdesign, this is a
commercial kit with realtime PCR technique with high threshold and specificity.
The results compare the sensitivity of Orientia tsutsugamushi detection RPA
reaction with the Genesig kit using realtime PCR technology shows that the