Plasmodium knowlesi is a simian parasite that can also cause malaria in humans [
1,
2]. Whereas this parasite is prevalent in the Malaysian Borneo, its geographical range extends into the mainland Southeast Asia, Indonesia, and the Philippines [
3]. Recently, the incidence of human infection of
P. knowlesi in Malaysia and southern Thailand appears to be on the rise, despite the overall decline of malaria [
3,
4]. The parasite is thus an emerging public health threat in affected areas.
Adaptation of
P. knowlesi to continuous in vitro culture in human RBCs has opened doors to new possibilities to study this parasite [
5,
6]. As common for long-term
Plasmodium culture,
P. knowlesi culture loses synchronicity over time. Freshly thawed
P. knowlesi culture becomes mixed stages within 4 to 5 days. However, a synchronized parasite culture is often needed in research, particularly when the aim is to examine stage-specific phenotypes, transcriptomes, and proteomes. Several synchronization methods developed for in vitro culture of
Plasmodium falciparum have the potential for synchronization of in vitro
P. knowlesi culture. These methods include magnetic separation to obtain mature trophozoites and schizonts [
7,
8], selective lysis of trophozoites and schizonts to obtain rings [
9], physical separation based on differential density [
10] or sedimentation [
11], and cold treatment to obtain rings [
12,
13]. Indeed, density gradient centrifugation and magnetic separation have been used successfully to obtain tightly synchronized
P. knowlesi culture [
5,
6]. However, these methods are either time-consuming or require special equipment. For
P. falciparum, lysis of trophozoite- and schizont-infected red blood cells (RBCs), first established in 1979, remains one of the most commonly used methods due to its high efficiency, simplicity and low cost [
9]. This approach exploits the increased permeability of the infected RBCs due to the new permeation pathways (NPPs) to kill trophozoites and schizonts. NPPs allow sorbitol to enter the host cell, causing influx of water which leads to cell lysis. Because NPPs are active only in the host membrane at the trophozoite and schizont stages [
14], the ring-infected RBCs are resistant to sorbitol treatment.
While the sorbitol method works well to synchronize
P. falciparum, its application to the recently human-erythrocyte adapted line of
P. knowlesi (A1-H.1) had limited success [
15]. In this study, alternative solutes were explored and guanidine hydrochloride (GuHCl) was found to selectively lyse trophozoite- and schizont-infected human RBCs to achieve synchronization of
P. knowlesi culture.