Investigation on possible transmission of monkeys' Plasmodium to human in a populations living in the equatorial rainforest of the Democratic republic of Congo.

Plasmodiums are protozoa that may infect various hosts. Only five species are now recognized as naturally parasitizing humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. This fifth species, P. knowlesi, previously identified as naturally parasitizing the monkey Macaca fascicularis, has been microscopically confused for a long time with P. malariae or P. falciparum and it was not possible to correctly differentiate them until the advent of molecular biology. To date, natural human infections with P. knowlesi only occur in Southeast Asia and a similar phenomenon of natural transmission of simian plasmodium to humans has not been reported elsewhere. This study was conducted to investigate a possible transmission of African small monkey's plasmodium to humans in populations living near the rainforest of the Democratic Republic of Congo (DRC) where several species of non-human primates are living. Two successive real-time PCRs were identified in the literature and used in combination for purpose. Only P. falciparum was found in this study. However, studies with larger samples and with more advanced techniques should be conducted.

Introduction

The first report on the existence of simian Plasmodium was published in 1908 by Daniels (1908). It was believed that simian Plasmodium spp. infect only monkeys and that human Plasmodium spp. infect only humans. Eyles et al. (1960) showed that a Plasmodium vivax-like species, Plasmodium cynomolgi, could infect humans in the laboratory. Warren et al. (1970) reported the first case Plasmodium knowlesi in humans, which had been detected in 1965. This species has long been known as a natural parasite of the macaque (Macaca fascicularis) and prior to the advent of molecular techniques was misidentified in humans as Plasmodium malariae or Plasmodium falciparum, which it resembles morphologically (Singh et al., 2004, Lee et al., 2009). In a large study in Sarawak, Malaysia, Singh et al. (2004) detected a high prevalence (57.7%) in humans. Since then, natural infections with this species, confirmed using molecular techniques, have been reported from many other countries in South-East Asia (Jongwutiwes et al., 2004, Vythilingam et al., 2006, Zhu et al., 2006, Cox-Singh et al., 2008, Luchavez et al., 2008, Ng et al., 2008, Khim et al., 2011). Thus, P. knowlesi has been declared to be the fifth species of Plasmodium infecting humans (White, 2008). More recently, Ta et al. (2014) have reported the first case of naturally acquired human infection with P. cynomolgi, in a woman in Malaysia. An initial microscopic diagnosis of P. malariae/P. knowlesi was established, followed by a second microscopic re-examination and a nested PCR that identified that species as P. vivax. However, a different PCR method and sequencing confirmed the infection as P. cynomolgi. These reports suggest that human infections by simian Plasmodium are probably under-reported because of misidentification. If P. knowlesi had been misdiagnosed for such a long time in Asia and if P. cynomolgi identification has been so difficult, it may be possible that in Africa, where many non-human primates live, natural transmission of simian Plasmodium spp. to humans has not been recognized. Indeed in Africa where malaria diagnosis is essentially microscopy-based, it is likely that some Plasmodium spp. in people have been misidentified.

Many studies have been reported that explore the presence of simian malaria parasites in human or human malaria parasites in other primates, but almost all of these studies were based on great apes, e.g., Pan paniscus (bonobo), Pan troglodytes (chimpanzee) or Gorilla gorilla (gorilla) (Contacos et al., 1970, Ollomo et al., 2009, Duval et al., 2010, Kaiser et al., 2010, Krief et al., 2010, Liu et al., 2010, Prugnolle et al., 2010). Very few studies included monkeys. For example, Prugnolle et al. (2011) analyzed blood samples from 338 monkeys of ten species and Pourrut et al. (2009) examined blood samples from 82 small monkeys (e.g., Chlorocebus aethiops and Erythrocebus patas) for Plasmodium. Many species of monkeys are present in the Democratic Republic of Congo (DRC) (Ladnyj et al., 1972, Colyn, 1994, Grubb et al., 2003) and some of these have been listed as hosts for Plasmodium species, especially P. gonderi, Plasmodium petersi, Plasmodium georgesi (Poirriez et al., 1993, Poirriez et al., 1995) and Plasmodium sp. DAJ- 2004 (Prugnolle et al., 2011). As P. knowlesi and P. cynomolgi naturally infect monkeys, and have been reported as able to also infect humans, the present study investigated the possible transmission of monkey Plasmodium to humans in a populations living in the equatorial rainforest of the DRC where several species of non-human primates are found.

Materials and methods

Sample collection

Three villages of the Equatorial Province of the DRC, Bolenge (N: 00° 24′ 89″, E: 18° 22′ 29″), Bongonde (N: -0.15° 42′ 42″, E: 18° 22′ 35″) and Wendji-Secli (N: -00° 06′ 56″, E: 18° 22′ 35″), were selected based on geographical location near the equatorial rain forest. One hundred individuals without symptoms of malaria were randomly selected from each village without consideration of age or sex. People with fever were not selected. A drop of blood from each individual using a finger prick and absorbed on to filter paper Whatman 3 MM (Sigma–Aldrich, St. Louis, MO, USA) was made in duplicate for each individual. People in these villages practice daily forest activities such as hunting and gathering. Monkeys are captured for commerce and for food.

DNA extraction

Parasite DNA was extracted using QIAamp® DNA mini kits (Qiagen, Hilden, Germany) according to manufacturer's instruction for filter paper and in a final elution volume of 150 μL.

Plasmodium species identification

Two real-time PCR were identified in the literature and were successively applied to the DNA templates. The first one allows the detection of all Plasmodium spp. and the second one allows the detection of the four common Plasmodium species that infect humans in Africa (P. falciparum, P. malariae, P. vivax and Plasmodium ovale).

As the species specific Real-Time PCR was more sensitive than the pan primer one, we hypothesized that if the first Real-Time PCR was positive and the second negative, it would suggest that a Plasmodium species different from the four human species was detected. In that case, sequencing would be considered.

First RT-PCR

This RT-PCR includes a pair of primers (Plasmo 1 and Plasmo 2) and one probe (plasprobe), designed to detect genes of the small subunit 18S rRNA of Plasmodium species, that allow the detection of all Plasmodium spp. (Table 1). The mix consists of 12.5 μl of 2X Taqman Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA), 200 nM of each primer, 50 nM of probe and water for a total volume of 25 μl including 5 μl of DNA. PCR conditions were as follows: an initial step at 50 °C for 2 min, 95C° for 10 min, and 45 cycles of 95 °C for 15 s followed by 1 min at 60 °C (Rougemont et al., 2004).

Table 1

Primers and probes sequences for the two RT-PCR.

RT-PCR	Forward primer	Reverse primer	Probes
plasmodiums sp	5′-GTTAAGGGAGTGAAGACGA TCAGA-3' (Plasmo1)	5′-TTATGAGAAATCAAAGTCTTTGGGTT-3' (Plasmo2)	5′-FAM-ACCGTCGTAA TCTTAACCAT AAACTATGCC GACTAG-TAMRA-3' (plasprobe)
Pfal	5′-CTAGGTGTTGGATG-3′	5′-FAM-AGCAATCTAAAAGTCACCTCGAAAGATGACT-DQ-3' (Falprobe)
Pova	5′-CGACTAGGTTTTGGATG-3′	5′-VIC-CGAAAGGAATTTTCTTATT-DQ-3’ (Ovaprobe)
Pviv	5′-GACTAGGCTTTGGATG-3′	5′-VIC-AGCAATCTAAGAATAAACTCCGAAGAGAAAATTCT-DQ-3' (Vivprobe)
Pmal	5′-GACTAGGTGTTGGATG-3′	5′-FAM-CTATCTAAAAGAAACACTCAT-DQ-3’ (Malprobe)

Second RT-PCR

Four species-specific forward primers, one universal reverse primer (Plasmo 2) and four species-specific probes were used in two simultaneous duplex reactions (Pfal + Pviv and Pmal + Pova) (Cnops et al., 2010). The probes and primers were designed to detect genes of the small subunit 18S rRNA of the four Plasmodium species. The mix consisted of 12.5 μl of 2X Taqman Universal PCR Master Mix (Applied Biosystems), 200 nM of each primer and probes except for P. vivax at 100 nM in a final volume of 25 μl including 5 μl of DNA. The following conditions were used: 2 min at 95 °C, followed by 50 cycles of 15 s at 95 °C and 60 s at 60 °C.

Both RT-PCR were run on a Stratagene Mxp 3000 Instrument (SABiosciences) and in the presence of positive controls (human Plasmodium) provided by the Parasitology Unit, Institute of Tropical Medicine, Antwerp and the Laboratory of Clinical Microbiology, University Hospital of Liège, Belgium.

To assess our methodology, P. berghei and P. vinckei cultures (provided by the Interfaculty Drug Research Center, University Hospital of Liege) have been used as non-human Plasmodium templates. Unfortunately, we were not able to test for the other monkey parasites P. gonderi, P. georgesi, P. petersi or Plasmodium sp. DAJ 2004.

Ethical considerations

This study has received the ethical approval of the Ministry of Public Health of the DRC and of the Institutional Committee of the Faculty of Medicine, University of Kinshasa.

Results and discussion

Assessment of the methodology

As template, P. vinckei and P. berghei were used together with the four human Plasmodium as positive controls for the two successive RT-PCRs. The first one detected all Plasmodium but the second only detected P. falciparum, P. malariae, P. ovale and P. vivax (Table 2).

Table 2

Cycle threshold obtained for the 6 plasmodium species.

	P. falciparum	P. malaria	P. ovale	P. vivax	P. vinckei	P. berghei
1st RT-PCR	16.98	26.01	26.08	30.15	15.18	20.33
2nd RT-PCR	18.07	26.69	26.66	30.48	No signal	No signal

As we were not able at the time of this study to get P. gonderi, P. petersi or P. georgesi samples, we aligned the primers and the probes used for both real-time PCRs with their genome to explore if they could match. Only primers and probe of the first RT-PCR matched.

Parasite identification

Of the three hundred samples analyzed, 139 (46,3%) were positive for both RT-PCRs. All 139 samples tested positive for P. falciparum. No other human Plasmodium species were detected. It is possible that the small sample size and the high prevalence of this species reported in the region (Ngimbi et al., 1980, Taylor et al., 2011, Mvumbi et al., 2013) could account for this. P. malariae and P. ovale are often found in association with P. falciparum and P. vivax is said to be very rare in that region because of the lack of the Duffy antigen in the population (Miller et al., 1976).

No monkey malaria parasites were found. However, the methodology used in this work is probably insufficient to confirm that there is no simian Plasmodium infecting humans in this area. Recent reports suggest that many simian Plasmodium species are phylogenetically close to those infecting humans. For example, Duval et al. (2009) described a P. ovale-like Plasmodium from chimpanzees in Cameroon, Kaiser et al. (2010) found a P. malariae-like infection in chimpanzees from Ivory Coast, and Krief et al. (2010) described new P. falciparum-like and P. vivax-like new parasites in DRC. These results indicate that some PCR assays cannot distinguish human Plasmodium species from those infecting simians. Further, the species-specific PCR primers described by Snounou et al. (1993) were unable to distinguish P. knowlesi from P. malariae (Cox-Singh et al., 2008) nor P. vivax from P. cynomolgi (Ta et al., 2014).

There is, therefore, the possibility that some of the P. falciparum we identified might be the P. falciparum-like parasite identified by Duval et al. (2010), by Krief et al. (2010) or by Liu et al. (2010). That is the major limitation of this study. The methodology used in the current study would allow us, however, to detect simian Plasmodium species that are phylogenetically distant from human species, e.g., P. gonderi, P. petersi, P. georgesi or Plasmodium sp. DAJ 2004.

Conclusion

In this study, we could not detect monkey Plasmodium species in a human population living near the rain forest of the Equatorial province of the DRC. We found only P. falciparum, which is the major species in this area. Though the methodology used here cannot discriminate simian P. falciparum-like from human P. falciparum, we can hypothesize that no other simian Plasmodium species were present in the individuals sampled. Studies on larger human populations using more advanced techniques (e.g., sequencing of mitochondrial genome of Plasmodium species or specific genes) would help to confirm this. To our knowledge, this work is the first to search for human infection with Plasmodium species from small apes in the DRC.