Asymptomatic malaria carriers and their characterization in hotpops of malaria at Mangalore.

OBJECTIVE: This study aimed to identify asymptomatic malaria carriers and study the differences in local and migrant population in Mangalore.
MATERIALS AND METHODS: This study was conducted using two-stage cluster sampling. In the first stage, wards were randomly selected. The sample size for each cluster (wards) was determined using the probability proportionate to size method. In the second stage, required number of households was selected using a simple random sampling technique from each cluster. From the selected clusters, samples from 140 participants were collected.
RESULTS: Of the 140 cases, 106 (75.7%) were male and 34 (24.3%) were female. Six cases (4.3%) of the 140 cases were positive for malarial parasites, of which gametocytes of Plasmodium falciparum were noted in three cases and schizonts of Plasmodium vivax in three cases. From the total number of cases, 56 (40%) constituted the migrant population and the remaining 84 (60%) were the local population. All the six patients with asymptomatic carriers belonged to the migrant population.
CONCLUSION: A more sound malaria elimination strategy needs to be implemented, for which active surveillance for cases would form a backbone. This study shows that the migrant population seems to show a predilection for asymptomatic malaria, thus targeting malaria elimination programs to areas with a high migrant population would help. Copyright:

INTRODUCTION

Asymptomatic malaria can be defined as “Plasmodium infections that do not lead to clinical symptoms and therefore remain undetected by fever based surveillance systems.”[1] Asymptomatic cases occur without eventual overt symptoms, and they do not come to clinical attention, thus representing a largely hidden reservoir of active infection that permits their persistence and eventual spread to other human hosts. As these cases do not get medical attention, they persist in the community and this expands the risk of transmission during much of this time, and overall, it constitutes a high proportion of transmission.[1] This is the case for many malaria infections in semi-immune individuals from endemic areas, which commonly cause a mild febrile illness or no apparent symptoms at all while keeping parasite numbers at low densities. Asymptomatic malaria infections are common in endemic areas, with prevalence being four to five times higher than clinically patent infections.[2] Studies based on South Africa reveal that migrants originating from areas with stable malaria transmission would have acquired some immunity to the disease. The local population, in turn, would have little or no immunity to malaria, and thus, the disease would be more virulent to them.[3]

Of the asymptomatic cases, some of them might be subpatent, i.e., of a density lower than the threshold needed for detection by microscopy or rapid diagnostic test.[4] Such cases are noted to occur more in low-transmission settings and have been estimated to result in 20%–50% of all transmission episodes.[5] As a result, managing asymptomatic infections would have considerable implications in malaria control. It could even help us design effective malaria elimination strategies.

Totally, 22% (275.5 million) of India's populace live in high transmission (>1 case for every 1000 populace) territories.[6] Around 95% of population in the nation dwell in malaria-endemic territories.[7] High transmission regions are present in parts of Odisha, Jharkhand, Chhattisgarh, Maharashtra, Madhya Pradesh, West Bengal, and Uttar Pradesh.[8]

Malaria is highly endemic and persistent throughout the year in several parts of Southwestern regions of India, including a substantial portion of Karnataka state.[9] Whereas the incidence of malaria is showing a downward trend in the rest of Karnataka, it is continuing in Mangalore, and in 2013, of the 13,302 cases reported from Karnataka, 4714 (35%) were from Mangalore, and in 2014, of the 12,335 cases from Karnataka, 7360 (59%) were from Mangalore.[6] In the past two decades, increase in building and road construction activities as a part of rapid urbanization resulted in a substantial number of immigrant laborers from other parts of India, prominently from the Northeastern regions, where malaria is highly endemic, migrating to Mangalore city. This could have resulted in the spread and high incidences of malaria in Mangalore city and the surrounding areas.[10]

To determine the prevalence of asymptomatic carriers in an endemic population, active case detection is employed. It takes advantage of the fact that parasite carriage tends to be spatially and temporally clustered [Figure 1].[9] Therefore, infections are found at a higher prevalence in households in close proximity to passively detected cases.[11] Hotpops can be defined as “Demographically discrete groups (populations) that maintain malaria transmission at higher rates than the surrounding population.” These areas seed transmission to the surrounding populations in receptive areas. Thus, targeting hotpops would immensely aid in malaria control.[12] Previous studies have shown imported malaria to pose difficulty to achieving the goal of malaria elimination.[131415] The objective of this study was to identify asymptomatic carrier and study the differences in local and migrant population in Mangalore.

Figure 1

Mangalore city map showing the malaria hotpops in the city

MATERIALS AND METHODS

Sources of participants

The burden of malaria is high in Mangalore in 2013; of the 13,302 cases reported from Karnataka, 4714 (35%) were from Mangalore, and in 2014, of the 12,335 cases from Karnataka, 7360 (59%) were from Mangalore.[6] Various hotpops in Mangalore city were marked and three of them were chosen by simple random sampling. The hotpops selected were Falnir, Urwa stores, and Mannagudda. Majority of the populations in these areas, especially Urwa stores, constituted largely of migrant workers.

Inclusion and exclusion criteria

Participants within the age group 18 years–60 years and those who were willing to participate and have consented were included in the study. Participants who were treated for malaria within the past 4 weeks or those presently on the treatment of malaria were not included. Participants not willing to participate and/or had not signed the informed consent were excluded from the study.

Sample collection

In the present study, a household survey was conducted using two-stage cluster sampling. In the first stage, wards were randomly selected. The sample size for each cluster (wards) was determined using the probability proportionate to size method. In the second stage, the required number of households was selected using a simple random sampling technique from each cluster. From the selected clusters, samples from 140 participants were collected. From the resulting population, we investigated the asymptomatic carrier with the characterization of clinicoepidemiological parameters. Blood samples were collected and epidemiological details were noted.

Statistical analysis

The data were analyzed using statistical software SPSS 25.0 IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp. A simple descriptive analysis was done to explore the prevalence of asymptomatic gametocyte carriage among the respondents along with subgroup analysis. The data were collected from the community using a pro forma (sociodemography details, history of respondent, observations, and diagnosis of respondent) by a researcher for clinicoepidemiological characterization.

Sample processing

After getting the consent, the study participants were asked to provide finger-pricked blood samples for malaria diagnosis. Thin and thick blood smear was prepared and stained with Leishman stain for the detection of malarial parasites. The thick smear was prepared using a larger drop of blood that was placed on the center of the slide and spreading the drop using a spreader to about 1.5 cm2. The smear was allowed to dry and once dried was checked for appropriate thickness by visualizing the printed page through the smear. Before staining with Leishman stain, these smears were subjected to de-hemoglobinization by placing the smear in distilled water for 10 min. Thin smears were prepared by placing the drop of the blood 1 cm from one end of the glass slide and using a spreader, a tongue-shaped smear was obtained, dried, and were later subjected to Leishman stain.

Detection of positive cases and estimation of parasite count: Microscopy was done by a consultant pathologist who has more than 8 years of experience in malaria diagnosis. Examinations of the smears were performed based on procedures described by the WHO 2015 guidelines.[16] Both thick and thin smears were examined under oil immersion objective using a specific pattern by moving along the edge of the smear, then shifting the slide inwards by one field to return in a lateral movement and repeating this sequence. The slides were viewed first at a low magnification (×400 objective lens) to identify the suitable fields for examination. A minimum of 100 fields were examined before labeling the case as negative for the malarial parasite. To establish the parasite count in positive cases, thick smears were used and number of parasites per 200 leukocytes were counted and recorded. In cases with 9 or less parasites/200 leukocytes, 500 leukocytes were counted and recorded as the number of parasites per 500 leukocytes. Thin smears were used to confirm the species identification. Parasites per μL of blood were calculated using a simple formula:

Ethics committee approval

The study was approved by the Institutional Ethics Committee, Kasturba Medical College, Mangalore. Informed consent was obtained from each participant and the purpose of the study was explained to them in the local language. Confidentiality was strictly maintained and the participants were assured of the same. The positive cases by slide microscopy have been contacted and advised appropriate treatment.

RESULTS

Of the 140 cases, 106 (75.7%) were male and 34 (24.3%) were female. On microscopy, slides were scrutinized for malarial parasites. Six cases (4.3%) of the 140 cases were positive for malarial parasites, of which, gametocytes of Plasmodium falciparum were noted in three cases and schizonts of Plasmodium vivax in three cases. All the cases which were positive for gametocytes or schizonts were males with a mean age of 33 years. Forty-six cases (32%) gave a history of malaria. From the six cases which showed gametocytes or schizonts on microscopy, only one had a past history of malaria.

From the total number of cases, 56 (40%) constituted the migrant population and the remaining 84 (60%) were the local population. All the 6 cases with asymptomatic carriers on microscopy belonged to the migrant population [Table 1]. The mean duration of their stay in Mangalore was 25 months and 17 days. From the 140 cases, 86 (61%) reported that indoor residual spray (IRS) was done at their residence. Of the six cases reported positive for gametocytes or schizonts, 4 reported that IRS was done at their residence.

Table 1

Gender demographics of the study population

	Asympomatic carrier
	Yes	No
Gender
Male	6	100
Female	0	34
Population
Migrant	6	50
Local	0	84
Past history of malaria
Present	1	45
Absent	5	89

The mean parasite density is obtained by calculating the average of the parasite densities (i.e., parasites/500 white blood cells [WBCs]) for the individual cases. The mean parasite density of the six positive cases, as obtained by microscopy, was found to be 16.5 parasites/500 WBCs. Table 2 depicts the results obtained from the positive cases.

Table 2

Microscopy of positive cases

Cases	WBC count ×109/L	Platelet count ×109/L	Thick and thin smear	Parasites/500 WBC’S	Parasite density (parasites/µL)
Case 1	6.40	226	Plasmodium falciparum gametocytes	16	256
Case 2	8.2	286	Plasmodium falciparum gametocytes	18	288
Case 3	12.4	198	Plasmodium vivax schizonts	16	256
Case 4	4.4	210	Plasmodium vivax schizonts	17	116
Case 5	6.0	254	Plasmodium vivax schizonts	15	240
Case 6	5.2	162	Plasmodium falciparum gametocytes	17	320

WBCs: White blood cells

DISCUSSION

The results of the present study show that hotpops of malaria transmission in Mangalore are having a significant number of patients with asymptomatic malaria who might be maintaining the transmission in these hotpops. Interestingly, all the patients with asymptomatic malaria were migrants and no one from the local population was a carrier. Asymptomatic malaria proportion in the migrant population was 10.7% even with the mean stay of more than 2 years away from the native area.

The city of Mangalore has seen sustained high levels of malaria transmission over the past two decades. In spite of efforts from the health authorities and advanced health care in the city, pockets of hotpops still persist in the city. The presence of a large number of patients with asymptomatic malaria might serve as a potential reservoir and lead to the persistence of transmission within these areas. Patients with symptomatic malaria are generally diagnosed immediately and treated aggressively, thereby reducing the possibility of transmission within the community. However, patients with asymptomatic malaria are not identified in the absence of active case detection and because of the long period of time and they continue to have parasitemia and hence form a major source of transmission of malaria in these hotpops. They remain infective even with subpatent parasitemia which can be detected by polymerase chain reaction (PCR).[4]

Endemic areas such as sub-Saharan Africa tend to have large number of patients with asymptomatic malaria.[17] A study based in Northeast India revealed a prevalence of 8.4% as asymptomatic malarial infection.[18] Most of the patients with asymptomatic malaria in the present study were from Northeast India. In general, patients with asymptomatic Malaria continue to have parasitemia for about 1½ years after migrating to the nonendemic area.[19] It is interesting to find that in our study, the average duration of stay in the city of Mangalore for these asymptomatic cases was 2 years which raises the possibility of the patients being infected and remaining asymptomatic even when they were in Mangalore itself. It is crucial to identify the patients by aggressive active case detection methods, especially in hotpops to achieve the total control of malaria.

Our study did not reveal any benefit from the use of IRS, as four of the six positive cases reported using IRS at their home, also suggesting that the infection could have been spread from their workplace or elsewhere. In a study done in South Africa, migrants from endemic sub-Saharan Africa showed about 5% asymptomatic parasitemia, which is similar to our observation. However, the difference between the native population and the migrant population was not studied in the South African study.[3]

Gametocytes are the transmissible stages of malaria. However, in reality, any stage of the malaria parasite is significant irrespective of gametocyte carriage since all of them have the potential to transmit the infection since gametocyte can be formed at a later stage. Hence, all stages of parasites should be viewed as potentially transmissible malaria.[20] In the present study, three patients had gametocytes and three patients had schizonts, and all six patients with asymptomatic parasitemia should be viewed as a potential threat for malaria transmission in hotpops.

Some of the asymptomatic malaria patients might have been in the preclinical stage of malaria. To avoid this, patients being labeled as asymptomatic malaria patients were followed up for 2 more weeks to ensure that they were truly asymptomatic malaria patients.

The overall prevalence was 4.7% of the patients screened. However, the positive cases were seen among migrants only and none of the local population were positive for asymptomatic carriers. Totally, 10.7% of migrants were asymptomatic. Furthermore, most of the positive cases, i.e., five of six cases, never suffered from malaria earlier in their native place and they continued to harbor gametocytes or schizonts even after 2 years of stay in the new locality.

Based on findings from this study and other studies, such as those conducted in South Africa, the migrant population seems to show a predilection for asymptomatic malaria.[3] This, as suggested in the previous studies, could be attributed to some immunities acquired by unknown mechanisms. These infections would be more virulent to the local population, who would have acquired less immunity to the infection.

At present, our understanding of malaria transmission and how host defenses and parasite factors interact to trigger the clinical spectrum of malaria infections remains limited. Achieving some control over malaria transmission is a mammoth task. A more sound malaria elimination strategy needs to be implemented for which active surveillance for cases would form a backbone. This study shows that the migrant population seems to show a predilection for asymptomatic malaria, thus targeting malaria elimination programs to areas with a high migrant population would help. As data available in asymptomatic malaria in India are scarce, more comprehensive research in this field is the need of the hour. Children and pregnant women should be included in such studies, as they constitute a more vulnerable population. Furthermore, more efficient diagnostic techniques such as PCR could supplement the detection of parasites by slide microscopy and detect cases with low parasitemia.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.