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Table of Contents
RESEARCH ARTICLE
Year : 2019  |  Volume : 56  |  Issue : 1  |  Page : 78-84

Prevalence of submicroscopic malaria in low transmission state of Punjab: A potential threat to malaria elimination


1 ICMR-National Institute of Malaria Research, Project site, Community Health Centre, Dhakoli, Zirakpur, Punjab, India
2 Directorate of Health Services, Punjab, Parivar Kalyan Bhavan, Chandigarh, India

Date of Submission27-Mar-2019
Date of Web Publication7-May-2019

Correspondence Address:
Dr Surya K Sharma
Former Scientist ‘G’ (Director Grade), Consultant (VBC), ICMR–National Institute of Malaria Research, Project Site, Ist Floor, Community Health Centre, Dhakoli, Zirakpur–160 104, District SAS Nagar, Punjab
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.257780

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  Abstract 

Background & objectives: Submicroscopic malaria infections with low parasite density serve as a silent reservoir for maintaining residual transmission in the population. These infections should be identified and targeted to be eliminated for sustained malaria control. The conventional methods of diagnosis such as light microscopy and rapid diagnostic kits often fail to detect low density infections. Therefore, the more sensitive molecular techniques should be employed to detect low density infections. The objectives of the study was to explore the prevalence of sub-microscopic infections in low transmission areas of Punjab using highly sensitive molecular tool.
Methods: A total of 1114 finger prick blood samples were collected through active surveillance and tested for malaria diagnosis using light microscopy, RDT and PCR. Nested PCR amplification was performed using a pair of Plasmodium genus-specific primers from the 18S rRNA small subunit gene (18S rRNA). The amplified PCR products were analysed using a 2% agarose gel, stained with ethidium bromide and observed under transilluminator.
Results: Test positive rate (TPR) by microscopy, RDT and PCR was 4.4, 3.95 and 5.75%, respectively. Microscopy and RDT failed to detect mixed infections whereas 0.26% cases were found to be mixed infection in PCR. Compared to LM and RDT, PCR has detected 1.3% additional positive cases. However, of the total positive cases detected by PCR, 23.4% infections were found to be submicroscopic, which could not be detected by conventional methods of diagnosis.
Interpretation & conclusions: The molecular study revealed the existence of submicroscopic malaria cases in the study population which would have remained undetected by conventional methods of diagnosis. This is particularly important because Punjab state is in malaria elimination phase and targeted to achieve elimination in 2021. However, such undetected parasite positive cases may pose bigger problem any time due to continued transmission. Therefore, application of more sensitive diagnostic tools like PCR and LAMP with conventional methods may be much more useful in case detection particularly in low transmission settings for malaria elimination.

Keywords: India; malaria; prevalence; Punjab; submicroscopic


How to cite this article:
Kaura T, Kaur J, Sharma A, Dhiman A, Pangotra M, Upadhyay A K, Grover GS, Sharma SK. Prevalence of submicroscopic malaria in low transmission state of Punjab: A potential threat to malaria elimination. J Vector Borne Dis 2019;56:78-84

How to cite this URL:
Kaura T, Kaur J, Sharma A, Dhiman A, Pangotra M, Upadhyay A K, Grover GS, Sharma SK. Prevalence of submicroscopic malaria in low transmission state of Punjab: A potential threat to malaria elimination. J Vector Borne Dis [serial online] 2019 [cited 2019 Jun 18];56:78-84. Available from: http://www.jvbd.org/text.asp?2019/56/1/78/257780




  Introduction Top


For successful control of malaria, accurate identification of Plasmodium infections in community surveys is essential. Microscopy which is considered as a gold standard and rapid diagnostic tests (RDTs) which allow for rapid diagnosis are the main techniques used to diagnose malaria in the field-based surveys. Microscopic diagnosis of malaria has various limitations, which include a lack of skilled microscopists, inadequate quality control, and the possibility of misdiagnosis due to low parasitaemia or mixed infections[1],[2]. Microscopy has also low sensitivity when performed by poorly trained personnel in malaria endemic areas, especially in primary and secondary healthcare facilities. In contrast, as malaria transmission declines and countries progress towards elimination, light microscopy (LM) and RDT are insufficiently sensitive to detect low level parasitaemia, thus, causing gross underestimation of parasite prevalence in areas where most infections are subpatent[3],[4],[5]. Although LM remains the gold standard for the diagnosis of malaria and quantification of Plasmodium parasites, the rapid advances in molecular biology and nucleic acid testing methods and their routine application in clinical studies and epidemiological surveys have enabled the detection of low-density submicroscopic infections as reported in several studies[6],[7],[8]. Moreover, the development of highly sensitive, specific and quantitative molecular diagnostic tests for malaria are becoming increasingly important as control strategies seek to eliminate asymptomatic infections that serve as reservoirs for transmission[9] especially in areas with low vector density and low transmission, asymptomatic and sub-patent malaria poses a challenge for malaria elimination[10]. Low-density parasite infections can be undetectable due to the limitations of routine diagnostic tools and thereby cause continued transmission in malaria endemic areas[10]. Previous studies have clearly suggested that polymerase chain reaction (PCR) detects more than double the number of Plasmodium falciparum infections compared to LM and RDT in low transmission settings[11]. In such areas, PCR apart from being a confirmatory tool for diagnosis of malaria can be useful to detect low parasite density[12].

Globally, India ranks fourth for total malaria burden and reports highest number of P. vivax cases in the world[13]. India has launched the malaria elimination drive and set the goal of malaria elimination by 2030[14]. Malaria surveillance and screening approaches play an important role in the elimination scenario. Plasmodium falciparum and P. vivax are two major parasites contributing to malaria, but their distribution is not uniform across India. Mixed species infection has been reported among different parts of India[15],[16],[17] but the diagnosis of mixed species infection in field setting using microscopy and RDT usually gets missed[18]. Thereby, PCR diagnostic assay can be utilized as a sensitive method to check Plasmodium infection. Furthermore, in the elimination setting when the parasitaemia is reduced, PCR may play a significant role in screening malaria parasites.

In view of launch of malaria elimination in India, the state of Punjab, which is reporting <1 annual parasite incidence (API) in all the 22 districts for the last five years has qualified for malaria elimination under category-1 as per National Framework of Malaria Elimination 2016–30. However, the actual burden of disease has not been estimated to strategize malaria elimination in the state. Therefore, this study was undertaken to assess the burden of sub-patent or submicroscopic malaria infections in a sample of the population in high, moderate and low malaria transmission areas of 9 districts of Punjab.


  Material & Methods Top


Study area

The selection of study area was based on API reported in 22 districts of Punjab in the last four years (2012–15). Based on API, the districts were stratified into three strata representing high, medium and low API zones. Accordingly, 8 districts were under strata-I reporting low API (<0.03), 9 districts were under strata-II having medium API (0.03 – <0.1), and 5 districts fall under strata-III reporting high API (>0.1). Three Blocks from three districts of each stratum were selected using PPS (Probability proportional to size) sampling. Overall, 9 block PHCs were selected from 9 districts allocated equal in high, medium and low API stratum. Thus, the total sample size of the study population was about 9 lakhs for the purpose of active and passive surveillance for estimation of malaria disease burden, however, only part of the samples were used for the present study. The location of nine study districts is shown in [Figure 1].
Figure 1: Map showing location of study areas in () Punjab. K—Kapurthala; M—Mohali.

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Blood collection for microscopy and molecular analysis

A total of 1114 finger prick blood samples were collected for this study through active surveillance in the study area during July 2017 to June 2018. From each malaria-suspected patient, finger-prick blood sample was collected only once (2 or 3 drops) and was utilized for all the three diagnostic assays. In the field, bivalent RDT kit, Falci-Vax (Zephyr Biomedical) was used for identification of either single or mixed infections due to P. falciparum and P. vivax. Simultaneously, thick and thin smears were prepared on a glass slide for microscopical examinations for the presence of different species of malaria parasites in the laboratory. The JSB-stained thick and thin blood smears were visualized under light microscope following the standard protocol by the expert technicians of the National Institute of Malaria Research (NIMR), for the identification of various stages of species of malaria parasite. The rest of the blood drops were used to prepare 2 to 3 spots on the Whatman filter paper for subsequent molecular analyses by PCR. These spots were dried and stored at 4°C and subsequently used for molecular diagnostic assay in the laboratory.

Genomic DNA extraction

DNA was extracted from the blood spots on filter papers using QIA amp mini DNA kit (Qiagen, Germany).

Nested polymerase chain reaction (Nested PCR)

Nested PCR amplification[12] was performed as follows: In the first step of nested PCR, a pair of Plasmodium genus-specific primers was used from the rRNA small sub unit gene (18S rRNA). Following this, the second step used two species-specific primers; P. falciparum and P. vivax primer pairs for amplifying specific gene products for these species [Table 1].
Table 1: Primers for nested-PCR of 18S rRNA gene in malaria parasites

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For each sample, PCR amplifications were carried out in a final volume of 25 μl, which included 10 pmol of each primer, 0.2 mM of dNTPs, 1 unit of Taq DNA polymerase (Bangalore, Genei), 1× Taq DNA polymerase buffer and 2 μl of DNA. Cycling conditions for the first step include initial denaturation at 95 °C for 5 min followed by 35 cycles of 30 sec denaturation at 94 °C, 1 min annealing at 55 °C and 1 min of extension at 72 °C followed by 10 min final extension at 72 °C. The cycling conditions for second step included an initial denaturation at 95 °C for 5 min followed by 35 cycles of 30 sec denaturation at 94 °C, 1 min annealing at 58 °C and 1 min of extension at 72 °C followed by 10 min final extension at 72 °C. The amplified PCR products were analysed using a 2% agarose gel, stained with ethidium bromide and observed under transilluminator. The presence or absence of different Plasmodium species was analysed with species-specific amplicon sizes. For this, 100 bp DNA ladder was used to identify the size of molecule run. Bands of 120 base pairs and 206 base pairs correspond to the presence of P. vivax and P. falciparum respectively [Figure 2].
Figure 2: Agarose gel electrophoresis pictures showing bands of representative PCR products. Lane 6–Gene ruler of 100–1000 bp; Lanes 1–3 display 206 bp PCR product signifying monoinfection of P. falciparum; Lanes 7–8 show 120 bp PCR product signifying monoinfection of P. vivax; and Lanes 4–5 Present both the bands of 120 bp (P. vivax) and 206 bp (P falciparum) size in a single sample indicating mixed species infection due to these two species of malarial parasites.

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Ethical Approval

The present investigation was part of a study on estimation of disease burden in Punjab and was approved by the Institutional Ethics Committee of ICMR-NIMR.


  Results Top


In the present study, 1114 blood samples collected from malaria symptomatic individuals were analysed by three different methods, viz. microscopy, Bivalent RDT kits and Nested-PCR to characterize the samples based on the type of infection caused by either single (mono) infection of either P. falciparum or P. vivax, or mixed species infections by these two species. The results are summarized in [Table 2]. Out of the 1114 samples, microscopy detected 49 positive malaria cases (47 P. vivax; 2 P. falciparum), bivalent RDT detected 44 positive malaria cases (42 P. vivax; 2 P. falciparum) and Nested-PCR detected 64 malaria cases (59 P. vivax; 2 P. falciparum; 3 Mixed [Pv + Pf]). The PCR technique detected 5.75% positive malaria cases that were comparatively higher than microscopy and RDT which detected 4.4 and 3.95%, respectively [Figure 3]. It is clear that out of three techniques used in the present study only PCR technique was able to detect mixed infection of 0.26%. With the use of PCR additional 15 cases of P. vivax were detected which were found to be negative by microscopy and RDT. In addition to this, microscopy and RDT techniques were also compared and it was found that microscopy detected additional 5 cases of P. vivax which were found negative by RDT. The results of microscopy were corroborated by two expert microscopists. Compared to LM and RDT, PCR has detected 15 (1.3%) additional positive cases. However, of the total positive cases detected by PCR, 23.40% infections were found to be submicroscopic. Thus, the molecular study revealed that a sizable proportion of sub-microscopic malaria cases was detected by PCR which otherwise had remained undetected by conventional methods of diagnosis.
Figure 3: Visual representation on the comparative assessment of the efficiency of three different malaria diagnostic methods (Microscopy, RDT and PCR assay).

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Table 2: Comparison of diagnostic results from microscopy, RDT and nested-PCR

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  Discussion Top


Quality-assured LM and RDTs are the basic diagnostic tools currently recommended for the confirmation and management of suspected clinical malaria, as well as for routine surveillance of clinical cases in malaria-endemic settings[19]. However, the threshold level of detection limit of these methods ranges to a higher limit of parasite density and is bound to miss low parasitaemic cases. Therefore, in low transmission intensity areas under elimination settings, use of more sensitive nucleic acid amplification (NAA) techniques should also be considered for the detection of low-density malaria infections such as those below the limit of detection (LOD) of LM or RDTs. Recent systematic reviews have concluded that LM misses approximately 50% of infections compared to PCR-based detection of parasitaemia7, 10. In malaria endemic areas, PCR-based detection of malaria infections has often been applied in surveys and research studies using active or reactive surveillance of affected populations. The most commonly used method for PCR-based detection in these surveys to find out missed or low parasitaemic infections is through amplification of the 18S rRNA gene from finger-prick blood samples[20]. However, the proportion of missed infections varied considerably in different epidemiological settings.

Low-density P. falciparum and P. vivax infections constitute a higher proportion of all infections in low transmission settings than in high transmission settings. However, the absolute number of infections is small in low and very low transmission settings, the absolute number of low-density infections is also smaller than in high transmission settings. In cross-sectional surveys, the proportion of low-density infections among all detected infections is higher in low transmission areas than in high transmission areas for both species[11]. High proportion of low-density infections have been reported from historically low transmission areas, such as Brazil[21],[22], Haiti[23] and the Pacific islands[24]. However, a small number of settings with very low transmission (PCR prevalence below 1%) in Haiti[25], China[26], and Solomon Islands[27] are exceptions to this general trend, as most P. falciparum infections were detectable by LM. It remains unknown whether after a prolonged period of low-level transmission a point is reached after which most infections become detectable again by conventional diagnosis or whether low-density infections that are undetectable by conventional diagnosis will persist in the community or not. The small numbers of infected individuals per survey in low to very low transmission settings, and the large uncertainty of estimates associated with these small numbers, remain a problem for determining trends at this very low level of transmission. In these very low transmission settings, the choice of population at risk may further influence prevalence estimates, as only a few positive cases may be found in small pockets or foci of transmission.

The occurrence of low parasitaemic cases in low transmission areas such as Punjab is much more common and such cases normally remain undetected through conventional methods of microscopy and RDTs. Such undetected cases may pose a greater risk of flaring up transmission and thus seriously affecting malaria elimination efforts. Therefore, high precision molecular techniques are required to be employed in such settings to provide reliable diagnosis and case detection to reduce parasite reservoirs in those areas progressing towards malaria elimination. The molecular study revealed the existence of a number of submicroscopic malaria cases in the study population which would have remained undetected by conventional methods of diagnosis. This is particularly important because Punjab state is in malaria elimination phase and is targeted to achieve elimination by 2021. However, such undetected parasite positive cases may pose bigger problem any time due to continued transmission. Therefore, application of more sensitive diagnostic tools such as PCR and LAMP in combination with conventional methods may be much more useful in case detection particularly in low transmission settings to achieve malaria elimination.

The relevance of low-density P. falciparum and P. vivax infections in maintaining malaria transmission needs to be reviewed on the basis of evidences as 15 states in India have reduced burden of malaria and are moving towards elimination. These states require clear-cut guidelines from the National Vector Borne Disease Control Programme (NVBDCP) on the case management and reporting of low-density infections identified during surveys or as part of research studies. However, there is no doubt that these highly sensitive tools will help in mapping low-density infections or for identifying foci to guide intervention measures aimed at achieving malaria elimination[19]. The role of low and high density infections in overall malaria transmission in a particular location may vary depending on their proportions. Mosquito feeding experiments may help to measure the infectiousness of low and high density infections. However, very limited data are available on the relative contributions of low-and high-density P. falciparum and P. vivax infections to the onward transmission to human populations at the community level. Therefore, it is important to understand the contribution of low-density infections to malaria transmission in order to plan effective malaria control strategies. Further studies are required to better understand and predict the public health importance of low-density malaria infections and the potential impact of detecting those using highly sensitive molecular techniques.


  Acknowledgements Top


We are also grateful to District Epidemiologists of the study districts in Punjab for providing manpower and technical support in collecting field samples for the study. The excellent technical support provided by the staff of the ICMR-NIMR project site, Dhakoli, Zirakpur, Punjab is gratefully acknowledged. The community in the study villages deserves our special thanks for their overwhelming response, co-operation and participation in the study. The study was funded by the Indian Council of Medical Research, New Delhi.



 
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