Recent records and this report show that the inherent risk of malaria transmission in Singapore demands continuous vigilance [2, 3, 22, 23]. However, with the possibility of relapse of vivax malaria cases among the large population of foreign workers from endemic countries, determining if a cluster is due to local transmission can be challenging. The hypnozoite stage of P. vivax can be dormant in infected liver cells for months or years and relapse of vivax cases are well known and documented. A study in India has shown that the northern P. vivax population was highly polymorphic and a high relapse rate of 40% has been observed . In Singapore, some recent local transmissions, such as the incidents in Jurong Island in 2006 and 2009 involved Indians and Bangladeshis only, and hence, despite the absence of any past medical history of malaria infection, the contribution of relapsed cases to these clusters remained uncertain.
The use of molecular techniques was instrumental in confirming two of the local clusters (Mandai-Sungei Kadut and Sembawang) to be outbreaks. Among the Mandai-Sungei Kadut local cases, RFLP analysis of the msp3α gene revealed that all, except one, shared the same RFLP profile, which is generated by at least two genotypes. Further cloning and sequencing of the msp1 gene confirmed the epidemiological link among all the cases, and the multiple genotypes carried by each individual case. The same situation was found in Sembawang. However, both the msp3α RFLP and msp1 sequence analysis of Jurong Island cases showed that only two cases were molecularly linked, and they were also linked to the Mandai-Sungei Kadut outbreak.
In contrast to the outbreaks at Mandai-Sungei Kadut and Sembawang, events at Jurong Island presented a more interesting enigma. There were nine cases that tested positive for P. vivax and detailed inquiry revealed no recent travel history. However, molecular epidemiology revealed unique PCR-RFLP patterns among the cases and vector findings showed only evidence of Anopheles separatus, a non-vector, as the only Anopheline found within 1 km from the cases. A similar incident occurred in 2006 in the same area, when 13 cases were found among foreign workers from malaria endemic regions, and no Anopheles was found in that location. The molecular and entomological data suggest a possible coincident relapse among the cases at Jurong Island, and data are in agreement with epidemiologic data, which showed that only foreign workers from endemic countries were involved in the Jurong island cluster. Alternatively, this may reflect the limit of the molecular techniques in finding common haplotypes when only a single blood specimen was obtained from each worker. The dynamics of disease transmission at Jurong Island are still not fully understood and clearly warrant further entomologic and molecular studies.
Interestingly, where there was substantial molecular evidence of local transmission, adult An. sinensis was predominant, and multiple breedings of the Anopheline were found around the vicinity. There are no reports of An. sinensis being a vector in Southeast Asia. In the Malayan region, including Singapore, An. sinensis has not been considered a vector, since it was found to be more zoophagic and no malaria parasite sporozoites have been found in the mosquito [16, 25]. In Thailand, An. sinensis has been considered only as a refractory vector with weak transmission capability [16, 26]. However, An. sinensis has been incriminated as a vector of malaria in Northeast Asia viz. Korea and China [27–32]. In a recent experiment carried out on the susceptibility of different strains of An. sinensis towards Korean and Thai strains P. vivax, it was found that An. sinensis from Korea was able to develop oocysts and sporozoites against local strain of P. vivax and those from Thailand. On the other hand, when fed with P. vivax (Thailand strain), An. sinensis from Thailand failed to develop sporozoites . This clearly demonstrated the possible variation in terms of the susceptibility of the different geographical strains of An. sinensis to malaria parasites. Park and co-workers reported that the sequences of ITS2 and COII among 10 Asian An. sinensis strains from China, Japan, Korea and Thailand were almost identical to one another with very small sequence variation (< 1.5% in both regions), despite its relatively wide geographical distribution range of > 2000 mi . In this current study, the local strains of An. sinensis has also been found to be very similar (genetic divergent 0.5 to 1.3%) to the single cosmopolitan species concluded by Park and coworkers. Despite the varying vectorial status of the species demonstrated by laboratory experiment and field epidemiological evidences, current molecular techniques are not able to discriminate between vector strain and non-vector strain. Genetic features that better represent vector status should be of research interest.
In the two recent Singapore outbreaks in Mandai and Sembawang, An. sinensis has been the predominant species and its apparent intrusion into the urban setting that fringes the rainforest may suggest some level of adaptation. Furthermore, its biting period appeared to coincide with the period (7:45 pm to 12 am) of human activity. Whether An. sinensis is a malaria vector in Singapore, facilitated by the adaptation, remains elusive and the vector of the two small concurrent outbreaks remains cryptic.
Studies in Korea have shown that human biting rate of An. sinensis has been 87.5 bites/man/night and the vectorial capacity was 0.081 . In our brief study, though the vectorial capacity was similar in Singapore (0.04 and 0.17), the bites/man/night was much lower, below 5. It thus seems that the high transmission potential may be due to the high human biting rate of the mosquito.
Results from this investigation strongly suggests that An. sinensis, which has never been implicated as a vector in Singapore, was the predominant Anopheline in Mandai-Sungei Kadut and Sembawang. In these areas, there was substantial molecular evidence of local transmission and multiple breedings of the Anopheline were found. Currently, work is ongoing to determine the vectorial status of An. sinensis. The human blood index is not known and no sporozoites have been found. The implication of An. sinensis as a vector would be significant, as this may result in the need to redefine boundaries of malaria receptive areas which are presently based on the geographical distributions in Singapore of An. maculatus and An. epiroticus (= An. sundaicus) .
Since it is critical to detect early any local transmission for control measures to be effective, the precautionary principle is applied by the public health authorities such that every clustering of cases in place and time is deemed to be a local outbreak until proven otherwise. Molecular tools and techniques can elucidate the parasite genotype or strain and the data can be used to track and link cases in an outbreak, but these techniques take time to be developed and validated. Meanwhile, interviews to determine case movements and previous infection remain the mainstay in outbreak prevention and control. Moving forward, this study shows how entomologic and molecular tools can be applied in epidemiologic investigation to provide an understanding of outbreaks. If conducted promptly and regularly, the data can be useful for assessment and mitigation of transmission.