The sensitivity and specificity of a diagnostic test define the extent to which a pathogen can be effectively identified in a patient specimen. For malaria, the examination of thin and thick blood smears by microscopy has been the gold standard diagnostic method for over a century. This test is simple to perform, requires only a microscope and has a sensitivity of 50 parasites/μL [
1]. The parasitaemia can be quantified and the species of
Plasmodium identified based on parasite morphology. When read by an experienced microscopist, the four major species of human malaria (
Plasmodium falciparum,
Plasmodium vivax,
Plasmodium ovale and
Plasmodium malariae) can usually be discriminated.
However, a major pitfall of microscopy was recently identified in the failure of this method to distinguish between the benign
P. malariae species and the potentially lethal primate species
Plasmodium knowlesi[
2]. Zoonotic transmission of
P. knowlesi from monkeys to humans was previously only observed in sporadic cases [
3,
4] and by blood passage from monkeys to humans in laboratory controlled experiments [
5‐
7] but was not routinely detected by microscopic analysis of patient specimens due to morphological similarities between
P. knowlesi and
P. malariae[
8]. As such,
P. knowlesi was not recognized as a cause of malaria in human populations, until recently. Using molecular diagnostic tools, including DNA sequencing and newly-developed
P. knowlesi-specific PCR primers, Singh
et al[
2] examined blood samples from 208 malaria patients in the Kapit division of Malaysian Borneo, and found that none of the cases identified as
P. malariae by microscopy were confirmed by PCR and 120 (58%) were identified as
P. knowlesi by PCR. These findings initiated a number of research studies into the epidemiological, clinical, ecological, and parasitological factors that determine the distribution and course of
P. knowlesi infection. It is now recognized that human
P. knowlesi malaria occurs in many countries in South-East Asia, causing locally-acquired malaria and infections in travelers returning from these regions [
9‐
17]. Of significant concern, approximately 1 in 10
P. knowlesi infections lead to severe malaria and seven deaths have been reported from this species [
18‐
20].
Plasmodium knowlesi has a 24-hour erythrocytic cycle, which is the shortest among the five species of
Plasmodium causing human malaria, and therefore correct identification and rapid treatment are essential, particularly when the parasitaemia is high.
Given the potential for misdiagnosis of
P. knowlesi by microscopy, alternative diagnostic tests must be employed to confirm this infection. Rapid diagnostic tests from two manufacturers have been evaluated for detection of
P. knowlesi antigens. Cross-reactivity was observed with both
P. falciparum and
P. vivax antigens, precluding the use of these tests for rapid diagnosis [
21]. Molecular diagnostics for detection of
P. knowlesi include nested PCR and/or sequencing [
2,
22], LAMP [
23], and real-time PCR [
24]. In clinical diagnostic and reference laboratories, particularly those in developed countries, real-time PCR is the method of choice by providing superior sensitivity, rapid results and low risk of false positives. It is also far less laborious than nested PCR, enabling high throughput screening of patient samples. A number of real-time PCR assays have been developed for malaria diagnosis but these can only detect
P. falciparum, P. vivax, P. malariae and P. ovale[
25‐
31]. Only one assay has been reported for detection of
P. knowlesi by real-time PCR [
24]. However, the validation of this assay was limited to 2 reference DNA samples from infections in monkeys and no human clinical samples were tested. In the current report, a real-time PCR assay for
P. knowlesi was developed and validated with clinical samples from 40 patients infected with
P. knowlesi. The
P. knowlesi assay developed uses the same reaction conditions of the real-time PCR assay described by Rougemount
et al that screens for
Plasmodium and identifies
P. falciparum, P. vivax, P. malariae and P. ovale using species-specific probes [
30].