Priobes

Arboviruses

2010-05-08T12:53:12Z

The arboviruses are a diverse group of over 500 RNA viruses that have in common their ability to cause infection after the bite of an arthropod vector. The most important vectors of arbovirus infection are mosquitoes, but some of these infections are transmitted by ticks or sandflies. The most important arbovirus infections in global terms include yellow fever, dengue and Japanese encephalitis.

Learning resources on arbovirus infections include study materials for the FACTM pt 1 exam and links out to other health information websites.

FACTM Arbovirus infections 1: clinical tropical medicine
FACTM Arbovirus infections 2: clinical entomology
WHO yellow fever resource
CDC arbovirus resource
ICPMR medical entomology website

Blood parasites

2010-08-29T11:31:11Z

Blood parasites

Lynne Garcia, LSG & Associates, Santa Monica, CA., USA.

Parasitology Masterclass, RMIT, Melbourne, 14^th August, 2010.

41% world’s population is exposed to endemic malaria. 2.7 million die from malaria each year. Malaria was eradicated in Australia as recently as 1981. Mosquito bite is the main method on transmission, but there are several other means of infection.

Life cycle. The early stages are not synchronised and lack the periodicity due to synchronised parasite release into the bloodstream. Laboratory diagnosis is difficult when parasitaemia is low. WHO defines any malaria parasite that originates from the liver again as a relapse. Emergence of gametocytes takes 1-2 weeks. Patients in the emergency department suspected of having malaria need blood drawing immediately, rather than waiting for the perfect timing. The need for a 24/7 service means that in many places malaria screening is done by Haematology rather than Microbiology. Whoever does them, good blood smears are an art and should be made as soon as the blood has been drawn. EDTA blood is better than heparinised blood.

CAP standard required both thick and thin films to be examined before a report can be issued. False negative smears can be caused by low parasitaemia, particularly after commencing treatment. So a travel history is mandatory. Malaria films should be a STAT preparation, the only other truly emergency parasitology being CSF examination for amoebae. Thick and thin films need 300 high power fields examined per film (i.e. x 1000 magnification)

Quality control for malaria films. This is now more practical and does not depend on sending out samples for review. The current approach is built into the system in use and judged by how good the leukocytes appear.

Variation & distortion of parasites. Beware real life variation e.g. P falciparum rings are often bigger then the textbook appearance. Distortion begins around 1hr after blood has been drawn. Typical appearance can be lost by 4-6hr. Gametocytes may round up and become unrecognisable, even exflagellating. Refrigeration of EDTA specimen is not recommended because parasites round up.

Stains for blood films. Giemsa is the stain of choice but may be difficult or costly. There are now many proprietary options e.g. DipStat stain. While colour is not critical to detection, the appearance of leukocytes is important (see QC comment above). There are pros and cons for both thick and thin films. Thick film is much more sensitive, picking up more infections than thin film but is more difficult to interpret and to use as means of species identification. A Coplin jar should not be used for stains. A quick double-dip in acetone once the blood smear has dried can produce a clearer film. A new has been developed technique for combining thick and thin smears.

Rapid tests.

Buffy coat smears. These can be more sensitive but P falciparum rings are less buoyant. The proprietary QBC (acridine orange) tubes are suitable for the classic stages.

A thick film will be positive at around 0.0001% or 5-20 parasites per uL, while a Binax Now card test will decline in sensitivity below 0.1% i.e. not better than a trained microscopist. External controls are needed but the result cannot be reported without performing films. These are quite good for P falciparum and P vivax but only at high level parasitaemia. There is a risk of false negative results if reliance is placed on a rapid test only. Failure to detect Plasmodium sp may result in patient death.

Problems

Oil on microscope slides
Need for standard precautions for occupational health & safety
Letting blood sample stand too long before preparing films
Sending report out without examination of a blood film

Species-specific notes (recognition chart) (CDC guide):

P vivax. Appearance is not always typical. There may be a steady low-grade fever in early infection.

P ovale. This infects young erythrocytes and can be easily confused with P vivax infection. The trophozoites moves more slowly than does P vivax and leaves a fimbriated edge to the infected RBC. The world distribution of P ovale infection is restricted.

P malariae. Band forms are characteristic but not limited to P malariae. Infection may remain for 20-40 years. Infection produces lots of malaria pigment and immune complex with time, contributing to kidney damage, leading to nephritic syndrome. The schizont is daisy head shape.

P falciparum. This causes most of the severe cases of infection, the majority of infections in the USA and produces greatest scope for failures. Mixed infections are easily missed. The early stages of infection don’t necessarily resemble malaria. Microscopic features that help distinguish this from other Plasmodium spp include accole and appliqué forms, gametocyte shape, ingestion of malaria pigment, double chromatin dots and exflagellation of gametocytes.

P knowlesi. This recently recognised Plasmodium infection of humans also causes infection of long tailed macaques and is found in Sarawak, Singapore, the Philippines, and along the Thai-Myanmar border. There is a 24hr cycle affecting all ages of erythrocyte. Rings can be delicate and Schuffner’s dots missing so that microscopic features of early infection resemble P falciparum, but later stages can mimic P malariae. 10% human infections are fatal and the severe complications resemble P falciparum. Rapid methods do not identify to species level.

Mixed infections. These are underestimated. E.g 30% may be mixed P falciparum/P malariae in Thailand. It appears that Anopheles species can transmit two Plasmodium species at one time.

Other blood parasites.

Babesiosis. Most often B.microti, mimics P falciparum with many ring forms on the blood film but no other stages. The characteristic Maltese Cross formation may not be visible on blood film. Lack of familiarity can cause long delays in arriving at a definitive diagnosis. This is a tick-borne infection in Europe and the USA, particularly in asplenic patients. Disease on the West Coast of the USA is more severe. Immediate thick and thin films need performing on patients with suspected Babesiosis. Babesia can be difficult to see on buffy coat smears by QBC and the ring forms can be outside RBCs.

Leishmaniasis. There are over 2 million cases of visceral and cutaneous leishmaniasis per annum worldwide. CL is now endemic in Texas. Clinical disease covers a spectrum of visceral, mucocutaneous and cutaneous. Following the first Gulf War there were 20 cases of CL caused by L major and 12 cases of VL caused by L tropica in returning troops In a survey from 2002-2004, over 500 cases of CL were detected due to L major in personnel returning from Iraq. It took up to around 60 days to develop skin lesions at up to 9 locations. Some showed evidence of reinoculation at multiple points consistent with slipping bandages. Bed nets and permethrin treatment of uniforms were used as control measures. Leishmania culture needs a positive control. True amastigote stage appearance can be seen when parasite released from macrophages on tissue squash prep. Samples best obtained by punch biopsy around the edge of the lesion after it has been carefully cleaned to avoid bacterial contamination of culture medium. A minimum of 3 samples is needed. The fine needle aspirate method has poor sensitivity. Culture works well on NNN or Schneider’s drosophila media. Best sensitivity is with real time PCR assay, where available. Patients probably have infection for life despite treatment, options for which include doing nothing, heat, cryotherapy, topical Paromomycin and pentostam. MCL in Mexico, Belize, Guatemala and the Southern USA due to L mexicana. VL associated with rise in immunoglobulins but antibodies do not eradicate infection. Recovery due to treatment may be accompanied by development of post kala-azar dermal Leishmaniasis.

Trypanosomiasis. American and African forms. The gambiense form of African trypanosomiasis is particularly difficult to diagnose. The American form (Trypanosoma cruzi) has caused infection in around 150,000 people, particularly in family donors in Teas & California. The Triatomid bugs leave an allergen causing scratching which leads to inoculation of parasite in bug faeces. Transmission also via intravenous drug use. Infection results in myocarditis and megacolon.

Notes by TJJI, AUG-2010

Burkholderia pseudomallei

2010-05-08T13:12:45Z

Burkholderia pseudomallei / melioidosis

Burkholderia pseudomallei is the Gram negative, environmental bacterium that causes the infection known as melioidosis. This potentially fatal infection occurs throughout the tropics but is most commonly encountered in Southeast Asia and northern Australia.

Learning resources include lecture notes, review articles, clinical and public health guidelines.

an introduction to melioidosis
lecture notes (the Burkholderias)
clinical guideline to melioidosis
public health aspects of melioidosis
treatment guide for B. pseudomallei infections/melioidosis
melioidosis as soil-borne disease (AUG-2010)
CDC melioidosis information
HPA melioidosis information
Life in the Fast Lane editorial (JUN-2010)

faecal parasite examination

2010-08-29T10:11:42Z

An introduction to faecal parasite examination

LS Garcia, LSG & Associates, CA., USA.

Parasitology Masterclass, RMIT, Melbourne, 14^th August, 2010.

There are considerable differences in practice between laboratories. The capability and confidence limits must be known. In the USA, CAP mandates a specific approach to faecal parasite examination; concentrate and permanent stain.

Sample transit. Samples deteriorate considerably during transit and therefore benefit from fixative. Various agents are used including formalin, PVA, mercury, copper and zinc based.

Sample number. 3 is better but we can live with a series of 2 samples. One study indicated up to 22% increase in yield on the third sample, and the minimum for Giardia FIA is 2). Various concentration devices are used.

Immunoassay. IA should not be used to confirm O & P examination. Assay wells need washing but should be washed gently. Sensitivity may be poor so that a negative result may be returned from sample with low level antigen present. Giardia and Crytosporidium assays can be read with FITC alone.

Rapid tests. These are helpful but not perfect e.g. kits usually do not distinguish between E histolytica and E dispar. Lateral flow devices do not cope well with lots of faecal sample. In real conditions any colour in a test well equates to a positive result.

Special stains. These are labour intensive especially if for Microsporidia and there is as yet no commercial reagent. They should be run with concentrated sediment. These are more easily detected in clean samples e.g. nasopharyngeal aspirate, and are particularly difficult to miss in stools. [Trichrome stain tips: 11 ways to make a better slide]

In view of the need for specific ordering, different patient groups trigger different tests:

immunocompromised with diarrhoea
municipal water-borne outbreak
history of international travel & diarrhoea

Reporting. Standardised comments are helpful. Free text should be avoided. With immunoassays, reports need to state what was tested, either present or negative, and must be clear about the findings. Names should be given as Genus, species and stage. Quantity of parasite present is still considered useful by some. Non-pathogens are reported as indicators of exposure to risky water or food source. Artifacts – report some, like Charcot-Leiden crystals, human polymorph nuclei but do not report pollen or chemical crystals.

Do not perform a parasite examination if the patient has been in hospital for more than 3 days. CAP dictates the need for a faecal concentrate and a stained faecal smear.

Cryptosporidium stools contain spores and are immediately infectious. Cyclospora is less infectious.

Notes by TJJI, AUG-10.

Leishmania

2010-08-09T14:21:41Z

Leishmania

Leishmania species are a group of apicomplexan protozoa responsible for a group of infections collectively known as leishmaniasis. They incude cutaneous, mucocutaneous and visceral leishmaniasis. Further resources on this group of infections can be found here:

Leishmaniasis 1. Clinical Tropical Medicine Lecture for FACTM exam preparation
Leishmaniasis 2. Clinical parasitology & Entomology Lecture for FACTM exam
Leishmania species developmental cycle downloadable diagram

Medical Entomology in Australia

2010-08-30T11:21:19Z

Medical Entomology in Australia

Richard Russell, University of Sydney.

Parasitology & Tropical Medicine Masterclass, RMIT, Melbourne, 14^th August, 2010.

Scope: lice, mites, bugs, fleas, biting flies.

University of Sydney Medical Entomology website

Medical significance

Antigen present in saliva, but some reactions are atypical
Urticarial – hairy caterpillars
Venoms – anaphylactic reactions, toxicosis (e.g. ticks bites)
Infections
Mental trauma

Lice. Many animals have resident lice. Few persist or survive on humans, but we have our own human lice species. Both male & females have a blood meal. Body & head lice difficult to distinguish –ecological species. Infectious agents are inoculated with louse faeces during scratching, but by body lice only. Head lice are common in children, have reduced motility in wet hair. Body lice prefer to live on clothing, increasingly resistant to insecticides. Infections associated with lice include:

Epidemic typhus (Rickettsia prowazecki)
Relapsing fever (Borellia recurrentis)
Trench fever (Bartonella quintana)
Head lice – nits = vacated eggs

Public lice may attach to eyelashes in children, following transmission from parents. No disease convincingly associated. Treatment with Lindane, permethrin based cream.

Mites. For humans this includes scabies mites, the follicle mite (Demodex folliculorum, not associated with human disease) and house dust mites. Also bird & rat mites, chiggers. Scabies may be difficult – may not be there in the first place, or may be there but impossible to prove. The rash does not occur at adult mite location. They burrow around wrists and hands. Crusted scabies (aka ‘Norwegian scabies’) causes very thick skin. Lowered immune response & unable to scratch. To demonstrate mite, keratin needs dispersing with KOH or lactic acid. Burrows difficult to see. Scraping around nodules. Treatment – permethrin = insecticide of choice. Itch may continue for more than a month afterwards.

Garden mites. Leptotrombidium deliense in wet tropical parts of northern Australia. Vector for Orienta tsutsugamushi, cause of scrub typhus. Rodents = part of life cycle. Infection often causes eschar at bite site.

Bugs. No blood-sucking Triatomid bugs in Australia, but related species are present. Still capable of causing pain and skin reaction at site of bite. Bedbugs are common, proliferating worldwide as a result of travel, particularly backpacker & budget hotels. No transmission of infection, but considerable nuisance value, allergic reactions, anaemia, asthma & mental trauma.

Fleas. “cat” flea, Ctenocephalides felis on cats & dogs. Human flea, Pulex irritans, also on pigs). Most other animals species have fleas close to the skin of the host. Xenopsylla cheopis, rat flea, is plague vector. Has no combs and a distinctive shape to bursa populatrix. Bite reactions vary from mild to severe. Murine typhus has been documented in Australia. Highly specialised species Tunga penetrans (chigoe) in Africa & South America. Female buries herself head first into skin and becomes an egg factory.

Ticks, arachnids. Soft ticks include vectors for relapsing fever (Borrellia duttoni) but not in Australia. Kangaroo soft tick around nesting areas. Hard ticks transmit arboviruses (not in Australia, rickettsias and possible Ehrlichia. Many eggs are laid since most die before finding a host. Male tick lacks blood sack, doesn’t take blood meal directly. In Australia, Ixodes holocyclus – rickettsias & toxicosis. Has caused deaths, mainly in children. Also unsteady gait, progressive paralysis, Bell’s palsy & respiratory paralysis. Doesn’t stop with removal of tick. Mild, severe or lethal anaphylactic reactions. Ix cornuatus – toxicosis only. Ix holocyclus also responsible for spotted fever rickettsioses including Queensland tick typhus. Flinders Island spotted fever and Australian spotted fever. Triad of fever, rash & eschar. Others include Amblyomma triguttatum – kangaroo hard tick in WA. Rhipicephalus sanguineus – can bite humans.

Butterflies & moths. Larval stages have urticating hairs of various structures, some hollow or with fragile tips, leading to different delivery mechanism. E.g cup moth & white-stemmed gum moth.

Biting flies

Biting midges (‘sandflies’)
Mosquitoes
March flies, following floods, Tabanidae, anaphylaxis in Pilbara & Kimberley
Black flies

Mosquitoes. Mosquito-borne diseases: local & introduced

Surveillance of mosquito vectors important in Australia

Aedes notoscriptus associated with domestic containers
Culex quinquefasciatus, brown house mosquito mainly of nuisance value, not that significant as disease vector
Culex annulirostris, important as main arbovirus vector in Australia
Aedes vigilax, salt marsh species left after high spring tides

Non-biting flies – chironomids, nuisance, moth flies; rat-tail maggots & hover flies

Myiasis. Invasion of tissues by larvae of non-biting flies. Dermatobia hominis, south & central America. Human bot fly causes lesion with appearance of a boil.This species captures biting fly and uses it to lay its own eggs. Cordylobia anthropophagia, Tumbu fly in Africa. Facultative myiasis – blowflies, use in debridement of wounds & ulcers, reintroduced due to antibiotic resistance. Avoid in fistulas & bleeding wounds [Lucilia sericata – sheep blowfly for maggot therapy]

Notes by MicroGnome, AUG-2010

Neglected bacterial diseases

2010-06-24T12:34:24Z

Lecture notes

Melioidosis, Leptospirosis & Scrub Typhus:

Neglected bacterial diseases 1

Neglected bacterial diseases 2

Plasmodium

2010-04-28T10:08:48Z

Plasmodium species (P falciparum, P vivax, P malariae and P ovale) are the mosquito-borne protozoan parasites responsible for malaria.

Teaching materials on Plasmodium spp. and malaria can be found via the following links:

Interactive world malaria risk map
Malaria, the raw facts
Malaria 1: clinical tropical medicine
Malaria 2: clinical parasitology
Malaria 3: public health, travel & expedition medicine
Malaria 4: Medical entomology
Malaria, quick test
Malaria, self assessment questions
Malaria medical history
Malaria review article
Rollback malaria campaign

Principia Aetiologica

2009-04-11T07:07:00Z

Principia aetiologica: taking causality beyond Koch's postulates.

TJJ Inglis. J Med Microbiol. 2007 Nov;56(Pt 11):1419-22.

There is no single accepted method to establish a causal relationship between an infective agent and its corresponding infectious disease. Different biomedical disciplines use a patchwork of distinct but overlapping approaches. To a greater or lesser extent these are based on criteria known as the Koch-Henle postulates, or 'Koch's postulates' for short. Deficiencies in Koch's postulates were recognized by their principal author shortly after their formulation. Now, over a century later, a more rigorous method to test causality has still to be finalized. One contender is a method that uses molecular methods to establish a causal relationship ('molecular Koch's postulates'). Recognizing the wider range of contemporary approaches used to build an argument for a causal relationship, the use of a more inclusive approach to establish proof of causality is proposed. This method uses an argument built from a series of assertions.

Assertion 1: congruence or reproducible correlation of a taxonomically defined life form with the clinico-pathological and epidemiological features of infection.

Assertion 2: consistency of the demonstrable biological response in the subject to an encounter with the prospective infective agent.

Assertion 3: progressive or cumulative dissonance as an explanation for pathophysiological processes at every known level of biological organization in the subject.

Assertion 4: curtailment of that pathophysiological process on the deliberate introduction of a specified biomedical intervention.

Evidence to implicate the candidate biological entity as an initiator of or primer for cumulative dissonance places it in a subcategory of micro-organisms to be known as 'priobes'. A priobe is the sufficient and necessary antecedent cause of a pathophysiological process evident as an infectious disease.

The full article can be found here: http://jmm.sgmjournals.org/cgi/content/full/56/11/1419

Rabies virus

2010-06-24T12:46:16Z

Rabies virus