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Background Information
The Manao (or Lime) Trial was first discussed at the Department of Health
in Bangkok on June 20th., 2003, when Dr Vallop Thaineua, the Permanent
Secretary and 6 members of his staff met with Dr Mechai Viravaidya and
Professor Roger Short (from the Department of Obstetrics and Gynaecology,
University of Melbourne), who put forward the initial proposal that the
Department of Health should run a clinical trial to evaluate the acceptability,
safety and efficacy of intravaginal lime juice first as a contraceptive,
and if successful, then as a microbicide in Thai women. Professor Short's
group in Melbourne would provide the scientific back-up for the trials.
Dr Vallop welcomed the proposal in principle, and this was confirmed in
a letter from Dr Vallop to Professor Short on September 15, 2003.
Professor Short approached the Australian Agency for International Development
(AusAID) for financial support for the Australian component of the study,
and this was rejected in a letter dated November 7th., 2003, from Mr Geoff
Adlide, Director of the Cambodia, Thailand and Burma Section of AusAID,
although no reasons were given. This funding has had to be obtained elsewhere.
The History of the Lime and Lemon
The wild Citrus plant is indigenous to India, China, Thailand and Malaysia.
It was first introduced into Europe by Alexander the Great, and was cultivated
by the Romans to produce the lemon, and the Arabs to produce the lime.
In 1753, lime juice was shown by the Scottish doctor, James Lind, to prevent
scurvy on long sea voyages because of its Vitamin C content; Linnaeus
in 1753 named the fruit Citrus medica.
Early in the 18th century it also came to be used as a contraceptive in
the Mediterranean region, either using half a lime or lemon, partially
squeezed out, as a cervical cap, as advocated by Casanova, or else soaking
a piece of sponge on the juice and placing it in the vagina before sex.
Subsequently it was shown to be the citric acid in the juice that immobilises
the sperm; lemon juice is 5% citric acid, and citric acid is a major component
of a man's ejaculate. This historical evidence is well reviewed in the
"Medical History of contraception" by N.E. Hines (1963), who
concludes that intra-vaginal lemon juice " is not surpassed in reliability
by any modern clinical contraceptive".
In 1995, Professor Short gave a lecture in London on the history of contraception
in Britain, and several elderly women in the audience informed him afterwards
that they had relied on lemons to control their fertility - there was
nothing else available. Recounting this to Mechai Viravaidya in Bangkok
in December 2001, it suddenly occurred to them that since the HIV virus
was known to be extremely susceptible to low pH, perhaps this historical
contraceptive might be the microbicide of tomorrow. In view of the S.E.Asian
origin of the citrus, it seemed sensible to start a Manao (lemon) Trial
in Thailand, provided that laboratory studies could confirm the contraceptive
and virucidal effects of the juice, and that animal studies could confirm
a lack of cervical and vaginal toxity after repeated application. That
moment has now arrived.
Laboratory studies of the contraceptive effect of lime and lemon juice
The Bangkok meeting of May 4, 2004, was shown ( hyperlink to the video
page) video which gave a dramatic demonstration of the almost instantaneous
immobilization of human sperm on contact with low concentrations of lemon
juice.The effect is thought to be due to inactivation of the dynein-ATPase
enzyme system in the midpiece sheath of the sperm, which is responsible
for the beating of the sperm tail.
Lemon or lime juice seem to be equally effective in producing this reaction;
they both have a pH of around 2.3. In response to questions from the audience,
it was agreed that there was a need for more information on the pH of
the juice at different seasons of the year and different stages of ripening
in Thailand.
If lime or lemon juice is mixed with a fresh human ejaculate and incubated
at 37 degrees Centigrade, a 5% concentration of juice in whole semen reduces
motility by half within 5 minutes, a 10% concentration causes a 30 fold
loss of motility within 5 minutes and complete motility by 4 hours, although
it can recover if the pH is restored to neutral, and a 20% concentration
caused complete, irreversible loss of motility in under 30 seconds.
The pH of a normal human ejaculate is about 8.4. 5% lemon juice reduced
it to 6.3, 10% to 5.1 and 20% to 4.1
Monkey trials of the toxicity of repeated daily intravaginal administration
of lime juice to the cervix and vagina
A study was carried out at the Primate Centre, Bogor Agricultural Institute
in Indonesia, from December 2002 - January, 2003.
Six treated Cynomologous monkeys ( Macaca fascicularis) were individually
housed, and each was given a cotton wool ball soaked in about 0.8ml fresh,
undiluted lime juice daily from the first day of menstruation for 28 days.
At the end of that time, the animals were killed and the complete reproductive
tracts removed and fixed in 10% formal saline.
Six control monkeys, individually housed, had a vaginal biopsy taken on
the first day of menstruation. This was then fixed in 10% formal saline.
The complete reproductive tracts of the six treated animals were bisected
down the mid-line; 2 of the animals still had cotton wool balls in place.
The tracts were embedded, sectioned and stained with haematoxylin and
eosin.
The vaginal epithelium of the treated animals did not appear to differ
in any way from the controls. The cervical epithelium of the treated animals
also appeared normal.Most of the treated animals showed isolated foci
of sub-mucosal infection, with aggregations of leucocytes. Since the overlying
epithelium was entirely normal, it was concluded that these lesions were
long-standing, and not related to the treatment. Such lesions are common
in wild-caught macaques, and are possibly due to chronic simian papilloma
virus infection. It was not possible to tell if these lesions were also
present in the control animals, as the biopsies did not extend deep into
the sub-mucosal tissues.
The sections were examined independently by two primate pathologists at
the University of California National Primate Research Centre in Davis,
California. They agreed that there was no cervical or vaginal lesions
in any of the treated animals that could be attributed to the lime juice
administration.
Since none of the treated animals were mated during the trial, the acidity
of the lime juice could not be neutralized by semen, as would normally
be the case. Thus the trial was exposing the animals to an extreme situation,
in which the cervix and vagina were repeatedly exposed to a relatively
large dose of highly acidic lime juice (pH 2.1-2.5).
These reassuring negative findings give us every confidence in proposing
that it is safe to carry out a clinical trial of intravaginal lime juice
in human volunteers. Representative photomicrographs were circulated to
all members of the May 4, 2004, Bangkok meeting.
Methods of applying lime juice to the vagina - Pain threshold
The average volume of a normal human ejaculate is 2-6ml. In order to guarantee
at least a 20% final concentration of lime juice in the ejaculate, we
recommend that 3ml lime juice needs to be introduced into the vagina -
a teaspoonful.
The following are three possible alternative modes of applying the juice:
- A piece of sea sponge. Can be rinsed out and re-used. This was how
it was applied traditionally. Need to avoid squeezing out the juice
during insertion. Should be easy to remove with the fingers.
- A small ball of cotton wool. Disposable. Very cheap. May not be
quite as easy to remove. Could tie a piece of thread round it to aid
removal. Need to avoid squeezing out the juice during insertion.
- A small ball of cotton wool encased in a piece of coarse muslin
tied in a knot. The knot provides a convenient handle for insertion
and removal. Disposable. Less problem with squeezing out juice during
insertion.
The three alternatives were demonstrated to the meeting. Important
to determine which method would be the most culturally acceptable
in Thailand. We would not recommend the traditional method of half
a lime partially squeezed out as a cervical cap because of its bulk.
The acidity of the juice is more important than the mechanical barrier
of the cap, at least as far as HIV prevention is concerned, since
HIV enters the vaginal epithelium rather than the cervix.
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Pain thresholds
A simple trial was conducted in Mexico City in August, 2003,
asking a group of University staff and students to apply neat
lemon juice to the penis or vagina and record the pain score on
a scale of 0 (no pain) to 10 (extreme pain), and to compare it
to the pain score of neat juice on the tip of the tongue.
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47 men aged between 20 - 73 and 27 women aged between 19 - 40
took part in the trial. In general, the juice caused little or
no discomfort. and the median score was zero for both sexes. The
circumcision status of the men had no effect on the pain score.
Two women did experience some discomfort; one was on the first
day of menstruation, and one was on anti-oestrogen treatment that
might have caused thinning of the vginal epithelium. The median
pain score on the tongue was zero, and there was no significant
difference between the scores on the genitalia and the tongue.
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The individual's age, and lifetime number of sexual partners, did not
influence the degree of genital discomfort.
It might be important for any investigators taking part in the Manao Trial
first to convince themselves that the juice is not painful when applied
to the genitalia, as this is people's first concern when hearing about
the project, and they need reassurance if they are to volunteer for the
study.
Effects of pH on growth of vaginal micro-organisms in culture
Unpublished American data from Reprotect shows that at a pH of 4, the
viability of Lactobacillus acidophilus is hardly affected even after
20 hours, whereas HIV-1, HSV2, Treponema pallidum and Chlamydia have
a 99% reduction in viability within minutes, and Haemophilus ducreyi
and Neisseria gonorrhoeae have a 99% reduction in viability within an
hour. Thus maintaining the vaginal pH at or below 4.0 is likely to kill
a variety of STD pathogens, including HIV, in a very short space of
time.
Effect of lime or lemon juice on survival of HIV in culture
Professor Short and his colleagues are presenting an Abstract on this
subject at the forthcoming World AIDS Conference in Bangkok in July.
The pH of lemon and lime juice ranged from 2.3 - 2.5. A 2% solution
of juice in the culture medium gave a pH of 5.7, and had no inhibitory
effect on HIV replication after 1 hour. A 5% solution (pH 4.0) halved
HIV replication after 1 hour. A 10% solution ( pH 3.4) reduced HIV replication
by 2/3, whereas a 20% solution (pH 2.9) reduced replication by 90% within
2 minutes. Studies are currently underway on the effects of lemon juice
on HIV in semen.
Evidence for use of lime or lemon juice as a contraceptive and/or
to control sexually transmitted infections in other countries
Muslim prostitutes in Kano, Nigeria, currently use lemon juice as a
pre-coital and/ or post coital douche to control STD's, and there is
no evdnce of any associated vaginal pathology. There's a (hyperlink)
detailed report by Dr Malcolm Potts and his colleagues in the Latest
News area in the Lemons and AIDS section of this website.
A personal account by a doctor working in Vientiane, Laos, in 1975 claimed
that lime juice was being used as a douche by prostitutes. It was also
put into the eyes of new-born babies in place of silver nitrate, which
was unavailable.
A recent book by Sian Rees, "The Floating Brothel", describes
the arrival of the first European women in Sydney in the 1790's. A total
of 250 women prisoners aged in their late teens to their 20's were shipped
from England on the Lady Julian, together with 30 sailors who had free
access to the women for the duration of the 11 month voyage. On arrival
in Sydney, only 12 of them were pregnant, so there seems to have been
a contraceptive aboard. Perhaps it was the limes, used for the prevention
of scurvy on long sea voyages following the discoveries of James Lind
in 1753.
Following a presentation to U.S.A.I.D. in Washington in 2002 about the
spermicidal and virudical effects of citrus juice, they commissioned
CONRAD to see if they could confirm our findings. They confirmed them
completely.
(CONRAD was established in 1986 under a co-operative agreement between
the Eastern Virginia Medical School (EVMS) and the U. S. Agency for
International Development (USAID)...and is dedicated to improving reproductive
health, particularly in developing countries where the need is greatest,
by supporting the development of better, safer and more acceptable methods
to prevent pregnancy and sexually transmitted infections (STI's), including
HIV/AIDS.)
The U.S. - based Microbicides Alliance, which has $100 million for
the development of new microbicides (Brown, H. (2004) Marvellous Microbicides,
The Lancet 363, 1042-3) is fully aware of our work, but chooses to ignore
it. They are not able to give details about the trials they are running,
because the compounds are patented and licensed to commercial companies,
hence the results are "commercial in confidence".
Citrus juice cannot be patented, and as a Natural product, F.D.A. has
no jurisdiction over its development or use.
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