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DENGUE DIAGNOSTICS
DENGUE DIAGNOSTICS
The Right Test at the Right Time
for the Right Group

edited by
Shamala Devi Sekaran
Published by
Jenny Stanford Publishing Pte. Ltd.
101 Thomson Road
#06-01, United Square
Singapore 307591

Email: [email protected]
Web: www.jennystanford.com

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

Dengue Diagnostics: The Right Test at the Right Time for the
Right Group
Copyright © 2024 by Jenny Stanford Publishing Pte. Ltd.
All rights reserved. This book, or parts thereof, may not be reproduced in any form
or by any means, electronic or mechanical, including photocopying, recording
or any information storage and retrieval system now known or to be invented,
without written permission from the publisher.

For photocopying of material in this volume, please pay a copying fee through
the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923,
USA. In this case permission to photocopy is not required from the publisher.

ISBN 978-981-4968-97-3 (Hardcover)

ISBN 978-1-032-66956-4 (eBook)
 Contents

Preface vii

1. Introduction to Dengue and Issues with

Current Diagnostics 1
Shamala Devi Sekaran, Rishya Manikam,
and Gaythri Thergarajan
1.1 Current Epidemiology 2
1.2 Diagnosis 5
1.3 Immune Responses to Dengue 6
1.4 Markers and Problems 8

2. Traditional and Current Diagnosis 13

Wang Seok Mui and Chandramathi Samudi Raju
2.1 Virus Isolation 15
2.1.1 Intracerebral Injection into Mice 15
2.1.2 Inoculation of Specimen into
Mosquitoes 16
2.1.3 Viral Culture Using Mosquito or
Mammalian Cell Line 17
2.2 Detection of Dengue-Specific Antigens and
Antibodies 18
2.2.1 Antigen Detection 18
2.2.1.1 Non-structural protein
(NS1 & NS5)-based assay 18
2.2.2 Antibody Detection 20
2.2.2.1 Hemagglutination
inhibition test 20
2.2.2.2 Envelope/membrane
(E/M)-antigen ELISA
(both IgM and IgG) 22
2.2.2.3 Rapid diagnostic tests 23
2.3 Viral Nucleic Acid Detection 23
2.3.1 Polymerase Chain Reaction (PCR) 23
2.3.2 Nucleic Acid Sequence‒Based
Amplification (NASBA) 24
vi Contents

2.3.3 Loop-Mediated Isothermal

Amplification (LAMP) 25
2.4 Diagnostic Limitation 26
2.5 Future Directions 27

3. Newer Diagnostics for Dengue Disease 35

Crystale Lim Siew Ying, Nur Haliza Hassan,
and Shamala Devi Sekaran
3.1 Biosensors in Dengue Diagnosis 35
3.2 Biosensor Platform 42
3.2.1 NS1 Antigen/Antibody Detection 42
3.2.2 Detection of Anti-Dengue IgM
Antibodies 43
3.2.3 Detection of Dengue Viral Genome 44
3.3 Limitations 45

4. Search for Newer Markers for Dengue Disease 51

Yong Yean Kong, Tan Hong Yen, Shamala Devi Sekaran,
and Rishya Manikam
4.1 Introduction 51
4.2 Viral Markers and Their Association with
Dengue Severity 52
4.3 Clinical Markers of Severe Dengue 55
4.4 Markers of Immune Activation and
Severe Dengue Disease 56
4.5 Cytokines in Dengue Infection 64
4.6 Metabolites Released During a Dengue Infection 72
4.7 Markers of Endothelial Activation and
Coagulopathy 74
4.8 Limitations 77
4.9 Research Aspects Needed 78

5. Research and Recommendations 91

Shamala Devi Sekaran
5.1 Research Aspects Needed 92

Selected Protocols 95

Index 103
 Preface

Dengue is a mosquito-borne viral infection that affects millions of

people worldwide every year. It can cause a range of symptoms,
from mild fever and rash to severe hemorrhagic fever and shock.
Dengue is a major public health problem, especially in tropical and
subtropical regions, where it poses a significant burden on health
systems and economies.
The diagnosis of dengue is crucial for the timely management
and treatment of patients, as well as for the prevention and
control of outbreaks. The diagnosis of dengue is challenging due
to the complexity of the disease, the diversity of the virus, and
the limitations of the available diagnostic methods. The clinical
manifestations are often nonspecific and overlap with other febrile
illnesses. Moreover, the laboratory confirmation of dengue requires
the detection of either the virus, its antigens, or its antibodies,
which have different sensitivities and specificities at different stages
of the infection. Therefore, there is a need for accurate, rapid, and
affordable diagnostic tests that can be used in various settings and
scenarios.
This book is based on my extensive experience and research in
the field of dengue diagnostics as well as that of the experts and
collaborators who have contributed to the chapters. It provides a
comprehensive overview of the current state of the art in dengue
diagnostics, covering the principles, techniques, applications, and
challenges of various methods. It also discusses future directions
and perspectives for the development and improvement of dengue
diagnostics.
The book is intended for researchers, clinicians, public health
professionals, students, and anyone interested in learning more
about dengue diagnostics, and I hope it will be a valuable resource
and reference for them and will inspire further research and
innovation in this important area of global health.
Shamala Devi Sekaran
Summer 2023
Chapter 1

Introduction to Dengue and Issues

with Current Diagnostics

Shamala Devi Sekaran,a Rishya Manikam,b

and Gaythri Thergarajana
aFacultyof Applied Sciences, UCSI University Kuala Lumpur Campus,
Malaysia
bEmergency and Trauma Centre, University Malaya Medical Centre,

Kuala Lumpur, Malaysia

[email protected]

Historically dengue is said to have been derived from the Swahili

phrase “Ki-denga pepo”, which basically translates to mean cramp-
like seizures caused by an evil spirit. The Swahili word may possibly
originate from the Spanish word “dengue”, which actually describes
the gait of a person suffering from intense bone pain (Hotta, 1952;
Skae, 1902). Today, however, we know dengue as a viral infection
caused by the dengue virus belonging to the family Flaviviridae.
The first record of this illness is recorded in the Chinese medical
encyclopedia during the Jin Dynasty (265–420) and the illness was
referred to as a water poison associated with flying insects. The

Dengue Diagnostics: The Right Test at the Right Time for the Right Group
Edited by Shamala Devi Sekaran
Copyright © 2024 Jenny Stanford Publishing Pte. Ltd.
ISBN 978-981-4968-97-3 (Hardcover), 978-1-032-66956-4 (eBook)
www.jennystanford.com
2 Introduction to Dengue and Issues with Current Diagnostics

first report of dengue disease was reported in Philadelphia in 1780.

First, recognized epidemics were noted to occur simultaneously in
Asia, Africa, and North America in the 1780s. Upon identification,
the disease was named with the first confirmed case report in the
same year by Benjamin Rush who was the one who coined the
name “breakbone fever” because of the symptoms of myalgia and
arthralgia. Japanese scientists were the first to discover the DENV-1
in 1943. Subsequently, in 1944, DENV-2 was discovered by Albert
Sabin. In Malaysia, Skae reported the first known published
outbreak of dengue in 1902, while severe dengue was first observed
in Georgetown Penang in 1962 (Skae, 1902).
Dengue is a mosquito-borne viral infection caused by the dengue
virus, the genus of which contains four serotypes. While this virus
causes mild infections with flu-like illness, it can occasionally
develop into a potentially lethal complication called severe dengue.
Found mainly in tropical and sub-tropical climates and mostly in
urban and semi-urban communities, it has, however, spread over the
temperate zones as a result of climate change. There is currently no
specific treatment but early detection and access to proper medical
care can lower the fatality rate to below 1%. The global incidence
as seen in Table 1.1 indicates the populations at risk and this has
been on the rise, and it is currently estimated that about half of the
world’s population is now at risk (Figure 1.1). Currently, prevention
and control depend on stringent effective vector control measures
with the help of community participation.

1.1 Current Epidemiology

In 2022, more than 2357301 cases and 1731 deaths were reported.
Most of the cases recorded were from Brazil, Vietnam, Peru, the
Philippines, and Indonesia; and most deaths were reported from
Brazil, Indonesia, the Philippines, Peru, and Timor-Leste. All four
dengue serotypes were detected. In Europe, no autochthonous
case of dengue was reported in 2022 (European Centre for Disease
Prevention and Control [ECDC] 2022).
 Current Epidemiology 3

Table 1.1 Dengue cases around the globe, as of July 2022

(Extracted from https://www.ecdc.europa.eu/en/dengue-monthly)

Year Date Country Number Number

of cases of deaths
2022 9 July Afghanistan 22 0
2022 26 July Bangladesh 6673 8
2022 14 July Cambodia 3322 9
2022 31 May China 5 0
2022 9 July Indonesia 52313 448
2022 14 July Laos 6393 10
2022 14 July Malaysia 26420 19
2022 31 May Maldives 344 0
2022 24 July Nepal 232 0
2022 7 April Oman 76 0
2022 4 July Pakistan 875 0
2022 25 June Philippines 64797 274
2022 25 July Singapore 21350 0
2022 25 July Sri Lanka 32404 0
2022 19 July Thailand 8660 0
2022 27 May Timor-Leste 4985 56
2022 14 July Vietnam 103433 37
2022 28 April Kenya 33 0
2022 14 July Australia 66 0
2022 28 May Cook Islands 3 0
2022 4 June Micronesia 16 0
2022 14 July Palau 22 0
2022 7 May Solomon Islands 34 0
2022 16 June Vanuatu 39 0
2022 16 June Wallis & Futuna 21 0
2022 27 July Brazil 1827617 737
2022 27 July Peru 56021 65
2022 27 July Colombia 32327 NA
2022 27 July Nicaragua 22887 NA
2022 27 July Ecuador 12279 NA
 4
Introduction to Dengue and Issues with Current Diagnostics

Figure 1.1 Geographical distribution of dengue cases reported worldwide, May to July, 2022.
 Diagnosis 5

1.2 Diagnosis
Dengue can be diagnosed clinically; however, laboratory tests are
required to confirm the infection. Definitive diagnosis is important
not only for the clinical management of patients, but also for
interventions during outbreaks, epidemiological surveillance, and
for vaccine development and monitoring. The development of assays
is ongoing and newer assays using more advanced technologies
have been developed, though not fully validated. The main hurdle
lies in the incompletely understood pathogenesis of dengue and
that multiple sequential infections occur in dengue-endemic areas,
notwithstanding the issues with primary and secondary infection
status that further complicate the diagnosis. Therefore, for a
diagnostic assay to be useful and effective, it is essential for users
to have some degree of confidence in the test in order to improve
disease management, especially in the acute early stage and for
detecting signs of severity. An ideal dengue diagnostic test would
be one that is simple, rapid, with high sensitivity and specificity,
preferably able to differentiate between primary and secondary
infections, serotype the virus, and most important of all affordable.
The optimal time frame for diagnosis would be from the onset to
10 days post-infection. In reality, more than half only consult the
physician when in dire situations, with many people in third-world
countries resorting to traditional healing. It is also important to note
that 2% do not seroconvert and a large number are asymptomatic.
Diagnostic tools currently are mainly serologically based,
nucleic acid-based, or antigen detection. A good understanding
of the clinical conditions of dengue patients is essential for the
appropriate usage of these tests. The highest confidence lies in the
isolation of the virus, thus fulfilling Koch’s postulate, detection by
direct immunofluorescence (IF), and genome amplification via
PCR (Medina et al., 2012; Prescott, Feldmann, & Safronetz, 2017;
Salles et al., 2018; World Health Organization, 2009), or detection
of viral antigens like NS1 using either ELISAs or rapid lateral flow
tests (Chong et al., 2020; Guzman, Jaenisch et al., 2010; Hunsperger
et al., 2009; Kumarasamy, Chua et al., 2007; Kumarasamy, Wahab et
al., 2007; Peeling et al., 2010; S. D. Sekaran, Lan, & Subramaniam,
2018; Shamala Devi, 2008; Tricou et al., 2010; Wang & Sekaran,
2010b, 2010a). However, more often than not we tend to use tests
6 Introduction to Dengue and Issues with Current Diagnostics

out of convenience and hence look for antibodies, and for these,
diagnostically useful results are best obtained using paired samples.
Currently, virus isolation, PCR, and direct IF are carried out only in
reference and research laboratories. NS1, however, is more user-
friendly, and apart from ELISAs, rapid flow tests have been developed
and validated. These, however, are useful in the first 5 days of
symptoms when viremia still exists. Serology can also be utilized
by demonstrating a seroconversion from negative to positive IgM
antibody to dengue, or a four-fold or greater increase in IgG antibody
titers in paired (acute and convalescent) serum specimens. Patients
who are IgM positive and PCR negative are usually classified as recent
probable dengue infection as IgM antibodies to dengue remain
elevated for 90 days after the illness and could have been from an
infection that occurred 2–3 months ago. In addition, it is important
to note that cross-reactivity with other flaviviruses including West
Nile virus (WNV), St. Louis encephalitis virus (SLE), Japanese
encephalitis virus (JEV), Zika virus, and yellow fever virus (YFV)
do occur and hence a review of the patient’s past medical history,
recent travel history, and vaccination record (especially yellow fever
vaccination) are needed to determine the likelihood that the current
acute febrile illness is actually due to an infection with dengue
virus. In many instances, a paired sample is best utilized to make
a diagnosis, especially for those who present late after the onset of
illness (> 5 days), as virus and viral antigens become undetectable,
and in these instances, the tests need to be carried out on all viruses
suspected. The current infection will be the only pair demonstrating
a four-fold rise (Schilling, Ludolfs, Van An, & Schmitz, 2004; Shamala
Devi Sekaran & Soe, 2017).

1.3 Immune Responses to Dengue

Dengue virus induces the production of all classes of antibodies
primarily targeting the virus envelope proteins. The level of
antibodies primarily depends on whether the individual has primary
or secondary dengue infection (S. D. Sekaran, Ew, Subramaniam, &
Kanthesh, 2009; Shamala Devi Sekaran & Artsob, 2007; Uno & Ross,
2018). Figure 1.2 shows the timelines of the disease and Figure 1.3
displays the production of the various aspects of the immune
response.
 Immune Responses to Dengue
7

Figure 1.2 Timeline of the various components of the dengue infection.

8 Introduction to Dengue and Issues with Current Diagnostics

Figure 1.3 Diagnostic timeline of primary and secondary immune responses.

In recent years, the development of rapid assays has allowed

patient specimens to be tested in point-of-care situations. Many
manufacturers of PCTs claim the tests are able to detect and
differentiate between primary and secondary infections with dengue
(Changal et al., 2016; Lovera et al., 2019). However, the diagnostic
accuracy of such assays has not been reliably established because
of the multiplicity of methodologies used in the evaluation. Newer
bio-diagnostic devices that can be quantitative and/or qualitative,
are the prototypes of the future rapid diagnostic test kits that will
be commercialized if they have desirable traits such as the ability
to be portable, automated, and easily disposed of. A survey of the
literature shows that most biosensors that are being developed
for dengue use piezoelectric, optical, and electrochemical methods
(Eivazzadeh-Keihan et al., 2019). More recently, nanoparticle beads,
mass spectrometry, as well as micro-sequencing have been utilized
and appeared promising. Generally, biosensor kits need to be
validated to be used as a rapid test for dengue.

1.4 Markers and Problems

Diagnosis begins with a clinical suspicion but has limited usefulness
for early diagnosis. Clinical symptoms have high sensitivities but
 References 9

have poor specificities and hence a confirmatory diagnosis is

essential. The current markers used are the non-structural antigen-1
(NS1), IgM, and IgG antibodies, while virus isolation and nucleic acid
amplification are only utilized in laboratories tracking infections or
public health laboratories. The titers of the IgM and IgG antibodies
depend on whether the infection is a primary or secondary infection.
During primary (first) dengue infection, IgM levels are very high, but
during secondary infection, IgM levels are lower. The levels of IgG
actually increase during secondary infection. Clinicians, therefore,
need to measure IgM and IgG quantitatively in order to determine
whether a patient has primary or secondary dengue infection. The
main issue with confirmatory assays is the validation of each assay.
Hence, early diagnosis remains a challenge, more so markers to
use, and the time frame of detection. Using a combination of clinical
markers and laboratory-confirmed assays may serve as predictive
markers at different stages of the disease.

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Chapter 2

Traditional and Current Diagnosis

Wang Seok Muia and Chandramathi Samudi Rajub

aDepartment of Medical Microbiology & Parasitology, Faculty of Medicine,

Universiti Teknologi MARA, Selangor, Malaysia

bDepartment of Medical Microbiology, Faculty of Medicine,

University of Malaya, Kuala Lumpur, Malaysia

[email protected], [email protected]

Dengue virus causes a spectrum of illnesses ranging from

undifferentiated mild febrile illness to severe dengue hemorrhagic
fever/dengue shock syndrome. Diagnosing dengue fever can
be challenging due to its signs and symptoms and can be easily
confused with other diseases such as influenza, chikungunya,
malaria, leptospirosis, and typhoid fever. Clinical diagnosis alone
is not always reliable, as dengue has no pathognomonic clinical
features from other febrile illnesses (Rathakrishnan & Sekaran,
2013). As there is no specific treatment for dengue, timely and
careful clinical management of patients by experienced healthcare
providers frequently saves lives. Hence, early definitive detection
of infection is essential, and this can be achieved by confirmatory
tests of the patient’s samples. Although various laboratory methods
are currently used for diagnosing dengue, none fulfill the ideal
requirement for both sensitivity and specificity, while also being

Dengue Diagnostics: The Right Test at the Right Time for the Right Group
Edited by Shamala Devi Sekaran
Copyright © 2024 Jenny Stanford Publishing Pte. Ltd.
ISBN 978-981-4968-97-3 (Hardcover), 978-1-032-66956-4 (eBook)
www.jennystanford.com
14 Traditional and Current Diagnosis

rapid, simple, and low cost. Newer detection methods for dengue
that can fill this acknowledged gap are urgently needed.
Common laboratory methods for diagnosing dengue include
virus isolation, detection of viral antigens, genomic detection by
molecular methods, and serology. Dengue diagnosis is divided
into two main phases: the early phase and the late phase. Different
laboratory methods are being used to diagnose dengue at different
phases of illness (Figure 2.1). In the early or febrile phase of illness,
the approaches of diagnosis are via viral isolation, RT-PCR, and
antigen detection, while the later phase is mainly through serology
(Shamala, 2015). For a diagnosis of ‘confirmed’ dengue, dengue virus
should be isolated or viral RNA amplified, or there should be a four-
fold rise in antibody titer in paired (acute and convalescent) serum
specimens, or demonstration of seroconversion from negative to
positive IgM antibody. Serological tests such as IgM Capture ELISA
are favorable, especially in hospitals of dengue-endemic countries
due to their inexpensiveness, simplicity, and narrow time frame of
viremia (Lee et al., 2011). Nevertheless, serological assays can be
a challenge in hyperendemic areas where pre-existing antibodies
complicate diagnosis (Sekaran et al., 2014). The choice of a test,
therefore, depends not only on the availability of facilities and
human resources but more importantly on the time of sampling.

Figure 2.1 Suitability of dengue diagnostics at different phases of illness.

 Virus Isolation 15

2.1 Virus Isolation

Virus isolation has been widely used as a gold standard in dengue
diagnosis (Velez, Kuno, Gubler, Oliver, & Sather, 1984). The method
involves the isolation of viruses using various approaches such as
inoculation into mosquito-derived cell lines or mammalian cell lines,
intrathoracic and intracerebral inoculation of adult mosquitoes,
intracerebral inoculation of mosquito larvae, and suckling mice
brains (De Paula & Fonseca, 2004; Guzmán & Kourí, 1996; Philip
Samuel & Tyagi, 2006; Shu & Huang, 2004). Virus isolation is highly
achievable when specimens are collected during the viremic phase,
which is 2–3 days prior to the onset of fever, and lasts for a further
2–3 days (Vorndam & Kuno, 1997). Therefore, samples for virus
isolation must be taken in the first few days of the disease. An acute
phase serum sample is the specimen of choice for routine dengue
virus isolation; however, dengue virus can also be detected in plasma,
peripheral blood, cerebrospinal fluid, pleural fluid, and tissues of
reticuloendothelial origin such as the liver, spleen, lymph nodes,
lung, and thymus (Guzmán & Kourı́, 2004; Vorndam & Kuno, 1997).
Following harvest, confirmation tests are then performed under a
UV microscope using an immunofluorescence method with dengue-
specific monoclonal antibodies or detection of viral nucleic acid
using a polymerase chain reaction. Thus, this method requires skill
and substantial equipment to be carried out and is not the choice for
many laboratories. This method depends heavily on the survival of
the sample. Hence, the timing of specimen collection, proper storage,
and transportation are pertinent, as these factors may affect virus
viability. However, virus isolation remains very useful and relevant
as a diagnostic tool, especially for monitoring dengue epidemiology
and evolution as well as determining its antigenic drift (Sekaran et
al., 2014).

2.1.1 Intracerebral Injection into Mice

Intracerebral inoculation of suckling mice (e.g., BALB/c mice) has
been used successfully in isolating all four dengue serotypes in
1-day-old suckling using 10–20 µl of serum (Singharaj, Simasathien,
Sukhavachana, Halstead, & Scanlon, 1966). These suckling mice
may exhibit encephalitis symptoms (Amaral et al., 2011). It is the
16 Traditional and Current Diagnosis

oldest and least sensitive method for virus isolation. This method
is only used when no other methods are available. Nevertheless, it
offers the advantage of evaluating the neurotropic characteristics of
the dengue virus particle in vivo. Viruses are generally identified by
the immunofluorescence method using dengue-specific monoclonal
antibodies applied to the brain tissue of mice. Not all laboratories
house animal facilities nor the ready availability of suckling. As such,
storage of samples till the availability of the suckling would result in
a lower sensitivity especially when viral loads are low. The advantage
of using suckling mice is that other arboviruses that cause dengue-
like symptoms may be isolated with this system (See Appendix 1 for
protocol).

2.1.2 Inoculation of Specimen into Mosquitoes

Mosquito inoculation remains the most sensitive system for dengue
virus isolation and this specific method for isolation of DENV takes
between 2 and 5 days in adults and 2 and 3 days in the 4th instar
larvae of Toxorhynchites splendens (Lam et al., 1986). In adults and
larvae, intracerebral and intrathoracic inoculation have yielded
comparable results with the intracerebral method being more
sensitive (Amaral et al., 2011; Guzmán & Kourí, 1996; Lam et al.,
1986). This method has been under-utilized because most dengue
laboratories do not have insectary facilities and equipment to breed
mosquitoes, and precautions need to be taken to avoid the release
of infected mosquitoes. Besides, the technique requires good eye-
hand coordination while doing manipulation under a stereoscopic
microscope. Extensive practice is needed to become proficient
at inoculating mosquitoes. These highly skilled techniques in
conducting direct mosquito inoculation make in vitro cell culture
a preferred option (Philip Samuel & Tyagi, 2006). Vectors for the
dengue virus such as Aedes aegypti, Aedes albopictus, and Aedes
polynesiensis, and non-blood feeding Toxorhynchites species can
be used for this method. Mosquitoes of both sexes are susceptible.
Generally, Toxorhynchites species are used because of their
comparatively larger size, which facilitates the easier introduction
of the inoculum (Donald, Siriyasatien, & Kohl, 2020). Besides its
unique feature of not being hematophagous, it allows the work to
be carried out with impunity. The drawback of this method is that
 Virus Isolation 17

between 5 and 20 mosquitoes are needed to produce results of

higher sensitivity. Following inoculation, mosquitoes are held in the
incubator at 32°C for 14 days to allow the dengue virus to replicate
and disseminate to tissues throughout the mosquitoes (Choy, Ellis,
Gubler, & Ellis, 2013). Surviving mosquitoes are then harvested
and examined for the presence of viral antigens in head tissue by
either immunofluorescence assay or polymerase chain reaction (See
Appendix 2 for protocol).

2.1.3 Viral Culture Using Mosquito or Mammalian Cell

Line
In vitro culture of cell lines is an alternative technique for isolating
viruses from specimens. Because of the technical skill and special
containment required for direct mosquito inoculation, cell culture
is preferable for routine diagnosis, despite the greater sensitivity
of using mosquitoes. The common cell lines currently utilized
include the mosquito cells from Aedes albopictus (C6/36), Aedes
pseudoscutelaris (AP-61, AP-64), and Tinissa amboinensis (TRA-
284); and mammalian cells such as Vero and BS-C-1 (both derived
from African green monkey kidney), rhesus monkey kidney
(LLCMK2), porcine kidney (PS), and baby hamster kidney (BHK21)
(Guzmán & Kourí, 1996; Igarashi, 1978). These cell lines are easily
available, stable, and can be kept at room temperature for up to a
week in maintenance media. Isolation of viruses typically takes
7–10 days with daily observation for cytopathic effect (CPE) before
confirmation by immunofluorescence assay or polymerase chain
reaction. Sometimes more than two passages may be required
to isolate the virus or to increase the virus load. Not all viruses
produce CPE. For these, a blind harvest may be carried out and
further confirmed by confirmatory tests. Various methods have
been conducted in an attempt of speeding up the isolation rate by
centrifuging serum samples onto cell monolayers prior to incubation
(Kao, King, Chao, Wu, & Chang, 2005; Roche, Alvarez, Guzmán, Morier,
& Kourí, 2000) and locating viral antigens using flow cytometry after
a few days of inoculation (Kao et al., 2001; Oliveira De Paula, Malta
Lima, Clotteau, Pires Neto, & Lopes da Fonseca, 2003). Rodriguez
et al. reported that 16.6% more isolates than with the conventional
method were obtained when using the centrifugation technique
18 Traditional and Current Diagnosis

(Roche et al., 2000). However, these methods may not always work
especially when the viral loads are low. In a comparative study,
the virus isolation rate was found to be the highest in TRA-284,
followed by AP-61 and C6/36 (Kuno, Gubler, Vélez, & Oliver, 1985).
Nonetheless, C6/36 cells were favored for detecting infected cells by
the direct fluorescent antibody test. Problems with AP-61 and TRA-
284 are due to the frequent cell clumping and difficulties in forming
a monolayer in a cell culture flask. For all these techniques, skilled
and experienced personnel are needed to be able to recognize the
CPE and the type of immunofluorescence observed upon staining
with dengue-specific monoclonal antibodies (See Appendix 3 for
protocol).

2.2 Detection of Dengue-Specific Antigens and

Antibodies

2.2.1 Antigen Detection

Samples that could be used to detect DENV antigens during the
febrile phase of dengue illness, include peripheral blood leukocytes,
liver (Couvelard et al., 1999), and lung at autopsy, and less often in
the thymus, lymph nodes, skin, spleen (Balsitis et al., 2009) bone
marrow, and serosa (Nakao, Lai, & Young, 1989). The detection of
virus antigens in frozen and paraffin-embedded tissue sections can
be done using immunohistochemistry and enzyme immunoassay
(EIA) techniques. These techniques include the use of labeled
monoclonal antibodies that bind with detection markers such as
fluorescent dyes (fluorescent antibody), enzymes (avidin-biotin
enzyme and immunoperoxidase), or colloidal gold. However, the
difficulty in sample collection would be the main limitation to
use these detection methods as routine laboratory tests. Hence,
other assay methods were developed to detect virus particles and
recombinant viral proteins using NS1 and E protein (Cuzzubbo et al.,
2001; Kumarasamy et al., 2007).

2.2.1.1 Non-structural protein (NS1 & NS5)-based assay

NS1 is a glycoprotein that is usually secreted during the replication
of flavivirus. Infected host cells synthesize NS1, which rapidly
 Detection of Dengue-Specific Antigens and Antibodies 19

homodimerizes in the lumen of the endoplasmic reticulum (ER) and

attaches to the ER membrane. It is transported to the cell surface
via the secretory pathway and released into the extracellular
environment as a hexamer with lipid cargo in its middle area. This
secreted NS1 (sNS1) is stable and exists at a high level in serum. It
is detectable in patients’ serum between day 1 and day 9 after fever
onset. Hence, sNS1 can be generally used as a marker for the early
diagnosis of flavivirus infections (Alcon-LePoder et al., 2008). At
present, commercial assays are available only for the detection of
DENV NS1 Ag. Although the assay can cross-react with NS1 antigens
of other flaviviruses namely ZIKV and YFV, the assay sensitivity
is high toward DENV. Also, the detection of NS1 antigen using the
ELISA technique or any other rapid kit cannot differentiate between
dengue serotypes.
NS5 is a potential biomarker for dengue virus infection, due to its
highly conserved and immunogenic properties. In a past study, two
recombinant protein products (104 kDa full-length NS5 and 70 kDa
NS5-C7) were found to react with the sera of patients infected with
DENV-2 suggesting its potential use as an effective antigen to detect
dengue virus infection (Zhang et al., 2019). However, as most studies
have focused on the detection of NS5 in only DENV-2, more data is
needed to test its sensitivity toward other serotypes especially when
it is used in rapid test kits.
Rapid tests are applicable in resource-limited settings where
suitable healthcare facilities might not be accessible. Usually, all
the rapid tests take around 30 min to perform. Nevertheless, all
the rapid tests have a significant decrease in sensitivity compared
to the FDA-approved Detect NS1 ELISA by InBios International.
The general interpretation of the rapid test is a positive NS1
supports a DENV infection, while a negative result does not rule
out infection possibility. The negative result is followed by an IgM-
based test. Also, one should take note that NS1-based tests are not
recommended after 7 days. There are seven commercially available
dengue NS1 Ag detection assays, with four of them being rapid
tests. They are Dengue NS1 Ag STRIP (BioRad, Marnes-la-Coquette,
France), Platelia Dengue NS1 Ag ELISA (BioRad), Dengue NS1 Detect
Rapid Test (InBios International), DENV Detect NS1 ELISA (InBios
International), Panbio Dengue Early Rapid (Alere, Waltham, WA,
USA), Panbio Dengue Early ELISA (2nd generation (Alere), and SD
20 Traditional and Current Diagnosis

Bioline Dengue NS1 Ag Rapid Test (Abbott, Abbott Park, IL, USA).
These tests are only used for diagnostic purposes and not as point-
of-care tests (Kabir, Zilouchian, Younas, & Asghar, 2021).

2.2.2 Antibody Detection

DENV-specific IgM antibodies are present in patient serum from
4 to 5 days of primary infection and are detectable for up to 3
months. IgG antibodies in serum can be seen about a week after
the onset of fever, which remains at high levels for several weeks
before declining. Usually, IgG can be detected for decades. Memory
B cells are stimulated in response to secondary DENV infection for
secreting DENV-specific IgG that is detectable even on the first day of
symptoms. The key point is, the IgG titer remains much higher (more
than two folds) compared to its level during primary infection. In
secondary dengue, the IgM response is variable and sometimes
may be undetectable. The most commonly used serology detection
methods for dengue diagnosis are hemagglutination inhibition (HI)
and ELISA for IgM or IgG (Kao et al., 2005).

2.2.2.1 Hemagglutination inhibition test

The HI assay is generally used to titrate the antibody response to
a specific viral infection in vitro. Some viruses have the ability to
hemagglutinate (bind) red blood cells and avoiding the red blood
cells from clumping. The basic principle of HI assay is, if antibodies
that cross-react with the virus are present in the infected person’s
sera sample, the antibodies will bind to the virus thus preventing the
virus from hemagglutinating the red blood cells. Using HI, the exact
titers of the antibodies in the sera can be determined. Primary and
secondary DENV infections can be differentiated using HI but not an
early infection. However, for secondary infections, the IgG-based test
is superior to the HI. The disadvantage of this assay is that it may not
be able to detect antibodies of other dengue virus subtypes. Also,
it will not be able to detect antibodies against other structures of
the virus but only the ones against the hemagglutinating protein. For
this, plaque reduction neutralization tests (PRNT) will be the option
(Bourgeois & Oaks, 2014; Fry et al., 2011).
The HI test (Figure 2.2) is based on the ability of dengue antigens
to agglutinate red blood cells (RBC) of ganders or trypsinized human
O RBC.
Detection of Dengue-Specific Antigens and Antibodies 21

Principle of HI test.
Figure 2.2
22 Traditional and Current Diagnosis

2.2.2.2 Envelope/membrane (E/M)-antigen ELISA (both IgM

and IgG)
The immune system produces antibodies to fight against an antigen.
As part of the adaptive immune response towards the antigen, T
cell will trigger B cells to produce immunoglobulin (Ig) proteins or
antibodies (IgD, IgM, IgG, IgA, Ig E). IgM antibodies are commonly
produced 5 days post onset of symptoms, and it can last for about
2‒3 months and at times even longer. During dengue primary
infection, IgM level is raised while raised level of IgG is seen during
secondary infection (Hunsperger et al., 2009).
Various enzyme-linked immunosorbent assay capturing the IgM
antibody (MAC-ELISA) testing kits that are developed and marketed
for testing dengue infection globally. The basic principle of the test
is that the anti-human IgM antibodies on a solid phase will capture
the dengue IgM antibody in patient’s serum. Apart from MAC-
ELISA, a sandwich-type immunoassay has also been developed. This
technique uses the recombinant dengue-derived antigen and DENV-
specific monoclonal antibody. One of FDA approved kit would be
the DENV Detecta IgM Capture ELISA by InBios International, Inc.
which uses a 96 well plate to detect the human Dengue-IgM antibody
(Namekar et al., 2013). After IgM, IgG will then be produced and this
antibody can last for many years or even lifelong.
In recent secondary dengue infection, a four-fold spike in
seen in the IgG level. Dengue IgG ELISA kits are developed and
commercialised by Standard Diagnostics (Seoul, South Korea) and
Panbio Inc (Brisbane, Australia now Alere Inc., Waltham, WA, USA)
(Blacksell et al., 2012). It is known that dengue IgG antibody cross-
reacts with IgG antibodies of other flaviviruses. This leads to difficulty
in determining primary or secondary infection by depending on IgG
level. To overcome this, IgM/IgG ratio is suggested to be used for
differentiating the primary from secondary infection. A ratio between
the optical density (OD) measured from the respective ELISA tests of
IgM and IgG is measured whereby a ratio more than 1.32 implicates
primary infection and below 1.32 implicates secondary infection
(Prince et al., 2011).
 Viral Nucleic Acid Detection 23

2.2.2.3 Rapid diagnostic tests

Immunochromatographic tests are used for the rapid diagnosis of
dengue. Such test is used to detect dengue NS1 antigen within 5 to 7
days of fever/symptom onset. A rapid test approximately takes about
30 minutes and very suitable for resource-limited settings. There
is also antibody-capture immunochromatographic test cassette
available for the detection of IgM and IgG which are basically to
view the bands that are formed after adding a patient’s whole blood,
serum or plasma. Commonly used RDTs are Duo IgM and IgG Rapid
Test Strip (Panbio); Bioline Dengue IgG/IgM (Standard Diagnostics);
VScan (Minerava); Smartcheck (GlobaleMed); Denguecheck-WB
(Tulip); and Dengue IgG/IgM (Core). Note that the Bioline™ Dengue
Duo kit is a rapid, immunochromatographic assay which detects both
dengue virus NS1 antigen and IgG/IgM antibodies. As the NS1 rapid
test kits’ sensitivity and specificity ranges (due to cross-reactivity
with other flaviviruses) among various manufacturers, the dengue
NS1 antigen is detected in combination with anti-glycoprotein E IgM
and IgG antibodies (Fry et al., 2011).

2.3 Viral Nucleic Acid Detection

Nucleic acid tests (NATs) are molecular tests that require costly
equipment (PCR machine) and skilled personnel. Dengue NATs
require viral RNA extraction from patient serum/plasma, which
in most cases, difficult to be performed in POC settings. NATs
quantify viral RNA from patient samples and this allows detection
of dengue during acute phase with higher accuracy and sensitivity.
This test is applicable for five days of symptom onset. Commonly
used methods are reverse transcription polymerase chain reaction
(RT-PCR), nucleic acid sequence-based amplification (NASBA), and
transcription-mediated amplification (TMA).

2.3.1 Polymerase Chain Reaction (PCR)

The most common DENV NAT test is PCR. In the one step reverse
transcriptase-polymerase chain reaction PCR (RT-PCR), the viral
RNA extracted from different patient samples (e.g., plasma, blood,
24 Traditional and Current Diagnosis

urine, and serum) will be added with primer pairs and probes
which are specific to each dengue serotypes (DENV1- DENV4). The
use of fluorescent probe (e.g., SYBR green, Taqman®) enables the
detection and quantification (qRT-PCR) of the amplified product in
real time using PCR machine. Generally, compared to the SYBR green,
the TaqMan real-time PCR is highly specific as it uses the sequence-
specific hybridization of the probe. The available qRT-PCR include
the “singleplex” (detecting only one serotype) or “multiplex” (able to
identify all four serotypes in each single sample). A multiplex RT-PCR
assay using SYBR green probe was developed to determine all four
serotypes from the blood sample (Yong et al., 2007). The developed
assay had a sensitivity and specificity of 98.18% and 100%,
respectively. Another multiplex one-step RT-PCR assay has been
developed using TaqMan probe (see Appendix 4 for protocol) (Kong
et al., 2006). The assay uses a set of forward and reverse primers
designed to target serotype conserved region at the NS5 gene, and
simultaneously target a variable region for all four serotypes (sets
of primers that were previously used to design the serotype-specific
TaqMan probes). This assay had sensitivity, specificity, and real-time
PCR efficiency of 89.54%, 100%, and 91.5%, respectively. Currently,
there are two NAT assays approved by the FDA: CDC DENV-1-4
Real-Time RT-PCR Multiplex assay and Trioplex rRT-PCR Assay. The
former (differentiates all four serotypes) is used in cases where the
primary infection cause would be the DENV. The Trioplex rRT-PCR
Assays are used when Dengue, Chikungunya, and Zika are suspected,
allowing one to screen for all three viruses at the same time (Kabir
et al., 2021).

2.3.2 Nucleic Acid Sequence‒Based Amplification

(NASBA)
NASBA is a one-step isothermal process for amplifying RNA that
does not require the use of a thermal cycler. This technique has
been proven to be successful in the detection of both viral and
bacterial RNA in clinical samples. The NASBA assay involves the
use of extracted nucleic acid and subsequently amplified at a
constant temperature of 41°C. The reaction mixture consists of
 Viral Nucleic Acid Detection 25

avian myeloblastosis reverse transcriptase (AMV-RT), T7 RNA

polymerase, and RNase H with two short single-stranded DNA
primers. The amplified product of RNA pools is then detected with
agarose gel electrophoresis and electrochemiluminescence (ECL)
signal count. NASBA has significant specificity and sensitivity,
comparable with that of virus isolation, and may be particularly
useful in field studies of dengue infection. Wu et al. reported that
the NASBA assay had a sensitivity of 98.5% (66/67 clinical samples),
and a specificity of 100% (21 normal human serum) when compared
with the cell culture method using C6/36 cells (Wu et al., 2001). The
effectiveness of this method has also been described for detecting
the dengue virus within mosquitoes (Jittmittraphap et al., 2006). It
is considered more efficient than PCR in that it bypasses the use of a
thermal cycler, which makes it both rapid (<1 day) and cost-effective
(Usawattanakul & P, 2002) (See Appendix 5 for protocol).

2.3.3 Loop-Mediated Isothermal Amplification (LAMP)

The reverse transcriptase (RT-LAMP) assay was first developed by
Notomi et al. (2000). The assay consists of four to six primers (2
outer primes, 2 inner primers, and 2 loop primers) which are able
to recognize eight distinct regions on the target. This method needs
only water bath or a simple heating block to amplify the viral target
at a persistent temperature (range of 60–65°C). The assay starts
with transcription of the target to DNA, followed by creating a loop
structure which generates target copies. The amplified target is
detected by the naked eye or sometimes under UV light.
The RT-LAMP assay is previously known to have 100% and 98.9%
success rate in detecting the virus in the clinical DENV strains and
infected patients, respectively compared to the RT-PCR that shows a
sensitivity of 93% for clinical strains and 84.2% for infected patients.
It has no false positives results (Hu et al., 2015). Therefore, RT-LAMP
has many advantages as it is rapid, cost-effective, isothermal, highly
sensitive, and specific. Nevertheless, PCR is superior to LAMP-based
assays as it is suitable for multiplexing and viral quantification
purposes. Usually, LAMP-based assay provides qualitative (detected/
not detected) results.
26 Traditional and Current Diagnosis

2.4 Diagnostic Limitation

Ideally, a laboratory test for the detection of dengue should be
rapid, simple, affordable, with high sensitivity and specificity, and
able to detect dengue at any phase of illness, preferably able to
distinguish primary and secondary dengue infections, as well as the
different serotypes. Therefore, the ideal diagnostic test should be
able to detect genuine dengue cases at any stage of the illness. The
challenges in developing ideal diagnostic tools have been hindered by
the (i) complexity of dengue pathogenesis; (ii) hyperendemicity and
multiple sequential infections; and (iii) clinical conditions of patients
including viremia and antibody response (Sekaran et al., 2014).
Although virus isolation is more specific, it is restricted by being
time-consuming, is expensive due to setting up and maintenance
of special infrastructures such as animal care facilities, insectary,
and cell culture, requiring expertise and skilled personnel, needing
good quality acute specimens, and the inability to differentiate
between primary and secondary infections (Guzmán & Kourı́, 2004).
Molecular detection of viral nucleic acid allows the detection of
concurrent infections by multiple serotypes and determining strain
variability with high sensitivity and specificity within a short time.
Despite many genomic detection methods for dengue detection, not
all have been evaluated and in one such external assurance study,
only 10.9% of participating laboratories with different molecular-
based methods met all criteria for optimal performance (Guzman
et al., 2010). Likewise, the technique is also limited by the need for
acute samples, specialized equipment, and skilled personnel, and
by its incapacity to distinguish between primary from secondary
infections (Clarke & Casals, 1958). While these limitations are being
overcome by the detection of NS1 antigen, its sensitivity compared
to genome detection methods is poor and cannot be guaranteed
(Kassim, Izati, TgRogayah, Apandi, & Saat, 2011). Nevertheless, this
antigen detection method is useful for low-resource settings, as the
kits are affordable and simple to use (Tricou et al., 2010). Serological
diagnosis has been the mainstay for dengue diagnosis; however, IgM
detection is only detectable by 4–9 days in primary infections and
by 3–5 days in secondary infections. IgG tends to cross-react with
other flaviviruses (Peeling et al., 2010). Combinations of different
methods such as the detection of both NS1 and IgM/IgG have proved
 Future Directions 27

to increase the overall performance (Blacksell et al., 2011). Hence, it

seems that the best way currently to diagnose and confirm dengue is
to run multiple assays or to obtain paired sera.

2.5 Future Directions

Overall, the development of diagnostic tool which is highly sensitive
and specific involves multiple challenges that need be addressed.
Combining the existing techniques could be one way of improving
both sensitivity and specificity of diagnosing dengue. Existing SD
BIOLINE Dengue Duo RDT (Abbott, Santa Clara, USA; former Alere
Inc, Waltham, USA) that could detect all three NS1, dengue IgM and
IgG is known to have good specificities, but low sensitivity, indicating
that it could be useful detecting rather than ruling out a dengue
diagnosis especially in endemic areas (Kikuti et al., 2019).
Therefore, numerous researchers have begun to focus on the use
of biosensors as effective tool for diagnosing infectious disease(s)
(Bhalla et al., 2016). A biosensor developed based on silicon nanowire
could detect the ‘reverse transcription polymerase chain reaction’
product of a sample within 30 min (Zhang et al., 2010). Sensitive and
specific results obtained from this system could counterpoise the
other labor-intensive and time-consuming laboratory techniques
namely ELISA, that usually requires well-trained laboratory staffs
to carry out the test. In dengue diagnosis, a biosensors-based
semi-quantitative anti-dengue IgG (immunoglobulin G) immuno-
magnetic agglutination assay for the diagnosis of both previous and
recent dengue infection in a single test has been developed. The
test’s good point-of-care diagnostic accuracy is suggested to ease the
conduct of dengue serosurveys for vaccination strategy and facilitate
pre-vaccination screening to ensure safety (Chong et al., 2021).
The biosensor technology can also be applied as point-of-care test
(POCT).
POCT technologies should be generally cheaper and reliable
in health care settings. Examples of such tests are electrochemical
impedance spectroscopy (EIS) based sensing, microarray-based
sensing, and surface plasmon resonance (SPR). Most recently,
nanoparticles are often combined with these technologies to
enhance their performance in diagnosing dengue in timely manner
28 Traditional and Current Diagnosis

which is essential for patient management. In addition, development

of all assays should utilise respective controls and proficiency testing
in order to warrant a high degree of assurance.

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Chapter 3

Newer Diagnostics for Dengue Disease

Crystale Lim Siew Ying, Nur Haliza Hassan,

and Shamala Devi Sekaran
Faculty of Applied Sciences, UCSI University Kuala Lumpur Campus,
Kuala Lumpur, Malaysia
[email protected]

3.1 Biosensors in Dengue Diagnosis

With all vector-borne diseases, the success of dengue surveillance
programs is determined by the speed and accuracy of diagnosis,
more so due to the lack of specific antiviral treatments for dengue.
Diagnosis in the laboratory (confirmatory testing) typically depends
on direct detection methods of virus isolation, genome detection,
and antigen detection, or indirect detection methods of serology IgG
or IgM. Other than virus isolation, the other methods usually utilize
ELISAs and reverse transcription real-time PCR (RT-qPCR). These
tests are generally not able to be carried out by the general public
and require specialized equipment, facilities, and expertise, both for
the technical and analytical aspects.

Dengue Diagnostics: The Right Test at the Right Time for the Right Group
Edited by Shamala Devi Sekaran
Copyright © 2024 Jenny Stanford Publishing Pte. Ltd.
ISBN 978-981-4968-97-3 (Hardcover), 978-1-032-66956-4 (eBook)
www.jennystanford.com
36 Newer Diagnostics for Dengue Disease

Biosensor technology can circumvent the limitations above while

retaining almost all the advantages of laboratory-based diagnostics.
Biosensors are analytical devices that involve integration between a
molecular recognition platform with a physicochemical transducer
to produce a single detection processing unit (Miserere & Merkoçi,
2015). In simpler terms, a biosensor is “a device that measures
biological or chemical reactions by generating signals proportional
to the concentration of an analyte in the reaction” (Bhalla et al.,
2016). In healthcare applications, lateral flow pregnancy tests and
glucose monitoring sensors are two of the most well-known and
well-used biosensor devices (Bhalla et al., 2016), with the most
recent success story being Covid-19 antigen rapid test kit (RTK-Ag).
The typical biosensor flow begins with the recognition or binding
of the analyte (the target of detection) to the bioreceptor (a molecule
specific to the analyte). This interaction between the analyte and
bioreceptor is termed “biorecognition”, which generates a signal
in the form of light, heat, pH, charge, or mass change (Bhalla et al.,
2016). The transducer then quantifies this biorecognition signal and
converts it into a measurable signal in a concentration-dependent
manner in a process called “signalization” via an electrical interface.
Electronics recognize, convert, and amplify the analog signal to a
digital one (signal conditioning) and process the signal for display
as an output signal, which can be in the form of numerical, graphical,
imagery, and other types of data. The biosensor workflow and
options are summarized in Table 3.1.
The suitability of biosensors is evaluated by a set of parameters
based on the International Union of Pure and Applied Chemistry
(IUPAC) guidelines, which include specificity, selectivity, limit
of detection (LOD), and sensitivity (den Boef & Hulanicki, 1983;
Persson, 2001). LOD, a quantifiably accurate amount of element
derived from the smallest signal, is usually calculated as 3s/b, with
s as the standard deviation of the signal of the blank sample and b
as the slope of the straight section of the calibration curve (Long
& Winefordner, 1983). The signal-to-noise ratio (S/N = 3) is an
alternative LOD calculation (Shrivastava & Gupta, 2011).
Another random document with
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 Alcohol is a State monopoly in Poland. It used to be in Russia. It is
a valued source of revenue to many European Governments. Who is
to manage this highly important Government industry? The peasants
are slow, ignorant and unreliable. They drink heavily. The Jews do
not drink. A drunken Jew is a thing unknown. The very words are a
contradiction in terms. It is a temperate and sober race. The Jews
must manage the liquor shops. To the Jews are given a very large
proportion of these positions in the interests of the State, and not
because of any partiality to the Jew. The drink-shop in a village very
naturally becomes the village store. The Jew is the storekeeper.
“We had to cease giving soap to the peasants in Czecho-Slovakia,
although they needed it so badly, because they would sell it to the
Jew for vodka,” said the lovely Countess Dŏbrenszky.
“Why not prohibit the sale of vodka?” I suggested. She smiled and
shook her head. “It could never be done.”
As the servant of the State the Jew is expected to encourage the
sale of drink in those countries where it is a State monopoly, and it is
easy to see how everything else follows.
The second of the two bottom facts of the Jewish side of the
controversy is the undoubted hatred and envy by the Gentile of the
superior Jewish intelligence, particularly in commerce, but as
certainly in everything else. Nothing can keep the Jewish race from
excelling. Ages of ancient wrong could not do it. Present-day
oppression cannot do it. In some countries still the Jew is not
allowed to own land. In others, Rumania for example, he is not
permitted to enter the profession of lawyer, doctor, or teacher. In the
old Russia he might not go to the Universities. In Poland he can
exempt himself from army service and consequently is denied
citizenship. Cruel as it all seems, and is, there is an underlying
instinct of self-preservation at the foundation of it, for, given equal
chances in the race of life, the Jew will ofttimes leave the Gentile
laggard far behind.
In the early ’forties an enterprising statesman of Vienna began to
train young Jews in journalism, and now all the important papers of
Vienna are run by Jews. Since the opening of new doors to them in
Germany they have dominated the artistic professions in Berlin, and
have contributed overwhelmingly to the intellectual life of Germany.
The greatest continental authority on Shakespeare, Professor Leon
Kellner, is a Jew. Professor Einstein is a Jew, Professor Ehrlich is a
Jew. These two great scientists are distinguished in a host of learned
Jewish men of science. Maximilian Harden, eminent journalist, is a
Jew. Max Reinhardt, composer, is a Jew. The list of famous living
Jews is too long to be given in full. In England they distinguish
themselves chiefly in politics—Lord Reading, Viceroy of India, Sir
Herbert Samuel, High Commissioner in Palestine. And the Jews are
dominant in the Socialist politics of Europe, not because of any deep
and treacherous design against humanity they possess, but for
precisely the reason they are dominant in other spheres, because of
their good brains, logical minds, keen perceptions and rare artistic
abilities.
If the economic domination of the world by the Jews should come
to pass it will be in no small measure due to the historic fact of the
persecution and exclusion which have necessitated to a great extent
the expression of the rich mental life of the race along one narrow
channel for two thousand years; and it will be due in some degree to
the comparative self-indulgence and contempt for hard intellectual
labour of the Gentile section of the world community.
This excursion into Poland, and the question of the Jews which the
discussion of Poland always invites, has postponed for several
pages the trip to Georgia. I had the intention to go to Warsaw this
month, but a charming young Pole, a lovely girl of twenty, has come
to stay with me for some months. Her cousin tells me she is Poland
in epitome and advises me to stay at home! Wanda is still too young
to be other than a fervid nationalist and patriot. She is full of the
poetry and romance of things, and the love of dainty dresses. She is
filled with the vague longings and sadness of youth, and likes the
autumn better than the spring, which is exactly as it should be in
sentimental twenty. My only trouble with my guest is one of race and
upbringing. I have an unconquerable and brutal British habit of
saying “yes” when I mean “yes.” She says “yes” when she means
“no,” because to her it is polite and proper to say the thing you
imagine you are wanted to say. The consequence is that I am in
danger of killing her by dragging her from her books over the hills
and dales of an English countryside, to put roses into the pale
cheeks, and a bright light into the grey eyes which have seen too
much of sorrow and suffering for one so young and fair.
 CHAPTER XII
GEORGIA OF THE CAUCASUS

M. Camille Huysmans persuaded me to accept the Georgian

invitation. “The Georgians want you to come very particularly
because you were in Russia recently. They want someone who can
make comparisons between the Bolshevik Government of Russia
and the Social Democratic Government of their own country. It would
be helpful to them, and would be interesting and useful to you.”
The delegation was selected from the Second International.
Besides myself, Mr. J. R. Macdonald and Mr. Tom Shaw were invited
from Great Britain; Messieurs Vandervelde, de Brouckere and
Huysmans from Belgium; Messieurs Renaudel, Marquet and Inghels
from France; and Herr Kautsky and his wife from Germany. Several
Georgians and Russians with their wives were also of the party, and
we were joined in Paris by Madame Vandervelde and Madame
Huysmans and her daughter. The Kautskys joined us in Rome,
travelling thither from Vienna.
Camille Huysmans would have to occupy a central position in any
picture of the personalities of the present-day European Socialist
Movement. His is a figure of more than ordinary interest. He is tall
and slender, with an attractive mop of fair, curly hair. He possesses a
keenly intellectual face, like that of Lasalle, delicate featured, but
with a slightly cruel mouth. His eyes are restless and his general
movements, except in speaking in public, are nervous. He has an
extraordinary capacity for organization, and speaks four or five
languages with equal fluency. His knowledge of the history and the
present position of the world movement for Socialism is unrivalled.
His knowledge of the private histories as well as the public records
of his Socialist colleagues in all lands is also very complete; which
makes him a terror to evildoers. I have heard attributed to this
knowledge the fact that the Russian Bolsheviks have left him
severely alone. It certainly cannot be because he has spared them,
for his hatred of their undemocratic form of government he has cried
from the housetops.
His is the artistic temperament, and he is passionately fond of
music and the drama. He loathes all the naked ugliness and stupid
self-repression that passes for Puritanism in the minds of the soured
and disappointed. He professes no personal religion, but
temperamental leanings towards the forms of Roman Catholic
worship are discernible in the expression of his general views of life.
The pictures, the colour, the incense, the music of the æsthetic
temples of every great Faith would probably be implicit in his scheme
of things, for the sheer beauty of them.
I have a great liking and admiration for the secretary of the Second
International; but it requires a sense of humour and a certain gift of
scepticism to make him understood of the great mass of his more
sober Saxon comrades. “You can as easily make an Englishman
musical as a Belgian moral,” he said gaily into the shocked ears of at
least two English persons present, delighted to be taken seriously
when he only wanted to draw us into a debate. His eyes twinkled
mischievously as he spoke. He is the Puck of the International, the
tormenting imp who likes nothing better than to stab with little darts
of irony the self-important people who take life too seriously.
On public occasions he appears the most self-possessed of men;
but he told me once that he suffers an agony of nervousness when
called upon to meet strangers. His public speech sparkles with wit.
He can laugh, sing, dance and shout with the abandon of a
schoolboy; but when some piece of stiff business arises and he has
to calm a raging storm of passion between two sets of nationals in a
conference his peculiar genius shows itself, and he restores order
and amity with the hand and voice of a master. Without Camille
Huysmans the ship of the International would sail very unsteadily
upon the turbulent waters of present-day politics. Huysmans is a
member of the Belgian Parliament, and if there be anything in
present signs and portents he is marked out by circumstance and his
own commanding abilities to play a prominent part in shaping the
future fortunes of his gallant little country.
“La petite Sara,” as his gifted young daughter was called by the
Georgians, helps her father, whom she adores. She has his
charming personality and marvellous facility for languages, with an
added seriousness and self-sufficiency, if not a slight stubbornness
of character, which will not detract but rather add to the quality of her
international work. She is a very pretty girl, with large, serious grey
eyes, dark fringed, and a complexion of cream and roses. All the
young men of the party fell in love with her and lived in hourly,
jealous fear lest some dancing Georgian rival should persuade her to
marry him and carry her off to his mountain home.
M. Louis de Brouckere, tall, handsome and dignified, another of
our Belgian companions, is the perfect scholar and gentleman.
Could more and better be added to that?

M. Emile Vandervelde, the Belgian Minister for Justice, is a portly

figure with a ruddy complexion and wonderful blue eyes, clear and
limpid as a child’s. He is slightly deaf, which obliges him to lean and
strain to catch the words of a speaker. He professes not to speak
English, but that is all nonsense. He both speaks and understands it
very well. His wife is an Englishwoman.
Of French he is a master. He is one of the greatest of living
orators. As chairman of the Delegation he spoke on almost every
occasion. So perfect is his art, so entirely matchless is his choice
and use of word and phrase, so magnificent the roll and crescendo
of his argument that his listeners stood fascinated as he spoke, or
leaned forward in their chairs, their faces aglow with enjoyment of
gesture and speech, even when they did not understand a word. To
the understanding the speech was ever a marvel of beauty and
delight, holding them spellbound to the last triumphant word and
overpowering gesture. The theme in Georgia was the same for us
all, and for all occasions: sympathy for the Georgians in their effort to
build up peacefully and on Social-Democratic lines the Socialist
Republic; offers of help in our various home countries; condemnation
of Bolshevism; praise of Internationalism.
M. Vandervelde is one of the most brilliant supporters of the
Temperance Movement. He is by preference a total abstainer,
although he is often placed by his public life and on foreign travel in
circumstances where it is very difficult to indulge his taste. In some
of those Eastern lands the water is tainted with germs and poisonous
to the last degree. When it comes to a choice between typhoid and
alcohol, the choice usually falls upon alcohol! Sometimes bitter
offence is given where it is highly important good feeling should be
maintained if a guest declines to drink wine with a host; incredible in
these days, but true; impossible in this country now, but in Eastern
Europe of the greatest frequency.
It was in the company of this distinguished statesman that I visited
the wine-cellars on the estate in central Georgia of an exiled Russian
Grand Duke. We entered the vast chambers led by smocked
peasants carrying torches. They bowed till their beards almost swept
the ground as we thanked them for their pains. Vast, gloomy,
mysterious in the light of the flaming torches, the cellars were not so
attractive, we thought, as the enchanting garden under the moon,
and the voices of the villagers singing their folk songs on the lawn;
so we left the rest of the company and sought the road back to the
palace ourselves.
“What do you think will happen at the next election in Belgium?” I
asked my companion.
He shrugged his shoulders and spread his small, white hands with
an expressive gesture. “I cannot tell. There will probably be little
change. I shall have to be home by then.”
The sound of the music came through the trees, guiding our steps.
“I should like to understand Belgian politics better,” was more than a
polite observation on my part. It represented a genuine regret that I
was so ignorant.
 “The Belgian Socialist point of view was not understood during the
war by the English comrades,” said the Minister. “And even now we
are roundly abused for joining the Government, even by a section in
Belgium. It is always the dividing line. Shall we stand outside and be
simply a propaganda body? Or, having secured a certain position
and membership, shall we take the responsibility for carrying out as
far as we can our political doctrines, recognizing that in a composite
Government we can go neither so fast nor so far as we might wish?
The workers’ party in Belgium is now the largest party in the State.
Can the largest party in the State refuse to share the responsibility of
helping in the country’s government? Camille thinks not. I have
thought not. Now I sometimes doubt the line we should take. We
shall see how things develop; what the result of the election is. But
you must come to Belgium and tell us about Russia, and we will
show you anything and tell you anything you wish to know.”
At this point we emerged from the thick wood into full view of the
palace. Servants were lighting paper lanterns. The clatter of plates
and cutlery spoke of the coming revel. The choristers burst into a
new song as we approached. The bright moon lit up the magnificent
range of mountains in the distance. It was fairyland come true,
making the things of this world, its dirty politics and mean diplomacy,
look small and poor.
A tall English blonde of very great charm of manner when she
chooses is Madame Vandervelde. When she does not so choose
she can be ruder in three languages than any woman of my
acquaintance knows how to be in one! I do not in the least complain
of her conduct to me. We got on extremely well. We were sufficiently
candid with each other to be able to maintain to the end a good
comradeship in spite of the very trying circumstance of joint sleeping
quarters. My one quarrel with my fellow-countrywoman was on
account of the number of trunks she carried. It was almost
impossible to turn round in that small state room because of the
array of bags, boxes, suit-cases, hat-trunks piled into the room and
occupying every available inch of space. One member of our party,
the little French bride of a Georgian physician, who was carrying her
trousseau to her new home in Tiflis, lost on the Italian railway a trunk
containing two thousand pounds’ worth of valuable hand-made
clothes, laces and household goods which she never recovered. An
old empty trunk with her original label attached was found in its
place. It may be the effect of the war. If four Prime Ministers in Paris
can steal several colonies in Africa, if fat profiteers can rob the dying
Austrian children in their thousands of their food, surely one little
Italian railway porter can annex one trunk without blame? Whatever
the reason, it is certainly true that, on more than one continental
railway at the present time, the only way you can assure the arrival
of your trunk at its destination is by sitting on it.
Madame Vandervelde contrived to bring all her goods safe into
port without sitting on them. She pressed into her service the gallant
men of the party. There are some women—and my friend is one of
them—who by reason of their presence of mind and absence of
conscience can command the services at all times and in all
circumstances of even the men who dislike them. And apparently
there are men who like being kicked!
But I do not want to imply that any man on this trip found his
service a trial. I am sure the beautiful Lalla commanded the whole-
hearted service of her numerous cavaliers. They liked her free
manners and fascinating personality. They delighted in her racy talk,
daring jests and semi-Bohemian tastes. The least that ought to be
said about her is that her impish delight in shocking people and in
saying teasing things kept the whole company titillating with
expectant amusement or nervous fear. Nobody could be dull in her
society; and, after all, dullness, which is always a nuisance,
becomes a positive crime on an excursion of this sort, which
compels twenty persons to live very closely together in ship or train
for fifty days and nights.

Of the remaining women of the party, Madame Huysmans is a

pretty dark woman, full of gentle kindnesses and not without the gift
of humour. Madame Dvarzaladze is a magnificent beauty of the
gipsy type. Madame Skobeloff, one time a prima donna at the
Petrograd Opera House, was the very incarnation of her favourite
heroine—Carmen—and by the skilful glances of her glorious black
eyes and her coquettish manner brought the passionate lady off the
stage to live amongst us for several days.
M. Dvarzaladze conducted the expedition on behalf of his
Government, and was the kindest of hosts. M. Skobeloff assisted
him. The latter is as fair as his wife is dark, with the Russian breadth
both of figure and of face, and a mass of light silky hair brushed back
from a square forehead. He was Minister of Commerce in the
Kerensky Government. Something in his speech and manner gave
the impression that he regretted a little the Bolshevik Government,
and would have liked to participate in it; but I was confidently
assured that I was mistaken.
M. Nazarov, as a student in Petrograd, embraced Bolshevism with
great enthusiasm. When student days ended he came back to his
original faith of Social Democracy. He acted as secretary to the
expedition and was, without a single exception, the most consistently
courteous and considerate person I have known who has ever
occupied so difficult and thankless a position. Early and late he was
engaged in looking after the comfort of everybody. Pestered to the
verge of insanity, as he must have been with the requests of various
members of the delegation, his manners never for an instant forsook
him, and the remembrance of him alone would make the visit to
Georgia unforgettable.
Of the three delegates from France, M. Inghels is the typical bluff
and substantial Trade Union leader, a representative of the textile
workers; M. Marquet is tall and slim and elegant, faultless in dress,
of impeccable manners, leaving on the mind the impression of easy
victories with women; M. Renaudel has already appeared in these
pages, the man of robust proportions and prodigious appetite, of
matchless eloquence in speaking, with a voice of great beauty.
There remain only the English delegates to describe, and one of
these was a Scotsman, Mr. J. R. Macdonald, of the dark eyes and
wavy hair of silvery grey, of the calm judgment and austere outlook
upon life so valuable to the leader of men, and so necessary for the
safeguarding of inexperienced Labour representatives in England
come new and defenceless against the seductions of wily enemies in
the House of Commons; and Mr. Tom Shaw of the Lancashire Textile
Unions, stout and ruddy complexioned, full of fun and good-natured
banter, the best of travelling companions and the kindest of men.

The delegates met in Paris at a dinner given to them by M.

Tseretelli, the Georgian Minister. Preliminary to this was the tiresome
and disgusting business of inoculation. The wily Georgians had said
nothing about this in Geneva. Had we known then of the ravages of
the pest, and had we been told we must be inoculated against
bubonic plague, it might have affected our decision about going. For
some time we resisted; but on the very earnest solicitations of our
friends, and because it was suggested that by not being vaccinated
we might endanger the lives of other people, we weakly yielded and
consented to allow ourselves to be ill-treated in this peculiarly
objectionable manner! I have never been able to reconcile myself to
the deliberate poisoning of my blood at intervals during my life, and
have always felt triumphant when the healthy blood I inherited from
plain-living and high-thinking ancestors refused to be poisoned by
the filthy injections.
The journey from Paris to Rome occupied two days, with a change
of train at Turin. The one memorable thing about this journey was the
descent through the Mont Cenis Tunnel into the Italian valley, with its
villas and vineyards and sun-steeped fields.
We stayed a couple of days in Rome awaiting the date for sailing
and to complete the passport business. Into those two swift days we
crowded as much sight-seeing as possible—the Forum, the
Coliseum, St. Peter’s Church and the Appian Way. There are some
travellers whose sole happiness lies in being able to boast of having
seen something which nobody else has seen, or to have got ahead
of the party by doing something it never occurred to the others to do.
You praise the sunset. “Ah, but you should have seen it an hour
ago,” is the remark which cools your enthusiasm. You are pleased
with the dinner. “But it is nothing like so good as yesterday’s,” is the
observation which robs you of half your pleasure. You are
enraptured with the song. “Oh, he’s gone off lately. You should have
heard him a year ago,” is the comment that leaves you flat and
disappointed.
“How wonderful is the Coliseum!” exclaimed one of the delegates
to the rest of us.
“But did you see it by moonlight? No? Then you have not seen it.
You must see it by moonlight if you really want to see the Coliseum.”
And we left Rome with the feeling that there was nothing to be done
but to return to Rome to see the Coliseum by moonlight, or our visit
to the city would be mere fruitless folly.
I discovered the Corso to be no place for a woman walking alone.
As a matter of fact, reputable Italian women do not walk in the
streets of Rome unattended, particularly at night. I was ignorant of
this, or had forgotten it, and did as I am accustomed to do in my own
country, when I speedily discovered one difference between an
English and an Italian city which pleasantly distinguishes the former;
for there are very few places in England where a modest woman
going about her legitimate business unattended would be stopped
and spoken to in a familiar way in a public thoroughfare. In the
streets of Rome the sun at midday is, apparently, no guarantee of
impunity for women from the annoying familiarities of unknown and
undesirable men.

Taranto, the port of sailing, is a quaint old city of antiquarian

interest situated on a beautiful bay. The museum is filled with ancient
statuary and pottery excavated from the ruins of a still older city,
dating back to the days of the ancients. We spent some hours in the
building, examining the tessellated tiles and old Greek vases under
the guidance of the elderly curator, who, as he said good-bye to us,
broke two delicious pink roses off the rose tree in the courtyard, and,
with a graceful old-world bow, his hand upon his heart, gave one
each to Miss Huysmans and myself.
Taranto comprises two towns, the old and the new. The new is set
upon a hill, the old lies about the port. The new has an American
look about its new white stone-fronted buildings, the old has the
stamp of the Middle Ages upon it. The streets of the old are winding
and so narrow that the people on opposite sides of the streets can in
some cases shake hands from their bedroom windows. They are
paved with cobblestones, and there are no sidewalks. The houses
have tiny windows and the top storeys project. The shops, as a rule,
have no windows at all, but are open to the street along the whole of
their front. Some of the cafés are underground cellars. Men and
women meet in the shops for gossip, and in the cafés for scandal
and politics. Work is leisurely. The men are mostly engaged in
fishing, net-making and basket-weaving. The women wear native
peasant dress, bright coloured, and attend to their houses or help
the men with the nets. Donkeys are numberless. Huge masses of
fruit, notably grapes and water melons, are piled up on the stalls and
barrows that line the street fronting the sea. It is a city of amazing
picturesqueness, astounding squalor and incredible smells.
Our ship was an Austrian vessel, part of the Italian share in the
spoils of war. Her commander was an easy-going Italian with a
tremendous admiration for Lord Fisher. He refused to promise us
fine weather, and, even as we entreated, the sun entered a cloud
which, before evening, had spread gloomily over the whole sky!
We sailed pleasantly amongst the Greek Islands, sighting Corinth
and Athens and the Hill of Mars. We steamed slowly through the
canal cut through the Isthmus of Corinth, a marvellous feat of
engineering. We crept gently past Gallipoli and gazed with dim eyes
on the graves of the gallant dead. The sea near the shore was full of
ships, sunk by the fire from the Turkish forts, and the captain told us
that here careful navigation was very necessary and we might not go
nearer the land; but with the aid of field-glasses we marked the
blasted hillsides and battered fortifications of the Turk. Here and
there a broken gun rusted on its side in the scorched and trampled
grass. Hearts felt sick for the sacrifice that the politicians were
threatening to make vain, and we silently renewed our vows to
devote our lives to the building up of such international organization
as should make such sacrifices unnecessary in the future.

On the fourth day after leaving Taranto we sighted Constantinople.

This city was the most completely satisfying of all my childhood’s
dreams come true. I recollect how disappointing to me was my first
glimpse of the Niagara Falls. So it has been with many of my friends.
Such beauty as that grows upon one, but at the first visit one expects
too much. One expects something more and bigger than can be
taken in with a single glance of the eye, a wilderness of waters,
something stupendous, to send one reeling! One sees a vast and
steady tumbling, a roar like a Tube train entering a tunnel, and feels
the lack of mystery. I am inclined to think the injury is done by the
aggressive and vulgar civilization all round: the tawdry town, the
eating-houses, the electric-power stations, the street cars, the
vendors of toys and ice-cream and picture post cards and penny
buns. Seen and heard in the vast spaces and awful silence of a
desert it would be altogether different.
Constantinople fulfilled every wish, satisfied every expectation.
Magnificently set upon its several hills it appeared the queen of cities
enthroned above the worshipping waters, crowned by the moon, and
glittering with ten thousand jewels of ten thousand shimmering lights.
By day her beauty changed. Unlike Moscow, whose domes and
minarets gleam golden in the sun, those of Constantinople have lost
their radiance, but they stand out nobly against the clearest of blue
skies, the mosques on the hills of Stamboul competing for praise
with the vast modern palaces at the water’s edge. The Golden Horn,
classic symbol of plenty, was crowded with shipping, a pleasing
contrast to the stagnation of Astrakhan.
The streets of Constantinople are a kaleidoscope, a mass of
jostling humanity, white and black and brown. The Turkish fez
predominates. The dark-skinned Jew and the cunning Greek vie with
the crafty Armenian in the business of stripping the guileless
stranger of his money. Thick-lipped Nubians are as common as flies.
Black-veiled Turkish women add a distinctive note to the scene.
Water-carriers sell their water to thirsty traders in carpets and
embroideries. Anatolian peasants bring their fruits to sell. Turkish
princes flash past in shabby automobiles. Gay French officers on
horseback menace the careless foot-traveller. Young British officers
on polo ponies rush laughingly by. The big hotels are filled with the
usual crowd of foreign Military Mission folk, big business men,
pseudo-politicians; youthful, very youthful diplomats and soldiers,
profiteers, adventurers, wives of officers and women of the
underworld—gay, charming, lovely and dangerous. No sign there of
the bitter hate that sits on the brow of the Turkish café habitué, who
deems the least tolerable part of his burden the position of
dominance over him given to his ancient insolent enemy, the corrupt
and perfidious Greek.
I shall write more about our doings in Constantinople later. We
sailed through the Bosphorus in a calm sea and into the dreaded
Black Sea after the third day. The beauty of the Bosphorus suggests
the exquisite reaches of the Rhine with its ancient castles and woody
crags, but with a gentle softness for the Rhine’s proud strength. The
Black Sea belied its name, and our passage was without a break in
its comfort and content.

We rested for a day outside the port of Trebizond. There, to our

amazement, was flying the red flag of the Bolsheviks, whose co-
operation with Kemal Pasha had evidently not been misreported by
the Press. Kemal’s headquarters were in Trebizond. Several boat-
loads of Bolsheviks in khaki uniforms and peaked caps came to
inspect the ship. Some came on board. They were perfectly civil. No
attempt was made to interfere with the passengers, who were
strongly urged by the chief officer on board not to risk a landing. We
took on board many new passengers here and at a previous
stopping-place, the name of which escapes me. These were of
various nationalities, chiefly Turks, with their carefully segregated
and veiled womankind carrying large quantities of fruit, and
themselves hauling on board loads of wonderful Turkey carpets. A
few long-bearded Greeks and swarthy Jews were amongst the new-
comers, and several fascinating black-eyed children. These people
shared the lower deck with the sheep and goats. The sheep were
penned, but the goats escaped, leaping all over the deck and
chewing to tatters the sailcloth and the ropes, to the anger of the
sailors, who, with all their nimbleness, were no match for the goats.
Below in the hold were the horned cattle, bellowing their protest at
two days and nights of painful thirst in their hot and crowded
quarters. The way in which these poor beasts were treated made us
sick. They were hauled from the small boats on to the ship and into
the hold suspended by the horns from the ship’s crane. Their eyes
bulged out of their heads, their legs beat the air as they swung up
and then down, their heavy bodies pulling at their horns. A young
Englishwoman expressed her detestation of the performance in a full
company, when, with a grin, a facetious foreign gentleman exclaimed
with his hand upon his heart: “Ah, mademoiselle, you English, you
have pity for ze poor animals but none for ze poor men. We break
our hearts for ze mademoiselle and she care not. But ze horses, ze
cats and ze dogs, she adores zem. It is desolating.” And he made a
frantic gesture of despair.
“What do you say to the idiots who talk like that?” I inquired, sorry
for the cause of that angry flush on her pretty face.
“I say nothing,” she replied; “but I begin to feel thankful that our
quarrel with the German people is only skin deep.”

One night more and we were in Batoum, beautifully situated on the

slopes and at the foot of great, wooded hills which make a sombre
background to the white houses. As the noise of the ship’s engines
ceased, distant strains of music crept into our ears. It came from the
shore, which was black with people. I grew nervous and
apprehensive. I opened the cabin door. I strained forward anxiously
to hear. I was not mistaken. My first fear was realized. It was the
“International,” the song which brought Russia back to mind, the
jingling melody that I had heard, at a modest computation, a
thousand times in Russia alone!
I rushed to the ship’s side and, borrowing a field-glass, stared out
to shore. Yes, yes, it was all there, the familiar circus; the bands, the
crowds, the carriages, the flowers, the red flags and bunting, the
photographer and cinema operator—all so kind and well-intentioned.
I looked at Tom Shaw; he grinned back at me. There was nothing to
be done but resign ourselves to the inevitable and look as pleased
as we could.
We clambered down the ship’s side on a shaky, swinging ladder to
the waiting tender and steamed away to shore. The kindest of
welcomes awaited us. Our arms were filled with flowers, and after
the usual courteous preliminaries we were led off amidst deafening
cheers to receive the official welcome at the City Hall.

The City Fathers gave us greeting in a few short and well-chosen

phrases to which Mr. J. R. Macdonald suitably responded. We then
proceeded for a similar function to the headquarters of the Social
Democratic Party. Five thousand people assembled in the street to
be introduced to us. The introductions were made from a balcony.
Each delegate was brought forward separately and named, with
certain of his gifts and exploits. Then the crowd yelled with delight.
M. Vandervelde on our behalf acknowledged the courtesy and struck
the international note, and we were released for lunch and a
subsequent tour of the city’s chief points of interest.
The tightness about my heart left me after the first hour amongst
these happy people. What, I asked myself, had I really been afraid
of? I had feared to see a starving company drawn up in stiff lines
giving us welcome by compulsion. I remembered how, in Petrograd,
loss of work or of ration was the punishment for non-attendance at
these formal ceremonies. The cruel fatigue of many hours of waiting
in biting wind or blistering sun was the price paid there by thousands
of underfed and underclad workmen and women for a sight of the
foreign delegates. I felt it quite impossible to endure this sort of thing
again.
But in Georgia it was different. The experience in Batoum was the
same everywhere. There was no compulsion to meet us. The people
came because they wanted to come. They moved freely amongst us,
without restraint of speech or manner, laughing, shouting, singing.
The brown-eyed children climbed into our laps. They shyly played
with our watches or examined our clothes. In all those merry faces
turned up at us on the balcony I saw not one look of bitterness, no
tightening of thin lips, no burning hate in the eyes. One jolly giant,
whose curly grey-black hair waved a head’s breadth above the
crowd, led the cheering, which was caught up by the crowd in
unmistakable sincerity. They ran by the side of our carriages, flinging
red roses into them and blowing kisses to us as we gathered up the
roses and pinned them to our coats as the red emblem of
international solidarity.
We spent a pleasant afternoon in the Botanical Gardens, rich with
every kind of tropical and semi-tropical fruit. The head gardener
boasted with joyous pride the possession of sixty different varieties
of orange. There they hung, yellow and tempting. Visions of
Southern California surged up, the blue Pacific at San Diego, and
the big glowing orange broken off the tree, ripe and delicious, for the
daily breakfast. From the figs and grapes, the lemons and bananas
of these gardens, we proceeded to the tea plantations and the
bamboo woods, and saw two infant industries developing
themselves, the one under the care of a skilled Japanese. Georgia’s
industry needs development on modern lines, with modern
machinery and by modern methods. At present production is slow
and old fashioned. A common sight on a Georgian landscape is a
wooden plough, hand guided, drawn by eight pair of stout oxen. This
is mediæval.
 In the evening we were entertained by the Batoum Municipality to
a dinner on the enclosed veranda of a large public ballroom. A
Georgian dinner is a thing to be remembered, and this, the first of
many, lingers pleasantly in the mind. Flowers and climbing plants
adorned the glass-covered veranda on the outside, palms and
flowering trees decorated and scented it within. The long table
accommodated two hundred guests. At one end of the room a choir
sang songs, and an orchestra made merry music whilst we ate.
Course followed course of the most deliciously cooked food.
Enormous epergnes, filled with glowing peaches of incredible size
and huge black grapes, adorned the table at frequent intervals of
space. There were sparkling wines of rich vintage and various
colours, exquisite in the soft light from the shaded lamps. This dinner
could not have been surpassed for the completeness of its
appointments by the most expensive mountain hotel in America.
Torrents of summer rain and vivid flashes of lightning added to the
sense of comfort and jollity within.
The speeches at a Georgian banquet are delivered between the
courses. After the speeches, before the speeches, furtively during
the speeches, the toasts are called. Never in the world was there
anything like this mad passion for toasting one another. Every guest
is toasted at least once. The health of every lady is drunk at least ten
times! If the wine does not give out, absent friends and popular
causes, the cook in the kitchen and the butler in the pantry supply
excellent excuses for a further riot of toasting. Conversation waxes
louder and more excited with every glass. Eyes begin to shine with
the moving spirit of alcohol. Strange stories of gallant adventure are
told aloud. Wild gestures are flung about. Out of the storm of
confused tongues and frantic gesticulations, from the far end of the
table comes a faint voice softly singing a slow song. Others take up
the strain. In less than two minutes the entire table is singing, each
person roaring his accompaniment at the very pitch of his voice. This
song sounds like a Scottish psalm tune, but it is the Georgian
equivalent to “He’s a jolly good fellow.” It is very impressive and runs
something like this; I give it from ear:
 Georgian “Toast” Song
Very slowly.
Perhaps twenty times in one evening this song is started and
taken up by the company. Each time it is a compliment directed at
some special guest, and concludes with the clinking of glasses and a
roar of cheers for the honoured one, who bows his appreciation of
the kindly courtesy.
A distinguished general of the ancien régime was my vis-à-vis. He
delicately complimented me upon the few words those gallant
Georgians would have me say, and afterwards sent to Tiflis a large
basket of delicious red roses for the ladies of our party. On my right
sat several young nobles in the handsome native costume. They
wore long grey coats, full skirted and with belts at the waist.
Underneath was a high-necked blouse, buttoned at the front. Each
side of the coat was ornamented at the breast with a row of pockets
for single cartridges. Ornamental cartridge-cases were fitted into
these pockets. The round hats were of white astrakhan, and they
wore soft leather Russian boots which came high in the leg and were
seamless and unlaced. Each carried a dagger at his side, with richly
chased silver handle. When the spirits of the company had risen
sufficiently high, two of these young princes rose and danced a
graceful Georgian dance down the whole length of the corridor and
back on the other side. The guests accompanied with a monotonous
clap, humming softly a suitable melody. One arm held gracefully
above the head, the left hand on the hip, the feet moving intricately
and delicately, the body swaying ever so slightly from the hips and
seeming to float upon the polished surface of the floor, there is
nothing that dance resembled so much as a sailing ship on a placid
lake gently moved by a soft wind.
The absence of rancour, the atmosphere of friendliness, the
fellowship and intimacy of it all, charmed us, and we left for the night