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Microscopy remains the cornerstone of laboratory testing for the identification of most blood parasites and many tissue parasites [ 289–291 ]. Expert microscopic examination of Giemsa-stained thick and thin peripheral blood films is used for detection and identification of the protozoan blood parasites Plasmodium , Babesia , and Trypanosoma , and the filarial nematodes Brugia , Wuchereria , Loa loa , and Mansonella , whereas microscopic examination, culture and/or nucleic acid amplification of ulcer samples, bone marrow, tissue aspirates, and biopsies are useful in the diagnosis of African trypanosomiasis, onchocerciasis, trichinosis, toxoplasmosis, and leishmaniasis. Although requiring a minimal amount of reagents and equipment, the accuracy of microscopic methods requires well-trained and experienced technologists. Even in the best hands, diagnosis may be hampered by sparseness of organisms on the slide and the subjective nature of differentiating similar-appearing organisms ( Plasmodium vs Babesia ; various microfilariae) or in identifying the species of Plasmodium present. The laboratory can enhance the sensitivity of these methods by employing a number of concentration procedures such as buffy coat examination, centrifugation, and filtration. In all of these procedures, samples must be properly obtained, transported to the laboratory as quickly as possible, and processed in a timely fashion to preserve organism viability and/or morphology. Organism viability and morphology may be adversely affected by a number of different factors including temperature, humidity, and exposure to fixatives or anticoagulants. Transportation requirements are described for each organism in the corresponding sections below.

Serologic assays for detection of antibodies are available as adjunctive methods for the diagnosis of a number of blood and tissue parasite infections. Unfortunately, none are sensitive or specific enough to be used to establish the diagnosis on their own. In particular, assays for infection with one helminth will often cross-react with antibodies to a different helminth [ 290 ]. When available, antibody titers may be used to determine the strength of the immune response or detect a trend in antibody levels over time. IFA can provide quantitative titer results but reading the slides is subjective and inherently prone to varying results. In contrast, EIAs typically provide only qualitative positive or negative results determined by an arbitrarily set breakpoint. Thus, clinicians will not be able to determine if a positive result was a very strong positive or a very weak one without calling the laboratory for more information. This can have important implications for interpretation of results that are not entirely consistent with the clinical picture. In some cases, it is desirable to confirm the result of an EIA by using a more specific immunoblot assay.

Based on the behavior of chromosomes during development of the Tetrahymena macronucleus, a model was proposed in which a palindromic sequence produces a break. Some possible mechanisms are in Figure 8 . A terminal snap back at this end can prime replication leading to the formation of a dicentric chromosome that initiates the BFB cycle. Asymmetric breakage of the dicentric at cell division leaves an inversion duplication centered on the palindrome. The telomeric ends of the original chromosome are lost. The final product carries inverse-order repeats of various size regions, whose junctions are symmetrical and subject to remodeling to form asymmetric junctions. Gene amplification by BFB cycles has been brilliantly reviewed ( Discount Fashionable JEWELLERY Bracelets Nick amp; Sons Cheap Sale Find Great Cheap Choice PuY1V6
). Palindrome-initiated amplification events of this type have been shown in yeast by Lobachev and coworkers ( Lobachev et al. 1998 ; BAGS Backpacks amp; Bum bags Kendall Kylie Discount Professional X1gHHVGP
). It should be noted that snap-back primer extensions of the types suggested in Figure 8 may also occur at single-strand nicks that generate TIDs as described in Clearance Store Cheap Official Site Small Leather Goods Wallets Mandarina Duck Cheap Online Store Buy Cheap For Nice O3g9S
.

View larger version:
Figure 7.

The breakage–fusion–bridge (BFB) cycle. Suggested many years ago by Barbara McClintock, this model explains the alternating orientation of copies seen in some amplification arrays. Issues are the source of the initial breaks, the forces that break a dicentric, the mechanisms of end fusions, and the stabilization of an array by blocking further end fusions. Several of these issues have been solved conceptually by the behavior of palindromic sequences.

View larger version:
Figure 8.

Use of palindromic sequences for induction of breaks and fusions in the breakage–fusion–bridge (BFB) model. The frequent association of palindromic sequences with amplifications in mammalian amplification suggested various ways in which they might contribute to the events in the BFB model. A break generated near a palindrome ( side, ) can leave ends whose snap-back primes repair synthesis, and serves to generate a dicentric chromosome ( side). A cruciform structure can be cut to leave snap-back ends that can similarly prime replication to form a dicentric. Heavy black lines denote duplex DNA and lighter black lines denote single strands. Ends lacking telomeres are likely to be subject to fusion and continued rounds of the cycle.

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Most models for duplication formation, like those for point mutations, propose a single discontinuous event or a cascade of immediately sequential events with intermediate structures that cannot be inherited. However, several other duplication models described here involve multistep processes in which intermediate forms are heritable and therefore subject to remodeling and selection over multiple cell generations. This is notably true of the TID formation process in which the initial symmetrical duplication can be remodeled by deletion and amplified over multiple generations. This is also true of the BFB model in which multiple cell generations may be required to increase repeat copy number. In such processes, duplications can form over several generations. Selection can progressively favor steps in their initial formation and later modification, as they lead to their higher amplification.

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