Objective During an epidemiological study (January–July 2012) on malaria in forest villages of Central India, Plasmodium malariae-like malaria parasites were observed in blood smears of fever cases. We aimed to confirm the presence of P. malariae using molecular tools i.e. species-specific nested polymerase chain reaction (PCR) and DNA sequencing.
Methods All fever cases or cases with history of fever in 25 villages of Balaghat district were screened for malaria parasite using bivalent rapid diagnostic test and microscopy after obtaining written informed consent. Nested PCR was employed on microscopically suspected P. malariae cases. DNA sequences in the target region for PCR diagnosis were analysed for all the suspected cases of P. malariae.
Results Among the 22 microscopy suspected P. malariae cases, nested PCR confirmed the identity of P. malariae in 19 cases. Among these 14 were mono P. malariae infections, three were mixed infection of P. malariae with Plasmodium falciparum and two were mixed infection of P. malariae with Plasmodium vivax. Clinically P. malariae subjects generally presented with fever and headache. However, the typical 3-day pattern of quantum malaria was not observed. The parasite density of P. malariae was significantly lower than that of P. vivax and P. falciparum.
Discussions Plasmodium malariae may have been in existence in forest villages of central India but escaped identification due to its close resemblance to P. vivax. The results re-affirm the importance of molecular methods of testing on routine basis for efficacious control strategies against malaria.
Au cours d'une étude épidémiologique du paludisme (janvier-juillet 2012) dans des villages forestiers du centre de l'Inde, des parasites du paludisme semblables à Plasmodium malariae ont été observés dans les frottis sanguins des cas de fièvre. Nous avons cherché à confirmer la présence de P. malariae à l'aide d'outils moléculaires spécifiques pour l'espèce: la réaction en chaîne par polymérase (PCR) nichée et le séquençage de l’ADN.
Tous les cas de fièvre ou les cas ayant des antécédents de fièvre dans 25 villages du district de Balaghat ont été testés pour le parasite du paludisme en utilisant le test bivalent de diagnostic rapide (TDR) et la microscopie après avoir obtenu le consentement éclairé écrit. La PCR nichée a été appliquée sur les cas suspects au microscope de P. malariae. Les séquences d’ADN dans la région cible pour le diagnostic par PCR ont été analysées pour tous les cas suspects de P. malariae.
Parmi les 22 cas suspects de P. malariae à la microscopie, la PCR nichée a confirmé l'identité de P. malariae dans 19 cas. Parmi ceux-ci, 14 étaient des mono infections à P. malariae, 3 étaient des infections mixtes de P. malariae et de P. falciparum et 2 étaient une infection mixte à P. malariae et P. vivax. Cliniquement, les sujets infectés par P. malariae présentent généralement de la fièvre et des maux de tête. Cependant, le profil typique de 3 jours de malaria quantique n'a pas été observé. La densité du parasite était significativement plus faible avec P. malariae qu'avec P. vivax et P. falciparum.
P. malariae pourrait avoir existé dans les villages forestiers de l'Inde centrale mais aurait échappé de l'identification en raison de son étroite ressemblance avec P. vivax. Les résultats réaffirment l'importance des méthodes moléculaires comme tests de routine pour les stratégies de lutte efficaces contre le paludisme.
Durante un estudio epidemiológico de malaria (Enero-Julio 2012) en poblados forestales de la zona central de la India, se observaron parásitos del tipo Plasmodium malariae en láminas de pacientes con fiebre. El objetivo de este estudio era confirmar la presencia de P. malariae utilizando herramientas moleculares: una PCR anidada especie-específica (PCR) y secuenciación del ADN.
A todos los casos de fiebre o casos con historia de fiebre en 25 poblados del distrito de Balaghat, se les realizaron pruebas en busca del parásito de la malaria, utilizando pruebas de diagnóstico rápido (PDR) y microscopía, después de haber obtenido un consentimiento informado por escrito. La PCR anidada se utilizó en los casos con sospecha microscópica de P. malariae. La secuencias de la región de ADN utilizada para el diagnóstico por PCR fueron analizadas en todos los casos con sospecha de P. malariae.
De los 22 casos sospechosos por microscopía de infección con P. malariae, la PCR anidada confirmó la identidad del parásito en 19 de ellos. Entre estos, 14 eran infecciones solo con P. malariae, 3 eran infecciones mixtas de P. malariae con P. falciparum y 2 eran infecciones mixtas de P. malariae con P. vivax. Desde un punto de vista clínico, los sujetos con infección por P. malariae generalmente presentaban fiebre y dolor de cabeza, pero no se observaba el típico patrón de 3 días de la malaria. La densidad parasitaria de P. malariae era significativamente menor que la de P. vivax y la de P. falciparum.
P. malariae puede haber estado presente en poblados forestales del centro de India y haber escapado a su identificación por su gran parecido con P. vivax. Los resultados reafirman la importancia de los métodos moleculares para realizar pruebas rutinarias a la hora de contar con estrategias eficaces para el control de la malaria.
Malaria is a global health problem with South-east Asia contributing nearly 40% of the world malaria burden (Snow et al. 2005). Among the 2.5 million reported cases in the Southeast Asia region, India alone contributes about 75% of the total cases. Human malaria is caused by five Plasmodium species, i.e. Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi (Singh et al. 2004). These species differ from each other in their morphological characteristics, clinical presentation, sensitivity to antimalarials, transmission potential and nature of immunity (Mohapatra et al. 2008). In India, both P. vivax and P. falciparum are prevalent in equal proportions. However, P. malariae and P. ovale are rare (Mohapatra et al. 2008), and P. knowlesi remains unreported.
Balaghat is a highly malaria-prevalent district in Central India (Madhya Pradesh). Both P. falciparum and P. vivax prevail, with preponderance of P. falciparum (Shukla et al. 2011). An epidemiological study for malaria was undertaken in Balaghat in 2010 to develop an evidence-based intervention model for malaria control in remote areas. During the recent cross-sectional surveys, we encountered morphologically distinct malarial parasites similar to P. vivax that differed from P. vivax because of host cell morphology. Notably, these malarial parasites resembled P. malariae. The study has been ongoing with microscopy as its main diagnostic tool since 2010; more than 3000 smears were examined each year with about 1000 cases positive for malaria. However, no peculiar forms of P. malariae-like parasites were observed in previous surveys. Accurate identification of malaria parasites is a crucial pre-requisite for successful treatment of malaria and effective control. Molecular methods of identification of Plasmodium are known to be more sensitive and specific (Snounou et al. 1993), hence for confirmation of P. malariae-like parasites, species-specific nested polymerase chain reaction (PCR) was employed on microscopically confirmed malaria parasites.
Materials and methods
Birsa Community Health Centre (CHC) in Balaghat is a forest area inhabited by the Baigas ethnic tribal group. There are 160 villages (population 142 032), bordering Chhattisgarh State, in Birsa CHC which are hot spots for epidemic malaria (Figure 1). These villages are small, located off the road, highly scattered and interspersed with streams and their tributaries. The inhabitants are mostly illiterate, scantily clothed and work mainly in forest nurseries. They spend the majority of their time outside their dwellings and sleep on the floor in verandahs or outdoors. Two vectors, Anopheles culicifacies and Anopheles Fluviatilis, breed throughout the year in streams and their tributaries. The villages are sprayed with two rounds of Alphacypermethrin in June and September each year. The local climate is characterised by a hot summer (April to June), a monsoon/rainy season (July to October), a cool autumn season (November to January) and a short spring season (February to March).
Active fever surveys were carried out door to door in 25 villages. All fever cases or cases with history of fever were screened for malaria parasite using bivalent rapid diagnostic test (RDTs) and microscopy after obtaining written informed consent. For children (<10 years) written consent was obtained from their parents. The study was approved by the centre’s ethical committee. The blood smears were examined under microscope under 1000× magnification. For quality control 100% of positive smears and 10% of negative smears were re-examined by second expert microscopist who was unaware of previous results. Spleen enlargement was determined by Hackett’s method in children between 2 and 9 years of age (Christophers et al. 1958). Two types of RDTs were used in this study: species-specific RDTs for diagnosing P. vivax and P. falciparum infections and pLDH- and HRP2-based RDTs designed to detect both falciparum and non-falciparum infections. These RDTs were FalciVax Rapid test for malaria Pv/Pf Device (Zephyr Biomedicals, Verna, Goa, India) and FIRST RESPONSE® Malaria Antigen pLDH/HRP2 Combo Card test (Premier Medical Corporation Ltd., Daman, Goa, India). During the cross-sectional surveys in January 2012, morphologically distinct malaria parasites resembling P. vivax were observed. However, these parasites differed from P. vivax in their morphological characteristics. The subsequent survey in February also revealed a sudden spurt of similar malaria parasites that led to blood collection for PCR-based detection methods beginning in March. The samples for PCR were collected on Whatman filter paper No. 3 along with smears for microscopy.
Genomic DNA was isolated from the filter paper blood spots and species-specific nested PCR was carried out using the 18s rRNA gene (Snounou et al. 1993). The DNA sequencing was carried out using PCR product of P. malariae positive samples. A partial sequence (144 bp) of the 18s rRNA gene was sequenced from both directions using the ABI PRISM® BigDye™ terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, USA) in an 3130xl DNA Analyzer (Applied Biosystems, USA). DNA sequences (forward and reverse) were edited and the consensus sequence was created using the BioEdit, DNASTAR (DNASTAR Inc., Madison, WI, USA). Consensus sequences were multiple-aligned with previously published sequences from the GenBank database using GENEDOC software (Genedoc, CA, USA). Student’s t-test was used to compare the mean parasite density between different type of malaria parasites and a P-value of ≤0.05 was considered statistically significant.
All parasite-positive subjects were treated as per guidelines of the National Vector Borne Disease Control Programme. Plasmodium vivax cases were treated with 1500 mg chloroquine followed by 15 mg primaquine daily for 14 days; cases with P. falciparum received Artemisinin-based combination (ACT) [Artesunate (200 mg) + Sulfadoxine (1500 mg) + Pyrimethamine (75 mg)] and 45 mg primaquine (single dose). Plasmodium malariae cases were given 1500 mg chloroquine for 3 days.
A total of 2779 fever cases ranging from 2 months to 75 years (mean age: 11.12 ± 11.27 years) were screened for malaria, of which 1506 were found positive (mean age: 8.4 ± 7.3 years) for malaria by microscopy (54.2%). Plasmodium falciparum was the most prevalent species (922/2779) followed by P. vivax, (467/2779) and P. malariae mono infection (28/2779). Mixed infections of P. malariae with P. vivax/or P. falciparum (12) and mixed infections of P. vivax with P. falciparum (77) were also recorded (Table 1). Malaria parasites were identified as P. malariae based on morphological features viz, the parasitised red blood cells were normal in size, carrying band form trophozoite across parasitised red blood cells with coarse stippling and number of merozoites was 7–10 per schizont. Clinically P. malariae subjects generally presented with fever and headache, but the typical 3-day pattern of quartan malaria was not stated by patients. Average spleen enlargment was 1.71 and pallor was generally observed in children. Nested PCR (species-specific) showed an amplification product of 144 bp confirming the presence of P. malariae in 19 out of 22 microscopically suspected cases collected during March and April. Of 19 PCR-positive cases, 14 were mono-infection, two were mixed infections of P. malariae with P. vivax (120 bp) and three were mixed infections of P. malariae with P. falciparum (205 bp) (Figure 2a). Interestingly, the majority of P. malariae cases were found during the spring season (February–March), when the temperatures range between 20 and 28 °C. This period coincides with the dry period of the year. When the temperature increased to about 30–40 °C with the onset of summer, P. malariae infections started declining sharply. Most of the malaria cases were children below 10 years. The mean age of P. malariae infected subjects was 7.78 ± 5.61 years while mean ages of P. vivax and P. falciparum infected cases were 8.13 ± 6.92 and 8.96 ± 7.87 years respectively. The parasite densities of P. malariae ranged from 46.4 to 11 400 parasites/μl, (mean: 263.33 ± 174.48 parasites/μl of blood). The mean parasite density of P. malariae was significantly lower than that of P. vivax (263.33 vs. 741.62; P =0.0210) and P. falciparum (263.33 vs. 669.75; P =0.0001). All 19 P. malariae-positive cases were also tested for P. knowlesi and P. ovale by PCR and found to be negative. DNA sequences in the target region for PCR diagnosis were analysed for all the 19 samples of P. malariae. All samples sequences obtained were identical and aligned with GenBank sequences representing the 18 s rRNA (from Africa and Myanmar; Figure 2b) thus confirming the presence of P. malariae.
Table 1. Malaria Slide positivity rate in study villages of CHC Birsa, district Balaghat (January–July, 2012)
Mixed (Pf + Pv)
Mixed (Pm + Pf/Pv)
+ve/BSE: positve for malaria/blood slide examined; Pf, Plasmodium falciparum; Pv, Plasmodium vivax; Mixed (Pf + Pv): P. falciparum + P. vivax; Pm, Plasmodium malariae; Mixed (Pm + Pf/Pv): P. malariae + P. falciparum/P. vivax; SPR, slide positivity rate; SFR, slide falciparum rate; Pf%, P. falciparum percentage; CHC, Community Health Centre.
Interestingly, the monoinfection of P. malariae was not detected by FalciVax while eight mixed infections with P. falciparum were detected accurately as P. falciparum and two mixed infection of P. malariae with P. vivax as P. vivax. However, two low density mixed infections of P. vivax with P. malariae were not detected. Notably, FIRST RESPONSE showed band for Pan for mono-infection of P. malariae or mixed infection of P. malariae with P. vivax and dual bands for both Pan and Pf in cases with P. falciparum infection.
Historically, there have been reports of sporadic occurrence of P. malariae in India based on microscopy (Knowles et al. 1930; Covell & Singh 1942; Panda et al. 1990). This is the first report of detection of P. malariae from Madhya Pradesh which has also been confirmed by PCR. The probable cause of the sudden spurt of P. malariae cases is not known. It is suspected that many of the P. malariae cases in areas of its prevalence in India may have been incorrectly diagnosed as P. vivax during routine microscopy. The ring forms of these two species are similar; moreover, P. malariae is difficult to identify if the typical morphology of the infected host cell is damaged (Mohapatra et al. 2008). Further, P. malariae is sustained at very low infection rates among the sparse and mobile human population (Mohapatra et al. 2008) and may remain for decades within a human host to infect mosquitoes (Carter & Mendis 2002), thus facilitating the transmission. In this area intense transmission of falciparum and vivax may have activated P. malariae, which warrants further studies. The clinical finding suggests that the range of P. malariae pathological manifestation is clearly underestimated. All the patients with malaria were symptomatic; however the typical 3 day fever periodicity of quartan malaria could not be established. Moreover, the presence of P. knowlesi and P. ovale were ruled out in view of the fact that microscopically identified P. malariae cases in Malaysia were found to be P. knowlesi by PCR detection (Singh et al. 2004; Rajahram et al. 2012).
Vector control measures applied in these areas were two rounds of Alphacypermethrin during the months of June and September as the main vector A. culicifacies is known to maintain malaria transmission between July and October (Vaid et al. 1974). These two rounds of indoor residual spray are being carried out under the national programme for interruption of malaria transmission (each round is effective for about 10 weeks). As detected in the present studies, the emergence of P. malariae in spring indicates no or suboptimal effect of June and September sprays. Thus an additional round is warranted in January to February to prevent transmission of P. malariae. The pattern of infection (mono or mixed) is also determined by the occurrence and the ability of the vector species to transmit different parasite species simultaneously (Mayxay et al. 2004). In the study area A. culicifacies and A. fluviatilis transmit both P. vivax and P. falciparum (data not shown). However, their role in P. malariae transmission is unknown.
In high malaria transmission areas screening by species specific RDT and subsequent treatment of P. vivax by Chloroquine and Primaquine and P. falciparum by ACT are being recommended in India. Under this screen and treat strategy, presence of P. malariae poses a difficulty if the screening is performed by RDTs alone. Further, in the present studies species specific RDTs failed to detect monoinfections of P. malariae or even low density P. vivax mixed with P. malariae. This could be due to low sensitivity of FalciVax for P. vivax (Singh et al. 2010). Notably, no evaluations of RDTs for P. malariae are presently being carried out in India. However, its sensitivity has been found to be low in countries where it was evaluated (Maltha et al. 2011).
Thus precise identification of the malaria parasite and its vectors are indispensable not only for successful treatment, but also for designing and development of effective malaria control measures. Further, misidentification of malaria parasite could prolong the parasite clearance time leading to anaemia and/or drug resistance.
The sudden emergence of P. malariae requires further investigation as it has been implicated in renal damage and nephritic syndrome (Harinasuta & Bunnag 1988). Furthermore, P. malariae also poses challenge to Roll Back Malaria Initiative as transmission of this parasite is tricky to control due to its characteristic persistence for decades within a human host and prolonged relapses (Siala et al. 2005). Finally, effective malaria surveillance using molecular methods is necessary on a regular basis to estimate the actual case load due to P. malariae for amenable diagnostic and treatment efforts.
We acknowledge and sincerely appreciate the support and inspiration received from the Department of Health and FW, Government of Madhya Pradesh. The Madhya Pradesh Technical Assistance and Support Team deserve special appreciation for their logistic support. We are grateful to Dr. S. Rajasubramanian for helping in revising the manuscript. The study was funded by Indian Council of Medical Research, New Delhi, India.