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.53946.1

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Research Article

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

[version 1; peer review: 2 not approved]

Munirah Munirah¹, Sitti Wahyuni², Isra Wahid², Firdaus Hamid³

PUBLISHED 23 Jul 2021

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: © 2021 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 1; peer review: 2 not approved]. F1000Research 2021, 10:645 (https://doi.org/10.12688/f1000research.53946.1) 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)

Introduction

Malaria is transmitted by the Plasmodium vector Anopheles mosquitoes. Four Plasmodium types, namely P. falciparum, P. vivax, P. ovale, and P. malariae cause pathologic conditions in humans. Recently in Southeast Asia, P. knowlesi infection cases have also been reported.¹^–³

Before molecular diagnostics development, only humans were assumed to be the primary host for Plasmodium. However, studies in the last two decades on Plasmodium reported that the parasites originated from animals. Further stating that P. falciparum originated in the gorilla⁴ and chimpanzee,⁵^,⁶ P. vivax was from African apes,⁷ P. malariae was from chimpanzees⁶ and P. knowlesi was from monkeys,⁸^,⁹ while P. ovale in humans and chimpanzees are genetically identical.¹⁰ The factors hypothesized to explain this situation include primate’s habitat loss and human’s aggressiveness in exploring forest.¹¹ A study from South Kalimantan reported the contribution of forest workers to malaria incidence.¹²

East Nusa Tenggara and West Papua are known as malaria-endemic areas in Indonesia as their annual parasite incidence (API) in 2015 was 31.29% and 7.04%, respectively,¹³ while in 2018, according to the health office in both the districts, the API rate in Fakfak, West Papua and East Nusa Tenggara, West Sumba was 4.85% and 12.9%, respectively.^{(unpublished data)} Due to this situation, we aimed to explore the presence of human Plasmodium among domestic animals that are a potential reservoir host.

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.

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 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 was dropped onto a filter paper (Whatman CAT No. 1442-090) until it absorbed to about 1.5 cm in diameter. The dry filter paper was put on a sterile plastic clip and stored at room temperature for a maximum of 10 days.

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 x 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. 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 of 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. vivax¹⁴ and P. knowlesi¹⁵ 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) 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 analysed using agarose gel 1.5% for electrophoresis. 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.

Considering the possibility of contamination, DNA was re-extracted from blood from the same filter paper. PCR was performed using the primers, rPF1 and rPF2, as well as rPV1 and rPV2¹⁶ 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 application¹⁷ 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 animal blood samples were collected from Gaura and Fakfak villages, respectively. These consisted of 92 buffalos, 21 horses, 121 goats, 18 dogs, and 18 pigs. Using the nested PCR method, 32 of the 270 animals were found to be P. falciparum and P. vivax positive. The percentage of Plasmodium positive animals included 20.7% buffalo, 14.3% horse, 5.8% goat, and 16.7% dog with one buffalo having a mixed infection (P. falciparum and P. vivax). There was no P. knowlesi found in any of the samples and no other Plasmodium was found in 18 pig blood samples. PCR gel products, DNA sequence results, and the sample’s quality can be seen in Figures 2, 3 and 4, respectively.¹⁸ Plasmodium distribution in the animals’ blood samples from Gaura and Fakfak are presented in Table 2, and it shows that blood containing Plasmodium was only found in Gaura. The results of the qPCR using rPF1–rPF2 and rPV1–rPV2 primers were similar to 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 DNA sequence alignments from blood samples taken in Gaura village, West Sumba, Indonesia by ClustalW multiple sequence alignment.

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

The presence of Plasmodium was suspected in domestic animals because malaria cases in these two villages remained high despite maximum preventive efforts having been applied including insecticide-treated bed nets. About 32 of the 270 blood (11.9%) samples contained human Plasmodium parasites, and this is the first data report and further study is therefore needed.

Previous studies found Plasmodium relictum in avian species,¹⁹ P. cephalophi in ungulates,²⁰ P. traguli in mousedeer,²¹ P. brucei in gray duiker,²²^,²³ P. bubalis in water buffalo,²⁴ and P. odocoilei in white-tailed deer.²⁵^,²⁶ Other parasites found included P. caprae in goats (ruminant),²⁷ P. bergei in Rodentia,²⁸ and P. cynomolgi, P. inui, and P. fragile in primates.²⁹ The five Plasmodium species that infect humans were originally parasites in primates.¹^,³^,⁶^–⁹ In this study, P. falciparum was found in buffalos, goats, dogs, and horses, while P. vivax was in buffalos, goats, and dogs. Initially, the presence of Plasmodium in these animals’ erythrocytes was not certain. However, the nested PCR showed the same results for all positive samples. The sequencing results of the positive bands in the nested PCR two analysis showed the bands were P. falciparum and P. vivax (Figure 3). This is the first investigation reporting human Plasmodium in domestic animals (ruminant, ungulate, and carnivore).

Plasmodium discovery among domestic animals in malaria-endemic areas raises the following questions. How do P. falciparum and P. vivax live in these animals? Are they intermediate hosts for this parasite? Did these Plasmodium species evolve to live in ruminants, ungulates, and carnivores? As a result of repeated exposure, have these animals become more permissive to Plasmodium, which generally lives in humans? Is this parasite pathogenic in animals? P. knowlesi is a commensal microbe in primates but pathogenic in humans¹^–³ and its migration from primates to humans is caused by forest loss or human invasion of primate habitat.¹¹ There is a possibility that animal and human proximity aids easier cross parasite transfer between both groups by mosquitoes.

Despite high API in Fakfak and Gaura village, West Sumba, only the animals from West Sumba had human Plasmodium. This difference is possibly due to the distance between the residents’ houses and animal enclosures as the enclosures are located approximately 50–500 meters from the main houses in Fakfak. Meanwhile, in Gaura, residents live in stilt houses where the ground floor functions as an animal shelter, allowing microbial transfer between humans and animals by mosquitoes. In Fakfak, the sampling locations were not easily accessible, and the steep geographical conditions made it difficult to collect many samples compared to Gaura.

Although Plasmodium can be detected microscopically due to erythrocyte size, which is smaller in animals than humans, molecular methods become significant in detecting Plasmodium presence. The nested PCR was used to detect Plasmodium because its sensitivity was equally as high as Real-Time PCR and the cost was relatively lower.³⁰^,³¹ The microscopic method of double fluorescent dye utilization with Giemsa stain is recommended for further studies.³²

Conclusion

In this study we found human Plasmodium in domestic animals. It is still not clear whether the animal had malaria, but this finding may be used as a reference for conducting malaria surveys in domestic animals in endemic areas. Human Plasmodium was only found in Gaura where the location of the animal enclosures is integrated with the residents’ houses. Local communities need to be educated about the possibility of malaria transmission due to the integration of animal enclosures and peoples’ homes. The discovery of human Plasmodium in domestic animals in this study may partly explain the persistence of the high prevalence of malaria in some endemic areas.

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.

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.v3.¹⁸

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.v3.¹⁸

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

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Comments on this article Comments (0)

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.

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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 1; peer review: 2 not approved]. F1000Research 2021, 10:645 (https://doi.org/10.12688/f1000research.53946.1)

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Reviewer Report 22 Dec 2022

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

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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
Report a concern
Respond or Comment

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

Views

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.
Competing Interests: I have no conflict of interest Close
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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.
Competing Interests: I have no conflict of interest Close
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Version 4

VERSION 4 PUBLISHED 23 Jul 2021

Open Peer Review

Reviewer Status

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

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

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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Competing Interests

No competing interests were disclosed.

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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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Reviewer Report

13 Views

28 Nov 2023 | for Version 2

13 Views Cite this report Responses(1)

Approved With Reservations

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 the authors enlist the assistance of an English editor to revise the article so that it meets the publication standards of the journal.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

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

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Reviewer Report

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

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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

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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

Keywords

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.

Figure 3.

Figure 4.

Table 2.

Figure 5.

Discussion

Conclusion

Acknowledgements

Data availability

Underlying data

Reporting guidelines

References

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