Beware Adult Onset CGD in Subjects with Pulmonary or Extrapulmonary Granulomas but Negative AFB & Tuberculous Culture

 Chronic Granulomatous Disease (CGD) is rare and affects 1 in 200,000 live births. While 75% have the X-linked form (and thus presenting in boys) due to a protein subunit of NADPH oxidase called gp91phox, the rest have an autosomal recessive form due to other protein subunits of the same enzyme, namely p22phox, p47phox, and p67 phox. Of these, p47phox is the second commonest and can present during adulthood occasionally. The involved gene is that of NCF-1 (Neutrophil Cytosolic Factor-1). The average age for presentation of the X-linked form is 3 years, and for the autosomal recessive forms, 8 years.

When CGD presents in adulthood, it is often confused with tuberculosis due to a pulmonary involvement (the most commonly affected organ in CGD), and presence of granulomas on biopsy. These granulomas are however, non-necrotising, and obviously without detectable AFB and yield negative cultures for M.tuberculosis. They are therefore sometimes diagnosed as pulmonary sarcoidosis, given the geographical setting and demographics.

Sputum or bronchoscopic washings growing the following 6 genus in a subject with pulmonary granuloma should lead to a suspicion of CGD- Aspergillus, Candida, Staphylococcus, Serratia, Burkholderia or Nocardia.

These patients often have hyperglobulinemia, with raised Ig levels, but this is not invariable. 

CGD can affect other areas, and thus cause abscesses or cellulitis in the skin, gingivitis (but not periodontitis, unlike leucocyte adhesion defects) Crohn's like granulomas in the intestines, and spinal abscesses. Granulomas can cause obstructive lesions in the urogenital & GI tracts. Delayed wound healing is often a notable feature.

Unlike LAD, which can also rarely present in adulthood, the neutrophil count tends to be normal between infectious episodes. Just for context, there are two forms of LAD, including LAD-1, caused by a defect of CD18, which is a part of the heterodimeric beta-integrins, and LAD-2, caused by a defect in fucosylation, and thus an absence of Sialyl Lewis-X , the latter being necessary for neutrophils to roll on endothelial cells prior to adhesion and diapedesis. Subjects with LAD tend to have severe periodontitis and perpetually high neutrophil counts. LAD-2 is associated with developmental abnormalities such as stunted growth.

The most convenient diagnostic test for CGD is flow cytometry with Dihydrorhodamine 123. Absence of fluorescent staining indicates abrogation of oxidative burst in neutrophils, which is characteristic of CGD. An alternative test is the Nitroblue tetrazolium test (NBT).

The only curative treatment is allogeneic BMT. Symptoms can be improved by treatment with gamma-interferon, and prophylactic administration of co-trimoxazole and itraconazole. Infective episodes should be treated with specific antibiotics.

Why Extensive Training May Not Help You Run Faster

Practice makes perfect. Right? Not always, it would seem.

I have often wondered why, despite running daily, I have not been able to improve my times. I would be classed as middle to long distance runner, averaging 6 miles daily during weekdays and 9.5 miles on weekends. In the spring of 2020, I thought I had hit a sweet spot, consistently running the longer weekend distance at between 73 and 75 minutes. At 54.5 years of age, that was a reasonable time.

But things went backwards in the autumn of that year, and this year I have struggled to get below 90 minutes for the 9.5 mile run.  In fact most runs have been in the mid to high 90s, with a few taking longer than 100 minutes.

Is this muscle fatigue, perhaps an inevitable consequence of the exercise addict's need to run daily? Or is it due to glycogen depletion?

The answer, it would seem lies elsewhere.

There are two broad categories of muscle fibres, the red, myoglobin rich, largely aerobic, mitochondria laden "slow" Type I fibre, and the larger, predominantly anaerobic, white, glycogen loaded "fast" Type II fibre, that predominantly use glycogen to generate its ATP. 

Type II fibres are further subdivided into Type IIA & Type IIB.

It seems that the determinant of the type of muscle fibre is the myosin heavy chain (MHC, not to be confused with Major Histcompatibility Complex) isotype. Thus Type I fibres are rich in MHC 1, type IIA in MHC 2A, and Type IIB in MHC 2X, also called MHC 2D.

You may well wonder what happened to MHC 2B and 2C? Well, MHC 2B is found in some ultrafast Type II fibres, mainly in cranial muscles, and MHC 2C is found in regenerating muscle fibres.

Biochemically, type I and type II fibres are distinguished by their ATPase staining and the speed of the sarcoplasmic reticulum (SR) in releasing calcium. Thus, Type I fibres stain for ATPase at low pH, while Type II fibres do so at an alkaline pH. Similarly Type II fibres possess SR that release Ca very quickly, while with Type I fibres, the release of Ca from SR is slower.

The distinction between Type IIA and Type IIB fibres is more than that of semantics. Type IIA has characteristics that are intermediate between Type I and Type IIB. Thus, they are versatile in being able to function as slow muscle fibres that serve sustained activity such as gaze, while, when called upon, they can also serve up the explosive speed of contraction, sustainable only for short periods, that epitomises Type IIB fibres. 

Muscle fibres can be "pure" and contain just MHC1 or 2A or 2X, but they can also be hybrid- ie. contain MHC1 & 2A, or 2A&2X, or all three, ie. MHC1, 2A &2X. 

Using immunohistochemical staining, long distance elite runners have a higher proportion of  MHC1 fibres than non-runners, roughly equivalent amounts of MHC2A, and almost no MHC2X fibres. Similarly, non-runners tend to have more hybrid MHC fibres in general than runners, which suggests that hybrid fibres can change their composition depending on the workload imposed on them.

Type I fibres are high maintenance. They tend to be smaller, have larger blood supply and higher oxygen consumption. Type II fibres are larger, but consume low oxygen, and at least the IIB fibres are relatively less used, "standing by" for the relatively less frequent occasions when explosive power is required.

A muscle fibre owes its "type" or allegiance to the alpha motor neuron that innervates it. Thus a motor unit (comprised of all the muscle fibres innervated by a particular motor neurone) will always innervate the same type of muscle fibres. Thus a motor unit can be classified as Fast twitch (F) or Slow twitch (S). The F units are further classified as Fatigue resistant (FR) or Fast fatiguing (FF). When there is denervation and cross re-enervation, the muscle fibres take on the characteristics of the "new" motor neurone. Thus S units can turn into FR or FF on cross-reinnervation and so on.

It follows from the above that with training, entire motor units change their character (as individual muscle fibres are unable to do so, being dictated by the motor unit they are part of). Thus, with prolonged training, FF units can change into FR and FR into S. Change in the other direction happens with deconditioning.

And herein, I believe lies my predicament. I believe that my FF units have slowly transitioned into FR and FR units into S units. Glycogen depletion would not be germane to this, as it would only affect the FF motor units, which are comprised principally of Type IIB muscle fibres. While the S units and FR units are fatigue resistant and ideal for long distance running, the muscle fibres they innervate are smaller, and lack the speed of FF units.

Not All Inherited Neurological Disorders are Untreatable

Hereditary Sensory and Autonomic Neuropathy Type 1 responds to large doses of L-serine. It is the only member of the HSAN group that has an effective pharmacologic treatment.

There are a number of inherited and metabolic disorders that respond to large doses of vitamins. These mostly present in adolescents and young adults but can occasionally present later in life.

Thus, a group of 4 disorders respond to large doses of riboflavin. These include 1. Multiple acyl CoA dehydrogenase deficiency, caused by a deficiency of one of 3 enzymes- Electron Transfer Flavoprotein A or B, and ETF dehydrogenase, also called ETF ubiquinone oxidoreductase, responsible for fully 97% of MADD cases. 2. Riboflavin Responsive Exercise Intolerance, caused by a deficiency of mitochondrial FAD transporter, 3. Lipid Storage Myopathy due to deficiency of FAD synthase, and 4. Acyl CoA dehydrogenase 9, or ACAD9 deficiency. All four present with muscle pain, exercise intolerance and a rise in plasma acylcarnitine level of all chain lengths. Episodes of stress associated rhabdomyolysis may occur.

There are two other riboflavin associated disorders: Riboflavin transporter deficiency (RTD) presents as a motor neuronopathy with bulbar involvement, mimicking MND, but can also cause optic and auditory neuropathy. CMTX4 is due to mutations in Apoptosis inducing factor mitochondria associated 1 gene and presents with the classic Charcot Marie Tooth phenotype of hereditary sensorimotor neuropathy. areflexia, pes cavus and additionally auditory neuropathy. Both RTD and CMTX4 show a delayed response to riboflavin supplementation.

Similarly, a number of inherited disorders respond to thiamine. Biotin-thiamine responsive encephalopathy (BTRE)  presents with Leigh syndrome- encephalopathy with T2 changes on MRI in basal ganglia, as does E1 alpha pyruvate dehydrogenase deficiency. In the latter, serum and CSF pyruvate and lactate are elevated in equal proportions.

Biotinidase deficiency presents with a neuromyelitic optica phenotype, comprising bilateral visual loss in combination with longitudinally extensive myelopathy. The disorder responds to oral biotin, which should be started ASAP.

Vitamin B12 deficiency and folate deficiency are probably the most frequently diagnosed vitamin deficiencies in clinical practice. They tend to present with similar phenotypes- gait difficulties, peripheral neuropathy, posterior column involvement and psychosis or dementia, with or without macrocytic anaemia.

It is not surprising therefore that inherited defects of B12 or folate pathways would present with similar manifestations. This can occur at any level from defects of absorption from the gut to deficiency of enzymes that produce the active forms of these vitamins. Thus, in Immerslund-Grasbeck syndrome, mutations in the small bowel mucosal transporter called Cubam, leads to megaloblastic anaemia and neurologic manifestations. Cubam consists of 2 subunits- Cubilin, which recognises the B12-IF complex, and Amnionless, which endocytoses them. Thus, defects in either of the putative genes CUB or AMN can lead to IGS.

B12 serves as a co-factor for 2 important reactions in the cell. Firstly, within the mitochondrion, as S-adenosyl cobalamin, it serves as a cofactor to convert methyl-malonyl Co-A to succinyl Co-A, catalysed by methyl-malonyl Co-A mutase. Succinyl Co-A then enters the TCA cycle. 

In the cytosol, B12, as Methyl cobalamin, is a methyl donor for the conversion of homocysteine to methionine, catalysed by methionine synthase. Methyl cobalamin is regenerated by donation of a methyl group from 5-methyl tetrahydrofolate, which latter itself becomes demethylated to tetrahydrofolate. THF spontaneously converts to 5,10 methylene tetrahydrofolate, which is then reduced by 5,10 methylene THF reductase to the active methyl donor 5-nethyl THF, thus completing the cycle.

Folate is also a cofactor in the latter reaction, but not the former. Thus both B12 and folate deficiency leads to a rise in serum homocysteine, but only B12 deficiency elevates methyl-malonic acid levels.

The B12/folate pathways overlap with the active form- pyridoxal phosphate (PLP)- of another vitamin- Pyridoxine, or Vitamin B6. Thus PLP is a cofactor for the enzyme cystathionine-beta synthase, which converts homocysteine into cystathionine, and eventually into cysteine. Deficiency of this enzyme leads to the hereditary condition homocystinuria, which has some features of Marfan syndrome. In homocystinuria, serum homocysteine levels and methionine levels are both elevated, unlike in deficiencies of the B12-folate pathway, where homocysteine is elevated but methionine levels are low.

Pyridoxal phosphate activity can also be disrupted by inborn defects of two other amino acid pathways- lysine and proline. In the first instance, deficiency of alpha-amino adipic acid delta-semialdehyde dehydrogenase results in conversion of that lysine metabolite to 6-piperideine carboxylate (P6C). In the second case, a condition called Hyperprolinemia Type II results from the deficiency of 5-Pyrroline carboxylate (P5C) dehydrogenase, leading to elevated levels of  P5C. P6C and P5C are both inhibitors of pyridoxal phosphate and thus can again lead to hyper-homocysteinemia.

Disorders of Vitamin E metabolism can lead to two ataxic disorders- Abetalipoproteinemia and Ataxia due to Vitamin E deficiency. Both conditions present with sensory neuronopathy. The former also features retinopathy while the latter features a tremor of the head. The former is due to deficiency of Microsomal triglyceride transfer protein (MTTP) while the latter is due to the deficiency of alpha-tocopherol transfer protein (TTPA) caused by involvement of the putative genes. These two disorders should be considered for all cases of chronic cerebellar ataxia or sensory neuronopathy, which latter presents as non-length dependent sensory neuropathy affecting thighs, arms, etc.

Usefulness of IgG Avidity to Diagnose Recent CMV Infection

 Primary CMV infection acquired during pregnancy is associated with a risk of foetal anomalies such as visual loss, sensorineural deafness, and cognitive impairment. Primary CMV infection is often asymptomatic, hence many women may not know they have been infected. Such infections are particularly common in women working with children such as primary school teachers and day care nurseries.

The prevalence of CMV infections rises with age. Subjects can be re-infected with a different strain or have re-activation of the original CMV strain- CMV is after all a herpesvirus and therefore establishes latency, just like EBV or HZV. It is important to clarify here that the risk of foetal anomalies only arises for primary CMV infections acquired during pregnancy rather than re-infections or re-activation of the virus. CMV infection picked up even shortly before pregnancy do not affect the foetus.

Why should this be so? It is thought that existing antibodies to CMV following a previous infection offer protection against the virus being passed on to the foetus. Thus, longstanding maternal antibodies against CMV bind the viral antigens with a high avidity and protect the foetus.

However, this does not apply for primary infections acquired during pregnancy. Antibodies formed following a recent infection initially bind the viral antigens with low avidity for the first 3-4 months. After this period, due to a process of B-cells with higher antigen specificity being positively selected, a process called affinity maturation, the avidity of CMV antibodies for the virus increases.

This differential avidity is used as an index (Avidity Index or AI) for the recency of CMV infection. Thus low avidity of IgG antibodies for a CMV viral lysate indicates a primary CMV infection within the last 3-4 months, while high avidity indicates one of 3 possibilities- either that the primary infection occurred more than 6 months ago, or that this was a re-activation of a previous primary infection, or that this is a new infection with a different strain of CMV.

The reason this metric is used to gauge the recency of CMV infection is because of the uselessness of IgM antibodies to CMV as a marker of recent infection. Not only can anti-CMV IgM be formed following other viral infections (cross-reactivity), but the IgM isotype can persist for several months after CMV infection, and as such would be of no value in determining if the infection picked up by a woman was acquired during pregnancy or before pregnancy. Thus, isolated IgM antibodies to CMV are of no diagnostic value.

When IgM antibodies to CMV co-exist with the IgG isotype however, the possibility of recent infection cannot be ruled out. It is in these instances that avidity of IgG is utilised. Low avidity (AI <50) is taken as an indication of primary infection within the last 4 months, while high avidity (AI >60) is compatible with infections> 6 months ago. Intermediate values of 50-60 can be seen with primary infections that happened 4-6 months ago.

High avidity tests obtained in the 1st trimester  are reassuring. However when the IgG antibodies picked up in 2nd or 3rd trimester display high avidity for the viral lysate, it is not possible to rule out the possibility that the primary CMV infection occurred within the 1st trimester. In such cases, amniotic fluid may have to be obtained and tested for CMV DNA.

Although the IgG avidity test is used mainly in pregnant women, there is no reason it cannot be used in other populations. An example of this would be a sexually active adolescent with unexplained transaminitis, perhaps following an episode of fever, who has tested positive for both IgG and IgM antibodies against CMV. A low avidity test makes it highly likely that the deranged LFTs were due to primary CMV infection.

A 41 year old male with a rapid heart rate

 How would you treat this?

a. If the patient was haemodynamically stable?
b. If he was haemodynamically unstable?

From the American College of Cardiology Archives.

S-Gene Target Failure is Proxy for VOC 202012/01 COVID-19 strain

 The new COVID-19 variant, designated "Variant of Concern" or VOC 202012/01 by Public Health England, now accounts for the majority of transmission in the UK. The strain is characterised by the fact that the PCR used in most places in the UK- TaqPath by Thermo Fisher, has a dropout where the S gene should be. This is called S-Gene Target Failure, or simply SGTF.

The new variant has 23 mutations- 13 non-synonymous mutations, 4 deletions and  6 synonymous mutations- compared with the wild type variant, and one extra stop codon. Most of these mutations are in the gene for Spike protein, as shown below from the relevant PHE document.. One of these mutations, present at the N-terminal domain of the S-protein, is a deletion of 6 nucleotides- from 21765-21770, resulting in the loss of two amino acids- Histidine and Valine at position 69-70 of the Spike protein. It is the latter deletion that causes the characteristic SGTF on the TaqPath PCR. 

(Synonymous mutations lead to base pair substitutions which leave the amino acid unchanged, while non-synonymous mutations alter the coded amino acid)

TaqPath uses a 3-target PCR- it amplifies the S-gene, the N-gene (called N) and and Open Reading Frame called Orf1ab. COVID-19 has a further Orf called Orf8, which is not a target for TaqPath and therefore not amplified. (Open Reading Frames are DNA sequences without stop codons. When they are long, they are likely to code for protein, rather than being non-coding sequences).

For the VOC, the PCR cannot read the S-gene sequence due to deletion at 69-70, and so the S-gene "drops out", resulting in S-gene target failure. Only the other 2 targets, i.e. N and Orf1ab are amplified. 

Fully 99.7% of variants sequenced in the UK with SGTF belong to the new VOC strain. SGTF is thus used as a reliable proxy for the new strain.

However, as referred to elsewhere in this blog, the increased transmissibility of the new strain- between 40%-70% more than the wild type strain- is owed to another mutation in the Spike protein, namely N501Y, which affects the RBD, and is thought to increase the strength with which the S-protein binds to its cognate receptor on human cells- ACE2. 

Incidentally, the 69-70 deletion was seen in the Danish Cluster 5 variant, the one that led to a cull of millions of minks. Its clinical significance, unlike that of N501Y, is unknown.