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Background
The development of the polymerase chain reaction (PCR) has greatly simplified DNA analysis and shortened laboratory time (ACOG, 2002). Polymerase chain reaction allows the exponential amplification of the targeted gene or DNA sequence. Only minute quantities of DNA, typically 0.1-1 mg, are necessary for PCR. DNA can be amplified from a single cell. One important prerequisite of PCR is that the sequence of the gene, or at least the borders of the region of DNA to be amplified, must be known.
The PCR procedure has three steps. First, DNA is denatured by heating to render it single stranded. Second, the PCR primers, which are short pieces of DNA (oligonucleotides) 20-30 base pairs in length exactly complementary to the ends of each piece of the double-stranded DNA to be amplified, anneal to their complementary regions of the DNA. Third, synthesis of the complementary strand of DNA occurs in the presence of the enzyme Taq polymerase and nucleotides triphosphates (dATP, dCTP, dGTP, and dTTP). The reaction cycle of denaturation, annealing, and extension is repeated 25-30 times to produce millions of copies of DNA. Typically, fragments several kilobases (kb) in size can be amplified, but sequences up to 10 kb have been successfully amplified. The exact cycling parameters and conditions for PCR must be determined empirically for each set of primers.
Polymerase chain reaction is very sensitive; therefore, extreme care must be taken to avoid amplification of contaminant DNA from aerosolized secretions or sloughed skin cells. These precautions are particularly important when DNA from a single cell is being amplified.
PCR amplification techniques raise considerable concerns regarding contamination from one specimen to another, creating the potential for false positive results. The clinical interpretation of PCR results may also be challenging. Amplification of organisms representing latent infection or colonization cannot be distinguished from active, clinically significant infections. Additionally, PCR may amplify fragments of nucleic acids, representing dead microorganisms, thus further clouding the clinical interpretation. Finally, specificities, sensitivities, and positive and negative predictive values of PCR have not been reported in large groups of patients for many of the microorganisms.
Polymerase chain reaction may be useful when culture is difficult due to the low numbers of the organisms, for fastidious or lengthy culture requirements, or when there is difficulty in collecting an appropriate sample. Quantification of viral load via PCR may be useful when the viral load can be used as a prognostic indicator, or when necessary follow the patient's response to therapy.
Vaginal Discharge
The three diseases most frequently associated with vaginal discharge are trichomoniasis (caused by T. vaginalis), bacterial vaginosis (caused by a replacement of the normal vaginal flora by an overgrowth of anaerobic microorganisms, mycoplasmas, and Gardnerella vaginalis), and candidiasis (usually caused by Candida albicans). C. trachomatis or N. gonorrhoeae can sometimes cause vaginal discharge, but can be distinguished in that the vaginal discharge is accompanied by a mucopurulent cervicitis.
Current Centers for Disease Control and Prevention Guidelines on management of diseases characterized by vaginal discharge (CDC, 2002) do not indicate any role for polymerase chain reaction (PCR) tests in the assessment of vaginal discharge unless the sexually transmitted diseases C. trachomatis or N. gonorrhoeae are suspected based on history of sexual activity and presence of mucopurulent cervicitis. Otherwise, the cause of vaginal infection can be diagnosed by pH and microscopic examination of the discharge.
Bacterial vaginosis:
Bacterial vaginosis is a condition in which the natural balance of organisms found in the vagina is changed from a predominance of Lactobacillus to an overgrowth of other bacteria including Gardnerella vaginalis, Mobiluncus and other anaerobes. Bacterial vaginosis can be diagnosed by the use of clinical or Gram-stain criteria (CDC, 2002; AGM-MSSVD, 2002). The most widely accepted method for diagnosis of bacterial vaginosis is the presence of three of the following four Amsel criteria: 1) a homogenous vaginal discharge; 2) a vaginal pH greater than 4.5; 3) the presence of clue cells; and 4) a fishy odor after addition of potassium hydroxide to the vaginal secretions (the amine test). Diagnosis on the basis of Amsel criteria has a reported sensitivity of 81% and a specificity of 94% (WHO, 1999). Gram stain of a vaginal smear has a sensitivity of 89% and a specificity of 93%. According to the CDC, culture of G. vaginalis is not recommended as a diagnostic tool because it is not specific. A DNA probe based test for high concentrations of G. vaginalis (Affirm™ VP III, manufactured by Becton Dickinson, Sparks, Maryland) may have clinical utility (CDC, 2002). This test has sensitivity for G. vaginalis of 94% and a specificity of 81% (WHO, 1999). Other commercially available tests that may be useful for the diagnosis of BV include a card test for the detection of elevated pH and trimethylamine (FemExam® test card, manufactured by Cooper Surgical, Shelton, Connecticut) and prolineaminopeptidase (Pip Activity TestCard™, manufactured by Litmus Concepts, Inc., Santa Clara, California). The FemExam is a rapid test that measures vaginal pH and volatile amines, corresponding to two of the four Amsel criteria. The proline aminopeptidase test is an indirect test for a chemical produced by the organisms associated with bacterial vaginosis. Prolineaminopeptidase assay has a reported sensitivity of 93% and a specificity of 93% for bacterial vaginosis (WHO, 1999).
Trichomoniasis:
Diagnosis of vaginal trichomoniasis (T. vaginalis) is usually performed by microscopy of vaginal secretions, but this method has a sensitivity of only about 60%--70% (CDC, 2002). According to the CDC, culture is the most sensitive commercially available method of diagnosis. The AGM-MSSVD (2002) states that up to 95% of female cases can be diagnosed by culture; 60-80% of male cases can be diagnosed by urethral culture or culture of first-void urine, and that sampling both simultaneously will significantly increase the diagnostic rate. According to the American Academy of Pediatrics (2006), culture of the organism and antibody tests using an enzyme immunoassay and immunofluorescence techniques for demonstration of the organism are more sensitive than wet-mount preparations but generally are not required for diagnosis. A rapid antigen detection assay for Trichomonas and Candidiasis has a sensitivity of 86% and a specificity of 99% for T. vaginalis (WHO, 1999). A commercially available, rapid, automated hybridization assay is available that uses DNA probes to directly detect Candida, Trichomonas and Gardnerella in vaginal swab samples (WHO, 1999). This assay has a sensitivity of 88-91% and a specificity of 100% (WHO, 1999) for T. vaginalis. PCR assay for trichomonas is somewhat more sensitive (93%) than antigen detection or hybridization assay for T. vaginalis but less sensitive than culture or microscopy (WHO, 1999). In addition, the PCR assay is less specific (96%) than microscopy, culture, antigen or hybridization assay, with the latter methods having sensitivities of 99-100% (WHO, 1999). The AAP (2006) stated that "[a] U.S. Food and Drug Administration-licensed polymerase chain reaction assay for T. vaginalis is not available in the United States but may be available as a research diagnostic test or from commercial laboratories."
Candidiasis:
A diagnosis of Candida vaginitis is suggested clinically by pruritus and erythema in the vulvovaginal area; a white discharge may be present (CDC, 2002). The diagnosis can be made in a woman who has signs and symptoms of vaginitis when either a) a wet preparation (saline, 10% KOH) or Gram stain of vaginal discharge demonstrates yeasts or pseudohyphae or b) a culture or other test yields a positive result for a yeast species (CDC, 2002; AGM-MSSVD, 2002). Candida vaginitis is associated with a normal vaginal pH (<4.5). According to the CDC (2002), identifying Candida by culture in the absence of symptoms is not an indication for treatment, because approximately 10% to 20% of women harbor Candida species and other yeasts in the vagina. Culture may be indicated in women with recurrent vulvovaginal candidiasis (defined as more than four episodes of vulvovaginal Candidiasis per year) to confirm the clinical diagnosis and to identify unusual species, including non-albicans species, including C. glabrata. A rapid antigen detection assay for Trichomonas and Candidiasis has a sensitivity of 61-81% and a specificity of 97% for C. albicans (WHO, 1999). A commercially available, rapid, automated hybridization assay is available that uses DNA probes to directly detect Candida, Trichomonas and Gardnerella in vaginal swab samples (WHO, 1999). This assay has a reported sensitivity 80% and specificity of 98% for Candida vaginalis.
Chlamydia trachomatis
Chlamydia trachomatis is an important cause of urethritis and cervicitis (WHO, 1999; AGM-MSSVD, 2002). Laboratory detection of C. trachomatis is necessary because as many as 70% to 80% of women and up to 50% of men who are infected do not experience any symptoms. The U.S. Preventive Services Task Force (USPSTF) strongly recommends that clinicians routinely screen all sexually active women 25 years and younger, and other asymptomatic women at increased risk of infection, for chlamydial infection (USPSTF, 2002). The conventional method for the laboratory diagnosis of C. tracomatis has been inoculation of a cell culture with a genital specimen. According to the WHO (1999), however, this method is expensive, labor-intensive, time-consuming, and requires considerable expertise. For these reasons, culture tests are now used less frequently and antigen and nucleic acid detection techniques have become common methods for detection of C. trachomatis infection.
The leukocyte esterase assay is a rapid urine dipstick test for the presence of an enzyme found in the urine when leukocytes are present due to inflammation. The LE test can diagnose urethritis but cannot identify the specific cause of the infection. The sensitivity and specificity of LE for the detection of chlamydial and gonococcal infection are 54%-97% and 36-95%, respectively (WHO, 1999). Microscopy detection of C. trachomatis has a reported sensitivity and specificity of 74%-90% and a specificity of 98%-99%. According to the American Academy of Pediatrics Committee on Infectious Diseases (AAP), nucleic acid amplification methods, such as PCR, transcription-mediated amplification (TMA), and strand displacement amplification (SDA) are more sensitive than cell culture and more specific and sensitive than DNA probe, direct fluorescent antibody (DFA) tests, or enzyme immunoassays (EIAs), although specificity is variable compared with culture (AAP, 2006). According to the USPSTF (2002), the sensitivities and specificities of nucleic acid amplification tests are all high, ranging from 82 to 100 percent. The sensitivity of antigen detection tests (EIA, DFA) is slightly lower (70 to 80 percent) but specificity remains high (96 to 100 percent). The AAP states that tests for detection of chlamydial antigen or nucleic acid are useful for evaluating urethral specimens from males, cervical specimens from females, and conjunctival specimens from infants. The PCR and LCR tests are useful for evaluating urine specimens from either sex. The Scottish Intercollegiate Guidelines Network (2000) states that nucleic acid amplification tests (LCR or PCR) are the recommended laboratory test for Chlamydia trachomatis.
Lymphogranuloma Venerum
Lymphogranuloma venerum (LGV) is caused by C. trachomatis serovars L1, L2, or L3. The disease occurs rarely in the United States. The most common clinical manifestation of LGV among heterosexuals is tender inguinal and/or femoral lymphademonpathy that is most commonly unilateral (CDC, 2002). Women and homosexually active men may have proctocolitis or inflammatory involvement of perirectal or perianal lymphatic tissues resulting in fistulas and strictures. According to the CDC (2002), the diagnosis of LGV is usually made by complement fixation and by exclusion of other causes of inguinal lymphadenopathy of genital ulcers. The diagnostic utility of serologic methods other than complement fixation is unknown (CDC, 2002). The AGM-MSSVD (2002) notes that although PCR and LCR methods have been used in diagnosis of C. trachomatis from urethral, cervical, or urine specimens, they have rarely been used in the context of LGV.
Gonorrhea
Infection of the genital tract with Neisseria gonorrhoeae can cause urethritis, cervicitis, proctitis, or bartholinitis (WHO, 1999; AGM-MSSVD, 2002). Complications of untreated disease include epididymitis, prostatitis, and infertility in men and pelvic inflammatory disease and infertility in women. Because most cases in females are asymptomatic, detection of infection using laboratory tests is needed to prevent sequelae and transmission to sexual partners and, for pregnant women, to neonates (WHO, 1999).
According to the AAP (2006), microscopic examination of Gram-stained smears of exudate from the eyes, the endocervix of postpubertal females, the vagina of prepubertal girls, male urethra, skin lesions, synovial fluid, and, when clinically warranted, cerebrospinal fluid (CSF) is useful in the initial evaluation. According to the World Health Organization (WHO), the sensitivity and specificity of Gram-stain for detection in urethral samples in symptomatic men is 90-95% and the specificity is 98-100% (WHO, 1999). However, Gram stain is not as useful for endocervical smears (sensitivity of 50-70%) because the presence of other Gram-negative diplococci makes interpretation difficult.
Neisseria gonorrhoeae can be cultured from normally sterile sites, such as blood, CSF, or synovial fluid, using specialized culture media. Selective media that inhibit normal flora and nonpathogenic Neisseria organisms are used for culture from non-sterile sites, such as the cervix, vagina, rectum, urethra, and pharynx. According to the WHO (1999), the sensitivity of culture for N. gonorrhea ranges from 81-100% with a specificity of 100%. An advantage of culture is that isolates are available for further testing. Disadvantages of culture are the need for stringent handling and up to 3 days for results.
The AAP (2006) notes that nucleic acid amplification methods by polymerase chain reaction (PCR), mediated amplification (TMA), and strand-displacement assays. are highly sensitive and specific when used on urethral (males) and endocervical swab assays. They also can be used with good sensitivity and specificity on first-void urine specimens, which has led to increased compliance with testing and follow-up in hard-to-access populations, such as adolescents. These techniques also permit dual testing of urine for C. trachomatis and N. gonorrhoeae. According to the WHO, the sensitivity and specificity of DNA hybridization assays for gonorrhea are 86-100% and 99% respectively. The sensitivity of PCR testing ranges from 89-97% and the specificity ranges from 94-100%. The sensitivity of LCR ranges from 95-100% and the specificity ranges from 98-100%.
According to guidelines from the CDC (2002), PCR tests for Neisseria gonorrhea are recommended for testing urethral swabs from males and endocervical swabs when conditions during holding and transport of inoculated culture media are not adequate to maintain the viability of organisms. The CDC guidelines note that commercial PCR assays have cross-reacted with nongonococcal Neisseria; such cross-reactivity has not been reported for commercial ligase chain reaction (LCR) and unamplified probe assays.
PCR testing of the urine may be indicated for urinary screening in women when pelvic examination is not indicated, and in men (CDC, 2002). The CDC guidelines note that the sensitivity of urine testing with PCR may be lower than with urethral (males) or endocervical swabs.
PCR tests may be indicated in screening vaginal swabs of prepubescent children for possible sexual abuse if culture is not available (CDC, 2002). According to CDC guidelines, additional revies is needed before a recommendation can be made for use of PCR tests in vaginal swabs of postmenarcheal adolescents and adults.
According to guidelines from the Centers for Disease Control and Prevention, PCR tests for gonorrhea are not recommended for vaginal, rectal, conjunctival or pharyngeal swabs, or for detecting disseminated gonococcal infection (CDC, 2002: AAP, 2006).
Chancroid
Chancroid is a genital ulcer disease caused by the bacterium Haemophilus ducreyi. In the United States, chancroid usually occurs in discrete outbreaks, although the disease is endemic in some areas. The accuracy of clinical diagnosis varies due to the atypical presentation of the ulcer. According to the AAP (2006), the diagnosis of chancroid usually is made on the basis of clinical findings and the exclusion of other infections associated with genital ulcer disease, such as syphilis or HSV, or adenopathies, such as lymphogranuloma venereum. Direct examination of clinical material by Gram stain may strongly suggest the diagnosis if large numbers of gram-negative coccobacilli, sometimes in "school of fish" patterns, are seen. Confirmation by recovery of H. ducreyi from a genital ulcer or lymph node aspirate is the more available alternative diagnostic test. According to the Association for Genitourinary Medicine (AGUM) of the Medical Society for the Study of Venereal Disease (MSSVD) (2002), in addition to culture or direct examination of gram stain, H. ducreyi may be identified by detection of nucleic acid (DNA) by amplification techniques such as PCR techniques, using nested techniques.
According to the CDC, a culture for H. ducreyi should be performed in patients with genital ulcer disease from regions where H. ducreyi is prevalent (CDC, 2002). According to the WHO (1999), the sensitivity and specificity of culture is 56-90% and 100% respectively; the WHO notes that the sensitivity of culture can only be estimated because there is no gold standard on which to base the diagnosis of chancroid. The resolved sensitivity of PCR using H. ducreyi compared to culture is 77-98%, and the specificity is 98-100% (WHO, 1999). Conversely, culture may be only 75% sensitive relative to PCR. Yet, PCR may be negative in a number of culture-proven chancroid cases, owing to the presence of Taq polymerase inhibitors in the DNA preparations extracted from genital ulcer specimens.
To circumvent the many problems of positive diagnosis of chancroid, the Centers for Disease Control and Prevention proposes that a "probable diagnosis", for both clinical and surveillance purposes, be made if the patient has one or more painful genital ulcers, and (a) no evidence of T. pallidum infection by dark field examination of ulcer exudates or by a serologic test for syphilis, and (b) the clinical presentation, appearance of the genital ulcers and regional lymphadenopathy, if present, is typical for chancroid and a test for HSV is negative (CDC, 2002).
Donovanosis
Donovanosis, or granuloma inguinale, is one cause of genital ulcer disease (WHO, 1999; AGM-MSSVD, 2002). The disease occurs rarely in the United States, although it is endemic in certain tropical and developing areas, including India; Papua, New Guinea; central Australia; and southern Africa. The disease is caused by Klebsiella granulomatis (previously known as Calymmatobacterium granulomatis), which can be seen in infected tissue as intracellular bacterial inclusions known as Donovan bodies but cannot be cultured on artificial media. Diagnosis of donovanosis is made by direct visualization under microscope of Donovan bodies. Alternatively, a piece of clean granulation tissue is removed from the leading edge of the genital ulcer with a scalpel, smeared on a slide, and stained. According to the WHO (1999), the sensitivity and specificity of tissue microscopy or swab is 60-80% and 100%, respectively. The AAP (2006) notes that diagnosis by polymerase chain reaction assay and serologic testing is available on a research basis. The sensitivities and specificities of antibody detection and PCR have not been determined (WHO, 1999).
Babesiosis
Babesiosis is a tick-borne disease caused by hemoprotozoan parasites of the genus Babesia. While more than 100 species have been reported, only a few have been identified as causing human infections. Babesia microti and Babesia divergens have been identified in most human cases, but variants (considered different species) have been recently identified. Little is known about the occurrence of Babesia species in malaria-endemic areas where Babesia can easily be misdiagnosed as Plasmodium.
Worldwide, but little is known about the prevalence of Babesia in malaria-endemic countries, where misidentification as Plasmodium probably occurs. In the United States, B. microti is the agent most frequently identified (Northeast and Midwest).
Infectious Disease Society of America Lyme disease guidelines (Wormser, 2000) state that the diagnosis of Babesiosis should be suspected in patients from areas where babesiosis is endemic who develop fever (especially if fever is very high (>37 degrees)) in the absence of erythema migrans after an Ixodes tick bite. Infection may also be suspected in patients who have residual symptoms after treatment for early Lyme disease.
According to the Centers for Disease Control and Prevention Guidelines on Identification and Diagnosis of Parasites of Public Health Concern (2001), PCR testing has a limited role in the diagnosis of Babesia infection. Diagnosis of Babesia infection should be made by microscopy detection of parasites in patients' blood smears. However, indirect fluorescent antibody (IFA) tests are useful for detecting infected individuals with very low levels of parasitemia (such as asymptomatic blood donors in transfusion-associated cases), for diagnosis after infection is cleared by therapy, and for discrimination between Plasmodium falciparum and Babesia infection in patients whose blood smear examinations are inconclusive and whose travel histories cannot exclude either parasite.
According to the CDC, molecular techniques, such as PCR, are necessary only in limited situations, specifically:
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Where the morphologic characteristics observed on microscopic examination of blood smears do not allow an unambiguous differentiation between Babesia and Plasmodium.
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In research investigations of new Babesia variants (or species) observed in recent human infections in the U.S. and in Europe.
Malaise and Fatigue (including Chronic Fatigue Syndrome)
"Shotgun" testing for a variety of infectious etiologies in patients with symptoms of fatigue is not medically necessary or appropriate. Testing for specific individual infectious etiologies is only appropriate when the patient exhibits signs or symptoms suggestive of active infection with that virus. (See also Aetna CPB 369 - Diagnosis of Chronic Fatigue Syndrome.)
According to the Centers for Disease Control and Prevention (2000), in clinical practice, no tests can be recommended for the specific purpose of diagnosing chronic fatigue syndrome. Tests should be directed toward confirming or excluding other possible clinical conditions.
In a statement on the "theoretical and experimental tests" for chronic fatigue syndrome, the Centers for Disease Control and Prevention (2000) states: "No diagnostic tests for infectious agents, such as Epstein-Barr virus, enteroviruses, retroviruses, human herpesvirus 6, Candida albicans, and Mycoplasma incognita, are diagnostic for CFS and as such should not be used (except to identify an illness that would exclude a CFS diagnosis, such as mononucleosis)."
A Clinical Practice Guideline from the Collège des médecins du Québec (1998) reached the same conclusion about the value of diagnostic testing for infectious agents in patients with chronic fatigue syndrome.
A review of the literature on the diagnosis of viruses in patients suspected of having chronic fatigue syndrome from Chronic Fatigue and Immune Dysfunction Syndrome (CFIDS) Association of America (2001) states that viral tests are only appropriate when a specific active viral infection is suspected based on clinical signs: "Because research has documented no clear association between a virus and CFIDS, testing patients for viral infection has limited use unless clinical signs indicate that an active viral infection may be present and requiring treatment. The results are also difficult to interpret, because the immune system in CFIDS may be up-regulated and latent viruses may not be fully suppressed. Tests to determine elevated antibody titers for EBV and other viruses are not considered diagnostic for CFIDS by most physicians, and are usually performed only when a specific viral infection is suspected as a cause of the patient's symptoms."
A review of diagnosis and management of chronic fatigue syndrome (2002) published in the journal American Family Physician explains that there is no clear evidence that chronic fatigue syndrome is caused by an infection: "Although a number of other viral pathogens (such as the Coxsackie virus, human herpes virus 6, cytomegalovirus, measles, and the human T-cell lymphotropic virus [HTLV-II]) have also been implicated as etiologic agents for CFS, there is no consistent or conclusive data to suggest any causal relationships. It is now believed that CFS is not specific to one pathogenic agent but could be a state of chronic immune activation, possibly of polyclonal activity of B-lymphocytes, initiated by a virus."
Regarding laboratory tests, the authors stated that "[l]aboratory tests should be limited to complete blood cell counts and tests specific for the patient's symptoms. For example, serologic and neurologic analyses for Lyme disease or multiple sclerosis need only be conducted if the patient presents with appropriate symptoms."
Thus, panels of PCR tests to detect various infections are not indicated in patients with symptoms suggestive of chronic fatigue syndrome. It is only appropriate for individual tests to be selected to detect particular infectious agents if the patient's clinical presentation suggests active infection with that infectious agent.
Genital Ulcer Diseases
According to guidelines from the Centers for Disease Control and Prevention (2002), a diagnosis of genital ulcer disease based only on the patient's medical history and physical examination often is inaccurate. Therefore, evaluation of all patients who have genital ulcers should include a serologic test for syphilis and a diagnostic evaluation for genital herpes; in settings where chancroid is prevalent, a test for Haemophilus ducreyi should also be performed.
According to the CDC, specific tests for evaluation of genital ulcers include:
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serology, and either darkfield examination or direct immunofluorescence test for T. pallidum;
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culture or antigen test for herpes simplex virus (HSV); and
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culture for H. ducreyi.
According to CDC guidelines, no FDA-approved PCR test for these organisms is available in the United States, but such testing can be performed by commercial laboratories that have developed their own PCR tests.
Herpes Simplex Virus
Herpes simplex virus (HSV) is one of the major causes of genital ulcer disease. Primary infection is followed by latency and variable periods of reactivation. Although clinical diagnosis may be accurate if based on the presence of typical vesicles, up to two-thirds of individuals acquire HSV asymptomatically, and most infected persons shed virus during latent periods Laboratory diagnosis is necessary to detect HSV in asymptomatically infected people to prevent transmission to sexual partners and to children born to infected mothers.
The Association for Genitourinary Medicine and the Medical Society for the Study of Venereal Diseases (2002) state that the clinical utility of HSV serologic tests has not been fully assessed, and that virus detection remains the method of choice. According to the Centers for Disease Control and Prevention's current guidelines for diagnosing herpes simplex virus (HSV) infection, type-specific serology for HSV type 2 may be helpful in identifying persons with genital herpes (CDC, 2002). Biopsy of ulcers may be helpful in identifying the cause of unusual ulcers or ulcers that do not respond to initial therapy.
According to the CDC, PCR testing for herpes virus DNA is indicated for diagnosis of HSV infection of the central nervous system, but other uses of polymerase chain reaction testing in suspected HSV have not been established (CDC, 2002). Neither guidelines from the AAP (2006) or CDC (2002) indicate any role for quantification of HSV viral load in the diagnosis or management of herpes simplex virus.
Human Immunodeficiency Virus (HIV)
Most individuals can be diagnosed as infected with human immunodeficiency virus (HIV) based on the detection of HIV specific IgG antibodies (WHO, 1999). For detection of early infection before seroconversion occurs, or to detect HIV infection in neonates, assays that detect HIV p24 protein or HIV DNA or RNA are used. Quantitative HIV RNA assays are not necessary for diagnosis of infection but are useful for monitoring treatment.
The laboratory diagnosis of HIV infection during infancy depends on detection of virus or virus nucleic acid. The transplacental transfer of antibody complicates the serologic diagnosis of infant infection. According to the AAP (2006), human immunodeficiency virus nucleic acid detection by PCR of DNA extracted from peripheral blood mononuclear cells is the preferred test for diagnosis of HIV infection in infants.
Plasma HIV RNA PCR may be used to diagnose HIV infection if the result is positive. However, this test result may be negative in HIV-infected persons. The test is licensed by the Food and Drug Administration only in quantitative format and, according to the AAP (2006), currently is used for quantifying the amount of virus present as a measurement of disease progression, not for diagnosis of HIV infection in infants.
According to the AAP (2006), enzyme immunoassays (EIAs) are used most widely as the initial test for serum HIV antibody. These tests are highly sensitive and specific. Repeated EIA testing of initially reactive specimens is required to reduce the small likelihood of laboratory error. Western blot or immunofluorescent antibody tests should be used for confirmation, which will overcome the problem of a false-positive EIA result.
Lyme Disease
According to available evidence-based guidelines, polymerase chain reaction has not been validated for either the diagnosis of Lyme disease or monitoring response to therapy.
American College of Physicians-American Society of Internal Medicine guidelines (1997) state that PCR of serum or cerebrospinal fluid "need[s] further validation" and that "[p]ublished experience with these techniques [PCR] is insufficient to allow development of guidelines for their use."
The Centers for Disease Control and Prevention (2001) states that "PCR has not been standardized for routine diagnosis of Lyme Disease."
The National Institute of Arthritis and Infectious Disease (2001) has explained the reasons why PCR has limited utility in the diagnosis of Lyme disease: "To be sure, the polymerase chain reaction (PCR) is an extremely sensitive laboratory test that is capable of detecting very few molecules of bacterial DNA. However, the numbers of Borrelia likely to be present -- if at all -- in patients suspected of having Lyme disease are too small to generate sufficient amounts of bacterial DNA to be detected by this procedure."
Guidelines from the Infectious Disease Society of America (2002) do not indicate any role for PCR in monitoring the treatment of patients with Lyme disease.
The American Academy of Pediatrics Committee on Infectious Diseases (2003) states: "New, more sensitive and more specific diagnostic tests such as the polymerase chain reaction assay, which may be able to identify the presence of even small quantities of spirochetal DNA, are in development. However, physicians should be cautious when interpreting the results of these investigational tests until their clinical usefulness has been proven."
American Lyme Disease Foundation states: "The polymerase chain reaction (PCR) test is a very sensitive assay that detects the DNA of B. burgdorferi. However, certain limitations prevent the PCR from being widely used. First, B. burgdorferi bacteria do not persist in easily obtainable fluids such as blood, synovial (joint) fluid or spinal fluid, but typically bind to joint and nerve tissues. A PCR done on spinal fluid may be positive in early neurologic disease (e.g., Lyme meningitis) but is usually negative in a patient with long-term central nervous system damage. Second, a PCR can be easily contaminated, producing false positive results. For this reason, a positive PCR in a patient whose standard blood tests (ELISA and Western blot) are negative must be viewed with skepticism."
Puotinen, et al. (2002) concluded that "[t]he Lyme multiplex polymerase chain reaction (PCR) has not been standardized; therefore, it is not employed currently in routine testing." Edlow (2001) explained that PCR remains a research technique, in part because labs performing PCR tests must be meticulous in technique to minimize the likelihood of false-positive results. "In addition" Edlow explained, "no large clinical series have been reported that assess the performance of the test in the non-research setting."
The AAP (2006) states that PCR tests for spirochete DNA have no role in diagnosis of Lyme disease.
Thus, the clinical utility of PCR in the diagnosis or monitoring of Lyme disease has not been established. In addition, current guidelines do not indicate any role for PCR quantification of spirochete load in the diagnosis or management of patients with Lyme disease.
Syphilis
Syphilis, a chronic infection with clinical manifestations occurring in distinct states, is caused by the spirochete Treponema pallidum. Diagnostic studies for syphilis include a targeted clinical history and physical examination, serologic tests, investigations of sexual contacts and, if appropriate, darkfield microscopic examination of fluids from lesions, cerebrospinal fluid (CSF) tests, and radiologic examination (CDC, 2002; Beer & Berkow, 1999).
According to the AAP, the non-treponemal antibody tests (VDRL, RPR, and ART) are useful for screening; the treponemal tests (FTA-ABS and MHA-TP) are used to establish a presumptive diagnosis (AAP, 2006). Quantitative non-treponemal antibody tests are used to assess the adequacy of therapy and to detect re-infection and relapse. Most current guidelines indicate no specific role for PCR testing in the screening or diagnosis of syphilis (AAP, 2006; CDC, 2002). The Association for Genitourinary Medicine and the Medical Society for the Study of Venereal Diseases (2002) suggests that either the direct fluorescent antibody test or the PCR test may be useful for used for oral or other lesions where contamination with commensal treponemes is likely.
Two classes of serologic tests for syphilis (STS) aid in diagnosing syphilis and other related treponemal diseases:
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Screening, non-treponemal tests using lipoid antigens detect syphilitic reagin and include the Venereal Disease Research Laboratory (VDRL) and the rapid plasma reagin (RPR) tests.
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Specific treponemal tests detect anti-treponemal antibodies and include fluorescent treponemal antibody absorption (FTA-ABS) test, microhemagglutination assay for antibodies to T. pallidum (MHA-TP), and Treponema pallidum hemagglutination assay (TPHA).
The VDRL test is a flocculation test for syphilis in which reagin antibody in the patient's serum reacts visibly with cardiolipin, the antigen. Reactive and weakly reactive VDRL tests are considered positive for T. pallidum, and should be confirmed by one of the more specific treponemal tests, and the reactive tests should be quantitated by serial dilution.
Before treatment (except in infections of < 1 yr), CSF examination is recommended to exclude neurosyphilis. The cell count and differential and total protein are usually measured, and VDRL or other nonspecific (reagin) serologic tests performed. Treponemal tests of CSF are not helpful.
Human Papillomavirus (HPV)
Human papillomaviruses (HPVs) produce epithelial tumors (warts) of the skin and mucous membranes (AAP, 2006). Cutaneous non-genital warts include common skin warts, plantar warts, flat warts, thread-like (filiform) warts, and epidermodysplasia verruciformis. Those affecting the mucous membranes include anogenital, oral, nasal, and conjunctival warts, as well as respiratory papillomatosis. Most cutaneous and anogenital warts are diagnosed by clinical inspection. Detection of cervical HPV infection may be enhanced by use of colposcopy with application of acetic acid (vinegar), which causes the lesion to turn white. This characteristic, however, is not specific for HPV infection, and false-positive test results are common. When the diagnosis is questionable, histologic examination of a biopsy specimen can be diagnostic.
Human papillomavirus cannot be cultured. According to the American College of Obstetricians and Gynecologists (ACOG, 2002), currently only one product, Hybrid Capture II, is FDA-approved for HPV DNA. This test uses nucleic acid amplification and hybridization to assess exfoliated cervical cells for the presence of one or more of 13 high- and intermediate-risk HPV types. Although this test appears to be very sensitive, rare cross-reactivity with low-risk HPV types and HPV types of undetermined significance has been reported.
Current guidelines from the Centers for Disease Control and Prevention (20002) and from the Association of Genitourinary Medicine and the Medical Society for the Study of Venereal Diseases (2002) mention no role for polymerase chain reaction (PCR) testing in the management of human papilloma virus (HPV) infection. The Institute for Clinical Systems Improvement (ICSI) (2001) states that polymerase chain reaction may be more sensitive and specific than hybridization assays, PCR testing has not been approved by the FDA for this indication, there is a risk of contamination, and methods have not been standardized.
Given the prevalence of HPV infection, the clinical benefit of testing for the presence of HPV, other than as an adjunct to cancer screening, is of unknown clinical benefit. According to the CDC, "[n]o data support the use of type-specific HPV nucleic acid tests in the routine diagnosis or management of visible genital warts." The Advisory Committee on Immunization Practices does not recommend HPV testing to select persons for HPV vaccination. The AAP (2006) explains that testing for HPV types is used in combination with Pap test to determine whether patients need to be sent for colposcopy; otherwise, screening for clinically inapparent HPV infection or evaluating anogenital warts using HPV DNA or RNA tests is not recommended.
The National Cancer Institute's interim guidelines for managing abnormal cervical cytology and the American Society of Colposcopy and Cervical Pathology recommend human papilloma virus (HPV) DNA testing for women with ASCUS (atypical cervical squamous cells of undetermined significance) using the a hybridization assay. The hybridization assay can distinguish low risk HPV (not usually found in precancerous lesions) from high-risk HPV (found in precancerous and cancerous lesions) in ASCUS lesions. According to the CDC, typing HPV DNA is not useful in types of cervical abnormalities other than ASCUS lesions. See Aetna CPB 359, HPV Testing in Cervical Cancer Screening. The CDC (2002) notes that screening for subclinical genital HPV infection using DNA or RNA tests is not recommended.
Ehrlichiosis
Ehrlichiosis is a febrile illness resembling Rocky Mountain spotted fever caused by rickettsial-like bacteria of the genus Ehrlichia and transmitted to humans by ticks (Merck Manual). Most cases have been identified in the southeastern and south-central parts of the United States. Two species of Ehrlichia are human pathogens in the United States: E. chaffeensis causes human monocytic ehrlichiosis and E. phagocytophilia or a related organism causes human granulocytic ehrlichiosis. E. canis is now regarded as human monocytic ehrlichiosis.
Regardless of the species causing the infection, the symptoms and signs are similar. Although some infections are asymptomatic, most cause an abrupt onset of illness with fever, chills, headache, and malaise, usually beginning about 12 days after the tick bite. Some patients develop a maculopapular or petechial rash involving the trunk and extremities, although rash is rare with E. canis. Abdominal pain, vomiting and diarrhea, disseminated intravascular coagulation, seizures, and coma may occur. Hematologic and hepatic abnormalities include leukopenia, thrombocytopenia, and abnormal liver function tests, especially elevated levels of transaminases.
According to guidelines from the Infectious Disease Society of America, ehrlichiosis may be suspected in patients with Lyme disease symptoms who have a very high fever (>38 degrees C) (Wormser, 2000), or patients who develop fever in the absence of erythema migrans after an Ixodes tick bite in areas where these infections are endemic.
The Centers for Disease Control and Prevention (CDC) defines a confirmed case of ehrlichiosis as a 4-fold or greater change in antibody titer by indirect immunofluorescence assay (IFA) between acute and convalescent serum samples (ideally collected 3 to 6 weeks apart), polymerase chain reaction (PCR) amplification of ehrlichial DNA from a clinical sample, or detection of intraleukocytoplasmic Ehrlichia microcolonies (morulae) and a single IFA titer of more than 64. A probable case is defined as a single IFA titer of more than 64 or the presence of morulae within infected leukocytes. Examination of peripheral blood smears to detect morulae in peripheral blood monocytes or granulocytes is insensitive, but this test is warranted for patients for whom a high index of suspicion exists. The PCR test is useful in detecting erlichiosis in the acute phase before immunohistochemical tests are positive (AAP, 2006). The use of the PCR test to amplify nucleic acid from acute phase peripheral blood of patients with ehrlichiosis seems sensitive, specific, and promising for early diagnosis but currently is unstandardized and is available only in research laboratories and at the CDC (AAP, 2006; CDC, 2000).
Doxycycline is the drug of choice for treatment of human ehrlichiosis and is also effective against Lyme disease. Ehrlichiosis may be severe or fatal in untreated patients, and initiation of therapy early in the course of the disease helps minimize complications of the illness.
Malaria
Malaria is infection with any of four different species of Plasmodia, causing periodic paroxysms of chills, fever and sweating, anemia, and splenomegaly.
Malaria is endemic in Africa, much of South and Southeast Asia, Central America, and northern South America. Malaria once was endemic in the United States but has been virtually eliminated from North America. The four important Plasmodium species are P. falciparum, P. vivax, P. ovale, and P. malariae.
P. falciparum infection is a medical emergency. Recurrent attacks of chills and fever without apparent cause should always suggest malaria, particularly if the patient has been in an endemic area within 3 to 5 yr, has an enlarged spleen, or has been recently transfused. Finding Plasmodium in a blood smear is diagnostic. The infecting species must be identified, since this influences therapy and prognosis.
Polymerase chain reaction, malarial ribosomal RNA, and species-specific DNA probes are under evaluation, as is a dipstick method for detecting an antigen of P. falciparum during acute illness (AAP, 2003). According to the AAP (2003), "[o]ther diagnostic tests, including polymerase chain reaction assay, DNA probes, and malarial ribosomal RNA testing, may provide rapid and accurate diagnosis in the future but currently are used in experimental studies only." Serology is only helpful in retrospect. IgM antibodies usually appear when parasites are first demonstrable in peripheral blood; later, the IgG response is marked.
Genital Mycoplasma Infections: Ureaplasma urealyticum and Mycoplasma hominis
Because detection of mycoplasma or ureaplasma is currently impractical, guidelines from the AAP and CDC recommend performing diagnostic tests for mycoplasmas and ureaplasmas when a patient presents with a clinical condition known to be caused by or associated with these organisms and when more common etiologies are excluded (AAP, 2006; CDC, 2002). The correct microbiological diagnosis takes on greater importance in patients who are immunosuppressed and at greater risk for disseminated infection with a poor outcome.
The standard method of diagnosing genital mycoplasma infections is by cell culture. Specialized culture media and growth conditions are necessary. Both M. hominis and U. urealyticum can be detected in culture within 2-5 days. According to the CDC, serologic studies are not useful for evaluating genital mycoplasma infections (CDC, 2002). Molecular techniques such as PCR are not needed when culture is available for M. hominis and Ureaplasma species.
Helicobacter pylori
Current guidelines do not provide any indication for PCR testing in the diagnosis of helicobacter pylori. To establish the presence of H. pylori, tests for antibody to H. pylori in blood are > 90% specific and sensitive. Other tests include a urea breath test or rapid urease test, and histology of antral biopsies obtained at endoscopy.
Helicobacter pylori infection can be diagnosed by culture of gastric biopsy tissue (AAP 2006). Organisms usually can be visualized on histologic sections using special stains. Because of production of urease by the organisms, urease testing of a gastric specimen can give a rapid and specific microbiologic diagnosis. Each of these tests requires endoscopy and biopsy. Noninvasive, commercially available tests include the breath test, which detects labeled carbon dioxide in expired air after oral administration of isotopically labeled urea, and serology for the presence of immunoglobulin G to H. pylori. According to the AAP (2006), each of the diagnostic tests has a sensitivity and specificity of 95% or more. A stool antigen test is also available commercially.
Mucosa-Associated Lymphoid Tissue (MALT) Lymphomas and Marginal Zone Lymphomas
MALT lymphomas have recently been reclassified as extranodal marginal-zone lymphomas of MALT-type. The most common and well-studied MALT lymphoma is gastric MALToma. This neoplasm is intimately associated with H. pylori, with the organism being present in more than 90% of pathologic specimens of MALTomas. This etiologic factor serves as the basis for treatment. Treatment of gastrointestinal MALT lymphomas is with antibiotics designed to eradicate H. pylori.
Guidelines from the National Comprehensive Cancer Network (NCCN, 2003) on non-Hodgkin's lymphoma indicate that PCR testing is useful in evaluating individuals with MALT lymphomas and marginal zone lymphomas who have non-diagnostic atypical lymphoid infiltrates that are positive for H. pylori infection. According to the NCCN guidelines, detection by PCR of a t(11;18) gene rearrangement, a specific translocation of genes 11 and 18, in these persons predicts no response to antibiotic therapy for H. pylori infection, and alternative treatment should be considered
Trichomoniasis
Trichomoniasis is the term for infection of the vagina or male genital tract with Trichomonas vaginalis (Beers & Berkow, 1999). T. vaginalis is a flagellated protozoan found in the GU tract of both men and women. The organism is more common in women, affecting about 20% during the reproductive years and causing vaginitis, urethritis, and possibly cystitis. T. vaginalis is more difficult to detect in men; probably causes prostatitis and cystitis; and may account for 5 to 10% of all cases of male urethritis in some areas. Asymptomatic infected men often infect their sex partners. The infection may coexist with gonorrhea and other sexually transmitted diseases.
The diagnosis of T. vaginalis in women is discussed above in the section above on vaginal discharge. In women, an immediate diagnosis of trichomoniasis can usually be made by examining vaginal secretions under microscopy (wet mount). The lashing movements of the flagella and striking motility of the oval-shaped organisms are readily observed. Cultures and antibody tests using an enzyme immunoassay and immunofluorescence techniques for demonstration of the organism are more sensitive and specific than direct examination, but according to the AAP, are generally not required for the diagnosis. Trichomoniasis is also commonly diagnosed on a Papanicolaou smear.
T. vaginalis may sometimes cause non-gonococcal urethritis in men. According to the CDC (2002), diagnostic procedures for T. vaginalis reserved for situations in which these infection is suspected (e.g., contact with trichomoniasis and genital lesions suggestive of genital herpes) or when non-gonococcal urethritis is not responsive to therapy. In men, an immediate diagnosis of T. vaginalis can be made by examining a wet mount of urethral secretions and by culture. Examining the centrifuged sediment of urine and prostatic secretions may also be helpful.
CDC guidelines indicate no role for PCR testing in the diagnosis of T. vaginalis. The AAP (2006) notes that an FDA-licensed PCR test for T. vaginalis is not available in the United States but may be available as a research diagnostic test or from commercial laboratories.
Rocky Mountain Spotted Fever
Rocky Mountain Spotted Fever is an acute febrile disease caused by Rickettsia rickettsii and transmitted by ixodid ticks, producing high fever, cough, and rash (Beers & Berkow, 1999). Rocky Mountain spotted fever (RMSF) is limited to the Western Hemisphere. Initially recognized in the Rocky Mountain states, it occurs in practically all states (except Maine, Hawaii, and Alaska) in the United States, especially the Atlantic states. In humans, infection occurs mainly from May to September, when adult ticks are active and persons are most likely to be in tick-infested areas. In southern states, cases occur throughout the year. The incidence is high in children less than 15 years of age and in others who frequent tick-infested areas for work or recreation.
Serologic tests, isolation and identification of Rickettsia rickettsii from blood or tissues, and identification of the agent in skin or other tissues by immunofluorescence help confirm the diagnosis, particularly in RMSF. To be useful, serologic tests require three serum samples, taken during the 1st, 2nd, and 4th to 6th weeks of illness. Polymerase chain reaction is useful in early identification of specific rickettsial nucleic acids.
According to the AAP (2006), the diagnosis of RMSF can be established by one of the multiple rickettsial group-specific serologic tests. A 4-fold or greater change in titer between acute- and convalescent-phase serum specimens is diagnostic when determined by indirect immunofluorescence antibody (IFA), enzyme immunoassay (EIA), complement fixation (CF), latex agglutination (LA), indirect hemagglutination (IHA), or microagglutination (MA) tests. The IFA is the most widely available confirmatory test. Antibodies are detected by IFA 7 to 10 days after onset of illness. According to the AAP, the nonspecific and insensitive Weil-Felix serologic test (Proteus vulgaris OX-19 and OX-2 agglutinins) is not recommended (AAP, 2006).
The AAP guidelines state that culture of R. rickettsii usually is not attempted because of the danger of transmission to laboratory personnel (AAP, 2006). Rickettsia rickettsii have been identified by immunofluorescent staining or PCR testing of tissue specimens. The AAP guidelines state that PCR for detection of R. rickettsii in blood and biopsy specimens during the acute phase of the illness confirms the diagnosis and is available from CDC reference laboratories.
Chlamydophila pneumoniae
Chlamydophila (formerly Chlamydia) pneumoniae is a species of Chlamydia that is antigenically, genetically, and morphologically distinct from Chlamydia species. C. pneumoniae has been found in 5 to 10% of older adults with community-acquired pneumonia and often produces disease severe enough to require hospitalization. This organism has also been implicated in 5 to 10% of cases of nosocomial pneumonia, but relatively little is known about its epidemiology. Transmission of C. pneumoniae is presumably by respiratory aerosol transmission between humans.
Clinical features of C. pneumoniae resemble those of mycoplasmal pneumonia, including pharyngitis, bronchitis, and pneumonitis, primarily in older children and young adults. Most patients have cough, fever, and sputum production but are not seriously ill.
In addition to acute respiratory tract disease, some investigators have associated C. pneumoniae with atherosclerotic cardiovascular disease. This association is based on the increased frequency of serum antibodies in patients compared with controls, the detection of antigen or DNA in atheromatous plaques, the production of arterial lesions in experimentally infected animals, and small human trials demonstrating that treatment of high-risk patients with macrolides decreases the risk of subsequent cardiovascular events. According to the AAP, large, prospective, randomized trials are underway to further explore this association and to determine whether treatment is beneficial (AAP, 2003). Other investigators have associated C. pneumoniae with asthma, Alzheimer disease, multiple sclerosis, and Kawasaki disease, but the AAP has concluded that the evidence supporting any of these associations is limited.
C. pneumoniae may be detected by cultivating it in embryonated egg cultures (as with other chlamydiae), by using direct stains with immunofluorescence or a polymerase chain reaction, or by using serial serologic tests to show seroconversion. However, these tests are usually unavailable in most clinical laboratories. The diagnosis is suspected in a patient who has typical symptoms, has no established alternative diagnosis, and does not respond to beta-lactam antibiotics. C. pneumoniae may be treated with erythromycin or tetracycline.
Cytomegalovirus
Cytomegalovirus (CMV) causes various infections, occurring congenitally, postnatally, or at any age, ranging from inconsequential silent infection to disease manifested by fever, hepatitis, pneumonitis, and, in newborns, severe brain damage, stillbirth, or perinatal death.
Especially in the immunocompromised host, CMV may be isolated from urine, other body fluids, or tissues. However, CMV can be excreted for months or years after infection without causing active disease, and a positive CMV culture must be interpreted with regard to the particular host and disease manifestation. Examination of cells shed in urine for intranuclear inclusions is an insensitive test. Biopsy showing CMV-induced pathology is often important in demonstrating invasive disease.
Recovery of virus from a target organ provides unequivocal evidence that the disease is caused by CMV infection. However, according to the AAP (2006), a presumptive diagnosis can be made on the basis of a 4-fold antibody titer rise in paired serum samples or by demonstration of virus excretion.
Complement fixation is the least sensitive serologic method for diagnosis of CMV infection and should not be used to establish previous infection or passively acquired maternal antibody. Various immunofluorescence assays, indirect hemagglutination, latex agglutination, and enzyme immunoassays are preferred for this purpose.
Techniques for detection of viral DNA in tissues and some fluids, especially cerebrospinal fluid, by polymerase chain reaction or hybridization are available from reference laboratories. Detection of pp65 antigen in white blood cells is used to detect infection in immunocompromised hosts.
CMV viral load tests using quantitative PCR are used to monitor disease progression.
Pneumocystis Pneumonia
Pneumocystis jiroveci (formerly P. carinii), now considered a fungus rather than a protozoan, causes disease only when defenses are compromised, most commonly when there are defects in cell-mediated immunity as in hematologic malignancies, lymphoproliferative diseases, cancer chemotherapy, and AIDS (Beers & Berkow, 1999). About 30% of patients with HIV infection have P. jiroveci pneumonia as the initial AIDS-defining diagnosis, and > 80% of AIDS patients have this infection at some time if prophylaxis is not given. Patients with HIV infection become vulnerable to P. jiroveci pneumonia when the CD4 count is < 200/µL. Most patients have fever, dyspnea, and a dry, nonproductive cough that may evolve subacutely over several weeks or acutely over several days.
A definitive diagnosis of PCP is made by demonstration of organisms in lung tissue or respiratory tract secretions (AAP, 2006). The most sensitive and specific diagnostic procedures have been open lung biopsy and transbronchial biopsy. However, bronchoscopy with bronchoalveolar lavage, induction of sputum in older children and adolescents, and intubation with deep endotracheal aspiration are less invasive and often diagnostic and have been sufficiently sensitive in patients with HIV infection who have an increased number of organisms compared with non-HIV-infected patients with PCP. According to the AAP (2006), "polymerase chain reaction assays for detecting P. jiroveci infection are experimental and are not approved by the U.S. Food and Drug Administration for diagnosis." The AAP notes that serologic tests are not useful. Guidelines from Cincinnati Children's Hospital Medical Center (2001) state that PCR testing for P. jiroveci infection is investigational.
Human Herpesvirus Type 6
Human herpesvirus 6 (HHV-6) is a T-cell lymphotrophic virus with high affinity for CD4 lymphocytes. HHV-6 has 2 variants, A and B. Variant B causes the childhood illness roseola infantum, while variant A has been isolated mainly from immunocompromised hosts. The disease manifestations of variant A still are undefined, but both variants may turn out to be pathogenic in the settings of transplantation and AIDS.
Primary HHV-6 infection usually occurs in infants and is the most common cause of febrile-induced seizures in children aged 6 to 24 months. Acute infection in immunocompetent adults is rare but may present as a mononucleosis-like illness with fever, lymphadenopathy, hepatitis or encephalitis, and negative test results for CMV or Epstein-Barr virus (EBV).
Infection remains latent in lymphocytes and monocytes and can persist in some tissues at low levels. In the immunocompetent host, this persistent infection generally is of no consequence. In the immunosuppressed host, HHV-6 may be associated with opportunistic disease. Whether this represents reactivation of latent infection or superinfection is unclear.
In transplant patients, HHV-6 infection has been linked with infection/reactivation and increased severity of CMV disease. In HIV patients, HHV-6 infection may up-regulate HIV replication and hasten the progression towards AIDS. HHV-6 also has been implicated in the pathogenesis of white matter demyelination in AIDS dementia complex. Causality has yet to be demonstrated clearly.
HHV-6 infection often is asymptomatic. Symptomatic disease occurs predominately after primary infection in infants and after either primary or reactivation disease in immunocompromised adults.
An Expert Working Group convened by the Health Canada Laboratory Centre for Disease Control (LCDC, 2000) concluded that the most appropriate clinical scenarios in which HHV-6 laboratory diagnosis may be indicated appear to be: a) primary infection in febrile children less than 3 years of age; b) primary infection or viral reactivation in immunocompromised individuals such as AIDS patients or transplant patients; c) mononucleosis-like syndrome in patients without heterophile antibodies or antibodies specific to Epstein-Barr virus (EBV).
The diagnosis of primary HHV-6 infection currently necessitates use of research techniques to isolate the virus from a peripheral blood specimen (AAP, 2006). A four-fold increase in serum antibody alone does not necessarily indicate new infection, as an increase in titer also may occur with reactivation and in association with other infections. However, seroconversion from negative to positive in paired sera is good evidence of recent primary infection. According to AAP guidelines (2000), commercial assays for antibody and antigen detection and polymerase chain reaction for detecting HHV-6 DNA are in development, but so far, none of these assays can differentiate reliably between primary infection and viral persistence or reactivation. The LCDC Expert Working Group (2000) concluded that serologic and PCR tests have been developed to diagnose an active or recent HHV-6 infection, "further evaluation in the clinical context (specificity, sensitivity, predictive values) needs to be done to improve confidence in and reliability of HHV-6 laboratory testing."
According to the AAP (2003), treatment for HHV-6 infection is supportive. Therapy usually is unnecessary with primary infection of immunocompetent patients. Therefore, HHV-6 testing in immunocompetent individuals is not necessary. For immunocompromised patients with serious HHV-6 disease, some experts recommend a course of ganciclovir. Therefore, HHV-6 testing may be necessary in immunocompromised patients such as AIDS patients and transplant recipients. In addition, HHV-6 testing may be necessary to rule out other potential diagnoses in patients presenting with a mononucleosis-like illness with fever, lymphadenopathy, hepatitis or encephalitis, and negative test results for CMV or Epstein-Barr virus (EBV).
Human Herpesvirus Type 7
According to the AAP (2006), recognition of the varied clinical manifestations of human herpesvirus 7 (HHV-7) infection is evolving. Many, if not most, primary infections with HHV-7 may be asymptomatic or mild; some may present as typical roseola and may account for second or recurrent cases of roseola. Febrile illnesses associated with seizures also have been reported. Some investigators suggest that the association of HHV-7 with these clinical manifestations results from the ability of HHV-7 to reactivate HHV-6 from latency.
An Expert Working Group convened by the Health Canada Laboratory Centre for Disease Control (LCDC, 2000) concluded that "presumably" the most appropriate clinical scenarios in which HHV-7 laboratory diagnosis may be indicated appear to be similar to those for HHV-6 - children with febrile illness and immunocompromised individuals. However, the LCDC Expert Working Group stated that HHV-7 "has not been linked to any specific clinical scenarios."
The AAP (2006) concluded that "[d]iagnostic tests for HHV-7 are also limited to research laboratories, and reliable differentiation between primary infection and reactivated is problematic." The LCDC Working Group stated that HHV-7 serologic assays must be carefully selected to avoid cross-reaction with antibodies to HHV-6. The LCDC Working Group commented that, while PCR-based assays can differentiate between HHV-6 and HHV-7, "further studies are required to determine the most appropriate samples and the most appropriate PCR method format (i.e., qualitative or quantitative) for detecting an active HHV-7 infection." Diagnosis of HHV-7 will not alter the patient's management because no effective treatment is known for HHV-7; treatment of HHV-7 is supportive (AAP, 2006).
Human Herpesvirus Type 8
Human herpesvirus 8 (HHV-8) is the most recently discovered member of the herpesvirus family (AAP, 2006). In adults, HHV-8 is etiologically associated with Kaposi's sarcoma. Evidence of HHV-8 infection in children is rare, and no clinical associations are known.
Diagnostic tests for detection of HHV-8 infections are limited to research laboratories, and reliable differentiation of primary versus latent infection is problematic (AAP, 2006). No effective treatment is known for HHV-8 (AAP, 2006). Thus, diagnosis of HHV-8 will not alter the patient's management.
Trichosporonosis
Trichosporon species are fungal soil inhabitants and common colonizers of human skin and GI tracts. Trichosporon beigelii causes the superficial dermatomycosis known as white piedra, a distal infection of the hair shaft. T beigelii can also cause onychomycosis, otomycosis, or superficial skin infections. This organism is also associated with summer-type hypersensitivity pneumonitis, a type of hypersensitivity pneumonitis commonly found in Japan.
Trichosporon has been implicated in severe, disseminated infections (trichosporonosis) associated with several immunocompromised states, particularly hematologic malignancies.
No role has been established for PCR for diagnosing trichosporonosis. According to the AAP (2006), trichosporon infection is diagnosed by blood culture and histopathological examination of tissue. The diagnosis of trichosporonosis is usually confirmed by a positive blood culture result obtained in the evaluation of a febrile (usually neutropenic) patient (Hale, 2002). The urine may be the first body fluid to grow Trichosporon in culture in the setting of disseminated disease, and it should not be presumed to be a contaminant or colonizer in the high-risk host (i.e., in the setting of neutropenic fever).
Saccharomyces Cerevisiae
Saccharomyces cerevisiae is a type of acospore-forming food yeast, also known as brewer's/baker's yeast. Yeasts are fungi that have a unicellular growth form and yield mucoid, bacteria-like colonies on laboratory media. Food yeasts, primarily S. cerevisiae, may cause allergic symptoms in occasional atopic persons but are rarely implicated with certainty (Middleton, 1998). Cases of vaginitis caused by Saccharomyces cerevisiae have been reported, and may be associated with baking (Mandell, 2000). Guidelines on treatment of vaginitis, however, do not include a recommendation for PCR testing for S. cerevisiae in patients suspected of having none.
Two serum antibodies, anti-neutrophilic cytoplasmic antibodies (ANCA) and anti-Saccharomyces cerevisiae (ASCA) have been investigated as a technique to improve the efficiency and accuracy of diagnosing inflammatory bowel disease in order to potentially decrease the extent of the diagnostic work up or to avoid invasive diagnostic imaging. See CPB 249 - Serological Diagnosis of Inflammatory Bowel Disease: ANCA and ASCA.
Parvovirus
Infection with parvovirus B19 is recognized most often as erythema infectiosum (EI), which is characterized by mild systemic symptoms, fever in 15% to 30% of patients, and, frequently, a distinctive rash (Cunningham & Rennels, 2002). Before onset of these manifestations, a brief, mild, nonspecific illness consisting of fever, malaise, myalgias, and headache, followed approximately 7 to 10 days later by the characteristic exanthema, may occur in some patients. The facial rash is intensely red with a "slapped cheek" appearance and often accompanied by circumoral pallor.
Infection with the causative agent of EI, human parvovirus B19, also can cause asymptomatic infection, a mild respiratory tract illness with no rash, a rash atypical for EI that may be rubelliform or petechial, arthritis in adults (in the absence of manifestations of EI), chronic bone marrow failure in immunodeficient patients, and transient aplastic crisis lasting 7 to 10 days in patients with hemolytic anemias (e.g., sickle cell disease, and autoimmune hemolytic anemia) and other conditions associated with low hemoglobin levels, including hemorrhage, severe anemia, and thalassemia (Cunningham & Rennels, 2002). Chronic parvovirus B19 infection has been detected in some human immunodeficiency virus (HIV)-infected patients with severe anemia. In addition, parvovirus B19 infection has been associated with thrombocytopenia and neutropenia. Patients with aplastic crisis may have a prodromal illness with fever, malaise, and myalgia, but rash usually is absent. The red blood cell aplasia is related to lytic infection in erythrocyte precursors.
Parvovirus B19 infection occurring during pregnancy can cause fetal hydrops and death but is not a proven cause of congenital anomalies. The risk of fetal death is probably between 2% and 6%, with the greatest risk when infection occurs during the first half of pregnancy.
Parvovirus B19 is distributed worldwide and is a common cause of infection in humans, who are the only known hosts. Modes of transmission include contact with respiratory tract secretions, percutaneous exposure to blood or blood products, and vertical transmission between a mother and her fetus. Parvovirus B19 infections are ubiquitous, and cases of EI can occur sporadically or as part of community outbreaks, which often occur in elementary or junior high schools during the late winter and early spring.
According to the American Academy of Pediatrics (2006), the most feasible methods of diagnosis are direct detection of parvovirus B19 antigen or DNA in clinical specimens and serologic tests. In the immunocompetent host, detection of serum parvovirus B19-specific immunoglobulin (Ig) M antibody is preferred, and detection indicates infection probably occurred within the previous 2 to 4 months. By using a radioimmunoassay or enzyme immunoassay, antibody may be detected in 90% or more of patients at the time of the EI rash and by the third day of illness in patients with transient aplastic crisis. Serum IgG antibody indicates previous infection and immunity. These assays are available through commercial laboratories and through some state health and research laboratories. However, their sensitivity and specificity may vary, particularly for IgM antibody. The optimal method for detecting chronic infection in the immunocompromised patient is demonstration of virus by nucleic acid hybridization or polymerase chain reaction (PCR) assay, because parvovirus B19 antibody is variably present in persistent infection. Since parvovirus B19 DNA can be detected by PCR in serum after the acute viremic phase for up to 9 months in some patients, PCR detection of parvovirus B19 DNA does not necessarily indicate acute infection. Less sensitive nucleic acid hybridization assays usually are positive for only 2 to 4 days after onset of illness. For HIV-infected patients with severe anemia associated with chronic infection, dot blot hybridization of serum may be a more appropriate assay. Parvovirus B19 has not been grown in standard cell culture, but the virus has been cultivated in experimental cell culture.
For most patients, only supportive care is indicated. Patients with aplastic crises may require transfusion. For the treatment of chronic infection in immunodeficient patients, intravenous immunoglobulin therapy has been used. Some cases of B19 infected hydrops fetalis have been treated successfully with intrauterine blood transfusions.
Cryptococcus
Primary Cryptococcus neoformans infection is acquired by inhalation of aerosolized fungal elements and often is unapparent or mild (AAP, 2006). Pulmonary disease, when symptomatic, is characterized by cough, hemoptysis, chest pain, and constitutional symptoms. Hematogenous dissemination to the central nervous system, bones and joints, skin, and mucous membranes can occur, but dissemination is rare in persons without defects in cell-mediated immunity (e.g., transplantation, malignant neoplasm, collagen-vascular disease, long-term corticosteroid administration, or sarcoidosis). Cryptococcal meningitis, the most common and serious form of cryptococcal disease, often follows an indolent course. Cryptococcal fungemia, without apparent organ involvement, occurs in patients with human immunodeficiency virus (HIV). Cryptococcosis is one of the acquired immunodeficiency syndrome (AIDS)-defining diseases.
Encapsulated yeast cells can be visualized by India ink or other stains of cerebrospinal fluid (CSF). Definitive diagnosis requires isolation of the organism from body fluid or tissue. The lysis-centrifugation method is the most sensitive technique for recovery of C. neoformans from blood cultures. According to the AAP (2006), the latex agglutination and enzyme immunoassay tests for detection of cryptococcal capsular polysaccharide antigen in serum or CSF are excellent rapid diagnostic tests. Antigen detection in CSF or serum is positive in 90% of patients with cryptococcal meningitis. The AAP guidelines (2000) state that cryptococcal antibody testing is useful, but skin testing is of no value. The guidelines do not indicate a role for PCR testing in the diagnosis of C. neoformans infection.
Adenovirus
Adenoviruses are DNA viruses. The most common site of adenovirus infection is the upper respiratory tract. Manifestations include symptoms of the common cold, pharyngitis, pharyngoconjunctival fever, tonsillitis, otitis media, and keratoconjunctivitis, often associated with fever. Life-threatening disseminated infection, severe pneumonia, meningitis, and encephalitis occasionally occur, especially among young infants and immunocompromised hosts. Adenoviruses are infrequent causes of acute hemorrhagic conjunctivitis, a pertussis-like syndrome, croup, bronchiolitis, hemorrhagic cystitis, and genitourinary tract disease. A few adenovirus serotypes can cause gastroenteritis.
Infection in infants and children may occur at any age. Adenoviruses causing respiratory tract infection usually are transmitted by respiratory tract secretions through person-to-person contact, fomites, and aerosols. Because adenoviruses are stable in the environment, fomites may be important in their transmission. Other routes of transmission have not been defined clearly and may vary with age, type of infection, and environmental or other factors.
According to AAP guidelines (2000), although PCR testing has been used to detect adenovirus DNA, detection of adenovirus infection by culture or antigen is the preferred diagnostic method. Adenoviruses associated with respiratory tract disease can be isolated from pharyngeal secretions, eye swabs, and feces by inoculation of specimens into a variety of cell cultures. Adenovirus antigens can be detected in body fluids of infected persons by immunoassay techniques, which are especially useful for diagnosis of diarrheal disease, because enteric adenovirus types 40 and 41 usually cannot be isolated in standard cell cultures. Enteric adenoviruses also can be identified by electron microscopy of stool specimens. Multiple methods to detect group-reactive hexon antigens in body secretions and tissue have been developed. Also, detection of viral DNA can be accomplished with genomic probes, synthetic oligonucleotide probes, or gene amplification by polymerase chain reaction. Serodiagnosis is based on detecting a four-fold or greater rise in antibodies to a common adenovirus antigen (e.g., hexon). According to the AAP (2006), serodiagnosis is used primarily for epidemiologic studies.
PCR has been used to diagnose adenovirus myocarditis (Martin, et al., 1994; Towbin, et al., 1994; Shirali, et al., 2001). Routine viral cultures and histopathology are rarely positive in cases of presumed viral myocarditis.
Treatment of adenovirus infection is supportive.
Enteroviruses
Enteroviruses are a subgroup of the Picornaviridae family. They are subclassified into polioviruses, Group A and B coxsackieviruses, and echoviruses (Dua & Berkowicz, 2003). Enteroviruses cause a wide range of infections. Poliovirus infections can be subclinical or can cause mild illness, aseptic meningitis, or poliomyelitis. Coxsackie virus, an RNA virus, is one of several non-polio enteroviruses that are responsible for significant and frequent illnesses in infants and children and result in protean clinical manifestations. Coxsackievirus infections are the most common cause of viral heart disease. Group A viruses cause flaccid paralysis, while group B viruses cause spastic paralysis. Other diseases associated with coxsackievirus infections are hand-foot-and-mouth (HFM) disease and hemorrhagic conjunctivitis, caused by group A, while group B coxsackievirus is associated with herpangina, pleurodynia, myocarditis, pericarditis, and meningoencephalitis. Aseptic meningitis and colds are associated with both group A and group B. Echovirus infections range from the common cold and fever to aseptic meningitis and acute hemorrhagic conjunctivitis (AHC). The enteroviruses are spread from person to person via the fecal-oral route (Marx, 2002).
All enteroviruses enter the body through the oropharynx and multiply in the tissues around the oropharynx (Marx, 2002). Most enteroviral infections are unapparent. The most common clinical manifestation is that of a nonspecific febrile illness. Young children may be admitted to hospitals with enteroviral fevers that simulate bacterial sepsis. Coxsackie B virus and some of the echoviruses may cause severe perinatal infection associated with fever, meningitis, myocarditis, and hepatitis. Immunocompromised patients with humoral deficiencies can have persistent central nervous system infections lasting for several months or more.
In patients with serious illnesses, viral isolation in cell culture as a means of diagnosis is particularly important (AAP, 2006). Specimens providing the highest rate of viral isolation are those obtained from the throat, stool, and rectal swabs. Specimens also should be obtained from any sites of clinical involvement, such as cerebrospinal fluid (CSF). Enteroviruses also may be recovered from blood during the acute febrile phase and, rarely, from biopsy material. Most viral diagnostic laboratories use cell culture techniques that are capable of recovering echoviruses, group B coxsackieviruses, and some group A coxsackieviruses. Polymerase chain reaction testing is most useful for detecting enterovirus RNA in the CSF (AAP, 2006; Dua & Berkowicz, 2003). The demonstration of a rise in titer of virus-specific neutralizing antibody can be used to confirm infection, particularly when the specific virus has been identified previously during a community outbreak. Serologic screening without a suspected serotype generally is not performed.
No specific treatments for the enteroviruses exist (Marx, 2002). Care is supportive. An antiviral agent, pleconaril, is undergoing clinical evaluation. For chronic enteroviral meningoencephalitis in an immunodeficient patient, intravenous immunoglobulin (IVIG) containing high antibody titer to the infecting virus has been used for treatment of persistent enterovirus infection (AAP, 2006).
Human T Cell Leukemia Virus
Human T-Cell Lymphotropic Virus Type I (HTLV-I) is a retrovirus that is endemic in Japan, the Caribbean, and parts of South America, and is associated with development of malignant neoplasms and neurologic disorders among adults (Armstrong, 2000). HTLV-I can be transmitted by sexual intercourse, inoculation of infected blood or blood products and perinatal exposure.
Only a small proportion of those infected with HTLV-I develop adult T-cell leukemia or HTLV-I-associated myelopathy: the lifetime risk of these diseases in HTLV-I-infected Japanese is estimated at 2-4% and 0.25%, respectively. The usual age at onset of is the fifth decade of life and more women than men are affected.
The myeloradiculopathy produced by HTLV-I mainly affects the pyramidal tracts and, to a lesser extent, the sensory system. HTLV-I-associated myelopathy is clinically characterized by a chronic syndrome with a combination of upper- and lower-motor neuron signs. Patients often complain of difficulty walking, dragging pains and stiffness of the legs, together with numbness and paresthesia, urinary retention and/or incontinence and impotence. About one-third of patients have weakness in the upper limbs, but the cranial nerves are only very rarely involved. Examination reveals a symmetric spastic paraparesis with mild sensory abnormalities indicative of posterior column involvement (diminished vibration and proprioception). Most patients progress gradually over months or years.
There may be confusion of HTLV-I-associated myelopathy with multiple sclerosis. There is, however, a lack of optic neuritis or ocular movement problems in the former and the latter tends to run a relapsing-remitting course. The World Health Organization has published diagnostic guidelines for HTLV-I myelopathy.
The diagnostic hallmark of HTLV-I infection is the presence of 'flower lymphocytes' (T-helper cells with multi-lobulated nuclei that are similar to the cells of ATL) in the blood. These cells only comprise about 1% of the circulating white cells, however, and the diagnosis of HTLV-I infection requires the demonstration of specific antibodies in the serum.
In HTLV-I CNS disease, the CSF examination may be normal or show a slightly elevated protein concentration and a mild lymphocytosis. Flower lymphocytes are found in a minority of cases. A definitive diagnosis of HTLV-I-associated myelopathy requires detection of HTLV-I DNA in the CSF by polymerase chain reaction or evidence of intrathecal synthesis of HTLV-I antibody.
No therapy has been proven to be of benefit in HTLV-I-associated myelopathy. At present the management of HTLV-I-associated myelopathy is similar to that of myelopathies of any cause, with supportive therapy of spasticity and urinary sphincter disturbance. Occasional patients have improved while receiving oral corticosteroids or systemic a-interferon, and plasmapheresis has also been claimed to lead to a temporary benefit.
Human T-cell lymphotropic virus type II (HTLV-II), also a retrovirus, has been detected among American and European injection drug users and some indigenous Native American groups. Limited data are available regarding the association of clinical disease with HTLV-II infection. In contrast to the clear association of adult T-cell leukemia with HTLV-I infection, no convincing link of HTLV-II to malignancy has been observed (Feigin, 1998). Although HTLV-II has been isolated in some patients with myeloneuropathies resembling HTLV-1 myelopathy, there is also no clear link between HTLV-II and myeloradiculopathies. Feigin (1998) concluded that "[t]he natural history and clinical manifestations of HTLV-II need further delineation in the context of ongoing prospective natural history studies."
Hepatitis G Virus
Although hepatitis G virus (HGV) can cause chronic infection and viremia, it is a rare cause of hepatic inflammation, and most infected persons are asymptomatic (AAP, 2003). Histologic evidence of HGV infection is rare, and serum aminotransferase concentrations usually are normal. Although high levels of HGV RNA are found in blood, the liver is not a significant site of replication. Currently, no conclusive evidence indicates that HGV causes fulminant or chronic disease, and co-infection does not seem to worsen the course or severity of concurrent infection with hepatitis B virus (HBV) or hepatitis C virus (HCV).
The HGV has been reported in adults and children throughout the world and is found in about 1.5% of blood donors in the United States. Infection has been reported in 10% to 20% of adults with chronic HBV or HCV infection, indicating that co-infection is a common occurrence. The primary route of spread is thought to be through transfusions, but HGV also can be transmitted by organ transplantation. Other important risk factors for infection include injection drug use, hemodialysis, and homosexual and bisexual relationships, indicating that sexual transmission also may occur.
Although PCR testing can detect HGV testing, such testing would not influence management because the disease is mild, and there is no known method to treat or prevent it. Currently, HGV infection can be diagnosed only by identifying viral genomes by using polymerase chain reaction assay, which is not widely available (AAP, 2003). No serologic test is available.
According to the AAP (2003), no treatment is indicated for this virus that causes mild, if any, disease. No method to prevent infection with HGV is known.
Hepatitis C Virus
The signs and symptoms of hepatitis C virus (HCV) infection usually are indistinguishable from those of hepatitis A or B. Persistent infection with HCV occurs in 75% to 85% of infected persons. Chronic hepatitis develops in approximately 60% to 70% of chronically infected patients, and cirrhosis develops in 10% to 20%; primary hepatocellular carcinoma can occur in these patients. Infection with HCV is the leading reason for liver transplantation in the United States.
The prevalence of HCV infection in the general population of the United States is estimated at 1.8%. Seroprevalence rates vary among individuals according to their associated risk factors. Infection is spread primarily by parenteral exposure to blood and blood products from HCV-infected persons.
The highest seroprevalence rates of infection (60% to 90%) occur in persons with large or repeated direct percutaneous exposure to blood or blood products, such as injection drug users and persons with hemophilia who were treated with clotting factor concentrates produced before 1987. Rates are moderately high among those with frequent but smaller direct percutaneous exposures, such as patients receiving hemodialysis (10% to 20%). Lower rates are found among persons with unapparent percutaneous or mucosal exposures, such as persons with high-risk sexual behaviors (1% to 10%), or among persons with sporadic percutaneous exposures, such as health care personnel (1%).
In persons with no risk factors, seroprevalence rates are less than 0.5%. For most infected children and adolescents, no specific source of infection can be identified.
According to the AAP (2006), there are two major types of tests available for the laboratory diagnosis of HCV infections: 1) antibody assays for anti-HCV, and 2) assays to detect HCV nucleic acid (RNA). Diagnosis by antibody assays involves an initial screening enzyme immunoassay (EIA); repeated positive results are confirmed by a recombinant immunoblot assay (RIBA), analogous to testing for HIV infection. The current EIA and RIBA assays are at least 97% sensitive and more than 99% specific. False-negative results early in the course of acute infection result from the prolonged interval between exposure or onset of illness and seroconversion that may occur. Within 15 weeks after exposure and within 5 to 6 weeks after the onset of hepatitis, 80% of patients will have positive test results for serum HCV antibody.
According to the AAP (2006), highly sensitive FDA-licensed polymerase chain reaction (PCR) assays for detection of HCV RNA are available from several commercial laboratories. Hepatitis C virus RNA can be detected in serum or plasma within 1 to 2 weeks after exposure to the virus and weeks before onset of liver enzyme abnormalities or appearance of anti-HCV. PCR assays for HCV infection are used commonly in clinical practice in the early diagnosis of infection, for identifying infection in infants early in life (i.e., perinatal transmission) when maternal serum antibody interferes with the ability to detect antibody produced by the infant, and for monitoring patients receiving antiviral therapy (AAP, 2006; CDC, 1998). However, false-positive and false-negative results can occur from improper handling, storage, and contamination of the test samples. Viral RNA may be detected intermittently, and, thus, a single negative PCR assay result is not conclusive. The AAP (2006) guidelines note that quantitative assays for measuring the concentration of HCV RNA also are available but are less sensitive than qualitative assays. These quantitative assays have primarily been used a prognostic indicator for patients undergoing or about to undergo antiviral therapy. A Consensus Conference convened by the Health Canada Laboratory Centre for Disease Control (1999) concluded that pretreatment quantitative HCV RNA assays provide important information with respect to the risks and benefits of treatment and duration of therapy and should be made available. In addition, the LCDC Consensus Conference concluded that pretreatment genotyping provides important information with respect to the risks/benefits and duration of treatment. Interferon given alone or in combination with ribavirin is FDA-approved for treatment of chronic HCV infection in adults.
Hepatitis B Virus
Hepatitis B virus (HBV) causes a wide spectrum of manifestations, ranging from asymptomatic seroconversion, subacute illness with nonspecific symptoms (e.g., anorexia, nausea, or malaise) or extrahepatic symptoms, and clinical hepatitis with jaundice, to fulminant fatal hepatitis (AAP, 2006). Chronic HBV infection with persistence of hepatitis B surface antigen (HBsAg) occurs in as many as 90% of infants infected by perinatal transmission, in an average of 30% of children 1 to 5 years of age infected after birth, and in 2% to 6% of older children, adolescents, and adults with HBV infection. Chronically infected persons are at increased risk for developing chronic liver disease (e.g., cirrhosis, chronic active hepatitis, or chronic persistent hepatitis) or primary hepatocellular carcinoma in later life.
Hepatitis B virus is transmitted through blood or body fluids, such as wound exudates, semen, cervical secretions, and saliva of people who are HBsAg-positive.
Commercial serologic antigen tests are available to detect hepatitis B surface antigen (HbsAg) and hepatitis B e antigen (HbeAg). Assays also are available for detection of antibody to HBsAg (anti-HBs), total antibody to hepatitis B core antigen (anti-HBc), IgM anti-HBc, and antibody to HBeAg. In addition, hybridization assays and gene amplification techniques (e.g., polymerase chain reaction, branched DNA methods) are available to detect and quantitate HBV DNA. Tests for HBeAg and HBV DNA are useful in the selection of candidates to receive antiviral therapy and to monitor the response to therapy. Quantitative PCR viral load tests are used to monitor response to therapy.
Varicella Zoster Virus
Primary varicella zoster virus (VZV) infection results in chickenpox, manifested by a generalized, pruritic, vesicular rash and mild fever and systemic symptoms (AAP, 2006). Most cases of varicella in the United States occur in children younger than 10 years of age. Immunity generally is lifelong. Immunocompromised persons with primary (varicella) or recurrent (zoster) infection are at increased risk of severe disease.
The virus establishes latency in the dorsal root ganglia during primary infection. Reactivation results in herpes zoster ("shingles"), which are grouped vesicular lesions appearing in a dermatomal distribution, sometimes accompanied by pain localized to the area. Zoster occasionally can become disseminated in immunocompromised patients, with lesions appearing outside the primary dermatomes and with visceral complications.
Varicella virus can be isolated from scrapings of vesicle base during the first 3 to 4 days of the eruption but rarely from other sites, including respiratory tract secretions. A significant increase in serum varicella IgG antibody by any standard serologic assay can retrospectively confirm a diagnosis. According to the AAP (2006), these antibody tests are reliable for determining immune status in healthy hosts after natural infection but are not necessarily reliable in immunocompromised. Many commercially available tests are not sufficiently sensitive to demonstrate a vaccine-induced antibody response.
According to the AAP (2006), rapid diagnostic tests (PCR, direct fluorescent antibody) are the methods of choice of diagnosing varicella virus infection. Varicella virus infection can be diagnosed using PCR testing of body fluid or tissue. The advantages of PCR testing over other methods is that it is very sensitive and can distinguish wild-type strains from vaccine virus.
Varicella and zoster may be treated with intravenous or oral acyclovir, valacyclovir, famciclovir, and foscarnet. The decision to use therapy and the duration and route of therapy should be determined by specific host factors, extent of infection, and initial response to therapy. Oral acyclovir is not recommended for routine use in otherwise healthy children with varicella, because it results in only a modest reduction in symptoms. Oral acyclovir should be considered for otherwise healthy persons at increased risk of moderate to severe varicella. Intravenous therapy is recommended for immunocompromised patients.
Influenza Virus
Influenza is characterized by the sudden onset of fever, frequently with chills or rigors, headache, malaise, diffuse myalgia, and a nonproductive cough (AAP, 2006). Subsequently, the respiratory tract signs of sore throat, nasal congestion, rhinitis, and cough become more prominent.
Influenza is spread from person to person by inhalation of small particle aerosols, by direct contact, by large droplet infection, or by contact with articles recently contaminated by nasopharyngeal secretions. In temperate climates, epidemics usually occur during the winter months and, within a community, peak within 2 weeks of onset and last 4 to 8 weeks or longer.
When viral cultures are performed, specimens should be obtained during the first 72 hours of illness because the quantity of virus shed subsequently decreases rapidly. Rapid diagnostic tests for identification of influenza A and B antigens in nasopharyngeal specimens are available commercially, although their sensitivity and specificity have been variable. Serologic diagnosis can be established retrospectively by a significant change in antibody titer between acute and convalescent serum samples, as determined by complement fixation, hemagglutination inhibition, neutralization, or enzyme immunoassay tests. Although the AAP guidelines (2006) note that reverse transcriptase-polymerase chain reaction (RT-PCR) testing of respiratory tract specimens may be available at some institutions, these guidelines do not indicate any specific role for PCR testing of influenza virus.
Amantadine and rimantadine are approved for treatment of influenza A; treatment with either drug diminishes the severity of influenza A infection when administered within 48 hours of onset of illness. Neither amantadine nor rimantadine is effective against influenza B infections. Two neuraminidase inhibitors, zanamivir and oseltamir, have been approved for treatment of influenza A and B.
According to the Centers for Disease Control and Prevention (2003), testing for highly pathogenic Avian influenza A (H5N1) virus can be performed by PCR. Testing of hospitalized patients for influenza A (H5N1) infection is indicated when both of the following exist: 1) radiographically confirmed pneumonia, acute respiratory distress syndrome (ARDS), or other severe respiratory illness for which an alternative diagnosis has not been established; and 2) a history of travel within 10 days of symptom onset to a country with documented H5N1 avian influenza infections in poultry or humans. Ongoing listings of countries affected by avian influenza are available from the World Organization for Animal Health at http://www.oie.int/eng/en_index.htm.
Testing for influenza A (H5N1) also should be considered on a case-by-case basis in consultation with state and local health departments for hospitalized or ambulatory patients with all of the following: 1) documented temperature of >100.4ºF (>38ºC); 2) cough, sore throat, or shortness of breath; and 3) history of contact with poultry or domestic birds (e.g., visited a poultry farm, a household raising poultry, or a bird market) or a known or suspected patient with influenza A (H5N1) in an H5N1-affected country within 10 days of symptom onset (CDC, 2003).
Bartonella
The predominant sign of Bartonella henselae (cat-scratch disease, CSD) is regional lymphadenopathy in an immunocompetent person. Fever and mild systemic symptoms occur in one-third of patients (AAP, 2006; CPS, 2002).
Bartonella henselae is the causative organism for most cases of CSD. Bartonella henselae are fastidious, slow-growing, gram-negative bacilli that also have been identified as the causative agent of bacillary angiomatosis and peliosis hepatitis, two infections that have been reported primarily in patients infected with the human immunodeficiency virus.
Infection with Bartonella henselae results in disease syndromes of variable severity, ranging from lymphadenopathy only (CSD) to systemic disease. The severity and presentation of disease are related to immune status. In general, immunocompetent patients who are otherwise healthy tend to present with classic CSD when infected with B. henselae. Patients who are immunocompromised by having AIDS, chronic alcoholism, immunosuppression, or other serious health problems tend to have systemic disease. However, there have been rare reports of systemic disease, including bacillary angiomatosis, in immunocompetent persons.
Cat-scratch disease is believed to be a relatively common infection, although the true incidence is unknown. Most cases occur in patients younger than 20 years of age. Cats are the common reservoir for human disease, and bacteremia in cats associated with patients with CSD is common. More than 90% of patients have a history of recent contact with cats, often kittens, which usually are healthy. No evidence of person-to-person transmission exists.
The indirect fluorescent antibody (IFA) test for detection of serum antibody to antigens of Bartonella species is sensitive and specific and useful for the diagnosis of CSD (AAP, 2006; CPS, 2002). Enzyme immunoassays for detection of antibody to B. henselae have been developed; however, they have not been demonstrated to be more sensitive or specific than the IFA test. If involved tissue is available, the putative agent of the disease may be visualized by the Warthin-Starry silver impregnation stain; however, this test is not specific for B. henselae. Pathologic and microbiologic examinations also are useful to exclude other diseases. Histologic findings in lymph node sections are characteristic but not pathognomonic for CSD. A cat-scratch antigen skin test, which was used formerly to confirm the clinical diagnosis, was prepared from aspirated pus from suppurative lymph nodes of patients with apparent CSD. The American Academy of Pediatrics states that this test should not be used (AAP, 2006).
Polymerase chain reaction assays are available in some commercial laboratories. According to the Canadian Paediatric Society (2002), PCR assays can differentiate between B. henselae and B. Quintana, the case of trench fever and of bacillary angiomatosis and bacillary peilosis hepatitis in HIV-infected patients.
Management is primarily symptomatic since the disease usually is self-limited, resolving spontaneously in 2 to 4 months. Painful suppurative nodes can be treated with needle aspiration for relief of symptoms; surgical excision generally is unnecessary.
Antibiotic therapy may be considered for acutely or severely ill patients with systemic symptoms, particularly persons with hepatosplenomegaly or persons with large painful adenopathy and immunocompromised hosts. No well-controlled randomized clinical trials have been performed that clearly demonstrate a clinically significant benefit of antimicrobial therapy for CSD. Reports suggest that several oral antibiotics (rifampin, trimethoprim-sulfamethoxazole, azithromycin, and ciprofloxacin) and parenteral gentamicin may be effective in CSD. Doxycycline, erythromycin, and azithromycin are effective for treatment of signs and symptoms associated with bacillary angiomatosis if administered for prolonged periods to immunocompromised persons.
Brucella
Brucella species are small, non-motile, gram-negative coccobacilli. The species that infect humans are Brucella abortus, B. melitensis, Brucella suis, and, rarely, Brucella canis (AAP, 2006).
Brucellosis in children frequently is a mild self-limited disease compared with the more chronic disease observed among adults. However, in areas where Brucella melitensis is the endemic species, disease can be severe. Onset of illness can be acute or insidious. Manifestations are nonspecific and include fever, night sweats, weakness, malaise, anorexia, weight loss, arthralgia, myalgia, abdominal pain, and headache. Physical findings include lymphadenopathy, hepatosplenomegaly, and, occasionally, arthritis. Serious compli |
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