1475-2875-3-34 1475-2875 Research <p>Infectious reservoir of <it>Plasmodium </it>infection in Mae Hong Son Province, north-west Thailand</p> Pethleart Aree pethleart@yahoo.com Prajakwong Somsak sprajakwong@hotmail.com Suwonkerd Wannapa suwannapa@yahoo.com Corthong Boontawee pethleart@yahoo.com Webber Roger rwebber@supanet.com Curtis Christopher chris.curtis@lshtm.ac.uk

London School of Hygiene and Tropical Medicine, London, WC1E 7HT, UK

Faculty of Medicine, Thammasat University, Pathumtani 12120, Thailand

Vector Borne Disease Section, Office of Disease Prevention and Control No.10, Chiang Mai, 52000, Thailand

Vector-borne Disease Control Unit No.8, Mae Hong Son Province, Thailand

 Malaria Journal 1475-2875 2004 3 1 34 http://www.malariajournal.com/content/3/1/34 15385050 10.1186/1475-2875-3-34 26 5 2004 22 9 2004 22 9 2004 2004 Pethleart et al; licensee BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

It was unknown whether the main reservoir of Plasmodium falciparum and Plasmodium vivax, which infects mosquitoes in Thailand, was (a) in people feeling sufficiently ill with malaria to come to a clinic or (b) in people who had remained in their home villages with some fever symptoms or with none.

Methods

Mass surveys were carried out in Thai villages to identify people with Plasmodium infections and with fever. Malaria patients were also located at a clinic which served these villages. Adults from both sources whose blood slides registered positive for Plasmodium spp. were requested to allow laboratory-bred Anopheles minimus to feed on them. Seven to nine days after the blood feeds the mosquitoes were dissected and checked for presence of oocysts.

Results and Discussion

There were higher rates of Plasmodium infection among people in the villages with fever than without fever and much higher rates of infection among clinic patients than among people who had remained in the villages. People with malarial infections identified via the clinic and the village surveys could infect mosquitoes, especially, but not only, if their blood slides showed visible gametocytes. Because only a very small minority of the village populations were visiting the clinic on any one day, assessment indicated that the main reservoir of infection was not primarily among clinic patients but among those in the villages, especially those feeling feverish.

Conclusions

Efficient use of an anti-gametocyte drug to suppress the parasite reservoir in a population requires that it be given, not just to clinic patients, but to infected people located by mass surveys of the villages, especially those feeling feverish.

Introduction

An estimate of the infectiousness of the Plasmodium reservoir to mosquitoes is of interest in understanding the epidemiology of malaria and its changes after application of certain types of control measures. Different approaches have been used to investigate this, including direct feeding of mosquitoes through the skin of human subjects 12345 or feeding through an artificial membrane 678910111213. The present study was designed to determine whether the reservoir of infection of vectors was mainly in people ill enough to go to a clinic or whether it was mainly in those with slight or no malarial symptoms who have remained in their villages.

Patients and Methods

Subjects

This study was approved by the Thai Ethics Committee and the Ethics Committee of London School of Hygiene and Tropical Medicine. Subjects were recruited from mass blood surveys in villages and from a clinic in Muang District, Mae Hong Son Province, which is in north-western Thailand. Anti-malarial drug use is tightly controlled in Thailand and these drugs are generally available on prescription. Self-medication is, therefore, unusual in this area. Mass blood surveys were conducted twice a year. After slides were examined for parasites, the people whose slides were positive and were more than 14 years of age were invited to take part in this study. The Thai ethical committee did not allow direct feeding of mosquitoes on children aged less than 15 years. Adults positive for malaria parasites were informed about the purpose of the study and, if they agreed, a consent form was signed and patients were interviewed.

Human subjects for the study were also enrolled from the Vector-borne Disease Control Unit No. 8 (VBDU: described as "Clinic" throughout this paper). In this Clinic, blood smears were taken by the clinic staff and stained with Giemsa to check for malaria. Patients who had presented with clinical malaria and/or parasitaemia and were more than 14 years of age were informed about the purpose of the study and asked to participate in it. If they agreed and signed the consent form, then the mosquito feeding was performed.

Mosquito feeding and dissection

Before feeding, patients' arms were cleaned with 70% alcohol. Then, 50 laboratory-bred female Anopheles minimus species A (aged 4–6 days), which had been starved for 9–12 hr, were allowed to feed on their arms for 30 minutes. After feeding was completed, anti-malarial drugs were given to all the human subjects by a malaria worker. Two hours after feeding the unengorged mosquitoes were removed from the cups using a sucking tube and destroyed, leaving only fully engorged mosquitoes in the cup. The mosquitoes were brought back to the Chiang Mai insectarium and maintained at 25 to 27 C and 70–80% relative humidity with permanent access to sucrose solution and without any further blood meals. Mosquitoes at 7–9 days post-feed were anaesthetized and the wings and legs were removed. Midguts were dissected on glass slides in a drop of 0.85% NaCl and examined at a 40× magnification. The number of oocysts present on the midgut to each mosquito was counted and recorded individually.

Quality control of blood slide data

Asexual parasitaemia and gametocytaemia were quantified as the number of aseuxal forms/200 white blood cells on a thick film. Throughout the period of the study, 20% of the negative blood smears and all positive slides which had been examined by the microscopist of the Clinic were chosen randomly and re-examined by a team from the Office of Vector-borne Disease Control No.2, Chiang Mai. All cases of positive slides without visible gametocytes and which led to oocyst production were re-examined by an expert team from the London School of Hygiene and Tropical Medicine to ascertain whether a few gametocytes might have been present but were initially missed.

Analyses

The database from mosquito feeding included 1) densities of gametocytes and trophozoites, 2) number of infected mosquitoes, and 3) mean number of oocysts per infected mosquito. The chi-square test or Fisher's exact test was used to examine the significance of differences in the proportion of people in various categories who transmitted malaria to the mosquitoes (i.e. yielded at least 1 oocyst). Regressions on the natural log of gametocyte density were also computed. Stata statistical software (version 6) was used for analysis.

Results

The upper part of Table 1 shows the results associated with 5,227 blood smears from children and adults in village surveys. 10.7% (561/5,227) of subjects reported fever within the previous seven days but the corresponding rate for slide positives was much higher (53.8%, 28/52). In children (age <15 yrs), 60% (9/15) of slide positives reported fever. Among 37 slide positive adults (age >14 years), 19 (51.4%) reported fever. Among these 19 cases, nine were infected with Plasmodium falciparum and 10 with Plasmodium vivax. The prevalence of infection of both species from the village surveys was higher in children (15/623 = 2.4%) as compared to adults (37/4604 = 0.8%) (χ2 = 12.8, P < 0.001). The lower part of Table 1 shows data from the Clinic where 101 patients aged >14 years were interviewed; 85.2% (86/101) reported fever. Among those, 39 were infected with P. falciparum and 47 with P. vivax.

<p>Table 1</p>

Data from village surveys and the clinic on reported fever and malaria infection.

Slide reading

Fever reported

Fever not reported

Fisher's exact tests of association of fever with infection

Village surveys, age <15 yrs

P. falciparum +ve

2 (3.2%)

3 (0.54%)

P = 0.082

P. vivax +ve

7(11.1%)

3 (0.54%)

P = 0.000007

Total slides

63

560

Village surveys, age >14 yrs

P. falciparum +ve

9 (1.8%)

10 (0.24%)

P = 0.00006

P. vivax +ve

10 (2.0%)

8 (0.19%)

P = 0.000003

Total slides

498

4106

Clinic (age >14 yrs)

P. falciparum +ve

39 (45.35%)

10 (66.67%)

χ2 = 0.39, P = 0.53

P. vivax +ve

47 (54.65%)

5 (33.33%)

χ2 = 0.14, P = 0.51

Total slides

86

15

As shown in Table 2, at least one mosquito became infected with oocysts in the batches fed on 10/28 people from the village infected with either species (35.7%). The proportion of individual patients from the Clinic who infected at least one mosquito was 43.4% (40/92). The difference between these two rates was not significant (χ2 = 0.26, p = 0.6). The probability of infection of mosquitoes from people infected with P. falciparum was significantly less than from people infected with P. vivax (24.7% versus 57.1%, χ2 = 11.6, P < 0.001). 77.1% (44/57) of P. falciparum cases reported fever. Among those, only 27.3% could infect mosquitoes. For P. vivax 84.1% (53/63) reported fever; among those 56.6% could infect mosquitoes. More than half of the infections from whose blood no mosquitoes developed oocysts had parasite density > 3,960/μl. There was no association of probability of mosquito infection with parasite density (χ2 = 1.3, P > 0.05).

<p>Table 2</p>

Results of feeding mosquitoes on human blood

No. people from whose blood some mosquitoes developed oocysts

No. people from whose blood no mosquitoes developed oocysts

Significance of differences

P. falciparum

Villages

3 (23.1%)

10 (76.9%)

Fisher not sig.

Clinic

11 (25.0%)

33 (75.0%)

 

Observable gametocytes

6 (46.2%)

7 (53.8%)

Fisher P = 0.06

No observable gametocytes

8 (18.2%)

36 (81.8%)

 

Fever reported

12 (27.3%)

32 (72.7%)

Fisher P = 0.32

Fever not reported

2 (15.4%)

11 (84.6%)

P. vivax

Villages

7 (46.7%)

8 (53.3%)

χ2 = 0.41, P = 0.52

Clinic

29 (60.4%)

19 (39.6%)

 

Observable gametocytes

31 (68.9%)

14 (31.1%)

χ2 = 7.3, P = 0.007

No observable gametocytes

5 (27.8%)

13 (72.2%)

 

Fever reported

30 (56.6%)

23 (43.4%)

Fisher P = 0.56

Fever not reported

6 (60%)

4 (40%)

Parasite density (P. falciparum and P. vivax)

<3961/μl

21 (35.9%)

38 (64.4%)

χ2 = 1.30, P = 0.25

>3960/μl

29 (47.5%)

32 (52.5%)

Considering each species separately, the probability of infectiousness to mosquitoes was significantly related to being observably gametocyte positive in P. vivax (P= 0.007), but the relationship was only of borderline significance for P. falciparum (Fisher's exact test, P = 0.06). Approximately 50% (7/13) of the P. falciparum cases with observable gametocytes failed to infect mosquitoes and 30% (14/45) of the P. vivax cases with observable gametocytes failed to infect. However, the difference was not significant (Fisher's exact test, P= 0.19). Approximately 21% (13/62) with no observable gametocyte of either species could infect mosquitoes.

58 of the infections with either species had observable gametocytes (11 cases from the village survey and 47 from the Clinic). Feeds on 37 of these 58 gametocyte carriers led to oocyst production. The regression of percent of mosquitoes infected on the natural log of gametocyte density was not significant (t = 0.87, df= 36, P= 0.39). Similarly, there was not a significant association of mean oocyst load per infected mosquito on the natural log of gametocyte density (t = 0.87, df = 36, P= 0.38).

Discussion

The results from the experiments with mosquitoes showed the infectiousness of subjects from the village surveys as well as from the Clinic. These indicated that some symptomatic and asymptomatic infections of each species could infect mosquitoes. The differences in percentages of infection in different studies 2414 might be explained by several possible factors. First, the method of feeding: a recent study comparing the infectivity of gametocyte carriers to mosquitoes, using membrane and direct feeding, found significantly higher proportions of mosquitoes infected and higher oocyst burdens in mosquitoes fed directly on the skin 15. Conversely, Vanderberg 16 reported that infectivity in mosquitoes fed through a membrane usually equaled or exceeded infections by direct methods. However, most studies gave better results for direct feeding than membrane feeding. Thus studies such as the present one using direct feeding may provide a more reliable estimate of the infectious reservoir. Second, recruited subjects: in several studies mosquitoes were fed on individuals selected randomly and not on the basis of gametocytaemia 51014. But in other studies mosquitoes were fed on selected gametocyte carriers 417 or parasitaemic cases with or without gametocytes (as in the present study).

Another relevant factor is variability in mosquito populations 1819, such as mosquito size 202122. The number of blood meals may also affect the infection rate 823. A careful comparison of Anopheles dirus, An. minimus and Anopheles maculatus infectivity in relation to size and blood-feeding behaviour would be of interest. Moreover, variability in susceptibility between different mosquito colonies is possible 424.

The results in the present study showed that some cases with undetectable gametocytes could infect mosquitoes. This apparent anomaly is presumably at least partly due to larger volumes of blood in mosquito bloodmeals than are observed on slides, so that sufficient gametocytes to infect a mosquito may have been below the level of detection on blood slides. An attempt was made to see if gametocyte densities are higher in bloodmeals than finger pricks. However, it was found that gametocyte density was not significantly higher (and actually appeared to be lower) in the blood taken up by mosquitoes than in the blood from finger pricks. In some cases high densities of gametocytes were not infectious and similar results have been reported in most studies assessing human malarial infectivity to mosquitoes 34112425. It has been suggested that the prevalence of gametocyte carriers is not a good indication of the infectiousness of a population to mosquitoes 10252627.

It is clear that, as the Ethical committee only authorized us to request people over 14 years of age to take part in our experiments, this excluded a sector of the reservoir population, the children, from the study. It is also clear that the occurrence of transmission-blocking immunity and prior histories of the subjects taking anti-malarial drugs might have influenced whether they infected mosquitoes. No data on these factors was collected and so no comment can be made on their possible influence on infectivity of people's blood to mosquitoes. However, the fact that availability of anti-malarial drugs is much more tightly controlled in Thailand than in most malarious countries should be emphasized.

From the results of the study, an attempt can be made to estimate the number of adults in the catchment area of the Clinic who were reservoirs of infection. On the basis of the number of patients visiting the Clinic per day and the catchment population from which the patients come (Figure 1), it was concluded that the main reservoir of infection for mosquitoes was not in adult patients feeling ill enough to be motivated to come to the Clinic. Among villagers, occurrence of fever is a strong indicator of likely malarial infection (Table 1). Thus, fever is an indicator of likelihood of being part of the infectious reservoir for mosquitoes. However, because there are far fewer people who are feverish than those who are not, the numbers of people in the infectious reservoir who are, or are not, feverish do not differ greatly (Fig. 1). The calculations in Figure 1 do not take into account the fact that the infected people found in the villages will remain infected for several days, whereas a new group of about nine people go to the Clinic every day. It would be useful to know for how many days people remain infectious to mosquitoes, but ethically one cannot leave detected infections untreated in order to test this. The data from Figure 1 indicate that directing an anti-gametocyte drug only to the clinic patients would be ineffective. Instead the drug would have to be targeted at the village populations after mass surveys for parasitaemia. Feverishness would assist to some extent in helping to identify people most likely to be infected. In view of the current interest in anti-gametocyte drugs 282930, these data may be of use in deciding how such drugs would have to be targeted to have an impact on transmission.

<p>Figure 1</p>

Diagram to show location of the reservoirs of infection (* data from the surveys, ** data from the Vector-borne Disease Control Clinic No. 8, Mae Hong Son Province)

Diagram to show location of the reservoirs of infection (* data from the surveys, ** data from the Vector-borne Disease Control Clinic No. 8, Mae Hong Son Province)

Authors' contributions

Aree Pethleart led the data collection team in the field and laboratory, analysed data and drafted the paper;

Somsak Prajakwong provided research facilities;

Wannapa Suwonkerd organized mosquito rearing and dissection;

Boontawee Corthong was in charge of Clinic data and provided facilities for village surveys;

Roger Webber was responsible for early planning of the work; and

Christopher Curtis supervised data analysis and edited the draft.

Acknowledgements

We would like to thank the staff from the Vector-borne Disease Control Unit No.8, Mae Hong Son Province for blood collection. We wish to acknowledge the help and co-operation of Mrs Suparp Chatchatreechan, Mr Pongnarin Dee-in, Mr Somkid Sumonmard and the staff in the Entomological Department of the Office of Vector-borne Disease Control No.2, Chiang Mai (VBDO) for their help in looking after and dissecting mosquitoes. We would also like to extend our thanks to staff in the Investigation Department of VBDO and to John Williams and his team from the London School of Hygiene and Tropical Medicine, UK, for re-checking the slides. We are also thankful to Dr Apinun Aramratana, Dr Walter RJ Taylor and Dr Ilona Carneiro for their suggestions on collection, analysis and presentation of data. This work was supported by the Royal Thai Government and World Health Organization (TDR).

 <p>Factors determining the true reservoir of infection of <it>Plasmodium falciparum </it>and <it>Wuchereria bancroffi </it>in a West African village</p> Muirhead-Thomson R Trans R Soc Trop Med Hyg 1954 48 208 225 10.1016/0035-9203(54)90067-X 13169237 <p>Measurement of malarial infectivity of human populations to mosquitoes in the Madang area, Papua, New Guinea</p> Graves PM Burkot TR Carter R Cattani JA Lagog M Parker J Brabin BJ Gibson FD Bradley DJ Alpers MP Parasitology 1988 96 251 263 3374964 <p>Infectious reservoir of <it>Plasmodium vivax </it>and <it>Plasmodium falciparum </it>malaria in an endemic region of Sri Lanka</p> Gamage Mendis AC Rajakaruna J Carter R Mendis KN Am J Trop Med Hyg 1991 45 479 487 1951856 <p><it>Plasmodium vivax</it>: gametocyte infectivity of naturally infected Thai adults</p> Sattabongkot J Maneechai N Rosenberg R Parasitology 1991 102 27 31 2038501 <p>The reservoir of <it>Plasmodium falciparum </it>malaria in a holoendemic area of western Kenya</p> Githeko AK Brandling Bennett AD Beier M Atieli F Owaga M Collins FH Trans R Soc Trop Med Hyg 1992 86 355 358 10.1016/0035-9203(92)90216-Y 1359683 <p>Studies in the feeding response of mosquitoes to nutritive solution in a new membrane feeder</p> Rutledge LC Ward RA Gould DJ Mosquito News 1964 24 407 419 <p>Studies on the use of a membrane feeding technique for infecting <it>Anopheles gambiae </it>with <it>Plasmodium falciparum.</it></p> Graves PM Trans R Soc Trop Med Hyg 1980 74 738 742 10.1016/0035-9203(80)90189-3 7010696 <p>Infectivity of cultured <it>Plasmodium falciparum </it>gametocytes to mosquitoes</p> Ponnudurai T Lensen AH van Gemert GJ Bensink MP Bolmer M Meuwissen JH Parasitology 1989 98 165 173 2668861 <p>Epidemiology of <it>Plasmodium falciparum </it>in a rice field and a savanna area in Burkina Faso: seasonal fluctuations of gametocytaemia and malarial infectivity</p> Boudin C Lyannaz J Bosseno MF Carnevale P Ambroise-Thomas P Ann Trop Med Parasitol 1991 85 377 385 1796877 <p>High human malarial infectivity to laboratory-bred <it>Anopheles gambiae </it>in a village in Burkina Faso</p> Boudin C Olivier M Molez JF Chiron JP Ambroise-Thomas P Am J Trop Med Hyg 1993 48 700 706 8517489 <p>Experimental infections of <it>Anopheles gambiae </it>with <it>Plasmodium falciparum </it>of naturally infected gametocyte carriers in Cameroon: factors influencing the infectivity to mosquitoes</p> Tchuinkam T Mulder B Dechering K Stoffels H Verhave JP Cot M Carnevale P Meuwissen JH Robert V Trop Med Parasitol 1993 44 271 276 8134766 <p>Effect of gametocyte sex ratio on infectivity of <it>Plasmodium falciparum </it>to <it>Anopheles gambiae</it></p> Robert V Read AF Essong J Tchuinkam T Mulder B Verhave JP Carnevale P Trans R Soc Trop Med Hyg 1996 90 621 624 10.1016/S0035-9203(96)90408-3 9015496 <p>The early sporogonic cycle of <it>Plasmodium falciparum </it>in laboratory – infected <it>Anopheles gambiae</it>: an estimation of parasite efficacy</p> Gouagna LC Mulder B Noubissi E Tchuinkam T Verhave JP Boudin C Trop Med Int Health 1998 3 21 28 10.1046/j.1365-3156.1998.00156.x 9484964 <p>The malaria infectivity of an African village population to mosquitoes <it>(Anopheles gambiae).</it></p> Muirhead-Thomson R Am J Trop Med Hyg 1957 6 971 979 13487967 <p>Comparison of artificial membrane feeding with direct skin feeding to estimate infectiousness of <it>Plasmodium falciparum </it>gametocyte carriers to mosquitoes</p> Bonnet S Gouagna C Safeukui I Meunier JY Boudin C Trans R Soc Trop Med Hyg 2000 94 103 106 10.1016/S0035-9203(00)90456-5 10748913 <p>The transmission by mosquitoes of <it>Plasmodia </it>in the laboratory</p> Vanderberg J Gwadz RW Malaria New York, Academic Press Kreier J 1980 4 <p>Gametocyte infectivity by direct mosquito feeds in an area of seasonal malaria transmission: implications for Bancoumana, Mali as a transmission-blocking vaccine site</p> Touré YT Doumbo O Touré A Bagayoko M Diallo M Dolo A Vernick KD Keister DB Muratova O Kaslow DC Am J Trop Med Hyg 1998 59 481 486 9749648 <p>Population dynamics of mosquito-borne disease: effects of flies which bite some people more frequently than others</p> Dye C Hasibeder G Trans R Soc Trop Med Hyg 1986 80 69 77 10.1016/0035-9203(86)90199-9 3727001 <p>Variability in the relationship between weight and wing length of <it>Anopheles gambiae </it>(Diptera: Culicidae)</p> Koella JC Lyimo EO J Med Entomol 1996 33 261 264 8742532 <p>Relationship between female <it>Anopheles dirus </it>(Diptera: Culicidae) body size and parity in a biting population</p> Kitthawee S Edman JD Upatham ES J Med Entomol 1992 29 921 926 1460629 <p>Relationship between body size of adult <it>Anopheles gambiae s.l. </it>and infection with the malaria parasite <it>Plasmodium falciparum.</it></p> Lyimo EO Koella JC Parasitology 1992 104 233 237 1594289 <p>Mosquito size and multiple transmission of avian malaria in the laboratory</p> Kelly R Edman JD J Am Mosq Contr Assoc 1992 8 386 388 <p>Interaction between bloodfeeding, fecundity and infection in mosquitoes</p> Hurd H Hogg H Renshaw M Parasit Today 1995 11 411 416 10.1016/0169-4758(95)80021-2 <p>Factors affecting the infection of anophelines with human malaria in Thailand</p> Rutledge LC Gould DJ Tantichareon B Trans R Soc Trop Med Hyg 1969 63 613 619 10.1016/0035-9203(69)90180-1 4898756 <p>Infectivity to mosquitoes of <it>Plasmodium falciparum </it>as related to gametocyte density and duration of infection</p> Jeffery GM Eyles DE Am J Trop Me Hyg 1955 4 781 789 Carter R Gwadz RW Infectiousness and gamete immunization in malaria New York, Academic Press 1980 Carter R Graves PM Gametocytes. In: Malaria: Principles and Practice of Malariology Edinburgh, Churchill Livingstone 1988 253 306 <p>Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant <it>Plasmodium falciparum </it>in vitro</p> Chavalitshewinkoon-Petmitr P Pongvilairat G Auparakkitanon S Wilairat P Parasitol Int 2000 48 275 280 10.1016/S1383-5769(99)00028-8 10725690 <p>Artemether-lumefantrine for the treatment of multidrug-resistant falciparum malaria</p> van Vugt M Looareesuwan S Wilairatana P McGready R Villegas L Gathmann I Mull R Brockman A White NJ Nosten F Trans R Soc Trop Med Hyg 2000 94 545 548 10.1016/S0035-9203(00)90082-8 11132386 <p>Artesunate reduces, but does not prevent, post-treatment transmission of <it>Plasmodium falciparum </it>to <it>Anopheles gambiae.</it></p> Targett G Drakeley C Jawara M von Seidlein L Coleman R Deen J Pinder M Doherty T Sutherland C Walraven G Milligan P J Infect Dis 2001 183 1254 1259 10.1086/319689 11262208