Background
Gametocytes, the precursors of male and female gametes, of malaria parasites are formed in the human host through the developmental switch from asexual replication in erythrocytes. Although gametocytes are not responsible for clinical symptoms, they ensure the transmission of malaria to another host. Upon taking a blood meal, gametocytes are transferred to a mosquito’s midgut lumen where they differentiate into male and female gametes. After complete sexual reproduction and successive processes of sporogonic development, mature sporozoites accumulate in the vector’s salivary gland, ready to be inoculated into a new host. Therefore, the presence of gametocytes in circulation of infected individuals is imperative for malaria to remain endemic in a given community. Malaria control strategies aiming at interruption of the malaria transmission process require knowledge on the status of gametocyte carriage in each endemic area [
1].
During acute malaria infection, the number of gametocytes in circulation of patients occurs at much lower densities than asexual stages and usually circulates at the level close to or below microscopic detection threshold, making it liable to be undiagnosed by microscopy [
2,
3]. Several epidemiological surveys have shown that only a subset of malaria-infected individuals possessed gametocytes upon microscopic examination of the blood smears [
3‐
7]. Importantly, these gametocyte-negative blood samples remain infective to anopheline vectors akin to those that have patent gametocytaemia [
6‐
8]. On the other hand, molecular detection of
Plasmodium gametocytes has revealed that a considerable number of malaria patients whose blood samples were gametocyte-negative by microscopy actually had sub-microscopic gametocytaemia [
3‐
7]. Therefore, microscopy detection of gametocytes could underestimate and thereby mislead evaluation of malaria transmission potential in endemic areas.
Molecular diagnostics of malarial gametocytes are based on amplification of mRNA transcripts that are exclusively expressed during gametocyte stages. Some of these transcripts are synthesized in co-ordination with specific periods of gametocyte development while some are sex-specific [
9,
10]. Therefore, specific mRNA transcripts could serve as appropriate markers for diagnosing stages of gametocytes. Of these, transcription of
Pfs25 begins when gametocytes of
Plasmodium falciparum become mature (stage V) and continues until the formation of ookinetes [
11]. Importantly, homologues of
Pfs25 have been identified in several other malaria, e.g.
Pvs25, Pbs25, Pgs25 and
Pys25 in
Plasmodium vivax Plasmodium berghei, Plasmodium gallinaceum and
Plasmodium yoelii, respectively [
12]
. To date, molecular epidemiological studies have been largely performed to assess
P. falciparum gametocyte carriage because it is the most prevalent and most pernicious malaria species that requires urgent effective control measures. On the other hand, a few reports have demonstrated molecular application for diagnosing
P. vivax gametocytes [
5,
13] despite the fact that it is the most widely distributed species with relapsing potential and is responsible for an important global public health burden [
14].
Both
P. falciparum and
P. vivax are major malaria species and sympatric in several endemic areas outside Africa [
15]. Therefore, epidemiological surveillance or assessment of malaria transmission by estimation of both
P. falciparum and
P. vivax gametocyte carriage in each endemic area is essential for malaria control policy. Because the expense of reagents and turnaround time in multiplex PCR are less than single PCR, a multiplex-nested RT-PCR assay targeting
Pfs25 and
Pvs25 is developed for rapid detection and differentiation of
P. falciparum and
P. vivax gametocytes simultaneously. Diagnostic performance of the method was evaluated using blood samples collected during both high and low transmission seasons from malaria patients in an endemic area of Thailand.
Discussion
Recent molecular epidemiological surveys of malaria in Thailand have revealed that all five
Plasmodium species circulate in this country. Most malaria cases are caused by
P. falciparum (~56.8% of all species) and
P. vivax (~41.3%). It is noteworthy that co-infections of both these species contributed ~12.5% of all malaria cases; the highest prevalence of ~23–24% occurred along the Thailand and Myanmar borders [
18,
19]. Therefore, malaria control by transmission-reducing interventions, such as gametocytocidal drugs or sexual stage vaccines, requires simultaneous assessment of human infectious reservoirs of both
Plasmodium species. The multiplex-nested RT-PCR developed herein offered high diagnostic performance with sensitivity and specificity over 97% when results from single-nested RT-PCR targeting each of these transcripts were used as standard. Multiplex-nested RT-PCR detected gametocytes of
P. falciparum and
P. vivax 9.50 and 2.73 times more patient samples, respectively, than was determined by microscopy. It is noteworthy that 87.4% of
P. falciparum and 91.1% of
P. vivax clinical isolates in Umpang District contained gametocytes based on detection of
Pfs25 and
Pvs25 mRNA by multiplex-nested RT-PCR and total malaria species identified by
18S rRNA PCR. Similarly, molecular surveys in Kenya and Tanzania revealed 89.3–92.3%
P. falciparum gametocyte prevalence despite differences in disease endemicity between these countries and Thailand [
22]. The high prevalence of submicroscopic gametocyte carriage has been hypothesized to ensure gametocyte survival by avoiding substantial stimulation for anti-gametocyte immune responses [
23,
24] and developmental success in vector by preventing damage of mosquito midgut during ookinete invasion through oocyst development [
24‐
26].
A recent study using
P. falciparum isolates from patients in Tak Province who had febrile symptom ~3 days (range one to eight days) before blood sample collection (n = 44) revealed that all isolates gave positive results for RT-PCR targeting
Pfg377, a female gametocyte-specific mRNA, whereas microscopy detected gametocytes in only six isolates. Subsequent
in vitro cultivation resulted in gametocyte production in 89% of isolates as detected by microscopy [
27]. Meanwhile, all except one malaria patients in this study had fever one day prior to blood sample collection. Although it is likely that there could be some expression of
Pvs25 and
Pfs25 at the early stages of gametocyte maturation, probably from leaky transcription or low-level transcription,
Pvs25 and
Pfs25 mRNA in blood samples are mainly from mature gametocytes. Taken together, it seems that almost all patients infected with
P. falciparum in Tak province had mature gametocytaemia within a few days after onset of febrile symptoms
albeit the majority existing below microscopic detection threshold. Likewise, similar findings were demonstrated in
P. vivax-infected patients, suggesting that malaria transmission could occur at the beginning or soon after the febrile onset in this endemic area. By contrast, a survey of
P. falciparum gametocytes among patients in an endemic area in Tak Province by RT-PCR using the method described by Babiker
et al has shown only 15 of 82 samples (18.3%) contained
Pfs25 mRNA whereas reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) provided slightly superior results (29.2%) [
28]. Discrepancy in prevalence of gametocyte carriage may not be directly compared between studies because of non-identical clinical samples and methodology. However, there remains a possibility that the detection methods conferred different diagnostic sensitivity. Importantly, the minimum diagnostic threshold of the multiplex-nested RT-PCR in this study was 10 copies of templates. Although estimation of detection limit of the multiplex-nested RT-PCR in this study did not directly reflect the actual number of gametocytes in the samples, it is likely that each mature gametocyte of
P. falciparum or
P. vivax possesses several copies of
Pfs25 mRNA or
Pvs25 mRNA; thereby a single gametocyte is potentially sufficient to give a positive result.
It has been noted that
P. falciparum gametocyte carriage is more common in areas with high malaria transmission intensity and the younger age group seems to have higher gametocyte prevalence than in an older age group. However, age-dependent patterns of gametocyte carriage for
P. falciparum and
P. vivax have not been documented in this study, consistent with previous findings that in areas with low transmission intensity, age groups of infected individuals seem not to be apparently associated with gametocyte prevalence [
29‐
31]. Meanwhile, the prevalence of gametocyte carriage relative to all malaria cases as detected by either microscopy or multiplex-nested RT-PCR in this study seems to show no significant seasonal fluctuation as previously noted [
32,
33]. Discrepancy could arise from the small sample size in this study, differences in intensity of malaria exposure among subjects, competency of routine microscopists in diagnosing gametocytes of
P. vivax or other unknown factors. Nevertheless, the high prevalence of gametocyte carriage detected by multiplex-nested RT-PCR for both
P. falciparum and
P. vivax in both high (wet season) and low (dry season) transmission periods could ensure sustainability of malaria endemicity in the study population.
The prevalence of individuals with gametocytaemia based on microscopy showed remarkable spatial variation. However, sensitivity of gametocyte detection by microscopy
per se could be influenced by the experience of microscopists, the quality of slide preparation and the number of microscopic fields examined [
1‐
3]. Herein, patients who carried microscopically patent
P. vivax gametocytaemia had significantly higher median parasite density than those without detectable gametocytes by microscopy. Therefore, a higher parasite density may offer a better chance for detecting gametocytes of
P. vivax prior to anti-malarial treatment. Importantly, a number of factors could influence microscopically patent gametocytaemia, e.g. exposure to anti-malarial drugs, increased in body temperature or anaemia [
1]. These constraints, compromising effective assessment of gametocyte carriage, could be alleviated by deployment of RT-PCR-based methods for detecting gametocyte-specific mRNA with remarkably higher sensitivity than conventional microscopy. Because the patterns and dynamics of gametocyte carriage in malaria-infected individuals have important contributions to the capability of malaria transmission and persistence in each endemic area, simultaneous detection of both
P. falciparum and
P. vivax gametocytaemia by multiplex-nested RT-PCR developed herein will be useful in areas where both malaria species co-circulate.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SJ and CP involved in study design, supervised and managed the project. NK, CP, UP and SJ carried out field work. NK, SJ and CP carried out molecular assay. NK and UP performed microscopy-based detection and estimation of parasite density. NK, CP and SJ analysed data. SJ and CP wrote the manuscript. All authors read and approved the final manuscript.