Plasmodium malariae Detected by Microscopy in the International Bordering Area of Mizoram, a Northeastern State of India

Northeastern states of India share international borders with Myanmar, China, Bangladesh, and Bhutan, contributing 7.45% of the overall malaria cases in the country. Mizoram accounts for the highest malaria burden in the northeastern states, with perennial transmission in the hilly and deep-forested areas. Plasmodium falciparum (93%) is the most prevalent human Plasmodium species, followed by P. vivax; however, information on P. ovale and P. malariae is negligible. Rapid diagnostic tests (RDTs) are the most preferred malaria diagnostic tool followed by microscopy in this high malaria-endemic region. The present epidemiological study was carried out in July and August 2019 to assess the malaria burden in and around the Chawngte primary health center, Lawngtlai District of Mizoram, using RDTs and microscopy as diagnostic tools. World Health Organization-certified level I microscopists examined the blood smears. Diagnosis using RDTs resulted in 151 malaria cases (P. falciparum: 136; P. vivax: 15) out of 948 screened fever cases. However, blood smear examination detected 179 cases (P. falciparum: 154; P. vivax: 17; mixed P. falciparum + P. vivax infection: 3; P. malariae: 5). Analysis revealed that the risk of malaria infection was higher in the ≥5-year-old subjects than in the under-5 age group. The mean parasite density of P. malariae (1455.00/μL blood) was the lowest; cf. with P. falciparum: 12,275.08/μL blood. Surveillance at the point-of-care level using microscopy was able to detect all the four human Plasmodium species and their mixed infections, including P. malariae, which were missed with RDTs. Thus, the quality of microscopy along with trained manpower should be strengthened to diagnose all human malaria parasite species (particularly P. malariae and P. ovale) until the molecular tools are deployed at the field level to achieve malaria elimination by 2030.


Introduction
India accounted for 83% of the estimated malaria cases reported from the World Health Organization (WHO) South-East Asia Region in 2020 [1]. India has varied transmission intensities across its landscape and high malaria endemic states in the center, east, and northeast, contributing 80% of the malaria cases [2]. Northeastern states of India contributed 7.45% of the total malaria cases in 2019. Mizoram alone contributes 2.52% of the overall malaria cases in the country and almost one-third of the patients from northeastern India. Plasmodium falciparum (93.76%) is the most prevalent malaria infection, having perennial transmission in the hilly and deep-forested area of Mizoram. Among 11 districts of the state, Lawngtlai, Lunglei, and Mamit have an annual parasite index (API) of above 10. In 2021, Lawngtlai District reported 2238 positive malaria cases and three deaths due to malaria [3]. Besides P. falciparum and P. vivax, limited data on regional and global transmission patterns of P. malariae, P. ovale, and P. knowlesi are available [4][5][6][7]. Diagnosis and treatment of non-P. falciparum/P. vivax species should also be focused on achieving the malaria elimination target [4,8,9].
In the early phase of malaria elimination, the focus on identifying and quantifying Plasmodium species can help better implement diagnosis strategies and treatment guidelines [10]. Accurate malaria diagnosis is essential for effective disease management. Epy World Health Organization (WHO) recommends universal testing of all suspected malaria cases before initiation of treatment. Rapid diagnostic tests (RDTs) have revolutionized malaria control efforts in the endemic regions where adequate microscopy facilities are unavailable [11]. RDTs may have variable consistency, quality control, and performance capabilities at the temporal and spatial levels [12]. Deletion of the histidine-rich protein 2 (HRP2) gene in P. falciparum results in false-negative RDTs, and a reduction in sensitivity owing to low parasitemia may complicate the diagnosis at the field level [13]. Polymerase chain reaction (PCR)-based diagnosis is the most reliable tool having high sensitivity, high specificity, and capability of diagnosing both human and zoonotic Plasmodium species [14,15]. Tahar et al. (1997) developed an alternative PCR method for identifying P. malariae using the circumsporozoite protein gene as the DNA target. It was reported to be a suitable add-on in clinical diagnosis and feasible for mass screening in the community [16]. Recent advances in molecular diagnostic tools are the most promising in detecting and quantifying human and zoonotic Plasmodium species, their mixed infections, and submicroscopic parasitemia in the community.
However, the need for advanced molecular biology laboratories and skilled biologists limits its application in rugged and inaccessible areas [17]. Microscopic diagnosis of the malaria parasite is a gold-standard technique used since the identification of malaria parasites. Malaria microscopy is easy to use and provides additional information on the gametocyte load in the community, which is critical for malaria transmission. Microscopic diagnosis has great importance in clinical research on the prevalence of non-P. falciparum/non-P. vivax malaria and therapeutic efficacy of antimalarials. However, it is a labor-intensive technique that requires skilled human resources, unable to detect submicroscopic objects and differentiate between recrudescence and reinfection of malaria. P. malariae and their mixed infections have been reported from other northeastern states and central India. In this region, Anopheles baimaii and An. minimus are evidenced as the primary vectors transmitting human malaria [18]. However, the distribution of these species varies considerably throughout the region [18,19]. Therefore, the present study investigated the prevalence of all human malaria parasites, including P. malariae infection, using microscopy from the high endemic district of Mizoram, a northeastern state, in terms of malaria elimination.

Description of the Study Area
Mizoram is one of the northeastern states of India, bordering Bangladesh and Myanmar, having a hilly terrain and inaccessible areas [20]. Lawngtlai District is located along the Myanmar and Bangladesh border, with a thick virgin forest and a hilly terrain ( Figure 1). Study site "Chawngte" (22 • 37 10 N, 92 • 38 11 E) has a population of 45,307 and is situated in the western part of Lawngtlai District, which shares an international border with Bangladesh.

Study Population and Malaria Diagnosis
Active fever surveys were conducted in July-August 2019 using bivalent RDTs and microscopy to diagnose malaria parasites. After the written consent/assent, thick and thin blood smears were prepared per the method reported elsewhere. The specimens were stained (Giemsa stain) to give the parasites a distinctive appearance. The thick and thin smears were examined with a 100× oil immersion objective. Two WHO-certified microscopists examined the smears at the field level. The third independent microscopist further assessed the thick and thin blood smears for quality control purposes. A blood smear was declared positive only after the concordant findings of two independent microscopists. The positive malaria cases were treated as per the National Drug Policy on Malaria (2013) [21].

Estimation of Parasitemia
Parasite density per microliter of blood was estimated by counting the number of asexual parasites per 200 (500 in case of low parasitemia) leukocytes in a thick blood smear using Equation (1).

Parasite density per μL
Number of parasite count Number of leucocyte count 6000 (1)

Quality Control
For quality control, another expert microscopist (WHO level 1), independently and blinded to previous microscopic findings, reassessed 100% of the positive cases and 10% of the negative smears.

Data Analysis
Statistical data analysis was performed using software package SPSS v.20.0.

Study Population and Malaria Diagnosis
Active fever surveys were conducted in July-August 2019 using bivalent RDTs and microscopy to diagnose malaria parasites. After the written consent/assent, thick and thin blood smears were prepared per the method reported elsewhere. The specimens were stained (Giemsa stain) to give the parasites a distinctive appearance. The thick and thin smears were examined with a 100× oil immersion objective. Two WHO-certified microscopists examined the smears at the field level. The third independent microscopist further assessed the thick and thin blood smears for quality control purposes. A blood smear was declared positive only after the concordant findings of two independent microscopists. The positive malaria cases were treated as per the National Drug Policy on Malaria (2013) [21].

Estimation of Parasitemia
Parasite density per microliter of blood was estimated by counting the number of asexual parasites per 200 (500 in case of low parasitemia) leukocytes in a thick blood smear using Equation (1).

Quality Control
For quality control, another expert microscopist (WHO level 1), independently and blinded to previous microscopic findings, reassessed 100% of the positive cases and 10% of the negative smears.

Data Analysis
Statistical data analysis was performed using software package SPSS v.20.0.

Results
A total of 948 cases were screened for malaria parasites using RDTs and microscopy, of which 151 were positive as per RDTs and 179 were positive as per microscopy. Using RDTs, 14.34% were detected as P. falciparum, 1.58%-as P. vivax. However, in the microscopic examination, the positivity of P. falciparum and P. vivax was found to be 16.24% and 1.79%, respectively. Further, mixed infection (P. vivax + P. falciparum) was found in three patients (0.32%), and P. malariae was detected in five cases (0.53%) from the study population. All the positive cases diagnosed with RDTs were also found to be positive by malaria microscopy. The results are summarized in Figure 2.

Results
A total of 948 cases were screened for malaria parasites using RDTs and microscopy, of which 151 were positive as per RDTs and 179 were positive as per microscopy. Using RDTs, 14.34% were detected as P. falciparum, 1.58%-as P. vivax. However, in the microscopic examination, the positivity of P. falciparum and P. vivax was found to be 16.24% and 1.79%, respectively. Further, mixed infection (P. vivax + P. falciparum) was found in three patients (0.32%), and P. malariae was detected in five cases (0.53%) from the study population. All the positive cases diagnosed with RDTs were also found to be positive by malaria microscopy. The results are summarized in Figure 2. Thick smears showed trophozoites of P. malariae, and their respective magnified images are depicted in Figure 3A-D. A thin smear and its magnified images of P. malariae are shown in Figure 4A-F. The developing schizonts, gametocytes, and band form of trophozoites were confirmed in thin smears. The Plasmodium parasites were differentiated as rings, trophozoites, schizonts, and male/female gametocytes based on their morphological characteristics. P. malariae were distinguished by the features of senescent RBC infection, band-like trophozoites, and rosette forms of merozoites which are lined up around the perimeter of a schizont. The unique part of P. malariae is the band/sash distinctive structure developed as the trophozoite matures. Other Plasmodium species also have specified characteristics, e.g., P. falciparum have thin and delicate ring-form trophozoites that possess one or two chromatin dots, preferably located on the periphery of RBCs and crescent-shaped gametocytes. In P. vivax, the infected erythrocytes are enlarged, and Schüffner's dots appear in the cytoplasm as the rings mature into trophozoites. P. ovale and P. vivax have similar morphological features, except Schüffner's dots are distorted into an oval shape and feathering around the edges of the RBCs.
Further analysis revealed that malaria infection is lowest among the under-5 age groups, and no significant difference was observed between the age groups. When the parasite infection was looked into, P. malariae and mixed infections were highest among the 5-15 years group than in other groups. However, P. vivax was most prevalent in the ≥ 15 years group. Moreover, Plasmodium parasite infections were not significantly different between male and female subjects ( Table 1).
The overall risk analysis of malaria infection among the different age groups is shown in Table 2. The 5-15 years and ≥15 years age groups had a 1.67 (95% CI 0.94-2.94) Thick smears showed trophozoites of P. malariae, and their respective magnified images are depicted in Figure 3A-D. A thin smear and its magnified images of P. malariae are shown in Figure 4A-F. The developing schizonts, gametocytes, and band form of trophozoites were confirmed in thin smears. The Plasmodium parasites were differentiated as rings, trophozoites, schizonts, and male/female gametocytes based on their morphological characteristics. P. malariae were distinguished by the features of senescent RBC infection, band-like trophozoites, and rosette forms of merozoites which are lined up around the perimeter of a schizont. The unique part of P. malariae is the band/sash distinctive structure developed as the trophozoite matures. Other Plasmodium species also have specified characteristics, e.g., P. falciparum have thin and delicate ring-form trophozoites that possess one or two chromatin dots, preferably located on the periphery of RBCs and crescent-shaped gametocytes. In P. vivax, the infected erythrocytes are enlarged, and Schüffner's dots appear in the cytoplasm as the rings mature into trophozoites. P. ovale and P. vivax have similar morphological features, except Schüffner's dots are distorted into an oval shape and feathering around the edges of the RBCs.
Further analysis revealed that malaria infection is lowest among the under-5 age groups, and no significant difference was observed between the age groups. When the parasite infection was looked into, P. malariae and mixed infections were highest among the 5-15 years group than in other groups. However, P. vivax was most prevalent in the ≥15 years group. Moreover, Plasmodium parasite infections were not significantly different between male and female subjects (Table 1). groups were 1.99 (95% CI 1.06-3.7) and 1.82 (95% CI 1.01-3.29) times higher, respectively, and varied significantly (p < 0.05). Among the three age groups, the risk of non-P. falciparum infection was found to be highest among the ≥15 years group-1.22 (95% CI, 0.40-3.67) times higher compared to the under-5 age group (    1.99 (95% CI 1.06-3.7) and 1.82 (95% CI 1.01-3.29) times higher, respectively, and varied significantly (p < 0.05). Among the three age groups, the risk of non-P. falciparum infection was found to be highest among the ≥15 years group-1.22 (95% CI, 0.40-3.67) times higher compared to the under-5 age group (Table 2).   The mean parasite density of P. falciparum was highest, i.e., 12

Discussion
Malaria remains a significant public health problem in Mizoram. The hilly and mountainous terrain, variable ecological habitats, heavy rainfall, and forest-dwelling provide a fertile ground for the prevalence of different human Plasmodium species. P. falciparum is the most prevalent human malaria species in Langwatali District. In the study district, no significant difference (p > 0.05) in the slide positivity rate and the slide Falciparum rate was observed even after the high annual blood examination rate of 29.54. The annual parasite index was between 20.4 to 30.4 from 2017 to 2020 ( Figure 5A). The monthly malaria data from 2017 to 2020 showed that malaria transmission was perennial (throughout the year) in Langwatali District ( Figure 5B). Early diagnosis, treatment, and introduction of long-lasting insecticidal nets as vector control strategies have significantly reduced malaria incidence in the northeastern states of India. The API of Mizoram decreased from 20.71 to 6.98 (2014-2019). The proper implementation of malaria control strategies by the State Vector-Borne Disease Control Programme has successfully combatted P. falciparum malaria, the deadliest among human Plasmodium species in the hard-to-reach areas of Mizoram.
P. malariae is considered a benign malaria parasite because of low parasite density and coinfections with other Plasmodium species. The density and infectivity of Plasmodium species depend on their potential for invasion into the different age stages of RBCs. The low parasite density of P. vivax is attributed to its invasion capacity only in reticulocytes (<1% of all erythrocytes). P. falciparum is efficient in invading all age stages of RBCs. However, P. malariae preferred to invade mature RBCs [22,23]. Thus, P. malariae infection is considered asymptomatic or sub-patent, and this low parasitemia level probably has substantial chronic transmission potential [24,25]. In the last two decades, the mono-infections of P. malariae and P. ovale and their mixed infections with P. falciparum and P. vivax have been reported in India and the neighboring countries (Table 3). In Myanmar, P. knowlesi and P. malariae were reported in about <3% of the tested cases [26,27], and P. malariae and P. ovale are also prevalent in Bangladesh [28,29]. In India, P. ovale was identified long back in Orissa [30], and P. ovale, as a single case, was reported for the first time from Titabor PHC, Jorhat, Assam, in 2003 [31]. Krishna et al. (2015) also reported the prevalence of P. malariae in many parts of India (Orissa, Chhattisgarh, Maharashtra, Madhya Pradesh, Tripura, Gujarat, and Rajasthan), and of P. ovale in Orissa, Jharkhand, Chhattisgarh, Madhya Pradesh, and Jharkhand. The prevalence of P. ovale was also reported in Jagdalpur, Chhattisgarh [32]. One published report from Mizoram indicates the presence of mixed infections of P. falciparum with other Plasmodium species [6].
Malaria RDTs are the diagnostic tool of choice at points of care in hard-to-access communities and the preferred option for programmatic deployment. However, low-threshold sensitivity, identification, and quantitation of the sexual/asexual stages of Plasmodium have given microscopy an edge over RDTs. RDTs fail to detect low-density parasitemia, deletion of P. falciparum histidine-rich protein 2 (HRP2) leads to false negativity, and the stable HRP2 in the blood results in false positivity. PCR is considered highly sensitive and specific for Plasmodium identification and quantification. However, this technique is expensive and requires regular power backup, trained human resources, high time expenditure, and has only limited application in low-resource settings or emergencies [17]. In summary, microscopy is more reliable in detecting four human Plasmodium species compared to RDTs. Yet, it has the limitations of skilled microscopists, quality of the blood smear, staining, and fixation.
Thus, to target all the Plasmodium species infecting humans, malaria detection using microscopy should be strengthened till the deployment of a molecular tool on the community/primary health center level.     Pf = Plasmodium falciparum; Pv = P. vivax; Pm = P. malariae; Po = P. ovale; Pk = P. knowlesi.

Limitations of the Study
In the present study, RDTs and microscopy were used for parasite detection. Molecular analysis was not performed due to the unavailability of blood samples. However, the availability of a WHO-certified level I microscopist strengthened the malaria diagnosis with microscopy.

Conclusions
Early and effective diagnosis of human malaria species, including low-density infection and asymptomatic malaria, is crucial in achieving the goal of malaria elimination. Presently, RDTs are used preferably to diagnose malaria, but they have several limitations in identifying P. falciparum, P vivax, and their mixed infection. In the present study, P. malariae cases were detected using microscopy as well as RDTs. Microscopy is the only choice of diagnosis for non-P. falciparum and P. vivax malaria until laboratories are equipped with molecular diagnostic tools at the community and primary health center levels. Thus, it is necessary to strengthen microscopy for accurate and early diagnosis of species-specific malaria until the deployment of molecular tools [5,10,37].
Author Contributions: K.S., P.K.B. and A.S. conceived the study. N.A., K.S. and N.C.D. evaluated the microscopic slides. K.S., N.A. and N.C.D. collected and analyzed the field data. K.S. and P.K.B. drafted the manuscript. A.S. and P.K.B. critically reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding:
The study was part of the study of therapeutic efficacy of artemether and lumefantrine, financially supported by India's World Health Organization country office (2018/851237).

Institutional Review Board Statement:
The study protocol was approved by the Institutional Ethical Review Committee, ICMR National Institute of Malaria Research, New Delhi (ECR/NIMR/EC/2018/28) Dated 26 February 2018, and conducted in accordance with the Declaration of Helsinki.

Informed Consent Statement:
The written informed consent was taken from each patient, and assent was obtained from the child's guardian.
Data Availability Statement: All the data generated or analyzed during this study are included in this published article.