Malaria Elimination in China: Innovative Three-Layer Strategy Applied to the Outbreak of Indigenous Cases in Sanya, Hainan

Yuchun Li Hainan provincial center for disease control and prevention https://orcid.org/0000-0001-6951-4403 Yingjuan Huang Tangshan City Center for Disease Control and Prevention Renqiang Chen Sanya city for disease control and prevention Weizhen Huang Wuzhishan city for disease control and prevention Huanzhi Xu Wuzhishan city for disease control and prevention Rongshen Ye Baoting county center for disease control and prevention Shaoling Huang wanning center for disease control and prevention Ji Zhen dongfang city for disease control and prevention Xiaodan Wen danzhou city for disease contol and prevention Guoyi Wang Qiongzhong county for disease control and prevention Yong Liu tunchang county for disease control and prevention Haishan Li ledong county for disease control and prevention Zaichun Zhen changjiang county for diease control and prevention Jian Wang Baisha county for disease control and prevention Guoshen Wang qionghai county for disease control and prevention Chong Chen lingshui institute of health supervision Wen Zeng hainan center for disease control and prevention Feng Meng hainan provicial center for disease control and prevention Xiaoming Huang hainan rovincial center for disease control and prevention Guangze Wang Hainan Provincial Center for Disease Control and Prevention Bing Yang Hainan Provincial Center for Disease Control and Prevention Yan Chen (  jkban702@163.com ) hainan Provincial Center for Disease Control and Prevention


Abstract
Background Forest-goers have been the most severely infected by malaria in Hainan since the 1990s. In 2015, there were successively 6 cases of P. malariae infection as an outbreak by forest-goers in Sanya city, Hainan Province. According to the 1-3-7 approach, active case detection(ACD) was performed with guidance from information from Case 1. Cases 2-4 had the same epidemiological history as Case 1 and were similarly con rmed to be infected with P. malariae. However, the clues from Case1 came to an end and it was uncertain whether there were other potential infection sources or whether transmission was established in local villages.

Methods
There was no detailed strategy for the outbreak until 2015, although the 1-3-7 approach was certi ed to be effective for sporadic case. A retrospective study was conducted on this epidemic of P. malariae transmission among forest-goers in Hainan, 2015. A three-layer strategy (TLS) that integrated malaria joint prevention and control strategies(JPCS) and the 1-3-7 approach was rst generated and applied to the disposal of the outbreak in Sanya in 2015, and its was also applied to strengthen epidemic measures to prevent emergence by residual foci from 2016 to 2018. The data were entered by Microsoft O ce Excel 2007 for collection and statistic analysis by SPSS.

Results
With guideline of TLS, three villages in Gaofeng town, Sanya city and Seven villages in Xinzheng Town, Baoting County, were selected as the rst layer with design of TLS. In the rst layer, 1,823 blood slides and PCR screening from villages in Baoting and Sanya were conducted on all residents by active case detection (ACD) and four cases were identi ed. In the second layers, passive case detection (PCD) and ACD for forest-goers were conducted and 7,831 blood slides were screened on all forest-goers. In the third layer, PCD was conducted for forest-goers, and 95,350 blood slides were screened on all forest-goers.
Meanwhile, vector surveillance and control were also conducted to support foci disposals and to minimize the possibility of transmission. From 2016 to 2018, TLS was also applied to strengthen epidemic measures to prevent emergence by residual foci. In the rst layer, chemoprophylaxis and ACD were conducted on residents in the villages of the foci and the surrounding villages, and 89.5% residents accepted chemoprophylaxis. Chemoprophylaxis and ACD were conducted and 79.1% received chemoprophylaxis while seeking forest-goers in the second layer. In the third layer, 89.6% received chemoprophylaxis while seeking forest-goers.

Conclusions
The innovative strategy of TLS not only was successfully applied in an epidemic response strategy in Sanya in 2015 but also was implemented in a strengthened intervention on forest-goers from 2016 to 2018 and nally achieved the goal of eliminating malaria in 2020 as scheduled. TLS is suitable for outbreak by indigenous case of unknown causes or for sporadic cases with defects in epidemic information, while 1-3-7 approach is suitable for sporadic cases which have clearly epidemic clues. Furthermore, TLS could be one of the major achievements after 1-3-7 approach, targeting outbreak in elimination malaria.

Background
Historically, malaria has been one of the most important infectious diseases in P.R.China. Hainan and Yunnan provinces were the main malaria transmission areas in P.R. China. Prior to 2010, indigenous cases of P. falciparum and P. vivax were still frequently detected in Hainan. Hainan is characterized geographically by mountains, hills, plateaus and plains. The tropical monsoon and marine climates jointly produce a generally warm temperature, within a suitable range not only for cultivating tropical plants (coconuts, areca nut and rubber trees, and more) and but also for the breeding of An. dirus and An. minimus [1]. Malaria cases in Hainan Province were mainly distributed in patches in the southwestern region of the island and a high-risk group for malaria infection can not be ignored in Hainan.
Forest-goers who sleep at night in the mountains include local residents and migrants are for picking and planting because of production and living. Forestgoers belong to a high-risk group of people who can become infected with malaria in Hainan [2]. Forest-goers have composed the main group of indigenous malaria infections in Hainan Province since 1990s. The infection rate was 30.9% (95/307), which is more than twice that of the non-forest-goer residents (15.2%) [3]. Malaria infection in forest-goers has been found to be related to the frequency of staying in the mountains and whether medicine was taken to prevent malaria, in addition the acceptance of the history of malaria control propaganda and the use of mosquito control measures [4][5][6]. Since the 1990s, Hainan Province has been engaged in malaria control and elimination for forest-goers for a long time. The strategy for the prevention and control of malaria, which had been supported by the Global Fund for ten years since 2003, involved focusing on treatment in patients, timely tracking the disposal of foci or outbreaks, and applying insecticide-treated nets (ITNs) or distributing long-lasting insecticidal nets (LLINs) to reduce the incidence of malaria in forest-goers [7]. In 2010, Hainan joined the National Malaria Elimination Programme (NMEP ) and involved eight Class I counties (endemic counties of P. falciparum) and ten Class II counties (endemic counties of P. vivax) in Hainan Province [8]. Since 2011, malaria elimination has been o cially implemented in Hainan Province. The last indigenous malaria case of P. vivax was reported and the "1-3-7"approach (case reporting within 1 day, case investigation within 3 days, and focus investigation and action within 7 days) launched in 2012.
Since 2013, there have been only imported malaria cases in Hainan Province, and every sporadic foci has been classi ed and disposed under the guidelines of the 1-3-7 approach. However, indigenous cases of P. malariae appeared in 2015 [9]. According to the 1-3-7 approach, active case detection (ACD) was performed with guidance from information from Case 1. Three co-workers had the same epidemiological history as Case 1, but whether other potential infection sources or transmission was established in local villages was not known. There was no detailed strategy for the outbreak until 2015, although the 1-3-7 approach was certi ed as effective for imported malaria cases [10]. In response to the epidemic in the elimination stage, an innovative three-layer strategy (TLS) that integrated malaria joint prevention and control strategies(JPCS) and the 1-3-7 approach was rst generated and applied to the disposal of the epidemic as an outbreak in 2015. Subsequently,TLS applied to the prevention of the emergence by residual foci from 2016 to 2018. Finally, Hainan Province achieved the goal of eliminating malaria in 2019 and a WHO eld certi cation of malaria elimination in 2021 [11]. This retrospective study on P. malariae transmission in 2015 as an example to illustrate and summarize the lessons learned about TLS processes from generation to application.

Study site
Sanya city is located at the southern tip of Hainan Island, and it is in the tropical climate zone of the surrounding Paci c Ocean, with an annual average temperature of 25.7°C. The tropical geographical climate environment is suitable for the reproduction of vectors of malaria throughout the year. Anopheles sinensis is considered to be a major vector in Sanya, but An. minimusis is also sometimes captured in mountainous regions. Sanya was the main endemic city and county of malaria in Hainan Province, with the recorded malarial parasites included P. vivax and P. falciparum [12]. Gaofeng township is located at the edge of Sanya city and borders Baoting, Ledong County and Wuzhishan City.

Design of the TLS
Due to the end of the clues from Case 1 and the dilemma of uncertain potential infection sources, a comprehensive strategy was essential for outbreak management rather than the disposal of each foci under the guidelines of the 1-3-7 approach. Case screening in these villages was the most effective method to solve this dilemma. Based on the geographical location of malaria cases, population characteristics, history of malaria joint defence and work requirements of the 1-3-7 approach, a three-layer strategy (TLS) that integrated malaria JPCS and the 1-3-7 approach was rst generated for screening. Villages with cases and adjacent villages were regarded as the rst layer in the prevention and control strategy. Gaofeng town and adjacent towns with cases were regarded as the second layer. Sanya city and adjacent counties or cities with cases were regarded as the third layer.
TLS in disposal of the outbreak TLS rstly applied to the disposal of the outbreak. Brie y, the detailed disposal strategies are as follows: In the rst layer, active case detection (ACD) was conducted mainly in the villages of the foci and the surrounding villages. In the second layer, the population of forest-goers was the main target, with an emphasis on the strategy of tracing clues, conducting spot visits to forest-goers in targeted townships, and conducting malaria blood slide screening for forest-goers who had a history of overnights in mountains with fever cases. In the third layer, the population of forest-goers was the main target, to provide them with health education and to alert them to the risk and to recommend to them not to overnight in the mountains at that time, while simultaneously reminding doctors to pay attention to forest-goers. To improve the accuracy, PCR detection was conducted on ltered blood collected from all samples in the rst layer or suspicious samples in others layers. The partial gene of the SSU rRNA gene was ampli ed by PCR with Plasmodium-speci c primers (rPLU1 and rPLU5) in Nest1 PCR and genus-speci c primers (rPLU3 and rPLU4) in Nest2 PCR [14] .

TLS in strengthening of epidemic measures
TLS was also applied to strengthen epidemic measures to prevent emergence by residual foci from 2016 to 2018, to achieve the goal of eliminating malaria in 2020. Brie y, the detailed strengthen epidemic strategies are as follows: In the rst layer, chemoprophylaxis and ACD were conducted in the villages of the foci and the surrounding villages. In the second layer, chemoprophylaxis and ACD were conducted mainly in the townships of the foci and the surrounding townships. In the third layer, preventive medication and health education were conducted in Sanya and the associated counties.

Data collection and statistical analysis
Basic and detailed malaria case information were gathered from the Infectious Diseases Information Reporting Management System (IDIRMS) and the information of 1-3-7 were gathered from Parasitic Diseases Information Reporting Management System (PDIRMS ) [15]. The historical malaria information data before 2004 were gathered from reports or archives. Detailed TLS information on the implementation for blocking P. malariae transmission from 2015 to 2018 was obtained from the annual reporting reports or archives of the local CDC. The population data and other relevant information on the villages were obtained from government annual reports or archives. The data were entered by Microsoft O ce Excel 2007 for collection and analysis by SPSS.

History of malaria surveillance in Sanya and in Hainan
Hainan had malaria endemic areas of P. falciparum and P. vivax. Since 1954, a parasite survey of local residents (PSLR), which acted as a type of surveillance for ACD, was conducted in Hainan Province every year. From PSLR recordings, 10 cases (CI: 2-42) of P. malariae were found every year in Hainan Province before 1964. However, Sanya also conducted PSLR but found only P. falciparum malaria and P. vivax since 1959 [16]. From IDIRMS, 27 malaria cases were reported in Sanya from 2009 to 2018, including 6 cases of P. falciparum malaria, 10 cases of P. vivax malaria, 6 cases of P. malariae malaria, 1 case of P. ovale malaria, and 4 cases of unclassi ed malaria. During the malaria elimination phase from 2011 to 2018, a total of 14 cases of malaria were reported in Sanya city, including 8 indigenous cases and 6 imported cases. The indigenous cases reported in 2011 were P. vivax. No cases were reported from 2012 to 2014 and 2017 [17]. In 2015, 6 indigenous cases of P. malariae were found (Fig. 1).
Established epidemic response for forest-goers in TLS in 2015 Three villages in Gaofeng town of Sanya city were selected as the rst layer, including Baolong, Lixin and Zhanan. Seven villages in Xinzheng Town, Baoting County, were also selected as the rst layer, including shirang, maowen, xinzheng, maopeng, baodao, shenna and nangai; All of the residents were targeted except for migrants and were not suitable for users, with active case detection (ACD). 1,823 blood slides and PCR screening from villages in Baoting and Sanya were conducted on all residents. Three positive samples were found from blood samples, and thirteen positive samples were found from PCR. After cross-checking, four cases were identi ed and other false positives were ruled out, including Case 2, 3, 4 and Case 6. Case 5 was found when he went to the hospital voluntarily. they were con rmed to be infected with P. malariae. In the second layers, ten townships were chosen, and passive case detection (PCD) and active case detection (ACD) for forest-goers were conducted mainly in Gaofeng townships of the foci and surrounding townships, including Gaofeng, Yucai in Sanya city, Daan, Zhizhong in Ledong County, Xiangshui, Maogan, Nanlin, Sandao, Xinzhen in Baoting County and Changhao in Wuzhishan City. 7,831 blood slides were screened from target townships and tests conducted on all forest-goers. In the third layer, 12 counties or cities were selected for health education, and passive case detection was conducted mainly around Sanya and the associated counties for forest-goers, including Ledong, Baoting, Wuzhishan, Dongfang, Lingshui, Qiongzhong, Baisha, Changjiang, Wanning, Qionghai, Tunchang and Danzhou. 95,350 blood slides were screened from target townships, and tests were conducted on all forest-goers (Table 1). Meanwhile, vector surveillance and control were also conducted to support foci disposals and to minimize the possibility of transmission (Table 2).   Tunchang  17  0  1249  2927  9  0  1081  3647  19  0  2253  3110  0  0  470   Danzhou  154  0  73  11  37  0  124  154  1  0  108  10  0  0  120   Total  277  37  3592  8874  59  61  3631  6133  22  37  4345  5592  27 21 1572 *The data of IRS didn't showed and IRS implemented only in Sanya for disposal six focis.

Strengthened intervention on forest-goers in TLS from 2016 to 2018
Target villages between 2016 and 2018 were included in the epidemic response strategy for forest-goers in 2015, but residents adopted the number of residents at that time. Piperaquine phosphate was selected as a chemoprophylactic drug. In March from 2016 to 2018, residents in the targeted villages were given a total dose of 1200 mg orally before bedtime for adults within 3 days, from 2016 to 2018. Starting in April from 2016 to 2018, forest-goers were administered 600 mg orally for adults before bedtime within a day. Doses for children decreased by weight or age. Villagers, including forest-goers, were required to sign informed consent before administration. At the same time, active case detection (ACD) should be conducted while seeking forest-goers. If fever is found in forest-goers, examination for malaria must be performed, including microscopy. (Fig. 2) In the rst layer, chemoprophylaxis and ACD were conducted on residents in the villages of the foci and the surrounding villages, and 89.5% residents accepted chemoprophylaxis. In the second layer, chemoprophylaxis and ACD while seeking forest-goers were conducted mainly in the townships of the foci and the surrounding townships. Of the 5,489 people who were forest-goers, 79.1% received chemoprophylaxis while seeking forest-goers. In the third layer, preventive medication and health education were conducted in Sanya and the associated counties for forest-goers. Of the 10,364 people who belonged to forest-goers, 89.6% received chemoprophylaxis while seeking forest-goers. Meanwhile, the training of clinicians, public health and laboratory personnel was strengthened to improve the capacity to respond to the epidemic from 2015 to 2018 (Table 3).  Technicians   2015  2016  2017  2018  2015  2016  2017  2018  2015  2016  2017  2018   Sanya  23  18  20  24  36  24  20  22  29  18  26  19   Ledong  25  26  46  0  51  68  34  23  25  26  46  23   Baoting  28  23  24  17  83  43  72  20  16  17  19  15   Wuzhishan  9  11  10  0  10  10  10  23  10  10  6  0   Dongfang  24  21  22  27  42  42  22  27  23  21  23  22   Lingshui  21  46  56  22  45  66  56  56  42  25  22  22   Qiongzhong  105  0  25  24  0  0  0  0  2  0  Case reports and epidemiological characteristics of this outbreak under TLS guideline A total of 6 P. malariae cases occurred in Sanya city in 2015 under TLS guideline. All cases except for the index case (case 1) were reported within 1 day through IDIRMS. From PDIRMS, None of the 6 patients had a history of travelling abroad, previous malaria or blood transfusion. Six cases were regarded as indigenous cases of P. malariae. All of the reported cases were con rmed by microscopy and sequencing. These cases were reported between September and November: 3 cases in September, 1 case in October and 1 case in November. The age group (19-40 years of age) belonged to the younger-middle age group.
They were all males and farmers in occupational categories. The total indigenous cases were reported at the Gaofeng township level (N=6, 100%) or in 3 villages. Two cases(Case 1 and 5) were found by passive case detection (PCD), but 4 cases were found by active case detection (ACD) in their villages (Table   4). We also collected information about the outpatient visiting behaviors and found that 100% of patients visited local village clinics after experiencing fever symptoms. However, only 16% (case 1) of patients were diagnosed with malaria in county-level hospitals, although 33% (cases 1 and 5) continued to higherlevel hospitals, including township-level or/and county-level hospitals.

Con rmation and epidemical investigation within 3 days 6 100%
Foci classi cation and response within 7 days 6 100% From the results of the epidemic survey, 5 patients had a history of night sleeping in mountains before the onset of fever and were classi ed as forest-goers.
However, one case (case 6) had no above mentioned history before the onset of fever. A total of 5 cases (Case1, Case2, Case3, Case4, Case5) had a history of collecting bodhi fruit (Daemonoropus margaritae Becc varpalawanica) or honey in the mountains in almost all months of the year. Case 1, who coworked with cases 2, 3 and 4, climbed mountains and slept overnight to collect bodhi fruit, and case 5, who coworked with the other 2 persons, did so also. However, Case 6 stayed in villages and had no history of staying in mountains overnight. From Case 1 to Case 5, infection with P. malariae was associated with the behaviour of overnights in the mountains. Moreover, infection also occurred in the village from case 6 ( Fig. 3).

Infection source
A total of 6 cases were found in this epidemic, among which 5 cases belonged to forest-goers, who had a history of overnight sleeping in the mountains, and 1 case did not belong to forest-goers and had no history of overnight sleeping in mountains. At present, there are three possible sources of infection in mosquito-borne malaria cases: 1) transmission caused by a long incubation period. 2) transmission caused by imported sources of infection and subsequent reintroduction. 3) transmission from an animal host in forests. Based on the epidemic characteristics of these cases, especially age characteristics, the second is more likely, the third is possible, and the rst is almost impossible.

Discussion
Malaria infection in forest-goers has also been an important and di cult point with regard to malaria elimination in Hainan. Forest-goers were the main group of malaria infections in Hainan, mostly due to the abundant forest products combined with human behaviour. Abundant forest products, such as wood, honey and wild animals, have attracted attention from local residents and mobile populations [18]. The behaviour of these people, especially going overnight without nets, leads to malaria infection. In malaria control stage, an investigation in Nanqiao, Wanning City showed that overnight behaviour in mountains, the low usage rate of mosquito nets and the lack of malaria prevention knowledge were the main factors that affected the epidemics and the control of malaria [19,20]. In the stage of elimination of malaria, this outbreak was a typical case of malaria transmission caused by forest-goers, and ve of them had a history of staying overnight in mountains, although they were infected with P. malariae, which was rst discovered in Sanya, Hainan.
Based on this outbreak and other outbreaks in other parts of China, outbreaks still occur during the malaria elimination phase [21,22], although its much less than during the control stage. Innovative TLSs were invented and rst applied in this outbreak, and Case 6 was importantly detected as an asymptomatic carrier. If only the 1-3-7 approach had been implemented, Cases 2-4 associated with Case 1 and two persons associated with Case 5 would be tested.
Comparing the results of the 1-3-7 approach to the results of this study, TLS improved the case detection ability and detected potential sources of infection, such as asymptomatic carriers and cases who did not seek medical treatment. TLS may also be suitable for outbreaks in the screening of unexplained indigenous cases or introduced cases in countries and regions that are undergoing malaria elimination and for current malaria outbreak areas.
From the results of the disposal outbreak in this study using TLS, the spread scope of the epidemic was further estimated, and four cases were distributed in different villages, but they belonged to the same township as GaoFeng township. No malaria cases have been found outside Sanya city. This information may be further applied in the management of high-risk groups and different intervention measures adopted in different layers, which are similar to the current strategies against COVID-19 in China [23].
This outbreak was caused by P. malariae and was highly likely an imported-introduced case. Sanya is a tourist city with a large number of migrant people, including people from abroad, and An. minimus, which is the main effective vector of malaria in Hainan, also exists in Sanya. According to epidemiological investigation data and the characteristics of P. malariae transmission, the present study concluded that there was a high possibility of human-to-mosquito-tohuman transmission in forest-goers. From the current release of P. malariae cases, primarily from Guangdong and Shanghai [24,25], these cases indicate that P. malariae has a long incubation period and may cause a life-long infection, with or without recurrent fever. However, these cases were in older age groups rather than young-to-middle age groups, which is the largest difference from this outbreak. The human-monkey mode of transmission is impossible because only 5 cases were found at this time. There have been no subsequent cases in the mountains, although forest-goers have existed since 2015. However, we cannot exclude macaque infections with P. malariae. All of the malaria parasites in this outbreak were identi ed as P. malariae, not P. simium, which led to zoonosis in forest-goers in Brazil [26]. Cities or counties in central Hainan province, where An. dirus and An. minimus exist, are actively developing tourism resources for economic development [26]. It is more challenging to prevent the retransmission of imported malaria, and forest-goers remain a high-risk group in preventing the retransmission of malaria.

Conclusions
TLS is suitable for outbreak by indigenous case of unknown causes or for sporadic cases with defects in epidemic information, while 1-3-7 is suitable for sporadic cases which have clearly epidemic clues, such as imported cases and coworker with imported cases. Moreover, TLS could be one of the major achievements after 1-3-7 approach, targeting outbreak in elimination malaria. The innovative TLS is effective in forest-goers in Hainan in this study and can be applied to southeast Asia, where An. dirus and An. minimus are transmitted, to address malaria elimination and control.