Diagnostic Technologies: Selected Tropical Diseases

Diagnostic Technologies:Selected Tropical Diseases

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Conventional Diagnostic Techniques . . . . . . . . . . . . . . . . .

Direct Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Serologic Diagnostic Techniques . . . . . . . . . . . . . . . . . . .

Genetic Tools for Improving Diagnostic Techniques . . . .Monoclinal Antibodies . . . . . . . . . . .Nucleic Acid Hybridization Probes.

Diagnosis: Current Status for SelectedMalaria . . . . . . . . . . . . . . . . . . . . . . . . .Schistosomiasis . . . . . . . . . . . . . . . . . .Trypanosomiasis . . . . . . . . . . . . . . . . .Leishmaniasis. . . . . . . . . . . . . . . . . . . .Filariasis . . . . . . . . . . . . . . . . . . . . . . . .Leprosy . . . . . . . . . . . . . . . . . . . . . . . . .Tuberculosis . . . . . . . . . . . . . . . . . . . . .Diarrheal and Enteric Diseases . . . . .Acute Respiratory Infections . . . . . . .Arbovira

Summary .

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. . . . . . . . . . . . . . . .Tropical Diseases. . .. . .. . .. . .. . .. . .. . .. . .. . .

‘and Related Viral Infections .. . . . . . . . . . . . . . . . . . . . . . . . . . .

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LIST OF FIGURES

Figure No,8-1.

8-2.

8-3.

Enzyme-Linked Immunosorbent Assay Technique forAntigen or Antibody . . . . . . . . . . . . . . . . . . . . . . . . .Thin-Layer Immunoassay Technique for Detecting Antigen or

. . .Detecting. . . . . . . . . . . . . . . . . . .

Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Reverse Passive Hemagglutination Technique for Detecting Viruses . . . . . . .

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8Diagnostic Technologies;

Selected Tropical Diseases

INTRODUCTION

Diagnosis has three important functions. Oneis to determine the nature of the individual’s dis-ease for the purpose of deciding on an appropri-ate course of treatment. A second function is todetermine the prevalence of specific disease-pro-ducing organisms or agents in populations, to al-low assessments of the impact of public health in-terventions. The third is to find out about therange of diseases affecting a population, the im-mune stages of populations, etc., for the purposesof research.

Diagnosis of tropical diseases is a challenge forthe clinician or epidemiologist, because many ofthe diseases mimic a wide variety of infectiousprocesses by presenting similar or ambiguoussymptoms. In countries where these diseases areendemic, symptomatic ambiguity can also resultfrom the common occurrence of multiple infec-tions in a single individual. The proper identifi-cation of the species of the organism that is caus-ing disease can be critical in clinical management,especially in serious illness.

Although each tropical disease can be diagnosedby a number of different methods, diagnostic tech-niques vary in their usefulness under different con-ditions. There are several parameters to considerin evaluating the usefulness of a diagnostic test:sensitivity, specificity, predictive value, cross-reactivity, and precision (388).

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Sensitivity is the ability of a test procedureto find a disease-producing agent or diseasewhen it is present. A very sensitive test cor-rectly identifies all infected individuals. Neg-ative tests for individuals who have the dis-ease are “false negatives. ”Specificity is the ability of a test procedureto correctly determine that a disease-produc-ing agent or disease is not present. A very

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specific test will correctly identify all un-infected individuals. Positive tests for indi-viduals who do not have the disease are “falsepositives.”Predictive value is an interaction of sensitiv-ity, specificity, and the prevalence of the in-fection in the population being tested. Thesensitivity and specificity of a test will givedifferent numbers of false negative and falsepositive results depending on the prevalenceof the infection in the population tested. Ifa test is conducted in a group of patientsstrongly suspected of having the disease, veryfew false positives would be expected, sim-ply because very few people tested will ac-tually be negative. Thus, positive results willbe readily accepted as true. Conversely, if thesame test is conducted in a population inwhich very few people are thought to havethe disease, the positives would have a higherprobability of being false positives, and infact, false positives may outnumber true posi-tives. The negative test results will mostly betrue negatives.Cross-reactivity is an aspect of immunodi-agnostic tests related to, yet different from,specificity. A cross-reaction occurs when thetest correctly identifies the appropriate chem-ical entity, but that chemical happens to ex-ist in a different organism than the one testedfor. A false positive results.Precision is reproducibility, the ability of atest procedure to give consistent results inrepeated trials of the same sample.

Broadly generalizing, there are two categoriesof conventional diagnostic technologies:

1. Direct examination of blood, stool, urine,sputum, tissue biopsy, or cultured isolates

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154 ● Status of Biomedical Research and Related Technology for Tropical Diseases

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using simple equipment (e.g., a light micro-scope) and reagents.Serologic examination to detect antibodiesto the pathogen or to detect the pathogen orits byproducts (antigen) in a sample of thepatient’s blood. Serologic methods have im-portant advantages over direct examination,but require in most cases specialized equip-ment and reagents and have never achievedwide practical use.

Biotechnology is leading to the development ofnew diagnostic methods based on the use of mon-oclinal antibodies (MAbs) and nucleic acid (DNAor RNA) hybridization and promises to revolu-tionize diagnosis by offering quick, simple, accu-rate tests when and where they are needed.

The conventional methods of diagnosing infec-tious diseases and identifying disease-producingorganisms include the following:

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the clinical impression of the physician orother health worker (this is the most widelyused method of diagnosis);direct examination of clinical specimens (e.g.,blood, stool, urine, sputum, or tissue biopsy)using light or electron microscopy to iden-tify the disease-producing organism (e.g.,malaria parasites, intestinal amebae and hel-minth eggs, leishmanial organisms, mycobac-teria that cause tuberculosis and leprosy,filaria);X-ray or computed tomography (CT) scanto image internal pathogens (e.g., amebic ab-scesses, echinococcal cysts); occasionally use-ful, but not routine;xenodiagnosis, by permitting an insect vec-tor to feed on a patient and then examiningthe insect by light microscopy to look for thedisease-producing organism after it has multi-plied to a detectable density (e.g., to diag-nose Chagas’ disease);culture of a specimen from a patient in a testmedium, tissue culture, or by inoculationinto an animal to allow it to multiply to adetectable density, followed by direct exam-

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ination for the disease-producing organism,or use of serologic techniques (e.g., for manybacteria and viruses);skin tests, demonstrating hypersensitivity re-actions to disease antigen (e.g., to diagnosetuberculosis);serologic methods to detect antibodies to thepathogenic organism in the patient’s serum;anddemonstration of a pathogen byproduct,such as an antigen by chemical methods (e.g.,schistosomes).

Photo credit: Office of Technology Assessment

Xenodiagnosis, by allowing laboratory-raised reduviidbugs to feed on suspected Chagas’ disease victim.

Ch. 8—Diagnostic Technologies: Selected Tropical Diseases ● 155

CONVENTIONAL DIAGNOSTIC TECHNIQUES

Direct Examination

Laboratory diagnostic methods that directlyidentify a disease-producing organism by micros-copy of clinical samples can provide a definitivediagnosis. Constraints on this approach includethe following:

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specially trained personnel are required;the work quickly becomes boring and repe-titious, while still demanding concentrationand attention to detail;procedures are relatively time-consuming andrequire equipment and materials that areoften inadequate in endemic countries;in some cases, the organisms are not detect-able either because:—they are not present in routine clinical sam-

ples (e.g., the organisms that cause trichi-nosis, toxoplasmosis, hydatid disease,hepatic capillariasis, toxocariasis);

—they are not detectable in routine clinicalsamples until a later stage of infection (e.g.,filaria); or

—their densities are very low, making sen-sitive diagnosis difficult (e. g., in chronicmalaria and chronic Chagas’ disease); and

identifying the disease-producing organismis difficult because some species appear simi-lar (e.g., intestinal amebae, malarial para-sites, the African schistosomes with termi-nally spined eggs, leishmanial organisms).

Serologic Diagnostic Techniques

Serologic diagnosis depends on two types of im-munologic methods, which are discussed furtherbelow:

methods for detecting specific antibodies (theimmunoglobulins IgG, IgM, IgA, IgE, or IgD)in a sample of the patient’s blood; andmethods involving the use of immunoglobu-lins for the detection of antigen (the disease-producing organism or its byproducts) in asample of the patient’s blood.

For the detection of antibodies, there are a va-riety of immunologic methods, all of which canbe adjusted by dilution of reagents to give a meas-ure of intensity of reaction (a titer). Because thevarious immunoglobulins have different roles anddevelop at different times in an infection, differ-ent titers will be obtained at various stages in theinfection. (For instance, the immunoglobulin IgMis usually an early but short-lived immune re-sponse to infection, whereas IgG production de-velops more slowly and then continues long af-ter resolution of the infection. ) A positive test forantibodies may indicate current infection or theimmunity that follows exposure to the infectiousagent. In some cases, the test may remain posi-tive for extended periods after the infectious agenthas been eliminated by drug treatment.

For the detection of antigen, methods have notbeen widely developed until recently, for techni-cal reasons. The current advances in MAb tech-nology make antigen detection more feasible.

Serologic Methods for Detection of Antibody

The principal serologic methods for detectingantibodies in a patient’s blood are describedbelow,

1. Complement fixation (CF) test. This methodis well established and has been applied to all vi-ral, bacterial, and parasitic diseases. Complement,a substance normally present in the blood, fixes. -or binds antigen to antibody. The CF test is per-formed by adding the known antigen and comple-ment (prepared in the laboratory) to an individ-ual’s serum in a test tube. If antibodies are presentin the patient’s serum, antigen-antibody comp-lexes are formed. No reaction indicates a lackof specific antibody. The CF test requires well-standardized reagents, some of which have limitedshelf-life or limited availability in the tropics.

The CF test is widely used for viral diagnosisand is most effective for parasite diagnosis ofAmerican trypanosomiasis (Chagas’ disease) and


schistosomiasis. It is highly specific, but relativelyinsensitive. It requires large amounts of antigenper test. The CF testis performed adequately onlyat specialized centers, and it is not considered atest for general, practical use in the tropics (388).

2. Agglutination assays. Clumping of particlesdue to the interaction of antibody and antigen isthe basis of all agglutination tests. In most varia-tions, the disease-producing organism or its by-product (antigen) is fixed to particles, and the par-ticles are added to a test sample that may containthe complementary antibody. If the complemen-tary antibody is present, the particles agglutinate.Each variation of this technique has acquired itsown name. The principal agglutination techniquesfor detection of antibody are described below.(The complementary techniques for detection ofantigen are noted in a later section. )

a. Direct agglutination test. In the direct agglu-tination test, whole organisms suspected of caus-ing disease are added to the individual’s serum.If antibody is present in the serum, the organismsclump together. The test is simple, but it requirespure, stable antigen preparations (from culture invivo or in vitro), the test result interpretation issubjective, and there can be complicating auto-agglutination reactions that result in false positives.

b. Indirect hemagglutination (IHA) test. In IHAtests, the antigen is attached to a carrier, such asspecially prepared red blood cells or latex parti-cles. When the individual’s serum is added, theparticles clump if specific antibody is present. II-1Atests have been developed for malaria, Chagas’disease, leishmaniasis, amebiasis, rotavirus, andhydatid disease. IHA tests are simple to perform,can be used to test minute volumes of serum, andcan be automated but the end-point reading is sub-jective. The preparation of test particles is not re-producible, the antigens have short shelf-lives,and there are problems in standardizing antigensfrom batch to batch.

c. Hemagglutination inhibition (HI) test. Vari-ous micro-organisms can clump or agglutinate redblood cells from test animals under specified con-ditions. In the HI test, prepared red blood cells,an appropriate micro-organism (e.g., a virus), anda test serum are combined. If the individual’sserum contains antibody specific to that virus, the

hemagglutination will be inhibited, because theantibody combines with the organism and cannotreact with the red blood cells. The lack of agglu-tination indicates a positive test.

3. Neutralization test. Many viruses damagecultured cells in certain detectable ways. Whentest serum containing a specific antibody is addedto a test well containing the cell culture and thevirus (both known quantities prepared in the lab-oratory), the damage is inhibited, indicating apositive test for the antibody. Neutralizing anti-body is usually the first to appear, early in theacute phase of the infection.

4. Precipitin (or immunodiffusion) test. Withthis method, two separate wells are cut into asemisolid substrate such as agar. Serum compo-nents are put in one well and antigen in the other.As they migrate, an observable precipitation lineis formed where antibody and antigen combine.No precipitation line forms in the absence ofantibody.

Counterimmunoelectrophoresis (CIE) and im-munoelectrophoresis are adaptations of the pre-cipitin test in which an electric current is passedthrough the substrate, causing the reactive com-ponents to migrate more rapidly and with greaterresolution.

The relatively simple diffusion methods aresuited to the tropics, but these methods are oflimited practical value because of their insensitiv-ity and the long period of time before results. Theelectrophoretic methods require equipment anda power supply, and the results are complex andsomewhat difficult to interpret (98).

5. Labeled immunodiagnostic reagent assays.The various types of labeled immunodiagnosticreagent assays all involve a similar procedure. Thetest antigen is attached to a slide or test well (e.g.,blood drops from malaria-parasitized monkeysare dried on microscope slides; in vitro culturedorganisms are fixed to plastic test wells). The pa-tients’ serum is incubated as a drop on the slideor in the well. If antibody is in the serum, it bindsto the fixed antigen. The excess serum is washedoff, leaving the antibody sticking to the antigenon the slide. Then a second antibody is added thatreacts with any antibody-antigen complexes that


were formed. This second reaction depends on theunusual structure of antibodies—one end of anantibody is a totally unique fit for just one anti-gen, and the other end is a totally generic mole-cule. The second lab-prepared antibody has aunique end which recognizes the generic end ofother antibodies. This second antibody is alsolinked to a chemical marker that is detectable—afluorescent molecule, a radioactive molecule, oran enzyme.

One type of labeled immunodiagnostic assay,the indirect fluorescent antibody (IFA) test, usesslides prepared with antigen attached (usually afixed, cultured organism). After serum antibodybinds to the antigen, the second lab-prepared an-tibody is added to react with any antibody-anti-gen complexes that were formed. This secondantibody is linked to a chemical marker thatfluoresces under ultraviolet light. A special micro-scope is used to detect fluorescence. If the slidefluoresces, it indicates that the original human se-rum contained the antibodies in question. Micro-scope slides are easily prepared with antigens sta-ble for long periods for a number of viruses,bacteria, and parasites. Disadvantages of IFA testsare that a well-maintained, carefully calibrated,and expensive fluorescent microscope is needed,the test is time-consuming and somewhat subjec-tive, and with many helminths, there are cross-reactions (388,421)

The enzyme-linked immunosorbent assay (ELISA)is complex to describe, yet simple and elegant inits determination. It is rapidly being adapted tothe diagnosis of a wide range of organisms. Asshown in figure 8-1, the ELISA for the detectionof antibody involves attaching specific antigen (aknown quantity of test organisms prepared in thelaboratory) to a test well or plate, then addingthe patient’s serum with suspected specific anti-body, then adding a lab-prepared second antibodywhich is linked to an enzyme. The final compo-nent added is a chemical substrate whose colorchanges by the action of the enzyme. The degreeof color change is read qualitatively by eye orquantitatively by photometric instrument to givean indication of the amount of antibody presentin the serum specimen.

The ELISA uses small sample volumes. Largenumbers of specimens can be processed, so theprocedure is useful for epidemiologic studies. Fordeveloping countries, the ELISA has clear advan-tages, since it can be done in simple laboratories,and the reagents are stable if refrigerated. Wide-spread field testing of ELISA procedures is underway for African sleeping sickness and Chagas’ dis-ease, leishmaniasis, amebiasis, malaria, filariasis,and schistosomiasis (82,353,389) One great ad-vantage of the ELISA procedure is that the resultcan often be judged positive or negative by thenaked eye. ELISA tests are rapidly being devel-oped and improved.

The radioimmunoassay (RIA), a procedure sim-ilar to ELISA, can be carried out using a secondantibody labeled with a radioactive compound,which is then read in a scintillation’ counter. RIAis very sensitive and very quantitatively accurate,but it requires a laboratory equipped to deal withradioactive isotopes, which carry a risk and havea short shelf-life. RIA has been a valuable researchtool, but not a practical diagnostic tool (335).

6. Other tests: circumoval precipitin tests(COPT) are examples of the variety of serologictests used for detection of specific pathogens. TheCOPT for schistosomiasis uses standardized pre-pared schistosome eggs obtained from animal in-fections. The eggs are incubated in patient sera,which causes a characteristic precipitate to formaround the egg, if antischistosomal antibodies arepresent.

The thin-layer immunoassay for the detectionof antibody is depicted in figure 8-2. This tech-nique uses antigen in test plates to capture spe-cific antibody from test serum. The antigen-anti-body complexes form a thin layer that attractswater. When the test plate is exposed to watervapor, water visibly condenses on the immunecomplexes in large droplets which form a patterndistinct from the background. The thin-layer im-munoassay has been used to detect the presenceof viruses, schistosomes, and amebae. It requiresrelatively large amounts of antigen, however, andin some cases has low sensitivity (421).


Figure 8-1 .—Enzyme=Linked Immunosorbent Assay (ELISA) Technique forDetecting Antigen or Antibody

1. Detection of antigen in a clinical sample:● Antibody to the antigen being tested for is adsorbed to a plastic plate.● The clinical sample is added. If the specific antigen is present, antigen-antibody complex forms on the

plastic plate.● A second antibody to the antigen, with a specific enzyme attached, is added to the plate. The enzyme-

Iabeled antibody adheres where antigen-antibody complexes were formed.● An enzyme substrate is added. If it comes in contact with the enzyme, a reaction occurs that produces

a visible color change, If no complexes are formed, there is no reaction.Il. Detection of antibody in a clinical sample:

● The process is the same as the one for detecting antigen, but includes one additional step: known an-tigen is added, resulting in one more layer in the final complex.

SOURCE: World Health Organization, Rapid Laboratory Techniques for the Diagnosis of Viral Infections, Technical Report Series#661 (Geneva: WHO, 1961).

Serologic Methods for Detection of Antigen after clearance of an infection. Thus, false nega-

The serologic techniques described above focustive and false positive results often occur.

on antibody-detection; which has two inherent Direct immunologic detection of the antigen it-disadvantages (388): 1) there is always a delay be- self can be preferable. A serious drawback of thistween infection and development of a detectable approach is that, compared to the presence of an-level of antibody; and 2) antibody levels persist tibodies, antigen presence is a short-lived occur-

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rence, especially for viral diseases. Current sero-logic methods for detecting antigen are describedbelow.

1. Labeled immunodiagnostic reagent assays.The principal methods for detecting antigen arelabeled reagent immunoassay that are modifica-tions of the ELISA, RIA, or IFA tests for antibod-ies (described above) and employ a known spe-cific, lab-prepared antibody as the first reagentto adhere to the plastic well. The ELISA techniquefor the detection of antigen is shown in figure 8-1.

Labeled immunodiagnostic assays for detectingantigens have been applied to viral infections,amebiasis, toxoplasmosis, onchocerciasis (93),schistosomiasis (84), malaria, Chagas’ disease,other protozoan infection (8), acute respiratoryinfectious (ARIs) caused by bacteria, and for de-tection of certain bacterial toxins causing diarrhealdiseases. These tests can identify minute quanti-ties of antigen, but their sensitivity has still beenless than the sensitivity of direct examination tech-niques. As newer methods using MAbs are de-veloped, sensitivity should improve greatly.

2. Agglutination assays. As indicated earlier,clumping of particles due to the interaction of an-tibody and antigen is the basis of all agglutina-tion tests. In the following variations, the disease-producing organism or its byproduct (an antigen)is detected by complementary antibody fixed toparticles which are added to a test sample.

In the coagglutination (COA) test, a bacterium(Staphylococcus aureus) is used as the earner par-ticle for a specific antibody. When the antibodyis mixed with a serum sample, it will agglutinateif the bacteria of interest are present. In a similartest, the latex agglutination (LA) test, latex par-ticles are the earners. Technical considerations de-termine which method is superior for the diag-nosis of any particular agent.

The reverse passive hemagglutination (RPHA)test, shown in figure 8-3, is used for rapid detec-tion of viruses (smallpox, arboviruses, and hepa-titis B). Here again, specific antibody is fixed toparticles that agglutinate if the appropriate anti-gen is present in the test serum.

Figure 8-3.—Reverse Passive Hemagglutination(RPHA) Technique for Detecting Viruses

Detection of viruses in a clinical sample:● Known antibody is added to and adheres to the surface of treated

erythrocytes (red blood cells).● A clinical sample, suspected of containing the specific virus, is

added. If the virus is present, complexes are formed linkingantibody-coated erythrocytes together, visible in the test tubeas an agglutination pattern.

SOURCE: World Health Organization, Rapid Laboratory Techniques for theDiagnosis of Viral Infections, Technical Report #661 (Geneva: WHO,1981).


Constraints on Serologic Diagnosis

Serologic diagnostic techniques provide someattractive technical options. Such methods can bevery effective in epidemiologic surveys (203).Some of these methods have been adapted for fielduse in tropical areas (e.g., by absorbing a fewdrops of blood onto a filter paper strip for laterexamination in the laboratory). Serologic methodsalso are more easily automated than techniquesof direct examination and thus have increased pre-cision. Despite these features, however, very fewsuch immunologic methods have proved to be ofpractical value in routine diagnostic laboratorypractice. Most have remained in the research lab-oratory (388).

Several constraints limit the usefulness of sero-logic techniques in medical practice or for publichealth measures:

● Serologic procedures require sophisticatedlaboratory instruments and equipment. Inmany developing countries, basic servicessuch as transport, electricity, and water areinadequate or unreliable, and trained person-nel are lacking.

● In industrialized countries, where the expen-sive, sophisticated tests are feasible, few

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centers specializing in “exotic” diseases havethe demand to justify doing the procedures.The result is that most medical facilities relyon the conventional techniques of direct ex-amination.Parasite antigens are highly cross-reactive.Cross-reactivity, coupled with the presencecommonly of more than one organism in anindividual, makes the interpretation of sero-logic tests difficult.Test reagents vary widely, and different lab-oratories can report grossly divergent resultsusing the same batch of antiserum. The needfor standardization and for high-quality anti-gens is acute (177,388).“Paired sera” (from patients in the acute andconvalescent stages of illness) may be re-quired if changing antibody titer is a criterionfor diagnosis.Many individuals are reluctant to have blooddrawn.

Many of the antigens needed for serodiagnos-tic tests can be cultured in vitro. In vitro cultiva-tion techniques are improving rapidly for manyof the agents of tropical diseases (296). The othermajor source of precisely defined antigens fordiagnosis will be recombinant DNA technology.

GENETIC TOOLS FOR IMPROVING DIAGNOSTIC TECHNIQUES

It is one thing for a university medical centerto undertake the diagnosis of a single patient re-cently returned from the tropics and quite anotherfor a survey team to apply diagnostic methodsunder field conditions to large numbers of localpeople in isolated, endemic, tropical areas. Foruse in the field, there is a great need for simple,reproducible, and inexpensive diagnostic meth-ods that can provide a clear-cut indication of thestatus of an infection, and a specific identifica-tion of the causative agent. This need is especiallyacute because of the current work on developingvaccines for a variety of tropical diseases (see ch.7). Without a means for accurate determination(or at least reliable estimation) of the prior ex-posure and immune status of vaccinees and con-trols, and their post-inoculation followup, validfield testing of these vaccines will not be possible.

Biotechnology (see ch. 5) is leading to the de-velopment of new diagnostic methods based onthe use of MAbs and recombinant DNA (or re-combinant RNA) techniques. These techniques,which are advancing rapidly, hold out a promiseof accuracy and simplicity. In addition, they gen-erally require smaller amounts of clinical samplesthan have heretofore been necessary. DNA andRNA hybridization tests hold great promise, butfurther development and testing are necessary be-fore many of the tests can achieve widespread usein the field (137).

Monoclinal Antibodies (MAbs)

Immunodiagnosis using MAbs is building onthe past 20 years of research in serologic diagnos-tics. The revolution is in making these diagnostic

162 . Status of Biomedical Research and Related Technology for Tropical Diseases

test procedures much more sensitive and specific,more reproducible, faster, and more economical.Three diagnostic uses of MAbs are being devel-oped (84):

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immunoassay of high specificity, i.e., mak-ing existing serologic diagnostic tests betterby production of purer reagents;“two-site” immunoassay of antigen (the patho-genic organism itself or its byproducts) fromappropriate body fluids, using two mono-clonals directed against different binding sitesof one antigen—one MAb to catch the anti-gen and another to label it for detection; andthe use of cross-reactive MAbs to absorb cer-tain common antigens out of crude mixtures,making possible the sensitive and specific de-tection of an antigen of interest.

MAbs have been used to distinguish betweenorganisms that cross-react in conventional sero-logic tests, e.g., Trypanosoma cruzi and Leish-mania braziliensis (71); between closely relatedspecies of New World Leishmania (283); betweenstrains within single species of Theeileria (277) andLeishmania (145); between genetic variants (43)or life cycle stages (111) of Trypanosoma rho-desiense; and between larval and adult Schisto-soma mansoni (332).

Assays of antigen in which MAbs are affixedto cellulose fibers or other solid substrates (e.g.,plastic test wells) have been referred to as “dip-stick technology” (136). Antigen present in blood,urine, stool, etc., binds to the MAbs, and the com-bination is detected by a second antibody throughan ELISA-like color reaction. Immunodiagnosisusing MAbs as probes for parasite antigen hasbeen applied to such diverse conditions as tuber-culosis (153), hydatid and other larval tapewormdiseases (78,79), Chagas’ disease (9), onchocer-ciasis (93), and schistosomiasis (83,84,239). ELISAprocedures using MAbs have been described forrotavirus infection (424), toxoplasmosis (8), andschistosomiasis (240). MAbs have been used toidentify malarial organisms within mosquitoeswithout the need for exhaustive dissection andmicroscopy.

Nucleic Acid Hybridization Probes

The use of a nucleic acid (DNA or RNA) hy-bridization probe to identify the actual DNA orRNA of a disease-producing organism (112) is apromising diagnostic technique that has some im-portant advantages, perhaps most important ofwhich is high specificity. Scientists are now ableto isolate and then reproduce (“clone”) DNA seg-ments from known organisms, label the segmentsradioactively, separate them from the normaldouble-stranded state to a single-strand state, andthen test them against unknown specimens ofDNA (e.g., in a stool sample) for the ability tohybridize (re-form double-stranded DNA). If thesuspected organism is present, then the labeledDNA and the sample DNA, being the same, willhybridize. The radioactivity or fluorescence or en-zymatic color change can then be detected.

The nucleic acid hybridization techniques fordifferent organisms vary, but such techniques canbe developed to be simple, practical, and rela-tively inexpensive. Using these techniques, inves-tigators can screen large numbers of samples atone time. Samples can be collected and stored forseveral weeks before the test is completed. Nu-cleic acid probes should be useful for large-scaleepidemiologic and surveillance studies (117,246).The major disadvantages of the tests that useradioactive labels are the radiation hazard and theshort shelf-life of the radiolabeled reagents. Theseproblems can probably be circumvented in mostcases by substituting tests that use color changemethods in place of radiolabels (196,202).

The uses of nucleic acid hybridization tech-niques to detect malaria parasites (122), Leishma-nia spp. (409), Escherichia coli (246), and rota-virus, and to detect Salmonella bacteria in foodproducts (116) are described below. The utilityof nucleic acid hybridization probes in the fieldin developing countries must be further evaluated,but good results have been reported in detectionof rotavirus and E. coli in stool sample dots. Al-though nucleic acid hybridization is still in its earlydevelopment phase, it may well progress to a pre-dominant role in diagnosis.


DIAGNOSIS: CURRENT STATUS FOR SELECTED TROPICAL DISEASES

Malaria

Since the emergence of drug-resistant malariaparasites in the late 1950s, the importance ofspecific diagnosis, including a determination ofwhether the malaria parasites are drug resistant,has been greatly elevated. Previously cheap, safe,effective, and completely standardized antimalar-ial treatment regimens were available, and, pre-sumptive treatment, without definitive diagnosis,was freely administered. Now, however, appro-priate treatment rests on accurate diagnosis.

Conventional Diagnosis

Diagnosis of malaria based on physical exami-nation alone can be difficult, because malaria’ssymptoms are protean. For that reason, labora-tory diagnosis of malaria is important. The stand-ard method of diagnosis is by microscope exami-nation of a stained blood smear made from afinger-prick. The presence of malaria parasites isdefinitive. Under field conditions, there are gen-erally quite a few false negatives, because peoplewith malaria do not always have large numbersof malaria parasites circulating in their blood. Inthe absence of microscopically confirmed infec-tion, a presumptive diagnosis of malaria may stillbe made, based on clinical symptoms. Treatmentis given to reduce the parasite load of the popu-lation and, in effect, to prevent mosquitoes fromacquiring malaria.

Although methods for the serologic diagnosisof malaria have long been available (CF, IHA, andIFA tests), they are not widely used for two rea-sons. First, the need for equipment and materialsto perform the tests cannot always be met. Sec-ond, the tests demonstrate the presence of anti-bodies, which persist after cure and may indicateprevious infection. In an endemic country whereinfection is always possible, such tests do notgreatly help the decision to treat. Nevertheless,these tests have been useful for epidemiologic sur-veys (especially IHA and IFA tests (168)), and forspecial purposes such as establishing the presenceof antibody in a particular patient in whom in-fection is suspected but cannot be demonstrateddirectly (e.g., in a blood donor who is suspected

of having transmitted malaria to a blood re-cipient).

Recent Progress

Serologic Diagnosis. —Improved serologicmethods for the detection of blood stage malar-ial antigens are being developed: RIA and ELISAtests for the detection of parasitized red blood cellshave been developed. Sensitivity of parasite de-tection with these tests is encouraging, and repro-ducibility should improve as standard reagents be-come available (353).

In the hope of establishing standard reagentsfor malaria serology, the World Health Organi-zation’s (WHO) Immunology Research and Train-ing Center, Geneva, has established a registry ofmalarial MAbs collected from other laboratoriesand evaluated for potential value as serodiagnosticreagents.

Sporozoite Diagnosis.—Immunologic work onplasmodial sporozoites has led to the developmentof two methods to test for sporozoites in mos-quitoes. Such testing has importance for epi-demiologic studies in determining the degree towhich various mosquitoes function as vectors ofspecies of malaria parasites.

In one method, MAbs are used to identifysporozoites in mosquito squashes by a direct bind-ing assay, which is species-specific. This methodwas developed as an RIA, but an ELISA proce-dure is being developed as well. Although the testprocedure must be carried out in a centralizedlaboratory, the samples are stable without refrig-eration and easily handled, and the results areavailable within an acceptable time interval forepidemiologic purposes.

A second method, another new immunoradio-metric test (inhibition of idiotype-anti-idiotype in-teraction, or “4 i-assay”), has been developed todetect circumsporozoite protein (280) and is basedon inhibition of binding of two MAbs. The firstMAb is against malarial antigen, and the secondMAb is against the first MAb (thus called an idio-type, which resembles the original antigen). Whenboth MAbs are mixed in a test well, they bind to-

164 . Status of Biomedical Research and Re/ated Technology for Tropical Diseases

gether in a detectable way, unless the test samplecontains antigen. If the sample contains antigen,the antigen will bind specific antibody, thus in-hibiting the two MAbs from interacting (inhibi-tion indicates the presence of true parasite anti-gen). This test is sensitive enough to distinguishbetween different species of malaria parasites. Themethod has general applicability. Furthermore,because the two immunoglobulin reagents areMAbs, this method does not require cultivationand purification of antigen from parasite, and thetwo immunoglobulin reagents are completely pure(422).

DNA Hybridization. —Work on developmentof a rapid diagnostic test using specific DNAhybridization is proceeding. A recent publicationdescribes experimental success in identifying Plas-modium fa]ciparum parasites in samples of bloodfrom in vitro culture and from malaria patients(122). This method is still at a very preliminarystage in relation to any practical use, because thesensitivity is no better than microscopic exami-nation of a stained blood film, and the procedureinvolves a number of laboratory steps that takeabout 24 hours to complete.

In Vitro Cultivation of Malaria Parasites andMicrotest of Drug Sensitivity .-With the currentsituation of widespread resistance to drugs by ma-laria parasites, diagnosis of drug susceptibility inparasite isolates is an important epidemiologictask. The method for in vitro cultivation of P.falciparum, the malaria species with widespreaddrug resistance, has been adapted to several tech-niques for testing drug susceptibility against theprimary antimalarial. The tests are available inkit form from WHO.

Research Needs

Greater standardization of serologic tests forthe detection of malaria antigens and antibodiesis needed. These tests need to be adapted for fieldapplications, both as quick and easy diagnosticsat remote or poorly equipped treatment posts andto assess any vaccination trials that may be at-tempted in the future. Assessment of vaccinationtrials will rely on detecting antibodies in in-dividuals who did not have antibodies before vac-cination as well as determining infection ratespost-vaccination.

A quick and easy field method is needed toestablish the existence of infection (to conservedrug for true cases) and to differentiate speciesof Plasmodium (for appropriate drug type). Sim-ilarly, a field method to detect infection in mos-quito vectors is needed to assess the impact of vac-cination on the overall risk of transmission.

A less urgent but important need is a methodof screening blood bank donations to preventtransfusion malaria.

Schistosomiasis

The major schistosomes infecting humans areSchistosoma mansoni, S. japonicum, and S. hae-matobium. Current control for schistosomiasiscalls for the identification and treatment of all in-fected persons at regular intervals (usually 6months) (353). Quick and reliable diagnosis is es-sential to identify people infected and for assess-ing the effectiveness of the treatment. To deter-mine optimal treatment, it is necessary not onlyto diagnose the presence of infection but also todetermine how heavy the parasite load is.


Direct examination of feces or urine for char-acteristic eggs is the classical method of diagno-sis of schistosomiasis. There are a number ofmethods for concentrating the sample to maximizethe chance of detection and a number of meth-ods for accurately estimating the number of eggsin order to estimate the parasite burden in thehost. Cytoscopy or sigmoidoscopy (viewing throughinstruments inserted into the body) is occasion-ally used to detect lesions, and rectal biopsy issometimes used. There is a CF test, an intrader-mal skin test, and the COPT to help confirmdiagnosis.

Recent Progress

Two new techniques have been developed forS. haematobium diagnosis. One is the filtrationof urine using a reusable plastic woven filter whichisolates excreted eggs. The second is a prototypeimage processing and pattern recognition appa-ratus for automated S. haematobium egg countsthat has been tested in the laboratory and is un-dergoing field trials in endemic areas (353).


A new diagnostic kit has recently been madeavailable by the Program for Appropriate Tech-nology in Health, a nonprofit, nongovernmentalorganization. The kit is for the diagnosis of S. hae-matobium, and it is designed for quick, practi-cal, and reliable field use.

In a special collaborative study sponsored jointlyby WHO and the Edna McConnell Clark Foun-dation, eight research laboratories evaluated anumber of procedures for immunodiagnosis ofschistosomiasis (247). Included were the COPT,ELISA, IHA, IFA, RIA, and other procedures,testing a pool of banked sera against a variety ofschistosomal antigens. A study of this type is ofgreat value in developing and standardizing ma-terials and methods for immunodiagnosis.

Several candidate antigens for immunodiagno-sis have been identified (59). There are severalmodifications of the ELISA procedure, includingan inhibition-ELISA used to detect and charac-terize schistosomal antigens (l). Many labora-tories have developed MAbs for identification ofrelevant antigens or for species diagnosis (e.g.,1,83,84,99,237,238,239,240) .

Many workers are attempting to identify andisolate relevant protective antigens, and at leasthalf of those are actively engaged in gene clon-ing experiments. Several laboratories are devis-ing improved diagnostic methods, primarily withELISA-based tests. Most schistosomiasis expertsbelieve that effective MAb-based diagnostic toolsare very near or within 5 years of introduction(16).

Research Needs

Field methods for quick and easy diagnosis ofschistosomiasis are being introduced but still re-quire evaluation and standardization.

Trypanosomiasis

African Sleeping Sickness(African Trypanosomiasis)

There are two forms of African sleeping sick-ness in humans. Trypanosoma brucei gambiensecauses the chronic form found in west Africa. T.b. rhodesiense causes the acute form in east Africa

and also infects livestock over large areas of thecontinent.

Conventional Diagnosis.—Direct examinationfor African trypanosomes is done from stainedblood preparations, but parasites are extremelydifficult to find. In the chronic form (T. b. gam-biense), fluid drawn by needle from lymph nodesin the neck is examined. The number of parasitesdetected varies daily. In the acute form (T. b.rhodesiense) too, parasites may vary in density,making diagnosis difficult. Cerebrospinal fluidmay also be examined. Inoculation of laboratoryanimals or culture on appropriate media to allowthe parasites to multiply is sometimes useful.

Immunodiagnostic techniques have been avail-able for many years (IFA and CF tests), but thesemust be performed in central laboratories. TheELISA has also been found effective in the lab-oratory but not for the field (353). All of thesetests are valuable for epidemiologic studies, butthe delay in reaching a diagnosis limits their use-fulness for patient care.

Recent Progress. —Tests for antibodies againstT. b. gambiense, which can be read within min-utes and carried out with blood obtained from afinger-prick, are under development and evalua-tion in the field. They are the card agglutinationtest for trypanosomiasis (CATT), the Cellognosttest (a commercial technique based on indirecthaemaggIutination), and the Tryptest. CATT isbeing evaluated on a large scale in west Africa.This test has been found to be as specific as theIFA test. It is easily transported to the field, re-quires little technical skill, and gives results withinminutes. The lack of stability of the antigen andstorage under field conditions are problems to beworked on. Antigens to diagnose T. b. rhode-siense are being sought (353).

Two other techniques are also being evaluated:the miniature anion exchange column techniqueand the microhaematocrit buffy coat centrifuga-tion method. Both have shown greater sensitiv-ity than blood film examination (208). Develop-ment of the double centrifugation technique forthe detection of trypanosomes in cerebrospinalfluid has improved diagnosis of central nervoussystem involvement (353).


The three subspecies of the Trypanosoma bru-cei species complex are morphologically indistin-guishable. Two species (T. b. rhodesiense and T.b. gambiense) are infective to humans, causingsleeping sickness; the third (T. b. brucei) is infec-tive to wild and domestic animals but not to hu-mans. Scientists’ inability to distinguish betweenhuman and animal forms has epidemiologic im-portance for determining risk to humans whereanimals are found infected. Culturing of trypa-nosomes in human serum is widely used for de-termination of infectivity to humans. Isoenzymeelectrophoresis, DNA hybridization, and compar-ative IFA with standardized sera have also beenused recently.

Research Needs. —Several avenues of researchmay be nearing fruition for the diagnosis of Afri-can trypanosomiasis. There is a need to developsensitive and specific techniques suitable for fielduse.

Chagas’ Disease (American Trypanosomiasis)

Chagas’ disease, caused by the protozoan para-site Trypanosoma cruzi, affects more than 12 mil-lion people in Latin America (229). Acute andchronic phases of the disease vary somewhat fromone region to another (166). Most damage is doneby tiny, nonflagellated forms within the cells ofheart muscle and certain nerve ganglia.

Conventional Diagnosis.—The clinical pictureof Chagas’ disease is variable. Examination ofblood during the acute phase of the disease mayreveal parasites. Because of commonly low para-site density in blood, several culture techniquesare used to allow the parasite to multiply to a de-tectable level: inoculation into laboratory animalsfollowed by periodic examination over 60 days;in vitro culture of blood; xenodiagnosis (allow-ing clean, uninfected reduviid bugs to feed on thesuspected patient and then examining the hind-gut of the bug for trypanosomes after 2 weeks).All these methods often require repeated attempts.

A CF test (the Machado-Guerreiro test) usingT. cruzi antigen is available, as is other serologicdiagnosis for the direct detection of antibody.Conventional serologic tests for Chagas’ diseasecross-react with leishmaniasis, leprosy, andsyphilis antigens and are not sufficiently sensitive

to detect Chagas’ disease with assurance. A num-ber of groups are working on development of animmunodiagnostic reagent, and optimism is gen-erally high. Immunodiagnosis of specific antibodyis available as a procedure in centralized labora-tories, but standardization between laboratoriesis a problem.

Recent Progress. —Standardization of serodiag-nostic techniques has been promoted by the Spe-cial Program for Research and Training in Trop-ical Diseases (TDR). A serum reference bank isnow providing standardized lyophilized (freeze-dried) serum samples to laboratories throughoutLatin America, and a network of collaborativelaboratories has developed protocols for thestandardization of reagents, techniques, and pro-cedures (353).

Many workers have developed MAbs that maybe useful for the detection of various trypanosomeantigens (9,259). Development of ELISA diagno-sis is under way in a number of laboratories. In-vestigators have shown that circulating antigensin acute infections can be detected (7).

A specific diagnostic test is being evaluatedusing purified cell membrane antigens of T. cruziwhich are fixed on polyamide strips. After ex-posure to suspected serum, the strips are treatedas in ELISA-type tests to detect any antigen-antibody complexes that would form if the serumwere from a person with Chagas’ disease. Prelimi-nary results indicate that the test greatly reduces,but does not eliminate, nonspecific reactions.

Blood-transfusion-transmitted Chagas’ diseasein Brazilian hospitals is a serious problem, andbetter screening methods for donated blood areneeded. An agglutination test for rapid screeningof donated blood for T. cruzi infection is under-going evaluation in Brazil, where it was devel-oped, and in a network of collaborating labora-tories (353).

A DNA-DNA hybridization probe showspromise of detecting the presence of T. cruzi orga-nisms or DNA fragments present in the blood. T.cruzi, has a unique, highly variable type of DNA(kinetoplast DNA) that can be used to distinguishspecies and strains within species. A technique forisolating DNA, cutting it into pieces, and then sep-


arating the DNA into recognizable patterns (re-striction endonuclease finger-printing of kineto-plast DNA) promises to provide a valuable newtool for epidemiologic and clinical purposes. Newsubdivisions of T. cruzi strains can be demon-strated using this method. These refinements inthe taxonomy of the parasites may help to explainthe variability of the disease in different locali-ties, including the variation in clinical symptomsand the variable susceptibility or resistance inhosts (353).

Research Needs.—A quick, easy, and reliablemethod is needed for diagnosis of acute Chagas’disease (when treatment might be prophylactic,and because the treatment is toxic). A means topredict prognosis in different geographic areasalso is desirable.

A sensitive test for screening donated blood isalso particularly important in Chagas’ disease, asinfection by transfusion is a serious problem inendemic areas.

.“

Leishmaniasis

Leishmaniasis is a disease with three clinicalpresentations depending on the leishmanialparasite species. Cutaneous leishmaniasis, causedby either L. tropica, L. mexicana, or L. brazilienis(depending on geographical location), is a self-lim-iting and usually self-resolving sore at the pointof infection. Mucocutaneous leishmaniasis, causedby L. brazilienis, begins as a sore but commonlymetastasizes and proliferates in the nasal andpharyngeal mucous membranes. Visceral leishma-niasis, or “kala-azar,” is caused by L. donovaniand affects the spleen, liver, bone marrow, andlymph glands.


Diagnosis of leishmanial organisms is compli-cated by the rather uniform appearance of differ-ent species under the light microscope, and cross-reactivity of different species with conventionalserologic diagnosis. Until recently, microscopicidentification and conventional serologic tech-niques were the only techniques available for di-agnosing leishmaniasis.

●

Photo credit: Office of Technology Assessment

Leishmanial organisms as seen through alight microscope.

The inability to distinguish correctly betweenspecies of Leishmania can have serious conse-quences for patients. For example, the lesions ofL. mexicana and L. braziliensis are very similarat first appearance, and both species overlap inmany parts of South America. Without treatment,L. mexicana is self-resolving, but L. braziliensisprogresses to gross destruction of the nose andthroat. Thus, treatment and followup for L.braziliensis infection are critical. But the treatmentis itself highly toxic and clearly not to be used forthose not requiring it.

Recent Progress

Investigators have developed a DNA hybridi-zation probe to distinguish between L. mexicana

168 ŽStatus of Biomedical Research and Related Technology for Tropical Diseases

and L. brazdiensis (409) using kinetoplast DNA,a unique form of DNA in Leishmania and Tryp-anosoma species. Kinetoplast DNA is extractedfrom growing cultures of various species of Leish-mania organisms, processed and labeled radioac-tively. Test material is collected from the patientas “touch preparations” on nitrocellulose filter pa-per from suspected lesions. Hybridization andanalysis are then carried out. Kinetoplast DNAhybridization is highly species-specific and pro-vides a relatively rapid means of diagnosis directfrom infected tissue. This has now been tested forthe diagnosis of human patients and shows promise.

Another test, called the “DOT-ELISA” test, hasbeen developed, and represents a major advancein the rapid field diagnosis of visceral leishmani-asis. It also is useful for field surveys for identify-ing infected vectors (267).

In recent years, scientists have prepared MAbsagainst a variety of antigenic determinants inLeishmania species (71,91,139,145,171) and usedthem to probe for specific morphologic and taxo-nomic differences. Among the many monoclonalsproduced, some recognize antigens common toall kinetoplastid (Leishmania and Trypanosoma)species tested; others bind only to certain strainswithin a single species. The process of sorting outthese specificities and defining the precise natureof the reactive leishmanial antigens should pro-duce advances in knowledge and diagnosis ofthese organisms.

Research Needs

The diagnosis of Leishmania species is very im-portant, because different species produce simi-lar lesions but have very different long-term con-sequences. DNA hybridization looks promisingbut needs development for practical use.

In the clinical-epidemiologic area, there aremany strains and types of Leishmania, but a lackof a good classification system. More field surveysto determine prevalence of disease are needed, andthose surveys will require practical field tests.

Filariasis

Filariasis is a collective term for several distinctparasitic infections by insect-transmitted, tissue-

dwelling nematodes. The principal species areWuchereria bancrofti and Brugia malayi, whichcause filarial elephantiasis; and Onchocerca vol-vulus, the cause of onchercerciasis (river blind-ness) in west Africa, also found in Central andSouth America.


Conventional diagnosis of filarial infectionshas severe limitations. For lymphatic filariasis(Wuchereria and Brugia), diagnosis is made bymicroscope examination of stained blood films tofind microfilariae. Density of microfilariae is usu-ally very low, especially during the early stagesof infection; and for several species, it is cyclicalaccording to a circadian rhythm, so at times theremay be no microfilariae present in the blood.Diagnosis of onchocerciasis (in which the sub-cutaneous tissues are infected) is made by usinga special surgical punch to take tiny snips of skinand then examining this skin under the micro-scope. Because of cross-reactions with other orga-nisms, no dependable serologic diagnostics forfilarial infections are available.

Microfilariae of Onchocerca volvulus were found inskin snips from these nodules.

Ch. 8—Diagnostic Technologies: Selected Tropical Diseases •169

Recent Progress

A number of MAbs to filarial antigens havebeen produced, but immunodiagnosis has beenhindered by a high level of cross-reactivity withother helminth antigens. Excretory-secretory anti-gens, which are released by the parasite and cir-culate in the host’s blood, and surface antigenson the parasite itself are being assessed for use inimmunodiagnosis of filaria. The demonstrationof circulating antigens in Onchocerca infectionshas made a diagnostic MAb feasible. A goodcorrelation between circulating antigen and pres-ence of parasites was found in one study, usinga MAb in an RIA (93). However, the false posi-tive rate was high, suggesting that further refine-ment of this test is needed. At a workshop heldin 1983, 26 researchers from around the worldbrought about 50 antifilarial MAbs representingthe total successful effort to that date (16).

Tests for detection of specific antibodies againstfilarial worms, for circulating parasite antigen,and for parasite surface antigen are being devel-oped. There is optimism about the possibility ofa breakthrough in filarial detection within the nextfew years. At least one Federal Government lab-oratory, one in industry, and one in a universityare conducting or planning recombinant DNAwork for expression of such filarial antigens.

A problem in diagnosing filariasis is the iden-tification of parasites found in wild-caught vec-tor insects. The problem of confusing intermedi-ate life stages of disease-producing organisms withother organisms of little public health significanceis common to vector-borne diseases, and repre-sents a special problem in diagnosis which isamenable to solution by modern methods. Inareas of west Africa with active vector controlprograms against onchocerciasis, new insects withpotential vectorial capacity are entering controlledareas from adjoining regions. Some of these in-sects are naturally infected with filarial larvae,possibly parasites of wildlife, that cannot be dis-tinguished by direct examination from larval On-chocerca. The development of probes, either byMAbs or possibly DNA hybridization, would per-mit identification of these nematode larvae anddetermination of whether they pose a threat tohumans.

Research Needs

Very generally, improvements in all aspects ofthe diagnosis of filarial infections are needed. Be-cause much remains to be learned about the epi-demiology and pathology of filariasis, useful diag-nostic tests are needed. It is important both forpatient care and for research purposes to be ableto distinguish animal and human filariae, a taskwhich at present is not always possible.

Leprosy (Hansen’s Disease)

Leprosy, caused by the bacillus Mycobactetiuznleprae, is the only bacterial infection among thesix diseases targeted by TDR. Since the 1950s, theWHO-recommended strategy of leprosy controlthrough early case finding, followup of contacts,and chemotherapy of patients has proved to bedifficult to implement and sustain in many coun-tries (353).


The initial diagnosis of leprosy is through rec-ognition of areas of skin that lack feeling (anes-thesia) and may be discolored or slightly raised.Definitive diagnosis is made by acid-fast stainingand microscope examination of skin biopsy smearsfrom these suspected lesions. This technique is use-ful in all forms of leprosy, but there maybe fewbacteria in milder cases, making detection un-certain.

The lepromin or Mitsuda test, a skin test simi-lar to the tuberculin skin test (see below), is usedas a prognostic test after leprosy is diagnosed. Ittells where the patient is along the immunopatho-logic scale of disease. A crude suspension of killedbacilli (derived from human leprosy patients) isinjected under the skin of the patient. About 3weeks later, the skin reaction is assessed, allow-ing a prognosis to guide treatment.

A lymphocyte transformation test has beenavailable for a decade as an indicator of M. lepraeinfection and the potential course of the disease(133). This test is an indicator of cell-mediated im-mune response. Despite its technical difficultiesand subjective interpretation, the lymphocytetransformation test has given useful results in ex-perimental field studies of exposure to leprosy an-


tigen, though it has not become a practical methodfor routine use. Another test of the cell-mediatedimmune response, microphage migration inhibi-tion, is also not used routinely (168). It also pos-sible to isolate and diagnose leprosy by inocula-tion of a clinical specimen from a suspected caseinto the footpad of a mouse.

Recent Progress

A phenolic glycolipid molecule has recentlybeen isolated and identified as a unique and spe-cific antigen that is abundant on M. leprae andin the skin lesions of leprosy patients. This anti-gen has been used in an ELISA and has provedto be specific for antileprosy antibody (56). It hasbeen tested experimentally to detect antibody inthe blood of leprosy patients, with excellent re-sults (433). This antigen has the potential to beused in a specific diagnostic test for leprosy. In-vestigators have found that contacts of knowncases are more likely than other people to developantibodies to this antigen. The phenolic glycolipidtest may therefore be useful in screening personsat increased risk of developing clinical disease.

A number of apparently M. leprae-specific an-tigens (other than the phenolic glycolipid antigen)have been reported (30,41,149), and MAbs havebeen produced, which may also prove useful asdiagnostics. Serologic tests using IFA, ELISA, orRIA are being developed for epidemiologic studies.The fluorescent leprosy antibody absorption testhas shown high specificity and is being evaluatedfor predictive value in long-term epidemiologicstudies (2). Skin tests have been developed formonitoring delayed-type hypersensitivity reac-tions to M. leprae after immunization, though thecurrent test antigens are crude extracts that lackspecificity. Other skin test preparations usingsoluble antigens are being evaluated in the fieldfor predictive value (224,232,346).

Research Needs

A method of culturing M. leprae in vitro is anurgent need. Such a method could lead to im-proved methods for early diagnosis, which in turnwould lead to earlier treatment and favorableprognosis. Practical techniques to diagnose lep-rosy for epidemiologic studies need to be devel-

oped to allow better understanding of transmis-sion and susceptibility.

Progress in development of serologic tests thatare sensitive and specific has raised optimismabout prospects for achieving practical diagnos-tic techniques for leprosy. The phenolic glycolipidantigen is most promising at present and may bedeveloped into a useful diagnostic test. At present,the main source of this antigen and others is fromM. leprae grown in armadillos. Obtaining anti-gens from M. leprae grown in armadillos is a slowprocess. It may be necessary or useful for diag-nosis to make important molecules from M. lepraeusing recombinant DNA.

A method for the differentiation of patientswith lepromatous leprosy (the severe form withpoor prognosis) and tuberculoid leprosy (the mildform with good prognosis) is needed to improveunderstanding of the epidemiology of the diseaseand for the early recognition of individuals at highrisk of developing disease. The evidence for agenetic basis to resistance needs further study.

Tuberculosis

Tuberculosis remains a major threat to healthin many parts of the world, causing several mil-lion deaths annually. Tuberculosis control pro-grams are built around vaccination coupled withearly case detection, treatment, and followup ofactive cases. Diagnostic methods and early diag-nosis before symptoms are overt are critical to thecontrol of this disease.


The consideration of clinical symptoms and ex-amination of direct sputum smears for the pres-ence of the causative tubercle bacilli are the con-ventional means of diagnosing tuberculosis. If nobacilli are found in a direct smear but tuberculo-sis still is suspected, culture isolation of bacteriafrom a clinical specimen (usually sputum, thoughurine, spinal fluid, or tissue biopsy may be appro-priate) is attempted. Culturing also is used to con-firm a diagnosis made by a direct smear. Isola-tion of bacilli from culture is the preferredmethod, but several serious problems arise: theneed to decontaminate the specimen to prevent


overgrowth by contaminating bacteria from theoral cavity; the need to collect and culture multi-ple samples from a suspected case; and the slowmultiplication of the bacilli, which means that itmay take 3 to 6 weeks for growth to appear.

Chest X-rays continue to be of great value inthe diagnosis of tuberculosis, particularly in areaswhere other diseases (like histoplasmosis) withsimilar X-ray appearance are absent. X-rays areparticularly useful for determining (by noting dif-ferences between earlier and later films) that a dor-mant case has been reactivated.

Microscope examination of stained sputumsmears can be used to quickly identify tubercu-losis-like bacteria, but does not distinguish viru-lent tubercle bacilli from look-alikes. In areas ofthe world where active pulmonary tuberculosisis common, a presumptive diagnosis can be madeon the basis of numerous bacteria with particu-lar staining qualities (acid-fast) in the sputum. Itis clear, however, that many different species ofMycobacterium, in addition to M. tuberculosis,can infect humans and cause tuberculosis-like dis-ease. Such environmental mycobacteria (called“atypical mycobacteria”) are diagnosed by theircharacteristics in culture, and cannot be distin-guished on direct sputum examination. Some ofthese (the M. avium-intracellulare complex) areassociated with acquired immunodeficiency syn-drome (commonly known as “AIDS”).

The tuberculin skin test is used to identify peo-ple infected with tubercle bacilli, by means of anallergic reaction to tuberculosis antigens (delayed-type hypersensitivity). A small amount of tuber-culoprotein is introduced into the skin, and theperson is observed for an inflammatory reaction2 to 3 days later. Old tuberculin (a crude concen-trate of tubercle bacilli in culture medium) hasbeen replaced by purified protein derivative(PPD), a purer, more standard material.

Three techniques can be used to introduce thetuberculin: 1) the Mantoux test, in which PPD isinjected intradermally; 2) the Vollmer patch test,in which tuberculin is applied to the skin on agauze adhesive strip; and 3) the Tine test, in whichthe tuberculin is dried on the points of a stand-ard puncture device that is pressed into the skin.

All of the tests have advantages and disadvan-tages. None of these tests can be used to test any-one already vaccinated against tuberculosis (alarge percentage of the population in many de-veloping countries and in a number of developedcountries), because vaccinated individuals shouldreact to the challenge. The Mantoux is the mostsensitive and reliable test, because a standard vol-ume and amount of tuberculin is injected directlyinto the skin; however, the procedure of injectionby hypodermic is a practical disadvantage. TheTine test is a rapid and easy test for use in largepopulation groups, but it gives a relatively highnumber of “false positives” (people who test posi-tive but who actually are not infected); positivesmust be followed up by a Mantoux test. TheVollmer patch testis useful for skin-testing infantsand children, but it is less sensitive than the Man-toux test.

A positive tuberculin skin test may be causedby infection with other species of mycobacteriaor as a result of previous vaccination. A nega-tive tuberculin test is strong evidence against thetuberculosis diagnosis, but can also result fromloss of potency in stored PPD, and is occasion-ally observed in far advanced cases.

Recent Progress

ELISAS have recently been applied to the diag-nosis of active tuberculosis pulmonary infection(183). Application of recombinant DNA methodsto M. tuberculosis is in its infancy, but severalinvestigators are planning projects. In a handfulof laboratories including the National Institutesof Health and several academic institutions, MAbsare being reacted with M. tuberculosis to isolateand purify antigens that may be useful as diag-nostic targets and may be the basis for a new tu-berculosis vaccine.

Attempts are being made to isolate specific an-tigens for skin tests in an attempt to make tuber-culin skin tests less cross-reactive to infectionswith other types of mycobacteria. Several tuber-culosis researchers also work with M. leprae, therelated bacterium that causes leprosy.


Research Needs

Tuberculosis may be the most serious infectiousdisease problem in many developing countries,as much a social as a scientific dilemma. Thereis an obvious need for better vaccination and diag-nosis. Diagnostic methods are needed to differen-tiate the “atypical mycobacteria, ” such as M. in-tracellulare and M. smegmatis, which hinderdiagnosis and seem to interfere with the effective-ness of BCG (Bacillus Calmette-Guerin) immuni-zations.

Diarrheal and Enteric Diseases

Diarrhea] and enteric diseases are caused by avariety of viruses, bacteria, protozoa, and worms,and a single individual often is infected with sev-eral at one time. Full understanding of the vari-ous etiologic agents will come only when simplertechniques of diagnosis are available. Clinicaldiagnosis of diarrheal disease calls for immediateinstitution of therapy. Fortunately, dehydrationtherapy is appropriate for all diarrheal disease (seech. 9). Nonetheless, identification of specific path-ogens is essential for understanding the distribu-tion of different agents and to establish prioritiesfor specific actions.

Viral Infections

Viruses are now recognized as important agentsof gastroenteritis. The principal agents are rota-viruses and Norwalk agents, though adenoviruses,astroviruses are enteroviruses, coronaviruses, andcalciviruses are also found in fecal specimens (15).

Conventional Diagnosis.—Most of the virusesthat cause diarrheal and enteric diseases do notgrow under ordinary cell culture conditions, sodirect diagnosis of viral antigen in stool specimensor detection of a serologic response is necessary.Electron microscopy is a sensitive and simplemethod for detecting the presence of virus (if anelectron microscope is available), but only limitednumbers of samples can be processed.

A variety of immunologic methods have beendeveloped for detection of viral antigen in stoolsamples. The ELISA, IFA test, RIA, and CIE pro-duce good results, though ELISA and RIA havethe greatest sensitivity.

Recent Progress.—Progress has been achievedin isolating and cloning rotavirus DNA and thendeveloping DNA hybridization assays (117). ClonedDNA hybridization probes have been used onstool specimens for rotavirus diagnosis. The utilityof DNA hybridization probes in the field in de-veloping countries must be further evaluated, butgood results have been reported in detection ofrotavirus in stool samples taken in remote areasof Venezuela (146).

A test “kit” for rotavirus based on an ELISAhas been developed and evaluated and is now inuse by more than 50 investigators in the field. Asecond generation ELISA test based on the use ofMAbs is being developed (427).

Bacterial Infections

Conventional Diagnosis.—Definitive identifi-cation of bacteria causing diarrhea is by the iso-lation of the agent through culture of clinical sam-ples (e.g., stool samples for Shigella and Vibriocholerae; blood or stool samples for Salmonella.Culture of V. cholerae is relatively simple andgives a result in 18 hours.

Serologic diagnosis of Salmonella typhi, thecause of typhoid fever, is possible because spe-cific agglutinins appear in the blood at 7 to 10 daysof illness (the Widal reaction). For cholera, risein titer of specific agglutinins or antibodies con-firms the diagnosis.

Two tests for the isolation and identificationof enterotoxigenic E. coli have been available fora number of years: one uses a miniculture of adrenalcells (302), and the other uses suckling mice (90).

Recent Progress. —Although E. coli is one ofthe most intensely studied of all organisms, withmany thousands of research publications on allaspects of its biology and biochemistry, much re-mains to be learned about its relation to diarrhealdisease. The several strains of E. coli that producediarrheal and intestinal disease in humans are rec-ognized and described on the basis of clinicalpathology (“enterotoxigenic,” “enteropathogenic,”“enteroinvasive,” see ch. 4). All of these charac-teristics are under genetic control, and investiga-tors in several laboratories are identifying andcloning the genes that code for attachment and


colonization, virulence, toxin production, and an-tibiotic resistance. With the genes in hand, diag-nostic procedures can be developed.

DNA hybridization probes for field identifica-tion and typing of E. coli are under developmentin several laboratories. A DNA hybridization testfor identification of enterotoxigenic E. coli hasbeen available for several years (41,42) and nowhas been tested in the field (103,312). The DNAhybridization test has been shown to be very spe-cific, reliable, stable, and sensitive (1,000 timesmore sensitive than the standard assays). It ap-pears to be a valuable tool for epidemiologicstudies.

The WHO Control of Diarrheal Diseases (CDD)program has evaluated the “Biken” gel diffusiontest for detection of certain strains of E. coli. Thetest is simple to perform, accurate, reproducible,and has potential for use in developing countries.Commercial production and marketing of the ma-terials and reagents used in the test is being pur-sued (427).

The CDD program is also evaluating ELISASfor diagnosis of strains of E. coli. Results indicatea potential for widespread application, thoughfurther development is needed to make it suitablefor routine diagnostic use (427).

A DNA-DNA hybridization probe was used byone team of researchers to detect Salmonella bac-teria not in stool samples but in food products(116). This type of application may provide farmore serotype-specific identification of contami-nating organisms than conventional culture meth-ods, without the need for incubators, sterile me-dia, and glassware.

Cholera is being studied by four or five re-searchers in the United States. Recombinant DNAlibraries are being constructed to collect gene se-quences from wild-type organisms. Studies are un-der way on the transmissible genetic elements iso-lated from V. cholerae from endemic areas suchas Bangladesh, and differences between toxigenicand nontoxigenic organisms are being defined (16).

Campylobacter jejuni is now recognized as animportant diarrheal agent, but epidemiologicstudy is hampered by lack of a serotyping tech-nique. Antigenic studies are under way to develop

a serotyping system. A simple slide agglutinationtechnique to identify antigens is under evaluationin a number of developing countries (49).

Acute Respiratory Infections (ARIs)

ARIs are caused by a range of etiologic agents—viruses, bacteria, rickettsia, and parasites—pre-senting great diagnostic complexity. Althoughmost of these agents can be specifically diagnosed,the process normally requires multiple serologictests of various kinds.

Many diagnostic procedures based on isolationand culture of the organism or based on compar-ison of an initial and a later serum sample (“pairedsera”) do not provide timely enough informationto be relevant to individual patient care. Also, toa large degree, the diagnosis of ARIs can be andis made on the basis of clinical symptoms, becauseno matter what the diagnosis, specific therapeu-tics are lacking, especially for the many viraldiseases.

Because much of the treatment of ARIs consistsof providing symptomatic relief, the identifica-tion of specific disease-causing agents is not so im-portant. In lieu of specific diagnosis flowchartsor decision trees, using a patient’s symptoms ascriteria may be more appropriate for use by med-ical staff at all levels of the health systems of de-veloping countries.

Still, in many cases, the etiologic agent needsto be identified. For the treatment of the individ-ual patient, diagnosis permits rational use of avail-able therapy. The nonspecific prophylactic orplacebo use of antibiotics for any undiagnosedARI is greatly abused. Bacterial infections can betreated with common antibiotics, but for viralagents, only symptomatic support and relief areproper (with the exception of viral influenza forwhich an antiviral drug is available).

For public health needs, specific diagnosis isnecessary to assess, plan, implement, and evalu-ate interventions against epidemic outbreaks andendemic infections.


Faced with an individual patient with respira-tory infection, the clinician or epidemiologist must

174 ŽStatus of Biomedical Research and Related Technology for Tropical Diseases

make simplifying judgments to decide which diag-nostic procedures are in order. Simple microscopeexamination of nose or throat secretions can beuseful, though limited, in ruling out possible etio-logic agents.

Culture and isolation of the pathogen leads toa definitive diagnosis, but the growth takes time(usually at least 2 days), delaying the diagnosis,For viral agents and even bacterial agents, thisprocedure requires careful collection; special han-dling; storage and transport; tissue culture facil-ities (including appropriate culture media, and celltypes for virus growth); and equipment for sero-logic diagnosis (e.g., immunofluorescent micro-scope, scintillation counter, electrophoretic equip-ment). Prior antibiotic treatment or contaminationof the sample at any of several points from col-lection to inoculation, in the mature medium caneasily lead to incorrect results.

Serologic diagnosis by most of the standardtests can demonstrate specific antibody in the in-dividual’s serum, but this is not definitive, sinceantibody from previous infections can persist af-ter cure. Active infection is demonstrated whenthe serum titer of antibody in a later convales-cent sample (after 1 to 3 weeks) is higher than anearly acute sample (“paired sera”). This means thediagnosis is often confirmed after the infection hasbeen resolved. This is acceptable for epidemio-logic use, but not so useful for individual patientcare.

Recent Progress

Viral Infections.—With the IFA test and ELISA,the diagnosis of respiratory viruses can now bemade in a few hours after specimen collection(52,123,125,418). This is a great improvementover isolation by tissue culture which takes sev-eral days. With appropriate antisera, the follow-ing viral antigens can be identified by the IFA test:influenza (types A and B), respiratory syncytialvirus (RSV), measles, adenovirus, parainfluenza1,2,3,4 (125,264). The economy of time permitslarge numbers of specimens to be processed.ELISA appears to be very useful for detecting sev-eral viruses, but additional experience is neededto evaluate it (264).

Even with a rapid serologic diagnosis, the evi-dence is only indicative because of possible con-tamination (i.e., if the clinical sample is negativebut the culture is positive, contamination of theculture is suggested rather than a positive diagnosis).

influenza.-Because influenza infection may befatal, and outbreaks occur annually, with major epi-demics and pandemics occurring sporadically, thethree main types of influenza, with numerous sub-types and strains, are monitored epidemiologically.Precise strain typing is carried out in order to pre-pare effective vaccines. Virus isolation and identifi-cation from tissue culture is available in 48 hours,but the definitive result may take a week or more.HI and CF tests produce a result in 24 hours, buttests of acute and convalescent serum (samples“paired sera”) separated by an interval of 2 to 3weeks provide definitive diagnosis. The neutraliza-tion test is useful but expensive and time-consuming.The direct or indirect immunofluorescent techniquecan be used with cells obtained from the respira-tory tract for a rapid diagnosis even when the pa-tient has no symptoms.

Respiratory Syncytial Virus (RSV).–Precise diag-nosis of RSV requires culture and isolation of thevirus from tissue culture, then identification withan IFA test. CF or neutralization tests can be usedfor serologic diagnosis in the patient. Completingthe positive identification may take up to 2 weeks,though preliminary results can be obtained by earlyexamination of the cell culture.

Fluorescent antibody tests have been developedto permit rapid diagnosis of RSV infections. If theblood sample is transported promptly to the lab-oratory and processed immediately, the diagnosiscan be made in 4 to 6 hours (264).

Parainfluenza. —Precise diagnosis of parainflu-enza viruses requires culture and isolation. Hemag-glutination tests identify the virus after 5 to 10 days,while immunofluorescence tests can make the diag-nosis in 24 to 72 hours. Detection of a rise in serumantibodies in the individual can be made with theHI test or CF titration, but nonspecific responsesmake serology an unreliable diagnostic tool. TheIFA test has been used for rapid diagnosis, but ithas not achieved widespread use.


Adenoviruses. -Definitive diagnosis is by culture,isolation, and use of CF, IHA, or neutralizationtests. These three tests can be used to establish thediagnosis using paired serum samples from thepatient.

Rhinoviruses. —For the most common agent ofthe common cold, with over 100 serotypes, rou-tine serologic diagnosis is not very practical, andis not routinely available even in developed coun-tries, though the neutralization test can be used.Even tissue culture isolation is often unsuccess-ful in detecting the infection, because the condi-tions for successful isolation require special han-dling (246).

Corona viruses. —Diagnosis of coronavirus byculture isolation is not a routine procedure andneeds specific types of culture cells. Serologic diag-nosis can be made using the CF test.

Bacterial and Mycoplasmal Infections.—Thediagnosis of bacterial agents of respiratory infec-tions has been greatly improved over the last dec-ade. Rapid and accurate diagnosis is now avail-able in hours rather than days through the useof a variety of diagnostic methods to detect in-tact bacteria, or in some cases soluble antigen, bythe COA and LA tests, ELISA, RIA, CIE, and theIFA test (264).

The COA test can be used for detection of bac-teria in specimens such as sputum, serum, urine,and cerebrospinal fluid. It can also be used forstrain typing of culture isolates. The test is sim-ple, rapid, sensitive, and specific when the anti-serum used is of good quality. Each bacterium tobe identified needs a specific antiserum reagentwith appropriate antibodies.

The LA test has been used to detect Strepto-coccus pneumonia and Hemophilus influenzain body fluids such as serum, cerebrospinal fluid,and urine. False positives result from nonspecificautoagglutinations and from reaction to antigenscommon to pathogenic and nonpathogenic or-ganisms.

ELISA can be used to detect bacteria in bodyfluids. It is highly sensitive for detecting H. in-fluenzae type B, as well as pneumococcal antigen.There are two variations: the direct assay uses aspecific antibody (against the bacterial antigen)

that is labeled with the enzyme. The indirect as-say uses unlabeled specific antibody. The antigen-antibody complex is then identified with labeledantibody that binds to it. The indirect method isvery sensitive and more useful, because it limitsthe need to just one labeled antibody.

RIA is extremely sensitive for detecting S. pneu-moniae and H. influenza. To evaluate the prac-tical use of RIA, however, more experience isneeded. CIE has been successfully used to detectpneumococci, streptococci, and H. influenza inrespiratory secretions and body fluids.

The IFA test can be used to identify bacteriabut not soluble antigens. It is very sensitive fordetecting H. influenzae, S. pneumonia, and Bor-detella pertussis. The IFA test can also be usedto identify various organisms in culture.

Streptococcus pneumoniae. —Direct microscopicexamination of sputum can indicate pneumococci,but the predictive value of this test is variable,because several organisms resemble pneumococci,healthy individuals can carry pneumococci, anda sputum negative for pneumococci does not ruleout infection. Sputum culture is the standard pro-cedure. Serologic diagnosis is not practical, be-cause antibodies persist for long periods of time.

Efforts to detect antigen in respiratory tract se-cretions, blood, and urine by CIE (97,345), as wellas by the COA and LA tests (342), have been suc-cessful.

Streptococcus pyogenes. —Diagnosis is madeby isolation of the streptococci from culture ofthroat samples. Group determination is made withspecific antiserum—the Lancefield precipitationtest is considered the standard, though direct fluo-rescent antibody test is also useful. IFA of throatswab isolates can be obtained after 2 to 24 hoursof culture. CIE can be used 6 hours after culture.The COA and LA tests have also been used. Thereare three convenient tests for detection of serumantibodies.

Bordetella pertussis.—Definitive diagnosis ismade 2 to 3 days after culturing a throat sampleon specific media, by agglutination with specificantiserum or the IFA test. A rapid diagnosis isavailable with the direct fluorescent antibody test.


Hemophilus influenzae. -After isolation andculture of a clinical sample, diagnosis is made bymicroscopically detecting a surface change in thebacterium (the Quellung test). Rapid diagnosis ofantigen in secretions and body fluids is availableby CIE, the LA test, and ELISA (264).

Mycoplasma pneumonia. —Definitive diagno-sis is made by culturing a throat sample on appro-priate media, with subcultures made weekly for8 weeks. When colonies appear, they can be iden-tified visually, though IFA staining confirms thediagnosis.

Rapid diagnosis of sputum by CIE has beendemonstrated, but more experience is needed tovalidate this technique (404). Serologic diagno-sis of paired sera is done with the CF test. Detec-tion of antibody in one sample is useless for diag-nosis, because high titers persist long after initialinfection.

Research Needs

There are many procedures for the diagnosisof ARIs, but various constraints reduce their prac-tical use. Direct demonstration of causative agentsis only now becoming an option. Serologic diag-nosis using paired sera and serologic diagnosis ofculture isolates do not provide timely informa-tion for the treatment of the individual patient.Requirements for well-equipped and well-suppliedlaboratory facilities are another constraint thatlimit the use of diagnostic procedures in tropicalcountries.

Continued development of the available andpromising technologies for the diagnosis of ARIsis needed. With rapid, simple, reliable diagnos-tic tests, both the needs of the patient and the com-munity could be better met. Ideally, such testswould not require expensive equipment, reagents,or highly trained operators. In some cases, how-ever, if the tests have certain sophisticated require-ments, it may be possible to use them in central-ized laboratories.

Arboviral and Related Viral Infections

Because of the general lack of effective treat-ment for arboviral infections, diagnosis is of lessimportance to individual patients than to the com-

munity, where it is critical for recognizing out-breaks and initiating vector control measures.


Clinical diagnosis of the diseases produced bythe arboviruses recognizes three syndromes: 1)fevers of an undifferentiated type, frequentlycalled “dengue-like,” with or without rash andusually relatively benign; 2) encephalitis, oftenwith a high case fatality rate; and 3) hemorrhagicfevers, also frequently severe and often fatal (418).

Conventional diagnosis of arboviral disease in-volves isolation of the virus in newborn mice orcell culture, followed by serologic diagnosis usingHI, CF, or neutralization tests. The serologic diag-nosis itself is quick and straightforward, but suc-cessful culture requires appropriate laboratoryequipment, tissue culture materials, and time (usu-ally at least 2 days). More sensitive cell cultureshave become available using mosquito cell linesfrom which early detection can be made using theIFA test (340). It is also possible to inoculate clin-ical samples into live mosquitoes and then iden-tify the virus using an IFA test of the salivaryglands. This technique is very sensitive, but slow(10 to 14 days) (190). Other new methods, suchas the RPHA test, are being developed to detectthe virus earlier in the culture cycle.

Serologic diagnosis with paired sera still usesconventional methods (HI, CF, and neutralizationtests). Single radial hemolysis has been introducedwith some important advantages (124). It is as sen-sitive as the HI test for dengue fever, tick-borneencephalitis, yellow fever, West Nile fever, andVenezuelan equine encephalitis, yet simpler to per-form. The method looks promising but needs fur-ther evaluation with different viruses.

Direct detection of virus from the patient is de-pendent on sufficient virus in specimens. The IFA.test has been used with some success for a fewdiseases (Japanese B encephalitis, Colorado tickfever, Rift Valley fever). ELISA and RPHA meth-ods are being evaluated for detection of viral an-tigen in body fluids and respiratory secretions.CIE has been used to detect dengue virus in serafrom patients with acute diseases but the test haslow sensitivity. In all cases of individual directdiagnosis, a negative result is not conclusive.

Ch. 8—Diagnostic Technologies: Selected Tropical Diseases “ 177

Detection of specific IgM antibody (which is anearly immune response in the acute phase of in-fection) is carried out by the HI test, ELISA, andthe IFA test. This is used for the diagnosis in con-valescent patients (when virus has disappeared)and for primary dengue virus.

Recent Progress

MAbs have been developed for certain of theTogaviruses, Bunyaviruses, and Arenaviruses,and nucleic acid hybridization techniques are invarious stages of development for some of theseviruses. ELISAS are also being developed for mem-bers of each group and field tests for some havebegun. MAbs to antigens common to groups ofviruses have been developed which allow “ge-neric” diagnosis of disease, frequently sufficientto initiate medical therapy and epidemic preven-tion measures (197).

MAbs for early type-specific identification ofthe four main serotypes of dengue viruses have

SUMMARY

There is great variability in the availability ofdiagnostic technologies for diseases of importancein developing countries. In general, however, lackof effective diagnosis is a major obstacle to healthcare only when health care systems are adequateto act on diagnoses. This situation is not the normtoday. While diagnosis is an integral part of med-

been developed by the U.S. Army at the WalterReed Army Institute of Research (197) and arenow generally available. However, dengue virusisolation is still difficult as most patients have lowvirus concentrations, and the viruses grow poorlyin cell cultures. The U.S. Public Health Servicelaboratory in Fort Collins, CO, also is produc-ing dengue monoclonals in collaboration withWHO. MAbs have also been made to specific sur-face antigens of several viruses, and ELISA testsbased on these reagents are being evaluated (16).

Research Needs

Development of synthetic peptides following se-quence analyses of the alphaviruses and flavi-viruses could be very important for developingdiagnostic reagents to detect serum antibodies (aswell as for vaccine development). With regard tothe needs of individual patients, rapid diagnosticmethods will become increasingly important asdrug treatments for arboviruses are developed.

ical care, in some cases, diagnostic technologieshave even greater value in providing informationabout the incidence, prevalence, and natural his-tory of diseases of importance to developing coun-tries. Development and use of diagnostic technol-ogies in research could lead to effective integrateddisease control strategies.

Diagnostic Technologies: Selected Tropical Diseases

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