First report of detection and molecular confirmation of Plasmodium ovale from severe malaria cases in central India

Authors


Abstract

Objective

We report for the first-time detection of Plasmodium ovale in central India.

Methods

From 2010, all hospitalised suspected malaria cases at the malaria clinic of the Regional Medical Research Centre for Tribals in Bastar district, central India, were screened. Plasmodium species were identified by microscopy and species-specific nested PCR of 18s rRNA.

Results

Of 256 enrolled cases of confirmed P. falciparum malaria by microscopy, P. ovale infection was detected in three cases (1.2%) by PCR and sequencing. Of these three cases, one had cerebral malaria and another had severe malaria anaemia. In both of these cases, P. ovale infection was mixed with P. falciparum, while in third case the infection was mixed with both P. falciparum and P. vivax. Phylogenetic analysis revealed that these isolates showed closed homology with West African genotypes.

Conclusion

All three hospitalised patients were originally residents of remote inaccessible forest villages and never moved out of their residence. This finding also has implications in malaria control and elimination as P. ovale causes relapses. This study highlights the need of molecular diagnosis of malaria species for appropriate treatment and control.

Abstract

Objectif

Nous rapportons la première détection de Plasmodium ovale dans le centre de l'Inde.

Méthodes

A partir de 2010 tous les cas suspects de paludisme hospitalisés dans la clinique du paludisme du Centre Régional de la Recherche Médicale pour les populations tribales dans le district de Bastar, dans le centre de l'Inde, ont été examinés. Les espèces Plasmodium ont été identifiées par microscopie et par PCR nichée spécifique à l'espèce sur l’ARNr 18s.

Résultats

Sur 256 cas inscrits de paludisme à P. falciparum confirmés par microscopie, l'infection à P. ovale a été détectée dans trois cas (1.2%) par PCR et séquençage. De ces trois cas, un était un paludisme cérébral et un autre était une anémie sévère du paludisme. Dans ces deux cas, l'infection par P. ovale était mélangée avec celle de P. falciparum alors que dans le troisième cas, l'infection était mélangée avec celle de P. falciparum et de P. vivax. L'analyse phylogénétique a révélé que ces isolats montraient une homologie proche des génotypes d'Afrique de l'Ouest.

Conclusion

Les 3 patients hospitalisés étaient originaires de villages forestiers éloignés et inaccessibles et ne s’étaient jamais déplacés hors de leur lieu de résidence. Cette découverte a des implications dans la lutte contre le paludisme et son élimination car P. ovale provoque des rechutes. Cette étude souligne la nécessité du diagnostic moléculaire des espèces de paludisme pour le traitement et la lutte appropriés.

Abstract

Objetivo

Reportamos por primera vez la detección de Plasmodium ovale en la zona central de la India.

Métodos

Desde el 2010 se evaluaron todos los pacientes hospitalizados con sospecha de malaria en la clínica para malaria del Centro de Investigación Médica Regional para Tribus del distrito de Bastar, India Central. Se identificaron las especies de Plasmodium mediante microscopía y una PCR anidada especie-específica del ARN18s.

Resultados

De los 256 casos incluidos con confirmación de malaria por P. falciparum mediante microscopía, se detectó infección por P. ovale en tres (1.2%) mediante PCR y secuenciación. De estos tres casos, uno tenía malaria cerebral y otro anemia severa por malaria. En dos casos la infección por P. ovale era una infección mixta con P. falciparum mientras que en el tercer caso la infección era mixta tanto con P. falciparum como P. vivax. El análisis filogenético reveló que estos aislados mostraban una estrecha homología con los genotipos de África del Oeste.

Conclusión

Los 3 pacientes hospitalizados eran originalmente residentes de poblados boscosos remotos, e inaccesibles y nunca habían salido fuera de su área de residencia. Este hallazgo también tiene implicaciones para el control de la malaria y su eliminación, puesto que P. ovale causa recaídas. Este estudio pone en evidencia la necesidad del diagnóstico molecular de las especies de malaria para un tratamiento y control adecuados.

Introduction

Plasmodium ovale, one of the five species of human malarial parasites, had been known from the African continent (Garnham 1966) and been occasionally reported from outside Africa (Sher et al. 1988; Bangs et al. 1992). Sporadic cases of P. ovale were found in India also (Jambulingam et al. 1989; Mishra et al. 1999; Marathe et al. 2006). Most of these cases were from hilly forested areas and in the paediatric age group.

Extensive studies on malaria were undertaken by Regional Medical Research Centre for Tribals (RMRCT) in the forest belt of Madhya Pradesh since 1986 (Singh et al. 1996, 2003). P. falciparum, P. vivax and P. malariae were found to be widely prevalent among the tribal communities (Bharti et al. 2013; Singh et al. 2013). However, for the first time, we found three cases of P. ovale during hospital surveillance. These cases are being reported for their rarity and clinical interest. Such uncommon cases of malaria are a diagnostic and clinical challenge.

Methods

Study site

A malaria clinic of RMRCT was established in a tertiary healthcare facility (Maharani Hospital and Associated Medical College Jagdalpur) in Bastar district of the state Chhattisgarh, central India in July 2010 to study the prevalence of severe malaria in Bastar, a highly malarious district. Bastar was recently divided into seven districts (Jagdalpur, Dantewada, Bijapur, Sukma, Narayanpur, Kondagaon and Kanker, Figure 1a). This hospital serves as referral health facility for the six districts adjacent to Jagdalpur. The inhabitants are predominantly Gond ethnic tribe members.

Figure 1.

(a) Map of India (i) showing RMRCT Jabalpur, Madhya Pradesh (B▲), Chhattisgarh (C) and locations of districts from where P. ovale was found in Chhattisgarh state (ii). (b) Gel picture showing the PCR amplification of Plasmodium ovale (788 bp). Samples are in lane 3–9, lane 1 is negative control, lane 2 is positive control and lane 10 is 100 bp DNA ladder.

Sample collection

All suspected hospitalised patients in the general ward were screened for malaria parasite after obtaining written informed consent. Finger prick blood samples from participants were taken by an expert technician. The blood smears were stained with Giemsa and examined under a microscope (1000 × magnification). Parasites were counted in 1800 RBCs, and per cent parasitemia was calculated following a standard technique (Abdalla & Pasvol 2004). Reports of the blood smears were immediately provided to the hospital staff for initiation of the treatment. Haemoglobin levels were measured by Hemocue cuvettes (HemoCueAB, Angelholm, Sweden), and random blood sugar was measured by commercially available strips (Contour TS- Bayer CropScience Ltd, India). Complications were defined following WHO criteria for malaria severity (World Health Organization 2000). For example, cerebral malaria was defined as unarousable coma in a patient (Glasgow coma score < 10) or >3 seizures in 24 h in exclusion of hypoglycaemia and other encephalopathies by appropriate examinations. Severe malaria was defined as presence of severe symptoms in a patient with no evidence of coma and seizures. Clinical, biochemical and demographic characteristics of the patients were noted from the hospital record. Treatment of malaria was administered by the physician on duty following guidelines of the National Vector Borne Disease Control Programme (NVBDCP 2011).

Ethics

This study was approved by the institutional review board of RMRCT. Written informed consent was obtained from all participants or the parents of children younger than 18 years, and assent was taken from children of age between 7 to <18 years as per Ethical Guidelines of Indian Council of Medical Research, New Delhi, India.

DNA template preparation and nested PCR assay

Genomic DNA was isolated from blood using a commercially available kit (HiPurA™ Blood Genomic DNA Miniprep Purification Spin Kit, HIMEDIA®) in accordance with the manufacturer's protocol. Species-specific nested PCR was carried out to diagnose the malaria parasite using the 18s rRNA gene (Snounou et al. 1993). Briefly, a two-step PCR approach was used. Primers used for primary PCR reaction were Pla – F: 5′ – TTA AAA TTG TTG CAG TTA AAA CG – 3′ and Pla – R: 5′ – CCT GTT GTT GCC TTA AAC TTC – 3′. Later, the amplified product was used for species detection in a nested PCR assay. The primary PCR was performed in a volume of 20 μl with 0.175 U of Taq DNA polymerase, 0.2 mm each dNTP, 0.4 μm each primer and 1.5 mm MgCl2. The reaction was allowed to proceed for 35 cycles after an initial denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min; final extension was at 72 °C for 10 min. Species-specific nested PCR was carried out for the detection of P. falciparum, P. vivax, P. malariae and P. ovale according to Snounou et al. (1993) and for P. knowlesi according to Lucchi et al. (2012). The primers used for the detection of P. ovale are ovaF: 5′ – ATC TCT TTT GCT ATT TTT TAG TAT TGG AGA – 3′ and ovaR: 5′ – GGA AAA GGA CAC ATT AAT TGT ATC CTA GTG – 3′. The nested PCR was performed in a volume of 20 μl with 0.15 U of Taq DNA polymerase, 0.2 mm each dNTP, 0.4 μm each primer and 1.0 mm MgCl2. The reaction was allowed to proceed for 35 cycles after an initial denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min and extension at 72 °C for 1 min and final extension was at 72 °C for 10 min. The amplified products (788 bp) were resolved by 1.5% agarose gel electrophoresis and stained with ethidium bromide for visual detection by ultraviolet transillumination.

DNA sequencing

DNA sequencing was carried out using PCR product of P. ovale-positive samples. The PCR products were purified from the agarose gel using HyYeld™ gel/PCR DNA extraction kit (Real Biotech Corp., Teipei Country, Taiwan), as per the manufacturer's recommended protocol. A partial sequence of the 18s rRNA gene was sequenced from both directions using the above-mentioned primers (ovaF and ovaR). The sequencing PCR was performed in a volume of 20 μl with 1 μl to Terminator Ready Reaction Mix (TRR), 3.2 pmol of gene-specific primer and 0.5 × sequencing buffer. Cycling conditions for the sequencing PCR included 25 cycles of denaturation at 96 °C for 10 s, annealing at 50 °C for 5 s and extension at 60 °C for 4 min. Templates were purified and sequenced in a 3130xl DNA Analyzer (Applied Biosystems, USA). DNA sequences were edited, and the consensus sequence was created using the BioEdit.

Phylogenetic analysis

Consensus sequences were multiple-aligned with previously published sequences from the GenBank database using MEGA4 software http://www.megasoftware.net. Phylogenetic analysis with the MEGA4 software and a distance-based neighbour-joining (NJ) analysis were carried out. The reliability of the trees was assessed by the bootstrap method with 1 000 replications. Similarity searches were carried out using the basic local alignment search tool (BLAST) (http://www.ncbi.nlm.nih.gov/).

Results

A total of 256 enrolled P. falciparum malaria cases under different severe categories of malaria were tested by PCR, of which P. ovale infection was identified in three cases (Figure 1b) and further confirmed by DNA sequencing (GenBank database accessing number KC 866363). All three patients had high fever associated with headache and vomiting during febrile episodes. Table 1 showed the clinical characteristics of these patients. Briefly, on the basis of clinical and parasitological investigations at the time of hospital admission, Case no. 1 was diagnosed as cerebral malaria (Glasgow Coma Score = 7, parasitaemia = 1%), whereas severe malaria anaemia was the main complication in Case no. 2 (parasitaemia = 1.8%). The third patient was diagnosed as mild malaria (parasitaemia = 2.4%). These cases were reported as P. falciparum by the microscopist, but molecular identification by PCR revealed that patients 1 and 2 had mixed infections of P. ovale with P. falciparum, whereas the third patient had a mixed infection of P. ovale, P. falciparum and P. vivax.

Table 1. Clinical characteristics of cases with mixed infection of Plasmodium ovale
Demographic/Hospital findingsPatient 1 (district Dantewada)Patient 2 (district Bijapur)Patient 3 (district Sukma)
  1. Some of the biochemical parameters were not measured of Patient 2 and 3 as there was no clinical indication of any complication.

Age (years)27435
GenderFemaleFemaleMale
EducationPrimaryNot beginHigher secondary
Mosquito preventive measurementsNoneNoneMosquito coil
Illness history (in days)1548
Important complaintsFever with Chills & Rigour, Headache, Vomiting, UnconsciousnessFever with Chills & Rigour, Headache, VomitingFever with Chills & Rigour, Headache
Temperature (Fahrenheit)99101.499.6
Pulse/minute105Not mentioned120
Systolic blood pressure (mm Hg)90Not mentioned114
Urine passed last 12 h (no of times)032
Random blood glucose (mg%)11182210
SeizureYesNoNo
Glasgow coma score at admission71414
Malaria diagnosis, % asexual parasiteCerebral malaria, 1%Non-cerebral severe Malaria, 1.8%Mild Malaria, 2.4%
Haemoglobin (g/dl)8.35.415.1
Serum creatinine (mg%)6.4Not measuredNot measured
Serum bilirubin (mg%) and Liver enzymes11.3 (Direct = 9.3; Indirect = 2) AST = 78; Alt = 30Not measuredNot measured
ShockMild hypotensionNoNo
Renal failureYesNoNo
JaundiceYesNoNo
Follow-up 48 hComa resolved, Afebrile, mild hypotensionImproved AfebrileComplete recovery, Afebrile
Duration of the hospital stay (in days)1046
OutcomeDischargeDischargeDischarge

The patients were treated with quinine (body weight-dependent dose) and intravenous fluids for 3 days followed by oral treatment. All three patients responded to treatment and made a complete recovery. Further follow-up was not carried out as these patients were from forest villages of Dantewada, Bijapur and Sukma districts and never returned with any clinical complications. P. ovale-positive isolates were sequenced and analysed on the basis of partial sequences of the 18s rRNA gene and found all three P. ovale isolates were identical. Phylogenetic analysis based on our sequences [JGI 093 (KC 866363)] and sequences from the GenBank representing the 18s rRNA showed closed homology with West African genotypes (HQ697267) (Figure 2).

Figure 2.

Phylogenetic diagram inferring evolutionary relationship. Phylogenetic analysis revealed that Indian isolate JGI 093 showed closed homology with West Africa (HQ697267) (GenBank accession number of each sequences has been shown).

Discussion

This is the first report of detection and molecular confirmation of P. ovale from central India in severe cases. This parasite is often found mixed with P. falciparum. The microscopist failed to report P. ovale as proper differentiation needs a lot of experience and expertise. Further, P. ovale is difficult to identify if the typical morphology of the infected host cell is damaged. Although all three patients were hospitalised in a referral hospital, they were residents of remote inaccessible forest villages that were very similar to villages of Koraput District, Orissa from where P. ovale was reported for the first time in India (Jambulingam et al. 1989). None of these patients visited Orissa or any other place prior to hospitalisation. It is also not clear whether P. ovale contributed any of the severe symptoms or played a role in increasing the clinical severity as exact pathogenesis, caused by P. ovale infection remains unrecognised and poorly studied because of a paucity of research in this area. Further, P. ovale is one of the two species of human malaria parasite other than P. vivax that has a dormant liver stage (hypnozoites) following primary infection. These hypnozoites are causing relapsing malaria even many months after the primary infection (Marathe et al. 2006). The treatment of P. ovale mono infection is not different from P. vivax.

P. ovale is much less researched than any other species of Plasmodium in India. It appears that the quantum of P. ovale is an underestimate through routine microscopy. The number of mixed infections is generally underestimated when optical microscopy is used for parasite identification compared with molecular-based tools (Mohapatra et al. 2008; Gupta et al. 2010). Further, misidentification of malaria parasite could prolong the parasite clearance time leading to anaemia and drug resistance. PCR-based identification is a better diagnostic tool due to its sensitivity and specificity at low levels of parasitaemia. However, the use of PCR is limited because of cost of equipment, trained manpower and good laboratory.

As most of the P. ovale cases are from hilly forested areas (Jambulingam et al. 1989), the link between the forested belt and detection of P. ovale needs further investigation.

Conclusion

The presence of P. ovale in remote inaccessible forest villages is of great concern from a transmission perspective as these cases will escape detection and continue to cause multiple disease relapses. Therefore, estimating the actual case load and relapse pattern is necessary not only for successful treatment but also for designing and developing effective malaria control measures.

Acknowledgements

We thank the hospital staff and technical members of the project for their kind cooperation and hard work. We are grateful to all the patients and their guardians for kind consent to take part in the study. This study was funded by the Indian Council of Medical Research. The funder had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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