Waheed et al.

Malaria Journal (2015) 14:146

DOI 10.1186/s12936-015-0660-0

CASE REPORT

Open Access

Vivax malaria and chloroquine resistance:

a neglected disease as an emerging threat
Anam A Waheed1, Najia K Ghanchi2, Karim A Rehman1, Afsheen Raza2, Syed F Mahmood3 and Mohammad A Beg2*

Abstract
In Pakistan, Plasmodium vivax contributes to major malaria burden. In this case, a pregnant woman presented with
P. vivax infection and which was not cleared by chloroquine, despite adequate treatment. This is probably the first
confirmed case of chloroquine-resistant vivax from Pakistan, where severe malaria due to P. vivax is already an
emerging problem.
Keywords: Malaria, Chloroquine resistance, Artemisinin combination therapy

Background
Plasmodium vivax malaria continues to be a global
threat, affecting 2.8 million people [1]. Chloroquine
(CQ) has remained the first-line of treatment for
P. vivax since the 1940s [2] and seemed to be universally
effective until the first case of CQ-resistant P. vivax was
reported from Papua New Guinea [3]. There have been
some reports of CQ resistance across the globe [4] but
CQ remains the mainstay of treatment in most regions.
This study probably reports the first case of CQresistant P. vivax found in Karachi, Pakistan.
Patient

A 26 years old female presented at 34 weeks of gestation

to the labour room at Aga Khan University Hospital
(AKUH) with a three-day history of fever. The patient
had been experiencing high grade fever for the past
three days, associated with chills and rigours, as well as
anorexia, malaise, myalgia, and back pain. These complaints had prompted her to go to Civil Hospital Karachi
where a Giemsa-stained peripheral smear had shown
P. vivax. She had then elected to come to AKUH
for treatment.
Clinical findings

On examination, she was alert and oriented to time

place and person. Vital signs showed her to be febrile
* Correspondence: [email protected]
2
Department of Pathology and Laboratory Medicine, Aga Khan University,
Stadium Road, PO Box 3500, Karachi 74800, Pakistan
Full list of author information is available at the end of the article

with a temperature elevation of 38C and tachycardia

with a heart rate of 140 beats/min. Her blood pressure
was 100/70 mmHg, respiratory rate of 14. She was saturating 99% on room air. Respiratory examination was
unremarkable with normal vesicular breathing bilaterally. Cardiac examination was normal. Abdominal
examination showed a fundal height of 34 cm, with a
longitudinal lie, cephalic presentation, and audible foetal
heart sounds.
Laboratory investigations prior to admission showed
haemoglobin of 10.0 g/dL, a platelet count of 285,000/mm3,
white blood cell (WBC) count of 4.6103. Thick and thin
blood films were positive for P. vivax mono-infection.
Dengue IgM and IgG were negative. Laboratory finding
on day of admission showed haemoglobin of 9.6 mg/dL, a
platelet count of 57,000. WBC count was 3.6103, with
a differential of 90% neutrophils and 8% lymphocytes.
Coagulation profile (prothrombin time, activated partial
tissue thromboplastin time) was also normal. Electrolytes
revealed sodium = 133 mmol/L, potassium = 3.3 mmol/L,
chloride = 107 mmol/L, bicarbonate = 13.2 mmol/L, and a
random glucose = 116 mg/dL. Liver function tests were
within normal ranges. Urinalysis revealed trace proteinuria
and haemoglobinuria. Chest X-ray was normal and foetal
cardiograph was reactive.
Microscopic examination of Giemsa-stained blood
smear showed trophozoites of P. vivax. Rapid diagnostic
testing using a Plasmodium falciparum/P. vivax antigen
detection kit (ICT Detection Kit, Sydney, Australia) revealed a P. vivax infection.

2015 Waheed et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
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unless otherwise stated.

Waheed et al. Malaria Journal (2015) 14:146

The patient was admitted and started on CQ regimen,

1,000 mg given immediately followed by 500 mg after six
hours and 500 mg for the next two days. She was also
given acetaminophen 1,000 mg for her fever and ferrous
sulphate and calcium tablets were continued. She was
monitored clinically and microscopy performed six hourly.
For the next three days, she continued to spike fevers on
the full CQ regimens and blood films remain positive for
malarial parasite. Her haemoglobin decreased progressively over the next three days to 8.0 mg/dL and her platelet count continued to drop to 48,000/mm3.
She was monitored intensively, with bleeding precautions initiated and the Infectious Diseases team was
consulted. When she failed to defervesce for greater than
12 hours for three consecutive days and remained positive for Plasmodium on Giemsa smears, her regimen
was changed to a combination therapy of artemether
and lumefantrine (40 mg/240 mg). This is a three-day
treatment schedule with one dose given immediately, another dose after eight hours and then subsequent doses
at 24 and 36 hours. On initiation of this regimen, the
patient defervesced rapidly, and remained afebrile. Subsequent Giemsa smears also revealed absence of parasitaemia, and P. vivax infection was successfully cleared.
On follow-up, there was no relapse of malaria, and the
patient delivered a healthy baby through spontaneous
vaginal delivery without any complications.

Page 2 of 3

Plasmodium vivax mono-infection was confirmed on microscopy, immuno-chromatography test (ICT) and polymerase chain reaction. Genotyping analysis revealed that
the sample carried pvmsp-1 Type 1 and pvcsp VK 210
repeat types. Furthermore, analysis of sulphadoxinepyrimethamine (SP) resistance associated mutations in
pvdhfr and pvdhps genes showed presence of 117 N, 50 I
and 119 K mutations. Both 117 N and 50I mutation have
been associated with emerging resistance against SP, implying that the patient was infected with SP-resistant
strain of P. vivax. Interestingly, no mutation was observed
in the pvcrt-o gene, however, the possibility of P. vivax
strain accumulating mutations in other CQ-binding regions cannot be ruled out in this study. Lack of validated
molecular markers to monitor CQ resistance is a major
limitation in surveillance of resistant strains of P. vivax
globally. With regard to cytokine levels, TNF, IL-10 and
ICAM-1 concentrations were found to be raised, indicating that respective cytokines and endothelial markers were
upregulated in response to treatment failure, and may have
led to further inflammation via parasite exposure.

number of cases range between 70 and 390 million

people [5,6]. These figures are suspected to be underestimates of the true burden of disease because of
limitations in the coverage for malaria notification and
diagnosis in many endemic areas [7]. There has been a recent increase in the severity and morbidity of malaria
caused by P. vivax and it is speculated to be associated
with infection with drug resistant strains of P. vivax [6].
There are reports documenting mutations predisposing to
CQ resistance present in P. vivax strains [8]. This portends
serious consequences for public health and global burden
of disease if CQ-resistant strains become widespread.
Malaria remains an endemic disease in Pakistan with an
estimated health care burden of 1.6 million cases annually
[9]. Plasmodium falciparum and P. vivax have been the
species documented to be responsible for this, with P.
vivax accounting for 67% of reported cases. In a developing
country where people struggle to afford mainstream antimalarial medication, the possible appearance and spread of
CQ-resistant P. vivax is cause for alarm. It would place a
huge burden on an already strained healthcare system, and
morbidity and mortality due to malaria would increase.
Resistance to CQ has been defined as parasitaemia detectable at 72 hours after initiation of therapy, or reappearance of parasitaemia within 28 days in spite of
CQ levels being maintained above 100 ng/ml [10]. In
this patient, parasitaemia was detectable while being on
the full therapeutic regimen, hence falling into the category of CQ resistance and treatment failure. However,
the absence of serum concentration of CQ or desethylchloroquine which is required to confirm the adequate
absorption of the drug is a limitation of this study.
Molecular genetic studies have revealed that P. vivax
from areas with a high incidence of CQ resistance carry single nucleotide polymorphisms in the multidrug resistance
gene (pmvdr1) [11]. Multiple studies have documented mutations in the pvcrt-o gene to be associated with tolerance
to CQ [12]. Genotypic variations in P. vivax dihydrofolate
reducatase gene (pvdhfr) as well as P. vivax dihyropteroate
synthetase (pvdhps) have also been implicated in drug resistance [8], including in isolates from Pakistan [13].
CQ resistance has not been reported previously from
Pakistan. However, a recent study carried out molecular
genetic analysis of strains of P. vivax from Pakistan,
sequencing pvdhfr, pvdhps and pmvdr1, which are associated with CQ resistance [14,15]. It reports a high prevalence of P. vivax mutant pvmdr1 mutant codon F1076L,
which points towards the possibility of the efficacy of CQ
plus primaquine being compromised in future, but no
clinical correlation has been made [16].

Discussion
Some 2.8 billion people across the globe are at risk of infection by P. vivax [1] and estimates of the total annual

Conclusion
It is suggested the possible presence of CQ-resistant
P. vivax strains in Pakistan and may be an emerging

Molecular analysis

Waheed et al. Malaria Journal (2015) 14:146

threat. Clinicians need to be aware that patients failing

treatment may be infected with CQ-resistant P. vivax
strains. In settings where resources allow, these strains
can be analysed with molecular and immunological
markers so that the extent and impact of drug pressure
may be monitored effectively.

Page 3 of 3

13.

14.

15.

Consent

Written informed consent was obtained from the patient

for the publication of this report and any accompanying
images.
Competing interests
The authors declare that they have no competing interests.

16.

ortholog of pfcrt, in Plasmodium falciparum and Dictyostelium discoideum.

Mol Biochem Parasitol. 2006;150:21928.
Raza A, Ghanchi NK, Khan MS, Beg MA. Prevalence of drug resistance
associated mutations in Plasmodium vivax against sulphadoxinepyrimethamine in southern Pakistan. Malar J. 2013;12:261.
Hawkins VN, Joshi H, Rungsihirunrat K, Na-Bangchang K, Sibley CH.
Antifolates can have a role in the treatment of Plasmodium vivax. Trends
Parasitol. 2007;23:21322.
Hastings MD, Maguire JD, Bangs MJ, Zimmerman PA, Reeder JC, Baird JK,
et al. Novel Plasmodium vivax dhfr alleles from the Indonesian Archipelago
and Papua New Guinea: association with pyrimethamine resistance
determined by a Saccharomyces cerevisiae expression system. Antimicrob
Agents Chemother. 2005;49:73340.
Khattak AA, Venkatesan M, Khatoon L, Ouattara A, Kenefic LJ, Nadeem MF,
et al. Prevalence and patterns of antifolate and chloroquine drug resistance
markers in Plasmodium vivax across Pakistan. Malar J. 2013;12:310.

Authors contributions
MAB, SFM and AAW were responsible for clinical work, designed and planned
the study. NKG, AR and KAR performed microscopy, PCR genotyping, ELISA
analysis, and interpretation. AAW and NKG prepared the initial manuscript.
MAB reviewed the final draft. All authors read and approved the final
manuscript.
Acknowledgements
All molecular studies for this project were funded by an Aga Khan University
Research Council grant.
Author details
1
Medical College, Aga Khan University, Karachi, Pakistan. 2Department of
Pathology and Laboratory Medicine, Aga Khan University, Stadium Road,
PO Box 3500, Karachi 74800, Pakistan. 3Section of Adult Infectious Diseases,
Department of Medicine, Aga Khan University, Stadium Road, PO Box 3500,
Karachi 74800, Pakistan.
Received: 19 December 2014 Accepted: 20 March 2015

References
1. Guerra CA, Howes RE, Patil AP, Gething PW, Van Boeckel TP, Temperley WH,
et al. The international limits and population at risk of Plasmodium vivax
transmission in 2009. PLoS Negl Trop Dis. 2009;4:e774.
2. Baird JK. Chloroquine resistance in Plasmodium vivax. Antimicrob Agents
Chemother. 2004;48:407583.
3. Rieckmann KH, Davis DR, Hutton DC. Plasmodium vivax resistance to
chloroquine? Lancet. 1989;2:11834.
4. Price RN, von Seidlein L, Valecha N, Nosten F, Baird JK, White NJ. Global
extent of chloroquine-resistant Plasmodium vivax: a systematic review and
meta-analysis. Lancet Infect Dis. 2014;14:98291.
5. Hay SI, Guerra CA, Tatem AJ, Noor AM, Snow RW. The global distribution
and population at risk of malaria: past, present, and future. Lancet Infect Dis.
2004;4:32736.
6. Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM. Vivax malaria:
neglected and not benign. Am J Trop Med Hyg. 2007;77:7987.
7. Goncalves LA, Cravo P, Ferreira MU. Emerging Plasmodium vivax resistance
to chloroquine in South America: an overview. Mem Inst Oswaldo Cruz.
2014;109:53.
8. Garg S, Saxena V, Lumb V, Pakalapati D, Boopathi PA, Subudhi AK, et al.
Novel mutations in the antifolate drug resistance marker genes among
Plasmodium vivax isolates exhibiting severe manifestations. Exp Parasitol.
2012;132:4106.
9. WHO. World malaria report 2012. Geneva: World Health Organization; 2012.
10. Baird JK, Sustriayu Nalim MF, Basri H, Masbar S, Leksana B, Tjitra E, et al.
Survey of resistance to chloroquine by Plasmodium vivax in Indonesia. Trans
R Soc Trop Med Hyg. 1996;90:40911.
11. Suwanarusk R, Russell B, Chavchich M, Chalfein F, Kenangalem E, Kosaisavee V,
et al. Chloroquine resistant Plasmodium vivax: in vitro characterization and
association with molecular polymorphisms. PLoS One. 2007;2:e1089.
12. Sa JM, Yamamoto MM, Fernandez-Becerra C, de Azevedo MF, Papakrivos J,
Naude B, et al. Expression and function of pvcrt-o, a Plasmodium vivax

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