Numeracy is a basic life skill so fundamental that we get our first introduction to numbers before going to primary school. Yet, as the saying goes, biologists don't do math. At least in this respect, clinical microbiologists and infectious disease physicians seem to fit the mould, their preoccupation being with the qualitative aspects of infection: the names of the disease causing agents (nouns) and their consequences (verbs).

But scratch the surface and you find a discipline teeming with numeric detail from the most basic statistical tests used to appreciate the strength of potential connections, through quantitative assessments of biological burdens to the sophisticated analyses that underly the burgeoning discipline of bioinformatics. Chemistry and physics have long exploited the boundaries of computational mathematics. It seems that biology is the waking giant, stretching its limbs before really learning how to play with numbers.

The problem with zero

Modern languages have an interesting variety of number conventions. Most are based on a decimal system, though there are often remnants of older, imperial currency systems such as the duodecimal. Much of our current numeracy leans on the infusion of the Arabic system, including the number zero. Zero, nothing, null is a difficult idea in the language of infection. There is far more interest in zero infection and particularly in the demonstration that zero infection is present, than is many other aspects of the biology of infection. You might say that the general view is a naive attachment to zero tolerance of a phenomenon that is widely misunderstood. But that is being a little harsh. This simple view of infection, and the presumed presence of microscopic life that is behind the infection process is actually a simple binary view: present or absent. Zero or One. In the popular imagination, there is no such thing as half an infection, let alone one tenth or one hundredth of an infection. Ironically, there is a whiff of science behind this simplification since the majority of single cell organisms multiply by division and thus increase on a binary scale; one becomes two, two become four, four become eight and so on. The potential for logarithmic expansion is wrapped up in their genes, and restricted only by external limits such as exhaustion of nutrients or attack by toxic substances like antibiotics.

The myth of one

Most microscopic life capable of causing the processes we collectively label 'infection' is conveniently described as unicellular i.e. having a single cell. The descriptive biology on which much of the classification of bacteria, yeasts and protozoa has been based relies on the single-cell concept. It is a useful idea, but has limited practical use. After all, what is a single staphylococcus? Infection with almost all priobes requires more than a single cell. The infective dose is usually multiple and often follows a threshold phenomenon. Agreed; some agents of infection are so potent that they can cause infection after encounter with a very small dose indeed. The single unit is also problematic because of the cell cycle in which each unit is either recovering from, or preparing for cell division. Maybe there are some forms of microscopic life that go through prolonged resting periods such as sessile bacteria in survival mode, but note that these are usually found in aggregates or communities that resemble the cellular consortia described as tissues in multicellular organisms. The single unit is even harder to nail when you try to count microscopic life. Bacteria can be dispersed in suspension and cultivated on solid media which are then incubated in the right conditions to generate a colony count (colony forming units per mL or CFL/mL), but when a range of different methods are used to measure the number of bacteria present in a suspension, they very rarely tally due to microaggregates, cells at varying stages of division and cell death. 

Making it count

The emergence of quantitative microbiology over the last two decades has caught some by surprise. Initially quantitative appreciation of bacterial load was the preserve of public health laboratories working to arbitrary safety standards for risk-prone food and drink such as seafood, dairy products and drinking water. Techniques such as the time-consuming most probable number method were used to determine the load of indicator organisms. These have been replaced by automated or semi-automated bacterial and viral counting methods. More recently a plethora of cell biology methods have come on line and have been quickly adapted for microbial counting tasks, though regulatory standards linked to traditional methods often slow down the adoption of promising new methods of quantitation. The other area in which quantitation has opened new analytic horizons is nucleic acid amplification and expression analysis. Quantitative PCR, in particular, provides rapid numeric insight into a huge range of clinically significant organisms. But there is a catch: PCR assays will only measure the known target i.e. dumb DNA (or if you hanker after certain viruses, dumb RNA). They do not necessarily measure the number of intact, living, metabolising and replicating cells. That requires at least some conventional, culture-based effort. From a contemporary position, the future for quantitative molecular and cellular microbiology is promising but has yet to show its full potential.

Another dimension

One the face of it, numbers allow us to take a measure of how many units of a given life form are present. They also give us a useful set of tools for other kinds of measurement. Size is probably one of the most significant since it determines what can and cannot be seen with the naked eye, and therefore what is by definition a microscopic form of life. Size has a bearing on what does and does not cause infection. Lower respiratory infections are usually caused by infective agents in a narrow size range capable of inhalation. Too large and they hit the back of the throat due to momentum during an inward breath. Too small and they are unlikely to contain any intact infective agent. There is more to this than a mere measurement of length, breadth and depth. These measurements combine to give volume, and with a measure of mass result in the derivative measure: density. Buoyant density, for instance, is likely to affect how long breathable particles can remain suspended in air. In the case of viruses, their small size dictates that they must be viewed with an electron microscope, whereas the larger size of typical bacteria and fungi allows viewing under a light microscope. The measured size of protozoa and helminths is of use in their identification. Time is another dimension capable of measurement. A binary pattern of growth results in many microscopic life forms going through a period of logarithmic growth in which a key measurement is the doubling time; a figure that is reproducible for a given species in controlled growth conditions. E.coli has a doubling time of only a few minutes, while Mycobacterium leprae takes days to double its number. Viruses can be very fast, and this is reflected in the speed of onset of acute viral infections. Another measure of microbial time is their so-called genetic clock; the amount of genetic variation accumulated by their genomes since their presumed species origin.  Although this concept can be a source of heated argument due to the assumptions and uncertainties on which the concept is based, it is a useful working hypothesis for developing ideas on the chronological time scale over which microbial life has developed.  

Getting your ducks in a row

Time has another numeric significance. It is one of the key forms of setting priorities. What came first, second, third and so on until the last. Lining up a series of observations or events in temporal sequence is a way to impose a sense of order. It gives meaning to those events. An early example of combining bacterial count and time sequence in order to investigate a complex causation was a study we performed on how bacteria get from the stomach into the lungs of mechanically ventilated intensive care patients. Temporal priority or getting in first is so important in our culture, that we often give pre-eminence to the first. First has champion status. Second is the first among losers, at least in competitive sports. But in a cascade of biological events, the first one is significant because it is the beginning and this idea lies close to the heart of the priobe concept, which regards the minute infective agent as the principal priming factor in the pathway or process that leads to what we eventually recognise as an infectious disease. In this we have to be a bit careful to avoid the post hoc ergo proper hoc error of logic in which we argue that just because something happened first, it must be the cause of a subsequent event. Thus the fact that my grandfather was a good fly fisherman does not necessarily explain why I know how to barrel cast with a fly rod. It might have been due to lessons given me by my grandmother, my father or a family friend. So, returning to lining things up in proper order, there are obviously other aspects of prioritisation such as ordering by size, mass, or as clearly happens in biology, according to order of name under the Linnean classification. Whatever feature has been used to list items in order, they can be given a number which is called an ordinal. Some of these are given above (first, second etc). It has been a source of grief in recent years that news announcers and other professional media communicators have chosen to drop the use of ordinals from dates, either to dumb down their delivery or to make it sound more decisive. Biologists have avoided this silliness if for no other reason than getting the credit for a discovery requires publishing first in order to establish priority. Far more important than that, on grounds of the number of clinical cases and thus burden of disease is the trio of malaria, tuberculosis and HIV/AIDS. These are priority diseases. The numbers say it all.

Clinical reality

And so to the pointy end of the numbers - patients with infection. The numbers come into heavy use for the analysis of infection data. Epidemiology has its origins in the study of epidemics, and continues to demonstrate new methods of numeric analysis in the investigation of emerging infections. In the clinical microbiology laboratory, work is still largely based on a series of qualitative value judgements. Look closely, though, and you will find pockets of quantitative biology (biometrics). Cell counts are conducted on sterile fluid samples, bacterial counts are performed on urine samples for which an interpretive threshold has been described, viral load measurements are made in viral hepatitis and HIV/AIDS. In the molecular microbiology lab quantitative PCR assays are performed and their numeric output used for result determination. In some molecular epidemiology methods, amplified fragment size is measured to determine the specific genotype, and antibiotic susceptibility determination relies heavily on quantitative or semi-quantitative methods. Numbers are necessary, particularly where consistency of results are important. Measurement and an orderly sequence of events play an increasing role in the clinical microbiology laboratory in future, as it already does in other pathology disciplines such as haematology and clinical chemistry.

9th January, 2011.

Verbos; the doing words

The commonest forms of greeting are short questions; opening gambits like ‘How are you?’ These are often followed by equally short questions, one of the commonest of which is ‘and what do you do?’  Our contemporary culture defines us according to what we do. Our identity and for some, their meaning in life, is based on occupation: doctor, pathologist, teacher, researcher. And what do you do?

Doing words

This question can be usefully applied to microorganisms. What can each one of them do?  The words used to speak of their actions are the classic doing words; verbs to the grammarian and linguist. Along with substantives (nouns, naming words) verbs lie at the heart of the most basic of sentences. They are so critical to the language we use that some sentences have a single verb as their only word. But before looking at the red-blooded action verbs, there are some basic verbs that hardly get noticed because they are there in everyday us; camouflaged by the mundane functions they serve. These verbs are used to refer to a state of being or having and usually only have a subject. Sounds a little complicated, but that’s because these action words fly under the radar. They include to be, to exist, to survive, to grow, to divide, and to die. The bacteria responsible for tuberculosis; Mycobacterium tuberculosis, therefore survives in phlegm.  It grows in laboratory culture, it divides in order to replicate and it dies when exposed to antimicrobial therapy. Viruses do not survive for long outside a suitable cellular host, in which they replicate and after treatment with antiviral therapy they die.

Intransigent intransitives

Verbs with a subject (the microorganism) but no object are sometimes called “intransitive”, to distinguish them from the vigorous actions packed into transitive verbs that have both subject (microorganism) and object (e.g. victim).  Examples of these red-blooded action words are to colonise, to invade, to infect, to transmit, to inoculate, to cause, to harm and to kill. So Plasmodium falciparum, the protozoan that causes malaria invades red blood cells and an Anopheles mosquito transmits the infective stage of the parasite, it harms many infected people every year and kills some of them. 

Speaking in code

There is something sinister about a microorganism that lurks in the shadows of human consciousness, hidden from view by virtue of its small size, waiting for an opportunity to pounce.  Perhaps this is why some of the early words for outbreaks of infectious disease were based on words that indicated a sudden strike. ‘Plague’ is one such word that carries a great burden of fear and loathing. The machinery that drives what these microorganisms do or are capable of doing is their genetic code. Bacteria, fungi, helminths (worms) and protozoa are all smart enough to have both main types of nucleic acid; DNA and RNA. Its organisation differs between and to a lesser extent among these different categories of microscopic life. But the essential idea common to all of them is that their full potential is wrapped up in a series of genetic letters, words, phrases and sentences in their DNA which gets converted first into RNA and then into proteins. Viruses are arguably even smarter and more efficient, it a little lazy. They have only one type of nucleic acid; either DNA or RNA, and rely on a range of mechanisms to fill the gaps left by lacking either RNA or DNA. These efficiency savings are made at the expense of the host cell they infect in order to continue living the life to which they have become accustomed.

The soul of the microbe

The reason this is all so important to the actions of microorganisms is that without DNA, RNA and proteins action there is no life. No verb ‘to be’. Nor ‘to survive’, let alone grow, divide or any of those vicious transitive verbs concerned with invasion, infection, harm and death. Note that this molecular machinery determines the action of the microorganism in question. DNA holds the plan and must be copied from generation to generation with a high degree of accuracy to guarantee continued survival and operation of the downstream molecular machinery. Changes obviously arise, either due to accidental reading errors, or the loss and gain of DNA from another source as these living things go about their daily business. Sometimes damage occurs to the DNA and it either reduces the microbe’s fitness for sustained survival, or it may alter its ability to adapt to changes in the external environment. DNA (and for the RNA virus, the RNA) contains the will or intent of the beast. It is about as close as we can get to the soul of the microorganism. By itself, DNA is still not enough to result in action, even in independent biological existence. There is still a need for conversion of the code locked away in strands of DNA into bite-sized chunks of readable message. This is done by the production of messenger RNA, which acts as a molecular go-between. Its message is read in the ribosome by ribosomal RNA, which converts genetic code into peptides and ultimately proteins. The generation of mRNA is the highest point upstream in the entire process that can be used as molecular evidence for life. Anything less (e.g. the presence of microbial DNA) is not proof of action, even at the intransitive level. 

Viral efficiency

This all applies to a greater or lesser extent to bacterial, fungi and parasites, which have both DNA and RNA. Viruses are that little bit more complicated on account of their smaller size, the smaller size of their genetic code and the specific type of interaction with their host cell. DNA viruses are generally on the bigger size as viruses go, and dine out on the host cell’s account, making use of their RNA to get the job of growth and replication done. RNA viruses, on the other hand, have to go into reverse gear and make a mirror image of their RNA template before they can crank the molecular machinery into action. Though this sounds a roundabout way of doing their stuff, it is so efficient that the RNA viruses are generally the most efficient at cellular infection, replication and subsequent transmission. The most successful are so efficient that they are responsible for the single largest infectious disease event to have affected the planet’s human population; the influenza pandemic of 1918-1919. Viruses add another twist to our consideration of action words in the language of infection. Their need to pursue a life as compulsory prisoners of the cells of more complex living things means that they are obligate invaders. They known their transitive verbs, demonstrating cellular, invasion, infection, subversion, harm and death, and appear to have a smaller repertoire of verbs of state. Yes, they replicate prodigiously, but being, existence and survival are of lesser concern to these lean and mean molecular machines.

Action by numbers

The verbs are so important to language that they are able to pack a lot more meaning by quite simple variations such as the number of subjects performing the action, the relative time it happens, whether the microorganism was caught up in the action and if there was an element of doubt or ambiguity. The number of bacteria (e.g. Staphylococcus aureus) that infect a surgical wound is likely to be a lot more than one solitary cell, but by convention we usually speak of monobacterial infection in the singular, and polymicrobial infections (e.g. enteric Gram negative bacilli such as E.coli and anaerobic bacteria such as Bacteroides fragilis) in terms of ‘they infect’ - plural. However, bacteria invade, harm or kill, while a single bacterium usually lacks the punch to do so. If it did, it invades, harms or kills, but usually just survives or dies. The one person normally left out of the language of infection is the first person (singular and plural i.e. ‘I’ and ‘we’), because we generally don’t leave microorganisms to speak for themselves.

A sense of occasion

That brings us onto the interesting question of relative time, revealed in language of infection through a variation in the action word called tense. The tense of a verb has nothing to do with pressure or tension. It is the way the action word changes to show that we’re talking about actions in the past, present or future. So this would look like the influenza virus caused millions of deaths during the 1918-1919 pandemic, it still causes annual epidemics today, and will infect many more people in future. There are further subtleties to the tense options open to us, particularly in the past with past historic, pluperfect and imperfect. Ross River virus was discovered in Northern Queensland in 1959, where it had probably been the cause of infection for some time. Other arbovirus infections were also a problem in the area and continue to cause periodic outbreaks to this day.

Turning the tables

A variation on the action words of infection is the passive voice, which we use to put the emphasis on the object of the action. This form is now under threat because the men of science prefer direct language, seeking the passive as a bit round about. Still, it has its use to show cause and effect relationships: the influenza pandemic of 2009 was caused by the H1N1/09 variant of the influenza virus. The passive turns the focus of the verb around, which is necessary if you need to indicate that a patient is the victim of an external biological agent. It is equally useful when you want to claim success for the antibiotic you prescribed: the S. aureus septicaemia was cured by a course of intravenous flucloxacillin [prescribed by me, of course].   

Terms & conditions

Action words can be very logical, revealing the use of a series of deductive steps that link one series of events to their predicted consequences. The tenses used for this are known as the conditional. We use this form of the verb to show our workings: if you need to confirm the S. aureus bacteraemia, you could collect a set of cultures. The workings of microorganisms are also subject to conditional actions: if S. aureus spreads via the blood stream, it could cause endocarditis. This can have a further sense of relative time added to provide a much richer and more specific sense of when these conditional events could occur: if S. aureus had spread via the bloodstream, it could have caused endocarditis (past conditional), or (future conditional) if S. aureus is going to spread via the bloodstream, it will have caused endocarditis by the time we get all the laboratory results back. Beyond conditional lies the murky world of the subjunctive, a subject many have managed to avoid encountering through secondary school and even university.  The subjunctive is used to give a sense of doubt, ambiguity or uncertainty that goes beyond the logical links of the conditional. Staying on the familiar ground of invasive staphylococcal infection, we encounter a subjunctive construction such as S.aureus might have been the cause of fatal septicaemia, but without any laboratory evidence it is unclear whether it were a virulent strain or not.

Simple sentence constructor

The simplest of sentences need an action word as an absolute minimum. The shortest sentences are commands; single doing words in the imperative: Survive! Grow! Divide! Replicate! As these commands are only issued directly to the subject and it would be very unusual to address microorganisms directly, the shortest meaningful sentence in the language of infection is a combination of an action word with a naming word: S.aureus grows; M.tuberculosis survives; influenza virus replicates. These are all intransitive verbs. To get into the more exciting actions of the intransitive verbs we need to add in at least another naming word as an object; Anopheles mosquitoes transmit Plasmodium species, S.aureus causes endocarditis. Short sentences these may be, but they can start to convey the processes we have come to know as infection. The following chapters will add some of the extra components of language that give breadth and depth to the living world under the microscope lens.

TJJI as at 1730hr, 3^rd April, 2010.

Before there was a language of infection there were words and phrases. Some of these go back a long, long way. So far, that their origins are obscure. It is possible, likely even, that what we believe about infection was shaped before we went to school.

This short essay is a highly selective, personal perspective on those ideas: undercurrents to our ideas of infection.

TJJ Inglis, 8th March, 2010.

Nouns are the naming words we use in everyday speech, effortlessly describing the things that populate our material existence. In the world under the microscope, which we first encountered in sudacoes (greetings) the early micronauts began to see different types or kinds of tiny, living things. This chapter is about the names we give to those things, how we go about naming them, what those names tell us and, by implication, what other types of words we are likely to encounter along with this group of nouns.

But first, a little about types of nouns. Their key quality is the noun’s ability to specify a given thing and thereby differentiate it from another. Nouns belong to groups, and those groups sometimes belong to other, larger groups in a hierarchy of naming words. One key feature that can sometimes be a bit hard to understand in different languages is gender. Many languages have only two genders; male and female, to the frustration of English speakers who are used to a third very useful gender; neuter. But that is only a whiff of the multiplicity of genders some languages possess. Think what you could do with 15 genders? If gender is the first point of difference between kinds of noun, then the four genders of the language of infection are bacteria, fungi, parasites and viruses. 

Saudações (greetings)

We are social animals. We meet and greet, and in doing so we use words or short phrases to verbally close the social distance. These are often the first words we use when travelling in distant places and meeting their inhabitants. They are the words we learn first when preparing to visit foreign lands. They also form the basis of our very first introduction, as adults, to another language. So, as we prepare to cross the frontier into a new world, it would be perfectly logical to make a start on the language we're going to use there by learning a few words of greeting. They're likely to be the equivalent of "Hello! Pleased to meet you. I'm XYZ. What's your name?"

You make need a few simple imperatives or command words such as "Look!" or "Listen!" so that your fellow travellers can share moments of recognition. Discoveries are not meant to be solitary experience. Communicating your expedition experience is one of the joys of travel and possibly your best way to get the show back on the road at a later date.

The first person to encounter microbes was almost certainly Antonie van Leeuwenhoek, working with the earliest microscope around 350 years ago. He was a self-educated draper who used homemade magnifying glasses to inspect the cloth he bought and sold. Using a process he never fully disclosed, he made lenses of progressively higher magnification until he was able to see single cells suspended in liquid. A man who had serious problems with the idea of spontaneous generation, van Leeuwenhoek used his microscopes to study the biology of reproduction and cellular generation. His findings were written up in his native Dutch and submitted to the Royal Society for verification by a sceptical scientific community. Gradually his ideas gained credence and wide recognition during his lifetime. His discoveries now stand alongside those of Isaac Newton and Robert Boyle as critical contributions to scientific knowledge. But before we get sucked into the universe under the microscope, we need to spare a thought for what van Leeuwenhoek's first impressions were and how he communicated them to his own household. Coming from a thoroughly Dutch Reformed tradition, it is unlikely he used any expletives, no matter how stunning his first sight of minute life might have been. As the first realisation of what he had seen dawned on him, the words are most likely to have been short, and quite probably commands: Hey! Look at this! Come and see what I've found!" or something similar. With a bit of repetition - and we know he spent a lot of time repeating experiments - he will have recognised patterns. He certainly saw movement among his "animalcules' or little animals. We now think from his drawings that these included various unicellular organisms such as protozoa and rotifers. He studied plant cells and spermatozoa, in his research into cellular generation. Though his work in a field we might want to call cell biology opened a window on what he called "wretched beasties", it appears that he got no further than "Hello! Pleased to meet you. What's your name?" and did not establish a formal nomenclature or functional analysis. These developments had to wait for others to catch up with van Leeuwenhoek and place his discoveries in context, a process that took the best part of two centuries.]]> tim.inglis@priobe.net (Tim Inglis) Language of Infection Wed, 03 Feb 2010 11:01:26 +0000