**5. Correct identification of malaria vectors and** *Plasmodium* **detection**

High levels of malaria transmission occurring in forest-fringe areas of Southeast Asia is explained by movements of people in search of forest products and exposure to many highly efficient vector species that have adapted to forest ecotypes [66, 85, 102, 103]. The wide diversity of both the deep-forest (e.g., Leucosphyrus Group of mosquitoes), forest-fringe and deforested area main vectors (e.g., *An. minimus*, *An. maculatus s.l.*, *An. culicifacies s.l.*, *An. fluviatilis s.l.*, *An. letifer*, *An. donaldi*), as well as their great potential to adapt to habitat changes, means that the consequences of deforestation on malaria transmission in Southeast Asia are difficult to predict and unlikely to be unidirectional [104]. Whilst *An. dirus* and *An. baimaii*, main vectors of the Dirus complex, can find tree-crop plantations suitable for breeding, a close association between malaria and rubber plantations has been demonstrated [4, 105–108], contributing to high larval and pupal density during the rainy season [90, 91] and low numbers during the cool-dry season [92, 109], or provide conditions that are similar to this vector's natural habitat [110]. This ecological adaptation in human settlements and shaded plantations contributes to outdoor transmission among rubber tappers.

The identification of secondary or incidental vector species poses new challenges as shown by mixed results of sporozoite-positivity using nested Polymerase chain reaction (PCR) and routine circumsporozoite enzyme-linked immunosorbent assay (CSP-ELISA) (**Table 2**). Confirmation of all positive CSP-ELISA results by a second CSP-ELISA test on the heated ELISA lysate, especially in zoophilic species showed a relatively high proportion of false positives (40%) [93]. On the other hand, PCR analysis of Deoxyribonucleic acid (DNA) extracted from the head and thorax alone, along with sequence data, revealed five *Anopheles* species (*An. hyrcanus*, *An. barbirostris s.s.*, *An. barbirostris* clade III, *An. nivipes*, and *An. peditaeniatus*) infected with *Plasmodium falciparum,* which are not considered major vectors in the GMS [94]. Similarly, out of 11 *P. falciparum* CSP positive samples from Bangladesh, seven turned out to be positive by PCR suggesting that *An. maculatus*, *An. jeyporiensis* and *An. nivipes* play important roles in malaria transmission in Kuhalong District [95]. In Vietnam, the role of a secondary vector, *An. pampanai* infected with *P. vivax*, was also reported in the Binh Phuoc Province [96]. Morphological misidentification of the closely related sympatric species, such as *An. aconitus*, *An. pampanai* and *An. varuna* are common [99, 100]. Morphological identification of *Anopheles* specimens prior to PCR assays allows them to be sort out at the group or complex level but does not permit species identification [85]. PCR assays must be applied for a reliable identification to the species level, which ensures that data received by malaria vector control programmes are suitable for targeting the correct vector species [101]. Given the low infection rates among many of these species especially in elimination phase, it is important for field entomologists to assess various

species' role in malaria transmission in the eco-epidemiological context. When changing objectives from control to elimination of malaria in Southeast Asia, the need to focus not only in the so-called main vector species, but also on secondary vectors is increasingly important.

Molecular identification was specifically conducted on *Anopheles barbirostris s.s*. and *An. barbirostris* clade III; *An. hyrcanus* and *An. hyrcanus s.s*; *An. peditaeniatus* and *An. nivipes*, and morphological identification for the other *Anopheles* species.

**Table 2.** Sporozoite infectivity rates of less known (secondary) vectors along the Bangladesh-Thailand-Cambodia

**Morphological**  *Anopheles* **species\***

*An. maculatus* 

*An. annularis* 

*An. barbirostris* 

*s.l*

*s.l.*

*s.s.*

*An. peditaeniatus*

*An. philippinensis*

\*

corridor.

**Nested PCR, Cambodia [93]**

> **Positive/ total**

**Total collection (%)**

*An. hyrcanus* 0.09 2/2

**Circumsporozoite ELISA, Thailand [97]**

**Total collection (%)**

*An. kochi* 0.93 1/44

*An. vagus* 41.9 25/1978

*An. karwari* 5.16 11/244 1.7

**Prior heating of eluate and circumsporozoite ELISA, Bangladesh** 

21.43 4/640 4.3 2/97

5.08 3/139

**Positive /total**

**PCR confirmation of ELISA-positives Bangladesh [95]**

> **Positive / total**

105

**Total collection (%)**

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**[98]**

**Total collection (%)**

Human and Simian Malaria in the Greater Mekong Subregion and Challenges for Elimination

**Positive /total**

14.43 3/431 0.78 1/19

6.6 3/55 3.52 1/105 2.9 1/140 7.4 1/186

*An. nigerrimus* 0.87 1/21 4.1 1/104

*An. nivipes* 10.8 1/264 *An. jeyporiensis* 3.1 1/142 18.9 2/479

3/219 24.7 25/1169

Deforestation may deplete the populations of deep-forest vectors and so initially reduce malaria transmission; in some localities this depletion may be followed by the invasion of other efficient vector species resulting in increased transmission. With the exception of two longitudinal studies examining the effects of progressive land use changes from pre-development forest to oil palm cultivation on the distribution of disease vectors and malaria incidence [111], there is a striking lack of primary research directly measuring the impact of deforestation on malaria in Southeast Asia [104]. Recent studies showed that *An. dirus s.l.* was abundant in rubber plantations in Myanmar [109] and *An. baimaii* (molecularly identified) adults were caught from human landing collections in Wae Kha Mi, Mon State, the site of an acceptability study of permethrintreated clothing [110]. In Lao PDR, a total of 46 *An. dirus s.l.* were collected, of which 31 were


cassava, fruit orchards). On return to their usual settlements, they contribute to the spread of malaria within and across international borders [41, 43]. By creating hot-spots of malaria and disproportionately affecting people with certain high-risk occupations [86, 89], residual trans-

High levels of malaria transmission occurring in forest-fringe areas of Southeast Asia is explained by movements of people in search of forest products and exposure to many highly efficient vector species that have adapted to forest ecotypes [66, 85, 102, 103]. The wide diversity of both the deep-forest (e.g., Leucosphyrus Group of mosquitoes), forest-fringe and deforested area main vectors (e.g., *An. minimus*, *An. maculatus s.l.*, *An. culicifacies s.l.*, *An. fluviatilis s.l.*, *An. letifer*, *An. donaldi*), as well as their great potential to adapt to habitat changes, means that the consequences of deforestation on malaria transmission in Southeast Asia are difficult to predict and unlikely to be unidirectional [104]. Whilst *An. dirus* and *An. baimaii*, main vectors of the Dirus complex, can find tree-crop plantations suitable for breeding, a close association between malaria and rubber plantations has been demonstrated [4, 105–108], contributing to high larval and pupal density during the rainy season [90, 91] and low numbers during the cool-dry season [92, 109], or provide conditions that are similar to this vector's natural habitat [110]. This ecological adaptation in human settlements and shaded plantations contributes to out-

The identification of secondary or incidental vector species poses new challenges as shown by mixed results of sporozoite-positivity using nested Polymerase chain reaction (PCR) and routine circumsporozoite enzyme-linked immunosorbent assay (CSP-ELISA) (**Table 2**). Confirmation of all positive CSP-ELISA results by a second CSP-ELISA test on the heated ELISA lysate, especially in zoophilic species showed a relatively high proportion of false positives (40%) [93]. On the other hand, PCR analysis of Deoxyribonucleic acid (DNA) extracted from the head and thorax alone, along with sequence data, revealed five *Anopheles* species (*An. hyrcanus*, *An. barbirostris s.s.*, *An. barbirostris* clade III, *An. nivipes*, and *An. peditaeniatus*) infected with *Plasmodium falciparum,* which are not considered major vectors in the GMS [94]. Similarly, out of 11 *P. falciparum* CSP positive samples from Bangladesh, seven turned out to be positive by PCR suggesting that *An. maculatus*, *An. jeyporiensis* and *An. nivipes* play important roles in malaria transmission in Kuhalong District [95]. In Vietnam, the role of a secondary vector, *An. pampanai* infected with *P. vivax*, was also reported in the Binh Phuoc Province [96]. Morphological misidentification of the closely related sympatric species, such as *An. aconitus*, *An. pampanai* and *An. varuna* are common [99, 100]. Morphological identification of *Anopheles* specimens prior to PCR assays allows them to be sort out at the group or complex level but does not permit species identification [85]. PCR assays must be applied for a reliable identification to the species level, which ensures that data received by malaria vector control programmes are suitable for targeting the correct vector species [101]. Given the low infection rates among many of these species especially in elimination phase, it is important for field entomologists to assess various

mission under these circumstances has so far hindered progress towards elimination.

**5. Correct identification of malaria vectors and** *Plasmodium*

**detection**

104 Towards Malaria Elimination - A Leap Forward

door transmission among rubber tappers.

\* Molecular identification was specifically conducted on *Anopheles barbirostris s.s*. and *An. barbirostris* clade III; *An. hyrcanus* and *An. hyrcanus s.s*; *An. peditaeniatus* and *An. nivipes*, and morphological identification for the other *Anopheles* species.

**Table 2.** Sporozoite infectivity rates of less known (secondary) vectors along the Bangladesh-Thailand-Cambodia corridor.

species' role in malaria transmission in the eco-epidemiological context. When changing objectives from control to elimination of malaria in Southeast Asia, the need to focus not only in the so-called main vector species, but also on secondary vectors is increasingly important.

Deforestation may deplete the populations of deep-forest vectors and so initially reduce malaria transmission; in some localities this depletion may be followed by the invasion of other efficient vector species resulting in increased transmission. With the exception of two longitudinal studies examining the effects of progressive land use changes from pre-development forest to oil palm cultivation on the distribution of disease vectors and malaria incidence [111], there is a striking lack of primary research directly measuring the impact of deforestation on malaria in Southeast Asia [104]. Recent studies showed that *An. dirus s.l.* was abundant in rubber plantations in Myanmar [109] and *An. baimaii* (molecularly identified) adults were caught from human landing collections in Wae Kha Mi, Mon State, the site of an acceptability study of permethrintreated clothing [110]. In Lao PDR, a total of 46 *An. dirus s.l.* were collected, of which 31 were from immature rubber plantations, nine from mature rubber plantations, five from secondary forests and one from the rural village [105] (Tangena Julie-Ann, personal communication).

which acquired infection from the macaques. Perhaps even likely given that confirmed vectors of human plasmodia in Southeast Asia also become naturally infected by the monkey malaria species [127]. A recent case control study conducted in Sabah revealed that the age group >15, predominantly males, working in farms, plantations, forested areas, and with travel history, were independently associated with the risk of acquiring knowlesi malaria [128]. It also highlighted

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107

There are only few investigations on record in understanding bionomics of vectors transmitting *P. knowlesi* malaria. In order to implement vector control activities, the bionomics of the vectors must be understood. Based on few studies, it has been shown that the vectors are biting in the early part of the night from 18:00 h to 21:00 h and mostly outdoors [121–123, 129]. In these rural areas, people go to bed by 22:00 h and they are up by 05:00 h. The results showed that only 39.79% of *An. balabacensis* [123], 43.8% of *An. latens* [121] and 12.8% of *An. cracens* [122] were found biting during this sleeping time. Thus, current vector control measures like IRS and ITNs are not appropriate for the exophagic and exophilic vectors. The forests in Southeast Asia is providing a favorable environment with high percentage of macaques being positive for *P. knowlesi* [130–132], and with the presence of the vectors, it is going to be a daunting task to eliminate malaria. On a global scale, malaria has been reduced to low levels due to the scaling up of ITNs, IRS, ACTs, and intermittent preventive treatment to infants and pregnant women [133]. Thus, it is obvious that new tools are urgently required for successful malaria elimination.

It is known that the two human malaria species (*P. falciparum* and *P. vivax*), which infects millions of people actually were of zoonotic origin (from the African apes), which evolved thousands of years ago [134, 135]. Thus, there is always a possibility that in the future *P. knowlesi* and other simian malarias may become established in humans, especially when human malaria is eliminated. However, currently human-to-human transmission of knowlesi malaria by mosquitoes has not been established. This is crucial in the light of malaria elimination and more focused research is needed on this topic if we are to succeed with malaria elimination.

Changing landscape affects *Anopheles* distribution, mosquito density and diversity in Malaysia, and more globally Southeast Asia [105, 111, 136–138]. It has been shown that with loss of forest cover, cases of *P. knowlesi* have increased in Sabah [119]. Land use change has also led to increase of malaria cases due to various factors such as increase of macaques in small forest patches along with the colonization of the main vectors [119, 136]. It is interesting to note that *An. balabacensis,* the predominant vector of human and simian malaria, was found in great abundance in logged forest, followed by thinly logged virgin jungle reserve and was lowest in primary forest [136]. This vector was also found to be biting humans more at ground level compared to canopy level [136]. It is therefore important to include both the public health and agro-forestry sectors in controlling malaria vectors in the country. Studies from Thailand also indicate that if landscape management should be used for malaria control in northern Thailand, large-scale reduction and fragmentation of forest cover would be needed [139, 140]. Such drastic actions, however, do not

align with current global objectives concerning forest and biodiversity conservation.

The vectors of simian malaria described to date were *An. hackeri* (Leucosphyrus Group) [141] recorded biting mainly the macaques and large numbers were collected resting on Nipah palm trees in Selangor in 1960s; *An. cracens* (Dirus Complex) [122] biting both macaques and humans and found mainly in the forest and farms; *An. latens* (Leucosphyrus Complex) [121] was the

that IRS was associated with decrease of risk [128].
