Anti-parasitic drugs:

susceptibility testing
Presenter: Dr. Arya Babu
Moderator: Dr. Nishant Verma
26/02/2024
 Bacteria Fungi Viruses Parasites
Culture methods Commonly done Commonly done Cultivable with difficulty Very difficult to grow

Susceptibility testing Well established standards Guidelines are available(CLSI & Not commonly done No established guidelines
and guidelines(CLSI & EUCAST)
EUCAST)

Treatment options Many Limited Limited Limited

Self-limiting No No Mostly No

Vaccine availability Few Nil Many Only available for malaria

Neglected tropical diseases Buruli ulcer Chromoblastomycosis Dengue Lymphatic filariasis

Leprosy Mycetoma Rabies Leishmaniasis
Trachoma Sporotrichosis Chikungunya Chaga’s disease
Yaws African trypanosomiasis
Noma Echinococcosis
Onchocerciasis
Scabies and other
ectoparasitosis
Schistosomiasis
Soil transmitted helminth
infections
Food-borne trematode
infections
Taeniasis
dracunculiasis

Targeted for elimination in Tuberculosis Malaria

India Filariasis
Kala-azar
Challenges in anti-parasitic susceptibility testing

• The complex life-cycles of various parasites having different stages and host
species requirements, particularly in the case of parasitic helminths, often make
parasite cultivation an uphill assignment

Source: Ahmed NH. Cultivation of parasites. Trop Parasitol. 2014 Jul

Genetu Bayih A,Pillai DR et al ;2017. Susceptibility testing of medically important parasites. Clin Microbiol Rev 30:647– 669
Need for anti-parasitic susceptibility testing
• Treatment options are limited in parasitic infections

• It allows surveillance for development of drug resistance

• To aid in elimination efforts

• For drug discovery

• Many parasitic diseases are among neglected tropical diseases

Malaria

• World Malaria Report 2023

249 million cases, 608 000 deaths
WHO African Region accounted for about 93.6% of cases and 95.4% of deaths
globally
The WHO South-East Asia Region had nine malaria endemic countries, accounting
for 5.2 million cases and contributing to 2% of the burden of malaria cases globally
India accounted for about 65.7% of all malaria cases in the region
• NCVBDC(National centre for vector borne disease control) report
0.18 million cases; 0.1 million P. falciparum cases(57%),83 deaths
Global trends in distribution of malaria cases and deaths by
country, 2022

Cases Deaths

Source: World Malaria Report 2023

Malaria

Source: Coronado LM; Malarial Hemozoin:

From target to tool;Biochim Biophys Acta.
2014 June
 Mechanism of action of anti-
malarials

Source: Haldar, K., Bhattacharjee, S. & Safeukui, I. Drug resistance in Source: Bassat Q. The use of artemether-lumefantrine for the treatment of
Plasmodium. Nat Rev Microbiol 16, 156–170 (2018) uncomplicated Plasmodium vivax malaria. PLoS Negl Trop Dis. 2011 Dec
History of malaria treatment

Coban C. The host targeting effect of chloroquine in malaria. Curr Opin Immunol. 2020 Oct
Drug resistance to anti-malarials
• Antimalarial resistance is defined as the ability of a parasite strain to survive
and/or multiply despite the administration and absorption of a drug given in doses
equal to or higher than those usually recommended but within tolerance of the
subject(WHO 1986)
• Resistance to currently available antimalarial drugs has been confirmed in only
two of the four human malaria parasite species, Plasmodium falciparum and P.
vivax.
• P. knowlesi is fully susceptible to chloroquine and other currently used drugs.

Source: Drug resistance in the malaria endemic world; Centre for disease control; 2018
 Quinine (QN),
Mefloquine
(MQ),
Halofantrine
(HF)
Chloroquine
(CQ),
Amodiaquine
(AQ) and
Artemisnin
(ART)
Primaquine
(PQ)
Doxycycline
(DOX),
Clindamycin
Source: Haldar, K., Bhattacharjee, S. & Safeukui, I. Drug Source: Khan J ;Molecular mechanisms of action and resistance of anti-malarial drugs;(CLI),
resistance in Plasmodium. Nat Rev Microbiol 16, 156–170 (2018) Bacterial adaptation to co-resistance; 2019 Tetracycline
(TET)
Factors determining probability of selection of de
novo antimalarial drug resistance

Source: White NJ. Antimalarial drug resistance. J Clin Invest. 2004 Apr
• In India resistance of Plasmodium falciparum to chloroquine, the cheapest and
the most used drug was first reported in the year 1973 from Diphu of Karbi-
Anglong district in Assam state

• Two instances of rapid development, spread, and natural selection of drug-

resistant mutant parasites in India (chloroquine across the country and
artesunate + sulfadoxine-pyrimethamine [AS+SP] in the northeastern states)
translated into drug policy changes for Plasmodium falciparum malaria in 2010
and 2013, respectively
Artemisinin resistance

• Artemisinin resistance typically refers to a delay in the clearance of malaria

parasites from the bloodstream following treatment with an ACT
• The mechanisms of resistance developed by the parasites against artemisinin
compounds affect only one stage of the malaria parasite cycle in humans: the ring
stage
• It is more appropriate to call the delayed clearance “partial resistance” to
highlight this time-limited and cycle-specific feature
• The majority of patients who have delayed parasite clearance are still able to
clear their infections following treatment with an ACT with an effective partner
drug or with an artesunate treatment lasting seven days
Source: www.who.int
Updates on antimalarial drug efficacy and resistance surveillance in the Americas; regional malaria
program 2019, PAHO/WHO
Artemisinin resistance contd.

• Artemisinin partial resistance likely emerged prior to 2001, and prior to the
widespread deployment of ACTs in the Greater Mekong subregion
• To date, it has been confirmed in 5 countries of the GMS-Cambodia, the Lao
People’s Democratic Republic, Myanmar, Thailand and Vietnam
• In late 2013, researchers identified a new molecular marker: mutations in the
Kelch 13 (K13) propeller domain were shown to be associated with delayed
parasite clearance in vitro and in vivo
WHO guidelines for treatment of uncomplicated
malaria

Source: WHO guidelines for malaria - 16 October 2023

WHO guidelines for treatment of uncomplicated
malaria contd.
Treatment of severe malaria

• Artesunate
intravenous or intramuscular artesunate for at least 24 h and until they can
tolerate oral medication
Treatment should be completed with 3 days of an ACT

• Parental alternatives when artesunate is not available

artemether should be used in preference to quinine
Indian guidelines

Source: Guidelines for diagnosis and treatment of malaria in India; NVBDCP; 2014
 Surveillance for anti-malarial drug resistance

In-vivo methods: In-vitro methods: Molecular methods

Therapeutic efficacy 1)Microscopy(WHO Microtest) 1)RFLP
studies 2)Ring-stage survival assay(RSA) 2)Sanger sequencing
3)ELISA(HRP2 & pLDH) 3)Real time PCR
4)Fluorescent markers
5)Flow cytometry
6)Isotopic test
Source: Nsanzabana, C., Djalle, D., Guérin, P.J. et al. Tools for surveillance of anti-malarial drug
resistance: an assessment of the current landscape. Malar J, 2018
Methods for
monitoring
antimalarial
drug efficacy
and drug
resistance
Basic follow-up schedule

Each national malaria control

programme should monitor its
first- and second-line
medicines according to
national treatment guidelines

Source: Assessment and monitoring of anti-malarial drug efficacy for the treatment of uncomplicated falciparum malaria; WHO;2003
 Severe malaria
• Coma
Classification of • Convulsions
• Renal failure
treatment • Jaundice
outcomes • Severe anaemia
• Pulmonary
oedema
• Hypoglycemia
• Metabolic
acidosis
• Shock
• DIC
• Hemoglobinuria
• Hyperpyrexia
Source: Assessment and monitoring of anti-malarial drug efficacy for the treatment of uncomplicated falciparum malaria; WHO;2003 • hyperparasitemia
Microscopy (WHO microtest)
• The in vitro microtest was developed by Rieckmann et al. based on culture
methods of Trager and Jensen
• Microtiter plates precoated with different concentrations of chloroquine and two
drug-free control wells per assay are used
• 5 μl whole capillary blood and 50 μl buffered RPMI 1640 medium added to each
well and incubated in a candle jar at 38–39°C for 24–30 h
• Immediately after incubation, thick blood films are prepared from each well, and
the number of schizonts is counted against 500 leukocytes
• The response of the drug expressed as the percentage of the number of schizonts
with each drug concentration, compared with the number of schizonts in drug-
free controls

Source: Maji AK. Drug susceptibility testing methods of antimalarial agents. Trop Parasitol. 2018 Jul-Dec
WHO Microtest

Source: Field application of in vitro assays for the sensitivity of human malaria parasites to antimalarial drugs; WHO;2007
 Ring-stage survival assay(RSA)

• Developed specifically to assess resistance of P. falciparum

parasites to artemisinin derivatives
• The decreased susceptibility to artemisinin affects the ring
stages only, hence resistance phenomenon cannot be well
detected in a culture that goes through all the parasites
stages
• Can distinguish culture-adapted isolates with fast clearance
or slow-clearing rates that can survive pharmacologically
relevant doses of ART
Ring stage survival assay (RSA)
• Parasites are cultured without any drug to reach a high
parasite density (≥ 0.2%), then a tight synchronization step
(ring-stages aged from 0 to 3 h) is performed to eliminate the
schizont stages, and the ring stage parasites are placed into
culture in the presence of 700 nM of dihydroartemisinin (DHA)
• This concentration represents the typical therapeutic
concentration found in patients treated with artemisinin
derivatives
• DHA is removed after 6 h (a physiologically relevant duration),
and the parasites are placed into a fresh culture mix for
another 66 h
• Survival rates-proportion of viable parasites that developed
into second-generation rings or trophozoites with normal
morphology; survival rate>1%- correlation with invivo
treatment failure
Leishmaniasis
• Encompassing a complex group of disorders, leishmaniasis is caused by unicellular
eukaryotic obligatory intracellular protozoa of the genus Leishmania and primarily
affects the host’s reticuloendothelial system.

• Leishmaniasis occurs in 98 countries in tropical and temperate regions

• More than 1.5 million cases occur annually, of which 0.7–1.2 million are CL and
200,000-400,000 are VL

• More than 350 million people are at risk, with an overall prevalence of 12 million

Source: Harrison's Principles of Internal Medicine, Twenty-First Edition

• India accounted for 18% of the global burden of kala-azar in 2020
• The four states in India endemic for kala-azar are: Bihar (33 districts, 458 blocks), Jharkhand
(4 districts, 33 blocks), West Bengal (11 districts, 120 blocks) and Uttar Pradesh (6 districts,
22 blocks)
• Sporadic cases also reported in other states including Assam, Gujarat, Himachal Pradesh,
Jammu & Kashmir, Kerala, Madhya Pradesh, Haryana, Puducherry, Sikkim, Tamil Nadu and
Uttaranchal

Source: https://www.who.int/india/health-topics/leishmaniasis
Operational guidelines on Kala-azar elimination in India – 2015;NVBDCP
Source: Garcia LS; diagnostic medical parasitology;6 th edition; 2016
 Mechanism of action of anti-leishmanial drugs

Limitations :Resistance,toxicity, high cost,

difficulty of administration and treatment
duration

Cellular targets of clinically used drugs against leishmaniasis and their limitations. The targets and action routes are still
subject to investigation
Source: Robles-Loaiza; Peptides to Tackle Leishmaniasis: Current Status and Future Directions. Int. J. Mol. Sci. 2021
 History of antimony resistance in Bihar

• The first indication of drug resistance came from North Bihar, in the early 80s, of
about 30% patients not responding to the prevailing regimen of antimonials, which
was a small daily dose (10 mg/kg; 600 mg maximum) for short duration (6 to 10 day)

• In 1984, the WHO expert committee recommended that pentavalent antimony be

used in doses of 20 mg/kg up to a maximum of 850 mg for 20 days, and a repetition
of similar regimen for 20 days in cases of treatment failures.

Source: Chakravarty J, Sundar S. Drug resistance in leishmaniasis. J Glob Infect Dis. 2010 May;
• However, by early 90s, extending the therapy to 30 days could cure only 64% of
patients in a hyperendemic district of Bihar

• In two studies carried out under strictly supervised treatment schedules, it was
observed that only about one-third of the patients could be cured with the then
prevailing regimen. The incidence of primary unresponsiveness was 52%,
whereas 8% of the patients relapsed.

Source: Chakravarty J, Sundar S. Drug resistance in leishmaniasis. J Glob Infect Dis. 2010 May;
• Incidentally, only 2% of the patients from the neighboring state of (Eastern) Uttar
Pradesh (UP) failed treatment.

• There are reports of antimony resistance spreading to the Terai regions of Nepal,
especially from the district adjoining the hyperendemic areas of Bihar, where up to
30% of the patients seems to be unresponsive, though in Eastern Nepal a 90% cure
rate has been reported.

• These studies confirmed that a high level of antimony resistance existed in Bihar,
whereas it was still effective in surrounding areas.

Source: Chakravarty J, Sundar S. Drug resistance in leishmaniasis. J Glob Infect Dis. 2010 May;
• In a study to determine whether acquired drug resistance was present in Bihar, L.
donovani isolates were taken from responders and nonresponders. In vitro
amastigote-macrophage assay showed that isolates from patients who did respond
to sodium stibogluconate treatment were threefold more sensitive, with 50%
effective doses (ED50) around 2.5 μg Sb/ml compared to isolates from patients who

did not respond (ED50 around 7.5 μg Sb/ml). The significant differences in
amastigote sensitivity supported the concept of acquired resistance in Bihar

Source: Chakravarty J, Sundar S. Drug resistance in leishmaniasis. J Glob Infect Dis. 2010 May;
• reason for the growing resistance in India while it still remained sensitive all over
the world could be due to the fact that leishmaniasis usually has zoonotic
transmission except in the Indian subcontinent and East Africa where the
transmission is largely anthroponotic

• In an anthroponotic cycle once resistance gets established, it spreads

exponentially and organisms sensitive to the drug get eliminated quickly, whereas
the drug-resistant parasites continue to circulate in the community
Source: Chakravarty J, Sundar S. Drug resistance in leishmaniasis. J Glob Infect Dis. 2010 May
Source: McConville MJ, Ralph SA. Chronic arsenic exposure
and microbial drug resistance. Proc Natl Acad Sci U S A.
2013 Dec 3
• Liposomal amphotericin B (LAmB) is recommended for treatment of Indian
visceral leishmaniasis (VL), with a cure rate of more than 95% in the Indian
subcontinent; The FDA-approved regimen for liposomal amphotericin B in
immunocompetent patients with VL is 21 mg/ kg in seven infusions over a 21-day
period
Source: Bennett: Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases
• Multidrug therapy for leishmaniasis is likely to be preferred in the future. Its
potential advantages in VL include
 better compliance and lower costs associated with shorter treatment courses
and decreased hospitalization
 less toxicity due to lower drug doses and/or shorter duration of treatment, and
 a reduced likelihood that resistance to either agent will develop
• In a study from India, one dose of LAmB (5 mg/kg) followed by miltefosine for 7
days, paromomycin for 10 days, or both miltefosine and paromomycin
simultaneously for 10 days (in their usual daily doses) produced a cure rate of
>97% (all three combinations).
• In Africa,combinstion of SbV and paromomycin for 17 days was as effective and
safe as antimonial given for 30 days
Source: Shyam Sunder; Harrison's Principles of Internal Medicine, Twenty-First Edition
Susceptibility testing

• The intracellular amastigote stage is the target of treatment for

leishmaniasis
• Drug susceptibility testing against amastigotes is considered the “gold
standard” for antimonial drugs
• For many antileishmanial drugs such as antimonials, pentamidine and
amphotericin B, in vitro amastigote susceptibility assays using
macrophage infection model are required as extracellular
promastigotes are intrinsically tolerant to the drug or depict
poor/partial correlation with amastigote drug susceptibility
Disadvantages of amastigote-macrophage model

• The amastigote system is labor intensive, hereby hampering the close

surveillance of drug susceptibility in the field.
• Lack of assay reproducibility and variations are also reported to occur
due to use of different cell lines
Genetu Bayih A et al: Susceptibility Testing of Medically Important Parasites. Clin Microbiol Rev. 2017 Jul
Giardiasis

• Giardia lamblia is a cosmopolitan protozoal parasite that inhabits the

small intestines of humans and other mammals
• Giardiasis is one of the most common parasitic diseases in both
developed and developing countries worldwide, causing both
endemic and epidemic intestinal disease and diarrhea

Source: Loscalzo J; Principles of Internal Medicine, Twenty-First Edition

Giardia
Anti-giardial drugs

• Cure rates with metronidazole (250 mg thrice daily for 5 days) are
usually >90%
• Tinidazole (2 g once by mouth) may be more effective than
metronidazole(currently drug of choice)
• In cases refractory to multiple treatment courses, prolonged therapy
with metronidazole (750 mg thrice daily for 21 days) or therapy with
varied combinations of multiple agents has been successful

Source: Loscalzo, Harrison's Principles of Internal Medicine, Twenty-First Edition

 • Treatment failure to giardiasis has been defined as the presence of the parasitic
protozoa in at least one of the three consecutive stool samples of the infected
patient along with persistence of symptoms such as diarrhoea, bloating,
abdominal pain, weight loss after completion of one or more courses of standard
treatment
• In cases refractory to multiple treatment courses, prolonged therapy with
metronidazole (750 mg thrice daily for 21 days) or therapy with varied
combinations of multiple agents has been successful

Source: Yadav P, Mirdha BR, Makharia GK. Refractory giardiasis: a molecular appraisal from a tertiary care centre in India. Indian J Med Microbiol. 2014 Oct-Dec
Loscalzo, Harrison's Principles of Internal Medicine, Twenty-First Edition
Susceptibility testing

• No standardized methods available

• Bioluminescence based method currently being tried-monitors viability of
trophozoites based on ATP content of G. lamblia trophozoites
Artemisinin and its derivatives

• Discovered by Chinese scientists in 1970s

• Nobel Prize in Medicine/Physiology awarded to Tu Youyou in 2015

• By screening compounds described in ancient Chinese medical texts, an extract of

sweet wormwood( Artemisia annua) found to be effective in curing simian and
murine Plasmodium infections

• The pure ingredient in this extract was termed qinghaosu or artemisinin

Source: Goodman & Gilman Artemisia annua

Malarial vaccines
• Since 2019, Ghana, Kenya and Malawi have been delivering the malaria vaccine
RTS,S/AS01 (RTS,S) through the Malaria Vaccine Implementation Programme, which is
coordinated by WHO and funded by Gavi, the Vaccine Alliance (Gavi)

• RTS,S vaccine has been administered to over 2 million children in Ghana, Kenya and
Malawi, and has been shown to be safe and effective, resulting in a drop of 13% in all-
cause early childhood deaths and a substantial reduction in severe malaria.

• In October 2023, the R21/Matrix-M (R21) malaria vaccine became the second vaccine
recommended by WHO to prevent malaria in children living in areas of risk
Amphotericin B unresponsiveness in
kala-azar

Source: Mechanism of
Amphotericin B Resistance in
Clinical Isolates of
Leishmania donovani
Authors: Bidyut Purkait;
msphere;2012
Plasmodium spp.:
characteristics of
the five infections

Source: Garcia LS; diagnostic medical parasitology;6 th edition; 2016

ELISA (HRP2 and pLDH)

• The principle of the assay is based on assessing parasite growth by measuring the
concentration of proteins produced by the cultured parasites
• Parasites are cultured with different concentrations of a drug for 72 h
• After 72 h, the culture plates are frozen at − 20 °C and thawed
• In parallel, ELISA plates are coated with monoclonal antibodies directed against the
Plasmodium spp. LDH (lactate dehydrogenase), or the P. falciparum-specific HRP2 (histidine-rich
protein 2) at 4 °C overnight
• The wells of the antibody coated plates are then incubated with parasite culture mixes
transferred from the thawed parasite culture plates
• After an incubation period of 2 h, biotinylated antibodies and colorimetric detection reagents
are added
• Finally, the plates are read on a spectrophotometer at 450 nm, and resulting absorbance values
are used to calculate the IC50 and determine the parasite susceptibility to anti-malarial drugs
• Based on the prevalence of
Pfdhfr+Pfdhps WHO-validated SP-
resistance markers across the
country
• Criterion for classifying a district
as a hot spot for a particular
mutation was prevalence of
mutation ≥ lower bound of the
95% CI of the pooled estimate for
the country
• Threshold is prevalence ≥4% for
single, ≥32% for double, ≥2% for
triple, >0% for quadruple, and
≥2% for quintuple and sextuple
mutations