Laboratory Diagnosis in Neurology, 1 Ed.

18 Parasitoses and Tropical Diseases

H. Reiber, E. Schmutzhard

Because of the increase in migration worldwide, it is essential that even European laboratories have a basic understanding of microbiological diagnostic tests for parasitoses and tropical infectious diseases. The present chapter is not a substitute for textbooks of parasitology and tropical medicine.

Direct and Indirect Methods of Detection

With respect to most protozoa and helminths that directly infect or indirectly involve the central nervous system (Schmutzhard, 2005), the gold standard for confirming a microbiological/parasitological diagnosis is direct detection of pathogens from the CNS in particular by CSF analysis. The neurological diagnosis (i. e., the clinical differential diagnosis) is also based on imaging procedures and, in many cases, directly on diagnostic aspiration biopsy. Tables 18.1and 18.2 provide an overview of the analytical methods available.

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Table 18.3 Eosinophilia in the blood and CSF associated with CNS parasitoses

Pathogen

Peripheral eosinophilia

CSF eosinophilia

Cysticercus cellulosae

Acute stage (usually prior to onset of neurocysticercosis)

+

Echinococcus spp.

+

Angiostrongylus cantonensis

++

+++

Gnathostoma spinigerum

++

+++

Trichinella spiralis

+++

+

Toxocara spp.

+++

++

Differential CSF Analysis

Eosinophilia. Many parasites invade the cerebrospinal fluid and/or the CNS parenchyma, usually at specific stages of their development. This facilitates direct detection of the pathogen by stereotactic brain puncture, aspiration of abscesses or cysts, or by lumbar puncture. The main indirect method of detection is characterization of the immune responses in the CSF. Eosinophilia in CSF and blood has an important part in directing suspicion toward parasitosis (Table 18.3).

Stage-dependent patterns of pathological response. CSF protein data can change dramatically between an early diagnostic puncture and the one performed 7 days after the onset of clinical symptoms, as illustrated in Fig. 18.1 for meningoencephalitis in children caused by Angiostrongylus cantonensis, a nematode endemic to Cuba. Apart from the eosinophilia which dominates the cytological picture, initially only a blood–CSF barrier dysfunction without a humoral immune response is observed. Just 7 days after the onset of clinical symptoms, however, the albumin quotient has normalized, and a humoral two- or three-class immune response has occurred.

This example is representative for early dynamics of CSF data patterns.

• Diseases with a dramatic fast appearance of symptoms (see also bacterial meningitis) are analyzed in the early stage of pleocytosis and severe barrier dysfunction.

• The humoral immune response appears no earlier than 7 to 14 days after onset of disease with normalizing barrier dysfunction.

• Chronic diseases with late recognition of symptoms are analyzed in the later stage with a humoral immune response but almost normalized barrier dysfunction (neurotrypanosomiasis, Fig. 18.2).

Trypanosomiasis. Trypanosomiasis, or African sleeping sickness (Lejon et al., 2003 a), is primarily recognized by infestation of the blood with trypanosomes; it is a treatable disease so long as only the blood is affected. Involvement of the CNS, by contrast, poses something of a diagnostic problem because at that stage the treatment is more risky. The conventional classification into early-phase and late-phase CNS involvement came about because of the uncertainty of the criteria for CNS involvement. The important question here is whether or not a slightly increased cell count, with or without detection of trypanosomes in the CSF, is enough to justify CNS treatment rather than systemic treatment. Recently, the intrathecal humoral immune response has provided far stronger evidence of CNS involvement (Lejon et al., 2003 a) than the demonstration of trypanosomes in the CSF: 96% of patients with CNS involvement show an intrathecal IgM class reaction. This highly sensitive response is supported by a simple test used in field analysis (latex card agglutination test; Lejon et al., 2002). Especially in the endemic regions of Central Africa, where there is also a high rate of infestation with tuberculosis and other infectious diseases of the CNS (e. g., HIV with opportunistic infections), characterization of immunoglobulin response patterns in the CSF could make a significant contribution to the differential diagnosis of neurological diseases (e. g., tuberculous meningitis, Fig. 18.2). The most modern diagnostic tests, which in the typical African setting are only practicable in the context of a scientific collaboration project, include the card agglutination test and “proteomic signature analysis,” both of which are highly specific and sensitive (Lejon et al., 2003 a; Papadopoulos et al., 2004). As trypanosomiasis is unexpected in the Northern hemisphere, it remains often undiagnosed or misdiagnosed (Lejon et al., 2003b).

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Fig. 18.1 Dynamics of the immune response in eosinophilic meningitis (Dorta and Reiber, 1998). (•) Data from a representative patient at the time of the first diagnostic lumbar puncture (within 3 days after the onset of symptoms): mild blood–CSF barrier dysfunction, but no intrathecal IgG, IgA, or IgM synthesis (70% eosinophils in the CSF). (■) Data from a second puncture 7 days later at the time of clinical recovery: the albumin quotient is close to normal, but an intrathecal three-class immune response with a dominant IgM fraction (86%) is observed.

Table 18.4 Stage-dependent immune response patterns in tuberculous meningitis (Kauffmann and Reiber, unpublished data)

Criteria

Early puncture (11 patients from Göttingen, Germany)

Late puncture (11 patients from Mumbai, India)

QAlb × 103 (median)

67

16

IgGIF*

28%

55%

IgAIF

82%

50%§

IgMIF

28%

55%

Lactate (> 3.5 mmol/L)

100%

65%

Cell count

233/μL

75/μL

* Usually identified as oligoclonal IgG.

† IgA synthesis, including cases where QIgA > QIgG in spite of IgAIF < 0.

‡ IgAIF was dominant in 85% of cases with a humoral immune response, and changing to a two-class or three-class response in the late course of treatment.

§ IgAIF was dominant in 80% of cases.

π IgMIF was dominant in 50% of cases.

Toxoplasma gondii infection. Infection of the CNS by Toxoplasma gondii is another important protozoan parasitosis, most often found as an opportunistic infection in HIV or immunosuppressed patients. The CSF immunoglobulin pattern may provide important clues to an opportunistic infection and indicate the need for more detailed specific antibody analysis (Fig. 18.3).

Tuberculous meningitis. In many parts of the world, such as Africa and India, tuberculous meningitis is very common. As in angiostrongyliasis, the CSF data pattern depends on the stage of the disease, i. e., a shift occurs between the early and late diagnostic tests. This is apparent when comparing European patients (relatively early puncture) and Indian patients (relatively late puncture) (Table 18.4):

• When puncture is early, an intrathecal dominant IgA class response and increased lactate levels in the CSF are frequently observed.

• When puncture is late, there is a shift to a lower albumin quotient, reduced lactate levels, a lower frequency of IgA synthesis, and a higher frequency of the IgM class response.

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Fig. 18.2 Differential diagnosis of trypanosomiasis and tuberculous meningitis (Lejon et al., 2003 a). (•) Typical immune response pattern in a 34-year-old patient with trypanosomiasis: normal barrier function, but a humoral three-class immune response with dominant IgM fraction; cell count, 139/μL; CSF lactate, 2.9 mmol/L; Trypanosoma-specific antibody index (AI) (IgG) = 4.5. (■) Data from a patient with tuberculous meningitis: severe blood–CSF barrier dysfunction (reduced CSF flow rate) and dominant IgA class response (IgAIF = 38%); cell count, 450/μL; lactate, 6.8 mmol/L.

The high sensitivity and specificity of the combined CSF data (intrathecal IgA synthesis with barrier dysfunction, increased lactate level, and moderate pleocytosis) are therefore much less pronounced if the first puncture is performed early in the course of the disease.

Bacterial meningitis. Because of the epidemiology and frequency of tuberculosis in India, tuberculous meningitis in that country is often diagnosed correctly even without CSF analysis. By contrast, without CSF analysis bacterial meningitis is often incorrectly interpreted. In European countries just the opposite occurs: bacterial meningitis is usually quickly treated, but the data combination typical of tuberculosis is not interpreted appropriately despite CSF analysis, and treatment is therefore often started far too late.

HIV encephalopathy with opportunistic infections. In large parts of Africa, up to 30% of people are HIV-positive, and neurological symptoms quickly prompt the suspicion of opportunistic infection. Since toxoplasmosis is a very common opportunistic infection and inexpensive to treat, it makes sense to offer treatment generously and without further CSF analysis for toxoplasmosis. Of course, this course of action interferes with the diagnosis of other infections. Unambiguous discrimination by CSF analysis between HIV encephalopathy and neurological symptoms due to opportunistic infections is shown in Fig. 18.3. The typical picture of a three-class immune response indicating toxoplasmosis (or cytomegalovirus infection, Reiber and Peter, 2001) is clearly distinguishable from pure HIV encephalopathy.

Relevance of CSF analysis. The CSF data patterns presented here show that differential CSF analysis can indeed contribute important information to the differential diagnosis of parasitoses seen in tropical neurology. However, this only is true under the high-tech conditions of clinical chemistry in the Northern hemisphere, but often not in those countries where tropical diseases are most common, where the economic conditions and structural framework often do not allow lumbar puncture—and even if it is carried out, determining no more than total protein content, cytology, and perhaps also glucose, represents an extensive program in these circumstances.

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Fig. 18.3 HIV encephalopathy and opportunistic toxoplasmosis (Reiber and Peter, 2001). (•) Data from a patient with HIV encephalopathy in an early phase; cell count, 22/μL; no oligoclonal IgG; HIV AI = 1.8; Toxoplasma AI = 0.9. (■) Data from a patient with chronic HIV encephalitis and opportunistic toxoplasmosis; increased albumin quotient, humoral three-class immune response; cell count, 140/μL; Toxoplasma AI = 9.2; HIV AI = 5.7; CMV AI = 1.0.

It is therefore of great relevance to develop more “lowtech” methods (Lejon et al., 2002), which are cheap and therefore not in the focus of industrial development.

Currently, the approach to differential diagnosis in developing countries differs dramatically from the system of differential diagnosis in Europe and North America, at least as far as specialized laboratories are concerned. Promising lowtech ideas for cost-effective and quick analytical tests without the need for large equipment (Lejon et al., 2002) are still much too rare.

References

Dorta AJ, Reiber H. Intrathecal synthesis of immunoglobulins in eosinophilic meningoencephalitis due to Angiostrongylus cantonensis. Clin Diagn Lab Immunol 1998;5:452–455

Lejon V, Legros D, Richer M, et al. IgM quantification in the cerebrospinal fluid of sleeping sickness patients by a latex card agglutination test. Trop Med Int Health 2002;7:685–692

Lejon V, Reiber H, Legros D, et al. Intrathecal immune response pattern for improved diagnosis of central nervous system involvement in trypanosomiasis. J Immunol Diseases 2003 a;187:1475–1483

Lejon V, Boelaert M, Jannin J, et al. The challenge of Trypanosoma brucei gambiense sleeping sickness diagnosis outside Africa. Lancet Infect Dis 2003 b;3:804–808

Papadopoulos MC, Abel PM, Agranoff D, et al. A novel and accurate diagnostic test for human African trypanosomiasis. Lancet 2004;363:1358–1363

Reiber H, Peter JB. Cerebrospinal fluid analysis—disease-related data patterns and evaluation programs. J Neurol Sci 2001;184:101–122

Schmutzhard E. Mikrobiologische Diagnostik im Liquor bei Parasitosen. In: Zettl UK, Lehmitz R, Mix E, eds. Klinische Liquordiagnostik. 2nd ed. Berlin: de Gruyter; 2005:329–346