**3. Parasite factors associated with infection duration**

After inoculation of sporozoites and the subsequent release of merozoites from the liver, there is a period of time when parasites are present in the blood at concentrations undetectable by conventional diagnostics. In many individuals, parasites then multiply to reach detectable densities, however in other individuals, parasites may remain at low densities that are undetectable. Human challenge studies on non-immune individuals in which parasites are monitored both by molecular methods and by microscopy have estimated that infections are detectable by PCR an average of 3.7 days (range 2–4 days) [35] or 3.1 days (range 0–4) [36] before being detectable by microscopy. Controlled human infections also suggest that the parasite stages that precede blood invasion might influence asexual blood stage dynamics. For example, Churcher and colleagues [37] observed that the inoculum size (the estimated number of sporozoites injected by infected *Anopheles* mosquitoes) influences the time it takes for infections to become patent: individuals receiving five bites from mosquitoes with more than 1000 sporozoites have detectable parasitaemia at least 2 days earlier than those volunteers infected by mosquitoes with 11–101 sporozoites. Quantification of sporozoite counts in wild-caught mosquitoes is necessary to confirm the relevance of this finding in natural settings. In Papua New Guinea, it was estimated that infected malaria vectors had on average (geometric mean) 4000 sporozoites [38], which is of the same order of magnitude as sporozoite counts in mosquitoes used in controlled infections.

gametocyte densities. Data from epidemiological studies confirm that most asymptomatic infections with patent or sub-patent asexual stage parasite levels have sub-patent gametocytaemia [42], only detectable by RNA-based molecular methods. However, a few asymptomatic individuals with low-density infections have relatively high gametocyte densities, which could be related to symptomless fluctuations in parasitaemia that result in higher gametocytaemia a few days later. The rate of commitment of asexually replicating parasites to sexual development is another factor that influences gametocyte levels in malaria infections. Adults, who on average carry lower asexual stage parasite densities, have a higher sexual to asexual density ratio [43]. This could be related to an unequal increase in clearance rates of asexual and sexual parasites with age, or potentially to changes in commitment to gametocytogenesis [43]. Consistent with the latter, parasite investment in transmission stages has been shown to vary in areas with different transmission levels, being higher in settings with lower endemicity. Recent data suggest that parasite variations in commitment to gametocytes are epigeneti-

Understanding the Importance of Asymptomatic and Low-Density Infections…

http://dx.doi.org/10.5772/intechopen.77293

135

As articulated above, the importance of asymptomatic infections for malaria transmission does not lie in their average sexual stage parasite densities but in the durations of gametocyte carriage and infectiousness over time. A mathematical model [45] fitted to both asexual parasite and gametocyte malariotherapy data estimated infectivity over the course of an infection based on gametocyte density data. This analysis concluded that the majority of infectivity was usually concentrated early in infection, although some patients were significantly infectious later on. However, in this model, it was assumed relatively low infectivity of low gametocyte densities compared with other analyses [46]. While these data are extremely detailed, it is not known whether these dynamics are similar to those in naturally infected individuals who have immunity. Furthermore, specific *P. falciparum* strains were selected for malariotherapy because they were 'benign' and may not exhibit the same behaviour in terms of parasite multiplication rates and gametocyte commitment as parasites in

Although asymptomatic infections do not prompt treatment-seeking behaviour, during community mass treatment campaigns that involve treatment regardless of symptomatology (e.g., mass drug administration (MDA) or mass screening and treatment), these infections are cleared with antimalarials. In a meta-analysis of trials with gametocyte density data [47], the combinations artesunate-mefloquine and artemether-lumefantrine were more effective in preventing the appearance of gametocytes and in clearing existing sexual stage parasites compared to dihydroartemisinin-piperaquine. The choice of drugs to be used during control interventions thus may be important to limit residual transmission from these infections. In Section 4, we discuss the impact of different interventions that target asymptomatic and

Two variables linked to blood sampling for parasite detection can influence prevalence and density estimates: volume and timing. Even sensitive molecular assays will not detect low parasite densities in samples if nucleic acids are isolated from small blood volumes. Highvolume PCR has been used in epidemiological studies in Southeast Asia to circumvent

cally imprinted and higher in parasites in lower endemicity settings [44].

endemic areas.

symptomatic infections.

**3.2. Underestimations of parasitaemia linked to sampling**

Microscopy has limited sensitivity to quantify low parasite densities and this will affect its utility for studying any chronicity in infection dynamics. Histidine rich protein 2 (HRP-2), a protein the parasite secretes in the plasma, is considered to be a more accurate measure of total falciparum parasite burden [39], however, this measure does not distinguish between monoclonal and multiclonal infections. Molecular tools are more sensitive and allow discrimination of different parasite genotypes. They have been used to assess the effects of super-infection and exposure to different parasite clones on clinical malaria risk. A study that involved daily blood sampling of children with initially asymptomatic infections [40] suggests that development of symptoms is often associated with appearance of a new parasite strain in the blood and increases in parasite levels. Correspondingly, recent data from Papua New Guinea [16] showed that incidence of infections by new clones correlates with clinical malaria risk. This indicates that clinical malaria is often associated with new infection, presumably by a parasite clone with a previously unencountered antigenic profile.

Consistent with this, genetic analysis of malaria parasite populations in Zambia [41] found that in some settings individuals with symptomatic infections had different parasite strains compared to asymptomatic individuals. One hypothesis for this is that symptomatic infections originated from imported or recently introduced strains and that immunity to these strains is insufficient. This indicates that the rate of importation may play a role in the proportion of infections that are asymptomatic and symptomatic, especially in areas approaching elimination. An infection with a clone to which immunity has been acquired might lead to infections with shorter duration (e.g., **Figure 1**, yellow line).

#### **3.1. Gametocytes in asymptomatic infections**

Gametocytes derive from a small percentage of asexual parasites that commit to sexual development; therefore, asymptomatic infections with low asexual levels may also have low gametocyte densities. Data from epidemiological studies confirm that most asymptomatic infections with patent or sub-patent asexual stage parasite levels have sub-patent gametocytaemia [42], only detectable by RNA-based molecular methods. However, a few asymptomatic individuals with low-density infections have relatively high gametocyte densities, which could be related to symptomless fluctuations in parasitaemia that result in higher gametocytaemia a few days later. The rate of commitment of asexually replicating parasites to sexual development is another factor that influences gametocyte levels in malaria infections. Adults, who on average carry lower asexual stage parasite densities, have a higher sexual to asexual density ratio [43]. This could be related to an unequal increase in clearance rates of asexual and sexual parasites with age, or potentially to changes in commitment to gametocytogenesis [43]. Consistent with the latter, parasite investment in transmission stages has been shown to vary in areas with different transmission levels, being higher in settings with lower endemicity. Recent data suggest that parasite variations in commitment to gametocytes are epigenetically imprinted and higher in parasites in lower endemicity settings [44].

**3. Parasite factors associated with infection duration**

134 Towards Malaria Elimination - A Leap Forward

After inoculation of sporozoites and the subsequent release of merozoites from the liver, there is a period of time when parasites are present in the blood at concentrations undetectable by conventional diagnostics. In many individuals, parasites then multiply to reach detectable densities, however in other individuals, parasites may remain at low densities that are undetectable. Human challenge studies on non-immune individuals in which parasites are monitored both by molecular methods and by microscopy have estimated that infections are detectable by PCR an average of 3.7 days (range 2–4 days) [35] or 3.1 days (range 0–4) [36] before being detectable by microscopy. Controlled human infections also suggest that the parasite stages that precede blood invasion might influence asexual blood stage dynamics. For example, Churcher and colleagues [37] observed that the inoculum size (the estimated number of sporozoites injected by infected *Anopheles* mosquitoes) influences the time it takes for infections to become patent: individuals receiving five bites from mosquitoes with more than 1000 sporozoites have detectable parasitaemia at least 2 days earlier than those volunteers infected by mosquitoes with 11–101 sporozoites. Quantification of sporozoite counts in wild-caught mosquitoes is necessary to confirm the relevance of this finding in natural settings. In Papua New Guinea, it was estimated that infected malaria vectors had on average (geometric mean) 4000 sporozoites [38], which is of the same order of magnitude as sporozoite counts in mosquitoes used in controlled infections. Microscopy has limited sensitivity to quantify low parasite densities and this will affect its utility for studying any chronicity in infection dynamics. Histidine rich protein 2 (HRP-2), a protein the parasite secretes in the plasma, is considered to be a more accurate measure of total falciparum parasite burden [39], however, this measure does not distinguish between monoclonal and multiclonal infections. Molecular tools are more sensitive and allow discrimination of different parasite genotypes. They have been used to assess the effects of super-infection and exposure to different parasite clones on clinical malaria risk. A study that involved daily blood sampling of children with initially asymptomatic infections [40] suggests that development of symptoms is often associated with appearance of a new parasite strain in the blood and increases in parasite levels. Correspondingly, recent data from Papua New Guinea [16] showed that incidence of infections by new clones correlates with clinical malaria risk. This indicates that clinical malaria is often associated with new infection, presumably by a parasite clone with a previously unencountered antigenic profile.

Consistent with this, genetic analysis of malaria parasite populations in Zambia [41] found that in some settings individuals with symptomatic infections had different parasite strains compared to asymptomatic individuals. One hypothesis for this is that symptomatic infections originated from imported or recently introduced strains and that immunity to these strains is insufficient. This indicates that the rate of importation may play a role in the proportion of infections that are asymptomatic and symptomatic, especially in areas approaching elimination. An infection with a clone to which immunity has been acquired might lead to

Gametocytes derive from a small percentage of asexual parasites that commit to sexual development; therefore, asymptomatic infections with low asexual levels may also have low

infections with shorter duration (e.g., **Figure 1**, yellow line).

**3.1. Gametocytes in asymptomatic infections**

As articulated above, the importance of asymptomatic infections for malaria transmission does not lie in their average sexual stage parasite densities but in the durations of gametocyte carriage and infectiousness over time. A mathematical model [45] fitted to both asexual parasite and gametocyte malariotherapy data estimated infectivity over the course of an infection based on gametocyte density data. This analysis concluded that the majority of infectivity was usually concentrated early in infection, although some patients were significantly infectious later on. However, in this model, it was assumed relatively low infectivity of low gametocyte densities compared with other analyses [46]. While these data are extremely detailed, it is not known whether these dynamics are similar to those in naturally infected individuals who have immunity. Furthermore, specific *P. falciparum* strains were selected for malariotherapy because they were 'benign' and may not exhibit the same behaviour in terms of parasite multiplication rates and gametocyte commitment as parasites in endemic areas.

Although asymptomatic infections do not prompt treatment-seeking behaviour, during community mass treatment campaigns that involve treatment regardless of symptomatology (e.g., mass drug administration (MDA) or mass screening and treatment), these infections are cleared with antimalarials. In a meta-analysis of trials with gametocyte density data [47], the combinations artesunate-mefloquine and artemether-lumefantrine were more effective in preventing the appearance of gametocytes and in clearing existing sexual stage parasites compared to dihydroartemisinin-piperaquine. The choice of drugs to be used during control interventions thus may be important to limit residual transmission from these infections. In Section 4, we discuss the impact of different interventions that target asymptomatic and symptomatic infections.

#### **3.2. Underestimations of parasitaemia linked to sampling**

Two variables linked to blood sampling for parasite detection can influence prevalence and density estimates: volume and timing. Even sensitive molecular assays will not detect low parasite densities in samples if nucleic acids are isolated from small blood volumes. Highvolume PCR has been used in epidemiological studies in Southeast Asia to circumvent this problem and less than 30% of all falciparum infections are estimated to be missed by this method [48]. The timing of blood sampling in parasitological surveys might also affect parasite detection and quantification because asexual falciparum parasites do not circulate continuously; sequestration of falciparum schizonts starts 12-18 hours after merozoite invasion and during this period they might not be detectable. An intensive longitudinal study in Tanzania showed that periodic changes in parasite densities are common. The periodicity of clone-specific detectability indicates that in natural infections, synchronised sequestration of clonal parasite populations occurs [49]. A study [50] that collected samples on two consecutive days found a prevalence disparity of approximately 25% between the two samples. Periodic changes in parasite levels could have a direct impact on the selection of diagnostics, for example by favouring assays that detect more persistent markers, such as HRP-2.

The detection of either asexual or sexual stage parasites is sufficient to establish the diagnosis of infection. Although gametocytes are not known to periodically sequester, there is evidence of periodic variation in gametocyte levels [51] in peripheral blood. For several decades now [52], accumulation of mature gametocytes in the skin [53] has been hypothesised as a possible mechanism of transmission enhancement. If confirmed, this would imply that subpatent gametocytaemias in peripheral blood might be associated with higher-than-expected infectivity.
