44

Parasitic infections

44

Parasitic infections

44

Parasitic infections

44

Parasitic infections

  44 Parasitic infections

Ingrid Reiter-Owona

44.1 Introduction

Parasites, unlike bacteria, have a complex life cycle. They can use the human body either as a definitive or intermediate host. The definitive host is where sexual reproduction of the parasite occurs. The final products of reproduction are passed to the outside world or to a vector. The intermediate host parasites remain after replication in a dormant developmental stage and can harm the intermediate host during their replication phase or due to the immune response they induce in their host. The period between infection and manifestation of parasite stages detectable with laboratory methods can vary between days to months (pre patency).

Parasitic infections are rather rare in central and northern Europe. The diagnosis of parasitic infection, especially involving the intestinal tract or the skin, may, at the extreme, create panic in the affected person and is associated with lack of hygiene and low social status. Although parasites do not know social or geographical boundaries, their propagation is promoted by inadequate disposal of human feces, poor drinking water quality and malnutrition in less developed countries. A correlation with dietary conditions, mental and physical development or morbidity presupposes a high degree of parasite infestation not reached in north European latitudes.

Therefore, parasitic infection plays different roles in different population groups in the context of diagnosis of ambiguous clinical symptoms. Risk groups include frequently traveling individuals, those with a migration background who regularly travel to their country of origin and immigrants from countries with lower hygiene standards. Very old and very young persons are also at risk, as well as those in close contact with animals. Immunocompromised patients assume a special status because opportunistic pathogens can cause life threatening diseases in such individuals.

Basic knowledge of the distribution and life cycle of parasites is the prerequisite for the target oriented and effective laboratory diagnosis of parasitic infections. The spectrum of potential parasitic pathogens can be limited by detailed information obtained from medical history and by taking into account past travels and abnormalities in the blood count, especially eosinophilia.

Laboratory detection of parasitic infections is based on two aspects:

  • Direct demonstration of parasites, parasite parts, their antigens or DNA/RNA
  • Evidence of contact with the pathogen by detection of specific antibodies (serology).

The efficacy of a specific detection method is decisively influenced by the clinical symptoms (e.g., diarrhea, fever), incubation period, pre patency, the body site which is colonized as well as their intracellular or extracellular localization. Parasites are classified into endoparasites (intestinal parasites, tissue parasites, blood parasites) and ectoparasites (insects).

Microscopy provides a general overview of all parasites and non pathogenic organisms present in the specimen at the time of sample collection. The diagnostic sensitivity of this method increases as a function of parasite density, number of samples analyzed (especially stool), sample volume (body fluids) and application of efficient concentration methods.

When searching for a specific pathogen, microscopy is usually less sensitive than antigen detection or DNA amplification. Moreover, conventional methods are not suited to detect virulence factors of morphologically identical species (e.g., Entamoeba histolytica, E. dispar) or the resistance factors. Serology is a useful method only when a tissue migrating or tissue invasive stage (e.g., E. histolytica) of the pathogen is present.

The primary objective of diagnostics is to provide direct evidence of the pathogen or one of its developmental stages. In many cases, the sole detection of antibodies or parasite specific DNA will not allow any conclusion as to the status or duration of the infection.

The following description only deals with parasitic infections endemic in central and northern Europe, those which are commonly acquired during trips abroad or imported by immigrants.

44.2 Sample collection, transport, diagnostic methods

Stool sample

Care should be taken to ensure proper collection of the stool sample because the diagnosis of most intestinal parasites is based on the search and detection of the eggs or larvae of worms or the trophozoites or cysts of protozoa. In most cases, intestinal parasites are not continuously excreted in the stool. Three stool samples taken at intervals of 2 days are required for an initial orientating examination for intestinal parasites. Reasonable clinical suspicion can even justify more than five samples. The interpretation of a negative result like no parasites detected which is based on the analysis of only one stool sample is only acceptable with reservations.

Stool samples are usually collected by the patient himself. The patient needs a suitable, wide necked container to do this and should be instructed to avoid contact of the sample with toilet water or soil. Entire worms, parts of worms or worm like structures should be collected unfixed in tap water and forwarded to the special diagnostic laboratory.

The sample should have the size of a walnut (20–40 g) in formed stool or have a volume corresponding to approximately 5–6 teaspoons in liquid stool. Formed stool can generally be sent unpreserved unless the transport time exceeds 24 h. Liquid stool must be either available in the laboratory within 30 minutes after excretion or, depending on further examination proceedings, preserved with a suitable fixative immediately at the patient’s and arrive at the laboratory within 24 h. For fixing, the fresh stool sample should be thoroughly mixed with the relevant fixative solution. Suitable fixatives include 5–10% formalin or SAF (sodium acetate-acetic acid-formaldehyde) solution. A merthiolate-iodine-formaldehyde (MIF) containing solution (MIF concentration technique), which was applied in the past with good results, should no longer be used because of the ecologically harmful properties of this mercury compound. For instance, the explosion prone ether has been replaced by ethyl acetate in some commercial concentration systems. Each fixative has a special effect on individual parasites or their developmental stages and can influence the specimen’s morphology, staining properties and concentration /12/.

Refrigerator temperatures are suited for extended storage of formed stool samples, whereas deep freezing destroys the parasite structure, thus rendering microscopic diagnosis impossible. The receiving laboratory must be informed of the kind and duration of sample processing in the accompanying document. This is necessary to allow adjustment between the fixative and further laboratory specific processing (concentration, staining, antigen detection, molecular biological method). The final laboratory result should include information on the concentration (low, medium, high) of the parasite, if possible.

Blood sample

Serum is sufficient for most analyses. The samples should be transported fast and non refrigerated.

Diagnostic methods

Methods for the detection and differentiation of parasites include microscopy, antibody and antigen detection using enzyme linked immunosorbent assay (ELISA), immunofluorescence test (IIFT), immunochromatography test (ICT), Western blot (WB), immunoblot (IB) and molecular biological analyses by polymerase chain reaction (PCR).

For the individual methods, refer to Chapter 52 – Selected analytical laboratory techniques.

44.3 Intestinal protozoan infection

Various laboratory methods are available for the detection of protozoa in the stool: microscopy, indirect evidence based on the detection of parasite antigens (copro antigen detection) and molecular biological detection. Soft samples containing blood, mucus or pus are selectively collected at different parts of the stool for analysis. If protozoa induced diarrhea is suspected and the stool passed by the patient is liquid, the laboratory needs the native stool sample immediately after excretion or not longer than 30 min. thereafter. This is necessary to be able to detect motile vegetative (trophozoite) stages (magna forms of E. histolytica, trophozoites of Giardia lamblia). If this prerequisite cannot be fulfilled, a suitable fixative should be used to preserve the morphology of the trophozoites and the parasitic antigens. Thus, trophozoites can be identified after staining (e.g., Giemsa stain) on their specific organelles.

44.3.1 Amebiasis

Amebiasis is an infection caused by the human pathogenic ameba species Entamoeba histolytica. E histolytica is transmitted by the fecal-oral route. It colonizes the colon where the trophozoites multiply by longitudinal binary fission and, after encystation, are passed in the stool to the environment as mature, infectious cysts (10–16 μm). Approximately 90% of infections are asymptomatic. An invasive form develops in 10% of cases, based on the ability of E. histolytica to kill cells using lytic components under certain circumstances. This results in extensive tissue lesions, especially in the intestine and liver, caused by large, hematophagous, species specific trophozoites, so-called magna forms. Other Entameba such as E. dispar and E. moshkovskii , the cystic stage of which is morphologically identical with E. histolytica and the somewhat larger species Entameba coli, are non invasive, are considered to be apathogenic intestinal inhabitants and do not require treatment.

Epidemiology

The precise epidemiology of parasitoses caused by E. histolytica, E. dispar and E. moshkovskii remains to be clarified because only a small number of studies have applied recognized methods of species differentiation to date /3/. It is assumed, however, that the ratio between carriers of E. histolytica and E. dispar is approximately 1 : 10 /4/. According to WHO estimates, there are about 500 million new cases of amebiasis every year, with the highest morbidity and mortality reported from Central and South America, Africa and India /5/.

In Germany, amebiasis is relatively rarely diagnosed and is primarily an imported infection. Only a small number of travelers fall ill with the invasive form of the disease after their return /6/. Asymptomatic carriers who may excrete cysts for months or years represent a special risk.

Whether and when an invasive form will develop from the carrier stage cannot be predicted. The mean lag time for development of an amebic liver abscess is 3–5 months /7/.

Incubation period

Highly variable (days, months to years)

44.3.1.1 Clinical symptoms

Amebic colitis, amebic dysentery

Abdominal pain and bloody mucoid diarrhea; in severe cases inflammation in the rectum, shaking chills and fever, possibly intestinal perforation. Intestinal amebiasis has to be considered when diarrhea persists for more than three weeks following journeys abroad.

Amebic liver abscess

This severe disease is characterized by abdominal pain associated with fever and partly extensive necrotic areas and subsequent abscess formation.

Mandatory reporting

This infection is not subject to mandatory reporting; however, there is an unofficial reporting office at the Bernhard Nocht Institute in Hamburg.

44.3.1.2 Laboratory findings

The diagnostic method in the laboratory is selected based on the clinical symptoms and the specimen received. Prior to sample collection, the sender should confer with a laboratory specialized in tropical medicine, especially in clinical suspicion of an invasive form of the disease.

Refer to Tab. 44-1 – Laboratory detection of infection with Entamoeba histolytica.

Microscopic stool examination

This complex analysis should be performed by experienced laboratory personnel. The detection limit of this method has been reported as approximately 60% compared with PCR (detection of cysts) /7/. The detection of hematophagous trophozoites (magna forms) is considered to be the only sufficiently reliable evidence for the diagnosis of E. histolytica infection.

Antigen detection

The availability of antigen detection assays has simplified diagnostics because the stool sample does not necessarily have to have body temperature. The available immunoassays (ELISA, IIFT, ICT) only detect genus specific Entameba antigens or E. histolytica-specific proteins. Species specificity must be explicitly guaranteed by the manufacturer. The assays usually require fresh, native stool stored at refrigerator temperatures.

Molecular biology (PCR)

These methods have the highest diagnostic sensitivity and specificity of all techniques /8/. They are recommended in reasonably suspected intestinal infection and negative microscopic findings or for species differentiation in positive microscopic stool findings. Moreover, PCR is used to verify successful treatment and represents the only useful method for analyzing biopsy and/or abscess specimens and bioptates.

Antibody detection

A variety of commercial and in-house assays like immunofluorescence test (IIFT), ELISA, indirect hemagglutination test (IHAT) and direct agglutination test (DA) are available for serology. Their diagnostic sensitivity can vary particularly during the early stage of the disease (i.e., manifestation of initial symptoms of a liver abscess). Although IIFT is considered to have the highest diagnostic sensitivity and specificity, it is advisable to combine two different immunoassays in order to increase the overall sensitivity.

Elevated to high concentrations of specific antibodies are always detected in invasive E. histolytica infection and at prolonged duration of the disease (sensitivity 3–100%) /4/. In clinically suspected liver abscess and short duration of the disease, negative and/or borderline test results must be verified at intervals of 10 days.

Persistently low antibody concentrations can indicate asymptomatic intestinal infestation or occur in individuals living or having lived in endemic regions who are frequently affected by reinfections.

44.3.2 Giardiasis (lambliasis)

G. intestinalis (G. lamblia, G. duodenalis) is the most common intestinal protozoon worldwide. Genotypes A and B are infectious for humans, but have also been isolated in pets and domestic animals such as dogs, cats and calves. The zoonotic significance of these animal isolates seems to be rather low. Infection occurs by ingestion of cysts (8–12 μm), followed by vegetative multiplication of the pear shaped trophozoites (10–20 μm) in the intestine. Cysts are directly infectious when passed in the stool.

Epidemiology

The pathogen occurs worldwide and is ingested with contaminated water or uncooked food. In addition, person-to-person transmission is also possible as seen in small scale epidemics in care homes and among male homosexuals. In Germany, approximately 3700 cases of giardiasis are reported every year, of which about 50% are acquired domestically. Risk factors include eating uncooked vegetarian food, having impaired immunity and male sex /9/.

Incubation period

Approximately 3 days to 3 weeks, mean of 7 days.

44.3.2.1 Clinical symptoms

Infection with G. lamblia can manifest in humans in various ways. Parasite carriers can be completely asymptomatic or present with acute self limited infections or chronic infections with changing conditions. Initial symptoms start with explosive, watery, foul smelling diarrhea not containing any blood or mucus. This is later on followed by abdominal pain and bloating, and the stool is yellowish, possibly fatty and can be formed. Symptomatic infections are more common in immunocompromised individuals, especially those with IgA deficiency. Individuals with chronic infection may suffer from malabsorption and weight loss.

Mandatory reporting

In compliance with Article 7 Section 1 No. 16 of the German Infection Protection Act (IfSG), direct or indirect evidence of G. lamblia, insofar as it indicates acute infection, is to be reported to the local health authority giving the name of the affected individual(s).

Specimen

Stool; duodenal fluid (5–10 mL) in chronic infection.

G. lamblia is excreted very irregularly. At least 3 stool samples collected on different days must be analyzed in order to exclude giardiasis.

44.3.2.2 Laboratory findings

Microscopy

In acute watery diarrhea, motile vegetative stages (trophozoites) may be detectable in the native sample within the first 30 min. after passing of the stool (phase contrast or interference microscopy). If the native sample (stool or duodenal fluid) cannot be microscoped within the specified time, it must be fixed immediately after collection. Sodium acetate-acetic acid-formaldehyde (SAF) solution is considered to be the optimal fixative for trophozoites because the sodium acetate proportion preserves G. lamblia trophozoites better and the morphology is maintained after concentration. Cysts are primarily found in formed or solid stool after concentration.

Antigen detection

Immunoassays (ELISA, IIFT) are usually based on the detection of cyst antigen. The individual assays follow different concepts. In order to attain the diagnostic sensitivity guaranteed by the manufacturer, it is important to observe the specified type and duration of storage because only a small number of products will be functional in deviation thereof. Contrary to microscopy which enables quantitative pathogen determination, the intensity of the immune response does not allow any conclusion as to the density of the pathogens in the specimen. A positive response can still occur after the end of treatment when pathogens are no longer detectable by microscopy.

Refer to Tab. 44-2 – Results of two different commercial assays for lamblia antigen detection depending on storage and processing of the stool sample.

Molecular biological methods

PCR, primarily performed as in-house assay, is the most sensitive and most specific method available. Moreover, molecular biological methods allow the investigation of infection chains in chronic carriers and group infections. Moreover, the determination of the gene sequence by special laboratories can be helpful in clinically suspected true treatment resistance.

Antibody detection

The detection of specific antibodies against G. intestinalis has not become established because of the high level of endemic infection and is not useful for the diagnosis of acute diarrheal disease.

44.3.3 Other intestinal flagellates

The microscopic examination of stool samples of any consistency may also reveal other flagellates not considered to be pathogenic to humans according to current knowledge, including, for example, Dientamoeba fragilis and Chilomastix mesnili. Their role as sole pathogens or when occurring in increased density in diarrheal stool remains to be clarified; there is no indication for treatment.

44.3.4 Cryptosporidiosis

Cryptosporidia (protozoa, sporozoa) are classified as coccidians and were first recognized as associated with human disease in 1976. Two species have been identified as being pathogenic to humans: Cryptosporidium hominis, which is transmitted from person to person, and Cryptosporidium parvum, which is transmitted to humans from animals (zoonosis) /11/. Numerous pet and livestock species can function as reservoirs. The pathogens invade the microvillus border in the upper intestine, where they multiply and then develop into oocysts, the infectious stage of approximately 4 × 6 μm in size. This stage is passed in diarrheal stool.

Epidemiology

Cryptosporidia occur worldwide and are primarily transmitted by the fecal-oral route. People-to-people transmission is also possible. Inadequately filtered drinking water has been identified as a source of infection in various outbreaks /12/. The extra intestinal stages (oocysts) are resistant to all disinfectants, including chlorine, and can survive in the environment for months.

Incubation period

1–14 days (mean of 7 days).

44.3.4.1 Clinical symptoms

In immunocompetent humans, oral infection usually causes asymptomatic, brief, self limited, watery diarrhea. Children aged 6–24 months are affected especially frequently compared to the normal population where the rate of parasite carriers is only approximately 0.2%. The infection takes a chronic course in immunocompromised patients and is feared as an opportunistic infection in HIV infected patients. In the pre HAART era (HAART, highly active antiretroviral therapy) , such patients have been reported to suffer from severe diarrhea and vomiting which in many cases became life threatening due to excessive loss of fluid and electrolytes. Chronic cryptosporidiosis is an AIDS defining condition. Approximately 1,000 cases of cryptosporidiosis are reported in Germany every year.

Mandatory reporting

In compliance with Article 7 Section 1 No. 10 of the German Infection Protection Act (IfSG), direct or indirect evidence of Cryptosporidium parvum, insofar as it indicates acute infection, is to be reported to the local health authority giving the name of the affected individual(s).

44.3.4.2 Laboratory findings

Since oocysts may be passed intermittently in the stool, at least three different stool samples should be collected on different days in all investigation methods.

The method of choice is the microscopic detection of oocysts in the stool applying a modified Ziehl-Neelsen staining technique (e.g., Kinyoun staining, carbol fuchsin staining according to Heine). The small, reddish stained cysts with their sporozoites are difficult to distinguish from fungal spores in the stool smear at low parasite density. Diagnosis can also be based on histological findings from endoscopically obtained tissue samples. The (larger and non sporulated) cysts of Cyclospora cayetanensis must be excluded by differential diagnosis.

Antigen detection

Antigen assays are based on the indirect detection of oocysts (IFT) or oocyst antigens (ELISA, NCT). They have a diagnostic sensitivity of 66–99% /13/ and detect on average 50 to 100 oocysts/100 μL of stool.

Molecular biological methods

These methods provide the highest diagnostic sensitivity and are the only laboratory tests to allow differentiation between C. parvum and C. hominis, which can be of special importance in outbreaks.

Antibody detection

Not suited for acute diagnosis, can be used in epidemiological investigations.

44.3.5 Other coccidia

Isospora belli and Cyclospora cayetanensis are Coccidia species which can cause severe, watery diarrhea in humans, especially in immunocompromised individuals. After concentration (where the flotation method yields better results than the sedimentation technique), the pathogens are detected either directly (I. belli) or after staining with a modified Ziehl-Neelsen staining technique (C. cayetanensis/14/.

44.3.6 Microsporidia

Microsporidia comprise a phylum of obligate intracellular protozoan parasites. Currently, at least 14 species in 8 genera are known to infect humans. Since the onset of the AIDS pandemia, they have gained in significance as opportunistic pathogens in AIDS patients and can also cause various disease patterns in patients with compromised immune systems, especially those who are immunosuppressed by drugs or have undergone organ transplants. Self limited diarrhea in immunocompetent adults, children and travelers have occasionally been reported.

The most frequently isolated Microsporidia are Enterocytozoon bieneusi and Encephalitozoon intestinalis. They invade the intestinal epithelium and cause chronic diarrhea.

E. bieneusi is said to occur at higher rates in the intestine of immunocompetent inhabitants of tropical countries.

Specimen

Stool, native; duodenal fluid (5–10 mL), if necessary; other body fluids in case of immunosuppression.

44.3.6.1 Laboratory findings

Microscopy is suited to detect very small parasitic spores (E. bieneusi 1.3 × 0.7 μm, E. intestinalis 1.7 × 1.0–1.1 μm). After direct staining (e.g., trichrome stain), Microsporidia are often mistaken for other opportunistic intestinal germs, especially fungi, in the stool smear. More specific screening is achieved by IIFT and monoclonal antibodies or by using molecular biological methods which, besides electron microscopy, allow identification to the species level. Identification to the species level is important for targeted therapy /1516/.

44.4 Intestinal nematode infections

Microscopic diagnosis is primarily based on the detection of worm eggs. The size of the eggs is determined with a micrometer and the morphology can be confirmed by comparison with reference material or images available at the workplace. The eggs of some worm species are very rarely detected in the stood, for example eggs of Taenia, and of Enterobius vermicularis.

44.4.1 Enterobiasis (oxyuriasis)

Enterobiasis (oxyuriasis, pinworm infection) is caused by the small roundworm Enterobius vermicularis (pinworm, threadworm, seatworm). The parasite mainly lives in the lower intestine, appendix and upper colon. The short lived males are only 3–5 mm; females are 9–12 mm long and have a longer life expectancy.

The white to whitish yellow females are occasionally passed with the stool, are visible with the naked eye and can be identified by their pointed tail. A female can lay up to 10,000 eggs during its lifespan. The eggs are deposited in the perianal area. Chimpanzees are the only other known hosts besides humans.

Epidemiology

The pinworm occurs worldwide, with the main areas being located in cool and temperate climatic zones. The highest prevalences have been observed in children aged 5–10 years, independently of their social status /17/. Re-infection (“ping-pong infection”) is seen especially frequently within families or members of groups living closely together (preschool, school, boarding school).

The parasites are primarily transmitted by the anus-hand-mouth route. The eggs are deposited in the perianal area by the females, where they embryonate and maintain the infection cycle. Another possibility of infection is by inhaling dust or ingesting embryonated eggs, with confined rooms and good housing conditions having a favorable effect. Water borne transmission or transmission via contaminated food can be virtually excluded because of the low resistance of the eggs; transmission by pets or livestock can be ruled out in general. Autoinfection has been postulated.

44.4.1.1 Clinical symptoms

As a rule, enterobiasis is a harmless and self limited infection. Many infected individuals are asymptomatic carriers of the parasite. Anal itching is the most common clinical symptom (more often reported from children and women). Vulvovaginitis can develop in girls in childhood and adolescence. Abdominal pain, diarrhea and tenesmus have been reported in severe infections. Persistent infections or reinfections have repeatedly been observed in individuals despite optimal hygiene and multiple treatment cycles. In such cases, the emotional stress due to exaggerated hygiene can cause more severe symptoms of the disease than the nematode infection itself. Eosinophilia usually does not occur if the infection is purely intestinal.

Mandatory reporting

None

Specimen

Enterobius eggs are very rarely shed in the stool and usually will not be detected in routine stool examinations. Eggs are sampled from the perianal skin by means of transparent cellophane tape which is the diagnostic method of choice. Immediately after the patient wakes up, the adhesive surface of clear tape is pressed several times against the perianal skin and then transferred to a microscope slide with the adhesive surface downward. This procedure is to be performed for at least three consecutive days (for up to 7 consecutive days in reasonable clinical suspicion) for increased diagnostic sensitivity /18/.

44.4.1.2 Laboratory findings

Microscopy

The eggs are well visible at 100-fold magnification and can be identified by their typical structure. In addition, patients should watch for defecated female pinworms and submit them for examination. If the result of the microscopic examination for pinworm eggs or pinworms is positive, all family members or close contacts should also be examined in order to identify and concurrently treat asymptomatic carriers.

Antibody detection: inexpedient.

44.4.2 Other intestinal nematodes

The whipworm Trichuris trichiuria, hookworms and Ascaris lumbricoides are the most frequently diagnosed intestinal nematodes worldwide. The distribution of whip worms and hookworms is primarily limited to warmer climate zones where contact with fecal contamination of agricultural soils provides the prerequisite for the obligate infection cycle (maturation of larvae in the eggs and/or development of free larvae in a moist and warm environment).

High prevalence is seen in children. Imported infections do not present a risk for contacts or communities as long as the stool does enter an environment where further development of the parasites is encouraged. Both whipworm and hookworm infections are diagnosed based on eggs detected in the stool.

44.4.2.1 Ascariasis

Ascaris lumbricoides is the largest nematode (roundworm) parasitizing the human intestine. Adult worms can live 1–2 years. The females (20–25 cm), which may produce up to 200,000 eggs per day, and the smaller males (15–30 cm) have a relatively rough surface and a yellowish to reddish color. This parasite is regarded as host specific to humans and transmitted by oral ingestion of eggs containing an infectious larva.

Larval maturation includes a development stage outside the host which takes at least two weeks (three to six weeks under Central-European climate conditions). In the subsequent migration phase, the infectious larva undergoes a maturation process passing through stomach, intestine, liver and lungs, until the fourth larval stage develops into a sexually mature adult worm in the intestine and oviposition can start. This so-called pre patency phase takes 2–2.5 months.

Epidemiology

Ascaris lumbricoides occurs worldwide. The main route of infection is oral by ingestion of contaminated soil (especially in children) or contaminated food. At moderate temperatures, the eggs are highly resistant and can remain infectious for years. Egg containing feces passed by a roundworm carrier does not represent a risk of infection for the environment as long as the feces is disposed of via a closed sewer system. Infection with the large roundworm of pigs (Ascaris suum) is rare.

Incubation period

Approximately 10–14 days

44.4.2.1.1 Clinical symptoms

The clinical symptoms depend on the infection dose and the worm’s developmental stage.

Intestinal infection

In mild infection, carriers are asymptomatic, more severe infection is associated with mild to colicky abdominal pain. Severe complications can occur due to mechanical obstruction (ileus) or migration into biliary or pancreatic ducts. Adult worms are very motile and have a marked tendency to migrate.

Migration phase

The migration phase occurs after about two weeks following infection (pulmonary ascariasis, Löffler syndrome). Blood eosinophilia is present.

Hypersensitivity

Hypersensitivity manifests as urticaria in many cases, mostly at the end of migration through the lungs; blood eosinophilia is high.

44.4.2.1.2 Laboratory findings

Infected, asymptomatic individuals can be surprised by the spontaneous output of an individual, young or mature worm. The stool of these individuals rarely contains eggs. In more severe infections, diagnosis is based on the microscopic evidence of the typical eggs in the stool sample.

Serology

Serology (IIFT, ELISA) plays no role in individual diagnosis, even though specific antibodies (IgG1, IgG4) may be detectable following infection. Antibodies are rarely detected in individuals with low parasite load during the intestinal stage. In numerous commercially available assays not using species specific antigens, a high degree of cross reactions with other worm species is to be expected /1920/.

44.4.3 Trematode infections

Immigrants from Asia and less frequently tourists occasionally import infections with small liver flukes (clonorchiasis, opisthorchiasis), also referred to as fish borne parasitic zoonoses. By contrast, schistosomiasis (intestinal or urogenital bilharziasis) is also a hazard to tourists who only take a short vacation in the tropics, especially Africa.

44.4.3.1 Schistosomiasis

Schistosomiasis or bilharziasis (named after Theodor Bilharz, who discovered the pathogen in 1851) is one of the main worm infections and one of the most prevalent tropical diseases. Approximately 250 million individuals are thought to be infected with Schistosoma worldwide. The disease is caused by trematodes of the Schistosomatidae family whose occurrence is dependent on the occurrence of their intermediate hosts (freshwater snails).

Infection takes place in fresh water via cercaria larval stages (0.3 to 0.6 mm). The cercariae penetrate the skin, enter the venous circulation via the lymph system and, after passing the pulmonary capillaries, finally reach the liver where they develop into sexually mature, adult worms (1–2 cm). Adult Schistosoma then travel to the rectal or mesenteric veins (intestinal schistosomiasis) or the small pelvic veins (urogenital schistosomiasis). The Schistosoma females produce 300–3000 eggs per day, depending on the species. If left untreated, the mean age of adult worms is 5–10 years; adult worms have been documented to live longer than 30 years /21/. The pre patent period between cercarial infection and the start of egg output varies and can be at least 2.5 months in S. haematobium, at least 1.5 months in S. mansoni and at least one month in S. japonicum. The eggs, which are passed to the environment with the stool or urine, are only infectious to the intermediate host (snail).

Epidemiology

The main human pathogenic species and their distribution are:

  • S. haematobium: Africa, Middle East (pathogen of urogenital bilharziasis)
  • S. mansoni: Africa, Subsaharan Africa, Saudi-Arabia, Yemen, South America and, in some cases, the Caribbean (pathogen of intestinal bilharziasis)
  • S. intercalatum: Central Africa (pathogen of intestinal bilharziasis)
  • S. japonicum: China, Philippines, Japan (pathogen of intestinal bilharziasis)
  • S. mekongi: Laos, Cambodia, Thailand (pathogen of intestinal bilharziasis).

Incubation period

Cercarial dermatitis: 4–48 h, Katayama fever: 4–6 weeks, chronic infection: months to years.

Mandatory reporting

None in Germany.

44.4.3.1.1 Clinical symptoms

Classical clinical manifestation of schistosomiasis is subdivided into 3 stages:

1. Cercarial dermatitis. Symptoms usually occurring after repeated exposure include pruritus, erythema, maculopapular rash

2. Katayama fever: acute feverish systemic disease at the start of oviposition 2–12 weeks after infection, may persist for several weeks. Usually occurs in immune non immune individuals. Results in the formation of immune complexes. Clinical symptoms include shaking chills, headache, cough, urticaria

3. Chronic granulomatous inflammation resulting from immune response to egg antigens: liver fibrosis, portal hypertension, obstructions in the urogenital tract and portal and pulmonary circulation.

The symptoms can manifest differently in returning travelers and in patients coming from endemic regions.

44.4.3.1.2 Laboratory findings

If there is reason to suspect past exposure, laboratory investigations are useful not earlier than 2–3 weeks following potential infection (serological testing) or after the prepatent period has elapsed (more than 6 weeks; egg detection). Diagnosing is performed by stages (Tab. 44-3 – Methods for detection of Schistosoma sp. according to a graduated scheme). Blood eosinophilia may serve as guidance, but has insufficient diagnostic specificity as a screening method.

In positive serology, the infection should be confirmed by the microscopic detection of eggs in the stool or urine. The sedimentation technique yields better concentration results than the flotation method. Diagnostic microscopy is the most reliable method of species identification. Eggs from unfixed specimens can also be used to determine larval vitality (miracidium) for conclusions as to the infection status.

Antibody detection

The analysis should be performed not earlier than 2, preferably 4, weeks after possible infection. All tests, for which sufficient diagnostic sensitivity during the early infection stage has been documented, are suited for antibody determination. The tests are based on using S. mansoni antigen, thus making the serological differentiation of Schistosoma species impossible. Antibodies of the IgM class are predominant in the early infection phase, and those of the IgG class are predominant later on. In non immune, initially infected travelers, IIFT (focal fluorescence, gut associated antigens of S. mansoni) can detect IgG or IgM antibodies earlier after exposure than IHAT or ELISA (adult antigen, egg antigen). Cross reactions with other helminths, especially with other trematodes, are possible. The combination of two different investigation methods will yield the best results /22/.

The antibody concentration is usually low after primary infection (low infection dose), especially if there is no egg output after the end of the pre patent period, a common finding in returning travelers /23/. It is also low in individuals with chronic infection (eggs are shedded) coming from endemic areas.

Serology is not suited for therapy monitoring because specific antibodies can persist for many years after the end of therapy. The detection of eggs, however, warrants repeated treatment. Serological activity markers of existing infection or cured infection are not available.

Assays for egg antigen detection and molecular biological methods are available in special laboratories, but play no significant role in individual diagnosis. A PCR from EDTA blood for the early diagnosis of Katayama fever has been tried and tested in special laboratories /24/.

The laboratory test results are to be assessed as follows:

  • A negative screening test (in primary infection and after the prepatent period has elapsed) indicates that infection is unlikely
  • A positive screening test, especially after primary infection and without treatment, indicates that infection is likely
  • The detection of specific antibodies and vital eggs confirms an infection.

44.4.4 Tapeworms

Tapeworms deposit their eggs either directly in the stool (for example Diphyllobothrium sp., Hymenolepis nana) ore more often multiple body segments (proglottides) filled with eggs in the stool where they are visible to the patient’s naked eye. In suspected tapeworm infection, especially those of the genus Taenia, excreted tapeworm segments proglottides allow differentiation between human pathogenic Taenia species (T. solium, T. saginata). A differentiation of the various Taenia species is not possible on the basis of the morphology of the eggs.

44.5 Parasites in tissue

Worm larvae and hemato tissue parasites are the main groups of parasites in tissues.

44.5.1 Worm larvae

Parasitic infections with worm larvae occur worldwide. Humans can serve either as definitive or intermediate hosts.

44.5.1.1 Echinococcosis

Taenia of the genus Echinococcus are the pathogens of echinococcosis in humans.

Infection caused by E. granulosus it is referred to as cystic echinococcosis (dog tapeworm disease).

Alveolar echinococcosis (fox tapeworm disease) is caused by E. multilocularis. In both cases, the human body serves as intermediate host for the parasite larvae. Infection is by the oral route via embryonated eggs in the environment or in the feces of carnivores. The larvae develop primarily in the liver, but may also spread to the lungs or other organs.

Epidemiology

Distribution of E. multilocularis is restricted to the northern hemisphere (endemic regions: southern Germany, eastern France, northern Switzerland, western Austria, Turkey, Russia, China, North America). E. granulosus is rather cosmopolitan. High incidences have been reported from southern and eastern Europe as well as the Balkan countries, Turkey and the states of the Russian Federation. Differentiation to species level is of special importance, especially in patients from areas co endemic for both species (Turkey, China, Russia).

In Germany, E. multilocularis is endemic with few small foci of infection, while cystic echinococcosis is usually imported by individuals from the Mediterranean region /25/.

Incubation period

Months to years.

44.5.1.1.1 Clinical symptoms

The two species have very different growth patterns, thus manifesting in different ways with different symptoms. The larvae of E. multilocularis invade the affected organs tumor like (involving the liver in approximately 97% of cases) and, if left untreated, may lead to the patient’s death, while the larvae of E. granulosus multiply within a confined cyst and can be considered to be benign (involving the liver in approximately 70% and the lungs in approximately 20% of cases).

In the early stage of the disease, patients are usually asymptomatic and coincidentally diagnosed during sonography or within the scope of screenings.

Later on in alveolar echinococcosis, feeling of abdominal pressure, pain, nausea or fever can occur; in cystic echinococcosis, cyst rupture is the most common complication. Especially severe courses of the disease are seen in immunocompromised patients.

Peripheral blood eosinophilia is not present in echinococcosis patients.

Mandatory reporting

In compliance with Article 7 Section 3 No. 3 of the German Infection Protection Act (IfSG), direct or indirect evidence of Echinococcus sp. is to be reported to the Robert Koch Institute without giving the name of the affected individual(s).

Specimen

Antibody detection: 2–3 mL of serum.

Direct evidence: aspirated cyst fluid, resection sample (native, if possible), fixed for histological analysis. Diagnostic cyst aspiration must be preceded by antibody determination.

44.5.1.1.2 Laboratory findings

To confirm diagnosis evidence of specific antibodies, histopathological detection of structures typical of Echinococcus (protoscoleces or hooks in the cyst fluid), parasite DNA or typical macroscopic changes in the surgical resection sample.

Antibody detection

Serology is performed in steps according to a graduated scheme. Assays such as IHAT, IIFT and ELISA are available for an initial antibody screening. As a rule, they use crude antigens of E. granulosus and are not suited for species differentiation (manufacturer specifications must be observed). Cross reactions in cysticercosis and nematode infections (filariae, ascaris, strongyloides) can occur. Differentiation of the species can be attempted using ELISA, which are based on the use of highly purified or recombinant E. multilocularis antigens (Em10, Em2, Em18) /26/, or by Western blot using larval extract of E. multilocularis as antigen.

Species differentiation by serological methods can be achieved at a diagnostic specificity of 80–95% in the presence of high genus specific antibody titers. By contrast, differentiation is difficult in low or borderline concentrations as seen, for example, in non-vital E. granulosus cyst or after treatment /27/. Antibodies can persist at a low level for years after drug or surgical treatment.

A positive serological result must be confirmed by clinical findings in order to allow the diagnosis of echinococcosis. A negative serological result cannot rule out echinococcosis, especially in the presence of a typical lesion. Approximately 5% of E. multilocularis infections and 20–40% of E. granulosus infections are seronegative.

Microscopy

The diagnosis is confirmed by the detection of structures typical of Echinococcus in cyst fluid (protoscoleces, hooks) or by histopathological findings (PAS positive, laminar membrane, protoscoleces). It should be noted that protoscoleces of E. multilocularis are very rarely produced in humans who are considered to be dead-end hosts.

Molecular biological detection

The detection of Echinococcus specific DNA and mRNA from solid specimens is possible, but no adequate methods have been validated to date. Analyses should be performed in special (consultant) laboratories.

44.5.1.2 Cysticercosis

In infection with Taenia solium (pork tapeworm), humans can serve as both definitive and intermediate host. After infection, the larvae (oncospheres) hatch in the intestine and, via the bloodstream, migrate to various organs (muscles, central nervous system, eyes), where they lodge within a cystic structure. Cysticercosis is encountered primarily in southern Europe, South and Central America, Africa and India. Cerebral, spinal or ocular infection can lead to severe symptoms (seizures, intracranial pressure, loss of vision). In clinically suspected neurocysticercosis (by imaging techniques), initial confirmation of the parasitic infection is sought by serological analysis.

Specimen

Serum, cerebrospinal fluid (1–2 mL)

44.5.1.2.1 Laboratory findings

Antibody detection

Screening can be performed with serum. A positive IIFT or ELISA result must be specified by immunoblot assay (strong cross reaction with Echinococcus sp.). A positive finding indicates infection with larvae of T. solium (also in the muscles) and may only be interpreted as neurocysticercosis in the context of corresponding cerebral masses. A negative serological result is obtained in approximately 30% of cases of neurocysticercosis with a singular, usually non viable, cyst.

Microscopic detection

The differentiation is possible in tissue section prepared from surgical specimen.

44.5.1.3 Trichinosis

Trichinosis is encountered worldwide. In Germany, epidemic outbreaks are very rarely observed and rare cases of affected individuals are seen who imported the infection, for example after ingestion of trichinous meat in other regions. The species Trichinella spiralis is the pathogen of human trichinosis in most cases. The source of infection is raw or inadequately heated meat of infected animals (e.g., pork, wild boar, horses, bears, seals) which has not, or has inadequately, been officially examined for trichinae. The severity of the disease is strongly dependent on the number of larvae ingested with the infectious meat. It is assumed that the ingestion of more than 70 larvae leads to clinical disease /28/.

Incubation period

5–28 days.

44.5.1.3.1 Clinical symptoms

A low infection dose can cause non specific symptoms. In higher infection doses, the following symptoms point to trichinosis:

  • Early, acute infection (3–14 days following infection): intermittent, high fever, gastrointestinal symptoms
  • Acute infection (9 days to approximately 4 weeks): muscular pain during movement, facial edema. Difficulty swallowing and breathing; complications include myocarditis and encephalitis
  • Chronic infection: rheumatoid muscular pain; autoimmune reaction.

Mandatory reporting

In compliance with Article 7 of the German Infection Protection Act (IfSG), direct or indirect evidence of Trichinella spiralis is to be reported by the head of the diagnosing laboratory insofar as the evidence points to acute infection.

44.5.1.3.2 Laboratory findings

Laboratory diagnostic confirmation of Trichinella infection is based primarily on antibody detection. A trichinosis case is considered to be confirmed upon detection of the parasites in muscle biopsy (microscopy, PCR) or detection of specific antibodies by adequate test methods in the presence or absence of clinical symptoms.

Blood count

Eosinophilia in peripheral blood approximately 2 weeks after infection (up to 80%); markedly elevated serum creatine kinase in some cases.

Antibody detection

Not before the 2nd to 3rd week of the disease because no antibodies are produced during the early migration phase of the pathogens. Positive IIFT or IHAT findings should be verified by ELISA or Western blot /2829/. The detection of acute infection presupposes a significant increase in specific antibodies. The antibodies may persist at a low level for years, depending on the test method and individual immune response.

Microscopy

Unstained muscle biopsy specimen preparation from M. deltoideus, M. pectoralis (pressed between two slides, 40–80 × magnification) after the 4th week of the disease. Adult worms or Trichinella larvae are very rarely detected in the stool during the early infection phase.

Molecular biology

Detection of Trichinella specific DNA can be performed in the reference laboratory, primarily to determine the species and/or subspecies.

44.5.1.4 Toxocariasis

Toxocariasis, also called visceral larva migrans, refers to human infection caused by the larvae of the dog roundworm (Toxocara canis), or, less commonly, the larvae of the cat roundworm (Toxocara cati). The pathogens occur worldwide. Infections tend to occur more frequently in countries with low hygiene standards where pets are irregularly dewormed or not dewormed at all.

Risk of infection is especially high in children (geophagy) or individuals frequently coming into contact with soil. The eggs are shed in carnivore feces. In the environment, they require 2–3 weeks to embryonate into infective larvae (no direct infection).

The larvae hatch in the upper part of the intestine and, via the circulating blood or lymphatic pathways, migrate into the liver and through the lungs from where they spread throughout the body. They penetrate the surrounding tissue, involving, in particular, the liver and the CNS (Larva migrans visceralis) and, less commonly, the eye (Larva migrans ocularis) /30/. This may cause tissue hemorrhage and inflammatory reactions. In Europe, 1–8% of the healthy population are considered to be seropositive.

Incubation period

Days to months.

44.5.1.4.1 Clinical symptoms

The infection is asymptomatic in most cases. In stronger infection and depending on individual disposition, non specific symptoms such as fatigue, fever, cough, asthmatic symptoms or urticaria can be present. Symptoms of the ocular form of the disease include endophthalmitis and uveitis. The key symptom eosinophilia to hyper eosinophilia can be absent in the ocular form.

Mandatory reporting

None.

Specimen

Serum.

44.5.1.4.2 Laboratory findings

Serology is the most reliable method for diagnosis because adult worms do not develop in the human host and detection of the larvae in tissue is very difficult. Assays such ELISA /31/ or immunoblot are used because cross reactions with other nematodes (Ascaris, Trichella, Filaria, Strongyloides, possibly also Echinococcus) can occur. Serum antibody concentrations are usually higher in acute, symptomatic than in asymptomatic infection. Decision for treatment must be based on the serological findings and the corresponding clinical symptoms.

44.5.1.5 Strongyloidosis

Strongyloidosis is caused by thread worms of the genus Strongyloides sp., from which the two species S. stercoralis and S. fuelleborni have been identified as pathogenic to humans. The pathogen occurs worldwide with a high level of endemic infection in tropical and subtropical regions and also becomes increasingly significant in travel medicine /32/. Strongyloidosis must especially be watched for in older and immunocompromised patients.

Infection in warmer climates is acquired through direct contact with soil contaminated with infective larvae that penetrate the human skin In temperate climates, the disease is primarily transmitted from people to people by smear infection, where the infective larvae are shed with the stool. Autoinfection is possible in an existing infection and can lead to high parasite loads (hyper infection), particularly in immunocompromised patients.

Incubation period

Several weeks to one year.

44.5.1.5.1 Clinical symptoms

Immunocompetent patients are usually asymptomatic or show mild and non specific intestinal symptoms, transient pulmonary infiltrates (Löffler’s syndrome) and unknown eosinophilia /33/.

Hyper infection syndrome (cough, shortness of breath, fever) with a mortality rate higher than 80% may be seen in immunocompromised patients.

Mandatory reporting

None in Germany.

Specimen

Stool (unrefrigerated), serum.

44.5.1.5.2 Laboratory findings

Antibody detection and stool analysis are to be performed concurrently or in steps according to a gradual scheme.

Antibody detection

Available immunoassays for screening include ELISA, IIFT and Western blot with crude antigens, all of which are to a high degree subject to cross reactivity with other helminths (Filaria, Ascaris, Toxocara, Schistosoma, Echinococcus) and therefore have limited diagnostic specificity. A high antibody titer indicates that infection is likely. The development of an ELISA using recombinant Strongyloides antigen has shown promise. This method allows specific diagnosis and detects significant decrease in antibodies after treatment, but is not yet available for routine laboratories /34/.

Stool analysis

Fecal culture (copro culture) or larval migration methods (Harada-Mori, Baermann Wetzel) are suited for stool analysis. At least 6 stool samples are to be examined in clinically suspected strongyloidosis and mild infection but seropositive result.

Molecular biological analysis

PCR is said to have higher diagnostic sensitivity than microscopy /35/.

44.5.2 Hemato tissue parasites

In hemato tissue parasites the causative pathogen can be detected in the blood.

44.5.2.1 Toxoplasmosis

Toxoplasmosis is the most common parasitic zoonosis in Europe. The pathogen Toxoplasma gondii is the only species under the genus Toxoplasma, but there are at least three different clonal lines (types I–III) of different virulence.

The moderately virulent type II has been isolated in 70–80% of cases in Europe. The single celled parasite has low host specificity and can infect humans as well as numerous different animal species. The life cycle of Toxoplasma consists of an asexual replication cycle in the intermediate host (final stage = tissue cysts) and a sexual replication cycle in the definitive host (final stage = oocysts). Human infection is by oral ingestion of cyst containing tissue (mainly raw or inadequately heated meat) or cat excreted stages (sporulated oocysts) from the environment. In rare cases, transplacental infection (congenital toxoplasmosis) can occur.

Epidemiology

Since most infections are subclinical, the source of infection (meat, oocyst) can only rarely be determined in retrospect. The risk of infection can vary regionally. Significant aspects include climate, hygiene standards, eating habits and the rate of infection in animals entering the food chain.

In Germany, the level of endemic infection in pregnant women was approximately 90% in 1974 and is now assumed to be about 22%. A decline in prevalence in pregnant women by at least 10% during the last decade has also been reported from other European countries.

Incubation period

1–3 weeks.

44.5.2.1.1 Clinical symptoms

Most infections in immunocompetent individuals have a clinically asymptomatic course; only about 15% of infected patients develop transient lymphatic toxoplasmosis.

Severe, disseminated courses of the disease may develop in immunocompromised patients (HIV, post transplants, tumor disease) following reactivation of latent infections and are usually fatal if left untreated. The incidence of cerebral toxoplasmosis, which was feared to occur during the pre HAART era, has declined by 50–60%.

Congenital infection results from primary maternal infection during pregnancy and is seen in approximately 30% of cases. Following intrauterine infection, most neonates are asymptomatic at birth. In Europe, the clinically severe form of the disease (hydrocephalus, chorioretinitis, intracranial calcifications) is diagnosed in only about 5% of these cases /36/.

Ocular toxoplasmosis, which in Germany usually results from subclinical congenital infection, can cause impaired vision or loss of vision.

Mandatory reporting

In compliance with Article 7 Section 3 of the German Infection Protection Act (IfSG), cases of congenital infection are subject to mandatory reporting to the Robert Koch Institute without giving the name of the affected individual(s).

Specimen

The specimen used for selective laboratory diagnosis depends on the clinical manifestation and immune status of the patient.

Refer to Tab. 44-4 – Specimen collection in clinically suspected toxoplasmosis according to a graduated.

44.5.2.1.2 Laboratory findings

Antibody detection

The detection of specific antibodies is proof of infection because the Toxoplasma parasites are thought to persist in the body for life as intracystic stages (latent infection). Cross reactions with other pathogens are not to be expected.

Available serological test systems include numerous commercial assays based on lysate or recombinant antigens /37/ where specific IgG antibodies are quantified and expressed in international units (IU/mL). Despite this standardization, the resulting quantitative values are assay specific. Inter assay results may vary to such an extent that the follow-up examinations must always be performed in the same system.

The screening for Toxoplasma antibodies includes the determination of specific IgM and IgG antibodies. The detection of specific IgM antibodies can point to recent infection or the presence of persistent IgM antibodies and requires further clarification (Tab. 44-5 – Results of serology and determination of Toxoplasma infection stage).

High avidity IgG antibodies exclude acute infection in immunocompetent individuals, whereas the detection of IgG antibodies of low or intermediate avidity does not allow final assessment of the infection stage.

If an additional test for specific IgA antibodies does not allow to determine the precise time of infection, monitoring at intervals of at least 2 weeks is necessary.

High antibody titers are to be expected in patients with lymphadenitis, while medium to low titers are seen in subclinically infected individuals (especially pregnant women).

Patients with reactivated toxoplasmosis (e.g., cerebral toxoplasmosis) tend to have higher IgG titers (possibly also IgA titers) than controls but no IgM antibodies in their serum.

The schematic approach shown in Tab. 44-5 – Results of serology and determination of Toxoplasma infection stage cannot be applied to immunocompromised individuals and neonates with congenital infection.

Prenatal infection is considered to be confirmed if the child’s serum contains specific IgM and/or IgA antibodies postnatally and 2–4 weeks after birth and/or if comparative immunoblot IgG analysis shows at least one additional band compared to the maternal serum.

Moreover, the persistence of specific IgG antibodies at 12 months of life confirms congenital toxoplasmosis /38/.

Direct parasite detection

In vivo cultivation of Toxoplasma in laboratory mice is only performed in special laboratories. Microscopic detection of tachyzoites (acute infection) in Giemsa stained preparations is only possible at high parasite density as seen after immunosuppression. Histological and/or immunohistological detection of the pathogen in tissue (CNS, placenta, umbilical cord) can provide information regarding the stage of infection.

Molecular biological analysis

PCR is the method of choice for pathogen detection. The detection limit of in-house or commercial methods is approximately 5–250 Toxoplasma/mL, depending on the specimen used. It increases as a function of increasing parasite density (immunosuppression together with extended duration of the disease, no specific treatment) and following specimen concentration. The diagnostic sensitivity is low in immunocompetent individuals, short duration of the disease and after treatment (Tab. 44-6 – Specimen and diagnostic value of Toxoplasma PCR under clinical conditions). The detection of Toxoplasma DNA in body fluids confirms acute infection; a negative result does not rule out latent infection.

44.5.2.2 Leishmaniasis

Infections in humans are caused by more than 20 species of the genus Leishmania. Corresponding to their clinical manifestation, these species are classified into viscerotropic groups (visceral leishmaniasis) and dermatotropic groups (cutaneous and mucocutaneous leishmaniasis). Most leishmaniases are zoonoses and, usually, the reservoir hosts are various species of mammals. In some endemic areas, the infection is also transmitted by people-vector-people contacts. Leishmania are in their pro mastigote form transmitted by small midges (Phlebotomus sp. or Lutzomyia sp.), also called sand flies. In the intermediate human or animal host, obligate intracellular, round/oval shaped amastigote parasites develop (3–5 μm) that primarily multiply in reticuloendothelial cells.

Epidemiology

Distribution of leishmaniasis is closely dependent on the occurrence of potential vectors. Cases of leishmaniasis have been reported from all continents and from at least 88 countries. In Australia and Oceania, infections in humans have been limited to imported cases to date /44/. The number of cases in the affected countries varies considerably. According to the WHO,

  • 90% of all visceral leishmaniases are recorded in Bangladesh, Brazil, India, Nepal and Sudan
  • 90% of all known cases of mucocutaneous leishmaniasis stem from Bolivia, Brazil and Peru
  • 90% of cutaneous leishmaniases occur in Afghanistan, Brazil, Peru, Iran, Saudi Arabia and Syria /45/.
  • In Germany, leishmaniasis is an imported disease, but the possibility of autochthonous infection has repeatedly been discussed. 38% of all cases of cutaneous leishmaniasis and 97% of all cases of visceral leishmaniasis are imported from the Mediterranean region /46/.
  • The pathogen Leishmania infantum is endemic to the Mediterranean region. The regional prevalence of the infection in dogs as reservoir hosts is 20–80%.

Rare paths of infection include direct parasite transmission from people to people, for instance, drug abusers, blood and organ donations, infection prenatal and perinatal or inoculation of ulcerative material into the skin (immunization).

Risk groups include individuals staying in endemic regions, such as travelers, soldiers, individuals with a migration background, refugees, asylum seekers and immunocompromised individuals.

Incubation period

Weeks to months; the infection may also remain latent for years; reactivation of a latent infection by immunosuppression is possible.

44.5.2.2.1 Clinical symptoms

Morbidity after primary infection is 5–10%. A wide spectrum of diseases occurs where the clinical symptoms and the course of the disease depend on the type of the pathogen and the immune status of the infected individual.

Cutaneous leishmaniasis is the most frequently diagnosed form of the disease.

  • Cutaneous leishmaniasis (oriental sore): chronic, painless, dry or wet skin ulcer (old world), occurring singly or multiply; nodular or diffuse, possibly ulcerative dermal lesions (new world)
  • Mucocutaneous leishmaniasis: progressing destruction of tissue (mucosal membranes, cartilage), primarily in the face, due to necrotizing granulomatous inflammation
  • Visceral leishmaniasis (Kala Azar): the main symptoms are fever, anemia, pancytopenia, hepatosplenomegaly, hypergammaglobulinemia; immunosuppression (malnutrition, HIV infection, immunosuppressive therapy) is a risk factor for visceralization. High mortality if left untreated.

Differential diagnosis: hemato oncological diseases.

Mandatory reporting

Not applicable. Unofficial reporting to the reference center on the diagnosis and treatment of imported leishmaniasis at the Institute of Tropical Medicine in Berlin is welcome.

Specimen

Suspected leishmaniasis warrants the use of all diagnostic methods available according to a graduated scheme (Tab. 44-7 – Methods for detection of infection with Leishmania). If possible, diagnostics should be initiated upon consultation with an institute for tropical medicine with the means to perform cultures and species differentiation.

44.5.2.2.2 Laboratory findings

The diagnostic sensitivity of conventional methods such as microscopy and culture is inferior to that of molecular biological methods (PCR). Nevertheless, the reproduction of the pathogen by culture is recommended to have sufficient parasitic DNA available for species differentiation. Determination of Leishmania species is useful and necessary in suspected cutaneous leishmaniasis of the new world (L. braziliensis) to allow selective treatment.

Microscopy

Direct detection of parasites is achieved by dabbing or smearing cellular material on a microscope slide and staining the preparation with Giemsa stain. The typical amastigote stages (3–5 μm) are located intra cellularly in cytoplasm and in some cases appear extra cellularly (artefact). The pathogens can be identified based on their morphological characteristics visible after Giemsa staining (reddish nucleus and small, compact kinetoplast). Differentiation between the various Leishmania species is not possible. Culture media suited for in vitro cultivation of promastigotes are, for example, Schneider’s Drosophila Medium, Leibovic and NNN medium. A culture is incubated at 27 °C and has to sit for at least 3 weeks before it can be assessed as negative.

Molecular biological analysis

The identification of the causative Leishmania sp. exist possible only by molecular biological analysis (PCR). This method can also be used to amplify part of the DNA from stained microscope slides and identify the amplicon at the species level in retrospect /47/. Differentiating in-house PCR is offered by tropical medicine institutes and laboratories specialized on parasitology.

Antibody detection

Specific antibodies are usually detected at high titers in visceral leishmaniasis, low titers in mucocutaneous leishmaniasis and very low titers (approximately 70%) in clinically apparent cutaneous leishmaniasis. Antibodies are rarely detectable in the presence of single cutaneous lesions (L. tropica). Serological monitoring is performed in children presenting with cutaneous L. infantum infection to detect possible visceralization in good time. IIFT, ELISA and IHAT are suitable tests for screening, while immunoblot is suited for confirmation. Cross reactions, especially with trypanosomes (Chagas’ disease, African trypanosomiasis), may occur. Positive antibody response indicates existing, acute, treated or asymptomatic infection. Falsely negative findings are to be expected if the primary Leishmania infection was contracted in a state of immunosuppression.

44.5.2.3 Malaria

Malaria occurs in tropical and subtropical regions. The disease is endemic to Asia, Africa, Oceania, Central and South America and affects more than 100 countries.

The burden of malaria is concentrated in Africa south of the Sahara where most highly endemic areas of the pathogen Plasmodium falciparum are located.

P. vivax is the only malaria pathogen in the eastern world (Turkey, Syria, Morocco, Afghanistan, Caucasus), also occurs in some South-American countries and is rare in West and Central Africa, where the local Duffy negative population is less susceptible to infection by this species. P. ovale is a parasite primarily in West Africa, but not in the New World.

P. malariae can be encountered worldwide, but its local occurrence is small compared to other species.

The fifth human malaria species, P. knowlesi, has been restricted to Malaysia and some Southeast Asian countries to date.

Epidemiology

Plasmodia are transmitted by the bite of an infected Anopheles mosquito. The distribution of malaria is defined by the presence of suitable vectors, specific climatic and geographical prerequisites and a minimum of parasite (gametocyte) carriers in the population. More than 500 cases of malaria are reported in Germany every year. With rare exceptions, all of these cases are imported from abroad. Infections with P. falciparum can be potentially fatal and account for the high portion of approximately 80% of malaria cases /48/. Most malaria infections are acquired because of inadequate malaria prophylaxis. Regionally restricted transmission of the disease can be observed during the summer months under special conditions, when an infected mosquito was imported (“airport malaria”). People-to-people transmission between mother and fetus (diaplacental transmission) and by blood donation (transfusion malaria) are possible.

Incubation period

Tropical malaria (P. falciparum) 7–30 days; tertian malaria (P. vivax/P. ovale) 12 days to more than a year; quartan malaria (P. malariae) 18–50 days, P. knowlesi 5–6 days.

44.5.2.3.1 Clinical symptoms

Initial symptoms include fever, in many cases up to 39 °C or higher, pain in the limbs and back, possibly diarrhea, nausea and vomiting, shaking chills and attacks of sweating. The relatively non specific symptoms in the early stage of the disease do not allow clear clinical diagnosis. The further course of the disease is decisively influenced by the pathogen species and the immunocompetence or level of infection (semi-immunity) of the affected individual.

Malaria is characterized by intermittent fever, with one (tertian malaria) or two (quartan malaria) fever free days between attacks of fever. In tropical malaria, the temperature curve can be intermittent or sustained.

The increased pathogenicity of P. falciparum also results from cytoadherence of infected erythrocytes to endothelial cells, leading to disseminated dysfunction of the microcirculation. The most common resulting complications include renal insufficiency, cerebral malaria, respiratory insufficiency and severe anemia.

Tropical malaria can rapidly develop into a life threatening condition with a mortality rate of approximately 20%. Therefore, it is important to exclude malaria in every patient who presents with fever after returning from an endemic area (6 days to a year), independently of the kind of fever.

Mandatory reporting

In compliance with Article 7 Section 3 of the German Infection Protection Act (IfSG), direct evidence of the pathogen is subject to mandatory reporting by the laboratory without giving the name of the affected individual(s). Evidence is reported directly to the Robert Koch Institute (RKI) using a RKI reporting form.

Specimen

Thin and thick smears of capillary or EDTA blood; EDTA blood: 3–5 mL.

44.5.2.3.2 Laboratory findings

Clinical chemistry: thrombocytopenia and, in most cases, elevated LD.

Microscopy

Parasite detection is the most important method for species differentiation, quantification and management of severe tropical malaria. The Giemsa stained blood preparation (fixed thin and thick blood smear) is considered to be the gold standard for diagnosis. The nucleus of the parasite stains reddish violet and plasma stains gray blue. Giemsa stain achieves reproducible staining and high stability at long term storage /49/.

The following must be observed in the production of appropriate blood preparations /50/:

  • The time period between collection of the blood sample and preparation of the blood smears should not exceed one hour. A longer transport time of EDTA blood or refrigeration of the sample will have a negative effect on parasite and white blood cell morphology
  • Schnüffner’s dots are only optimally visible in recently collected blood samples and after a staining time of ≥ 40 minutes. In stored EDTA blood, they are poorly visible or not visible at all.
  • Mature gametocytes can ex flagellate already after 15 min. and thus be confused, for example, with spirochetes.

Definitive diagnosis of malaria is based on the detection of Plasmodia in the blood smear. In low parasite density (below 1%), thick blood smears with a 10-fold (or higher) concentration factor are better suited for parasite detection. The non hemolyzed blood smear is then used for species differentiation and quantification. Identification of the Plasmodium species is based on the typical morphological appearance of the pathogens in different developmental stages and the infected erythrocytes (Tab. 44-8 – Plasmodium species and their developmental stages in blood smear). Microscopic assessment is to be performed with oil immersion (400–1,000-fold magnification). Platelets and stain particles deposited on erythrocytes represent a common source of error. In patients from southeast Asia diagnosed with tertian/quartan malaria by light microscope, the potentially fatal infection with P. knowlesi must also be considered /51/.

In the strongly suspected presence of malaria and negative tests on microscopy (200 visual fields examined on thick smears at a magnification of 1,000 x), repeat tests for Plasmodium detection are needed at 12–24 hour intervals, independently of the pattern of the fever curve. In positive findings, especially in P. falciparum infection, the blood parasite density is to be determined. In parasitemia above 5% (i.e., 5 in 100 erythrocytes contain parasites, not counting the non dividing stage of gametocytes), the infection is considered to be life-threatening and requires close clinical monitoring.

In low parasite density (below 1%), the density can be determined on thick smears based on the calculation of the parasites counted in the visual fields per leukocyte against the leukocyte count which was either determined in the laboratory or estimated at approximately 8000 cells per μL of blood.

Antigen detection

Malaria rapid diagnostic tests (RDTs), which are usually based on immunochromatography, are available for the fast detection of malaria. RDTs use whole blood or serum/plasma to detect the P. falciparum specific antigens histidine rich protein-2 (HRP-2) and lactate dehydrogenase (PfLD) as well as pan plasmodium antigens such as aldolase or the parasite specific LD (pLD). According to the WHO recommendations, a detection limit of at least 200 parasites/μL or 95% referred to microscopic detection is acceptable.

However, the quality of the RDTs of individual manufacturers varies significantly /52/. A high detection rate can be assumed for the P. falciparum antigen at a parasite density above 1,000 parasites/μL and above 500/μL for P. vivax/P. ovale but performance is poor for P. malariae /53/.

Species differentiation is not possible in many cases because, for instance, the pan malaria antigens pLD and aldolase are also expressed by P. falciparum gametocytes. It must also be noted that antigen specific lines may remain positive for a long time after treatment (e.g., HRP-2 up to 28 days). Falsely negative results have been reported in very high and low parasitemia. The tests are suited for initial emergency diagnosis if no personnel is available experienced in microscopy. However, both positive and negative RDT results under these conditions must be verified by another, microscopic or molecular biological method.

Molecular biological analysis

Molecular biological analysis is not applied in acute diagnosis of malaria (detection limit) under clinical conditions.

In special cases, however, PCR is helpful to confirm the microscopic diagnosis:

  • In low parasite density during the very early stage of infection or in semi-immune individuals /54/.
  • For differentiating between morphologically similar forms (P. vivax, P. ovale, P. malariae, P. knowlesi)
  • For detecting mixed species infection or in samples collected post mortem.

Quantitative real-time PCR methods are suited to control parasitemia. The detection limit of PCR is considered to be inadequate for the screening of blood donors.

Antibody detection

The determination of specific antibodies plays no role in acute diagnostics because antibodies are not detectable until 2–3 weeks after infection. Antibody tests are useful to confirm a recently treated infection in retrospect. (antibodies are detectable for up to 3–6 months after infection). Antibody tests are also useful in excluding subclinical infection, especially in potential blood donors who originate from or spent some time in endemic areas. Suitable serological assays include IIFT using culture derived P. falciparum antigen or ELISA using culture derived and/or recombinant antigens. Cross reactions with other pathogens are not to be expected, except in the very rare case of Babesia infection. Cross reactions between P. falciparum and P. malariae are more pronounced than between P. falciparum and P. vivax/P. ovale. The detection limit of tests based solely on P. falciparum antigen is considered to be inadequate for the screening of blood donors /55/. Since a borderline result will already imply exclusion of the blood donor, the quality of screening assays needs to be verified by using weak positive reference sera (P. falciparum, P. vivax).

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Table 44-1 Laboratory detection of infection with Entamoeba histolytica

 

Methods

Clinical
presentation

Sample

Micros-
copy

Antigen
detection

Serology

PCR

Asymptomatic intestinal infection

Formed stool (cysts)

x

x

(x)

In positive stool test results

Intestinal amebiasis, amebic colitis

Hemor-rhagic mucoid stool, serum

(x)

x

x

x

Extra intestinal amebiasis

Tissue abscess sample,
serum

(x)

x

x

x, recommended; (x), partially recommended; (–), not recommended

Table 44-2 Results of two different commercial assays for lamblia antigen detection depending on storage and processing of the stool sample

 

 

Results

Days after
collection
of the stool
sample

Test

Native
Refrige-
rator

Native
–20 °C

SAF*-fixed
RT

Formalin-
fixed (10%),
RT

2

ICT

+++

n.d.

+++

+++

ELISA

+++

n.d.

negative

negative

4

ICT

+++

n.d.

++

++

ELISA

+++

n.d.

negative

not def.

7

ICT

+++

n.d.

+

++

ELISA

+++

n.d.

negative

not def.

14

ICT

+++

++++

+

++

ELISA

+++

++

negative

negative

The stool sample examined under the microscope contained numerous cysts.

ICT: qualitative immunochromatographic test; recommendation for stool sample examination: as soon as possible after sample collection; storage for not more than 24 h at 2–8 °C; long-term storage at –20 °C; no processing with formalin or solutions containing formalin derivatives.

ELISA: qualitative cassette enzyme immunoassay; recommendation for stool sample examination: fresh or just frozen; storage for not more than 72 h at 2–8 °C; deep freezing for long term storage; unfixed.

Abbreviations: RT, room temperature.; n.d., not done; + to ++++, intensity of positivity.

* SAF solution from a commercial concentration.

Table 44-3 Methods for detection of Schistosoma sp. according to a graduated scheme (step 1–3)

Clinical
manifestation

Pathogen

Speci-
men

Sero-
logy

Micros-
copy

Histology

Cercarial dermatitis
(swimmers itch)

Schistosoma sp.

Avian
schistosomes

(1)

Katayama
syndrome (fever
> 38.4 °C, eosinophils > 10%)

Especially

S. mansoni,

S. japonicum

Serum

(EDTA

blood)°

1

2

In positive serology

Urogenital
schisto-somiasis

S. haema-tobium

Serum, random urine* 10–50 mL, 24-hour*

1

2

In positive serology

(3)

Biopsy in repeated egg negative results and strong clinical suspicion

Intestinal
schisto-somiasis

S. mansoni

S. japonicum

S. intercalatum

S. mekongi

Serum, 3 stool samples

1

2

In positive serology

(3)

Biopsy in repeated egg negative results and strong clinical suspicion

* Late in the morning, if possible, and after physical effort (e.g. stair climbing).° In special institutions possibly PCR from 7.5 mL EDTA blood. The numbers 1, 2 ,3 dedcribe the sequence of investigations; Abbreviations: (1) low grade; (2) medium grade; (3) high grade; (–) not recommended.

Table 44-4 Specimen collection in clinically suspected toxoplasmosis according to a graduated (step 1–3)

Manifestation

Specimen

Serum

EDTA
blood

CSF

Other
body fluids

Tissues

Lymphadenitis

1

(2)

(2)* (lymph nodes)

Ocular
toxoplasmosis

1

2 (anterior chamber fluid, vitreous body fluid)

Toxoplasmosis
during pregnancy

1

(3)

(2) (amniotic fluid)

Congenital
toxoplasmosis

1 (from mother and child)

1 (child)

(1) (in
cerebral
infection)

2 (umbilical cord blood, amniotic fluid)

2 (caul, placenta, umbilical cord)

Reactivated
toxoplasmosis

2

1

1
(in ence-
phalitis)

2 (e.g. BAL)

* Unless the serological result provides evidence of acute infection. ° Depending on the focus of the center of infection. ( ) In special cases, only.

Abbreviations: (1) low grade; (2) medium grade; (3) high grade; (–) not recommended.

Table 44-5 Results of serology and determination of Toxoplasma infection stage

Infection

IgM antibodies

IgG antibodies

IgG avidity

No infection

Negative

Negative

Suspected
acute
infection

Positive

Negative

Positive

Positive

Low

Acute infection

Positive

Positive, significant
increase

Low

Latent infection

Negative

Positive

Positive

Positive

High

Unclear

Positive,
constant
concentration

Positive,
constant
concentration

Low,
intermediate

Table 44-6 Specimen and diagnostic value of Toxoplasma PCR under clinical conditions

Specimen

Clinical significance

CSF in cases of clinically suspected toxoplasma encephalitis (especially in AIDS patients)

≥ 1 mL

Mean diagnostic sensitivity 50–62% (81% in untreated and 20% in treated patients) /39/.

Amniotic fluid (following seroconversion in pregnancy)

≥ 5 mL

Maternal infection must have persisted for > 4 weeks; amniocentesis should not be performed before the 16th gestational week; no treatment (especially with pyrimethamine and sulfadiazine) to be started beforehand. Diagnostic sensitivity: 92– 97% /4041/ and/or 58–78% /42/.

Aqueous humor

≥ 200 μL

Higher positive rate in large, atypical, acquired lesions and under immunosuppression; diagnostic sensitivity 25–75% /43/.

Table 44-7 Methods for detection of infection with Leishmania sp. in immunocompetent individuals according to a graduaded scheme (step 1–3)

Clinical
manifestation

Pathogen

Specimen

Sero-
logy

PCR

Micros-
copy

Cutaneous
leishmaniasis

L. major, L. tropica, L. infantum, L. aethiopica, L. braziliensis, L. mexicana, L. amazonensis, L. panamensis, L. guyanensis

Skin (punch
biopsy*), serum

2

1

3

Mucocutaneous
leishmaniasis

L. braziliensis, L. guyanensis (rare),
L. panamensis (rare),
L. infantum (very rare)

Tissue°

Serum

1

2

3

Visceral
leishmaniasis

L. infantum,
L. donovani,
L. amazonensis
(rare)

Serum,
Bone marrow° EDTA blood

1

2

3

* 4 mm, taken from the margins of the most recent active lesion; ° Tissue (skin, connective tissue, bone marrow, spleen, liver, lymph node); EDTA blood at least 5 mL.

Abbreviations: (1) low grade; (2) medium grade; (3) high grade; (–) not recommended.

Table 44-8 Plasmodium species and their developmental stages in blood smear

 

P. falciparum

P. vivax

P. ovale

P. malariae

P. knowlesi

Incubation period in days

10–15, rarely several weeks

12–20, in some cases several months to a year

12–20

18–50

5–6?

Recurrence

No

Yes

Yes

Recrudescence

No

Key characteristics in peripheral blood smear

Red blood cells are not enlarged; most only ring forms (often multiply infected) and gametocytes in peripheral blood; delicate rings; crescent-shaped gametocytes; elongated membrane of the red blood cells not always visible

Red blood cells are visibly enlarged (1½ to 2-fold); fine Schnüffner’s dots may be present; mostly in various developmental stages; markedly boid trophozoites; large, gametocytes appear early contain scattered granules of pigment

Red blood cells are enlarged (1¼- to 1½-fold); often oval with fringed edges; Schnüffner’s dots are coarser; gametozytes resemble those of P. vivax, but are mostly smaller and fewer in number

Red blood cells are not enlarged; small compact ring forms, partly band shaped conspicious pigment granules; gametozytes resemble those of P. vivax, but smaller and fewer in number with coarser pigment

Similar to P. malariae, red blood cells may have some dots; gametozytes have spread pigment

Parasitemia

Unlimited; rarely up to 50%

Limited, 2–4%, rarely above 5%

Limited, 2–4%, rarely above 5%

Limited, rarely above 1%

Several 100 to 10,000 and more per μL of blood

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