Dossier sodium chlorite - Dióxido de Cloro scabelum consumidores

1) Can chlorine dioxide prevent the spreading of coronavirus
or other viral infections? Medical hypotheses

Can chlorine dioxide prevent the spreading of
coronavirus or other viral infections? Medical
hypotheses
K. KALY-KULLAI1
, M. WITTMANN1
, Z. NOSZTICZIUS1
and
LASZLO ROSIVALL2
*
1
Department of Physics, Group of Chemical Physics, Budapest University of Technology and
Economics, Budapest, Hungary
2
Institute of Translational Medicine and International Nephrology Research and Training Center,
Semmelweis University, Budapest, Hungary
© 2020 The Author(s)
INTRODUCTION
Motivation
Viruses have caused many epidemics throughout human history. The novel coronavirus [10] is
just the latest example. A new viral outbreak can be unpredictable, and development of specific
defense tools and countermeasures against the new virus remains time-consuming even in today's
era of modern medical science and technology. In the lack of effective and specific medication or
vaccination, it would be desirable to have a nonspecific protocol or substance to render the virus
inactive, a substance/protocol, which could be applied whenever a new viral outbreak occurs. This
is especially important in cases when the emerging new virus is as infectious as SARS-CoV-2 [4].
Aim and structure of the present communication
In this editorial, we propose to consider the possibility of developing and implementing antiviral
protocols by applying high purity aqueous chlorine dioxide (ClO2) solutions. The aim of this
proposal is to initiate research that could lead to the introduction of practical and effective
antiviral protocols. To this end, we first discuss some important properties of the ClO2 molecule,
which make it an advantageous antiviral agent, then some earlier results of ClO2 gas application
*
Corresponding author: Prof. emer. Laszlo Rosivall, MD, PHD, DSc, FERA, FAPS, Institute of Translational
Medicine, International Nephrology Research and Training Center, Semmelweis University, Budapest,
Nagyvarad ter 4., H-1089, Hungary. Tel/Fax: 36-1-2100-100, E-mail: rosivall.laszlo@med.semmelweis-univ.hu
Physiology International
DOI: 10.1556/2060.2020.00015

against viruses will be reviewed. Finally, we hypothesize on methods to control the spread of
viral infections using aqueous ClO2 solutions.
PREVIOUS EXPERIENCE AND BACKGROUND OF USING ClO2 AS AN
ANTIVIRAL AGENT
Inactivating viruses with ClO2 in aqueous phase
To our present knowledge, an aqueous solution of ClO2 is able to inactivate all types of viruses.
Disinfectants (in water phase) are compared by their CT values, which is the concentration
(measured in mg/L) multiplied by the contact time (measured in minute). In CT tables, ClO2 is
indicated for viruses in general, without mentioning any exemptions. For example, according to
[6], a CT value of 8.4 mg 3 min/L is needed to achieve a four-orders-of-magnitude (“4 log” or
“99.99%”) inactivation of viruses in an aqueous medium at 25 8C.
Chemical mechanism of virus inactivation: reaction of ClO2 with amino acid residues
In 1986, Noss et al. [19] proved that the inactivation of bacterial virus f2 by ClO2 was due to its
reactions with the viral capsid proteins, and almost no inactivation of the infectious viral RNA
occurred [8] when that was treated with ClO2 separately. They found [19], however, that three
discrete chemical moieties in the viral protein, namely the cysteine, tyrosine, and tryptophan
amino acid residues were able to react with ClO2 rapidly. In 1987, Tan et al. [28] tested the
reactivity of ClO2 on 21 free amino acids. ClO2 reacted only with six amino acids dissolved in 0.1
M sodium phosphate buffer, pH 6.0. The reaction with cysteine, tryptophan, and tyrosine was
too rapid to be followed by their technique. Three further amino acids (histidine, hydroxy-
proline, and proline) reacted with ClO2 much more slowly, at a measurable rate.
The reactivity of the three fast-reacting amino acids (cysteine [12], tyrosine [17], and
tryptophan [27]) was studied in Margerum's laboratory between 2005 and 2008. They found
that cysteine had the highest reactivity among these amino acids. From their experimental data
they calculated second order-rate constants (at pH 7.0, 25 8C and 1 M ionic strength) and
obtained the following sequence: cysteine 6.9 3 106
MÀ1
sÀ1
tyrosine 1.3 3 105
MÀ1
sÀ1

tryptophan 3.4 3 104
MÀ1
sÀ1
guanosine 50
-monophosphate 4.5 3 102
MÀ1
sÀ1
. (They
studied guanosine 50
-monophosphate [18] as a model compound for guanine in nucleic acids.
Data presented here are taken from Table 3 of ref. [18]).
In 2007, Ogata [22] found that the antimicrobial activity of ClO2 is based on denaturation of
certain proteins, which is primarily due to the oxidative modiﬁcation of the tryptophan and
tyrosine residues of the two model proteins (bovine serum albumin and glucose-6-phosphate
dehydrogenase) used in his experiments. In 2012, it was again Ogata who showed [23] that the
inactivation of inﬂuenza virus by ClO2 was caused by oxidation of a tryptophan residue (W153)
in hemagglutinin (a spike protein of the virus), thereby abolishing its receptor-binding ability.
In this context it is interesting to remark that the spike protein of the new coronavirus
SARS_CoV-2 contains 54 tyrosine, 12 tryptophan, and 40 cysteine residues [29]. If we assume
that in an aqueous solution all of these residues are able to react with ClO2 just like the free
amino acids, then the inactivation of the viruses can be extremely rapid even in a very dilute
(e.g., in a 0.1 mg/L) ClO2 solution.
2 Physiology International

ClO2 is a water soluble gas
Although chlorinedioxide in itself is a gas, it is highly soluble in water. When both air and water
are present, ClO2 is distributed between the two phases in an equilibrium ratio determined by
the temperature. This distribution coefﬁcient of ClO2 was determined by Ishi [11] in 1958. The
distribution coefﬁcient, g 5 [ClO2]G/[ClO2]L gives the ratio of the concentrations expressed in
the same units in the gas and the aqueous phases (e.g., g/L) and changes as a function of the
temperature. For example, at 20 8C g 5 0.0316, indicating that in equilibrium 1 cm3
aqueous
phase contains (0.0316)À1
5 31.6 times more ClO2 molecules than 1 cm3
gas phase.
In practice, the concentrations in the two phases are usually given in ppm. However, these
dimensionless numbers are defined in a different way in the gas and liquid phases as ppm (V/V)
and ppm (m/m), respectively. Therefore, for practical purposes, we need a distribution coeffi-
cient, which gives the ratio between these concentrations. Straightforward calculation yields that
the distribution coefficient in terms of ppm is 357 times the distribution coefficient in terms of
(g/L), so at 20 8C gppm 5 11.3. Thus, the following formula can be used to calculate the ClO2
concentration of the gas phase being in equilibrium with a ClO2 solution at 20 8C:
½ClO2Šgas in ppmðV=VÞ ¼ 11:3 3 ½ClO2Šaq in ppmðm=mÞ
Inactivating viruses with ClO2 in gas phase
The virus-inactivating reactions (the reactions of ClO2 with the three amino acids) take place in
an aqueous medium; consequently, ClO2 can inactivate microbes in their wet state only.
Therefore, ClO2 gas that is moisturized can be an ideal agent against viruses both in their wet
and dry states. Viruses that are carried by water droplets could be easily inactivated even by
ClO2 gas owing to the high solubility of ClO2 in water [11]. A dry ClO2 gas would be inap-
propriate as the water content of the aqueous droplet could evaporate, and in the absence of
aqueous medium the reactions of ClO2 slow down extremely. Indeed, Morino et al. [16] reported
that when applying a low concentration of ClO2 in the gas phase against FCV in dry state,
atmospheric moisture – at least a 75–85% relative humidity – is indispensable to inactivate
viruses. The advantage of using a moisturized ClO2 gas is that its water content is also able to
wet viruses in dry environment. Most viruses are found on hard surfaces indoor, but a small
fraction of viruses are “airborn”, attached to dust particles, which can also carry a single microbe
or an aggregate of microbes. Therefore, it is a prerequisite of an effective disinfection that all
microbes in all parts of the room should be wet and should be in contact with ClO2. If enough
aqueous ClO2 solution is sprayed into the room, the droplets will saturate the atmosphere with
water vapor everywhere, moreover, the atmosphere will also contain gaseous ClO2 everywhere.
The great advantage of this method is that H2O and ClO2 molecules of the gas phase can reach
the microbes in every small corner of the room. Finely dispersed water droplets containing
dissolved ClO2 can create an advantageous environment to maintain such conditions for a
longer time.
This method using high ClO2 concentration allows fast disinfection of rooms when people
are not present, e.g., intensive care units, buildings used as quarantine, or public transport
vehicles. However, the application of ClO2 gas is limited when people are present, as it is
harmful for humans and animals above certain concentrations. The US Occupational Safety and
Health Administration (OSHA) limits the concentration of ClO2 gas allowed in workplace air to
Physiology International 3

0.1 ppm (V/V) time-weighted average (TWA) for an 8-h exposure, and to a temporarily higher
0.3 ppm Short-Term Exposure Limit (STEL) only for a 15-min period [30].
Previous research on preventing viral infections with gaseous ClO2
Ogata [21] realized first that ClO2 is able to inactivate viruses even under the 0.1 ppm (OSHA
TWA) limit that is in concentrations which are not harmful to humans. In 2008, Ogata and
Shibata [25] demonstrated that infection of mice with influenza A virus applied in an aerosol
can be prevented by ClO2 gas present at 0.03 ppm concentration in the air, which is only 30% of
the permissible TWA exposure level for humans at a workplace. They concluded that “ClO2 gas
could, therefore, be useful as a preventive tool against influenza in places of human activity
without necessitating evacuation.” They have even made attempts to decrease the incidence of
flue infections among schoolchildren by applying low concentrations of ClO2 gas in a classroom
[24].
In spite of these promising early results we are not aware of any wider-scale application of
this method in the last decade. There are two problems which could hinder the widespread
adoption of this method:
1. With the technique applied by the above-cited authors it is not an easy task to achieve and
maintain a very low ClO2 concentration in a large space and for a long time, which is a
prerequisite of achieving a satisfactory level of virus inactivation.
2. It is not understood why low ClO2 levels are not harmful to humans or animals and still
effective against viruses.
Size selective effect of ClO2
Although cysteine, tyrosine, and tryptophan residues can also be found in human tissues, ClO2
is much less toxic for humans or animals than for microbes (bacteria, fungi, and viruses).
Noszticzius et al. [20] found that the main reason for this selectivity between humans and
microbes is based not on their different biochemistries but on their different sizes. Based on
experiments and calculations using a reaction-diffusion model Noszticzius et al. [20] found that
the killing time of a living organism is proportional to the square of its characteristic size (e.g., its
diameter), thus small ones will be killed extremely fast. Their calculations indicated that a
bacterium 1 mm in diameter would be killed in a 300 mg/L ClO2 solution within 3 ms, and even
in a much more dilute, 0.25 mg/L ClO2 solution it would be eliminated in only 3.6 s. During this
time, ClO2 reaches all parts of the cell and kills it by destroying its cysteine-, tyrosine-, and
tryptophan-containing proteins, which are essential for life processes.
The protective role of glutathione against ClO2 oxidation in a living cell
According to Ison et al. [12] glutathione reacts with ClO2 at a rate, which is even higher than the
rate of the very fast ClO2 – cysteine reaction. When ClO2 contacts a living cell containing
glutathione, at first the ClO2 concentration remains very low even at the point of entry into the
cell due to this rapid reaction. As a small molecule, glutathione can also diffuse rapidly to the
point of entry from other parts of the cell consuming most of the ClO2 there, and preventing it
from reaching the cysteine, tyrosine, and tryptophan residues of the proteins in the bulk of the
cytoplasm. Consequently, the initial low ClO2 concentration cannot make too much harm.

However, continuous ClO2 entry can finally exhaust the cell's glutathione (and other antioxi-
dant) capacity even if the cell produces such antioxidants continuously. At this point, ClO2 can
enter into the previously protected zones of the cell and react with the reactive amino acid
residues, causing denaturation of the affected proteins and ultimately cell death.
The effect of glutathione and other small antioxidant molecules present in living cells was
not taken into account in the theoretical calculations of Noszticzius et al. [20]. Their experi-
ments were done on a non-living and washed animal membrane, where membrane-fixed
reactive proteins were present, but glutathione and other small molecules were absent. A living
cell, however, continuously produces these antioxidants, thus their role cannot be neglected.
Indeed, looking at the experimentally measured disinfection dynamics of a 0.25 mg/L ClO2
solution against Escherichia coli bacteria [2] we can see a disinfection rate, which is surprisingly
fast but still about one order of magnitude slower than the theoretical estimate. It is reasonable
to assume that the delaying effect of these small reducing molecules is responsible for that
deviation.
Protection of human tissues against the oxidative effect of ClO2
Human cells also contain glutathione in mM concentrations, as well as other antioxidants like
vitamin C and E, which work together with glutathione to reduce ClO2 [7]. As a human cell is
much larger than a bacterium, consequently its glutathione reserve and glutathione production
potential are also greater, so even an isolated human cell can survive much longer in a ClO2
environment than a planktonic bacterium. Considering that human cells are not isolated but
form tissues, their glutathione stock may be many orders of magnitude greater than that of a
planktonic bacterium. Additionally, in multicellular organisms circulation transports antioxi-
dants continuously to the cells of the tissue affected by a ClO2 attack, helping them to survive.
This strengthens the size-selectivity effect, and explains the surprising observation [15] that
ClO2 solutions that are able to kill planktonic bacteria in a fraction of a second may be
consumed, because they are safe for humans to drink in a small amount (e.g., drinking 1 L of 24
mg/L ClO2 solution in two portions on a single day caused no observable effects in humans
[15]).
The effect of ClO2 on the lung
While human tissues are not very sensitive to ClO2 in general, lungs should be considered
differently. This is because the interalveolar septum separating the airspace of an alveolus from
the blood stream of a capillary lumen is very thin. That diffusion barrier in the human lung is a
mere 2 mm thick [1] in order to facilitate an efficient diffusional exchange of oxygen and carbon
dioxide between the air and blood. The alveolus is covered by a thin layer of lining fluid called
epithelial lung lining fluid (ELF) or hypophase. The ELF is only 0.2 mm thick in rat alveoli [1,
13]. It contains glutathione [3] and other antioxidants such as ascorbic and uric acids [5]. It is
remarkable, that the ascorbic acid concentration is 2.5 times, and the glutathione concentration
is more than 100 times higher in the ELF than in the plasma. The normal function of these non-
enzymatic antioxidants in the ELF is to protect the epithelial cells from reactive oxygen species
(ROS) like superoxide radicals or hydrogen peroxide, which are toxic products of the meta-
bolism. They can also defend the lung against other toxic gases such as ozone (O3), nitrogen
dioxide (NO2) or ClO2. However, high amounts of ClO2 can consume all reducing agents in the

ELF, at which point ClO2 starts to react with the epithelial cells causing a continuously growing
damage to these cells. It is known that higher concentrations of ClO2 gas can be lethal. However,
it is reasonable to assume that the effect of ClO2 on the lung depends not only on its con-
centration in the gas phase but also on the contact time. Thus, when considering the impact of
ClO2 on the lung, it would be logical to regard the CT (concentration) 3 (contact time) product
in a similar way as in the case of the microbes.
Estimating the inactivation time of viruses
In the case of viruses, the inactivation mechanism differs from that of bacteria or other cells. It is
feasible to assume that the inactivation time of a virus is probably much shorter than the
inactivation time of a bacterium under the same conditions (ClO2 concentration, temperature,
etc.). The following arguments support this assumption:
1. Viruses are about one order of magnitude smaller than bacteria e.g., the diameter of SAR-
S_CoV-2 is about 120 nm [9]. The killing time of a virus as introduced in [20] would be 1–2
orders of magnitude shorter than that of a bacterium, i.e., the diffusion-controlled reaction
with ClO2 would happen on a shorter time scale in the entire volume of the virus.
2. It is not necessary for ClO2 gas to penetrate the virus in order to inactivate it. It is enough if
ClO2 reacts with one or some of the cysteine, tyrosine, and tryptophan amino acid residues of
the spike, which are located on the surface of the virus. This means that the theoretical
approach of ref. [20] overestimates the inactivation time of viruses. On the one hand, because
diffusion is extremely fast over a 0.1 mm length scale, thus probably it is not limiting the rate
of the reactions. On the other hand, ClO2 can reach a large part of the reactive amino acid
residues of the spike without permeating through the protein envelope of the virus.
3. Viruses do not contain protective small molecular thiols like glutathione or other small
molecular protective metabolic products, because viruses have no metabolism. In this respect
viruses should be much more vulnerable than bacteria to an attack by ClO2.
These facts all suggest that once ClO2 contacts the surface of a virus, its inactivation is quick.
However, a virus ready to infect a cell is typically in aqueous phase, e.g., in a ﬂuid droplet, or in the
epithelial lining ﬂuid covering the mucous membranes. The size of these aqueous phases is much
larger than that of the virus. Therefore, in such cases, the rate-limiting step probably is the diffusion of
ClO2 in the water and the reaction with other substances. The time required to inactivate the virus
itself would be short compared to the time needed to transport enough ClO2 molecules to the virus.
SUGGESTIONS FOR PREVENTING THE SPREAD OF VIRAL INFECTIONS
USING ClO2
Based on the previous arguments, some propositions will now be put forward on how aqueous
ClO2 solutions could be applied for global and local (personal) disinfection purposes. Many of
these propositions are based on hypotheses, and therefore can only be applied after careful
research. It is a goal of the present work to initiate research to check these hypotheses and
proposals experimentally, which could lead to new applications of high-purity ClO2 solutions
against viral or other infections. These ideas might be further matured in time, but due to the
threat of a global pandemic, we have chosen to move fast.

Global prevention
Disinfection of air spaces, hard surfaces, and persons simultaneously with aqueous ClO2
solutions. What we are proposing here is basically the same idea what has been already pro-
posed by Ogata et al. [21, 24, 25]: it is possible to create ClO2 atmospheres which can be safe for
humans but at the same time harmful for microbes. There are differences, however, between
their proposals and ours. Ogata's group regarded the ClO2 concentration (C) of the atmosphere
as the sole important parameter of the treatment. They proposed to apply a ClO2 concentration
below the 0.1 ppm (V/V) OSHA limit, for a time that is necessary to inactivate the microbes.
With such a method, however, the necessary contact time (T) can be very long. Here we suggest
regarding the CT product as the parameter of disinfection. In this way it is possible to apply
ClO2 concentrations above the OSHA limit but for a limited time only. The advantage of this
method is that as higher C values are applied, the necessary contact times can be much shorter.
The idea will be illustrated by a numerical example below.
Another important difference is that Ogata's method focuses mostly on the role of the ClO2
gas, whereas we emphasize the importance of the simultaneous usage of ClO2 and H2O gases, as
conﬁrmed by the observations of Morino et al. [16]. For this purpose, we suggest a new way of
creating a ClO2 atmosphere: to apply aqueous ClO2 solutions which can establish equilibrium
ClO2 and H2O concentrations in the atmosphere, when these are sprayed into the air. Aqueous
solutions are also easier to handle than to maintain stable and very low ClO2 levels in
continuous gas streams.
It is advisable to apply high-purity ClO2 solutions for spraying to avoid any unwanted side
effects to the persons or the surfaces treated. High-purity ClO2 solutions evaporate without any
residue or trace.
An illustrative numerical example. Let us assume that we want to disinfect a closed space by
spraying some aqueous stock ClO2 solution into it. The equilibrium ClO2 concentration cair in
the gas and cw,e in the liquid phase can be calculated from the vapor-liquid equilibrium dis-
tribution measured by Ishi [11]:
g ¼
cair
cw;e
and from the component balance for ClO2:
Vw$cw0 ¼ Vw$cw;e þ Vair$cair
where Vair is the volume to be disinfected, Vw is the volume of the ClO2 stock solution, and cw0 is
its ClO2 concentration.
With the help of the above two equations cw;e can be calculated as
cw;e ¼
cw0
1 þ g$Vair
Vw
Suppose we apply Vw 5 20 mL of cw0 5 40 ppm (m/m) aqueous stock ClO2 solution in a
Vair 5 1 m3
closed space.
At 20 8C the value of the distribution coefficient is g 5 0.0316 (data of Ishi [11]) when all
concentrations are given in the same units (e.g., in mg/L), and it is gppm 5 11.3 (see section ClO2

is a water soluble gas) when ppm (m/m) and ppm (V/V) are used in the aqueous and gaseous
phases, respectively. Substituting our data the results are
cw;e ¼ 0:025 ppmðm=mÞ;
cair ¼ gppmcw;e ¼ 0:29 ppmðV=VÞ
This result is just below the OSHA STEL (Short Term Exposition Limit) value which is 0.30
ppm (V/V) for 15 min. According to OSHA, STEL is the acceptable average exposure over a
short period of time – usually 15 min – as long as the time-weighted average (TWA) is not
exceeded. If a person is exposed to 0.30 ppm for 15 min, and right after that he or she stays in a
ClO2-free atmosphere for 30 min, then the TWA value for this whole 45 min period is just the
acceptable 0.10 ppm. All this means that an exposure of a person to a 0.30 ppm ClO2 atmo-
sphere for 15 min on a single occasion, or even applying that treatment periodically with 30 min
pauses within an 8 h period, should not cause any health problems.
Questions and remarks.
1. It is a major question, whether or not a 15-min stay in a 0.29 ppm (V/V) ClO2 atmosphere is
enough to inactivate the viruses present? Regarding the wet atmosphere we can assume that
the viruses are also wet, or even that they can be found in small water droplets containing
0.025 ppm (m/m) ClO2. We have no direct data for the inactivating time of viruses in such a
solution, but we have an estimated 15 ± 5 s killing time value for an E. coli bacterium in a
0.25 ppm (m/m) ClO2 solution [2]. It is reasonable to assume that the killing time would be
10 times longer in a 10 times more dilute solution, i.e., 150 s 5 2.5 min. As viruses are
probably inactivated faster than bacteria, and 15 min is six times longer than the estimated
2.5 min for E. coli, such a method can be successful, at least in theory.
2. To test such a method, we suggest constructing special disinfecting rooms with larger vol-
ume. Starting such experiments would be highly desirable, because this method could be an
effective nonspecific defense against all types of viruses and could help to contain viral
outbreaks.
Local prevention. Personal disinfection techniques against viral infections
Disinfection of the mouth and the upper respiratory track with gargling. The current epidemic
coronavirus is known to be present in the mouth and both in the upper and lower respiratory tract,
but causes severe infections only in the lower respiratory tract, especially in the lung. The incu-
bation period of the disease is several days, but the virus can often be detected in samples taken
from the upper respiratory tract a few days before symptoms appear. As discussed in a previous
chapter, chlorine dioxide will certainly inactivate the virus. With gargling, the upper respiratory
tract is accessible except for the nasal cavity, but that is also accessible using e.g. nose drops or
impregnated tampons. These parts can be disinfected by rinsing them regularly with high-purity
chlorine dioxide solutions available commercially [31], thus the number of the viruses can be
significantly reduced in the mouth and in the upper respiratory tract. We cannot be sure that such a
treatment would be enough to prevent the development of the illness, as viruses living in other parts
of the body can survive. However, inactivating part of the viruses with such a treatment surely helps
the immune system to fight against the disease. In this respect, it is interesting to remark that

Japanese researchers have proven [26] that regular gargling with drinking water has reduced the
incidence of upper respiratory tract infections to a statistically significant extent. The effect was
explained by the fact that the drinking water used in the experiments contained 0.5 mg/L of
chlorine, which was used to disinfect the water. We remark here, that in certain places chlorine
dioxide is applied for the disinfection of drinking water instead of chlorine.
Disinfection of the lower respiratory track. The first problem is how ClO2 can be safely
introduced into the lower respiratory tract. For this purpose any inhalation technique could be
applied using aerosols of water droplets containing ClO2 [14].
The second and more important problem is how much ClO2 can be inhaled without damaging
the lung? It would be helpful to know the dose of ClO2 that is not yet harmful for the lung. To our
knowledge such direct data are not available in the literature, but can be calculated from other
data. The starting point for such a calculation is the OSHA STEL value [30], according to which
0.30 ppm ClO2 in the workplace atmosphere is tolerable for a 15 min period without any damage.
The volume of air inhaled by a worker during 15 min is 15 times the so-called “minute volume
ventilation” [32]. According to Table 3 of ref. [32], during light activities e.g., when sitting in a car
the minute volume is around 12 L, thus the total inhaled air is about 180 L. In the case of 0.30 ppm
concentration the total inhaled amount of ClO2 is 54 mL, which is (at 20 8C) 2.25 mmol ≈ 0.15 mg
ClO2. Assuming a more vigorous activity it can be two times more, 0.30 mg.
This rough calculation indicates that approximately this is the amount of ClO2, which can be
tolerated by the lung. The OSHA limit probably applied high safety factors, thus the real limit should be
higher.
We suggest that animal experiments should be performed to obtain experimental values for
the pulmonary toxicity of ClO2. Furthermore, it would be important to check in additional
animal experiments, whether ClO2 applied in a nontoxic amount is able to treat infections of the
lung caused by bacteria or viruses.
CONCLUSION
In this editorial, we summarized the unique properties of chlorine dioxide, which make it an
ideal and nonspecific antimicrobial agent at concentrations harmless to humans, and we
reviewed previous research on preventing viral infections with gaseous ClO2. Based on this
background, we suggested some novel hypothetical methods using chlorine dioxide to disinfect
rooms, prevent human infection, and slow down viral spread. These are nonspecific methods,
which could be used against any newfound virus as a first line of protection until effective
specific countermeasures are developed.
Conflict of interest: Zoltan Noszticzius, Maria Wittmann and Kristof Kaly-Kullai are co-in-
ventors of the European patent 2069232 “Permeation method and apparatus for preparing fluids
containing high purity chlorine dioxide”. Zoltan Noszticzius is a founder and owner of the
Solumium Ltd (a company producing chlorine-dioxide), while Kristof Kaly-Kullai is its payed
employee.

REFERENCES
1. Bastacky J, Lee CY, Goerke J, Koushafar H, Yager D, Kenaga L, et al. Alveolar lining layer is thin and
continuous: low-temperature scanning electron microscopy of rat lung. J Appl Physiol 1995; 79(5): 1615–28,
https://doi.org/10.1152/jappl.1995.79.5.1615.
2. Benarde MA, Snow WB, Oliveri VP, Davidson B. Kinetics and mechanism of bacterial disinfection by
chlorine dioxide. Appl Microbiol 1967; 15(2): 257–65.
3. Cantin AM, North SL, Hubbard RC, Crystal RG. Normal alveolar epithelial lining ﬂuid contains high levels of
glutathione. J Appl Physiol 1987; 63(1): 152–7, https://doi.org/10.1152/jappl.1987.63.1.152.
4. Cao Z, Zhang Q, Lu X, Pfeiffer D, Jia Z, Song H, et al. Estimating the effective reproduction number of the
2019-nCoV in China. Available online at http.medxriv.org; Jan. 29; 2020; https://doi.org/10.1101/2020.01.27.
20018952.
5. Cross CE, van der Vliet A, O’Neill CA, Louie S, Halliwell B. Oxidants, antioxidants, and respiratory tract lining
ﬂuids. Environ Health Perspect 1994; 102(Suppl. 10): 185–91, https://doi.org/10.1289/ehp.94102s10185.
6. EPA Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water
Sources (AWWA; 1991). Table C-9: CT values for inactivation of viruses by chlorine dioxide. Available at
http://www.opssys.com/InstantKB/article.aspx?id514495.
7. Forman HJ, Zhang H, Rinna A. Glutathione: overview of its protective roles, measurement, and biosynthesis.
Mol Aspects Med 2009; 30(1–2): 1–12, https://doi.org/10.1016/j.mam.2008.08.006.
8. Hauchman FS, Noss CI, Olivieri VP. Chlorine dioxide reactivity with nucleic acids. Water Res 1986; 20(3):
357–61, https://doi.org/10.1016/0043-1354(86)90083-7.
9. https://www.britannica.com/science/coronavirus-virus-group.
10. Hui DS, Azhar EI, Madani TA, Ntoumi F, Kock R, Dar O, et al. The continuing 2019-nCoV epidemic threat
of novel coronaviruses to global health – The latest 2019 novel coronavirus outbreak in Wuhan, China. Int J
Infect Dis 2020; 91: 264–6, https://doi.org/10.1016/j.ijid.2020.01.009.
11. Ishi G. Solubility of chlorine dioxide. Chem Eng Japan 1958; 22(3): 153–4, https://doi.org/10.1252/
kakoronbunshu1953.22.153.
12. Ison A, Odeh IN, Margerum DW. Kinetics and mechanisms of chlorine dioxide and chlorite oxidations of
cysteine and glutathione. Inorg Chem 2006; 45: 8768–75, https://doi.org/10.1021/ic0609554.
13. Knudsen L, Ochs M. The micromechanics of lung alveoli: structure and function of surfactant and tissue
components. Review Histochem Cell Biol 2018; 150: 661–76, https://doi.org/10.1007/s00418-018-1747-9.
14. Koren E. A suggestion. Personal information; 2020.
15. Lubbers JR, Chauan SR, Bianchine JR. Controlled clinical evaluations of chlorine dioxide, chlorite and
chlorate in man. Environ Health Perspect 1982; 46: 57–62, https://doi.org/10.1289/ehp.824657.
16. Morino H, Fukuda T, Miura T, Lee C, Shibata T, Sanekata T. Inactivation of feline calicivirus, a Norovirus
surrogate, by chlorine dioxide gas. Biocontrol Sci 2009; 14: 147–53, https://doi.org/10.4265/bio.14.147.
17. Napolitano MJ, Green BJ, Nicoson JS, Margerum DW. Chlorine dioxide oxidations of tyrosine, N-ace-
tyltyrosine, and Dopa. Chem Res Toxicol 2005; 18: 501–8, https://doi.org/10.1021/tx049697i.
18. Napolitano MJ, Stewart DJ, Margerum DW. Chlorine dioxide oxidation of guanosine 50
-monophosphate.
Chem Res Toxicol 2006; 19: 1451–8, https://doi.org/10.1021/tx060124a.
19. Noss CI, Hauchman FS, Olivieri VP. Chlorine dioxide reactivity with proteins. Water Res. 1986; 20(3): 351–6,
https://doi.org/10.1016/0043-1354(86)90083-7.
20. Noszticzius Z, Wittmann M, Kaly-Kullai K, Beregvari Z, Kiss I, Rosivall L, et al. Chlorine dioxide is a size-
selective antimicrobial agent. PloS One 2013; 8(11): e79157, https://doi.org/10.1371/journal.pone.0079157.

21. Ogata N. Chlorine dioxide gas for use in treating respiratory virus infection. European Patent Specification EP
1955719 B1. Priority to JP 2005342503; 2005.
22. Ogata N. Denaturation of protein by chlorine dioxide: oxidative modification of tryptophane and tyrosine
residues. Biochemistry 2007; 46: 4898–911, https://doi.org/10.1021/bi061827u.
23. Ogata N. Inactivation of influenza virus haemagglutinin by chlorine dioxide: oxidation of the conserved
tryptophan 153 residue in the receptor-binding site. J Gen Virol 2012; 93: 2558–63, https://doi.org/10.1099/
vir.0.044263-0.
24. Ogata N, Shibata T. Effect of chlorine dioxide gas of extremely low concentration on absenteeism of
schoolchildren. Int J Med Med Sci 2009; 1(7): 288–9.
25. Ogata N, Shibata T. Protective effect of low-concentration chlorine dioxide gas against influenza A virus
infection. J Gen Virol 2008; 89: 60–7, https://doi.org/10.1099/vir.0.83393-0.
26. Satomura K, Kitamura T, Kawamura T, Shimbo T, Watanabe M, Kamei M, et al. Great cold investigators:
prevention of upper respiratory tract infections by gargling. A randomized trial. Am J PrevMed 2005; 29:
302–7, https://doi.org/10.1016/j.amepre.2005.06.013.
27. Stewart DJ, Napolitano MJ, Bakhmutova-Albert EV, Margerum DW. Kinetics and mechanisms of chlorine
dioxide oxidation of tryptophan. Inorg Chem 2008; 47: 1639–47, https://doi.org/10.1021/ic701761p.
28. Tan H, Wheeler BW, Wei C. Reaction of chlorine dioxide with amino acids and peptides: Kinetics and
mutagenicity studies. Mutat Res 1987; 188(4): 259–66, https://doi.org/10.1016/0165-1218(87)90002-4.
29. Tao Y, Queen K, Paden CR, Zhang J, Li Y, Uehara A, et al. Severe acute respiratory syndrome coronavirus 2
isolate 2019-nCoV/USA-IL1/2020, complete genome. NCBI GenBank; 2020. Available at https://www.ncbi.nlm.
nih.gov/nucleotide/MN988713.1?report5genbanklog$5nuclalignblast_rank51RID5304U21XH016.
30. US Occupational Safety and Health Administration. Determination of chlorine dioxide in workplace atmo-
spheres; 1991. Available at https://www.osha.gov/dts/sltc/methods/inorganic/id202/id202.html.
31. www.solumium.com.
32. Zuurbier M, Hoek G, van den Hazel P, Brunekreef B. Minute ventilation of cyclists, car and bus passengers:
an experimental study. Environ Health 2009; 8: 48, https://doi.org/10.1186/1476-069X-8-48.
Open Access statement. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0
International License (https://creativecommons.org/licenses/by/4.0/),which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited, a link to the CC License is provided, and changes
– if any – are indicated. (SID_1)

2) ATC Malachlorite® for treatment of patients with acute
Plasmodium falciparum infection: A pilot study
incorporating 500 patients in the rural area of Cameroon

GSC Biological and Pharmaceutical Sciences, 2018, 02(02), 006–017
Available online at GSC Online Press Directory
GSC Biological and Pharmaceutical Sciences
e-ISSN: 2581-3250, CODEN (USA): GBPSC2
Journal homepage: https://www.gsconlinepress.com/journals/gscbps
Corresponding author
E-mail address: enno.freye@ uni-duesseldorf.de
Copyright © 2018 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0.
(RESEARCH ARTICLE)
ATC Malachlorite® for treatment of patients with acute Plasmodium falciparum
infection: A pilot study incorporating 500 patients in the rural area of Cameroon
Enno Freye 1*, Hans-Peter Strobel 2 and Olivia M. Weber 3
1 University Clinics of Düsseldorf, Germany.
2 Department of Energy Development Davos-Platz, Switzerland
3 Center of Novel Therapeutic Strategies (NTS), Luzern, Switzerland.
Publication history: Received on 17 January 2018; revised on 27 January 2018; accepted on 07 February 2018
https://doi.org/10.30574/gscbps.2018.2.2.0004
Abstract
Malaria is one of the most widespread infectious diseases with over 2 million deaths per year in Central Africa. Since
new drug combinations are characterized by the development of tolerance as well as serious side effect there is an
increasing demand for a new medication. We therefore set out to study the efficacy of a sublingual tablet with a new
mode of action consisting of sodium chlorite (NaClO2) plus citric acid and an artimisin extract. By adding sodium
bicarbonate, an effervescent composition is attained, which when being exposed to saline, chlorine dioxide (ClO2) the
active ingredient is being released. Being a potent oxygen radical, it attaches to the layer of a trophozoite resulting in
the disintegration and its demise. This reaction is terminated within one hour and ClO2 dissolves into the two end
products, sodium chloride (table salt) and water. Following informed consent 500 patients (240 male, 260 female mean
age 27 +/-19 SD) within the rural area of Cameroon and demonstrating acute symptoms of malaria infection, were given
5 sublingual tablets of ACT Malachlorite® 3 times per day at an 8 hour interval. The medication was able to reverse
acute symptoms of malaria within the first 2 days. Using thick blood films, trophozoite count under the microscope
demonstrated a highly significant reduction (p 0.001) of count of Plasmodium falciparum. From a mean of 6430 (+/-
816 SD) trophozoites per mm3, prarasitemia dropped to a mean of 4932 (+/- 842 SD) on day two and further declined
to 1454 (+/- 401 SD; p 0.001) on day four. No trophozoites could be detected in the blood on day six. Side effects were
reported from the intestinal tract consisting of bloating and cramping while the majority of patients complained of the
pungent chlorine taste as being unpleasant. The new ATC Malachlorite® tablet presents a promising new approach in
malaria treatment with a different mode of action than all present antimalarial agents.
Keywords: Malaria treatment; Plasmodium falciparum; Chlorine dioxide; Pilot study Cameroon
1. Introduction
Malaria affects about a hundred countries in the world, particularly the disadvantaged tropical areas of Africa, Asia and
Latin America. Africa is by far the most affected continent with 90% of malaria cases in tropical areas. Malaria is a
disease caused by parasites of the plasmodium type, which according to WHO, causes around 1 million deaths per year
worldwide. About 40% of the world population is exposed to the disease and 500 million clinical cases occur each year.
On the other hand, Europe is experiencing cases of so-called imported malaria. Especially countries like Britain and
France show the highest number of imported malaria as reported recently [1]. The situation is particularly worrying in
that for several years the parasites develop resistance to many commonly used anti-malarial agents while in addition,
mosquitoes demonstrate an increase of resistance to the present available insecticides. So far, no vaccine is available
for this life-threatening disease resulting in every new exposure into a full-blown course of the malaria disease. While

Enno et al. / GSC Biological and Pharmaceutical Sciences 2018, 02(02), 006–017
7
it is generally accepted that malaria is one of the most widespread infectious diseases representing over 2 million deaths
per year in Central Africa and although every medical and hygienic effort is undertaken to reduce the number, reports
of WHO show a steady increase in the number of infections as well as the development of tolerance to conventional
drugs such as resochine (Chloroquine®), the antibiotic tetracycline Doxycycline® or the combination Malarone®
(Atovaquon-Proguanil), all of which are not only characterized by resistance development but also of severe side
effects [2]. Although the latest generation of agents in the treatment of malaria such as Lariam® (a chinine derivative)
showed little resistance development they are however characterized by serious side effects. And while treatment is
readily available in large cities where hospital doctors as well as medical staff are able to treat acute symptoms of
malaria, the inhabitants of suburban and rural areas lack such advanced care [3]. It is because in such remote areas, the
general medical practitioner (GP) is only visited by patients who demonstrate acute symptoms of Plasmodium
falciparum infection, having reached a critical state. Especially, development of tolerance to conventional medication
presents a problem in treatment of an acute infection when using the ACT (additional combination therapy) consisting
of a two-drug approach, one of which is always an artemisinin derivative [2-4].
In spite of such new strategies in the treatment of malaria, there is an ongoing demand for alternative therapeutic agents
which do not present the problem of tolerance development. Therefore, the objective of the study was to ascertain, that
this newly formulated effervescent tablet is easily accessible to patients in urban areas, demonstrates a high therapeutic
efficacy, is easily administered to patients, and does not demonstrate the usual side effects usually seen with the
conventional medications.
2. Material and methods
2.1. Composition of ATC Malachlorite® - mode of action
We therefore set out to study the efficacy of a totally new medication which so far had only been used for the
manufacture of safe drinking water in a number of small city water plants in rural areas and for trekker and boot
camping and which already is in practical use for the past 10 years. This compound by the name of sodium chlorite
(NaClO2), when getting into contact with an acidic acid, dissolves into chlorine dioxide (CLO2) which by itself acts as a
potent oxygen radical releaser. When it attaches to the layer of a trophozoite, it induces the disintegration of the outside
covering of the protozoon and its immediate demise. By adding sodium bicarbonate an effervescent composition was
developed, which when being exposed to water or to saline, results into the release of ClO2 being the active ingredient.
This chlorine dioxide is readily taken up by the mucous membranes of the oral cavity and after entering the blood cycle
it attaches to any kind of preexisting trophozoite. This reaction is terminated within one hour after chlorine dioxide has
reached the blood where it dissolves into the two end products, sodium chloride (NaCl or table salt) and water, showing
the following chemical reaction:
5 NaClO2 + C6H8O7 = 4 ClO2 + 5 NaCl + 2 H2O
Thus, the compressed tablet, aside from containing the prodrug sodium chlorite (NaClO2) and the additive citric acid
(C6H8O7) which later serves as the ignition key for the production of ClO2, are kept in a compressed tablet to which
artimisin extract was added. This addition deemed necessary being an agent already known for a long time in traditional
Chinese medicine as an efficient antimalarial agent but also since WHO demands that any new agent contains parts of
artimisin or its active ingredient artemisinin [5]. All other compositions added to the tablet, however, are inert and are
used solely either to increase solubilization, the reabsorption through the mucous membranes or to increase tastiness
(table 1).
The gas chlorine dioxide (ClO2) is the active compound, being reabsorbed through the mucous membranes and/or the
intestinal lining inducing a sufficiently high serum level. Because of the negative charge of chlorine dioxide, the agent is
attracted by the positive charge of any kind of parasite, and by closely attaching to the layer to the Plasmodium
falciparum it releases a highly aggressive oxygen radical (O2-). This radical in order for the sake of stability, tears out
electrons from the outer lining of the parasite resulting in the disintegration of its layer which is followed by its demise.
All of the active compounds of the tablets were crushed into tiny pieces of 250 pm in diameter. Thereafter, the raw
powder was added to the auxiliaries and the whole was compressed into tablets of 0.7 g (table 1). These tablets were
given to patients having symptoms of an acute malaria infection characterized by acute fever and a high number of
trophozoites in their blood count under the microscope.

8
Table1 Composition within ATC Malachlorite® tablet given to patients for treatment of parasite infection with
Plasmodium falciparum
Sr.
No.
Composition Content in mg Role in treatment
1 Sodium bicabonate 44.0 To get an effervescent formulation, at the same time being a
penetration enhancer through mucous membranes of oral cavity
2 Sodium chlorite 15.0 The active ingredient which later is converted into ClO2
3 Arabic gum 197.9 An important agent to increase solubilization and absorption
through the mucosal lining
4 Povidon 25 7.0 A synthetic polymer vehicle for dispersing suspending drugs
and also a binder for tablets
5 Xylitab 376.0 A natural sweetener to increase tastiness
6 Acesulfame-Potassium 7.0 An artificial sweetener and also an enhancing agent for
absorption
7 Prosol 30.0 Silicified microcrystalline cellulose, a combination of
microcrystalline cellulose (MCC) and colloidal silicon dioxide
(CSD) for binding properties of the tablet
8 Citric acid (540 + 110) 65.0 The ignition key for the formation of chlorine dioxide
9 Magnesium stearate
40%
50.0 An inert compound for reducing stickiness during the
production process
10 Artemisin 1.9 An extract of Artemisia annua with oxygen radical scavenging
properties, an antinflammatory and an enhancer in efficacy of
antimalarial agents
The production of ACT Malachlorite® tablets followed good standard pharmaceutical practices (GSP) at an officially
accredited and authorized laboratory in Switzerland by the name of Merisana® Company, in Cotébert, Switzerland..
2.2. Dosing, inclusion, exclusion criteria, and drop outs
A total dose of 75 mg of sodium chlorite within 5 ACT Malachlorite® tablets was given 3 times per day with an interval
of 8 hours. Starting off with five tablets on the first day this was followed by two times 3 tablets (= 45 mg of sodium
chlorite) at an interval of 8 hours over the following 5 days. Patients were instructed to place the tablet under their
tongue (sublingual) and allow it to dissolve for about 10-15 minutes. The remnants of the sublingual tablet that
remained in the buccal cavity was swallowed by the patient drinking a glass of water, not expecting any kind of residual
effect. The patients were not allowed to suck, chew or swallow the tablet as this would have resulted in lower plasma
concentrations of chlorine dioxide.
All doses of medication were administered under the supervision of a qualified member of the staff designated by the
principal doctor on site of various health care facilities within the rural country of Cameroon. The study patients were
observed for 30 min after administration for any kind of adverse reactions or vomiting. Any patient who vomited during
this observation period was re-treated with the same dose of the medication and observed for an additional 30 min. If
the patient vomited again, he or she was withdrawn and offered a rescue therapy. The patients were advised to stay in
the health facility until they completed the treatment course for malaria infection with ACT Malachlorite®.
2.3. Rescue medication in case of incompatibility
If a patient vomited twice during the treatment phase he/she received parenteral therapy based on national treatment
guidelines (IV saline, electrolytes etc.). Once a patient did not tolerate the trial medication he/she discontinued the
treatment and an alternative anti-malaria medication was started. In this case, the reason for discontinuation was
recorded in the case record form (CRF) as Adverse Reaction and the patient was withdrawn from the study and
received parenteral therapy with Artemether or Quinine 7 days (IV) plus Tetracycline for 7 days as well as relevant
supportive treatments.

9
If a patient met one of the criteria for a therapeutic failure, he/she received Artemether at day 1 using 3.2mg/kg (max
160mg) intramuscularly, on day 2 - 1.6 mg/kg (max 80mg) IM, and from day 3 to day 5 - 1.6 mg/kg (max 80mg) per day
intramuscularly. At day 6 Mefloquine 25mg/kg was added which was spread over 3 days (max 1250 mg) according to
the current national recommendation guidelines.
2.4. Parameters measured for demonstration of efficacy
Valid study end-points included either treatment failure, or completion of the follow-up period without treatment
failure reflected in an adequate clinical and parasitological response, loss to follow-up, withdrawal from the study,
and/or protocol violation. At all times, well-being of the patient had a priority over his or her continuation within the
study.
Treatment outcomes were classified on the basis of an assessment of the parasitological and clinical outcome of the
antimalarial treatment using the latest WHO guidelines [6]. Since elimination from the plasmodium parasites was the
goal of the antimalarial therapy, all patients within the study who did show a treatment failure (non-effectiveness) were
given a rescue medication with a follow-up until recovery. Also, the incidence of any adverse event was documented.
All patients were asked routinely about previous symptoms and about symptoms that had emerged since the previous
follow-up visit.
2.5. Informed consent
A standard physical examination was performed at baseline (day 0 before dosing) and on days 1, 2, 3, 4 and 6. Also,
complete medical history including prior and concomitant medication, demographic data and contact details were taken
at baseline.
Patients were only included in the study if adults and children (parents or guardians of children) had given their
informed consent. The principal investigator(s) also obtained and documented the assent of children between the age
of 12 and 18 years, but their assent had to be accompanied by the consent of a parent or a guardian. A child aged between
12 and 18 years who did not agree to participate was not enrolled in the study and was referred to the health facility
staff to be treated according to the standard of care established by the Ministry of Health of Cameroon. Also, a written
consent statement for the pregnancy test deemed necessary since a pregnant participant was not eligible to participate
in the study. In addition, the need for contraception was required for female participants of child-bearing age who were
sexually active.
2.6. Patients inclusion and exclusion criteria
The following criteria have to be met in order for a patient to participate in the study while on the other hand patients
were excluded from the study as outlined in the following once he or she patient did not meet the criteria for enrollment
or did not want to comply the conditions.
2.6.1. Inclusion criteria of patients to participate in the study
i. An age between 10 and 60 years of age;
ii. A mono-infection with P. falciparum;
iii. The presence of an axillary temperature ≥ 37.5°C or a history of fever during the past 24 h;
iv. The ability to swallow oral [by mouth] medication;
v. The ability and willingness to comply with the study protocol for the duration of the study and to comply with
the study visit schedule;
vi. Signing of an informed consent by the patient or by a parent or guardian in the case of children aged less than
12 years;
vii. An informed assent from any participant aged more than 12 years and less than 60 years; and
viii. A consent for pregnancy testing from females of child-bearing age or from their parent or guardian if under
18 years.
2.6.2. Exclusion criteria of patients not to participate in the study
i. The presence of signs of severe falciparum malaria in children aged less than 10 years of age, according to the
definition of the WHO
ii. An age less than 10 years of age;
iii. A mixed or mono-infection with another plasmodium species being detected by microscopy;

10
iv. The presence of severe malnutrition (defined as a child whose growth standard is below –3 z-score, has
symmetrical edema involving at least the feet or has a mid-upper arm circumference of 110 mm);
v. A febrile condition due to any kind of disease other than malaria (e.g. measles, acute lower respiratory tract
infection, severe diarrhea with dehydration due to intestinal parasites, viral, or protozoa infection) or other
known underlying chronic or severe diseases (e.g. cardiac, renal and hepatic disease, HIV/AIDS);
vi. Any regular medication which may have interfered with antimalarial pharmacodynamics and its kinetics;
vii. A history of a hypersensitivity reaction or a contraindication to the medication being tested or used as a
rescue medication(s);
viii. All unmarried women 12-18 years of age with a positive pregnancy test or breastfeeding mothers;
ix. Being unable to or unwilling to take a pregnancy test or unwilling to take contraceptives for women of child-
bearing age.
2.7. Parasite count before and during therapy with ACT Malachlorite®
Thick and thin blood films for parasite counts were obtained and examined at screening on day 0 to confirm adherence
to the inclusion and exclusion criteria. Thick blood films were examined every once every 12 hours on day 1, 2, 3, 4 and
6, once the patient returned spontaneously and parasitological reassessment was required.
Giemsa-stained thick and thin blood films were examined at a magnification of 1000 times to identify the parasite
species and to determine parasite density. Three blood slides per patient were obtained consisting of two thick blood
smears and one thin blood smear. One of the slides was stained immediately (10% Giemsa for 10–15 min) for initial
screening, while the others was kept for possible later screening and stored in a refrigerator.
For initial screening the thick blood smear was used to count the numbers of asexual parasites and white blood cells
within a circumscribed microscopic area (number of parasites/mm3). Parasitemia was defined once at least one parasite
for every 16 white blood cells was found, corresponding to approximately 500 asexual parasites per microliter,
reflecting a low-to-moderate parasitemia.
The second blood smear was used to calculate parasite density, by counting the number of asexual parasites in a set
number of white blood cells (typically 200) with a hand tally counter. Once a field was started for calculation, it had to
be counted until completion. Parasite density, was defined as the number of asexual parasites per µl of blood. It was
calculated by dividing the number of asexual parasites by the number of counted white blood cells and then multiplied
by an assumed white blood cell density (typically 6000 per µl) using the following equation.
Parasite density (per µl) =
Number of parasites counted × 6000
Number of leukocytes counted
The same technique was used to establish parasite count on each subsequent blood film. When the number of asexual
parasites was less than 100 per 200 white blood cells in any follow-up smear, counting had to be done again using a
field containing at least 500 white blood cells (i.e. using a new field with at least 500 white blood cells).
A blood slide was considered negative when examination of 1000 white blood cells or 100 fields containing at least 10
white blood cells per field revealed no asexual parasites. Presence of gametocytes in a blood smear or on a follow-up
slide were noted- however, this information was not used for basic calculation.
In addition, 100 fields of the second thick blood film were examined to exclude mixed infections on day 0; in case of
doubt, the thin film was reexamined on a later time for confirmation purposes. If the examination of the thin film was
not conclusive, the patient was excluded from the analysis and the following complete treatment regime within the
follow-up period.
Two qualified microscopists read all the slides independently, while parasite densities were calculated by averaging the
two counts. Blood smears with discordant results (differences between the two microscopists in species diagnosis, in
parasite density of 50% or in the presence of parasites) were re-examined by a third and independent microscopist,
and parasite density were calculated by averaging the two closest counts.
2.8. Statistical Analysis
Failure of treatment to ACT Malachlorite® was considered positive if a decline in parasite count was not within the area
of 80%. Thus, the study power is 85% which is powerful enough to detect 20% difference between efficacy and non-

11
efficacy within the one-sided treatment arm. The total sample size for the one-sided study arm was 500 with the
adjustment of the recruitment period (one month and a half) and the duration of the proposed study 12 months. Due to
the sample size with P. falciparum, statistical power exponential is 0.70 to 0.90, resulting in an overall power of 0.85
during the period of 12 with a frequency period of 1.5.
In case of drop-out or withdrawal, this patient was replaced by the next one in order to reassure that at the end of the
study a minimum of 500 patients had completed the treatment phase. In addition to the reasons for withdrawal listed
above, patients were considered withdrawn from the analysis if the decline in parasite count was unclassifiable or if the
results of the analysis indicated that failure was due to a reinfection with P. falciparum.
For statically evaluation of efficacy the number of trophozoites within the treatment period were compared to baseline
using the Wilcoxon matched-pair signed rank test. Significance is defined once p 0.05. Before study initiation, official
approval for conducting the study was obtained from the National Ethical Committee at the Institute of Public Health in
Cameroon. The study key information will be posted on a public clinical trial registry (http://www.ANZCTR.org.au).
3. Results
Of the 579 patients recruited for the study 79 dropped out or were excluded from the analysis because of different
reasons. Of these dropouts 29 did not show up for the next evaluation, 30 dropped out because of violation not adhering
to the study protocol and 10 were misdiagnosed as they had fever of different origin. In addition, the two youngest
patients in the group 1-4 years of age (table 2) were not included in the study analysis because they had additional
infections which needed special medical treatment in the neonatal care unit. All other 500 adhered to the protocol and
they were used for the analysis demonstrating the effectiveness of ATC Malachlorite® in the treatment of malaria.
Although ATC Malachlorite® tablets were given to patients of different sex and age who all lived in the rural area, there
were not differences in side effects attributing them to gender difference (table 2).
Table 2 Summary of demographic data of patients taking part in the study using ATC Malachlorite® tablets and the
incidence of side-effects during the treatment of malaria infection (n=500; mean +/- SD)
Age groups in years
1-4 5-20 21-50 50
Parameters
Number of patients 2 180 240 80
Sex
Male 2 80 100 60
Female 0 100 140 20
Body weight (kg) 25 ± 5 65 ± 7 61 ± 8 55 ± 5
Height (cm) 80 ± 7 171 ± 9 175 ± 9 160 ± 7
Side effects Gastric upset with
painful cramps
Unpleasant pungent
chlorine taste
Abdominal discomfort,
cramping bloating
Chlorine taste
Severity* +++ ++ ++ +
Results are expressed as mean ± SD. *- mild +, medium ++, severe +++
All patients included within the study showed acute symptoms of varying degrees affecting the whole person, showing
a close correlation of parasite blood count done at the first visit by their local doctor. Symptoms included undulant fever,
joint pain, throbbing headache, loss of appetite, muscle aches and general weakness.
Demographic data revealed that the majority of patients were in the middle age group (21-50 years), as depicted in
Table 2.
With a mean number of 6430 (+/- 816 SD) of trophozoites per mm3 reflects a high infection rate also explaining the
severity of their symptoms. Following the intake of ATC Malachlorite®, blood count of Plasmodium falciparum
significantly declined (p 0.001) to a mean of 4932 (+/- 842 SD) trophozoites on day two and further declined to 1454
(+/- 401 SD; p 0.001) on day four, while no trophozoites could be detected in the blood on day six (Fig. 1). Every count

12
demonstrated a highly significant decline to the previous count (p 0.001) suggesting high efficacy in the use of ATC
Malachlorite® for the eradication of Plasmodium falciparum in malaria patients.
Parallel to the decline of microbes, patients quickly recovered regaining their well-being and on day six no pre-existing
symptoms were reported by patients. But apart from the quick recovery of acute malaria infection, it should be noted
that none of the patients suffered extreme side effects nor abandoned the cycle of medication due to an intolerance of
ATC Malachlorite® medication.
Figure 1 Results of treatment with ATC Malachlorite® tablets in patients with acute malaria infection, as evidenced
by the number of trophozoites/mm3 in the blood samples (sample size n = 500; mean ±SD)
4. Discussion
This so far is the first publication of data on patients with infection of Plasmodium falciparum who were treated with
the active ingredient chlorine dioxide (ClO2) plus artemisin, demonstrating a highly significant reduction in parasite
count while at the same time resulting in marked reduction of their clinical symptoms. The efficacy of anti-malarial
principle of chlorine dioxide, as demonstrated in the present pilot study, is due to killing of all asexual blood stages of
parasites that are responsible for the disease manifestations (schizontic activity of the blood). The data also suggests
that the antimalarial effect involves lysosomal trapping in the intra-erythrocytic parasite, followed by binding to the
toxic hemin which is produced during the digestion of hemoglobin [7] and where heme acts as an oxidant sensitizer of
chlorine dioxide [8, 9].
As to the mode of action, sodium chlorite (NaCl2) presents a totally new avenue in the treatment of this disease as it
inherits a number of advantages. Use of the prodrug sodium chlorite (NaCl2) compressed together with dry powdered
citric acid in a readily soluble tablet, which only when getting into contact with water, saliva or gastric acid, results in
the formation of chlorine dioxide (ClO2). After being taken up by the mucous membranes and getting into the circulation,
due its negative charge chlorine dioxide selectively attaches to the positively charged plasmodium parasite, thus
releasing a highly aggressive, unpaired oxygen radical (O2-). And in order to complete itself, the oxygen radical tears out
electrons from the outside layer of the plasmodium, inducing a disruption and a killing of the parasite. Therefore, the
mode of action of chlorine dioxide is based on the formation of the oxygen radical, disrupting the outside layer of the
plasmodium, initiating a chain reaction which ultimately results in a disintegration of the whole parasite turning its
inside to the outside.
While it is well known that chloride dioxide can quickly kill many types of bacteria [10-11] and protozoa such as the
asexual blood stages of Plasmodium falciparum that are responsible for disease manifestation (schizontic activity of
blood) in malaria [12-15] and Plasmodium vivax, Plasmodium falciparum, Plasmodium ovale and Plasmodium malariae
just like bacteria, are indeed sensitive to oxidants like chlorine dioxide resulting in a quick destruction of Plasmodium
falciparum and P. vivax, including strains resistant to multiple drugs. Since free radicals from ACT Malachlorite® are
responsible for killing malaria parasites, the selectivity of their toxicity is likely related to the fact they reach high
concentrations in the human body in infected red blood cells while at the same time attacking selectively the plasmodia.
This is because they are especially sensitive to oxidants (electron grabbers) and upon sufficient removal of the parasite's

13
life sustaining thiols by oxidation, the parasite rapidly dies [16-20]. Such a connotation is underlined by the current
data where a rapid decline of parasites in the blood is observed. And once the concentration remains high for at least
three to four days, there is little chance for any other parasite to escape this deadly force of ClO2. This assumption is also
underlined by data from other areas in human life, where chlorine dioxide is used for the decontamination of drinking
water getting rid of residual virus, bacteria or protozoa [18, 21-24] a reason, why it is important that high blood
concentrations appear to be more effective than all other known antimalarial drugs in use since this day.
While it was estimated that treatment with conventional medications reduces the parasite biomass by a factor of about
10 on each asexual cycle of the parasite from 36 to 48 hours and by a factor of 106-108 on a 3-day course treatment, the
current findings indicate a much higher efficacy by a factor of almost 100% over a period of three hour, it is believed
that chlorine dioxide, like quinine, very much unlikely results in a tolerance development, which so far always has been
the major obstacle in fighting malaria in the long run.
There is less information about the effect against the other two human malaria parasites, Plasmodium malariae and
Plasmodium ovale, but they are likely to be attacked in a similar way [25-32]. Moreover, it is known that chlorine dioxide
has a certain effect on gametocytes, a stage which is infectious for mosquitoes ingesting a blood meal from an infected
person [7]. What is also known from animal studies, that ClO2 has effect on hypnozoites which are in the liver and can
cause relapses in Plasmodium vivax and Plasmodium ovale malaria [33]; however, conclusive evidence of such effects in
human is still lacking.
Biochemical studies suggest that the antimalarial effect of oxygen radicals from hydrogen peroxide as well as from
chlorine dioxide involves lysosomal entrapment of the drug in the intra-erythrocytic parasite followed by the link to the
toxic hemin which is produced during the digestion of hemoglobin [34-35]. This binding prevents polymerization of
nontoxic hemin pigment of malaria [36]. The mechanism of action differs from that of any other anti-malaria drug and
is closely related to the release of oxygen free radicals. Such a release appears at a peroxide bridge which bursts within
the molecule and occurs when the compound meets high levels of heme, a hemoglobin degradation product produced
by malaria parasites living in red blood cells [37]. In living things including parasites, iron is a necessary cofactor for
many enzymes. Thus it is reasonable to estimate that any damage to plasmodia caused by oxides of chlorine is
compounded by conversion of ferrous (Fe++) cofactors to ferric (Fe+++) [38-39]. All these events occur within three to
four days of treatment as demonstrated in this present set of patients.
As for the pharmacokinetics, the rapid absorption and the large volumes of distribution, together with a short half-live
are two main characteristically traits of chlorine dioxide [40]. Since it also penetrates the blood-brain-barrier and
accumulates in erythrocytes it explains why there is an immediate effect also related to malaria induced CNS symptoms
[41].
Despite the potential neurotoxicity of chlorine dioxide, which has been demonstrated in animal studies at doses that far
exceed those used in the present treatment of malaria in patients, no such side-effects were observed in the present set
of patients. Also, animal studies using much higher oral or topical doses have proven relatively safety [42-43], so it is
not unexpected that we did not encounter any serious side effects. This is also underlined by others who had
demonstrated no serious side effects in humans when chlorine dioxide is being used for water decontamination [44].
The potential side effects of treatment with chlorine dioxide used for water disinfection, as reported in the literature at
even high therapeutic doses are rare, and mostly involve the formation of methemoglobin [42, 45], with a low incidence
of dizziness, fatigue and palpitations, while myalgia, sleep disorders are practically absent, and joint pain, headaches
and rashes can be related to a dysbalance of gut bacteria [46]. The latter can be expected especially when part of the
sodium chlorite is being swallowed and where the newly formed oxygen radical results in the elimination of beneficial
gut bacteria which effects the response of the immune system being located by up to 80% within the gut [47]. In this
context, it is important to note that many the above mentioned side effects as reported in the literature, have not been
observed in our series of patients and when the medication was taken according to instructions. Especially, and in
contrast to the present available combination therapy for the treatment of malaria, ACT Malachlorite® is less expensive
in manufacturing, it has demonstrated practically no serious side effects and it has a high acceptance in patients and
showed a significant therapeutic efficacy after 3 to 4 days of treatment. Therefore, it can be recommended as a first-line
routine treatment in countries where resistance to chloroquine or agents of the newer generation containing
artemisinin present a problem, especially in countries with endemic malaria such as Cameroon, Gambia or the south of
Senegal where on site visits of health care personal in the rural area are rare and patients often, even once diagnosed
with malaria, are left unattended. Since there is already a resistance against sulfadoxinel pyrimethamine, the useful life
of a combination therapy is limited life quality of a combination therapy is very much limited.

14
5. Conclusion
Higher priority should be given to the search for alternative methods in malaria treatment today. This demand, which
had realized in the present study especially as scientists have recently been warning of a “supermalaria” parasite that
is quickly spreading throughout Southeast Asia and will pose a worldwide health threat if it makes its way to Africa.
This parasite is resistant to the most common first-line malaria treatment, artemisinin [48]. Also, the high level of
genetic diversity between the different species of mosquitoes and their ability to swap their genes is making it very
difficult to prevent insecticide-resistant groups from forming. While new insecticides are being developed – such as
those that kill mosquitoes with genetic approaches, stop them from breeding or prevent them from transmitting the
parasite responsible for malaria – experts are worried that the mosquitoes will simply develop resistance to these
methods as well. According to the World Health Organization, the gains seen in malaria prevention in recent years are
under threat from growing insecticide resistance that could ultimately lead to a significant rise in malaria cases and
deaths. They’ve called on the global malaria community to act urgently to keep insecticide resistance under control. 212
million people a year are infected with malaria, and children are at particular risk of dying from it [49]. Since 2010, 61
countries have reported mosquitoes developing resistance to one or more insecticide class, and 50 of those countries
report on mosquitoes developing resistance to two or more classes. The problem could actually be worse than the
statistics indicate as many countries are not reporting their data quickly or are failing to monitor for insecticide
resistance on a routine basis. In addition, drug resistance making the problem even worse. This parasite is resistancy is
evident to the most common first-line malaria treatment, artemisinin [50]. With 438,000 malaria deaths around the
world in 2015, it’s frightening to think of what kind of mortality figures a super malaria could cause.
In summary, this new sublingual ATC Malachlorite® tablet formulation presents a promising new approach in malaria
treatment as it is based on a completely new mode of action. It proved to be safe and effective and should be introduced
to replace the combination therapy such as artesunate plus sulfadoxinel pyrimethamine. The replacement would very
likely allow a longer useful life, because so far there is no reported resistance to the components in ATC Malachlorite®.
And finally, the wide distribution as a package, such as a blister (blister pack) where only one component is kept,
improves patient compliance, while newer fixed combination formulations would take more years to develop.
Compliance with ethical standards
Acknowledgments
The work and assistance of all doctors and nurses in the rural areas of Cameroon who had participated in the study and
who by means of their meticulous data acquisition is gratefully acknowledged, making the study a valuable contribution
in the treatment of malaria.
Disclosure of conflict of interest
The authors of the paper declare that there is no conflict of interest, that they have seen and approved the manuscript
being submitted and that the paper has not been published in total or in part elsewhere. Also, the authors listed on the
title page attest that they have significantly contributed to the work have read the manuscript, attest to the validity and
the legitimacy of the data and its interpretation and agree for the submission to the journal. All authors agree that the
authors list is correct in its content and order and no modification of the authors list will be made.
Statement of ethical approval
Before study initiation, official approval for conducting the study was obtained from the National Ethical Committee at
the Institute of Public Health in Cameroon.
Statement of informed consent
Patients were only included in the study if adults and children (parents or guardians of children) had given their
informed consent. The principal investigator(s) also obtained and documented the assent of children between the age
of 12 and 18 years, but their assent had to be accompanied by the consent of a parent or a guardian.

15
References
[1] Tatem AJ, Jia P, Ordanovich D, Falkner M, Huang Z, Howes R, Hay SI, Gething PW and Smith DL. (2017). The
geography of imported malaria to non-endemic countries: a meta-analysis of nationally reported statistics. The
Lancet Infectious Diseases, 17(1), 98-107.
[2] Basco LK, Ndounga M, Keundjian A and Ringwald P. (2002). Molecular epidemiology of malaria in cameroon. IX.
Characteristics of recrudescent and persistent Plasmodium falciparum infections after chloroquine or
amodiaquine treatment in children. The American journal of tropical medicine and hygiene, 66(2), 117-23.
[3] Maitland K, Bejon P and Newton, CRJC. (2003). Malaria. Current Opinion in Infectious Diseases, 16, 389-395.
[4] James S and Miller L. (2003). Malaria vaccine development status report. In Doherty AM (Ed), Annual Reports in
Medicinal Chemistry, Academic Press, New York, 203-211.
[5] World Health Organization. (2012). WHO position statement on effectiveness of non-pharmaceutical forms of
Artemisia annua L. against malaria. WHO Press, Geneva, 4.
[6] Ringwald P, Shallcross L, Miller JM, Seiber E and World Health Organization. (2005). Susceptibility of Plasmodium
falciparum to antimalarial drugs: report on global monitoring 1996-2004, WHO Press, Geneva.
[7] Jefford CW. (2001). Why artemisinin and certain synthetic peroxides are potent antimalarial- implications for
the mode of action. Current medicinal chemistry, 8(15), 1803-1826.
[8] Francis SE, Sullivan Jr DJ and Goldberg ADE. (1997). Hemoglobin metabolism in the malaria parasite Plasmodium
falciparum. Annual Reviews in Microbiology, 51(1), 97-123.
[9] Saliba KJ, Allen RJ, Zissis S, Bray PG, Ward SA and Kirk K. (2003). Acidification of the malaria parasite's digestive
vacuole by a H+-ATPase and a H+-pyrophosphatase. Journal of Biological Chemistry, 278(8), 5605-5612.
[10] Young SB and Setlow P. (2003). Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine
dioxide. Journal of Applied Microbiology, 95(1), 54-67.
[11] Harakeh S, Illescas A and Matin A. (1988). Inactivation of bacteria by purogene. Journal of Applied
Microbiology, 64(5), 459-463.
[12] Winiecka-Krusnell, J and Linder E. (1998). Cysticidal effect of chlorine dioxide on Giardia intestinalis cysts. Acta
tropica, 70(3), 369-372.
[13] Korich DG, Mead JR, Madore MS, Sinclair NA and Sterling CR. (1990). Effects of ozone, chlorine dioxide, chlorine,
and monochloramine on Cryptosporidium parvum oocyst viability. Applied and environmental
microbiology, 56(5), 1423-1428.
[14] LeChevallier MW, Au KK. (2004). Water treatment and pathogen control. Iwa Publishing, Geneva, 52-54.
[15] Driedger AM, Rennecker JL and Mariñas BJ. (2000). Sequential inactivation of Cryptosporidium parvum oocysts
with ozone and free chlorine. Water Research, 34(14), 3591-3597.
[16] Müller S, Gilberger TW, Krnajski Z, Lüersen K, Meierjohann S and Walter RD. (2001). Thioredoxin and glutathione
system of malaria parasite Plasmodium falciparum. Protoplasma, 217(1-3), 43-49.
[17] Rahlfs S, Nickel C, Deponte M, Schirmer RH and Becker K. (2003). Plasmodium falciparum thioredoxins and
glutaredoxins as central players in redox metabolism. Redox report, 8(5), 246-250.
[18] Jaeger T and Flohe L. (2006). The thiol-based redox networks of pathogens: unexploited targets in the search for
new drugs. Biofactors, 27(1-4), 109-120.
[19] Olagunju O, Siegel PD, Olojo R and Simoyi RH. (2006). Oxyhalogen− sulfur chemistry: kinetics and mechanism of
oxidation of N-Acetylthiourea by chlorite and chlorine dioxide. The Journal of Physical Chemistry A, 110(7),
2396-2410.
[20] Müller S. (2004). Redox and antioxidant systems of the malaria parasite Plasmodium falciparum. Molecular
microbiology, 53(5), 1291-1305.
[21] Dowling LT. (1974). Chlorine dioxide in potable water treatment. Water Treatment and Examination, 23(2), 190-
204.
[22] Granstrom ML and Lee GF. (1958). Generation and use of chlorine dioxide in water treatment. Journal (American
Water Works Association), 50(11), 1453-1466.

16
[23] Augenstein H. (1968). Use of chlorine dioxide to disinfect water supplies. Journal – American Water Works
Association, 60(939).
[24] LeChevallier MW and Au . (2004). Water treatment and pathogen control in process efficiency in achieving safe
drinking water, world health organization, editor, IWA Publishing, Geneva. p. 52-54.
[25] Biot C, Dessolin J, Grellier P and Davioud-Charvet E. (2003). Double-drug development against antioxidant
enzymes from Plasmodium falciparum. Redox report, 8(5), 280-283.
[26] Turrens JF. (2004). Oxidative stress and antioxidant defenses: a target for the treatment of diseases caused by
parasitic protozoa. Molecular aspects of medicine, 25(1-2), 211-220.
[27] Oliveira PL and Oliveira MF. (2002). Vampires, Pasteur and reactive oxygen species. FEBS letters, 525(1-3), 3-6.
[28] Allison AC and Eugui EM. (1983). The role of cell-mediated immune responses in resistance to malaria, with
special reference to oxidant stress. Annual review of immunology, 1(1), 361-388.
[29] Vennerstrom JL. (1989). Amine peroxides as potential antimalarials. Journal of medicinal chemistry, 32(1), 64-
67.
[30] Senok AC, Nelson EA, Li K, Oppenheimer SJ. (1997). Thalassaemia trait, red blood cell age and oxidant stress:
effects on Plasmodium falciparum growth and sensitivity to artemisinin. Transactions of the Royal Society of
Tropical Medicine and Hygiene, 91(5), 585-589.
[31] Dockrell HM and Playfair JH. (1984). Killing of Plasmodium yoelii by enzyme-induced products of the oxidative
burst. Infection and immunity, 43(2), 451-456.
[32] Clark IA, Butcher GA, Buffinton GD, Hunt NH, Cowden WB. (1987). Toxicity of certain products of lipid
peroxidation to the human malaria parasite Plasmodium falciparum. Biochemical pharmacology, 36(4), 543-546.
[33] Siddiqi NJ and Pandey VC. (1999). Studies on hepatic oxidative stress and antioxidant defense systems during
arteether treatment of Plasmodium yoelii nigeriensis infected mice. In Stress Adaptation, Prophylaxis and
Treatment, Springer, Boston, 169-173.
[34] Mishra NC, Kabilan L and Sharma A. (1994). Oxidative stress and malaria-infected erythrocytes. Indian journal
of malariology, 31(2), 77-87.
[35] Dockrell HM and Playfair JH. (1983). Killing of blood-stage murine malaria parasites by hydrogen peroxide.
Infection and immunity, 39(1), 456-459.
[36] Clark IA and Hunt NH. (1983). Evidence for reactive oxygen intermediates causing hemolysis and parasite death
in malaria. Infection and Immunity, 39(1), 1-6.
[37] Rosenthal PJ and Meshnick SR. (1996). Hemoglobin catabolism and iron utilization by malaria
parasites. Molecular and biochemical parasitology, 83(2), 131-139.
[38] Cabantchik I, Glickstein H, Golenser J, Loyevsky M and Tsafack A. (1996). Iron chelators: mode of action as
antimalarials. Acta haematologica, 95(1), 70-77.
[39] Padmanaban G and Rangarajan PN. (2000). Heme metabolism of plasmodium is a major antimalarial
target. Biochemical and biophysical research communications, 268(3), 665-668.
[40] Abdel-Rahman MS, Couri D and Bull RJ. (1979). Kinetics of Cl02 and effects of Cl02, Cl02-, and Cl03-in drinking
water on blood glutathione and hemolysis in rat and chicken. Journal of environmental pathology and toxicology,
3(1-2), 431-449.
[41] Toth GP, Long RE, Mills TS and Smith MK. (1990). Effects of chlorine dioxide on the developing rat brain. Journal
of Toxicology and Environmental Health, Part A Current Issues, 31(1), 29-44.
[42] Moore G and Calabrese EJ. (1980). The effects of chlorine dioxide and sodium chlorite on erythrocytes of A/J and
C57L/J mice. Journal of environmental pathology and toxicology, 4(2-3), 513-524.
[43] Bercz JP, Jones L, Garner L, Murray D, Ludwig DA and Boston J. (1982). Subchronic toxicity of chlorine dioxide
and related compounds in drinking water in the nonhuman primate. Environmental Health Perspectives, 46, 47.
[44] Heffernan WP, Guion C and Bull RJ. (1979). Oxidative damage to the erythrocyte induced by sodium chlorite, in
vivo. Journal of environmental pathology and toxicology, 2(6), 1487-1499.
[45] Moore GS, Calabrese EJ, DiNardi SR and Tuthill RW. (1978). Potential health effects of chlorine dioxide as a
disinfectant in potable water supplies. Medical hypotheses, 4(5), 481-496.

17
[46] Martinez KB, Leone V and Chang EB. (2017). Western diets, gut dysbiosis, and metabolic diseases: Are they
linked?. Gut microbes, 8(2), 130-142.
[47] Vighi G, Marcucci F, Sensi L, Di Cara G and Frati F. (2008). Allergy and the gastrointestinal system. Clinical and
Experimental Immunology, 153(s1), 3-6.
[48] Fouet C, Atkinson P and Kamdem C. (2018). Human interventions: driving forces of mosquito evolution. Trends
in parasitology.
[49] World Health Organization. (2017). Framework for a national plan for monitoring and management of insecticide
resistance in malaria vectors. Malaria Vector Control Technical Expert Group of WHO, Editor, World Health
Organization, Geneva, 39.
[50] Imwong M, Hien TT, Thuy-Nhien NT, Dondorp AM and White NJ. (2017). Spread of a single multidrug resistant
malaria parasite lineage (PfPailin) to Vietnam. The Lancet Infectious Diseases, 17(10), 1022-1023.
How to cite this article
Enno F, Hans-Peter S and Olivia MW. (2018). ACT Malachlorite® for treatment of patients with acute Plasmodium
falciparum infection: A pilot study incorporating 500 patients in the rural area of Cameroon. GSC Biological and
Pharmaceutical Sciences, 2(2), 06-17.

3) Controlled Clinical Evaluations of Chlorine Dioxide,
Chlorite and Chlorate in Man

Environmental Health Perspectives
Vol. 46, pp. 57-62, 1982
Controlled Clinical Evaluations
of Chlorine Dioxide, Chlorite
and Chlorate in Man
by Judith R. Lubbers,* Sudha Chauan,* and Joseph
R. Bianchine*
To assess the relative safety of chronically administered chlorine water disinfectants in man, a
controlled study was undertaken. The clinical evaluation was conducted in the three phases
common to investigational drug studies. Phase I, a rising does tolerance investigation, examined
the acute effects of progressively increasing single doses of chlorine disinfectants to normal
healthy adult male volunteers. Phase II considered the impact on normal subjects of daily
ingestion of the disinfectants at a concentration of 5 mg/l. for twelve consecutive weeks. Persons
with a low level of glucose-6-phosphate dehydrogenase may be expected to be especially
susceptible to oxidative stress; therefore, in Phase III, chlorite at a concentration of 5 mg/l. was
administered daily for twelve consecutive weeks to a small group of potentially at-risk
glucose-6-phosphate dehydrogenase-deficient subjects. Physiological impact was assessed by
evaluation of a battery of qualitative and quantitative tests. The three phases of this controlled
double-blind clinical evaluation of chlorine dioxide and its potential metabolites in human male
volunteer subjects were completed uneventfully. There were no obvious undesirable clinical
sequellae noted by any of the participating subjects or by the observing medical team. In several
cases, statistically significant trends in certain biochemical or physiological parameters were
associated with treatment; however, none of these trends was judged to have physiological
consequence. One cannot rule out the possibility that, over a longer treatment period, these
trends might indeed achieve proportions of clinical importance. However, by the absence of
detrimental physiological responses within the limits of the study, the relative safety of oral
ingestion of chlorine dioxide and its metabolites, chlorite and chlorate, was demonstrated.
Introduction
Chlorine dioxide is currently under serious con-
sideration in the United States as an alternative to
chlorine water treatment. Before chlorine dioxide
may be used routinely as a water disinfectant, the
safety of oral human ingestion of chlorine dioxide
and its by-products must be assessed. For this
purpose, a controlled clinical evaluation of chlorine
dioxide, chlorite and chlorate was undertaken under
the auspices of USEPA HERL #CR805643.
The study was conducted in three parts. Phase I
was designed to evaluate the acute physiological
* The Department ofPharmacology, The Ohio State Universi-
ty, College of Medicine, 333 W. 10th Avenue, Columbus,
OH 43210
effects ofprogressively increasing doses ofdisinfec-
tants administered to normal healthy adult males.
Chronic ingestion by normal male volunteers was
studied in Phase II. Phase III assessed the physio-
logical response of a small group of potentially
susceptible individuals, those deficient in glucose-6-
phosphate dehydrogenase, to chronic ingestion of
chlorite.
Methods
Subject Selection
For Phase I and for Phase II, normal healthy
adult male volunteers were selected. No prospec-
tive study participant who exhibited a significant
abnormality in the routine clinical serum analysis,

58
blood count, urinalysis, or electrocardiogram was
selected. Subjects manifested no physical abnor-
malities at the pretreatment examination, were 21
to 35 years of age, and weighed within ± 10% of
normal body weight for their frame and stature. A
history of disease or any medical or surgical condi-
tion which might interfere with the absorption,
excretion, or metabolism of substances by the body
precluded inclusion. Regular drug intake prior to
the start of the investigation, either therapeutic or
recreational, resulted in exclusion from the study.
Normal methemoglobin levels, thyroid function, and
glutathione levels were mandatory. Written informed
consent was obtained from each subject prior to
initiation of treatment.
For Phase III, volunteers were defined as glucose-
6-phosphate dehydrogenase (G-6-PD)-deficient on
the basis of a hemoglobin G-6-PD level of less than
5.0 IU/GM hemoglobin in the pre-study screening.
Phase III subjects were normal in all otherrespects.
Water Disinfectant Preparation
A detailed description of the water disinfectant
preparation techniques has been presented by Lub-
bers and Bianchine (1). In general, freshly pre-
pared stock solutions of chlorine dioxide, sodium
chlorite, sodium chlorate, chlorine and chloramine
were assayed by the colorimetric techniques of
Palin (2) then diluted with organic-free demineral-
ized deionized water to appropriate concentrations.
Individual bottles were capped and stored in the
dark under refrigeration until use. All bottles were
coded by an independent observer and the identity
of each bottle remained double-blind to both the
investigative staff and the volunteer subjects.
Study Design: Phase I
The 60 volunteers in Phase I were divided at
random into six treatment groups (1). Ten persons
were assigned to receive each of the disinfectants;
the ten members of the control group received
untreated water. The study involved a series of six
sequences of three days each. Treatment concen-
trations were increased for each treatment. The
specific concentrations or disinfectant administered
to the study participants are listed in Table 1. A
clinical evaluation of the collection of blood and
urine samples for determination of pretreatment
baseline laboratory values preceded the first treat-
ment. On the first day of each three day treatment
sequence, each volunteer ingested 1000 ml of the
water in two portions. The second 500 ml portion
aliquot was administered 4 hr after the first. Each
500 ml portion was consumed within 15 min. Only
two doses of disinfectant were administered on the
LUBBERS, CHAUAN AND BIANCHINE
first day of each treatment sequence. No disinfec-
tant was administered on the second and third day
of each sequence, since these two days were to
serve as followup observation days. The second day
of the treatment sequence consisted of a physical
examination and collection of blood and urine sam-
ples for determination of posttreatment laboratory
values. On the third day, each volunteer was given
a physical examination to determine residual effects
of treatment with the water disinfectants and
byproducts.
Taste evaluations were obtained at each dose
level. Study participants were asked to rate the
treated water as very unpleasant, slightly unpleas-
ant, not pleasant, pleasant, or tasteless.
Study Design: Phase II
The sixty volunteers of Phase II were divided at
random into six treatment groups of ten subjects
each (3). In order to assure efficient management of
the 60 subjects, they were randomly assigned to
three subsets. These subsets were sequentially
entered into the study on three successive days and
exited from this study in a similar fashion. For all of
the treatment groups, the concentration of disin-
fectants ingested was 5 mg/l. The control group
received untreated water. Each subject received
500 ml daily for 12 weeks. Physicals, collection of
blood and urine samples for laboratory assays, and
taste evaluations were conducted on a weekly basis
during the treatment period and for 8 weeks follow-
ing cessation of treatment.
Study Design: Phase III
The three glucose-6-phosphate dehydrogenase-
deficient subjects of Phase III were given sodium
chlorite at a concentration of 5 mg/I. chlorite (4).
The treatment protocol was identical to that of
Phase II, with daily administration of 500 ml of
solution to each volunteer.
Evaluation Procedures
An extensive battery of parameters was moni-
tored to assess the biochemical and physiological
response to the oral ingestion of the water disinfec-
tants and water treatment by-products (Table 2).
All laboratory determninations of biochemical param-
eters were conducted by a licensed medical labora-
tory, Consolidated Biomedical Laboratories, Inc.
(CBL), Columbus, Ohio, HEW license number
34-1030. For each volunteer, pretreatment baseline
values and six sets of posttreatment values were
compiled. Laboratory tests were carefully chosen.

ORAL INTAKE OF CHLORINE DISINFECTANTS IN MAN
Table 1. Concentration of disinfectants in phase I: acute rising dose tolerance.a
Disinfectant concentration, mg/l.
Water disinfectant Day 1 Day 4 Day 7 Day 10 Day 13 Day 16
Chlorate 0.01 0.1 0.5 1.0 1.8 2.4
Water control 0 0 0 0 0 0
Chlorine dioxide 0.1 1.0 5.0 10.0 18.0 24.0
Chlorite 0.01 0.1 0.5 1.0 1.8 2.4
Chlorine 0.1 1.0 5.0 10.0 18.0 24.0
Chloramine 0.01 1.0 8.0 18.0 24.0
aFor each dose, two portions of 500 ml each were administered at 4-hr intervals.
Table 2. Biochemical parameters assayed in the controlled clinical evaluation ofchlorine dioxide, chlorite and chlorate in man.
Serum chemistry
Blood count
Urinalysis
Special tests
Plasma glucose, sodium, potassium, chloride, urea nitrogen, creatinine, BUN/creatinine ratio, uric
acid, calcium, phosphorus, alkaline phosphatase, gamma glutamyl transferase, total bilirubin, serum
glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase, lactic dehydrogenase,
cholesterol, triglycerides, total protein albumin, globulin, albumin/globulin ratio, iron
Platelet count, white blood cell count, red blood cell count, hemoglobin, hematocrit, mean
corpuscular volume, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular
hemoglobin concentration, high peroxidase activity, neutrophils, lymphocytes, monocytes, eosino-
phils, basophils, large unstained cells
Color,a appearance,a specific gravity, pH, protein, sugar,a acetone, blood,a white blood count, red
blood count, casts,a crystals,a bacteria,a mucus*, amorphous cells,a epithelial cells
Serum haptoglobin, sickle cell,a methemoglobin, glucose-6-phosphate dehydrogenase, Coombs
test,a hemoglobin electrophoresis,a T-3 (uptake), T-4 (RIA), free thyroxine index, electrocardiograma
Physical exam Systolic blood pressure, diastolic blood pressure, respiration rate, pulse rate, oral temperature
aThese parameters yielded qualitative data only; no statistical analysis was performed.
On the basis of the literature (5-8), areas of sus-
pected biochemical response to ingestion ofchlorine
oxidants were defined; a portion of the test battery
was specifically devoted to monitoring this response.
Red blood cell surface antibody formation was clini-
cally monitored by the qualitative Coombs test;
thyroid function by T-3 (uptake), T-4 (RIA), and
free thyroxine; and response to oxidative stress by
glucose-6-phosphate dehydrogenase, methemoglobin
and glutathione levels. Hemoglobin electrophoresis
was used to detect possible hemoglobin abnormali-
ties. A battery ofperipheral parameters was assayed
to provide supplementary information and to assist
in evaluation of overall physiological well-being.
The specific serum, blood and urine parameters
assayed have been discussed by Lubbers and
Bianchine (1).
The numerical values obtained were collected
and analyzed by utilizing the facilities of The Ohio
State University Division of Computing Services
for Medical Education and Research. Specially
designed programs facilitated rapid clinical feed-
back. Any value for an individual subject which
differed from the group mean by more than two
standard deviations was noted. In addition, every
individual value which fell outside normal labora-
tory ranges for that parameter was designated as
abnormal. Chemical parameters for volunteers who
exhibited abnormal values were subjected to care-
ful scrutiny; the safety and the possibility of hyper-
sensitivity to the disinfectant agents were evalu-
ated for each of these individuals on a continuing
basis throughout the study.
Statistical analyses utilized commercially avail-
able computer packages, specifically, the Biomedi-
cal Computer Programs (BMDP) and the Statistical
Package for the Social Sciences (SPSS). Two-way
analyses of variance with repeated measures uti-
lized BMDP2V. BMDP1R was used to perform
multiple linear regression analyses. For pairwise
t-tests and simple t-tests, SPSS-T-test was employed.
Results
Qualitative
An important aspect of this study was the careful
and continued medical observation of all subjects.
The general clinical histories and physical examina-
tions alone with subjective observations and quali-
tative laboratory tests throughout this study were
accumulated in each subject's medical file. A careful
59

Dossier sodium chlorite - Dióxido de Cloro scabelum consumidores

Dossier sodium chlorite - Dióxido de Cloro scabelum consumidores

Recommandé

Recommandé

Contenu connexe

Tendances

Tendances (17)

Similaire à Dossier sodium chlorite - Dióxido de Cloro scabelum consumidores

Similaire à Dossier sodium chlorite - Dióxido de Cloro scabelum consumidores (20)

Plus de Neuromon 21

Plus de Neuromon 21 (20)

Dernier

Dernier (20)

Dossier sodium chlorite - Dióxido de Cloro scabelum consumidores