Original Article
Effect of Tetracycline therapy on Microfilaraemia and
Antigenemia in Raipur city, Chhattisgarh state, India
Santosh Kumar Agrawal
Dept. of Zoology, Vivekanand Govt. P.G. College, Manendragarh, Korea, (C.G.) India
Keywords: Lymphatic filariasis, Wuchereria bancrofti, Microfilaraemia, Antigenemia
ABSTRACT
Background: Lymphatic filariasis caused by Wuchereria bancrofti is one of the major health problems and is
transmitted in India by Cx. quinquefasciatus. Diethylcarbamazine remains the drug of choice in selective as well as
mass chemotherapeutic programs against lymphatic filariasis. Treatment of filarae infected animals with tetracycline
resulted in the elimination of Wolbachia endosymbiont from the filarial tissues, prevented parasite establishment, filarial
growth and rendered adult worm sterile.
Method: Tetracycline capsules (200 mg/day) were given to microfilaraemic patients for 30 days and mf count and
antigen level in their blood and infection & infectivity rates in their house hold Culex mosquitoes were assessed post
tetracycline therapy.
Results: It was found that mf counts and antigenemia in the blood & infection & infectivity rates in the mosquitoes were
reduced gradually up to 24th months post tetracycline therapy.
Conclusion: Targetting endosymbiont Wolbachia using antibiotics might be effective controlling measure in lymphatic
filariasis.
*Corresponding author:
Dr. Santosh Kumar Agrawal, Dept. of Zoology, Vivekanand Govt. P.G. College, Manendragarh, Korea, (C.G.) 497442, India
Phone: +91 9827122495
E-mail: [email protected]
This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)
Original Article
A-38
Introduction
Bancroftian filariasis is a mosquito-transmitted parasitic
disease of humans characterized by lymphangitis,
hydrocele, lymphoedema, and elephantiasis, and is one of
the most common causes of global disability. The disease
has been considered to be potentially eradicable due to the
fact that anti filarial drugs currently could break the cycle
of transmission in endemic areas. Diethylcarbamazine plus
albendazole are proved more effective than DEC alone
[1, 2] for treating lymphatic filariasis. It is an effective
microfilaricidal compound in vivo and its adulticidal
function is yet to be proved. However, the drug is nonfunctional in vitro either against microfilariae or adult
filarial parasites. Ivermectin is another potential filaricidal
drug tested for efficacy and side reactions [3]. At a single
dose of 200-400 µg/kg body weight, ivermectin showed
promising results [4]. Tetracycline has been demonstrated
to have anti-filarial function against microfilariae as well
as adult filarial parasites in vitro as well as in vivo [5].
The tetracycline is primarily targeted to the intracellular
bacteria viz., Wolbachia in filarial worms. Wolbachia are
intracellular microorganisms that form maternally inherited
infections with numerous arthropod species [6]. These
bacteria have drawn much attention due to the reproductive
alterations they induce in their hosts including cytoplasmic
incompatibility, feminization and parthenogenesis. The
bacteriostatic activity of tetracycline on Wolbachia
symbionts appears responsible for altered fertility and
embryogenesis [7]. Targeting Wolbachia is becoming an
active area of research for control of lymphatic filariasis.
Oxytetracycline exhibited macrofilaricidal activity against
O. ochengi [8].
Materials And Methods
Study population: Individuals enrolled from a colony
of Raipur city (C.G.) in the study. A total of 10 (6 males
and 4 females) microfilaraemic patients were included in
this study. Written informed consent was obtained from
all participants. Individuals eligible for participation were
adults of both sexes aged 18-50 years, with a minimum body
weight of more than 40 kg, in good health and without any
clinical condition requiring chronic medication. Hepatic
and renal functions were assessed by urinal test.
Treatment regimen: Participants received 2 x 100 mg
capsule of tetracycline for a total of 4 weeks. Treatment
was done and monitored by a trial clinician in the form of
daily observed treatment (DOT).
The drug regimen was assessed for microfilaricidal effect
by assessing the following parameters
1. The rate of successful treatment: the proportion of mf
positive persons treated in whom there was a reduction in
the microfilarial count.
2. The percentage of cure rate- the proportion of mf positive
persons treated who became negative for microfilariae after
treatment.
Determination of microfilarial load: For a quick screening
in the night, the microfilarial load was determined
by microscopic examination of finger-prick blood
samples. Subsequently, eligible patients donated
10 ml of venous blood for accurate quantification
using Whatman Nucleopore filter method. The
same volume of blood was taken from each patient
1, 6, 12, 24 months after the commencement of
tetracycline treatment. At each time point, plasma
was taken from the remaining sample and frozen at
-80 °C for later analysis of antigenemia (filarial adult
worm antigens).The mean microfilaraemia load was
found 830 (175-1600) / ml of blood.
Determination of circulating filarial antigenemia: For
determina­tion of circulating filarial antigenemia
(CFA), W. bancrofti antigen was measured with
the TropBio ELISA test kit (TropBio, Townsville,
Australia). The manufacturer’s protocol was
followed except that the samples were diluted (1:20
ratio) with the diluent before pipetting into the
TropBio ELISA test plates. Samples were tested
in duplicate before treatment and at 6, 12 and
24-months follow-ups. The optical density at 414
nm was recorded from plasma samples. Antigen
units were calculated with a standard curve from
standards provided by the manufacturer, and the
final units multiplied by the dilution factor of 20.
USG examination: Ultrasonography (by professional
ultrasonography centre) of the scrotal area was undertaken
to detect adult W. bancrofti. Each patient’s scrotum was
scanned in transverse and longitudinal section of the right
and left sides. Worm nests were detected by the typical
movement of the adult worms (known as Filarial Dance
Signs-FDS).
Determination of pre and post therapy vector
infection & infectivity rates of Cx. quinquefasciatus
of the microfilaraemic patient’s houses: Indoor resting
Cx. quinquefasciatus will be collected before and after
tetracycline therapy from all 10 microfilaraemic patient’s
houses, dissected and assessed for mf (infection rates)
and L3 larva (infectivity rates) load in the mosquitoes post
tetracycline therapy.
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Result
Four weeks course of tetracycline (200 mg/day) was given
to 10 microfilaraemic patients, and they were monitored
for mf count and antigenemia up to 24 months following
tetracycline therapy. The treatment was well tolerated.
There were no major adverse reactions of tetracycline
therapy to any of the subjects. Some of them faced loose
motion for 1 or 2 days which was controlled automatically
without any treatment.
Table1.1 shows the significant change in microfilaraemia
and antigenemia from baseline and at follow-up time points.
The mean mf count and antigenemia (CFA) units per ml of
blood before treatment were 830 ± 145 and 9447 ± 2397
respectively. Both of these units decrease substantially one
month after the start of treatment. The infection reduces
gradually up to 24 months. When assessed, the rate of
successful treatment was 100% throughout the study
period up to 24 months follow up. The cure rate was found
0% when assessed one month after the treatment. 6 months
after treatment it was 10% and was 20% after 12 and 24
months post therapy.
Measurement of Adult Worm Vitality in Microfilaraemic
patients by USG: Presence or absence of worm nest in
ultrasound examination was used to asses’ adult worm
vitality in microfilaraemic patients before and after
tetracycline therapy. Ultrasonography to detect the filarial
dance signs (FDS) was performed only in male patients since
FDS is detected less frequently and probably less reliably in
women. FDS in the scrotal region before treatment showed
that three of the five male patients examined had between
1-4 worm nests. All three male patients became FDSnegative at 24 months after treatment.
Determination of pre and post therapy vector
infection & infectivity rates of Cx. quinquefasciatus
of the microfilaraemic patient’s houses: The impact of
tetracycline therapy on the vector infection & infectivity
rates of Cx. quinquefasciatus of the microfilaraemic patient’s
houses was examined. Indoor resting Cx. quinquefasciatus
were collected before and after tetracycline therapy from
all 10 microfilaraemic patient’s houses. The mean vector
infection & infectivity rates before therapy were 40.3
& 5.23 respectively. Gradual reduction the rates were
found after tetracycline therapy. One month post therapy
the vector infection & infectivity rates were found to be
33.07 (82%) & 4.48 (86%), 6 month after 14.35 (36%)
& 1.83 (35%), 12 months after 7.31 (18%) & 0.84 (16%)
and 24 months after treatment 4.08 (10%) & 0.45 (9%)
respectively. (Table: 1.2).
Discussion
The past decades has seen major advances that have changed
lymphatic filariasis from a neglected disease in to a disease
now accepted as potentially eradicable. The main reason
is the identification of ivermectin, DEC and albedazole, as
effective antifilarial agents. a 4-week course of tetracycline
against W. bancrofti induced both sustained reductions in
microfilaraemia and, most notably, macrofilaricidal activity
according to reductions in antigenemia and absence
of adult worms by ultrasonography. This is especially
important since the adult worms cause the disease
pathology in lymphatic filariasis and no safe, effective
macrofilaricidal treatment exists. In this study, tetracycline
treatment resulted in almost complete elimination of
microfilaraemia, which was sustained from at least 6 to
24 months after treatment. This gradual reduction over
months is different to the rapid efficacy seen within a few
days with diethylcarbamazine and ivermectin. This slow
rate of microfilarial loss is most probably due to the effect
of Wolbachia depletion on embryogenesis and the loss of
microfilariae from the circulation through natural attrition,
as recorded in onchocerciasis and lymphatic filariasis [9].
Table 1.1: Effect of Tetracycline Treatment on Microfilaraemia & Antigenemia
Terms
Mean Mf count ± SE
% Mean Mf count
p-value*
No. of Mf +ve patients
treatment rate
No. of cured patients
Cure rate
Antigenemia Mean ± SE
% Antigenemia Mean
p-value*
Before
Treatment
830 ± 145
100%
10
100%
0
0%
9447 ± 2397
100%
1 month
post treatment
495 ± 82
60%
0.00043
10
100%
0
0%
4887 ±1277
52%
0.0014
6 month
post treatment
300 ± 63
36%
0.00009
10
100%
1
10%
1956 ± 489
21%
0.0019
12 month post
treatment
140 ± 38
17%
0.00010
10
100%
2
20%
717 ± 206
8%
0.0016
24 month post
treatment
85 ± 22
10%
0.00014
10
100%
2
20%
187 ± 53
2%
0.0017
*p-value = t-Test
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e-ISSN: 2349-6991; p-ISSN: 2455-0396
Original Article
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Table 1.2: Pre & post therapy vector infection & infectivity rates
Megaloblastic
anemia
[n, (%)]
Leukemia
[n, (%)]
Aplastic
anemia [n,
(%)]
Hypersplenism
[n, (%)]
Infection
[n, (%)]
MDS
[n, (%)]
HIV
[n, (%)]
Fatigue
51(82.26)
22(81.48)
9(69.23)
8(80)
7(77.78)
6(75)
5(100)
Shortness of breath
37(59.68)
12(44.44)
5(38.46)
5(50)
4(44.44)
4(50)
3(60)
Dizziness
15(24.19)
3(11.11)
2(15.32)
4(40)
2(22.22)
1(12.5)
3(60)
Palpitation
29(46.77)
9(33.33)
6(46.15)
5(50)
1(11.11)
5(62.5)
2(40)
Headache
14(22.58)
7(25.93)
3(23.08)
1(10)
3(33.33)
0(0)
3(60)
Fever
19(30.65)
20(74.07)
5(38.46)
4(40)
8(88.89)
2(25)
3(60)
Frequent infections
26(41.93)
17(62.96)
4(30.77)
3(30)
2(22.22)
3(37.5)
5(100)
Bleeding manifestations
27(43.55)
14(51.85)
6(46.15)
7(70)
5(55.56)
2(25)
4(80)
chronic diarrhea
10(16.13)
0(0)
0(0)
0(0)
1(11.11)
1(12.5)
3(60)
One benefit of a gradual decline in microfilaraemia and
the depletion of endosymbionts would be the avoidance of
inflammatory adverse events after the rapid destruction of
parasites and release of bacterial symbionts into the blood
and tissues [10, 11, 12].
Conclusion
In this study, tetracycline treatment resulted in almost
complete elimination of microfilaraemia, which was
sustained from at least 6 to 24 months after treatment.
Advantages of antibiotic treatment are that tetracycline
and other antirickettsial antibiotics are already licensed
for human use and are available in endemic areas.
Additionally, the drugs are cheap and have known safety
and pharmacological activities.
Acknowledgements
[It should include persons who provided technical help,
writing assistance and departmental head that only
provided general support. Financial and material support
and conflict of interests must be written in this section.]
Funding
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following statements into your manuscript: None
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