Follow-up brain imaging of 37 children with congenital Zika syndrome: case series studyBMJ 2017; 359 doi: https://doi.org/10.1136/bmj.j4188 (Published 13 October 2017) Cite this as: BMJ 2017;359:j4188
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We congratulate Natacha Petribu and colleagues for their paper reporting the follow-up study of brain CT scan imaging among 37 children with congenital Zika syndrome (CZS) . In this case series study, the authors highlight that the cerebral calcifications presented on the initial scan have diminished in number, size, density or in combination in 92% of the children, were no longer visible in 1 child, and remained unchanged in 2 children over a one-year follow-up study period.
Interestingly, the observed changes concerned predominantly the calcifications found at the cortical-white matter junction, while the pattern of calcifications previously observed in basal ganglia and cerebellum remains unchanged. This was particularly obvious for the cortical-white matter junction calcifications observed in the parietal and occipital lobes. Curiously, for this pattern, there were 2 children for whom the calcifications in the frontal and temporal lobes occurred secondarily . These new data raise questions about the pathogenesis of cerebral calcifications as a hallmark for diagnosis of CZS, as well as their prognostic significance.
Based on current knowledge, the authors advance that CZS-related brain calcinosis may be the result of an apoptotic process followed by microglial reaction, and not an exudative mechanism involving neuroinflammation after breakdown of the blood-brain barrier (BBB). In addition, they propose for the most severe calcifications accompanied by hydrocephalus, the possibility that chronic venous hypertension contribute to calcinosis and progression to hydrocephalus .
We were intrigued both by these observations and hypotheses and wish to discuss here some likely mechanisms based on genetic conditions presenting inherited brain deposits of calcium.
Congenital calcifications were previously known to belong to three leading aetiology groups including autosomal dominant diseases (Stürge-Weber syndrome, tuberous sclerosis, neurofibromatosis), autosomal recessive syndromes (Gorlin, Cockayne) and intracranial lipoma, TORCH-type infections, and calcifications related with hormonal and metabolic disorders .
Despite upregulation of GNAQ, TSC1 or BSL2 genes, or downregulation of FOS gene, in neural progenitor cells [3,4], phacomatoses and intracranial lipoma seem to be ruled out in the pathogenesis of CZS-related brain calcinosis given different pattern and location of calcifications.
Notwithstanding negative serologic testing (i.e. all the children of the case series were negative for syphilis, toxoplasmosis, rubella, cytomegalovirus and herpesvirus), the authors could have ruled out other TORCH-type infections merely for the same reason, rather than dismissing breakdown of the BBB as possible explanation for CZS-related brain calcinosis. Despite absence of major neuroinflammation in the brain of children with CZS, we believe that the assertion excluding the role of peripheral inflammatory cells is somewhat misleading. Indeed, presence of inflammation at the blood-retina barrier , evidences of BBB leakage or basolateral release of Zika virus (ZIKV) from human brain microvascular endothelial cells in mouse models [6,7], and possible role of blood monocytes as vehicle for ZIKV  should not rule out BBB breakdown as another plausible mechanistic hypothesis for CZS-related brain calcinosis, even and especially, in the absence of marked neuroinflammation.
Primary familial brain calcifications (PFBC), also known as idiopathic basal ganglia calcifications (IBGC) or Fahr disease , or calcifications secondary due to hormonal disorders, such as pseudohypoparathyroidism (another genetic disease), suggest that basal ganglia and cerebellum are two of the brain regions the most vulnerable to calcinosis [10,11]. In this context, calcinosis has been linked to four pathomechanisms: inorganic phosphate transporter defect, dysregulation of pericyte homeostasis and BBB regulation, resistance to parathyroid hormone, and difficulties or impossibility to maintain the integrity of the BBB . Importantly, among the four causative genes hitherto reported in PFBC/IBGC, PDGFRB, a gene involved in maintaining the integrity of the BBB  was found upregulated in ZIKV-infected neural progenitor cells . Together with PDGFB, a gene involved in pericyte homeostasis and BBB regulation , this gene could explain some of the persistent calcifications observed in the follow-up of CT scan imaging of children with CZS through a disruption of BBB integrity. In addition, XPR1, a gene driving the transport of inorganic phosphate  was also found upregulated in the same ZIKV infection cellular model , adding a possible role of the co-repression of calcinosis inhibitors, like phosphatase alkaline, as a complementary mechanism for less efficient calcium clearance .
Persistent calcifications of basal ganglia and the subcortical white matter could also be found in ZIKV-induced Aicardi-Goutieres-like syndromes, an encephalopathy of post-natal onset that might be triggered by the downregulation of TREX1, RNASEH2A, or RNASEH2C genes, owed to the infection [3,4]. Coarse (band-like) calcifications could be observed in the setting of the microangiopathy secondary to ZIKV-induced pseudo-TORCH-like syndromes. This calcinosis process could involve downregulation of OCLN or JAM3 by the virus [3,4], two genes known to encode endothelial cell adhesion proteins aimed at maintaining the tight junctions responsible for the BBB integrity . Last, prominence CZS-related brain calcinosis at the cortical-white matter junction, a richly vascularized area with a coiling architecture, should evoke the process of vascular calcinosis, which implies an imbalance during embryo life between apoptosis and osteogenic transformation of vascular smooth muscle cells, and their development under the control of the NOTCH signalling pathway , strongly repressed in ZIKV-infected neural progenitor cells .
In conclusion, we welcome the follow-up of CT scan imaging of children with CZS and emphasise the possibility of gene-driven processes explaining the cerebral calcifications. Next step is to link symptoms to the calcification patterns, to see whether these could explain the clinical phenotypes or indicate persistent infection, as for hypertonia and impaired neurodevelopment associated with basal ganglia calcifications in a case of CZS with long-lasting viremia .
João Ricardo Mendes de Oliveira 1,2*, Roberta R Lemos 2, Wilmer Ernesto Villamil-Gomez 3, Alfonso Javier Rodriguez-Morales 4, Patrick Gérardin 5
1 Neuropsychiatric department, Federal University of Pernambuco (UFPE), Recife, Brazil;
2 Keizo Asami Laboratory, Federal University of Pernambuco (UFPE), Recife, Brazil;
3 Infectious Diseases and Infection Control Research Group, Hospital Universitario de Sincelejo, Sincelejo, Sucre, Colombia; Programa del Doctorado de Medicina Tropical, SUE Caribe, Universidad del Atlántico, Barranquilla, Colombia;
4 Public Health and Infection Group of Research, Faculty of Health Sciences, Universidad Tecnológica de Pereira, Pereira, Risaralda, Colombia;
5 Pôle Femme Mère Enfant, Centre d'Investigation Clinique (INSERM CIC1410), UM 134 PIMIT "Processus Infectieux en Milieu Insulaire Tropical" (Université de La Réunion, CNRS 9192, INSERM U 1187, IRD 249), CHU Réunion, Saint Pierre, Reunion.
* Correspondence to: Prof Joao Ricardo Mendes de Oliveira, Neuropsychiatric department, Federal University of Pernambuco (UFPE), Av. Prof. Moraes Rego, 1235 - Cidade Universitária, Recife, Pernambuco, Brazil - CEP: 50670-901; E-mail : email@example.com
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Competing interests: No competing interests
Dear Josephine C. Weaver:
Thank you very much for your comments.
At first, we tried to quantify calcium using existing software that evaluates coronary arteries, as you mentioned, but they are not optimized to quantify calcifications so dispersed throughout the brain and it was not feasible. In view of easily perceivable tomographic changes, even by non-radiologists (as can be seen in figure 2) we decided to share this information with the scientific community to alert that the absence of calcification should not exclude the late diagnosis of congenital Zika syndrome. However, we continue to seek an appropriate means for the accurate quantification of calcium so scattered in the brain.
Competing interests: No competing interests
Firstly I would like to thank the authors for writing this most interesting article on an this issue which I’m sure will continue to problematic in the international medical community for many years to come.
My main critique is regarding the lack of a subjective quantification of cerebral calcifications. The authors state that they are unaware of any specific software and tried to minimise bias by consensus. I think consensus of 4 experts is likely to be less accurate than the commonly used Agaston Score. This is a standardised scoring system for quantifying calcification using the open access software ImageJ . Calcification has also been quantified in other studies using custom-made programs such as MATLAB . Furthermore, programs designed primarily for 2D image interpretation of histological specimens could also be used similarly for radiological quantification. Given that this is the focus of the article’s investigation it might be helpful to offer a quantitative analysis rather than an subjective assertion of “diminished in number, size, or density or a combination … were no longer present in one child, and remained unchanged in two children. No child had an increase in number, size, or density of calcifications”.
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Competing interests: No competing interests