Feature Articles

Cutaneous manifestations of neonatal bacterial infection


v5_i1_a22Skin forms a dynamic interface with the external environment and is a complex organisation of cell types and associated structures that performs many essential functions. Although the stratum corneum of full-term neonates is analogous to that of adult skin, structural and compositional differences of the skin renders the newborn more susceptible to bacterial colonisation. Particularly for the preterm neonate, impaired cutaneous barrier function and an immature immune system reduce the capacity to defend against bacterial pathogens. The majority of cutaneous bacterial infections are localised to the skin and are easily treated, however, systemic bacterial infection and disseminated disease in the neonatal period may be life-threatening.

Differences in neonatal skin

Newborn skin is fundamentally different from that of the adult and adapts to the extrauterine environment during the first year of life through ongoing structural and functional changes. [1] Skin is a complex, selectively permeable membrane that performs a number of roles, including protection from infection and external stressors such as ultraviolet (UV) light damage, temperature regulation, sensation, and physical appearance. Protection against the external environment is primarily due to the most superficial layer of the epidermis, the stratum corneum, and recent advances in fluorescence spectroscopy and electron microscopy have helped elucidate the differences between adult and newborn skin. [1-3] The stratum corneum is a layer of lipid-depleted, protein-rich corneocytes embedded in a matrix of extracellular lipids, resulting from the continuous proliferation of keratinocytes in the basal epidermis. [3] Compared with adult skin, newborn skin produces smaller corneocytes, a thinner epidermis, and an increased density of microrelief grooves (Table 1). [1] The corneocytes of newborns also have a higher degree of irregularity and decreased organisation in both development and subsequent desquamation phases. [2] The change in neonatal skin pH from neutral, at birth, towards a more acidic mantle is also likely to impact on stratum corneum integrity, as incomplete skin surface acidification is linked to variable rates of desquamation. [3,4] Overall, decreased corneocyte size and a thinner, less cohesive stratum corneum has negative implications for skin barrier function, as indicated by increased transepidermal water loss (TEWL) in newborn skin. [5]

Compared with full-term infants born at 37-42 weeks’ gestation, preterm newborns do not develop the same level of protection provided by the stratum corneum until 2-4 weeks after birth. [6,7] The most significant difference is an increase in the stratum corneum from two to three cell layers at 28 weeks’ gestation to the equivalent of adult skin with 15 layers by 32 weeks’ gestation. [8] Due to the role of the stratum corneum in barrier protection, the premature newborn is at considerably greater risk of cutaneous complications.

Changes in TEWL, skin pH, and sebaceous activity all lead to the creation of a skin environment that promotes colonisation of certain microbial skin flora. [9] Colonisation of resident flora commences at birth, but newborns have a unique skin microbiome profile that develops throughout the first year of life and beyond. [9] The protection offered by resident commensal and mutualistic skin flora in the adult is therefore not immediately present in the newborn, leading to different patterns of subsequent infection.

Table 1. Differences between newborn and adult skin. [1,2]
Table 1. Differences between newborn and adult skin. [1,2]

Skin defences and immune response of the newborn:

Skin has antimicrobial function afforded by the innate immune system and antigen presenting cells (APCs) of the epidermis and dermis, as well as circulating immune cells that migrate into the dermis. This innate system works together with the adaptive immune system to defend against infection. In the newborn, innate immunity is the most important mechanism of defence, as this system is present at birth as a result of pattern recognition receptors encoded by germline DNA. This system responds to biochemical structures common to a number of pathogens, producing a rapid response with no residual immunity or memory. In contrast, adaptive immunity develops slowly and involves specific antigen receptors of T- and B-lymphocytes as part of a system that develops memory for faster successive responses.

Innate immune defences comprise the physical barrier formed by the skin itself, antimicrobial peptides (AMPs), complement pathways, and immune cells including monophages, macrophages, dendritic cells, and natural killer cells. [10,11] While the keratinocytes of the skin are typically considered to be static ‘bricks’ of the physical skin barrier, they are also dynamically involved in immunity by their secretion of cytokines and chemokines, AMPs, and complement components. AMPs secreted by keratinocytes are cationic proteins termed cathelicidins and defensins, and they appear to be particularly important in neonatal immunity, with selective bactericidal activity against common cutaneous pathogens. [12,13] Cathelicidins (LL-37) and beta-defensins (BD-1, BD-2, BD-3) are attracted to negatively charged bacteria, viruses, and fungi and exert their influence by membrane insertion and pore formation. [13] Neonates display higher baseline concentrations of cutaneous AMPs than adults, suggesting a greater role for these proteins in newborn skin defences. In the absence of specific antibodies, pattern recognition receptors such as Toll-like receptors (TLRs) play a pivotal role, with subsequent cytokine production changing with increasing age and correlating to age-specific pathogen susceptibility. [14]

Figure 1. Innate immunity of the skin in the newborn
Figure 1. Innate immunity of the skin in the newborn

Bacterial infections

Cutaneous staphylococcal and streptococcal infections cause a variety of clinical presentations depending on site of infection, strain of organism, and neonatal immunity. Impetigo is a superficial bacterial cutaneous infection that may present with or without bulla formation, as described by the conditions non-bullous and bullous impetigo. The bullae of bullous impetigo are invariably due to infection with S. aureus and the subsequent production of epidermolytic toxin, which is also responsible for the widespread bullae and desquamation in staphylococcal scalded skin syndrome (SSSS). Development of resistant strains, overcrowding, and poor infection control have been linked to nosocomial outbreaks of S. aureus and is of particular concern in neonatal intensive care units where neonates are more susceptible to infection. [15]

Non-bullous impetigo

Both Streptococcus pyogenes and S. aureus are associated with the non-bullous form of impetigo, which presents as an erythematous macular rash before developing eroded lesions with a honey-coloured crust. [16] Isolated staphylococcal pustules and paronychia are also common in neonates. Although mild non-bullous impetigo has the capacity self-resolve, treatment with topical mupirocin and fusidic acid limits the opportunity for disease to persist. [16]

Bullous impetigo

Localised cutaneous S. aureus infection presents with an erythematous vesiculopustular rash that preferentially affects the diaper area and skin folds, coalescing to form large flaccid bullae that rupture easily and appear as honey-crusted erosions. [16,17] Bacteria are present in the lesions, and the infection usually responds to first-line systemic flucloxacillin, which may be used in conjunction with topical fusidic acid. [16,17] Certain strains of S. aureus are associated with epidermolytic toxins, which facilitate pathogen entry beneath the stratum corneum and limit disease to the superficial epidermis. [18,19] The distinct bullae present in bullous impetigo are due to the toxin-induced cleavage of desmosomal cadherin proteins in the granular layer of the epidermis, which are normally responsible for maintaining functional adhesion between keratinocytes. [18] These same toxins are produced in SSSS.

Staphylococcal scalded skin syndrome

Haematogenous spread of S. aureus is facilitated by inoculation at a distant site such as the conjunctiva, umbilicus, or perineum, and the effects of bulla formation and desquamation are the direct result of circulating epidermolytic toxins. [20] This haematogenous spread results in a widespread infection that is more severe than the localised infection of bullous impetigo. Generalised erythema and skin tenderness are the initial clinical features, with evolution into large flaccid bullae and desquamation of the entire cutaneous surface. The Nikolsky sign is present, where blistering can be elicited by light stroking of the skin. [21] Bacterial cultures of cutaneous lesions are typically negative and S. aureus is only found at the distant sites of infection. Skin biopsy is considered the gold standard of diagnosis and is particularly relevant when considering toxic epidermal necrolysis (TEN) as a differential. In contrast to SSSS, TEN results in subepidermal blisters and keratinocyte necrosis rather than epidermal cleavage and typically involves oral mucous membranes. [20,21] Although biopsy is helpful in providing a definite diagnosis, neonatal biopsies are rarely performed due to the characteristic clinical presentation of both conditions. Despite the apparent polarity of the cutaneous and haematogenous forms of S. aureus infection, a handful of mild SSSS cases have been reported, lending support to a likely clinical spectrum ranging from a mild form to the classic severe disease. [22]


After birth and separation of the umbilical cord, necrosis of the stump is followed by epithelialisation. The healing stump may become colonised, with the exposed umbilical vessels forming a potential portal of entry for pathogenic bacteria. [23] Omphalitis is characterised by stump erythema and periumbilical oedema, with or without discharge, and is frequently due to S. aureus. It is more common in developing countries, and the risk is increased in cases of protracted labour, non-sterile delivery, and prematurity. [23] A recent Cochrane systematic review identified significant evidence to support the use of topical chlorhexidine on the umbilical stump to reduce omphalitis and neonatal mortality in developing countries. However, this benefit could not be demonstrated in developed countries, possibly owing to reduced risk factors for omphalitis. [24]

Necrotising fasciitis

Infection of the fascia and overlying soft tissues is a rapidly progressive neonatal emergency. Pathogens gain entry by cutaneous breaches such as omphalitis, birth trauma, and superficial skin wounds, with group A streptococci most commonly implicated as the causative organism. [25] Infection may also be polymicrobial, with a combination of organisms detected on wound cultures. [26] The infection follows the fascial plane, causing thromboses in the blood supply to overlying tissues and leading to tissue necrosis, and the skin becomes progressively more discoloured, tender, and warm. [26] While the initial presentation may not appear concerning, neonates rapidly become disproportionately tender and toxic. [26] Necrotising fasciitis has a high morbidity and mortality and requires immediate identification for surgical debridement. [25]

Ecthyma gangrenosum

Pseudomonas aeruginosa septicaemia is the most common underlying cause for this cutaneous manifestation, which typically presents with macules that progress via a necrotising vasculitis to form indurated necrotic ulcers with surrounding erythema. [27,28] Prematurity, immune deficiencies, and neutropaenia are the main predisposing factors, but lesions may develop in the absence of immunodeficiency when direct inoculation occurs through a breach in the skin barrier. [28]

Antimicrobial resistance and prevention

The treatment of neonatal bacterial infection depends on the pathogen and sensitivities to antibiotic treatments. In the Australian healthcare setting and internationally, antibiotic resistance poses a growing problem in this ‘post-antibiotic’ era. Methicillin-resistant S. aureus (MRSA) has become increasingly prevalent, particularly in the intensive care setting such as the neonatal intensive care unit (NICU). [29] As colonised neonates are continually admitted, the introduction of many unique sources and various strains over time adds to the ongoing burden and is likely to contribute to difficulties in fully eradicating MRSA from the NICU. [30] Transmission of organisms such as S. aureus most commonly occurs secondary to direct contact with colonised caregivers, and this problem is compounded when hand hygiene and barrier protection is inadequate. Premature infants in the NICU are particularly susceptible, due to their immature immune systems and the increased risk of nosocomial infection with invasive monitoring and frequent healthcare worker contact. [31] The identification of previous treatment with third-generation cephalosporins and carbapenem as independent risk factors for the development of multidrug-resistant Gram-negative bacteraemia in the NICU highlights the issue of antibiotic resistance and underscores the importance of judicious antibiotic use. [32]


Skin is the first line of defence against invading pathogens, and there are a number of unique cellular, functional, and immunological factors that underpin an increased susceptibility to bacterial infection in the newborn. Premature newborns are at particular risk of infection, owing to potential deficits in cutaneous barrier function. Future practice in treating bacterial infections is likely to be influenced by the emergence of multi-resistant strains and may shift the focus toward improved prevention measures.

Conflict of Interest

None declared.


J Read:


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[2] Fluhr JW, Lachmann N, Baudouin C, Msika P, Darlenski R, De Belilovsky C, et al. Development and organization of human stratum corneum after birth. Electron microscopy isotropy score and immunocytochemical corneocyte labelling as epidermal maturation’s markers in infancy. Br J Dermatol. 2014 Feb 7. DOI:10.1111/bjd.12880.

[3] Stamatas GN, Nikolovski J, Mack MC, Kollias N. Infant skin physiology and development during the first years of life: a review of recent findings based on in vivo studies. Int J Cosmet Sci. 2011;33(1):17-24.

[4] Fluhr JW, Man MQ, Hachem JP, Crumrine D, Mauro TM, Elias PM, et al. Topical peroxisome proliferator activated receptor activators accelerate postnatal stratum corneum acidification. J Invest Dermatol. 2009;129(2):365-74.

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[7] Fluhr JW, Darlenski R, Lachmann N, Baudouin C, Msika P, De Belilovsky C, et al. Infant epidermal skin physiology: adaptation after birth. Br J Dermatol. 2012;166(3):483-90.

[8] Taeusch HM, Ballard RA, Gleason CA, editors. Avery’s diseases of the newborn. 8th ed. Philadelphia: Elsevier Saunders; 2005.

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[12] Nelson A, Hultenby K, Hell E, Riedel HM, Brismar H, Flock JI, et al. Staphylococcus epidermidis isolated from newborn infants express pilus-like structures and are inhibited by the cathelicidin-derived antimicrobial peptide LL37. Pediatr Res. 2009;66(2):174-8.

[13] Yoshio H, Lagercrantz H, Gudmundsson GH, Agerberth B. First line of defense in early human life. Semin Perinatol. 2004;28(4):304-11.

[14] Kollmann TR, Levy O, Montgomery RR, Goriely S. Innate immune sensing by Toll-like receptors in newborns and the elderly. Immunity. 2012;37(5):771-83.

[15] Bertini G, Nicoletti P, Scopetti F, Manoocher P, Dani C, Orefici G. Staphylococcus aureus epidemic in a neonatal nursery: a strategy of infection control. Eur J Pediatr. 2006 Aug;165(8):530-5.

[16] Sladden MJ, Johnston GA. Current options for the treatment of impetigo in children. Expert Opin Pharmacother. 2005;6(13):2245-56.

[17] Johnston GA. Treatment of bullous impetigo and the staphylococcal scalded skin syndrome in infants. Expert Rev Anti Infect Ther. 2004;2(3):439-46.

[18] Hanakawa Y, Schechter NM, Lin C, Garza L, Li H, Yamaguchi T, et al. Molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin syndrome. J Clin Invest. 2002;110(1):53-60.

[19] Yamasaki O, Yamaguchi T, Sugai M, Chapuis-Cellier C, Arnaud F, Vandenesch F, et al. Clinical manifestations of staphylococcal scalded-skin syndrome depend on serotypes of exfoliative toxins. J Clin Microbiol. 2005;43(4):1890-3.

[20] Stanley JR, Amagai M. Pemphigus, bullous impetigo, and the staphylococcal scalded-skin syndrome. N Engl J Med. 2006;355(17):1800-10.

[21] Berk DR, Bayliss SJ. MRSA, staphylococcal scalded skin syndrome, and other cutaneous bacterial emergencies. Pediatr Ann. 2010;39(10):627-33.

[22] Hubiche T, Bes M, Roudiere L, Langlaude F, Etienne J, Del Giudice P. Mild staphylococcal scalded skin syndrome: an underdiagnosed clinical disorder. Br J Dermatol. 2012;166(1):213-5.

[23] Fraser N, Davies BW, Cusack J. Neonatal omphalitis: a review of its serious complications. Acta Paediatr. 2006;95(5):519-22.

[24] Imdad A, Bautista RM, Senen KA, Uy ME, Mantaring JB 3rd, Bhutta ZA. Umbilical cord antiseptics for preventing sepsis and death among newborns. Cochrane Database Syst Rev. 2013;5:CD008635.

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[26] Jamal N, Teach SJ. Necrotizing fasciitis. Pediatr Emerg Care. 2011;27(12):1195-9.

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[30] Gregory ML, Eichenwald EC, Puopolo KM. Seven-year experience with a surveillance program to reduce methicillin-resistant Staphylococcus aureus colonization in a neonatal intensive care unit. Pediatrics. 2009;123(5):e790-6.

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[32] Tsai MH, Chu SM, Hsu JF, Lien R, Huang HR, Chiang MC, et al. Risk factors and outcomes for multidrug-resistant Gram-negative bacteremia in the NICU. Pediatrics. 2014;133(2):e322-9.

Feature Articles Articles

Approaching autism

Autism Spectrum Disorder is a social communication disorder in someone displaying repetitive and restrictive interests. Diagnosed in early childhood, children struggle to develop social relationships required for further learning and independent living. This article discusses changes to the diagnosis, how the diagnosis is made, the prevalence, causes and interventions. Importantly, this review guides medical students towards an understanding of what to expect in individuals with autism spectrum disorder and how to interact with them.

What is autism?

Autism is diagnosed according to the American Psychiatric Association (APA) guidelines in the Diagnostic and Statistical Manual of Mental Disorders (DSM), [1] or alternatively, according to the International Classification of Diseases (ICD) published by the World Health Organization. [2] Mostly because of convention, the DSM is more often used in the diagnosis of autism in Australia.

From 2013, ‘autism’ would be a short form of the term Autism Spectrum Disorder (ASD). A diagnosis of ASD applies where there is evidence of functional impairment caused by:

Problems reciprocating social or emotional interaction, including difficulty establishing or maintaining back-and-forth conversations and interactions, inability to initiate an interaction, and problems with shared attention or sharing of emotions and interests with others.

Severe problems maintaining relationships, ranging from a lack of interest in other people to difficulties in pretend play and engaging in age-appropriate social activities, and problems adjusting to different social expectations.

Nonverbal communication problems such as abnormal eye contact, posture, facial expressions, tone of voice and gestures, as well as an inability to understand these. [1]

Additionally, two of the four symptoms related to restricted and repetitive behaviour need to be present:

Stereotyped or repetitive speech, motor movements or use of objects.

Excessive adherence to routines, ritualised patterns of verbal or nonverbal behaviour, or excessive resistance to change.

Highly restricted interests that are abnormal in intensity or focus.

Hyper- or hypo-reactivity to sensory input or unusual interest in sensory aspects of the environment.

Symptoms must be present in early childhood, but may not become fully manifest until social demands exceed limited capacities. There must be an absence of general developmental delay. Finally, symptoms should not be better described by another DSM-5 diagnosis.

From the criteria it is important to note that there are many combinations of symptoms that can lead to a diagnosis of ASD. Therefore, not only do individuals with ASD share the same degree of uniqueness as the rest of the general population, but even the type of ASD they experience can be unique.

Confusion can arise in the use of the terms ‘autism’ and ‘autism spectrum disorders.’ Part of the reason for this emerges from the subtypes of Pervasive Developmental Disorders (PDD) previously recognised by the APA. PDD previously contained the subtypes Asperger’s Disorder, Childhood Disintegrative Disorder, Autistic Disorder and PDD-Not otherwise specified (NOS). While the ICD also recognizes Atypical Autism, recent changes to the DSM should simplify the nosology. [2]

The current ASD diagnosis criteria came into effect from May 2013 and is expected to identify approximately 90% of children with a clinical diagnosis from the previous DSM-IV. [3] A new diagnosis called Social Communication Disorder (SCD) has been created. Simply put, SCD is ASD, without the restrictive and repetitive interests. It is not yet clear how many children will receive a diagnosis of SCD, nor how many children previously identified with a DSM-IV diagnosis of PDD would meet the criteria for SCD only.

Diagnoses made under DSM-IV guidelines are still valid. A new diagnosis is not required simply because of the change in criteria. However, individuals being assessed for ASD now, and in the future, will be diagnosed under the new criteria.

Features of ASD

Social communication, interaction and motivation

ASD is principally characterized by social communication and interaction deficits. Individuals often experience difficulty with interpreting facial expressions, tone of voice, jokes, sarcasm, gestures and idioms. Imagine the literal meanings of “Fit as a fiddle,” “Bitter pill to swallow” and “Catch a cold.” Some people with ASD may have only limited speech or may be completely non-verbal. [4,5] Echolalia, pronoun reversal, unusual vocalisations and unusual accents are common. [4,5] Alternative, augmentative and visually based communication techniques may help when a child is unable to consistently follow verbal instructions. In this regard, touch-screen portable devices may appeal to their visual and pattern-orientated learning strengths (see Figure 1). [6]

Difficulties with social interactions mean that affected individuals often grow up without a social circle, and as a consequence, miss out on peer-initiated learning opportunities. [7] Such challenges include difficulty with understanding unwritten social rules such as personal space and initiating conversation. [7] They can appear to be insensitive, because they are unable to perceive how someone else is feeling. Turn taking and sharing is not intuitive or learned, and individuals need to be trained how to do this. An inability to express feelings, emotions or needs results in inappropriate behaviour such as unintentionally aggressive actions. [8] This can lead to isolation, a failure to seek comfort from others and signs of low self-esteem. [7] Individuals can also suffer from hypersensitivity to sensory stimuli, which may lead them to prefer limited social contact. Individuals do feel enjoyment and excitement; however, this tends to be a personal experience and often goes unshared, which may be due to a failure to need the reward of another’s attention and praise. [9]


Individuals with ASD may experience challenges with social imagination. [10] Individuals are less likely to engage in make-believe play and activities. They are less likely to determine and interpret other’s thoughts, feelings and actions, and as a consequence unable to appreciate that other people may not be interested in their topic of interest.

Individuals with ASD are often unable to predict outcomes, or foresee what might occur next, including hazards. [11] This leads to a difficulty in coping in new or unfamiliar situations, or making plans for the future. Parents, carers and health professionals often need to stick to routines to avoid unpredicted events that could cause distress.

Sensory and motor processing

Sensory information processing is heightened for tactile input, but reduced for social input, in individuals with ASD. [12] Changes in these inputs are understood to contribute to the repetitive and restrictive interest criteria of the diagnosis. Hence, the state of the tactile environment is important to the wellbeing of individuals with ASD, potentially serving as an aggravation by, or as a refuge from, incomprehensible cues. Changes in sensory information processing may present as an inability to distinguish context-relevant stimuli, and varying capabilities and capacity to respond to a stimulus (e.g. ignoring some sounds but over-reactive to others). They may also experience difficulty with proprioception and responding to pain, including temperature extremes. [12,13] Individuals may explore their environment by smelling or mouthing objects, people and surfaces, and as a consequence, develop eating behaviours that relate to smell, texture or flavor, including inedible objects. Participating in repetitive movements such as rocking, bouncing, flapping arms and hands, or spinning with no apparent dizziness is sometimes as a means of coping with stress, or alternatively, could be used as a means of self-stimulus, providing pleasure. [12] Contrary to popular perception, savant skills are uncommon in individuals with ASD. [14]

What causes ASD?

ASD is a multi-factorial disorder. There is no one cause of ASD. The most prominent risk factor is genetics, both familial and de novo. [15] Studies have shown that among monozygotic twins, if one child has ASD, then the other will be affected about 36–95% of the time. [16] In dizygotic twins, if one child has an ASD, then the other is affected up to about 31% of the time. [16] Parents who already have a child with an ASD have a 2%–18% chance of having a second child who is also affected. [17]

In addition to the well-documented increase in chromosomal abnormalities associated with advanced maternal age, the risk of ASD is also associated with advanced paternal age. [18] The current hypothesis to explain this observation is that small, de novo genetic mutations and rearrangements accumulate in the sperm, which are then incorporated into the DNA of the child. [19,20] Other risk factors include premature birth or low birth weight, preeclampsia [21] and in utero exposure to medications, particularly sodium valproate. [22] There is no evidence to support the theory that the measles, mumps and rubella vaccine causes autism. [23]

Three cognitive theories have evolved to explain the behavioural challenges of ASD: the theory of mind deficit, executive dysfunction, and weak central coherence. The first posits that individuals with ASD are unable to recognize mental states in others, leading to social behaviour discordance. [24,25] The second attempts to explain a lack of goal directed behaviour, and lack of behavioural flexibility. [26] Both these cognitive explanations may be underpinned by an early deficit in social motivation, whereby underdevelopment of the brain regions involved in social recognition and response leads to a failure to learn social cues and their contexts. [27] This has been traditionally thought to impact on imitation learning from which social and food reward by infants is derived and goal-directed behaviour emerges, although research in the area is equivocal. [28] Whether this same underdevelopment also delays acquisition of receptive and expressive language is unclear. [29] Finally, the weak central coherence theory is a means of understanding why individuals focus on details and neglect the context. [30] Yet, it could be argued that a neglect of the context emerges from a deficit in social motivation.


In the community there is considerable concern of an ASD epidemic. Parental reports indicate ASD prevalence may have increased from 1 in 88 in 2007 to 1 in 50 children in 2012. [31] Currently, the prevalence of ASD in Australia is about 1 in 165 children aged 6-12 years. [32] In the USA this prevalence is slightly higher with 1 in 50–88 eight-year olds receiving a diagnosis; [31,33] this breaks down to 1 in 31–54 boys and 1 in 143–252 girls. [31,33]

A true appreciation of the changes in ASD prevalence can only come from understanding the historical basis of the diagnosis. [34,35] In this regard, marked changes in prevalence have been caused by nosology changes (e.g. autism was once called childhood schizophrenia) and a number of changes to the APA PDD criteria in the DSM over the past 30 years. [36] Other contributing factors include demographic variables (e.g. where older individuals may have missed receiving a diagnosis, but receive one now with the help of a retrospective investigation such as archival home video), increased awareness of normal childhood development and developmental disorders, changes in testing and protocols, and the sampling of data, such as parents versus clinicians, or state schools versus all children. Other factors that often go unreported include socioeconomic factors (e.g. those with sufficient knowledge and resources are able to seek out professional assistance and are more likely to receive a diagnosis than those without) and pressure on clinicians to provide a diagnosis, thereby assisting struggling parents access services and financial support. Trying to account for all these factors in an epidemiological study is very difficult. Hence, the true historical prevalence of ASD is difficult to establish. Studies that have tried show no, or a slight, increase in ASD. [35]

How is a diagnosis made?

While parents often suspect developmental delay or ASD, the variability in child development during the first four years can lead to variability in the age of first diagnosis – typically around three years of age. [34] Reliability of the diagnosis also suffers as a consequence. [37] Restrictive and repetitive interests can be difficult to identify before the age of four because even typically developing two- and three-year-olds can show repetitive behaviours. Since the new diagnosis also requires behaviours to be demonstrably incompetent (such as during a child’s interaction at day care), a lag between symptoms and diagnosis is likely to continue.

In Australia, paediatricians, clinical psychologists, psychiatrists and speech pathologists specialising in the field of paediatrics or adolescence make a formal diagnosis of ASD. Further, within these specialisations, diagnoses are likely to be made by practitioners with experience in testing and diagnosing ASD.

A typical diagnostic evaluation involves a multi-disciplinary team including pediatricians, psychologists, speech and language pathologists. Testing takes a number of hours and can be exhausting for subjects, parents and clinicians. Because of this and other factors, waiting times for diagnosis can be up to 24 months across the country, with particular difficulties in rural and remote areas. [38]

In initial consultations, screening tools may be used such as the Autism Behavior Checklist (ABC), Checklist for Autism in Toddlers (CHAT), Modified Checklist for Autism in Toddlers (M-CHAT), Childhood Autism Rating Scale (CARS) and Gilliam Autism Rating Scale (GARS). However, for a diagnosis, the Autism Diagnostic Interview-Revised (ADI-R) and Autism Diagnostic Schedule (ADOS) are used. [39,40]

Differential diagnoses and comorbidities

There is no single test for ASD and there are no unique physical attributes. For this reason differential diagnoses such as hearing and specific language impairments, mutism, environmental conditions such as neglect and abuse, and attachment and conduct disorders need to be excluded. Disorders similar to ASD include Social Communication Disorder and Social Anxiety Disorder. In the latter, communication is preserved but a degree of social phobia persists.

Since ASD diagnoses are made according to a description of a set of behaviours rather than a developmental abnormality or genetic condition, it is not uncommon to find a diagnosis of ASD comorbid with another pre-existing condition. For example, approximately 20% of children with Down syndrome meet the diagnosis for ASD. [41] Theoretically, all children with genetic abnormalities such as Angelman syndrome or Rett syndrome would also meet criteria for a diagnosis for ASD. In the event of an existing condition, a diagnosis of ASD may also be warranted in order to guide the child’s behavioural management and education.

ASD often co-occurs with another developmental, psychiatric, neurologic, or medical diagnosis. [42] The co-occurrence of one or more non-ASD developmental diagnoses with ASD is approximately 80%. Co-morbidities often occur with attention deficit hyperactivity disorder, Tourette syndrome, anxiety disorders and dyspraxia. Although common, the majority (~60%) of children with ASD do not have an intellectual disability (ID; intelligent quotient ≤70). [33] Some individuals with an intellectual disability are likely to always remain dependent on health care services.

There is an increased risk of epilepsy in individuals with ASD. However, the increased co-morbidity of epilepsy is strongly linked to ID – 24% in those with ASD and ID, and 2% with ASD only. [43] There is no evidence that a particular epileptic disorder can be attributed to ASD (or vice versa). [44] The more common presentations include late infantile spasms, partial complex epilepsies and forms of Landau-Kleffner syndrome. Mutations in the tuberous sclerosis genes are particularly associated with ASD and epilepsy. [45]

Gastrointestinal disorders (GID) are a common complication in ASD. [46] Given that some “cognitive” genes of the brain are also expressed in the enteric nervous system, decreased visceral sensitivity, myogenic reflexes or even CNS integration of visceral input may be exacerbated in genetically susceptible individuals. Language impairments may be associated with toilet training difficulties, which can lead to constipation with overflow incontinence and soiling. However, medications and diet are not significantly associated with GID in individuals with ASD. [47]

What treatments are available?

At this point in time, it is thought that biological changes affect the function or structure of the brain over time, leading to different developmental, psychological and behavioural trajectories. There is no cure for ASD, but early intensive behavioural intervention (based on Advanced Behavioural Analysis) is somewhat successful towards promoting learning and independent living. [48] This intervention aims to addressing the core deficits of ASD in a structured, predictable setting with a low student-teacher ratio (initially 1:1). It promotes behavioural systems for generalization and maintenance, promotes family involvement and monitors progress over time. There is some evidence to suggest that participation in social skills groups also improves social interaction. [49] Other intervention strategies are pedagogical approaches to matching faces and actions to meanings.

The Australian Government has made available a support package called Helping Children with Autism. The package includes information, workshops and financial assistance for early intervention services.

At present, pharmacological intervention targets some symptoms associated with ASD. These include serotonin reuptake inhibitors, anti-psychotics, anti-epileptics, mood stabilisers and other medications to treat hyperactivity, aggression and sleep disruption. Given the degree of notable side effects in these pharmacotherapeutics, new generation compounds continue to be tested. There are high rates of complementary and alternative diet use in children with ASD, but a lack of rigorous studies means that the evidence for efficacy is poor. [50,51]

Guidelines on dealing with children with ASD

As medical students and interns, there may be opportunities to participate in a diagnostic clinic or therapy session. Community placements also provide insight into understanding special education interventions, respite and support. However, most interactions would occur during resident training or paediatric rotations. In these situations students will be observing or assisting specialists.

Children with ASD are only likely to be admitted into hospital with a medical problem distinct from their behavioural features. Nevertheless, children with ASD are twice as likely to become inpatients. [52] The more common reasons for hospital admission and general practice visits are seizures, sleep disturbances and constipation. [53]

Parents may describe their child as high-functioning, which tends to imply an IQ >70. This doesn’t usually reflect the social capacity of the child. Ultimately, as with other children, it is important to know the child’s strengths and weaknesses; for example, whether they respond more to visual or verbal communication. Most considerations when working generally with typically developing children also apply to children with ASD and developmental disorders. Suggestions for interacting with individuals with ASD can be found in the Table.

Ultimately, by engaging the parents and working with the child’s strengths, most issues can be resolved. Even for experienced clinicians, interactions can present challenges. It helps to be patient and adaptable. In some cases it may be pointless performing some examinations if doing them would be disruptive and not provide critical medical information. In such cases, working from the collaborative history will have to suffice.

ASD workshops and training opportunities

Workshops and training opportunities for community members range from one-hour presentations to nationally certified training programs. There are currently two nationally accredited training programs: CHCCS413B ‘Support individuals with ASD,’ and CHCEDS434A ‘Provide support to students with ASD.’ Associations around Australia and New Zealand also deliver community education programs and recognized training. The Autism Centre of Excellence (Griffith University, Queensland) provides tertiary training in ASD studies. The Olga Tennison Autism Research Centre (La Trobe University, Victoria) provides behavioural intervention strategy (Early Start Denver Model) training to qualified professionals.

Conferences of note include the annual International Meeting for Autism Research, the biennial Asia Pacific Autism Conference and the Australian Society for Autism Research conferences.

Concluding remarks

Autism is a spectrum disorder. As such, each child is unique. For this reason it is best not to get caught up with the ‘label’, but to focus on the individual’s abilities or disabilities, with an understanding that simplicity, patience and adaptability may be needed. Work with the parents or the carer to achieve the desired outcomes.


Thanks to Dr. Elisa Hill-Yardin and Dr. Naomi Bishop for a critical reading of a draft of this manuscript.


Dr. Moldrich is Adjunct Research Fellow of The University of Queensland investigating biological causes of autism. Together with Drs. Hill-Yardin and Bishop, he is co-organiser of autism research conferences. Dr. Moldrich is on the scientific advisory board of the Foundation for Angelman Syndrome Therapeutics.


R Moldrich:


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Letters Articles

Pentraxin 3 – A new player in twinning frequency

The conception of dizygotic twins is a complex trait.

It is thought to be influenced by a variety of environmental and genetic factors and displays significant regional variation in prevalence worldwide. [1] For example, in Sub-Saharan areas of Africa, twinning is very common (~23 per 1000 pregnancies), while in Asia twinning is much rarer (~5-6 per 1000 pregnancies). [2] Recent research has sought to determine the reasons behind the increased frequency of twinning in regions of Sub-Saharan Africa. Independent studies of women from Gambia and Upper East Ghana have given insight into gene mutations which may possibly increase the fertility of women and hence the frequency of twinning. Specifically, it was found that certain single-nucleotide polymorphisms (SNPs) in the gene of pentraxin 3 (PTX3), a key player in human fertility and innate immunity, occurred in higher frequency amongst the mothers of twins. [3] This report will review the known functions of PTX3 in immunity and fertility and their relation to twinning frequency.

Pentraxin 3 in innate immunity

PTX3 is a soluble pattern recognition receptor, which belongs to the acute phase reactants superfamily. [4] In the innate immune response, PTX3 is produced in response to primary pro-inflammatory signals such as interleukin 6 (IL-6) release or toll-like receptor activation. [5] It participates in immunity by recognising pathogens, facilitating complement activation and opsonisation. [6] Indeed, it is involved in immune defence against Aspergillus, Pseudomonas, Salmonella, Mycobacterium tuberculosis, cytomegalovirus and influenza. [7-9] Known mechanisms of anti-pathogenic action include the binding of sialylated ligands on PTX3 to membrane proteins such as haemagglutinins found in influenza viruses and cytomegaloviruses. As haemagglutinins are used by viruses for fusion and entry to host cells, the binding of PTX3 ligands to the haemagglutinins can block this function and hence lower the chance of viral infection.  [7,8] The anti-viral actions of PTX3 against cytomegalovirus can also activate downstream immune components such as interferon regulatory factor 3 (IRF3) and the interleukin-12/interferon gamma (IL-12/IFN)-γ-dependent effector pathway, which in turn heighten anti-fungal defences against species such as Aspergillus. [8] Previous experiments performed by Garlanda et al. also show that PTX3-null mouse models were more susceptible to fungal infections, suggesting that PTX3 plays a non-redundant antifungal role. [10]

Pentraxin 3 in fertility

PTX3 is not only a major player in immunity, it has also been demonstrated to be linked to fertility in various studies. Specifically, PTX3 interacts with proteins such as TNF-stimulated gene 6 (TSG6) and inter-alpha-trypsin inhibitor (IαI) to form multimolecular constructs which facilitate cross-linking in the hyaluronan matrix that surrounds the cells of the cumulus oophorus. [11] This is crucial to the stability and organisation of the cumulus matrix, as shown in animal studies where PTX3-null mice produced ova with abnormal cumulus oophorus, which led to lower litter counts. [12,13] The infertility resulting from PTX3 knockout is not surprising as a functional cumulus oophorus is required for oocyte maturation, movement to oviduct and penetration by sperm. [14-16] Notably, mouse and human PTX3 are highly conserved, suggesting that PTX3 may play a similar role in humans. [4] Further supporting the key, non-redundant roles of PTX3 in fertility is the finding that PTX3 is one of the most highly upregulated genes during the pro-inflammatory cascade at the foetal-maternal interface, which is crucial to decidualisation, blastocyst invasion, anchorage and implantation. [17-20]

Pentraxin 3 in twinning

It is clear that PTX3 plays a crucial role in immunity and fertility. Tying all these findings together is  research by Sirugo et al. and May et al. which demonstrate associations between twinning, female fertility and PTX3 SNPs in humans. [3,21] Sirugo et al. demonstrated that the frequency of certain PTX3 haplotypes differed in frequency between mothers of twins and mothers without twins in a sample of 130 Gambian sister pairs (p = 0.006– 3.03×10-6, depending on haplotype). [3] In concordance with this, data from May et al. based on a population study suggest that those findings may indeed be due to increased fertility conferred by the PTX3 mutations. [21] It was found that women with more than12 children had SNPs in PTX3 causing the highest production of PTX3 and that women with less than 2 children had SNPs which conferred the lowest production of PTX3. Specifically, rs6788044 SNP, which was associated with the highest PTX3 production (p = 0.003), was also associated with the highest fertility (p = 0.043). In addition, increased ex vivo LPS-induced PTX3 production, suggesting better immunity, was also associated with increased fertility (p = 0.040). [21]


Taken together, the data suggests that PTX3 may contribute to the high rates of twinning in Sub-Saharan Africa.  As increased PTX3 expression confers improved innate immune response, local selective pressures due to disease may skew epigenetic controls to favour these particular variants in particular populations where a strong immune response is crucial.  [3] Certain SNPs of PTX3 which are selected for also confer increased fertility, via mechanisms such as increased cumulus oophorus stability and regulation of the pro-inflammatory cascade of implantation.  While the role of PTX3 in multiple ovulations – a primary factor of dizygotic twinning – is still unclear, the contribution of PTX3 to successful implantation is also vital to twinning, by increasing the chance of survival of multiple blastocysts. In conclusion, the available evidence suggests that PTX3 may be an important contributor to twinning, at least in some African populations.

Conflict of interest

None declared.


I thank God, my family, the Brisbane research team I worked with and my Griffith University lecturers for their guidance and support of me in pursuing a career in medicine and research.


G Yeung:


[1] Hoekstra C, Zhao ZZ, Lambalk CB, Willemsen G, Martin NG, Boomsma DI, et al. Dizygotic twinning. Human reproduction update. 2008;14[1]:37-47.

[2] Bulmer M. The biology of twinning in Man. Oxford, United Kingdom: Oxford Clarendon Press, 1970.

[3] Sirugo G, Edwards DRV, Ryckman KK, Bisseye C, White MJ, Kebbeh B, et al. PTX3 genetic variation and dizygotic twinning in The Gambia: could pleiotropy with innate immunity explain common dizygotic twinning in Africa? Annals of Human Genetics. 2012.

[4] Garlanda C, Bottazzi B, Bastone A, Mantovani A. Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility. Annual review of immunology. 2005;23:337-66.

[5] Bottazzi B, Garlanda C, Salvatori G, Jeannin P, Manfredi A, Mantovani A. Pentraxins as a key component of innate immunity. Current opinion in immunology. 2006;18[1]:10-5.

[6] Bottazzi B, Garlanda C, Cotena A, Moalli F, Jaillon S, Deban L, et al. The long pentraxin PTX3 as a prototypic humoral pattern recognition receptor: interplay with cellular innate immunity. Immunological reviews. 2009;227[1]:9-18.

[7] Reading PC, Bozza S, Gilbertson B, Tate M, Moretti S, Job ER, et al. Antiviral activity of the long chain pentraxin PTX3 against influenza viruses. The Journal of Immunology. 2008;180[5]:3391-8.

[8] Bozza S, Bistoni F, Gaziano R, Pitzurra L, Zelante T, Bonifazi P, et al. pentraxin 3 protects from MCMV infection and reactivation through TLR sensing pathways leading to IRF3 activation. Blood. 2006;108[10]:3387-96.

[9] Olesen R, Wejse C, Velez DR, Bisseye C, Sodemann M, Aaby P, et al. DC-SIGN [CD209], pentraxin 3 and vitamin D receptor gene variants associate with pulmonary tuberculosis risk in West Africans. Genes Immun. 2007;8[6]:456-67.

[10] Garlanda C, Hirsch E, Bozza S, Salustri A, De Acetis M, Nota R, et al. Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response. Nature. 2002;420[6912]:182-6.

[11] Scarchilli L, Camaioni A, Bottazzi B, Negri V, Doni A, Deban L, et al. PTX3 interacts with inter-alpha-trypsin inhibitor: implications for hyaluronan organization and cumulus oophorus expansion. The Journal of biological chemistry. 2007;282[41]:30161-70.

[12] Salustri A, Garlanda C, Hirsch E, De Acetis M, Maccagno A, Bottazzi B, et al. PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization. Development. 2004;131[7]:1577-86.

[13] Varani S, Elvin JA, Yan C, DeMayo J, DeMayo FJ, Horton HF, et al. Knockout of pentraxin 3, a downstream target of growth differentiation factor-9, causes female subfertility. Mol Endocrinol. 2002;16[6]:1154-67.

[14] Wassarman P. The mammalian ovum. Knobil E NJ, editor. New York: Raven Press; 1988.

[15] Yanagimachi R. Mammalian fertilization. Knobil E NJ, editor. New York: Raven Press; 1988.

[16] Tesarik J MOC, Testart J. Effect of the human cumulus oophorus on movement characteristics of human capacitated spermatozoa. J Reprod Fertil. 1990;88:665-75.

[17] Garlanda C, Maina V, Martinez de la Torre Y, Nebuloni M, Locati M. Inflammatory reaction and implantation: the new entries PTX3 and D6. Placenta. 2008;29 Suppl B:129-34.

[18] Hess AP, Hamilton AE, Talbi S, Dosiou C, Nyegaard M, Nayak N, et al. Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators. Biology of reproduction. 2007;76[1]:102-17.

[19] Popovici RM, Betzler NK, Krause MS, Luo M, Jauckus J, Germeyer A, et al. Gene expression profiling of human endometrial-trophoblast interaction in a coculture model. Endocrinology. 2006;147[12]:5662-75.

[20] Tranguch S, Chakrabarty A, Guo Y, Wang H, Dey SK. Maternal pentraxin 3 deficiency compromises implantation in mice. Biology of reproduction. 2007;77[3]:425-32.

[21] May L, Kuningas M, Bodegom Dv, Meij HJ, Frolich M, Slagboom PE, et al. Genetic Variation in Pentraxin [PTX] 3 Gene Associates with PTX3 Production and Fertility in Women. Biology of reproduction. 2010;82[2]:299-304.


Case Reports Articles

Metastatic melanoma: a series of novel therapeutic approaches

The following report documents the case of a 63 year old male with metastatic melanoma following a primary cutaneous lesion. Investigation into the molecular basis of melanoma has identified crucial regulators in melanoma cell proliferation and survival, leading to the inception of targeted treatment and a shift toward personalised cancer therapy. Recently, the human monoclonal antibody ipilimumab and the targeted BRAF inhibitor vemurafenib have demonstrated promising results in improving both progression-free and overall survival.


A diagnosis of metastatic melanoma confers a poor prognosis, with a median overall survival of six to ten months. [1-3] This aggressive disease process is of particular relevance in Australia, owing to a range of adverse risk factors including a predominantly fair-skinned Caucasian population and high levels of ultra-violet radiation. [4-6] While improved awareness and detection have helped to stabilise melanoma incidence rates, Australia and New Zealand continue to display the highest incidence of melanoma worldwide. [4-7] Clinical trials have led to two breakthroughs in the treatment of melanoma: ipilimumab, a fully human monoclonal antibody, and vemurafenib, a targeted inhibitor of BRAF V600E.

Case Presentation

The patient, a 63 year old male, initially presented to his general practitioner ten years ago with an enlarging pigmented lesion in the centre of his back. Subsequent biopsy revealed a grade IV cutaneous melanoma with a Breslow thickness of 5mm. A wide local excision was performed, with primary closure of the wound site. Sentinel node biopsy was not carried out, and a follow-up scan six months later found no evidence of melanoma metastasis.

In mid-2010, the patient noticed a large swelling in his left axilla. A CT/ PET scan demonstrated increased fluorodeoxyglucose avidity in this area, and an axillary dissection was performed to remove a tennis ball- sized mass that was histopathologically identified wholly as melanoma. A four week course of radiotherapy was commenced, followed by six weeks of interferon therapy. However, treatment was discontinued when he developed acute abdominal pain caused by pancreatitis.

CT/PET scans were implemented every three months; in early 2011 pancreatic metastases were detected.

The tumour was tested for a mutation in BRAF, a protein in the mitogen activating protein kinase (MAPK) signaling pathway. BRAF mutations are found in approximately half of all cutaneous melanoma, and this is a target for a recently developed inhibitor, vemurafenib. [8-11] The patient’s test was negative, and he was commenced on a clinical trial of nanoparticle albumin bound (nab) paclitaxel. He completed a nine month course of nab-paclitaxel, and experienced many adverse side effects including extreme fatigue, nausea, and arthralgia. A CT/PET scan demonstrated almost complete remission of his pancreatic lesions. Despite this progress, three months after completing treatment, a follow-up CT/PET scan revealed liver metastases that were confirmed by biopsy.

In 2012 he was commenced on the novel immunotherapy agent ipilimumab, which involved a series of four infusions of 10mg/kg three weeks apart. One week after his second dose, he was admitted to hospital with a two day history of maintained high fevers reaching above 40oC, rigors, sweats, and diffuse abdominal pain. These symptoms were preceded by a week long mild coryzal illness. On investigation he had elevated liver enzymes, more than double the reference range, and his blood cultures were negative. His symptoms settled within eight days, and he was discharged after an admission of two weeks in total.

The patient remains hopeful about his future, and is optimistic about the ‘fighting chance’ that this novel therapy has presented.


The complexity of the melanoma pathogenome poses a major obstacle in developing efficacious treatments; however, the identification of novel signaling pathways and oncogenic mutations is challenging this paradigm. [12,13] The resultant development of targeted treatment strategies has clinical importance, with a number of new molecules targeting melanoma mutations and anomalies specifically. The promise of targeted treatments is evident for a number of other cancers, with agents such as trastuzumab in HER-2 positive breast cancer and imatinib in chronic myelogenous leukaemia now successfully employed as first-line options. [14,15]

This patient’s initial treatment with interferon alpha aimed to eradicate remaining micro-metastatic disease following tumour resection. While interferon-alpha has shown disease-free survival benefit, studies have failed to consistently demonstrate significant improvement in overall survival. [16-18]

Favourable outcomes in progression-free and median survival have been indicated for the taxane-based chemotherapy nab-paclitaxel that he next received; however, it has also been associated with concerning toxicity and side effect profiles. [19]

Ipilimumab is a promising development in immunotherapy for metastatic melanoma, with significant improvement in overall survival reported in two recent phase III randomised clinical trials. [20,21] This novel monoclonal antibody modulates the immune response by blocking cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), which competitively binds with B7 on antigen presenting cells to prevent secondary signaling. When ipilimumab occupies CTLA-4, the immune response is upregulated and host versus tumour activity is improved. Native and tumour-specific immune response modification has led to a profile of adverse events associated with ipilimumab that is different from those seen with conventional chemotherapy. Immune- related dermatologic, gastrointestinal, and endocrine side effects have been observed, with the most common immune specific adverse events being diarrhoea, rash, and pruritis (see Table 1). [20,21] The resulting patterns of clinical response to ipilimumab also differ from conventional therapy. Clinical improvement and favourable outcomes may manifest as disease progression prior to response, durable stable disease, development of new lesions while the original tumours abate, or a reduction of baseline tumour burden without new lesions. [22]

Recently discovered clinical markers may offer predictive insight into ipilimumab benefit and toxicity, and are a key goal in the development of personalised medicine. Pharmacodynamic effects on gene expression have been demonstrated, with baseline and post-treatment alterations in CD4+ and CD8+ T cells implicated in both likelihood of relapse and occurrence of adverse events. [23] Novel biomarkers that may be associated with a positive clinical response include immune- related tumour biomarkers at baseline and a post-therapy increase in tumour-infiltrating lymphocytes. [24]

Overall survival was reported as 10 and 11.2 months for the two phase III studies compared with 6.4 and 9.1 months in the control arms. [20,21] Furthermore, recently published data on the durability of response to ipilimumab has indicated five year survival rates of 13%, 23%, and 25% for three separate earlier trials. [25]

Somatic genetic alterations in the MAPK signaling cascade have been identified as key oncogenic mutations in melanoma, and research into independent BRAF driver mutations has resulted in the development of highly selective molecules such as vemurafenib. Vemurafenib inhibits constitutive activation of mutated BRAF V600E, thereby preventing upregulated downstream effects that lead to melanoma proliferation and survival. [26,27] A multicentre phase II trial demonstrated a median overall survival of 15.9 months, and a subsequent phase III randomised clinical trial was ended prematurely after pre-specified statistical significance criteria was attained at interim analysis. [8,9] Crossover from the control arm to vemurafenib was recommended by an independent board for all surviving patients. [8] Conversely, in patients with mutated upstream RAS and wild-type BRAF mutation status, the use of vemurafenib is unadvisable on the basis of preclinical models. For these mutations, BRAF inhibition may lead to paradoxical gain-of-function mutations within the MAPK pathway, and drive tumourigenesis rather than promoting downregulation. [13] The complexity of BRAF signaling and reactivation of the MAPK pathway is highly relevant in the development of intrinsic and acquired drug resistance to vemurafenib. Although the presence of the V600E mutation generally predicts response, acquisition of secondary mutations has resulted in short-lived treatment duration. [28]

Ipilimumab and vemurafenib, when used individually, clearly demonstrate improvements in overall survival. Following the success of these two agents, a study examining combination therapy in patients testing positive to the BRAF V600E mutation is currently underway. [29]

With the availability of new treatments for melanoma, the associated health care economics of niche market therapies need to be acknowledged. It is likely that the cost of these drugs will be high, making it difficult to subsidise in countries such as Australia where public pharmaceutical subsidies exist. Decisions about public subsidy of drugs are often made on cost-benefit analyses, which may be inadequate in expressing the real life benefits of prolonging a patient’s lifespan in the face of a disease with a dismal prognosis. Non-subsidy may lead to the availability of these medicines to only those who can afford it, and it is concerning when treatment becomes a commodity stratified by individual wealth rather than need. This problem surrounding novel treatments is only expected to increase across many fields of medicine with the torrent of medical advances to come.


This case illustrates the shift in cancer therapy for melanoma towards a model of personalised medicine, where results of genomic investigations influence treatment choices by potentially targeting specific oncogenes driving the cancer.

Conflict of interest

None declared.

Consent declaration

Informed consent was obtained from the patient for publication of this case report.


J Read:


Review Articles Articles

The influence of vitamin D on cardiovascular disease

Background: Vitamin D is essential for many biological functions in the body. Populations that are deficient in vitamin D have increased cardiovascular morbidity and mortality. Current research is controversial, and the evidence base is still developing. This review looks at the interaction between vitamin D levels and cardiovascular disease, including the major cardiovascular risk factors – diabetes, obesity, hyperlipidaemia and hypertension. Methods: A literature review was undertaken through MEDLINE / PubMED / Ovid / Springerlink / Web of Science databases. The terms, “vitamin D”, “vitamin D deficiency”, “cardiovascular risk”, “cardiovascular disease”, “structure”, “function”, “ergocalciferol”, “cholecalciferol”, “calcitriol”, “vitamin D receptors”, “1α-hydroxylase”, “diabetes”, “obesity”, “hypercholesterolaemia”, “hyperlipidaemia” and “hypertension” were used. Sixty-eight articles were selected and analysed, with preference given to studies published in English and published within recent years. Results: There is a correlation between adequate vitamin D levels and type two diabetes mellitus, but limited research to support this. Obesity, physical inactivity and elevated circulating lipids are more common in vitamin D deficiency. These relationships have not been shown to be causal. Some studies have shown an inverse correlation between hypertension and vitamin D levels, while others have shown no relationship. Conclusion: The studies analysed show there is limited evidence to suggest that cardiovascular disease may be prevented by adequate vitamin D levels. There are few well-designed studies that demonstrate the relationship between the cardiovascular risk factors – diabetes, obesity, hyperlipidaemia, hypertension, and vitamin D. Further research is needed to clarify the infl uence of vitamin D on cardiovascular disease.

What is vitamin D, and how do you get it?

Vitamin D is a group of secosteroids, derived from steroid precursors by the opening of the steroid B-ring between carbons nine and ten. Vitamin D has a cis-triene structure which is susceptible to oxidation, ultraviolet (UV) light-induced conformational changes, heat-induced conformational changes and attack by free radicals. [1,2]

Cholecalciferol, also known as vitamin D3, is a 27-carbon molecule derived from cholesterol. [2] It is available through diet and through synthesis in the skin. [1] 7-dehydrocholesterol found in skin is converted to previtamin D3 following exposure to ultraviolet B (UVB) light. Previtamin D3 is unstable and breaks down to vitamin D3. This binds to vitamin D binding protein (VDP) and is delivered to the liver and other sites of action via the circulatory system. [3,4] Vitamin D levels are regulated in the body in a number of ways. While exposure to UVB radiation causes vitamin D3 production in the skin, excessive exposure to sunlight degrades it into inactive photoproducts. [5]

Ergocalciferol, also known as vitamin D2, is a 28-carbon molecule produced by irradiation of ergosterol found in plant and fungi, which is available through diet. [2,4] Vitamin D2 and D3 (available via diet) are absorbed with fat in the gastrointestinal system into chylomicrons, which are delivered to the liver or storage sites outside the liver, such as adipose tissue. [1]

The liver converts vitamin D3 to biologically inactive 25-hydroxyvitamin D3 (calcidiol). This is converted to biologically active 1,25-dihydroxyvitamin D3 (calcitriol) under the infl uence of renal 1α-hydroxylase predominantly in the kidney. [5,6] 1α-hydroxylase is under the control of parathyroid hormone (PTH). Calcitriol is regulated by negative feedback on itself, by increasing production of 25-hydroxyvitamin D-24 hydroxylase. This enzyme catabolises calcitriol to its biologically inactive form, calcitroic acid, which is excreted in the bile and urine. Other factors such as serum phosphorus, calcium and fibroblast growth factor 23 (FGF-23) can increase or decrease production of calcitriol. Increased serum calcium levels reduce PTH, causing down-regulation of 1α-hydroxylase, reducing calcitriol, and therefore calcium levels. [5,6] A simplified diagram of the biological function of vitamin D is outlined in Figure 1.

1α-hydroxylase is the rate-limiting step in production of calcitriol. Although calcidiol is the most abundant form of vitamin D in the blood, it has minimal capacity to bind to vitamin D receptors (VDRs). 1α hydroxylation of calcidiol to calcitriol causes vitamin D to gain affinity for VDRs. [7] In recent years, 1α-hydroxylase has been found to exist at many extra-renal sites. The role of extra-renal vitamin D activation remains controversial, but may play a role in the hypothesised actions of vitamin D. [8]

VDRs are found in almost every cell in the body. Calcitriol actions occur through intracellular receptors and interaction with DNA via the classic steroid pathway. These receptors were originally thought to regulate genes responsible for regulation of serum calcium and phosphate. [1] More recently, they have been found to regulate transcription in many tissues and cells, including immune cells, bone marrow, skin, muscle and intestine. [1,9]

How does vitamin D affect cardiovascular disease?

Vitamin D deficiency has been associated with high blood pressure, risk for cardiovascular-related deaths, symptoms of depression, cognitive deficits and mortality. [10] Calcitriol inhibits renin synthesis, increases insulin production and increases myocardial contractility. [11-13] Vitamin D deficiency reduces serum calcium levels, causing an increase in PTH, which promotes atherosclerosis and cardiovascular risk. [14,15]

The majority of evidence for the role of vitamin D in cardiovascular disease (CVD) has arisen from studies involving patients with end stage renal disease. Cardiovascular mortality is ten to twenty times higher in patients undergoing dialysis. [16] In patients using dialysis, the risk of death from CVD can be reduced with vitamin D replacement. [17,18]

As kidney function deteriorates, calcitriol levels decline. [19] Reduced calcitriol production can lead to hypocalcemia, and in turn, compensatory elevated PTH. Overstimulation of the parathyroid gland eventually leads to secondary hyperparathyroidism (SHPT). [20] Patients with ESRD are thought to suffer from reduced cardiac inotropy, increased heart weight, increased myocardial collagen content, and increased vascular smooth muscle cell proliferation as a result of the vitamin D depletion. PTH excess may impair intracellular calcium metabolism of the cardiomyocyte and promotes chronic atherosclerosis. Elevated PTH may increase cardiac contractility, insulin resistance, calcium and phosphate deposition in vessel walls, chronic myocardial calcification, and chronic heart valve calcification. [14,15] In patients with SHPT, treatment advice usually consists of correction of calcitriol deficiency using calcitriol or vitamin D analogues. [6]

Mechanisms for cardiovascular risk reduction with vitamin D supplementation include the inhibition of smooth muscle proliferation, the suppression of vascular calcification, the down-regulation of inflammatory cytokines, the up-regulation of anti-inflammatory cytokines, and the negative regulation of the renin-angiotensin-aldosterone system (RAAS). [21-26] Inappropriate stimulation of the RAAS is associated with hypertension, myocardial infarction and stroke. [14] Calcitriol treatment has been shown to reduce blood pressure, renin activity and angiotensin II levels. [27] The effects of vitamin D deficiency on the cardiovascular system are outlined in Figure 2.

A systematic review and meta-analysis looked at the relationship between the naturally occurring level of vitamin D and cardiometabolic disorders including CVD, diabetes and metabolic syndrome. [28] Twenty-eight studies were selected, including nineteen crosssectional studies, three case-control studies and six cohort studies, analysing 99,745 patients. [28] High vitamin D levels were associated with a 43% reduction in cardiometabolic disorders. [28] There was a significant association between high levels of vitamin D and risk of having cardiovascular disease (33% reduction), type two diabetes (55% reduction) and metabolic syndrome (51% reduction). [28] Vitamin D supplementation has been shown to have a protective effect in limited studies of CVD, but further research is needed. [29]


The research surrounding the interaction between vitamin D supplementation and type two diabetes mellitus is controversial. To date, there have been no adequate, large and prospective, randomised controlled trials to test the efficacy of vitamin D supplementation for the prevention and treatment of type two diabetes mellitus. The current available data allows a recommendation that further research be conducted to determine whether adequate vitamin D levels may prevent the onset of type two diabetes. Type one diabetes mellitus will not be discussed in this review.

Insulin resistance has been associated with low serum vitamin D, which improved after treatment with vitamin D. [30-36] One study demonstrated a positive relationship between calcitriol and insulin sensitivity, and a negative effect of vitamin D deficiency on beta cell function. [12] These studies are limited by small sample size, subject selection and lack of randomisation. However, there was a clinical correlation and it is worthwhile investigating further the possibility of improvement in insulin sensitivity with vitamin D supplementation. Serum blood sugar levels and prevalence of type two diabetes mellitus increases with age, and vitamin D levels tend to fall with age. [37,38] Type two diabetes is associated with systemic inflammation, which may induce beta-cell dysfunction and death. [39] Several studies show that vitamin D could directly affect beta-cell growth and differentiation via modulation of systemic inflammation and the immune response. [39-42] One of these was a double-blinded 39-week follow-up study of interleukin-1 blockade with anakinra. [40] Although being limited by small sample size and limitations in subject selection, the study showed improvement in markers of systemic inflammation 39 weeks after treatment withdrawal. [40]

Several studies indicate that calcitriol regulates beta-cell function by regulating intracellular calcium levels. This is thought to influence insulin secretion, increase beta-cell resistance to apoptosis and increase beta-cell replication. Calcitriol is thought to bind to nuclear VDRs in the beta-cell to increase preproinsulin mRNA level. Research to support this hypothesis is limited, due to being conducted in rats. [39,43-45]

Obesity and hyperlipidaemia

Studies have shown that high body mass index (BMI) is associated with low serum vitamin D levels. [46] Vitamin D is fat soluble and readily stored in adipose tissue. [1,47] Sequestration of cholecalciferol in adipose tissue reduces bioavailability in obese individuals. [1,48,49] The distribution of fat may be associated with vitamin D status, but this relationship may be dependent on metabolic factors. [49]

Vigorous physical activity is a strong and modifiable contributor to vitamin D status. This may be due to sun exposure correlated with physical activity, however, a number of studies have shown the positive effect on vitamin D status may be independent of sun exposure. [50-54] Further research is needed to clarify this.

A large, prospective study of the modifiable predictors of vitamin D status was conducted using 2,621 healthy individuals aged 55-74 in the USA. [46] Predictors of low vitamin D status were found to be low dietary vitamin D intake, BMI > 30kg/m2, physical inactivity and low milk and calcium supplement intake. [46] There is an inverse relationship between apolipoprotein A-I and high density lipoprotein cholesterol with vitamin D levels in a survey of 358 Belgian people. [55] This relationship was not shown to be causal, but further research is warranted to see if vitamin D provides this cardioprotective link.

Vitamin D deficiency may increase insulin resistance and thereby increase circulating lipids, but supplementation has not been shown to improve circulating lipid levels. [56,57] Statin therapy increases the circulating levels of 7-dehydrocholesterol, leading to an increase in conversion to vitamin D (in the presence of UVB radiation), and therefore vitamin D levels. [58-61]


To date, there are few good quality randomised controlled trials looking at the relationship between vitamin D levels and blood pressure. There is weak evidence to suggest that there may be a relationship between the two, however, further research is needed to draw any conclusions that may change the management of blood pressure.

Vitamin D may regulate blood pressure via an interaction with the RAAS, which is often activated in hypertension. Calcitriol is a known negative regulator of the RAAS. [11,21] The effects of vitamin D on the suppression of renin activity may be due to increased intracellular calcium levels. [62] It is hypothesised that vitamin D regulation of renin is independent of calcium metabolism, by regulating renin mRNA production with VDRs. [11] This study was completed using a line of cells derived from transgenic mice kidney tumours. [11]

There are some studies which show an inverse correlation between vitamin D levels and blood pressure. [63-66] A meta-analysis which included eleven randomised controlled trials (small, variable methodological quality) found weak evidence to support a small effect of vitamin D on blood pressure in studies of hypertensive patients. [67] There was a small statistically significant reduction in diastolic blood pressure, and no significant reduction in systolic blood pressure in hypertensive subjects supplemented with vitamin D or UV radiation. [67]

Several studies have shown differing results when trying to establish a relationship between vitamin D intake and hypertension. [10] There are two cross-sectional studies that have been completed using the Third National Health and Nutrition Examination Survey data. One study demonstrated a significant difference in systolic blood pressure and pulse pressure between the highest and lowest quintile groups divided by vitamin D level. [10,63] Participants with hypertension were excluded from analysis. [63] Another study revealed increased systolic blood pressure with reducing levels of vitamin D, and a twenty percent reduction in systolic blood pressure in those with vitamin D levels greater than 80 nmol/L compared with those with less than 50 nmol/L. [64] Both of these studies had a good sample size, but were limited by the methods of the study. [10] A cross-sectional study using a different data set with low prevalence of vitamin D deficiency showed no association between systolic blood pressure and vitamin D level. [10,65] A different study did not show any significant relationship between vitamin D levels and blood pressure after adjusting for confounding variables, however, this may have been due to low estimated vitamin D intake. [10,68]


Vitamin D is an important molecule to consider in the pathogenesis of cardiovascular disease. Current research shows that vitamin D deficiency contributes to cardiovascular morbidity and mortality. The mechanisms proposed for this include direct actions on the heart and vasculature, as well as by increasing the risk of cardiovascular risk factors such as diabetes, obesity, hyperlipidaemia and hypertension. Further research is needed to clarify the influence of vitamin D on cardiovascular disease and its risk factors, and whether vitamin D is an efficient, cost-effective and safe intervention to prevent cardiovascular morbidity and mortality.


Dr Ruan Lakemond, for his kind assistance in proof-reading this article and technical support. Prof Rick Jackson, for his generous support and help finding a suitable topic.

Confl icts of interest

None declared.


R Lakemond:


The justice of melancholia

In a previous issue of this journal, Nguyen [1] succinctly identified a high incidence of mental health conditions in Australian medical students.

The increased rates of depression and suicidal ideations experienced by this population depict a bleak future for the medical profession in this country. Of great concern is the fact that the barriers preventing medical students from accessing support are not only unique, but despairingly fraught with immeasurable difficulty and stigmatisation; stigma that is entrenched and perpetuated through the core of the medical culture. [2] Despite our existence in an apparently enlightened and diverse cultural framework, the disconcerting stigma branded upon mental health exists and it is truly deplorable…