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Column: Sleepless in the liver: would revisiting the past give any directions on how to deal with hypnozoite carriers of P. vivax?

February 6, 2014 - 20:51 -- Bart G.J. Knols
Many of the recent strategies to improve malaria control have been focusing on improving the detection of very low parasitaemias to identify asymptomatic carriers of Plasmodium sp. to reduce the infective reservoir. Although these strategies are extremely rational and sensible to tackle most of malaria infections, they will probably not be sufficient to achieve elimination for parasites that present relapses, as is the case for Plasmodium vivax. The need to look for new and sophisticated methods has been exhaustively stressed by researchers, however, at this time, revisiting a simple and efficient strategy successfully employed in the past could prove to be a path to be followed...

After a long period of dormancy, health policy makers and scientists now have their eyes wide-opened for Plasmodium vivax infection. Until recently seen as a benign infection, this parasite's importance has increased considerably within the scientific community because of increasing evidence of a much higher proportion of severe manifestations than previously believed; and, especially, due to its complex biology and the substantial additional barriers it presents to controlling transmission. Particularly in the context of the renewed and reinforced agenda to achieve malaria eradication, the higher difficulty to achieve control of this infection has been attracting a great deal of attention, as many say that P. vivax will be “the last man standing”. 
The evidence for the resilience of this parasite comes from a long way, through the observation of the ability of this parasite being able to cause disease during winter in temperate areas, when mosquitoes and P. falciparum would disappear almost completely. As pointed out by professor Marcus Lacerda, the persistence of malaria during the ice age could well he attributed to the existence of hypnozoites. The biology behind this includes an earlier production of gametocytes (thus rendering that early diagnosis and treatment is not as effective as for P. falciparum) and the presence of a dormant stage, the hypnozoite, which is responsible for causing relapses. Currently there is only one drug available to treat the hypnozoite, primaquine, which requires a long-course regimen of treatment (seven to fourteen days) and harbours the risk of severe haemolysis in G6PD (glucose-6-phosphate dehydrogenase) deficient individuals (a topic which could render several columns of its own).
Briefly speaking, the hypnozoite is a stage of the parasite that after invasion of the liver remains dormant and has the ability to, when being activated, cause a new episode of malaria without requiring inoculation by an Anopheles mosquito, which can occur from one month to until two years after the initial episode. There are still huge knowledge gaps in how the hypnozoite is formed and what determines the number and periodicity of relapses. There is no better resource to whoever is interested in the state of art of the hypnozoites than the two brilliant reviews by professors Nick White and Dennis Shanks [1,2]. Amongst the species causing human disease, only P. vivax and P. ovale share the unique characteristic of presenting the hypnozoite as part of their life cycle, with P. cynomolgi infecting non-human primates serving as an experimental alternative to trying to understand its physiology and to test new therapeutic alternatives.
The importance of the hypnozoite in terms of control tends to be higher as the transmission intensity is reduced and the proportion of episodes occurring as a direct consequence of relapses increases. So, in the context of elimination, surveillance systems and control programmes in areas where vivax malaria is endemic have to deal with both low sensitivity of point-of-care diagnostic methods to detect low parasitaemic infections and the lack of methods to detect hypnozoite carriers. High sensitivity methods, such as PCR and LAMP, if proved to be valid for use in the field, will be valuable tools to tackle the first problem but, unfortunately, of virtually no impact on the latter. And to further complicate matters for policy makers, bednets, a highly effective control measure, have no impact on preventing relapses and the fact that an individual sleeping under a net can still present repeated episodes of disease, the trust in and compliance to use bednets can suffer severely in vivax-endemic areas.
Amongst the possibilities to detect hypnozoite carriers, the use of proteomics and metabolomics have been proposed as approaches to discover a reliable marker for the carrier state, but this will probably take considerable time as we currently do not have any knowledge on the metabolism of this stage of the parasite. The exciting prospect of an animal model using humanised mice, with the observation of small parasite forms inside hepatocytes is still stuck by its current lack of ability to infect the erythrocytes hampering the observation if these forms are able to cause relapses. In this context it is very useful to revisit a successful elimination experience performed in the early 1980s in the Southern region of Brazil, which I recently had the fantastic opportunity to discuss with professor Pedro Luiz Tauil of the University of Brasilia (see picture), one of the greatest Brazilian malariologists.
In the 1950-60s, the malaria eradication campaign successfully eliminated transmission from most of Southern and Central Brazil, with transmission being restricted almost entirely to the Northern Amazon region. However in the 1980s two municipalities of the Southern state of Santa Catarina, São Francisco do Sul and Araquari, with estimated 30,000 inhabitants, still reported around 1,600 yearly cases of P. vivax. Several control measures had been deployed in the region, where Anopheles (Kerteszia) cruzii was the vector, such as IRS and mass screening and treatment (MSAT), without achieving successful elimination. By that time Professor Pedro was the Director  of the Endemic Diseases Surveillance Control of the Ministry of Health and a political decision was made by regional and national authorities to eliminate malaria in that region.  It was hypothesised that transmission was being maintained by one or both of the following factors: low and undetectable infections in asymptomatic individuals; and infections caused by parasites presenting long interval between relapses. The options discussed at the time included mass drug administration to the entire population – which was rapidly dismissed due to the size of the population and predicted lack of acceptability – and the screening of the population with serology. 
The decision made was then to screen the whole population and to treat with chloroquine and primaquine any individual with either IgM or IgG to whole-parasite antigen titre (a very simple and rustic method) above 1:64. In the first year, having achieved an impressive coverage of 90% of the population, about 500 individuals were found positive and treated, with the incidence of episodes falling drastically to 20 in the following year. In the second year survey 200 individuals were found positive and treated, with no autochthonous cases occurring in the following 25 years! This is a fantastic success story, which deserves to figure amongst the great experiences of P. vivax elimination for its innovative and original approach. 
One of the main problems with applying MDA for P. vivax endemic areas is that it requires a full course of primaquine, which can cause severe haemolysis in G6PD deficient individuals, mentioned above. Depending on the prevalence of the deficiency, there is a high public health cost of exposing uninfected person to a harmful intervention, rendering this strategy to be unacceptable in most places. At the same time, conducting surveys with methods that detect the parasite in the blood are also insufficient as they miss to detect the hypnozoite carriers, which can be activated within one month or even after two years, causing a new fever episode and even initiating an outbreak in some areas. By using serological methods, one could be using an in-between strategy that could be spot on for vivax. In terms of specificity, it would be far-from-perfect, but it could provide a high sensitivity of not missing any hypnozoite carrier allowing for effective elimination of any parasite reservoir within the screened population. A limitation of this approach would still be administering primaquine (or even tafenoquine), which could require an additional G6PD deficient detection test for point-of-care use.
The property of presenting relapses is a highly remarkable characteristics of P. vivax that will require a great parcel of interest and inventiveness for one to deal with it properly. The discovery and full understanding of the hypnozoite is the dream for vivax malariologists (the sleep and dreaming references are never too much for vivax aficionados). Today we have much more sophisticated serological methods and what one may have to do is to be inspired by this historically successful experience and try to improve it by applying more advanced current methods and technologies, such as the identification of hotspots where to target interventions and the selection of antibodies with high sensitivity and specificity (with clearance time an important aspect to look for) through well-conducted epidemiological studies. In view of our current knowledge this seems a sensible strategy that deserves to be properly evaluated in the different vivax-endemic areas aiming for elimination. Paulinho da Viola probably did not think he might be giving an important clue for tackling vivax malaria when he composed his famous beautiful samba, with its lyrics remind us “When I think about the future, I do not forget my past”. 
1. White NJ (2011) Determinants of relapse periodicity in Plasmodium vivax malaria. Malar J 10: 297.
2. Shanks GD, White NJ (2013) The activation of vivax malaria hypnozoites by infectious diseases. Lancet Infect Dis 13: 900-906.

Andre is a Brazilian Infectious Diseases Doctor who have been working with clinical and epidemiological aspects of malaria in Brazilian Amazon  region since 2009 at the University of Amazonas State in Manaus. He has mainly been involved with P. vivax studies and is interested in improving the clinical management and control of the disease in the Amazon.



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As regards the current state of knowledge concerning hypnozoites in relation to malarial relapse, see the 2015 article in "Trends in Parasitology" that is entitled "Do Hypnozoites Cause Relapse in Malaria?". It can be located at: