Malaria Skin: Recognizing and Understanding Cutaneous Manifestations of Malaria

12.11.197820.11.2021 by alexxlab

What are the skin signs associated with malaria. How does malaria affect the skin. What cutaneous features may indicate malaria infection. Can malaria cause skin rashes or other dermatological symptoms. How is malaria diagnosed through skin examination.

Содержание

The Global Impact and Epidemiology of Malaria

Malaria remains a significant global health challenge, particularly in tropical and subtropical regions. The World Health Organization’s 2019 World Malaria Report estimated a staggering 229 million cases and 409,000 deaths in that year alone. This mosquito-borne disease disproportionately affects sub-Saharan Africa, accounting for approximately 90% of global cases as of 2016.

Why is malaria prevalent in certain regions. The Anopheles mosquito, the vector for malaria transmission, thrives in specific environmental conditions found in 91 countries worldwide. Factors such as altitude, climate, and vegetation play crucial roles in determining disease incidence within these areas.

High-Risk Groups for Malaria Infection

Children under 5 years old
Pregnant women
Immunosuppressed individuals

While mosquito bites are the primary mode of transmission, it’s important to note that malaria can rarely be contracted through blood transfusions or shared needles, as it is a blood-borne disease.

Understanding the Causative Agents of Malaria

Malaria is caused by parasitic protozoa of the genus Plasmodium. Four species are primarily responsible for human infections:

P. falciparum
P. vivax
P. malariae
P. ovale

How does malaria infection occur. The female Anopheles mosquito, active between dusk and dawn, transmits the Plasmodium protozoa through its bite. These microorganisms are initially deposited in the intervascular skin matrix before invading the bloodstream and rapidly establishing an infection in the liver.

The parasites undergo a complex life cycle within the human host:

Maturation and multiplication in the liver over several days
Invasion of red blood cells for final growth phase
Bursting of infected red cells, releasing protozoa into the bloodstream
Potential collection by another mosquito during a blood feed, continuing the transmission cycle

The phase of parasitemia, when numerous parasites circulate in the blood, corresponds to an acute attack of symptoms.

Clinical Presentation of Malaria: Beyond the Skin

Malaria symptoms typically emerge 10-15 days after an infectious mosquito bite, though this incubation period can range from 7 days to 3 months. An acute attack of uncomplicated malaria presents with a constellation of symptoms:

Fever
Chills
Headache
Cough
Myalgia (muscle pain)
Abdominal pain
Vomiting
Diarrhea

How long does an acute malaria attack last. An uncomplicated malaria episode typically persists for 6-10 hours. However, the disease can progress to severe malaria, involving various organs and systems:

Brain (cerebral malaria)
Kidneys (acute renal failure)
Lungs (pulmonary edema and acute respiratory distress)
Blood (severe anemia, metabolic acidosis, hypoglycemia, or disseminated intravascular coagulation)

Cutaneous Manifestations of Malaria: Rare but Significant

While skin findings in malaria are generally rare and non-specific, they have been reported in cases of P. falciparum and P. vivax infections. These cutaneous manifestations can provide valuable diagnostic clues for healthcare providers.

Reported Skin Signs in Malaria

Urticaria (hives)
Erythema multiforme-like lesions
Petechiae
Purpura
Disseminated intravascular coagulation (DIC)
Flushing
Urticarial rash
Jaundice

It’s important to note that mucosal involvement has not been reported in malaria cases. However, certain skin signs may indicate complications of the disease:

Jaundice: yellowing of the skin and eyes, indicating liver involvement
Pallor: a sign of severe anemia
Petechiae or purpura: potential indicators of disseminated intravascular coagulation (DIC)

Dermatological Side Effects of Malaria Treatment and Prophylaxis

While treating or preventing malaria is crucial, it’s essential to be aware of potential skin-related side effects associated with antimalarial medications:

Tetracyclines (e.g., Doxycycline)

How do tetracyclines affect the skin. Tetracyclines can cause photosensitivity, including photo-onycholysis (separation of the nail from the nail bed due to light exposure). This side effect is particularly significant in tropical countries where sun exposure is high. Healthcare providers should recommend appropriate sun protection measures when prescribing doxycycline for malaria chemoprophylaxis in travelers.

Hydroxychloroquine

What skin reactions can hydroxychloroquine cause. Hydroxychloroquine may lead to morbilliform (measles-like) or psoriasiform (resembling psoriasis) rashes in up to 10% of individuals taking the medication.

Complications of Malaria: A Multisystem Threat

Malaria can progress from an uncomplicated infection to severe disease, affecting various organ systems:

Respiratory Complications

Pulmonary edema
Acute respiratory distress syndrome (ARDS)

Renal Complications

Acute renal failure

Neurological Complications

Cerebral malaria
Epilepsy
Reversible post-malaria neurological syndrome
Permanent visual, motor, or language disorders

Splenic Complications

Hyper-reactive malarial splenomegaly (severely enlarged spleen)
Splenic rupture

Pregnancy-Related Complications

How does malaria affect pregnancy. P. falciparum malaria in pregnancy can lead to severe maternal disease, resulting in premature delivery or low birth-weight infants. Two forms of malaria can affect newborns:

Congenital malaria: Due to transplacental infection from the mother or during delivery, presenting within the first 48 hours after birth
Neonatal malaria: Presents three weeks after birth following an infected mosquito bite

Both forms of neonatal malaria carry a high mortality rate, emphasizing the importance of prevention and prompt treatment in endemic areas.

Relapse Potential

Can malaria recur after initial treatment. P. vivax and P. ovale infections have the potential for relapse months or even years after the initial infection. This is due to the reactivation of dormant liver parasites, highlighting the importance of complete eradication and follow-up care.

Diagnostic Approaches for Malaria: From Skin to Laboratory

Accurate and timely diagnosis of malaria is crucial for effective treatment and prevention of complications. Healthcare providers should consider malaria in any patient presenting with a febrile illness in an endemic area or after visiting such a region.

Clinical Suspicion

When should malaria be suspected. A high index of suspicion is warranted in the following scenarios:

Fever in a patient from or recently traveled to an endemic area
Cyclical fever patterns (although not always present)
Presence of associated symptoms (headache, myalgia, fatigue)
Relevant skin findings (urticaria, petechiae, jaundice)

Laboratory Diagnosis

What tests confirm malaria infection. Several laboratory methods are employed to diagnose malaria:

Microscopy: Examination of thick and thin blood smears remains the gold standard for malaria diagnosis. This method allows for species identification and quantification of parasitemia.
Rapid Diagnostic Tests (RDTs): These immunochromatographic tests detect specific malaria antigens in the blood. They provide quick results but may not distinguish between all Plasmodium species.
Polymerase Chain Reaction (PCR): Highly sensitive and specific, PCR can detect low levels of parasitemia and accurately identify Plasmodium species. However, it’s not widely available in resource-limited settings.
Serology: Detection of antibodies against malaria parasites. This method is primarily used for epidemiological studies rather than acute diagnosis.

Additional Diagnostic Considerations

What other tests support malaria diagnosis. While not specific to malaria, the following tests can provide valuable information:

Complete blood count (CBC): May reveal anemia, thrombocytopenia
Liver function tests: To assess for hepatic involvement
Renal function tests: Important in monitoring for complications
Blood glucose: Hypoglycemia is a potential complication
Coagulation studies: If DIC is suspected

In cases where cutaneous manifestations are present, a skin biopsy may be considered to rule out other dermatological conditions. However, skin biopsies are not routinely performed for malaria diagnosis.

Prevention Strategies: Protecting the Skin and Beyond

Preventing malaria involves a multi-faceted approach, with skin protection playing a crucial role. Key strategies include:

Vector Control

Use of insecticide-treated bed nets
Indoor residual spraying
Environmental management to reduce mosquito breeding sites

Personal Protection Measures

Wearing long-sleeved clothing, especially during peak mosquito activity hours
Applying EPA-registered insect repellents containing DEET, picaridin, or oil of lemon eucalyptus
Using permethrin-treated clothing and gear

Chemoprophylaxis

For travelers to endemic areas, antimalarial medications may be prescribed. Options include:

Atovaquone-proguanil
Doxycycline
Mefloquine
Chloroquine (in areas without chloroquine-resistant P. falciparum)

It’s important to note that while these medications reduce the risk of malaria, they do not provide complete protection. Adherence to personal protection measures remains crucial.

Vaccination

The RTS,S/AS01 malaria vaccine has shown promise in clinical trials and is being implemented in pilot programs in select African countries. While not yet widely available, it represents a significant step forward in malaria prevention efforts.

Treatment Approaches: Addressing Systemic and Cutaneous Manifestations

Effective treatment of malaria requires a comprehensive approach, considering both the systemic infection and any cutaneous manifestations that may be present.

Antimalarial Medications

The choice of antimalarial drug depends on several factors:

Plasmodium species identified
Severity of infection
Local resistance patterns
Patient characteristics (age, pregnancy status, comorbidities)

Common antimalarial medications include:

Artemisinin-based combination therapies (ACTs)
Chloroquine (for chloroquine-sensitive infections)
Quinine
Mefloquine
Atovaquone-proguanil

Management of Cutaneous Symptoms

While skin manifestations in malaria are generally self-limiting and resolve with treatment of the underlying infection, symptomatic management may include:

Antihistamines for pruritus or urticaria
Topical corticosteroids for inflammatory lesions
Appropriate wound care for any ulcerative lesions

Supportive Care

Severe malaria often requires intensive supportive care, which may include:

Fluid and electrolyte management
Blood transfusions for severe anemia
Respiratory support in cases of pulmonary edema or ARDS
Renal replacement therapy for acute kidney injury
Management of seizures and other neurological complications

Follow-up and Monitoring

After initial treatment, follow-up is essential to ensure parasite clearance and to monitor for potential relapses, particularly in P. vivax and P. ovale infections. This may involve repeat blood smears and clinical assessments.

In conclusion, while cutaneous manifestations of malaria are relatively rare, they can provide valuable diagnostic clues when present. A comprehensive approach to malaria management, encompassing prevention, accurate diagnosis, and effective treatment, is crucial in reducing the global burden of this potentially life-threatening disease. Healthcare providers should maintain a high index of suspicion for malaria in patients presenting with fever and relevant travel history, even in the absence of specific skin findings.

Malaria and its skin signs

Authors: Ellie Dodson, Medical Student, University of Bristol, United Kingdom; Dr Lauren Thomas, RMO, Royal Darwin Hospital, Darwin, NT, Australia; Niket Shah, Medical Student, University of Otago, Wellington, New Zealand. Copy edited by Gus Mitchell. November 2020.

What is malaria?

Malaria is a blood-borne tropical infection caused by parasitic protozoa of the genus Plasmodium transmitted between people via infected female Anopheles mosquitoes.

Who gets malaria?

Malaria only occurs in the 91 tropical and subtropical countries where the Anopheles mosquitoes can survive, multiply, and complete their growth cycle. Disease incidence within these countries further depends on environmental factors including altitude, climate, and vegetation. In 2016, the World Health Organisation (WHO) estimated 90% of malaria cases in the world occurred in sub-Saharan Africa, but other regions at risk included South-East Asia, Central and South America, and parts of the Middle East. The WHO World Malaria Report for 2019 estimated 229 million cases and 409,000 deaths in that year.

Children under 5, pregnant women, and the immunosuppressed are at the highest risk of contracting malaria.

As malaria is a blood-borne disease, it is possible to contract it from blood transfusions and the sharing of needles, but this is rare.

What causes malaria?

Malaria is caused by just four of the many species of Plasmodium protozoa. These are:

P. falciparum
P. vivax
P. malariae
P. ovale.

The Plasmodium protozoa are spread by the female Anopheles mosquito which bites between dusk and dawn. The mosquito deposits the protozoa in the intervascular skin matrix, from where the microorganisms invade the bloodstream and rapidly establish infection in the liver. The parasites mature and multiply in the liver over several days before entering red blood cells for their final growth phase. The infected red cells burst open to release protozoa into the bloodstream where another mosquito can incidentally collect them during a blood-feed. This phase of parasitaemia, when many parasites are circulating in the blood, corresponds to an acute attack of symptoms.

What are the clinical features of malaria?

Symptoms of malaria usually develop 10–15 days after an infectious mosquito bite (range 7 days to 3 months). An acute attack of uncomplicated malaria lasts 6–10 hours and presents with fever, chills, headache, cough, myalgia, abdominal pain, vomiting, and diarrhoea. Progression to severe malaria involves organs including the brain, kidney, or lungs, or blood disturbances such as severe anaemia, metabolic acidosis, hypoglycaemia, or disseminated intravascular coagulation (DIC).

What are the cutaneous features of malaria?

Apart from the visible mosquito bites, cutaneous findings are rare and non-specific in malaria, but have been reported with P. falciparum and P. vivax malaria. Reported cutaneous findings include:

Mucosal involvement has not been reported.

Skin signs may indicate complications such as jaundice, anaemia, and DIC.

Skin side effects of malaria treatment and chemoprophylaxis

Tetracyclines can cause photosensitivity including photo-onycholysis. Photosensitivity is an important adverse effect in tropical countries, and appropriate sun protection should be recommended when doxycycline is prescribed for chemoprophylaxis in travellers.

Hydroxychloroquine can cause morbilliform or psoriasiform rashes in up to 10% of people.

What are the complications of malaria?

Uncomplicated malaria can progress to severe disease, with organ involvement including:

Pulmonary oedema and acute respiratory distress
Acute renal failure
Cerebral malaria, epilepsy, reversible post-malaria neurological syndrome, and permanent visual, motor, or language disorders
Hyper-reactive malarial splenomegaly (very enlarged spleen) and splenic rupture.

P. falciparum malaria in pregnancy can cause severe disease in the mother resulting in premature delivery or low birth-weight baby. Congenital malaria is due to transplacental infection from the mother or during delivery and presents in the first 48 hours after birth. Neonatal malaria presents three weeks after birth following an infected mosquito bite. Both forms carry a high mortality rate.

Relapses can occur months or years after P. vivax or P. ovale infection following reactivation of dormant liver parasites.

How is malaria diagnosed?

Malaria should be considered in anyone with a febrile illness in an endemic area, or after visiting an endemic area in the previous 12 months.

Malaria is diagnosed on microscopic examination of a blood film. Thick blood film examination provides sensitivity. Thin blood film examination allows speciation and quantitation. If the initial film is negative, blood should be reassessed every 6–12 hours for 36–48 hours before malaria can be confidently excluded.

Antigen-based rapid diagnostic tests (RDT) detect specific antigens produced by the parasite. Some are species specific, while others can detect multiple species.

What is the differential diagnosis for malaria?

Malaria presents as a nonspecific acute febrile illness. The differential diagnosis therefore is long and includes many other tropical infectious febrile illnesses, including:

What is the treatment for malaria?

The best treatment option for malaria is prophylaxis including clothing, netting over beds, mosquito repellents, and chemoprophylaxis with medications such as doxycycline and hydroxychloroquine, depending on the geographic location. Work on vaccine development continues.

Vector control to prevent the spread of mosquitoes can include:

The destruction of areas of stagnant water where mosquitoes breed
Treatment of houses and netting with insecticide
Release of sterile male mosquitoes
Genetic modification of mosquitoes to reduce disease susceptibility.

General measures

Treatment may be required for:

Secondary infection
Electrolyte and fluid imbalance
Anaemia
Hypoglycaemia.

Antihistamines have been reported to ease the symptoms of cutaneous involvement.

Supportive measures for complications may also include ventilation and/or dialysis.

Specific measures

The medication used for prophylaxis and treatment will depend on drug resistance and severity of disease. Drugs used include:

Doxycycline
Clindamycin
Hydroxychloroquine and chloroquine
Artemisinin derivatives, such as artesunate and artemether.

What is the outcome for malaria?

Full recovery from malaria is expected with appropriate prompt treatment. However, treatment is not always curative due to drug resistance, high parasite density, treatment noncompliance, low host immunity, and poor drug bioavailability. Recurrent symptoms with detectable parasitaemia can occur 2–6 weeks after apparently successful treatment.

Urticaria is reported to subside 12–48 hours after starting antimalarial treatment. All skin symptoms resolve within days of treatment without recurrence.

Cerebral malaria and anaemia in children can result in persistent movement disorders, speech difficulties, deafness, blindness, behavioural issues, and epilepsy.

Severe malaria can deteriorate rapidly, with death within hours or days due to missed or delayed diagnosis, but also sometimes despite appropriate treatment and intensive care.

P. falciparum malaria can be rapidly fatal within 24–48 hours of presentation, especially in children.

In 2018, there were an estimated 228 million cases of malaria, and 405,000 deaths (mostly children) worldwide.

Where Malaria Infection and the Host Immune Response Begin

Semin Immunopathol. Author manuscript; available in PMC 2014 Feb 25.

Published in final edited form as:

PMCID: PMC3934925

NIHMSID: NIHMS411807

Johns Hopkins Bloomberg School of Public Health, Malaria Research Institute, 615 North Wolfe St. , Baltimore, MD 21205

See other articles in PMC that cite the published article.

Abstract

Infection by malaria parasites begins with the inoculation of sporozoites into the skin of the host. The early events following sporozoite deposition in the dermis are critical for both the establishment of malaria infection and for the induction of protective immune responses. The initial sporozoite inoculum is generally low and only a small percentage of these sporozoites successfully reach the liver and grow to the next life cycle stage, making this a significant bottleneck for the parasite. Recent studies highlight the importance of sporozoite motility and host cell traversal in dermal exit. Importantly, protective immune responses against sporozoites and liver stages of Plasmodium are induced by dendritic cells in the lymph node draining the skin inoculation site. The cellular, molecular and immunological events that occur in the skin and associated lymph nodes are the topic of this review.

Keywords: malaria, dermis, sporozoites, plasmodium, dendritic cells, CD8+ T cells

Introduction

Malaria, one of the most important infectious diseases worldwide, is caused by protozoan parasites of the genus Plasmodium. These parasites cycle between a vertebrate and mosquito host and experience a significant reduction in numbers during transmission. Sexual or asexual reproductive cycles follow transmission and restore parasite numbers in the mosquito or vertebrate host, respectively. Thus, infection in the vertebrate host has two phases: an asymptomatic pre-erythrocytic stage, when parasite numbers are low, and a symptomatic erythrocytic stage, composed of iterative cycles of replication in host red blood cells. The pre-erythrocytic stage is short-lived, yet critical for the establishment of malaria infection. It is comprised of sporozoites, which are inoculated by infected mosquitoes, and the liver stages (or exoerythrocytic stages) into which they develop. In this review we will focus on the early stages of malaria infection, following the fate of inoculated sporozoites and outlining how the immune response to these stages is initiated and sustained. We will conclude with some thoughts as to how this knowledge can inform the generation of improved malaria vaccine candidates.

The skin stage of malaria infection

Until recently, our knowledge of the molecular interactions between host and parasite during the early stage of malaria infection was limited due to the small numbers of sporozoites and liver stages present in the mammalian host. Indeed, a review of the literature between 1970 to 1995 indicates that malariologists labored under the assumption that sporozoites rapidly left the inoculation site and significant interactions between host and parasite did not begin until sporozoites invaded hepatocytes and began to develop. Recent studies have now shown that sporozoites spend several hours at the inoculation site [1] and initiate an immune response in the lymph nodes which drain this site [2], thus bringing this early stage of infection into the limelight.

Sporozoites reside in mosquito salivary glands and exit with the mosquito’s saliva into the skin of the vertebrate host as the mosquito probes for blood. Salivation stops when the mosquito locates and begins to imbibe blood, thus sporozoites are primarily deposited into the skin and are not inoculated directly into the blood circulation [3, 4]. In most cases, the skin compartment into which sporozoites are inoculated is the dermis since this is the depth to which the mosquito’s proboscis reaches. However, skin thickness varies from one location to another and a minority of sporozoites likely find themselves in the epidermis or sub-cutaneous tissue. The average number of sporozoites inoculated by a single infected mosquito varies enormously and is, to some extent, a function of the salivary gland load [5]. Studies using mosquitoes infected with rodent malaria parasites showed that a single infected mosquito injects between 0 and 1,300 sporozoites with the average inoculum being approximately 125 sporozoites [5]. These studies, however, used laboratory-raised mosquitoes where infection is optimized and salivary gland sporozoite numbers tend to be high. In the field, mosquitoes harbor lower numbers of parasites and it is likely that the inoculum is generally under 100 sporozoites [6, 7].

After their deposition in the skin, sporozoites must locate and penetrate blood vessels in order to reach the liver. Intra-vital microscopy studies show that sporozoites move randomly in the skin until they contact either endothelial cells of the blood or lymphatic system [8, 9]. Sporozoites glide around and along these vessels, enter by an as yet unknown mechanism and are carried away, either rapidly by the blood circulation, or slowly by the lymphatic system [8]. Although some sporozoites rapidly leave the injection site, many take hours to exit and enter the bloodstream. That transit to the bloodstream could take hours was initially suggested by experiments in monkeys with the primate malaria parasite Plasmodium cynomolgi, in which it was demonstrated that transplantation of skin containing the inoculation site, up to 2 hours after sporozoite injection, resulted in infection of naïve recipients [10]. More recently, using the rodent malaria parasite Plasmodium yoelii, which enables a more quantitative and comprehensive analysis, it was demonstrated that sporozoites exit the dermis and enter the blood circulation in a slow trickle extending for 2 to 3 hours after their inoculation [1]. Some sporozoites do not enter the bloodstream and instead enter the lymphatic circulation and go to the draining lymph node. Studies have shown that approximately 15–20% of the inoculum ends up in the draining lymph node [1, 2, 8]. These sporozoites, though at least initially alive, ultimately do not continue further and likely become fodder for the immune response [2, 8].

It is likely that the remainder of the inoculum is destroyed at the site of deposition, likely by the innate immune response of the host, although this has, to date, not been well-studied. Recently, however, it was shown that a small proportion, between 0.5 and 5%, of the inoculated sporozoites remain and begin to develop into exoerythrocytic stages at the inoculation site [11, 12]. Thus far this has only been studied using rodent malaria parasites so it is possible that the development of exoerythrocytic stages in an aberrant location may result from a non-optimal host-parasite combination since the natural hosts of rodent malaria parasites are African thicket rats. Equally possible is that this may be an evolutionary relic since the avian malaria parasites, with whom the rodent and primate parasites share a common ancestor, develop into exoerythrocytic stages in mesodermal tissue including the skin. Importantly, these aberrantly developing parasites are not able to initiate a blood stage infection [12]. This is possibly because the merozoites within the exoerythrocytic forms do not fully mature although imaging data argues that this is not the case [11]. More likely it is because these parasites cannot easily access the blood circulation from this location: the liver sinusoids with their fenestrated endothelia provide a more direct route to the blood circulation than the closed endothelia of the blood. Nonetheless, it will be important to determine whether these skin exoerythrocytic stages contribute to the adaptive immune response that targets infected hepatocytes and whether this is a feature of malaria infection that is shared by all Plasmodium species.

Sporozoite exit from the dermis

Two sporozoite behaviors are required for dermal exit: motility and an ability to traverse cells. Sporozoites move by gliding motility, which is powered by an actin-myosin motor beneath their plasma membrane (reviewed in [13]. This motor is connected to the sporozoite surface via the cytoplasmic domain of a transmembrane surface protein called TRAP, (thrombospondin related anonymous protein) which has extracellular adhesive domains that bind to matrix such that the force of the motor translocates TRAP posteriorly, propelling the sporozoite forward. Previous studies have demonstrated that sporozoites actively invade hepatocytes and gliding motility is required for cell invasion [14]. More recently it has been shown that robust gliding is critical for sporozoite exit from the skin: sporozoites with mutations in TRAP which result in slow staccato movement have a much more pronounced effect on infectivity after intradermal inoculation than after intravenous injection [15]. Another critical property for dermal exit is the ability of sporozoites to traverse host cells, wounding these cells as they enter and exit [16, 17]. Mutants deficient in proteins required for cell traversal have normal infectivity when placed directly on hepatocytes in vitro yet are substantially less infective in vivo where they must exit the dermis and traverse the liver sinusoid to reach their target cell [16, 18–20]. In vivo imaging of fluorescent cell traversal mutants demonstrates that they are not able to efficiently move through the skin, becoming immobilized after contacting cells [16]. These data raise the possibility that cell traversal may also be a mechanism by which sporozoites escape phagocytic cells that arrive at the site in response to the mosquito’s saliva [16]. Importantly migrating sporozoites must switch to an invasive phenotype once they reach the liver. Recent studies have shown that the major surface protein of sporozoites, the circumsporozoite protein or CSP, is critical for this switch [21]. CSP has a cell adhesive domain in its carboxy-terminus which is masked in salivary gland sporozoites. This domain remains masked as sporozoites migrate through the skin and then upon contact with hepatocytes, CSP is proteolytically processed by a parasite protease, revealing this domain and changing a migratory sporozoite into an invasive one. Although the signal for CSP cleavage and the switch to an invasive phenotype are incompletely understood, the highly sulfated heparan sulfate proteoglycans specific to hepatocytes likely play a role [17]. Thus, shortly after their arrival in the liver, cell traversal activity is stopped and invasion, with development into the next life cycle stage, proceeds.

Induction of protective anti-Plasmodium CD8+ T cell responses

Early studies using experimental models clearly demonstrated that protective immunity against sporozoite-induced infection requires antigen-specific CD8⁺ T cells [22, 23]. Some of these CD8⁺ T cells were specific for defined epitopes in CSP, and these T cells strongly inhibited the development of liver stage parasites [24]. Subsequent studies using T-cell receptor transgenic CD8+ T cells specific for a CSP epitope, demonstrated that these T cells were primed primarily in lymph nodes draining the skin where sporozoites were deposited [2]. Forty-eight hours after immunization, either by the bites of irradiated infected mosquitoes or via intradermal inoculation of irradiated sporozoites, epitope-specific CD8⁺ T cells producing IFN-γ were first detected only in the lymph nodes draining the inoculation site. Once CD8+ T cells are activated in lymph nodes, they migrate to other lymphoid and non-lymphoid organs including the liver. The importance of T cell priming in skin draining lymph nodes was demonstrated in experiments in which these lymph nodes were surgically ablated or through pharmacological inhibition of T-cell egress from lymph nodes. Under these experimental conditions the number of T cells reaching the liver was drastically reduced and the protective capacity of the anti-parasite CD8+ T cell-mediated protection was diminished.

An intriguing observation made in early studies indicated that protective immunity could be induced with irradiated yet live sporozoites [25]. Consistent with this observation, it was later shown that the induction of effector CD8+ T cell responses also requires immunization with live sporozoites [2, 26]. The strict requirement of viable sporozoites was previously interpreted as evidence that sporozoite invasion of hepatocytes was required for the induction of protective immune responses and this led to the idea that liver stage antigens were critical to induce protective immunity. However, the demonstration that protective CD8+ T cells are induced in the skin draining lymph node suggests that these responses are induced after complex interactions between professional antigen presenting cells and sporozoites.

Antigen presentation by dendritic cells (DCs)

It is well established that CD11c+ DCs play a critical role in the priming of Plasmodium specific CD8⁺ T cells. Studies have shown that DCs incubated in vitro with sporozoites or obtained from lymph nodes of mice previously injected with sporozoites present parasite epitopes to T cells [2, 27, 28]. Moreover, in vivo depletion of the CD11c+ DCs abolishes the induction of parasite specific CD8+ T cell responses [29]. The skin is a tissue that harbors large numbers of DCs belonging to phenotypically and functionally distinct groups which are likely to interact with parasites. In addition, there are large numbers of lymph node-resident DCs which may also play a role in inducing T cell responses, particularly considering that a significant number of parasites migrate to lymph nodes draining the inoculation site.

The mechanisms by which DCs acquire antigen from live parasites is an intriguing yet poorly understood process. It is well known that the CSP and other parasite molecules are shed as sporozoites move and conceivably, these secreted molecules may be endocytosed and processed by DCs, and peptides within these antigens eventually presented to T cells. Alternatively, as was outlined earlier, the critical role of cell traversal for exit from the dermis means that sporozoites may also directly deposit antigen in the cytosol of DCs.

While the precise mechanisms of DC antigen uptake are unknown, experimental evidence indicates that DC priming of CD8⁺ T cells occurs by antigen cross-presentation. This notion is supported by studies in which Toll-like receptor ligands administered prior to sporozoite immunization, inhibit the induction of CD8+ T cell responses [2]. It is known that TLR ligands hasten the maturation of DCs, a process that is accompanied by an inhibition of their endocytic activity [30, 31]. Recently, these studies were expanded using new methodological approaches and novel transgenic parasites. P. berghei parasites expressing mutant CSP containing the H-2K^b SIINFEKL epitope have enabled studies in genetically modified C57Bl/6 (H-2^b) mice deficient in molecules involved in antigen processing and presentation [32]. These studies demonstrated that priming of CD8+ T cells required intact endocytic function as experiments performed in mice lacking the endosomal protein Unc93B1 failed to develop robust CD8+ T cell responses. Unc93B1 is believed to mediate translocation of endocytosed molecules to the ER which are subsequently transported to the cytosol where they are processed to generate peptide epitopes [33]. A critical role for cross-presentation is further supported by in vivo experiments in which treatment of mice with cytochrome-c resulted in a severely reduced CD8+ T cell response [32]. Studies in other systems show that after cytochrome-c is internalized by endocytosis, it is translocated to the cytosol where it induces apoptosis, thus depleting antigen cross-presenting cells [34–36]. Finally, no CD8+ T cell responses were observed in mice lacking the TAP1 molecule which mediates the transport of the proteosome-processed CSP peptides from the cytosol to the ER. This clearly indicates that CSP must reach the cytosol where it is processed, generating epitope-containing peptides that are transported by TAP from the ER to where they bind to class l MHC molecules.

The precise tissue compartment where the capture of parasite antigen occurs and the identity of the DC subpopulation involved in this process, are critical matters that remain to be defined. As discussed in the preceding sections, intradermally inoculated sporozoites remain in the skin for over one hour and exit the skin in a slow trickle [1]. In addition a significant proportion of sporozoites migrate to the draining lymph nodes [1, 2, 8]. These findings raise an important question: where do DCs acquire sporozoite antigen? An obvious possibility is that sporozoite antigen is acquired in the dermis by skin-resident DCs that then migrate to the lymph nodes where they present antigen directly to naive CD8⁺ T cells. At least three distinct subsets of skin-resident migratory DCs have been characterized: Langerhans cells, dermal DCs, and langerin⁺CD103⁺ dermal DCs [37]. Langerin⁺CD103⁺ dermal DCs are a subset of migratory DCs that play a key role in cross-presenting viral and self antigens [37–40] and their possible involvement in cross-presentation of sporozoite antigens requires further investigation. Alternatively, dermal DCs could transfer skin-derived antigen to lymph node resident DCs for CD8⁺ T cell priming as shown in studies using herpes simplex virus [41]. Finally, it is also possible that CD8⁺ T cell priming does not require skin-derived DCs, but instead occurs via direct acquisition of sporozoite antigen by lymph-node resident DCs. In this regard it is important that skin-inoculated sporozoites can be found associated with DCs in the lymph nodes [8].

Antigen persistence and maintenance of memory responses: another role for skin draining lymph nodes

Prolonged antigen presentation is crucial for maximal expansion of effector CD8+ T cell responses and recently it was demonstrated that continuous antigen presentation occurs for up to two months after immunization with irradiated sporozoites [42]. This observation is quite striking considering that irradiated sporozoites are not able to undergo proliferation and do not differentiate beyond early liver stages [43, 44]. Apparently, the parasite antigen does not persist as a dormant form of the parasite because treatment with primaquine to eliminate early liver stage parasites has no effect on continuous antigen presentation [42]. Antigen-presenting cells are responsible for trapping antigens, although the precise identity of the cell types involved in presenting persisting antigens is unclear and remains an area of further investigation. Persistent antigen is detected mostly in skin draining lymph nodes although it can also be found in spleen and liver. Continuous antigen presentation to CD8+ T cells is required for renewing and maintaining the memory CD8⁺ T cell population and in fact naive cells such as recent thymic emigrants, are primed by persisting antigens. It is important that this prolonged antigen presentation does not induce CD8⁺ T cell exhaustion as described in some chronic viral infection models [45, 46], on the contrary, persistent antigen induces effector T cell differentiation and is necessary to develop or maintain optimal memory responses.

Relevance of the skin stage to the malaria vaccine effort

Several decades ago it was demonstrated that immunization with attenuated sporozoites could protective rodents, primates and humans from challenge with infected mosquito bites. Initially studies in rodents utilized irradiated sporozoites inoculated intravenously. In humans, immunization has always relied upon the bites of irradiated infected mosquitoes with ≥ 950 infected bites required for protection [47]. Calculations based upon the demonstration that individual mosquitoes inoculate on average 125 sporozoites [5], indicate that the dose required for protection in humans is not significantly different from rodent models. Thus, studies in humans clearly demonstrate that sporozoites inoculated into the skin can induce protective immune responses. More recently it has also been shown in the rodent models that sporozoites delivered by mosquito bite or intradermal injection can induce protection equivalent to that observed after intravenous immunization [2, 48, 49].

That attenuated sporozoites inoculated into the dermis induce protection should not be surprising given what we now know of sporozoite biology and priming of the immune response in the dermis and in the lymph nodes that drain this site. Moreover, a significant number of sporozoites, perhaps as high as 50%, may remain in the skin as non-viable parasites or undergoing further development to exoerythrocytic stages [1, 8, 11, 12]. Of the parasites that reach the bloodstream, most do not productively infect hepatocytes; studies with the rodent malaria parasites P. yoelii and P. berghei suggest that at most 25% and 10% of the inoculum, respectively, develops into liver stages [50, 51]. The skin, therefore, is the site where sporozoites may have their greatest exposure to the host immune system and it is not unexpected that a strong immune response is induced first in lymphoid organs associated with this tissue compartment.

Conclusions

Research conducted in several laboratories over the past 10 years has demonstrated that the skin, a compartment originally thought to be irrelevant to malaria infection, is a critical barrier for the sporozoite. Both robust gliding motility as well as the ability to traverse cells are required for dermal exit, yet even under optimal conditions, well-over 50% of sporozoites do not leave the inoculation site. Thus, this is a vulnerable time for the parasite suggesting that antibodies targeting critical processes such as gliding and cell traversal, could have a dramatic impact on infection. The recent explosion in our understanding of sporozoite biology at the inoculation site sets the stage for discovering and testing these new antibody targets.

This initial stage of malaria infection is also critical for the induction of T cell responses that ultimately target infected hepatocytes as priming occurs in the lymph node draining the inoculation site. Ultimately, the immunogenic properties of sporozoites combined with the rich array of immunologically active cells in the skin, confers upon give the dermis a critical role in determining the magnitude and quality of the anti-parasite immune response. As we move forward with candidate malaria vaccines, the biological and immunological importance of this early stage of infection is likely to play a role in vaccine design.

Acknowledgments

PS is supported by NIH R01 AI056840 and FZ by NIH R01 AI44375. The authors are grateful for the support of the Bloomberg Family Foundation.

Footnotes

¹This article is published as part of the Special Issue on Immunoparasitology [35:1]

The authors declare no financial conflicts of interest.

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