The discovery of human&nbsp;<i>Plasmodium</i>&nbsp;among domestic animals in West Sumba and Fakfak, Indonesia

Munirah Munirah; Sitti Wahyuni; Isra Wahid; Firdaus Hamid

doi:10.12688/f1000research.53946.3

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Research Article

Revised

The discovery of human Plasmodium among domestic animals in West Sumba and Fakfak, Indonesia

[version 3; peer review: 1 approved, 1 approved with reservations, 1 not approved]

Munirah Munirah¹, Sitti Wahyuni ², Isra Wahid², Firdaus Hamid³

PUBLISHED 09 Apr 2024

Author details Author details

¹ Doctoral Study Program, Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi, 90245, Indonesia
² Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi, 90245, Indonesia
³ Department of Microbiology, Faculty of Medicine, Hasanuddin University, Makassar. Jln. Perintis Kemerdekaan 10 Tamalanrea, Makassar, South Sulawesi, 90245, Indonesia

Munirah Munirah
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Writing – Original Draft Preparation, Writing – Review & Editing

Sitti Wahyuni
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

Isra Wahid
Roles: Data Curation, Formal Analysis, Methodology, Supervision, Writing – Original Draft Preparation

Firdaus Hamid
Roles: Formal Analysis, Supervision, Visualization, Writing – Original Draft Preparation

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background

In Indonesia, malaria incidence is at a high rate despite maximum preventive efforts. Therefore, this study aims to determine the possibility of a Plasmodium reservoir among domestic animals in malaria-endemic areas.

Methods

Animal blood was collected using EDTA tubes, then smeared and stained with Giemsa for Plasmodium microscopic identification. About 10 μl of blood was dropped on to a filter paper to capture Plasmodium DNA. Nested PCR was used for parasite molecular detection, while Plasmodium species were identified using the sequenced DNA.

Results

A total of 208 and 62 animal blood samples were collected from Gaura village, West Sumba and Fakfak village, West Papua, Indonesia respectively. In total, 32 samples from Gaura contained P. falciparum or P. vivax, while the Plasmodium percentage in buffalo, horse, goat, and dogs were 20.7%, 14.3%, 5.8%, 16.7%, respectively. P. knowlesi was not found in any of the samples, and no other species were detected in 18 pig blood samples.

Conclusion

Human Plasmodium existence among domestic animals in Indonesia partly explains the high prevalence and persistence of malaria in some endemic areas due to a reservoir host presence. Therefore, future studies need to ascertain the cause.

Keywords

Plasmodium falciparum, Plasmodium vivax, malaria, animals, host reservoir, PCR.

Corresponding author: Sitti Wahyuni

Competing interests: No competing interests were disclosed.

Grant information: The research was supported by Directorate of Research and Community Service. Directorate General of Research and Development Strengthening Ministry of Research, Technology and Higher Education, according to the Research Contract Number: 149 / SP2H / LT / DPRM / 2018 August 21, 2018.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Munirah M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Munirah M, Wahyuni S, Wahid I and Hamid F. The discovery of human Plasmodium among domestic animals in West Sumba and Fakfak, Indonesia [version 3; peer review: 1 approved, 1 approved with reservations, 1 not approved]. F1000Research 2024, 10:645 (https://doi.org/10.12688/f1000research.53946.3) First published: 23 Jul 2021, 10:645 (https://doi.org/10.12688/f1000research.53946.1) Latest published: 10 Dec 2024, 10:645 (https://doi.org/10.12688/f1000research.53946.4)

Revised Amendments from Version 2

In this revised iteration of the article, significant enhancements have been implemented compared to its previously published counterpart. Primarily, meticulous attention has been devoted to refining the clarity and precision of language throughout the manuscript, addressing ambiguities and convoluted expressions to ensure a more coherent and accessible presentation of the research findings.

Moreover, the organization of content has been optimized to establish a more logical and seamless flow of information. Sections have been restructured, and transitions between different subsections have been strengthened to enhance the overall coherence of the narrative, facilitating a clearer understanding of the research methodology, results, and implications.

See the authors' detailed response to the review by Jaishree Raman
See the authors' detailed response to the review by Paul Cliff Simon Divis
See the authors' detailed response to the review by Alfred Amambua-Ngwa

Introduction

Indonesia is striving toward becoming a malaria-free country through the Malaria Control Programme. Despite the implementation of several efforts, such as early detection, treatment, and mosquito vector eradication, the disease remains a persistent challenge. Factors including parasite resistance to antimalarial drugs, mosquito resistance to insecticides, inadequate health system performance, and host reservoir presence contribute to continuous malaria prevalence.

Plasmodium falciparum and P. vivax commonly infect humans, leading to high morbidity and mortality. Only humans were considered the primary host for Plasmodium species including P. falciparum, P. vivax, P. malariae, and P. ovale before molecular diagnostics development. However, studies over the past two decades reported that the parasites originated from animals. Specifically, P. falciparum was traced back to gorillas (Liu et al., 2010) and chimpanzees (Duval et al., 2010; Krief et al., 2010), while P. vivax was associated with African apes (Liu et al., 2014). Other sources stated were the origination of P. malariae from chimpanzees (Duval et al., 2010) and P. knowlesi from monkeys (Knowlesi, 1935; Zhang et al., 2016), while P. ovale found in humans and chimpanzees had genetic similarities (Duval et al., 2009). Factors such as habitat loss among primates and human encroachment into forested areas are believed to have facilitated the transmission of these parasites (Davidson et al., 2019). An investigation performed in South Kalimantan reported the contribution of forest workers to malaria incidence (Rahayu et al., 2016). Therefore, this study was conducted in Gaura village, West Sumba and Fakfak, West Papua, which contain a high percentage of forestry workers.

West Papua and East Nusa Tenggara are known as malaria-endemic provinces in Indonesia, with annual parasite incidence (API) of 7.04% and 31.29%, respectively in 2015 (Kemenkes, 2016), while the rates were 3.42% and 8.49% in 2018. Even though there was a decrease in API numbers, Fakfak, West Papua and East Nusa Tenggara, West Sumba, continued to report high API rates of 4.85% and 12.9%, respectively (Public Health Office of Fakfak and West Sumba, unpublished data). The trend of malaria cases varies, and despite reduced API rates recorded in several provinces, the two areas still experience a high incidence. Therefore, this study aimed to explore the presence of human Plasmodium among domestic animals, which might serve as a potential reservoir host in endemic areas. The exploration would provide insights into the reasons for high endemicity rates in the examined areas.

Methods

Study area and population

This study was conducted in October 2018 in Gaura village, West Sumba Regency, an area 29.96 km² in size inhabited by 9,584 people, and Fakfak, West Papua Province, in August 2019 with an area of 11,036 km² inhabited by 84,692 people (Figure 1). The residents’ main occupation is farming, while livestock such as goats, horses, cows, pigs, and buffalos are commonly found in their enclosures located around the owner’s residence. Furthermore, they also own pets such as dogs and cats. The average distance between enclosure and home in Fakfak was 225 meters while in Gaura village the distance was 0-10 meters.

Figure 1. Study area: Map of Gaura Village, West Lamboya, West Sumba, Indonesia and Fakfak Regency, West Papua, Indonesia.

Sample collection

Sampling was carried out by the veterinarian and staff from West Sumba and Fakfak Animal Husbandry Office. The buffaloes, goats, pigs, and horses’ blood samples were collected in 5 ml EDTA tubes from the jugular vein located in the ventrolateral area of the neck using vacutainer needles, size 16–18. Meanwhile, the dog’s blood was drawn from the cephalic antebrachial vein in the leg using a size 21 vacutainer needle. By using a sterile micropipette, approximately 10 ul of EDTA blood was dropped onto a microscope slide, then smeared and stained with Giemsa (MERCK Millipore, Germany) for Plasmodium microscopic identification, while the remaining blood was dropped by sterile micropipet (about 20 ml) onto a filter paper (Whatman CAT No. 1442-090) until it absorbed to about 1.5 cm in diameter and hold back until blood at filter paper dry. The dry filter paper was put on a sterile clip seal plastic bags and stored at room temperature for a maximum of 10 days. All the sample collection process was done by sterile conditions.

DNA extraction

A dried blood spot (DBS) isolation kit for DNA extraction on filter paper (Cat. no. 36000) from Norgen Biotec was used. A 6 × 3 mm piece of blood-stained filter paper was put into a 1.5 ml tube containing 100 μl of digestion buffer B. It was vortexed and incubated at 85°C for 10 minutes. Afterwards, 20 μl of proteinase K and 300 μl of lysis buffer B were added to the tube and then vortexed before incubation at 56°C for 10 minutes. About 250 μl of 95% ethanol was added to the tube and then vortexed, while the DNA content was washed by adding 500 μl of WN wash solution and centrifugated for one minute at 8,000 rpm. Washing was carried out again using 500 μl of WN wash solution and centrifugated at 14,000 rpm. For DNA elution, 90 μl of elution buffer B was put into the tube and centrifuged at 8,000 rpm for one minute, and the purified DNA was stored at -20°C.

DNA amplification and electrophoresis

DNA amplification by nested PCR and qPCR were performed as directed by Tiangen Biotech (Beijing). Plasmodium DNA amplification was carried out using the nested PCR method with a 2× Tag Plus PCR mix enzyme (Tiangen). The final volume of 12.5 μl contained 6.25 μl enzyme, 2.25 μl ddH₂O, 1 μl forward primers, 1 μl reverse primers, and 2 μl DNA sample. For sequencing, the PCR mixture’s volume was doubled, with the final volume being 25 μl, while the primer sequences of P. falciparum, P. vivax (Snounou et al., 1993a) and P. knowlesi (Lee et al., 2011) can be seen in Table 1.

Table 1. Primer sequences for nested PCR.

Nested PCR	Species	Sequences primers	Size (bp)
Nested 1	Plasmodium	rPLU6: 5′-TTAAAATTGTTGCAGTTAAAACG-3′ rPLU5: 5′-CCTGTTGTTGCCTTAAACTTC-3′	1200
Nested 2	P. falciparum	rFAL1: 5′-TTAAACTGGTTTGGGAAAACCAAATATATT-3′ rFAL2: 5′-ACACAATGAACTCAATCATGACTACCCGTC-3′	205
Nested 2	P. vivax	rVIV1: 5′-CGCTTCTAGCTTAATCCACATAACTGATAC-3′ rVIV2: 5′-ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA-3′	120
Nested 2	P. knowlesi	Kn1f: 5′-CTCAACACGGGAAAACTCACTAGTTTA-3′ Kn3r: 5′-GTATTATTAGGTACAAGGTAGCAGTATGC-3′	296
Other primers	P. falciparum	rPF1: 5′-AGAAATAGAGTAAAAAACAATTTA-3′ rPF2: 5′-GTAACTATTCTAGGGGAACTA-3′	918
Other primers	P. vivax	rPV1: 5′-CCGAATTCAGTCCCACGT-3′ rPV2: 5′-GCTTCGGCTTGGAAGTCC-3′	714

The nested one DNA amplification temperature was set at 94°C denaturation (one minute), 55°C annealing (one minute) and 72°C extension (one minute) for 35 cycles. For nested two, denaturation was carried out at 94°C (30 seconds), 55°C annealing (one minute) and extension was at 72°C (30 seconds) in 35 cycles. There was a difference in the annealing temperature for each species in nested two, namely 55°C (one minute) for PCR multiplex P. falciparum and P. vivax, but 56°C (one minute) for P. knowlesi. Nested one products were used as templates for nested two and both were run on agarose gel 1.5% and 2%, respectively, while qPCR was run on agarose gel 1.5% and view in a gel documentation system. All the stage of DNA amplification was carried out in sterile media and places such us laminar airflow. Molecular work was not performed for P. ovale and P. malariae due to difficulties in finding the positive control, and according to the local health office these species have never been reported from Sumba and Fakfak.

Preventing the possibility of positive contamination of DNA Plasmodium in the first extraction, DNA was re-extracted from the same blood spot at filter paper. The extraction room used was confirmed to have never been used for Plasmodium extraction sample previously. Filter papers were cut by scissors which has been sterilization. PCR was performed using the primers, rPF1 and rPF2, as well as rPV1 and rPV2 (Snounou et al., 1993b) to detect P. falciparum and P. vivax, respectively. The same extraction and amplification method were used as described above.

Sequencing and alignment

To determine the Plasmodium species, in the second round of nested PCR, products having positive band targets were sent to the 1^st BASE, Axil Scientific Pte Ltd Singapore for sequencing. The DNA sequence result was adjusted using multiple alignments found in the BioEdit 7.0 application (Hall et al., 2011) and then read by the BLAST program from the NCBI website.

Ethical clearance

This study was approved for ethical clearance by the ethics committee of the Faculty of Medicine, Hasanuddin University (734/H4.8.4.5.31/PP36-KOMETIK/2018). All efforts were made to ameliorate any suffering of animals. To prevent stress, animals were comforted by their owners while blood samples were taken, and sampling was performed by experienced officers. Second and third blood samples were taken if there was a failure in the first sample and only if the animals were cooperative. About 20% of animals were sampled more than once.

Results

A total of 208 and 62 blood samples were collected in Gaura and Fakfak villages, respectively, from 92 buffalos, 21 horses, 121 goats, 18 dogs, and 18 pigs. Tests conducted using the nested PCR method identified 32 of the 270 animals as positive for P. falciparum and P. vivax. Furthermore, 20.7% buffalo, 14.3% horse, 5.8% goat, and 16.7% dog were Plasmodium-positive, with one buffalo showing mixed infections of P. falciparum and P. vivax. P. knowlesi was absent in all the blood samples and no form of malaria parasites was found in the 18 pigs. Additionally, PCR gel products, DNA sequence results, and the quality of samples can be seen in Figures 2, 3, and 4, respectively (Munirah et al., 2021). Plasmodium distribution in samples from Gaura and Fakfak are presented in Table 2, showing that blood containing the malaria parasites was only found in Gaura. The results of qPCR performed using rPF1–rPF2 and rPV1–rPV2 primers were similar to those of the nested PCR.

Figure 2. Gel view of PCR product from Plasmodium vivax and Plasmodium falciparum in domestic animals in Gaura, West Sumba (LD = DNA ladder, PS = positive samples, CN = control negative, CP = control positive) by nested PCR (multiplex PCR).

120 bp for positive Plasmodium vivax, 205 bp for positive Plasmodium falciparum.

Figure 3. Plasmodium falciparum and P. vivax DNA sequence alignments from blood samples taken in Gaura village, West Sumba, Indonesia by ClustalW multiple sequence alignment (Kr = buffalo, Ku = Horse, A/An = dog, Ka = goat, P.f = P. falciparum, P.v = P. vivax).

Figure 4. Example of Plasmodium PCR product quality from a blood sample taken in Gaura village, West Sumba, Indonesia.

Table 2. Distribution of animal blood samples and Plasmodium species found in Gaura village, West Sumba, Indonesia and Fakfak, West Papua, Indonesia.

No	Domestic animals	Number of samples		Positive Plasmodium				Total positive
No	Domestic animals	Gaura (2018)	Fakfak (2019)	Pf	Pv	Pk	Mix Pf & Pv	Total positive
1	Buffalos (Bubalus bubalis)	92	0	15	3	0	1	19
2	Horse (Equus caballus)	21	0	3	0	0	0	3
3	Goat (Capra aegagrus hircus)	72	49	6	1	0	0	7
4	Dog (Canis lupus familiaris)	10	8	2	1	0	0	3
5	Pig (Sus scrofa domesticus)	13	5	0	0	0	0	0
	Total	208	62	26	5	-	1	32

Microscopically, trophozoites, schizonts, and gametocyte forms at 100× magnification can be seen in Figure 5. P. falciparum gametocytes found in buffaloes were sausage and crescent-shaped (a, b), while schizonts found in horses were smaller or the same size as the red blood cells (c). The P. vivax gametocyte was larger than the red blood cells found in buffalo (d). P. falciparum gametocyte and trophozoites (ring-shaped) with one or two nuclei was found in goats (e) and P. falciparum trophozoite found in horses had one nucleus (f).

Figure 5. Morphology of Plasmodium in animals from Gaura village, West Sumba, Indonesia.

Gametocytes (a,b,d) in buffalo, schizont in horse (c), gametocyte and trophozoite in goat (e) and trophozoite in horse (f) with magnification 1000 ×.

Discussion

Plasmodium presence was suspected in domestic animals because malaria cases in both Gaura and Fakfak villages remained high despite applying maximum preventive efforts including insecticide-treated bed nets. The results showed that 32 of the 270 blood (11.9%) samples contained human Plasmodium, serving as the first data report regarding this parasite, hence further investigation should be conducted.

Previous studies found P. relictum in avian species (Cox, 2010), P. cephalophi in ungulates (Bruce et al., 1913), P. traguli in mousedeer (Garnham and Edeson, 1962), P. brucei in gray duiker (Bruce et al., 1915; Templeton et al., 2016a), P. bubalis in water buffalo (Sheather, 1919), and P. odocoilei in white-tailed deer (Garnham and Kuttler, 1980; Perkins and Schaer, 2016). Other parasites detected included P. caprae in goats (ruminant) (Kaewthamasorn et al., 2018), P. bergei in Rodentia (Vincke and Lips, 1948), as well as P. cynomolgi, P. inui, and P. fragile in primates (Dixit et al., 2018). The five Plasmodium species infecting humans were originally parasites in primates (Duval et al., 2010; Knowlesi, 1935; Liu et al., 2014; Ng et al., 2008; Singh and Daneshvar, 2013; Zhang et al., 2016). This study identified P. falciparum in buffalos, goats, dogs, and horses, as well as P. vivax in buffalos, goats, and dogs. Initially, Plasmodium presence in the erythrocytes (RBCs) of these animals was uncertain, but the nested PCR produced the same results for all positive samples. Sequencing analysis of positive bands in the nested PCR confirmed the presence of P. falciparum and P. vivax (Figure 3). The number of positive Plasmodium cases in buffalos was higher because of susceptibility to parasitic infections. This was consistent with the report of 10 parasitic infections observed by Mursyid in Central Lombok (Mursyid et al., 2020). Another source declared age as one of the factors predisposing buffalos to high risk of infections (Nurhidayah et al., 2019), with the average age exceeding seven years. This study provides the first report of human Plasmodium in ruminant, ungulate, and carnivorous domestic animals.

Investigations conducted by Templeton in Thailand in 2008 and 2015 showed the presence of P. bubalis among buffalos. The microscopic appearance of P. bubalis was depicted in the journal published by Templeton et al. (2016b). However, this current study did not detect any similarity between P. bubalis and P. falciparum gametocytes in buffalos. A molecular examination method targeting the cytochrome b (cytb) gene identified the presence of P. caprae in goats from Thailand, Myanmar, Iran, Kenya, and Sudan. The trophozoites of P. caprae can be observed in a journal published by Kaewthamasorn et al. (2018).

The discovery of Plasmodium among domestic animals in malaria-endemic areas raises the following questions. How do P. falciparum and P. vivax survive in these animals? Can animals serve as intermediate hosts for these parasites? Have both species evolved to live in ruminants, ungulates, and carnivores? Due to repeated exposure, have these animals become more susceptible to Plasmodium, which generally infects humans? Is Plasmodium pathogenic in animals?

P. knowlesi is identified as a commensal microbe in primates but pathogenic in humans (Jongwutiwes et al., 2004; Ng et al., 2008; Singh and Daneshvar, 2013), and the transmission to humans can be attributed to forest loss or the invasion of primate habitats (Davidson et al., 2019). There is a possibility that the proximity of animals and humans facilitates easier transfer of parasites between both groups through mosquitoes. In humans, P. falciparum and P. vivax infect by initially growing in liver cells before moving to RBCs. The parasites multiply in RBCs, leading to medical conditions characterized by fever, chills, headache, profuse sweating, weakness, rheumatic pain, symptoms of anemia or lack of blood, and nausea or vomiting. Plasmodium found in non-primates remains undetermined as pathogenic or commensal. However, sporozoites of P. brasilianum identified in animals migrate directly to the liver where multiplication occurs, releasing merozoites. The merozoites infect RBCs, initiating symptomatic disease in these animals (Erkenswick et al., 2017). Organisms infected by Plasmodium become symptomatic when the parasite cycle advances to the erythrocyte stage or causes malaria due to the rupture of RBCs. The identification of intermediate hosts for P. falciparum and P. vivax in livestock is still a challenge. Furthermore, various studies attempted to provide evidence for the origination of these two species from chimpanzees and gorillas.

The investigation conducted by Prugnolle in 2013 signified that P. vivax detected in monkeys and humans had similarities. The results showed the possibility of natural transfer between the two organisms, particularly in environments where animals and humans coexist, facilitating continuous parasite transmission through vectors (Prugnolle et al., 2013). Similarly, a study by Mu in 2005 described P. vivax as a zoonotic parasite (Mu et al., 2005).

High API was observed in Fakfak and Gaura villages, but only animals from Gaura had human Plasmodium. This difference may be attributed to the long distance of approximately 50–500 m between the residential houses and animal enclosures in Fakfak. Meanwhile, in Gaura, residents live in stilt houses, with the ground floor and surroundings serving as a shelter for animals, which facilitate microbial transfer between humans and animals through mosquitoes. The inaccessibility of sampling locations and steep geographical conditions in Fakfak posed challenges during sample collections. Moreover, the vector census conducted in both areas identified 11 Anopheles mosquito species in Gaura village, including An. vagus, An. sundaicus, An. aconitus, An. Kochi, An. flavirostris, An. indefinitus, An. maculatus, An. minimum, An. annularis, An. nivipes, and An. subpictus. Molecular examination results showed that two An. sundaicus were infected by Plasmodium, while no Anopheles was found in Fakfak. The species An. sundaicus was identified as zoophilic in India (Vidhya et al., 2019) and anthropophilic in Mekong, Vietnam (Trung et al., 2005). These different reports from both areas suggested variations in the behavior of An. sundaicus, showing the diversity of mosquito biting patterns influenced by environmental factors. The proximity between domestic animals and humans in Gaura village may increase the tendency of bites from An. sundaicus, thereby necessitating further investigations.

The application of livestock for zooprophylaxis in malaria-endemic areas offers several advantages but tends to increase the survival of mosquitoes, which become potential disease vectors. Proximity between infected and uninfected organisms is a significant factor driving parasite transmission (Kuris et al., 1980). The study by Hasyim suggests that livestock in endemic areas have a high potential to increase malaria incidence in Indonesia (Hasyim et al., 2018). Moreover, zoopotentiation often occurs when livestock are kept indoors or near houses (Donnelly et al., 2015). Locating livestock away from humans tends to reduce malaria cases (Franco et al., 2014). Strategies for separating livestock from humans to minimize mosquito bites in both groups are essential. Mosquitoes that have fed on the blood of animals tend to not suck blood from humans. However, closeness between animals and humans can lead to massive pathogen transmission through intermediary vectors. Addressing factors such as vector abundance, mosquito survival, mosquito behavior, and local environment is crucial in mitigating zoonotic potential.

Plasmodium can be detected microscopically due to the smaller size of RBCs in animals compared to humans, but the molecular method is more significant for identifying the presence of this parasite. Nested PCR which offered high sensitivity similar to Real-Time PCR at a low cost was applied for the detection process (Green and Sambrook, 2019; Perandin et al., 2004). The microscopic method using double fluorescent dyes with Giemsa stain is recommended for subsequent studies (Guy et al., 2007).

Further investigation is needed to confirm the presence of domestic animal Plasmodium by amplifying the cytochrome B sequence and sequencing the invasion ligands such as DBL protein. Additionally, other challenges in malaria elimination shown by the results of this study should be addressed.

Conclusion

In conclusion, Plasmodium species previously recognized as human parasites were detected among domestic animals in this study. The obtained results provided insights into malaria elimination challenges in the explored areas and were expected to promote public health improvement while guiding the development of malaria prevention strategies.

Data availability

Underlying data

Figshare: Underlying data for ‘The discovery of human Plasmodium among domestic animals in West Sumba and Fakfak, Indonesia’, https://doi.org/10.6084/m9.figshare.14703012.v3 (Munirah et al., 2021).

This project contains the following underlying data:

• Gel photo: Result of rPF1–RPF2 primers
• Gel photo: Result of RPV1–rPV2 primers
• Gel photo: Nested PCR P. falciparum and P. vivax

Reporting guidelines

Figshare: ARRIVE checklist for ‘The discovery of human Plasmodium among domestic animals in West Sumba and Fakfak, Indonesia’, https://doi.org/10.6084/m9.figshare.14703012.v3 (Munirah et al., 2021).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgements

The author acknowledges the assistance of the West Sumba District and Fakfak Animal Husbandry Service, who helped carry out animal blood collection. We thank the people of Gaura village and Fakfak, who allowed us to take blood samples from their animals. We also thank Syahruni and Handayani Halik from Hasanuddin University Medical Research Center (HUM-RC) for their help with molecular work.

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Version 4

VERSION 4 PUBLISHED 23 Jul 2021

Author details Author details

Munirah Munirah
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Writing – Original Draft Preparation, Writing – Review & Editing

Sitti Wahyuni
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

Isra Wahid
Roles: Data Curation, Formal Analysis, Methodology, Supervision, Writing – Original Draft Preparation

Firdaus Hamid
Roles: Formal Analysis, Supervision, Visualization, Writing – Original Draft Preparation

Competing interests

No competing interests were disclosed.

Grant information

The research was supported by Directorate of Research and Community Service. Directorate General of Research and Development Strengthening Ministry of Research, Technology and Higher Education, according to the Research Contract Number: 149 / SP2H / LT / DPRM / 2018 August 21, 2018.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (4)

version 4

Revised

Published: 10 Dec 2024, 10:645

https://doi.org/10.12688/f1000research.53946.4

version 3

Revised

Published: 09 Apr 2024, 10:645

https://doi.org/10.12688/f1000research.53946.3

version 2

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Published: 15 Feb 2023, 10:645

https://doi.org/10.12688/f1000research.53946.2

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Published: 23 Jul 2021, 10:645

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© 2024 Munirah M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Munirah M, Wahyuni S, Wahid I and Hamid F. The discovery of human Plasmodium among domestic animals in West Sumba and Fakfak, Indonesia [version 3; peer review: 1 approved, 1 approved with reservations, 1 not approved]. F1000Research 2024, 10:645 (https://doi.org/10.12688/f1000research.53946.3)

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Key to Reviewer Statuses VIEW HIDE

ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 3

VERSION 3

PUBLISHED 09 Apr 2024

Revised

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Reviewer Report 22 Nov 2024

Paul Cliff Simon Divis, Malaria Research Centre, Faculty of Medicine and Health Sciences, University of Malaysia, Sarawak, Malaysia

Approved with Reservations

https://doi.org/10.5256/f1000research.164578.r337062

General Comments

The manuscript explores the intriguing possibility of domestic animals serving as reservoir hosts for human Plasmodium species. While the research question is thought-provoking, significant issues in the rationale, methodology, and interpretation of results undermine the conclusions. The manuscript would benefit from a stronger theoretical framework that considers parasite-host coevolution and the evolutionary barriers to host switching.

Specific Comments

In the introduction, the authors discuss the evolutionary origins of human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale), tracing them to non-human primates, but this discussion is not effectively connected to the hypothesis that domestic animals could serve as reservoir hosts. Including a discussion of parasite-host coevolution would strengthen the argument. This discussion could highlight how human Plasmodium species are highly adapted to their primate hosts and emphasize the evolutionary and ecological barriers that make host-switching from primates to non-primate domestic animals unlikely. Addressing these barriers could challenge the hypothesis that primate Plasmodium species can adapt and persist in non-primate hosts.

The rationale for hypothesizing that domestic animals might harbor human Plasmodium species needs to be more robust. Empirical or theoretical evidence should support why such a host switch might occur and whether this is biologically plausible.

The qPCR methodology used in this study is inadequately described. It is important to clarify whether SYBR Green dye or probe-based assays were employed, as these methods differ in sensitivity and specificity. Details about primer design and validation should also be provided. When the primers were initially designed, they were not tested on Apicomplexan parasites from non-primate hosts, leaving their potential cross-reactivity with other parasites unknown. This is a critical limitation that should be addressed to ensure the validity of the findings.

The authors should consider whether observed DNA signals might result from contamination, cross-reactivity, or amplification of related but non-Plasmodium DNA. Parasite-host coevolution poses significant challenges to the hypothesis that primate-adapted Plasmodium species can easily switch to non-primate hosts. The evolutionary pressures and physiological barriers that would make such a host switch unlikely should be discussed.

The conclusion that domestic animals harbor human Plasmodium species is premature and unsupported by the data. A more robust genetic approach, such as sequencing longer DNA fragments across multiple genes, is necessary to confirm parasite identity. Short sequences are prone to misidentification, and the limitations of the current data should be acknowledged.

Microscopy findings should be reported cautiously. It is inappropriate to label observed parasites as P. falciparum or P. vivax without further genetic confirmation. More neutral language, such as “resembling Plasmodium species,” is recommended. The title of the manuscript may also be misleading, as it suggests that human Plasmodium species have been conclusively identified in domestic animals. The title should reflect the preliminary nature of these findings.

The reference to Mursyid et al. (2020) is inconsistent with the claims made in this study. Mursyid et al. did not report Plasmodium species infections in buffaloes, and this reference should be clarified or omitted.

In my opinion, the authors should introduce parasite-host coevolution as a framework for evaluating the plausibility of host-switching events. This discussion could challenge the hypothesis and strengthen the manuscript’s theoretical basis. A more detailed and transparent description of the qPCR methodology is necessary, including primer validation to ensure specificity, especially given the lack of prior testing on Apicomplexan parasites from non-primate hosts. Genetic analyses should be expanded by sequencing longer DNA fragments from multiple genes to confirm species identity.

The manuscript title and conclusions should better reflect the preliminary nature of the findings. Proof-reading is also needed to improve clarity and ensure that terminology and interpretations align with the presented data.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: molecular epidemiology, population genetics, genomics, zoonotic malaria

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 11 Dec 2024

Sitti Wahyuni, Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, 90245, Indonesia

11 Dec 2024

Author Response

We sincerely appreciate your insightful feedback regarding the need for stronger empirical support for our hypothesis. In response to your suggestion, we have revised the section discussing host-switching and included ... Continue reading We sincerely appreciate your insightful feedback regarding the need for stronger empirical support for our hypothesis. In response to your suggestion, we have revised the section discussing host-switching and included additional evidence that explores similar host shifts observed in other parasite species, as well as the potential evolutionary constraints that may apply to Plasmodium species. Furthermore, we have incorporated recent studies on parasite-host coevolution, which we believe will provide greater depth to our argument.

We also wish to thank you for your careful attention to the details of the qPCR methodology. To clarify, in this study, we utilized a SYBR Green-based qPCR approach, which is widely recognized for its sensitivity and cost-effectiveness in detection. We have now provided a more detailed description of the methodology in the revised manuscript, including the specific validation steps we undertook to ensure the accuracy of our results.

As per your request, we have also revised the title and conclusion of the manuscript. While the primers were initially tested using Apicomplexan parasites from primate hosts, subsequent BLAST analysis suggested that the target sequences were highly conserved across various host species. Additionally, we employed the rPF1-rPF2 primers for P. falciparum and rPV1-rPV2 primers for P. vivax to amplify DNA fragments of 918 bp and 714 bp, respectively. These larger amplicons offer additional confidence in the assay's specificity. The results obtained from these primers can be accessed via the following link: https://doi.org/10.6084/m9.figshare.14703012.v3.

While we were unable to perform sequencing of the PCR products with the rPF1-rPF2 and rPV1-rPV2 primers due to budgetary constraints, the PCR bands observed were consistent with the expected base pair lengths for P. falciparum and P. vivax.

We greatly appreciate your constructive comments and believe these revisions have strengthened the manuscript.
We sincerely appreciate your insightful feedback regarding the need for stronger empirical support for our hypothesis. In response to your suggestion, we have revised the section discussing host-switching and included additional evidence that explores similar host shifts observed in other parasite species, as well as the potential evolutionary constraints that may apply to Plasmodium species. Furthermore, we have incorporated recent studies on parasite-host coevolution, which we believe will provide greater depth to our argument.

We also wish to thank you for your careful attention to the details of the qPCR methodology. To clarify, in this study, we utilized a SYBR Green-based qPCR approach, which is widely recognized for its sensitivity and cost-effectiveness in detection. We have now provided a more detailed description of the methodology in the revised manuscript, including the specific validation steps we undertook to ensure the accuracy of our results.

As per your request, we have also revised the title and conclusion of the manuscript. While the primers were initially tested using Apicomplexan parasites from primate hosts, subsequent BLAST analysis suggested that the target sequences were highly conserved across various host species. Additionally, we employed the rPF1-rPF2 primers for P. falciparum and rPV1-rPV2 primers for P. vivax to amplify DNA fragments of 918 bp and 714 bp, respectively. These larger amplicons offer additional confidence in the assay's specificity. The results obtained from these primers can be accessed via the following link: https://doi.org/10.6084/m9.figshare.14703012.v3.

While we were unable to perform sequencing of the PCR products with the rPF1-rPF2 and rPV1-rPV2 primers due to budgetary constraints, the PCR bands observed were consistent with the expected base pair lengths for P. falciparum and P. vivax.

We greatly appreciate your constructive comments and believe these revisions have strengthened the manuscript.
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 11 Dec 2024

Sitti Wahyuni, Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, 90245, Indonesia

11 Dec 2024

Author Response

We sincerely appreciate your insightful feedback regarding the need for stronger empirical support for our hypothesis. In response to your suggestion, we have revised the section discussing host-switching and included ... Continue reading We sincerely appreciate your insightful feedback regarding the need for stronger empirical support for our hypothesis. In response to your suggestion, we have revised the section discussing host-switching and included additional evidence that explores similar host shifts observed in other parasite species, as well as the potential evolutionary constraints that may apply to Plasmodium species. Furthermore, we have incorporated recent studies on parasite-host coevolution, which we believe will provide greater depth to our argument.

We also wish to thank you for your careful attention to the details of the qPCR methodology. To clarify, in this study, we utilized a SYBR Green-based qPCR approach, which is widely recognized for its sensitivity and cost-effectiveness in detection. We have now provided a more detailed description of the methodology in the revised manuscript, including the specific validation steps we undertook to ensure the accuracy of our results.

As per your request, we have also revised the title and conclusion of the manuscript. While the primers were initially tested using Apicomplexan parasites from primate hosts, subsequent BLAST analysis suggested that the target sequences were highly conserved across various host species. Additionally, we employed the rPF1-rPF2 primers for P. falciparum and rPV1-rPV2 primers for P. vivax to amplify DNA fragments of 918 bp and 714 bp, respectively. These larger amplicons offer additional confidence in the assay's specificity. The results obtained from these primers can be accessed via the following link: https://doi.org/10.6084/m9.figshare.14703012.v3.

While we were unable to perform sequencing of the PCR products with the rPF1-rPF2 and rPV1-rPV2 primers due to budgetary constraints, the PCR bands observed were consistent with the expected base pair lengths for P. falciparum and P. vivax.

We greatly appreciate your constructive comments and believe these revisions have strengthened the manuscript.
We sincerely appreciate your insightful feedback regarding the need for stronger empirical support for our hypothesis. In response to your suggestion, we have revised the section discussing host-switching and included additional evidence that explores similar host shifts observed in other parasite species, as well as the potential evolutionary constraints that may apply to Plasmodium species. Furthermore, we have incorporated recent studies on parasite-host coevolution, which we believe will provide greater depth to our argument.

We also wish to thank you for your careful attention to the details of the qPCR methodology. To clarify, in this study, we utilized a SYBR Green-based qPCR approach, which is widely recognized for its sensitivity and cost-effectiveness in detection. We have now provided a more detailed description of the methodology in the revised manuscript, including the specific validation steps we undertook to ensure the accuracy of our results.

As per your request, we have also revised the title and conclusion of the manuscript. While the primers were initially tested using Apicomplexan parasites from primate hosts, subsequent BLAST analysis suggested that the target sequences were highly conserved across various host species. Additionally, we employed the rPF1-rPF2 primers for P. falciparum and rPV1-rPV2 primers for P. vivax to amplify DNA fragments of 918 bp and 714 bp, respectively. These larger amplicons offer additional confidence in the assay's specificity. The results obtained from these primers can be accessed via the following link: https://doi.org/10.6084/m9.figshare.14703012.v3.

While we were unable to perform sequencing of the PCR products with the rPF1-rPF2 and rPV1-rPV2 primers due to budgetary constraints, the PCR bands observed were consistent with the expected base pair lengths for P. falciparum and P. vivax.

We greatly appreciate your constructive comments and believe these revisions have strengthened the manuscript.
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 14 May 2024

Jaishree Raman, UP Institute for Sustainable Malaria Control, WRIM,, University of Pretoria, Pretoria, Gauteng, South Africa; National Institute for Communicable Diseases, University of the Witwatersrand Johannesburg, Johannesburg, Gauteng, South Africa

Approved

https://doi.org/10.5256/f1000research.164578.r264595

I have reviewed the revised article and think the authors have ... Continue reading

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Author Response 30 May 2024

Sitti Wahyuni, Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, 90245, Indonesia

30 May 2024

Author Response

Thank you for taking the time to review our revised article. We appreciate your constructive feedback and are pleased that we were able to address your concerns to your satisfaction. ... Continue reading Thank you for taking the time to review our revised article. We appreciate your constructive feedback and are pleased that we were able to address your concerns to your satisfaction. We look forward to the acceptance of our article.
Thank you for taking the time to review our revised article. We appreciate your constructive feedback and are pleased that we were able to address your concerns to your satisfaction. We look forward to the acceptance of our article.
Competing Interests: No competing interests were disclosed. Close
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Respond or Comment

COMMENTS ON THIS REPORT

Author Response 30 May 2024

Sitti Wahyuni, Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, 90245, Indonesia

30 May 2024

Author Response

Thank you for taking the time to review our revised article. We appreciate your constructive feedback and are pleased that we were able to address your concerns to your satisfaction. ... Continue reading Thank you for taking the time to review our revised article. We appreciate your constructive feedback and are pleased that we were able to address your concerns to your satisfaction. We look forward to the acceptance of our article.
Thank you for taking the time to review our revised article. We appreciate your constructive feedback and are pleased that we were able to address your concerns to your satisfaction. We look forward to the acceptance of our article.
Competing Interests: No competing interests were disclosed. Close
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Version 2

VERSION 2

PUBLISHED 15 Feb 2023

Revised

Views

Reviewer Report 28 Nov 2023

Approved with Reservations

https://doi.org/10.5256/f1000research.144004.r163587

I thank the authors for revising the article based on the comments of two reviewers. While the scientific content of the manuscript has improved through the review process, it still requires correction in terms of English scientific writing. I recommend ... Continue reading

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Author Response 30 May 2024

Sitti Wahyuni, Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, 90245, Indonesia

30 May 2024

Author Response

Thank you for the feedback.
We have submitted the manuscript to an English scientific writing service provider for language refinement. Subsequently, we have re-uploaded the revised file.

Sincerely
Author
Competing Interests: No competing interests were disclosed.
Thank you for the feedback.
We have submitted the manuscript to an English scientific writing service provider for language refinement. Subsequently, we have re-uploaded the revised file.

Sincerely
Author
Thank you for the feedback.
We have submitted the manuscript to an English scientific writing service provider for language refinement. Subsequently, we have re-uploaded the revised file.

Sincerely
Author
Competing Interests: No competing interests were disclosed. Close
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Respond or Comment

COMMENTS ON THIS REPORT

Author Response 30 May 2024

Sitti Wahyuni, Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, 90245, Indonesia

30 May 2024

Author Response

Thank you for the feedback.
We have submitted the manuscript to an English scientific writing service provider for language refinement. Subsequently, we have re-uploaded the revised file.

Sincerely
Author
Competing Interests: No competing interests were disclosed.
Thank you for the feedback.
We have submitted the manuscript to an English scientific writing service provider for language refinement. Subsequently, we have re-uploaded the revised file.

Sincerely
Author
Thank you for the feedback.
We have submitted the manuscript to an English scientific writing service provider for language refinement. Subsequently, we have re-uploaded the revised file.

Sincerely
Author
Competing Interests: No competing interests were disclosed. Close
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Version 1

VERSION 1

PUBLISHED 23 Jul 2021

Views

Reviewer Report 22 Dec 2022

Not Approved

https://doi.org/10.5256/f1000research.57382.r156443

This manuscript describes the interesting finding of human malaria parasites in domestic pets and farm animals in Indonesia. While this finding has potential implications for countries trying to stem residual transmission with the aim of being certified malaria free, this article needs some revision to ensure clarity and a logical flow of ideas. More information on the malaria burden and interventions in place in the study area need to be provided. Some possible reasons for the study findings need to be provided. The ideas for future studies should be presented as statements rather than questions.

Below are some more specific comments:

Introduction:

It is far too short. It needs to be expanded to explain the issue of residual malaria transmission in the study area and why it is concern. What interventions are in place to stop transmission and why you think they are failing to stem residual transmission.
There are four species of Plasmodia, not types that primarily affect humans
Is the statement on forest workers included because the study area has large numbers of forest workers? If so please provide this information.
From the data presented it looks like malaria transmission has decreased in both study areas so it is not clear to me what situation needs to be investigated. More clarity needs to be provided here.
A clear reason for the study needs to be provided as well as possible uses of the research findings.

Methods:

I am correct in assuming that the animal enclosures are in close proximity to the farmers homesteads? Please give an average distance between enclosures and homes – as mosquitoes will only travel as far as they need to, to get their next blood meal.
The remaining is either blotted or spotted onto the filter paper. I do not understand what “the dry filter paper was put on a sterile paper clip” means.
Where the houses geo-located so you could map the infected animals?
How long was the incubation at 85 degrees?
It is DNA amplification by PCR not of PCR
For nest two is there no annealing stage?
Please explain why nest one products were run on a gel.
Where the qPCR products really analysed using gel electrophoresis? The benefit of qPCR is real time data generation through the detection of fluorescence in the amplification stage. The analysis of qPCR products is generally done by reading fluorescence outputs.
Please provide a bit more information on the rationale for the re-extraction of the sample. In addition please state if the additional blood spot was from the same blood spot or different blood spot?

Results

It is not clear whether the pigs were not infected will any form of malaria parasites or only not infected with human malaria parasites. Please clarify.

Discussion

Please expand on the questions presented in the discussion in terms on possible results and their implication for malaria elimination in Indonesia and globally.
Please clarify if only infected were found in West Sumba region or only one study area within West Sumba. The reason provided for only one area having infected animals is unclear to me. Please rephrase for more clarity.
Please some guidance for future studies.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Antimalarial drug and diagnostic resistance, molecular biology, molecular epidemiology, malaria elimination and control

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

CITE

Report a concern

Author Response 20 Mar 2023

Sitti Wahyuni, Hasanuddin University, Indonesia

20 Mar 2023

Author Response
This manuscript describes the interesting finding of human malaria parasites in domestic pets and farm animals in Indonesia. While this finding has potential implications for countries trying to stem residual ... Continue reading
This manuscript describes the interesting finding of human malaria parasites in domestic pets and farm animals in Indonesia. While this finding has potential implications for countries trying to stem residual transmission with the aim of being certified malaria free, this article needs some revision to ensure clarity and a logical flow of ideas. More information on the malaria burden and interventions in place in the study area need to be provided. Some possible reasons for the study findings need to be provided. The ideas for future studies should be presented as statements rather than questions.

Below are some more specific comments:

Introduction:

It is far too short. It needs to be expanded to explain the issue of residual malaria transmission in the study area and why it is concern. What interventions are in place to stop transmission and why you think they are failing to stem residual transmission.

Author’s response: Thank you, we have made a revision.

There are four species of Plasmodia, not types that primarily affect humans

Author’s response: Thank you for your input, we already revised.

Is the statement on forest workers included because the study area has large numbers of forest workers? If so please provide this information.

Author’s response: Thank you, we already revised.

From the data presented it looks like malaria transmission has decreased in both study areas so it is not clear to me what situation needs to be investigated. More clarity needs to be provided here.

Author’s response: Thank you, we already revised.

A clear reason for the study needs to be provided as well as possible uses of the research findings.

Author’s response: Thank you for your comment, we have revised accordingly.

Methods:

I am correct in assuming that the animal enclosures are in close proximity to the farmers homesteads? Please give an average distance between enclosures and homes – as mosquitoes will only travel as far as they need to, to get their next blood meal.

Author’s response: Thank you, we have already given the average distance in the method.

The remaining is either blotted or spotted onto the filter paper. I do not understand what “the dry filter paper was put on a sterile paper clip” means.

Author’s response: Thank you, more clarity has been made.

Where the houses geo-located so you could map the infected animals?

Author’s response: We apologize, because we don’t have the data about geo-location. But, in future research, we will apply this method. Thank you for your input.

How long was the incubation at 85 degrees?

Author’s response: Thank you for your question, the incubation at 85 degrees was 10 minutes.

It is DNA amplification by PCR not of PCR

Author’s response: Thank you for your comment, we have revised accordingly

For nest two is there no annealing stage?

Author’s response: Thank you for your question, the annealing nested two was 55°C in one minute.

Please explain why nest one products were run on a gel.

Author’s response: Thank you. Actually, the nested one does not need to run on a gel, but it was done just to notice if there were any bands in the nested one. However, it is not the standard for determining whether positive at that stage, because we used nested two for determining Plasmodium positive.

Where the qPCR products really analysed using gel electrophoresis? The benefit of qPCR is real time data generation through the detection of fluorescence in the amplification stage. The analysis of qPCR products is generally done by reading fluorescence outputs.

Author’s response: Thank you for your question. Both products of nested and qPCR were analysed using gel electrophoresis and then run on agarose gel (nested one in 1.5%, nested two in 2% agarose gel and qPCR in 1.5% agarose gel) in order to identify fluorescence band wave in a gel documentation system.

Please provide a bit more information on the rationale for the re-extraction of the sample. In addition please state if the additional blood spot was from the same blood spot or different blood spot?

Author’s response: Thank you, we already revised.

Results

It is not clear whether the pigs were not infected will any form of malaria parasites or only not infected with human malaria parasites. Please clarify.

Author’s response: Thank you, we already revised.

Discussion

Please expand on the questions presented in the discussion in terms on possible results and their implication for malaria elimination in Indonesia and globally.

Author’s response: Thank you, we already revised.

Please clarify if only infected were found in West Sumba region or only one study area within West Sumba. The reason provided for only one area having infected animals is unclear to me. Please rephrase for more clarity.

Author’s response: Thank you, we already revised.

Please some guidance for future studies.

Author’s response: Thank you, we already revised.
This manuscript describes the interesting finding of human malaria parasites in domestic pets and farm animals in Indonesia. While this finding has potential implications for countries trying to stem residual transmission with the aim of being certified malaria free, this article needs some revision to ensure clarity and a logical flow of ideas. More information on the malaria burden and interventions in place in the study area need to be provided. Some possible reasons for the study findings need to be provided. The ideas for future studies should be presented as statements rather than questions.

Below are some more specific comments:

Introduction:

It is far too short. It needs to be expanded to explain the issue of residual malaria transmission in the study area and why it is concern. What interventions are in place to stop transmission and why you think they are failing to stem residual transmission.

Author’s response: Thank you, we have made a revision.

There are four species of Plasmodia, not types that primarily affect humans

Author’s response: Thank you for your input, we already revised.

Is the statement on forest workers included because the study area has large numbers of forest workers? If so please provide this information.

Author’s response: Thank you, we already revised.

From the data presented it looks like malaria transmission has decreased in both study areas so it is not clear to me what situation needs to be investigated. More clarity needs to be provided here.

Author’s response: Thank you, we already revised.

A clear reason for the study needs to be provided as well as possible uses of the research findings.

Author’s response: Thank you for your comment, we have revised accordingly.

Methods:

I am correct in assuming that the animal enclosures are in close proximity to the farmers homesteads? Please give an average distance between enclosures and homes – as mosquitoes will only travel as far as they need to, to get their next blood meal.

Author’s response: Thank you, we have already given the average distance in the method.

The remaining is either blotted or spotted onto the filter paper. I do not understand what “the dry filter paper was put on a sterile paper clip” means.

Author’s response: Thank you, more clarity has been made.

Where the houses geo-located so you could map the infected animals?

Author’s response: We apologize, because we don’t have the data about geo-location. But, in future research, we will apply this method. Thank you for your input.

How long was the incubation at 85 degrees?

Author’s response: Thank you for your question, the incubation at 85 degrees was 10 minutes.

It is DNA amplification by PCR not of PCR

Author’s response: Thank you for your comment, we have revised accordingly

For nest two is there no annealing stage?

Author’s response: Thank you for your question, the annealing nested two was 55°C in one minute.

Please explain why nest one products were run on a gel.

Author’s response: Thank you. Actually, the nested one does not need to run on a gel, but it was done just to notice if there were any bands in the nested one. However, it is not the standard for determining whether positive at that stage, because we used nested two for determining Plasmodium positive.

Where the qPCR products really analysed using gel electrophoresis? The benefit of qPCR is real time data generation through the detection of fluorescence in the amplification stage. The analysis of qPCR products is generally done by reading fluorescence outputs.

Author’s response: Thank you for your question. Both products of nested and qPCR were analysed using gel electrophoresis and then run on agarose gel (nested one in 1.5%, nested two in 2% agarose gel and qPCR in 1.5% agarose gel) in order to identify fluorescence band wave in a gel documentation system.

Please provide a bit more information on the rationale for the re-extraction of the sample. In addition please state if the additional blood spot was from the same blood spot or different blood spot?

Author’s response: Thank you, we already revised.

Results

It is not clear whether the pigs were not infected will any form of malaria parasites or only not infected with human malaria parasites. Please clarify.

Author’s response: Thank you, we already revised.

Discussion

Please expand on the questions presented in the discussion in terms on possible results and their implication for malaria elimination in Indonesia and globally.

Author’s response: Thank you, we already revised.

Please clarify if only infected were found in West Sumba region or only one study area within West Sumba. The reason provided for only one area having infected animals is unclear to me. Please rephrase for more clarity.

Author’s response: Thank you, we already revised.

Please some guidance for future studies.

Author’s response: Thank you, we already revised.
Competing Interests: I have no conflict of interest Close
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COMMENTS ON THIS REPORT

Author Response 20 Mar 2023

Sitti Wahyuni, Hasanuddin University, Indonesia

20 Mar 2023

Author Response
This manuscript describes the interesting finding of human malaria parasites in domestic pets and farm animals in Indonesia. While this finding has potential implications for countries trying to stem residual ... Continue reading
This manuscript describes the interesting finding of human malaria parasites in domestic pets and farm animals in Indonesia. While this finding has potential implications for countries trying to stem residual transmission with the aim of being certified malaria free, this article needs some revision to ensure clarity and a logical flow of ideas. More information on the malaria burden and interventions in place in the study area need to be provided. Some possible reasons for the study findings need to be provided. The ideas for future studies should be presented as statements rather than questions.

Below are some more specific comments:

Introduction:

It is far too short. It needs to be expanded to explain the issue of residual malaria transmission in the study area and why it is concern. What interventions are in place to stop transmission and why you think they are failing to stem residual transmission.

Author’s response: Thank you, we have made a revision.

There are four species of Plasmodia, not types that primarily affect humans

Author’s response: Thank you for your input, we already revised.

Is the statement on forest workers included because the study area has large numbers of forest workers? If so please provide this information.

Author’s response: Thank you, we already revised.

From the data presented it looks like malaria transmission has decreased in both study areas so it is not clear to me what situation needs to be investigated. More clarity needs to be provided here.

Author’s response: Thank you, we already revised.

A clear reason for the study needs to be provided as well as possible uses of the research findings.

Author’s response: Thank you for your comment, we have revised accordingly.

Methods:

I am correct in assuming that the animal enclosures are in close proximity to the farmers homesteads? Please give an average distance between enclosures and homes – as mosquitoes will only travel as far as they need to, to get their next blood meal.

Author’s response: Thank you, we have already given the average distance in the method.

The remaining is either blotted or spotted onto the filter paper. I do not understand what “the dry filter paper was put on a sterile paper clip” means.

Author’s response: Thank you, more clarity has been made.

Where the houses geo-located so you could map the infected animals?

Author’s response: We apologize, because we don’t have the data about geo-location. But, in future research, we will apply this method. Thank you for your input.

How long was the incubation at 85 degrees?

Author’s response: Thank you for your question, the incubation at 85 degrees was 10 minutes.

It is DNA amplification by PCR not of PCR

Author’s response: Thank you for your comment, we have revised accordingly

For nest two is there no annealing stage?

Author’s response: Thank you for your question, the annealing nested two was 55°C in one minute.

Please explain why nest one products were run on a gel.

Author’s response: Thank you. Actually, the nested one does not need to run on a gel, but it was done just to notice if there were any bands in the nested one. However, it is not the standard for determining whether positive at that stage, because we used nested two for determining Plasmodium positive.

Where the qPCR products really analysed using gel electrophoresis? The benefit of qPCR is real time data generation through the detection of fluorescence in the amplification stage. The analysis of qPCR products is generally done by reading fluorescence outputs.

Author’s response: Thank you for your question. Both products of nested and qPCR were analysed using gel electrophoresis and then run on agarose gel (nested one in 1.5%, nested two in 2% agarose gel and qPCR in 1.5% agarose gel) in order to identify fluorescence band wave in a gel documentation system.

Please provide a bit more information on the rationale for the re-extraction of the sample. In addition please state if the additional blood spot was from the same blood spot or different blood spot?

Author’s response: Thank you, we already revised.

Results

It is not clear whether the pigs were not infected will any form of malaria parasites or only not infected with human malaria parasites. Please clarify.

Author’s response: Thank you, we already revised.

Discussion

Please expand on the questions presented in the discussion in terms on possible results and their implication for malaria elimination in Indonesia and globally.

Author’s response: Thank you, we already revised.

Please clarify if only infected were found in West Sumba region or only one study area within West Sumba. The reason provided for only one area having infected animals is unclear to me. Please rephrase for more clarity.

Author’s response: Thank you, we already revised.

Please some guidance for future studies.

Author’s response: Thank you, we already revised.
This manuscript describes the interesting finding of human malaria parasites in domestic pets and farm animals in Indonesia. While this finding has potential implications for countries trying to stem residual transmission with the aim of being certified malaria free, this article needs some revision to ensure clarity and a logical flow of ideas. More information on the malaria burden and interventions in place in the study area need to be provided. Some possible reasons for the study findings need to be provided. The ideas for future studies should be presented as statements rather than questions.

Below are some more specific comments:

Introduction:

It is far too short. It needs to be expanded to explain the issue of residual malaria transmission in the study area and why it is concern. What interventions are in place to stop transmission and why you think they are failing to stem residual transmission.

Author’s response: Thank you, we have made a revision.

There are four species of Plasmodia, not types that primarily affect humans

Author’s response: Thank you for your input, we already revised.

Is the statement on forest workers included because the study area has large numbers of forest workers? If so please provide this information.

Author’s response: Thank you, we already revised.

From the data presented it looks like malaria transmission has decreased in both study areas so it is not clear to me what situation needs to be investigated. More clarity needs to be provided here.

Author’s response: Thank you, we already revised.

A clear reason for the study needs to be provided as well as possible uses of the research findings.

Author’s response: Thank you for your comment, we have revised accordingly.

Methods:

I am correct in assuming that the animal enclosures are in close proximity to the farmers homesteads? Please give an average distance between enclosures and homes – as mosquitoes will only travel as far as they need to, to get their next blood meal.

Author’s response: Thank you, we have already given the average distance in the method.

The remaining is either blotted or spotted onto the filter paper. I do not understand what “the dry filter paper was put on a sterile paper clip” means.

Author’s response: Thank you, more clarity has been made.

Where the houses geo-located so you could map the infected animals?

Author’s response: We apologize, because we don’t have the data about geo-location. But, in future research, we will apply this method. Thank you for your input.

How long was the incubation at 85 degrees?

Author’s response: Thank you for your question, the incubation at 85 degrees was 10 minutes.

It is DNA amplification by PCR not of PCR

Author’s response: Thank you for your comment, we have revised accordingly

For nest two is there no annealing stage?

Author’s response: Thank you for your question, the annealing nested two was 55°C in one minute.

Please explain why nest one products were run on a gel.

Author’s response: Thank you. Actually, the nested one does not need to run on a gel, but it was done just to notice if there were any bands in the nested one. However, it is not the standard for determining whether positive at that stage, because we used nested two for determining Plasmodium positive.

Where the qPCR products really analysed using gel electrophoresis? The benefit of qPCR is real time data generation through the detection of fluorescence in the amplification stage. The analysis of qPCR products is generally done by reading fluorescence outputs.

Author’s response: Thank you for your question. Both products of nested and qPCR were analysed using gel electrophoresis and then run on agarose gel (nested one in 1.5%, nested two in 2% agarose gel and qPCR in 1.5% agarose gel) in order to identify fluorescence band wave in a gel documentation system.

Please provide a bit more information on the rationale for the re-extraction of the sample. In addition please state if the additional blood spot was from the same blood spot or different blood spot?

Author’s response: Thank you, we already revised.

Results

It is not clear whether the pigs were not infected will any form of malaria parasites or only not infected with human malaria parasites. Please clarify.

Author’s response: Thank you, we already revised.

Discussion

Please expand on the questions presented in the discussion in terms on possible results and their implication for malaria elimination in Indonesia and globally.

Author’s response: Thank you, we already revised.

Please clarify if only infected were found in West Sumba region or only one study area within West Sumba. The reason provided for only one area having infected animals is unclear to me. Please rephrase for more clarity.

Author’s response: Thank you, we already revised.

Please some guidance for future studies.

Author’s response: Thank you, we already revised.
Competing Interests: I have no conflict of interest Close
Report a concern

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Reviewer Report 06 Jan 2022

Alfred Amambua-Ngwa, Medical Research Council Unit, The Gambia at London School of Hygiene and Tropical Medicine, Fajara, The Gambia

Not Approved

https://doi.org/10.5256/f1000research.57382.r101607

The authors present data showing microscopically identified Plasmodium species in non-primate domestic animals in a region of Indonesia. They followed on to confirm infections with Plasmodium species with molecular methods targeting human malaria parasites P. falciparum, P. vivax, and P. knowlesi. They sequenced the amplified fragment, indicating that they indeed detected the genus. If this claim is validated, it will have significant consequences on malaria interventions and the goal of its elimination across the globe. The authors need to pay attention to the following to validate their claim:

Several Plasmodium species infect a wide range of animal species and there are other protozoa with similar cellular features that can also be microscopically misidentified for human Plasmodium. Thus, the identification of human Plasmodium will require species-specific distinguishing features by comparing the infected erythrocyte of the Plasmodium-infected domestic animals to those of other protozoan parasites including non-human Plasmodium.
A single sequence of each of the PCR products is shown per species and these sequences are similar to those of Plasmodium falciparum small rRNA. The alignment could include all samples sequenced and also how this was used to determine other species beyond P. falciparum. For speciation to be better validated, the authors could amplify and sequence cytochrome B, which is more divergent for Plasmodium.
More detail on the control protocols to limit contamination at sampling, extraction, and amplification of parasite DNA from the samples will help.
Given that humans and the family of domestic animals listed are largely divergent on RBC surface receptors for parasites, how these putative human-specific parasites invade the non-human hosts and develop will be of interest. The authors could therefore further validate their claim by sequencing an invasion ligand, such as the DBL proteins: EBA 175 for P. falciparum and DBA for P. vivax.
While the proximity between humans and animals is thought to be an explanation for the potentially zoonotic transmission, this will be surprising as the sharing of houses between humans and their domestic animals is a common occurrence in malaria-endemic tropical regions, where such a zoonosis has not been previously described. A census of the vector species between the two sites could enlighten on whether this potential zoonosis is driven by the vector.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Malaria, genomics, genetics, immunology, epidemiology, invasion, immunology

CITE

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Author Response 16 Feb 2023

Sitti Wahyuni, Hasanuddin University, Indonesia

16 Feb 2023

Author Response

Thank you for the suggestions and corrections. The corrections and suggestions were compiled and a revised version of the document is written. We believe that the method used has followed ... Continue reading Thank you for the suggestions and corrections. The corrections and suggestions were compiled and a revised version of the document is written. We believe that the method used has followed the malaria detection standards. The used methods have provided sufficient positive evidence so far. As for suggestions for validating our findings in several ways such as amplifying and sequencing cytochrome B, and sequencing an invasion ligand (such as the DBL proteins: EBA 175 for P. falciparum and DBA for P. vivax), we have included all in the discussion for further research because the method requires research funds which, for us, is quite a lot. Therefore, we are open to collaborate with other researchers from anywhere to work with us. We really appreciate the time spent by the reviewer to provide helpful comments and corrections.
Thank you for the suggestions and corrections. The corrections and suggestions were compiled and a revised version of the document is written. We believe that the method used has followed the malaria detection standards. The used methods have provided sufficient positive evidence so far. As for suggestions for validating our findings in several ways such as amplifying and sequencing cytochrome B, and sequencing an invasion ligand (such as the DBL proteins: EBA 175 for P. falciparum and DBA for P. vivax), we have included all in the discussion for further research because the method requires research funds which, for us, is quite a lot. Therefore, we are open to collaborate with other researchers from anywhere to work with us. We really appreciate the time spent by the reviewer to provide helpful comments and corrections.
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COMMENTS ON THIS REPORT

Author Response 16 Feb 2023

Sitti Wahyuni, Hasanuddin University, Indonesia

16 Feb 2023

Author Response

Thank you for the suggestions and corrections. The corrections and suggestions were compiled and a revised version of the document is written. We believe that the method used has followed ... Continue reading Thank you for the suggestions and corrections. The corrections and suggestions were compiled and a revised version of the document is written. We believe that the method used has followed the malaria detection standards. The used methods have provided sufficient positive evidence so far. As for suggestions for validating our findings in several ways such as amplifying and sequencing cytochrome B, and sequencing an invasion ligand (such as the DBL proteins: EBA 175 for P. falciparum and DBA for P. vivax), we have included all in the discussion for further research because the method requires research funds which, for us, is quite a lot. Therefore, we are open to collaborate with other researchers from anywhere to work with us. We really appreciate the time spent by the reviewer to provide helpful comments and corrections.
Thank you for the suggestions and corrections. The corrections and suggestions were compiled and a revised version of the document is written. We believe that the method used has followed the malaria detection standards. The used methods have provided sufficient positive evidence so far. As for suggestions for validating our findings in several ways such as amplifying and sequencing cytochrome B, and sequencing an invasion ligand (such as the DBL proteins: EBA 175 for P. falciparum and DBA for P. vivax), we have included all in the discussion for further research because the method requires research funds which, for us, is quite a lot. Therefore, we are open to collaborate with other researchers from anywhere to work with us. We really appreciate the time spent by the reviewer to provide helpful comments and corrections.
Competing Interests: I have no conflict of interest Close
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Version 4

VERSION 4 PUBLISHED 23 Jul 2021

Open Peer Review

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Reviewer Reports

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Version 4 (revision) 10 Dec 24				read
Version 3 (revision) 09 Apr 24		read	read
Version 2 (revision) 15 Feb 23		read
Version 1 23 Jul 21	read	read

Alfred Amambua-Ngwa, The Gambia at London School of Hygiene and Tropical Medicine, Fajara, The Gambia
Jaishree Raman, University of Pretoria, Pretoria, South Africa; University of the Witwatersrand Johannesburg, Johannesburg, South Africa
Paul Cliff Simon Divis, University of Malaysia, Sarawak, Malaysia
Didik Sumanto, Universitas Muhammadiyah Semarang, Semarang, Indonesia

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Reviewer Report

7 Views

09 Jan 2025 | for Version 4

Didik Sumanto, Universitas Muhammadiyah Semarang, Semarang, Central Java, Indonesia

7 Views Cite this report Responses(0)

Approved

1. About claim: "the first data report". Comment: This claim seems unfounded because there have been previous findings in Etawa crossbred goats in Purworejo and cattle in Jayapura. Please check the following refer 1 and refer 3

2. Figure 5: The microscopic image shown is not clear enough. The image cannot prove the morphological characteristics written in real terms. It is recommended that the re-shoot be done with a higher resolution. It would be better if each image of Plasmodium findings was accompanied by its microscopic ruler.

3. It is recommended to create a table showing the results of microscopic tests and nested PCR tests for 32 positive samples to support this statement.

4. The authors place more value on the genetic similarity between humans and animals in arguing to strengthen the finding of h-Plasmodium in domestic livestock. This is not wrong but the presence of zoophilic Anopheles plays an important role in transmitting h-Plasmodium from humans to animals. There needs to be a more explicit statement about the importance of zoophilic Anopheles' role as a cause of h-Plasmodium transmission in domestic livestock. Please see the following refer 2

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

References

1. Faculty of Medicine, Universitas Diponegoro Semarang, Indonesia, Sumanto D: Human-Plasmodium Like in Domestic-goat Blood in Malaria Endemic Areas in Purworejo Indonesia. Journal of Communicable Diseases. 2021; 53 (04): 148-152 Publisher Full Text
2. Sumanto D, Sayono: Penularan Penyakit Malaria Oleh Vektor Zoofilik Dengan Sumber Pakan Darah Non Manusia. PROSIDING SEMINAR KESEHATAN MASYARAKAT. 2023; 1 (Oktober): 35-43 Publisher Full Text
3. Department of Community Medicine, Sri Devaraj Urs Medical College, Sri Devaraj Urs Academy of Higher Education & Research Tamaka, Kolar, Karnataka, India., Kumar S J N: Utilisation and Delivery of Healthcare Services as Factors in Malaria Control in Kolar: A Community-Based Study. Journal of Communicable Diseases. 2023; 55 (03): 1-6 Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Epidemiology, Parasitology, Malaria disease, Helminthiasis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report

14 Views

22 Nov 2024 | for Version 3

Paul Cliff Simon Divis, Malaria Research Centre, Faculty of Medicine and Health Sciences, University of Malaysia, Sarawak, Malaysia

14 Views Cite this report Responses(1)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

molecular epidemiology, population genetics, genomics, zoonotic malaria

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Author Response

11 Dec 2024

Sitti Wahyuni, Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, 90245, Indonesia

We sincerely appreciate your insightful feedback regarding the need for stronger empirical support for our hypothesis. In response to your suggestion, we have revised the section discussing host-switching and included additional evidence that explores similar host shifts observed in other parasite species, as well as the potential evolutionary constraints that may apply to Plasmodium species. Furthermore, we have incorporated recent studies on parasite-host coevolution, which we believe will provide greater depth to our argument.

We also wish to thank you for your careful attention to the details of the qPCR methodology. To clarify, in this study, we utilized a SYBR Green-based qPCR approach, which is widely recognized for its sensitivity and cost-effectiveness in detection. We have now provided a more detailed description of the methodology in the revised manuscript, including the specific validation steps we undertook to ensure the accuracy of our results.

As per your request, we have also revised the title and conclusion of the manuscript. While the primers were initially tested using Apicomplexan parasites from primate hosts, subsequent BLAST analysis suggested that the target sequences were highly conserved across various host species. Additionally, we employed the rPF1-rPF2 primers for P. falciparum and rPV1-rPV2 primers for P. vivax to amplify DNA fragments of 918 bp and 714 bp, respectively. These larger amplicons offer additional confidence in the assay's specificity. The results obtained from these primers can be accessed via the following link: https://doi.org/10.6084/m9.figshare.14703012.v3.

While we were unable to perform sequencing of the PCR products with the rPF1-rPF2 and rPV1-rPV2 primers due to budgetary constraints, the PCR bands observed were consistent with the expected base pair lengths for P. falciparum and P. vivax.

We greatly appreciate your constructive comments and believe these revisions have strengthened the manuscript.

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Reviewer Report

18 Views

14 May 2024 | for Version 3

18 Views Cite this report Responses(1)

Approved

I have reviewed the revised article and think the authors have adequately addressed my concerns so am happy if this article is accepted.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Antimalarial drug and diagnostic resistance, molecular biology, molecular epidemiology, malaria elimination and control

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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13 Views

28 Nov 2023 | for Version 2

13 Views Cite this report Responses(1)

Approved With Reservations

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Reviewer Expertise

Antimalarial drug and diagnostic resistance, molecular biology, molecular epidemiology, malaria elimination and control

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40 Views

22 Dec 2022 | for Version 1

40 Views Cite this report Responses(1)

Not Approved

It is far too short. It needs to be expanded to explain the issue of residual malaria transmission in the study area and why it is concern. What interventions are in place to stop transmission and why you think they are failing to stem residual transmission.
There are four species of Plasmodia, not types that primarily affect humans
Is the statement on forest workers included because the study area has large numbers of forest workers? If so please provide this information.
From the data presented it looks like malaria transmission has decreased in both study areas so it is not clear to me what situation needs to be investigated. More clarity needs to be provided here.
A clear reason for the study needs to be provided as well as possible uses of the research findings.

Methods:

I am correct in assuming that the animal enclosures are in close proximity to the farmers homesteads? Please give an average distance between enclosures and homes – as mosquitoes will only travel as far as they need to, to get their next blood meal.
The remaining is either blotted or spotted onto the filter paper. I do not understand what “the dry filter paper was put on a sterile paper clip” means.
Where the houses geo-located so you could map the infected animals?
How long was the incubation at 85 degrees?
It is DNA amplification by PCR not of PCR
For nest two is there no annealing stage?
Please explain why nest one products were run on a gel.
Where the qPCR products really analysed using gel electrophoresis? The benefit of qPCR is real time data generation through the detection of fluorescence in the amplification stage. The analysis of qPCR products is generally done by reading fluorescence outputs.
Please provide a bit more information on the rationale for the re-extraction of the sample. In addition please state if the additional blood spot was from the same blood spot or different blood spot?

Results

It is not clear whether the pigs were not infected will any form of malaria parasites or only not infected with human malaria parasites. Please clarify.

Discussion

Please expand on the questions presented in the discussion in terms on possible results and their implication for malaria elimination in Indonesia and globally.
Please clarify if only infected were found in West Sumba region or only one study area within West Sumba. The reason provided for only one area having infected animals is unclear to me. Please rephrase for more clarity.
Please some guidance for future studies.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Antimalarial drug and diagnostic resistance, molecular biology, molecular epidemiology, malaria elimination and control

Respond to this report

Responses (1)

Author Response

20 Mar 2023

Sitti Wahyuni, Hasanuddin University, Indonesia

It is far too short. It needs to be expanded to explain the issue of residual malaria transmission in the study area and why it is concern. What interventions are in place to stop transmission and why you think they are failing to stem residual transmission.

Author’s response: Thank you, we have made a revision.

There are four species of Plasmodia, not types that primarily affect humans

Author’s response: Thank you for your input, we already revised.

Is the statement on forest workers included because the study area has large numbers of forest workers? If so please provide this information.

Author’s response: Thank you, we already revised.

From the data presented it looks like malaria transmission has decreased in both study areas so it is not clear to me what situation needs to be investigated. More clarity needs to be provided here.

Author’s response: Thank you, we already revised.

A clear reason for the study needs to be provided as well as possible uses of the research findings.

Author’s response: Thank you for your comment, we have revised accordingly.

Methods:

I am correct in assuming that the animal enclosures are in close proximity to the farmers homesteads? Please give an average distance between enclosures and homes – as mosquitoes will only travel as far as they need to, to get their next blood meal.

Author’s response: Thank you, we have already given the average distance in the method.

The remaining is either blotted or spotted onto the filter paper. I do not understand what “the dry filter paper was put on a sterile paper clip” means.

Author’s response: Thank you, more clarity has been made.

Where the houses geo-located so you could map the infected animals?

Author’s response: We apologize, because we don’t have the data about geo-location. But, in future research, we will apply this method. Thank you for your input.

How long was the incubation at 85 degrees?

Author’s response: Thank you for your question, the incubation at 85 degrees was 10 minutes.

It is DNA amplification by PCR not of PCR

Author’s response: Thank you for your comment, we have revised accordingly

For nest two is there no annealing stage?

Author’s response: Thank you for your question, the annealing nested two was 55°C in one minute.

Please explain why nest one products were run on a gel.

Author’s response: Thank you. Actually, the nested one does not need to run on a gel, but it was done just to notice if there were any bands in the nested one. However, it is not the standard for determining whether positive at that stage, because we used nested two for determining Plasmodium positive.

Where the qPCR products really analysed using gel electrophoresis? The benefit of qPCR is real time data generation through the detection of fluorescence in the amplification stage. The analysis of qPCR products is generally done by reading fluorescence outputs.

Author’s response: Thank you for your question. Both products of nested and qPCR were analysed using gel electrophoresis and then run on agarose gel (nested one in 1.5%, nested two in 2% agarose gel and qPCR in 1.5% agarose gel) in order to identify fluorescence band wave in a gel documentation system.

Please provide a bit more information on the rationale for the re-extraction of the sample. In addition please state if the additional blood spot was from the same blood spot or different blood spot?

Author’s response: Thank you, we already revised.

Results

It is not clear whether the pigs were not infected will any form of malaria parasites or only not infected with human malaria parasites. Please clarify.

Author’s response: Thank you, we already revised.

Discussion

Please expand on the questions presented in the discussion in terms on possible results and their implication for malaria elimination in Indonesia and globally.

Author’s response: Thank you, we already revised.

Please clarify if only infected were found in West Sumba region or only one study area within West Sumba. The reason provided for only one area having infected animals is unclear to me. Please rephrase for more clarity.

Author’s response: Thank you, we already revised.

Please some guidance for future studies.

Author’s response: Thank you, we already revised.

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Competing Interests

I have no conflict of interest

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Reviewer Report

63 Views

06 Jan 2022 | for Version 1

Alfred Amambua-Ngwa, Medical Research Council Unit, The Gambia at London School of Hygiene and Tropical Medicine, Fajara, The Gambia

63 Views Cite this report Responses(1)

Not Approved

Several Plasmodium species infect a wide range of animal species and there are other protozoa with similar cellular features that can also be microscopically misidentified for human Plasmodium. Thus, the identification of human Plasmodium will require species-specific distinguishing features by comparing the infected erythrocyte of the Plasmodium-infected domestic animals to those of other protozoan parasites including non-human Plasmodium.
A single sequence of each of the PCR products is shown per species and these sequences are similar to those of Plasmodium falciparum small rRNA. The alignment could include all samples sequenced and also how this was used to determine other species beyond P. falciparum. For speciation to be better validated, the authors could amplify and sequence cytochrome B, which is more divergent for Plasmodium.
More detail on the control protocols to limit contamination at sampling, extraction, and amplification of parasite DNA from the samples will help.
Given that humans and the family of domestic animals listed are largely divergent on RBC surface receptors for parasites, how these putative human-specific parasites invade the non-human hosts and develop will be of interest. The authors could therefore further validate their claim by sequencing an invasion ligand, such as the DBL proteins: EBA 175 for P. falciparum and DBA for P. vivax.
While the proximity between humans and animals is thought to be an explanation for the potentially zoonotic transmission, this will be surprising as the sharing of houses between humans and their domestic animals is a common occurrence in malaria-endemic tropical regions, where such a zoonosis has not been previously described. A census of the vector species between the two sites could enlighten on whether this potential zoonosis is driven by the vector.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Malaria, genomics, genetics, immunology, epidemiology, invasion, immunology

Respond to this report

Responses (1)

Author Response

16 Feb 2023

Sitti Wahyuni, Hasanuddin University, Indonesia

Thank you for the suggestions and corrections. The corrections and suggestions were compiled and a revised version of the document is written. We believe that the method used has followed the malaria detection standards. The used methods have provided sufficient positive evidence so far. As for suggestions for validating our findings in several ways such as amplifying and sequencing cytochrome B, and sequencing an invasion ligand (such as the DBL proteins: EBA 175 for P. falciparum and DBA for P. vivax), we have included all in the discussion for further research because the method requires research funds which, for us, is quite a lot. Therefore, we are open to collaborate with other researchers from anywhere to work with us. We really appreciate the time spent by the reviewer to provide helpful comments and corrections.

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Competing Interests

I have no conflict of interest

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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The discovery of human Plasmodium among domestic animals in West Sumba and Fakfak, Indonesia

Abstract

Background

Methods

Results

Conclusion

Keywords

Revised Amendments from Version 2

Introduction

Methods

Study area and population

Figure 1. Study area: Map of Gaura Village, West Lamboya, West Sumba, Indonesia and Fakfak Regency, West Papua, Indonesia.

Sample collection

DNA extraction

DNA amplification and electrophoresis

Table 1. Primer sequences for nested PCR.

Sequencing and alignment

Ethical clearance

Results

Figure 2. Gel view of PCR product from Plasmodium vivax and Plasmodium falciparum in domestic animals in Gaura, West Sumba (LD = DNA ladder, PS = positive samples, CN = control negative, CP = control positive) by nested PCR (multiplex PCR).

Figure 3. Plasmodium falciparum and P. vivax DNA sequence alignments from blood samples taken in Gaura village, West Sumba, Indonesia by ClustalW multiple sequence alignment (Kr = buffalo, Ku = Horse, A/An = dog, Ka = goat, P.f = P. falciparum, P.v = P. vivax).

Figure 4. Example of Plasmodium PCR product quality from a blood sample taken in Gaura village, West Sumba, Indonesia.

Table 2. Distribution of animal blood samples and Plasmodium species found in Gaura village, West Sumba, Indonesia and Fakfak, West Papua, Indonesia.

Figure 5. Morphology of Plasmodium in animals from Gaura village, West Sumba, Indonesia.

Discussion

Conclusion

Data availability

Underlying data

Reporting guidelines

Acknowledgements

References

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