QA Discussion
The following highlights the questions and responses made bysymposium participants at the end of all the sessions. Drs. Ron P.
Marchand and Shusuke Nakazawa led the discussion encouraging
participants to share thoughts and ideas on where to take the current
research in Khanh Phu, while resolving the issues of species
identification in the area. Individual names of those who commented
are not included and where applicable, information is added to provide
context in this summary.
The Khanh Phu Malaria Center faces the challenge of unambiguously
identifying all the malaria species maintained in the area, especially inthe advent of reports of past markers used for identifying P. vivax toalso cross-react with many monkey malarias. The Khanh Phu Malaria
Center would like to resolve the species identification issues bypromptly analyzing samples obtained onsite using reliable molecularbiology techniques.
The floor opened to discuss the immediate diagnostic capabilities ofpatients in the field using the Loop-mediated Isothermal Amplification(LAMP) method. As portable polymerase chain reaction (PCR)
machines have been designed by the Centers for Disease Control andPrevention (CDC) in the past; could LAMP be a more suitable
technique for field applications?
First comment:
LAMP is very simple and quite appropriate for field use. The
procedure is very simple, so minimal training is required in the field.
There are freeze dry kits developed for LAMP. LAMP can accurately
distinguish species; LAMP for genus identification can be followed bya species specific, second LAMP step.
Comments on LAMP use in the field:
Using LAMP now would be a mistake, especially at a time when there
appears to be a struggle with application of current traditional

techniques, ie). PCR, real-time PCR, and sequencing in determining
species. LAMP offers a cheap, readily trainable system, but a solid
foundation on current molecular techniques must be established,otherwise everyone involved would be wasting a lot of time andresources with inconclusive data. First, identify the parasites using thebest available techniques, then move onto developing simpler
techniques involving LAMP use in the field.
Additional comments to field development:
Since a field detection system that can be applied in the field iscompletely lacking in Khanh Phu, LAMP may just be the appropriatetechnology to develop along with establishing molecular applications
to producing consistent data. This comment was prefaced with thecurrent state of rapid detection kits in the field, where problems offalse positives and false negatives with the use of ICT(immunochromatographic test) rapid detection kits in the field havebeen further developed to improve its technology.
Field applications may be required, but bad solutions that are cheap
are not the way to resolve the present problems in Khanh Phu.
Suggestions for suitable collaborators; although Khanh Phu may beconsidered remote, it is only 20-30km away from the Pasteur Institutewith a lot of experience in many areas of molecular biology.
What is clear at present is that the current 18s rRNA primer sets to P.
vivax detection is not capable of distinguishing among several knownmonkey malaria species. Generating primer sets for monkey malaria
which can differentiate itself from human malaria is an immediate
interest.
In Vietnam, PCR remains as the final confirmatory test applied tospecies identifications.
In Kyoto, molecular biologist and primatologists, entomologists,
discussed which methods to use in the field (Khanh Phu?) to detect
the parasites. As there are really no standards for field activities, weare at a point in establishing standards of procedure.
Among the challenges in the field where the number of parasites arevery little and multiple malaria species exist between human andmonkey, it is important to be able to distinguish the parasites thathave existed in humans, not only for diagnostic purpose.
LAMP is useful, but false positive results must be managed. PCR
techniques can be further improved, but developing a panel to screen
for all possible malaria species is needed right now.
Comment on how to work in parallel with recent malaria findings in thearea:
Identifying what other parasites may exist in the Khanh Phu area may
be of great interest. This will not only complement the malaria
research, but also help understand the ecology of all diseases present
in Khanh Phu beyond malaria, as there has not been a report
identifying the impacts of other factors involved in the zoonoses. A
global approach to understand the impact of certain factors to theecology of disease could provide a better picture of the whole system
in Khanh Phu. Addressing such a comprehensive approach may offer
a way to obtain further support to studies that may have a broader
impact.
Annual deworming intervention in the area was brought up as amethod to understanding the re-infection rates of certain disease. Thismight be a great opportunity to carry out a survey right before anintervention to gain insight to all the microorganisms present in thearea.
Expensive studies on transmission between humans and pet monkeys
have been appearing in the literature, but not from Vietnam. Is this a
topic that can be explored?
Malaria remains a problem here in the forest and is still a topic toattract funding. How important is this threat involving monkey malaria
in Khanh Phu? Are there other areas to focus our attention? Thismight be an opportunity to reevaluate our current understanding
regarding persistence of malaria, especially with respect to zoonotic
forest malaria.
Sarawak data system described, outbreaks are monitored intensively
with integration of GPS data.
Does Vietnam have a systematic surveillance data or patient record
reporting system?
Field data in Vietnam may still require collection efforts to be done
individually for scientific validity.
Description of data management at Khanh Hoa General Hospital:
At present, Khanh Hoa General Hospital has a record of all severe
malaria cases. Patients that have been admitted are properlyrecorded. There is a designated infectious disease section in thehospital dedicated to collecting patient data.
Response on the possibility of establishing a recording system:
Speaking from sixteen years of experience living in Vietnam, theJapanese funding agencies will need to come through with the timecommitment and necessary funds/investments to make systematic
data collection a possibility. GPS mapping of patients and carrying out
extensive survey is quite possible. The Vietnamese personnel arevery much interested in engaging in long term surveillance measures.
Additional comments:
In Binh Phuoc, 22 diseases are being reported to the central
government with detailed diagnostics information. In some provinces,viral infections detected in patients are GPS mapped and sequence
data obtained to follow the molecular evolution over time and spaceon pathogens of interest.
Description of a small scale surveillance system set up in Lao, PDR:

It is difficult to provide a nationwide surveillance system at this time,
but based on a small scale Lao experience that began 3-4 years ago,
handheld devices were implemented using village health volunteers(VHV) to carry out a household survey, covering a population of
approximately 7000. There is potential to collaborate on setting up asimilar system on a small scale first, and later consider expanding.
Discussion moved onto the next topic of interest involving antibodies
detection methods in urine. What can be done with detecting
antibodies in humans, beyond epidemiological relevance?
Comments on current capabilities with urine analyses:
The discrimination of species specific malaria is very difficult. Urine
analyses and antibody detections are ideal in mass surveys, mainly toscreen for various pathogens of concern. A realistic application is inscreening school children on their health status as a monitoring tool.
Using antibody detections for species identification remains to beinvestigated.
Further comments:
Serology studies are important. However, to know the serologicalresponses to pathogens within a person requires understanding awhole lot, including not only the understanding of the disease, but also
the dynamics of IgG and IgM in the blood in each specific population
over time, and then translating the immunologic responses to the IgG
and IgM dynamics found in the urine. Specific serological responses
between populations are quite different even among the Vietnamese
populations; IgG responses among the people in Khanh Hoa will becompletely different from the people living in Ho Chi Minh City. Many
challenges exist in validating the antibody-related responses todisease of interest.
Based from studies detecting malaria DNA in the urine, capturing theshort window in which molecules of great importance such as DNA
can be found excreted in the urine might offer an explanation as towhy we see clinical variations in P. knowlesi infections among thosereported in Malaysia and other parts of Asia.
How can DNA be detected in the urine?
During the course of an infection, an immunological response follows
with apoptosis of cells resulting in shedding of proteins and DNA. Cell
contents of apoptosis passes through the permeable kidneys withurine in patients under stress and fever.
Single and multiple species infections, why is this an unusual effect tofurther understand the ecology surrounding disease transmission?
When considering an environment where all of these elements areavailable, the issue of surrounding mixed or single infection is quite
strange.
Response to the above question:
Co-infections are common. What is so peculiar is that falciparum
infections cannot be detected with knowlesi. P.v. + P.f./ P.v. + P.k. arecommon co-infections, but P.f. + P.k. has not been found. Even inpositive mosquito salivary glands; with one exception of P.f+P.v.
sample, most were of mixed infections. Could falciparum and knowlesi
parasites exhibit competition? Parasite interactions still need to beinvestigated. Also, what we see as vivax, may not be entirely vivax.
Could there be a monkey malaria species that has evolved in a way
we have not identified yet? This is still unknown and brings attention
to the interesting incident with detecting vivax in monkeys. For futurestudies P. knowlesi mono-infections must be detected in humans, theco-infection with vivax muddles the confirmatory testing, as we know
now that the P. vivax markers also cross reacts with many monkey
malarias.
Looking at the malaria phylogeny, P. vivax is much closer to monkey
malarias, than P. falciparum.
Closing statement provided by Dr. Ron P. Marchand.
As a small group working on addressing some big questions, we will
now be working to answer the questions addressed within the limited

resources available. Regarding identifications of species, doubts
remain, but concerns will be worked out for solutions.
The organizers thank all participants for their active participations andpresentations and comments. Special thanks to Dr. Nakazawa andProf. Kazuhiko Moji for taking the initiative to making the 2nd
International Symposium a reality providing the necessary funds; Dr.
Quang for preparing all the symposium materials along with membersof the Khanh Phoa Health Department and Khanh Phu Malaria
Center; Dr. Masuda for making the symposium available on-line to apublic audience; and all participants traveling from Ho Chi Minh Cityand afar.