Diagnosis of Viral Infections
Saturday, August 16, 2008
INTRODUCTION
It is becoming increasingly important for the practitioner to perform viral diagnostic studies because prognostic, epidemiologic, and therapeutic considerations may be greatly influenced by knowledge of the specific virus causing a given illness. Even if no therapy is available, the establishment of a definite diagnosis of viral infection is often beneficial in (1) epidemiologic monitoring, (2) educating physicians and patients, (3) defining the disease process, and (4) evaluating therapeutic implications, both positive and negative. Moreover, identification of a virus as the cause of a patient's illness may be cost effective, because expensive diagnostic procedures and antibiotic therapy may be avoided or discontinued.
The virology laboratory can confirm the suspected diagnosis by cytologic examination of clinical specimens; attempting to isolate the virus; detecting the presence of viral antigens, or nucleic acids or evaluating the patient's immune response to the virus (serology).
CYTOLOGY
The simplest technique for viral diagnosis is cytologic examination of specimens for the presence of characteristic viral inclusions, but this approach is insensitive and applicable to only a few viruses, especially herpes viruses. These intracellular structures may represent aggregates of virus within an infected cell or may be abnormal accumulations of cellular material resulting from the virus-induced metabolic disruption. Papanicolaou (Pap) smears may show these inclusions in single cells or in large syncytia (aggregates of cells containing more than one nucleus), as in a patient with herpes simplex infection of the cervix. Cytology can be used to detect infections with herpes simplex virus, varicella-zoster virus, cytomegalovirus, human papillomavirus, and adenoviruses. Rabies infection may also be detected by finding Negri bodies (rabies virus inclusions) in brain tissue.
CULTURE-BASED METHODS
Introduction
The historical "gold standard" of viral diagnosis is recovery of the agent in tissue culture, embryonated eggs, or experimental animals. Embryonated eggs are still used for the growth of virus for some vaccines but have been replaced by cell cultures for routine virus isolation in clinical laboratories. Likewise, the use of experimental animals rarely occurs in most clinical laboratories. Just as multiple media are used in bacteriology, several different types of tissue culture cells (eg, monkey kidney, human fetal lung, human amnion, or human cancer cells) are inoculated with each viral specimen.
Most clinically significant viruses can be recovered in at least one of these cell cultures, but several clinically important viruses are not isolated in these cells. For example, specimens submitted for the identification of viruses such as human immunodeficiency virus, coxsackie A virus, and rubella virus require, respectively, cocultivation with normal human peripheral blood mononuclear cells, inoculation of suckling mice, and the use of specialized cell cultures, which are not generally available. Therefore infections caused by these and several other viruses are most frequently diagnosed serologically or by detection of virus-specific antigens or nucleic acids.
Detection of the growth of a virus is by observation of changes in the cell culture monolayer [cytopathic effect (CPE)]. Characteristic CPEs include changes in cell morphology, cell lysis, vacuolation, syncytia formation, and presence of inclusion bodies. Inclusion bodies are histologic changes in cells caused by the presence of viral components or changes in cell structures. With experience a technologist can distinguish CPE characteristics of the major virus groups. The observation of which cell culture exhibits CPEs and the rapidity of viral growth can be used for the presumptive identification of many clinically important viruses. This approach for identifying viruses is similar to bacterial identification based on growth and morphology of colonies on selective, differential media. Some viruses do not readily cause CPEs in cell lines typically used in clinical virology laboratories. However, some of these can be detected by other techniques, such as (1) erythrocyte hemadsorption onto cells infected with paramyxoviruses or mumps virus or (2) interference with the replication of other viruses (eg, picornaviruses cannot replicate in cells previously infected with rubella virus; this is known as heterologous interference).
In contrast with the intrinsic delay of antibody studies, the results of viral culture can be surprisingly rapid. Almost 50% of all viral isolates can be reported within 3 to 4 days of culture, with herpes simplex virus and influenza A virus usually detected within 1 to 3 days (Table 6-6).
The selection of the appropriate specimen for viral culture is complicated because several different viruses may cause the same clinical disease (Table 6-7). For example, several types of specimens should be submitted from patients with viral meningitis to enhance the recovery of the possible etiologic agents: CSF (enteroviruses, mumps virus, and herpes simplex virus), throat swabs and washings (enteroviruses), and stool or rectal swabs (enteroviruses). Also, serum should be collected as an acute-phase specimen in case subsequent serologic tests are indicated (eg, acute and convalescent sera for mumps virus or arbovirus infections). Many considerations, however, allow the physician to select the most appropriate specimens (Table 6-8). For example, during the summer, when enteroviral meningitis is prevalent, CSF, throat, and stool specimens should be submitted. On the other hand, the development of encephalitis in children after being bitten by mosquitos in wooded areas endemic for California encephalitis virus suggests that a serum specimen for antibody testing would be preferred. Central nervous system disease after parotitis would suggest collection of CSF and urine for the isolation of mumps virus. The specimens that should be collected for other viruses are summarized in Table 6-7.
Timing of Specimen Collection
Proper timing of specimen collection is essential for adequate recovery of viruses. Specimens should be collected early in the acute phase of infection. Studies with respiratory viruses indicate that the mean duration of viral shedding may be only 3-7 days. Also, herpes simplex virus and varicella-zoster virus may not be recovered from lesions beyond 5 days after onset. Isolation of an enterovirus from the CSF may be possible for only 2-3 days after onset of the central nervous system manifestations.
Transport to Laboratory
The shorter the interval between collection of a specimen and its delivery to the laboratory, the greater is the potential for isolating an agent. When feasible, all specimens other than blood, feces, urine, and tissue, which need special processing, should be inoculated directly onto cell cultures at the patient's bedside (Table 6-9). These should then be transported to the laboratory promptly.
For specimens that cannot be inoculated onto cell cultures immediately, several types of transport media have been used. It is generally believed that protein (serum, albumin, or gelatin) incorporated into a transport medium enhances survival of viruses.
Improper storage of specimens before processing can also adversely affect viral recovery. Significant losses in infective titer occur with enveloped viruses (eg, herpes simplex virus, varicella-zoster virus, or influenza virus) after specimens have been frozen and then thawed. This is not observed with nonenveloped viruses (eg, adenoviruses or enteroviruses). Therefore, when it is impossible to process a specimen immediately, it should be refrigerated but not frozen and packed in shaved ice for delivery to the laboratory if delays in transit are anticipated. Storage of specimens for the recovery of viruses at 4oC is far superior to storage at ambient temperature.
Interpretation of Culture Results
In general the detection of any virus in host tissues, CSF, blood, or vesicular fluid can be considered highly significant. Recovery of viruses other than cytomegalovirus in urine may be diagnostic of significant infection. For example, both mumps virus and adenovirus type 11 (associated with acute hemorrhagic cystitis) may be recovered in urine and indicate acute infection. However, the presence of cytomegalovirus in urine is difficult to interpret because this may reflect asymptomatic virus replication long after infection or indicate a significant active infection in the patient. In the newborn, viruria (isolation of virus in urine) in the first 3 weeks of life establishes a diagnosis of congenital cytomegalovirus infection, whereas the onset of viral excretion after 3-4 weeks of life reflects intrapartum or postpartum infection. Diagnosis of acquired cytomegalovirus in older patients usually requires a combination of findings, including positive cultures, illness compatible with cytomegalovirus disease, reasonable exclusion of other potential etiologic agents, and support by specific serologic or histologic data.
The significance of viruses isolated in upper respiratory tract, vaginal, or fecal specimens varies greatly. At one extreme, isolates such as measles, mumps, influenza, parainfluenza, and respiratory syncytial virus are significant because asymptomatic carriage and prolonged shedding of these viruses are unusual. Conversely, other viruses can be shed continually or intermittently without symptoms for periods ranging from several weeks (enteroviruses in feces) to many months or years (herpes simplex virus or cytomegalovirus in the oropharynx and genital tract; adenoviruses in the oropharynx and intestinal tract). Herpes simplex virus, cytomegalovirus, varicella zoster virus, and Epstein-Barr virus may remain latent for long periods and then become reactivated in response to a variety of stressful stimuli, including other infectious agents. In this setting their detection may not be significant, may merely represent a secondary problem complicating the primary infection (eg, herpes simplex virus "cold sores" in patients with bacterial sepsis), or may be associated with significant disease, especially in the immunocompromised patient.
Based on the epidemiology of adenovirus infections and observed serologic responses, the simultaneous isolation of these viruses from throat and feces is significantly associated with febrile respiratory disease. Isolation of viruses from the throat alone is less frequently associated with disease, and isolates from feces alone are probably nondiagnostic in a patient with respiratory disease.
Enteroviruses are generally found in infants and children, particularly during the late summer and early autumn. A knowledge of the relative frequency of virus shedding among various age groups in a particular locale is extremely helpful in assessing the significance of results of throat or stool cultures. For example, the peak prevalence of enteroviruses in the stools of toddlers during the late summer may range from 5% in temperate zones to > 20% in subtropical climates. Even in temperate areas, rates may approach 30% in infants during periods of enterovirus activity. Shedding of enterovirus in the throat usually occurs for 1-2 weeks, whereas fecal shedding may last 4-16 weeks. Thus, in a clinically compatible illness, isolation of an enterovirus from the throat supports a stronger temporal relationship to the disease than does an isolate from only the feces.
Herpes simplex virus is unusual in a fecal culture. In such cases it usually represents either severe disseminated infection or local infection of the perianal areas. Detection of herpes simplex virus in the upper respiratory tract may mean nothing other than nonspecific reactivation of virus caused by fever unless typical vesicles or ulcers are also present. Because of the fever-related phenomenon, isolation of herpes simplex virus in the throat or mucocutaneous lesions of patients with encephalitis cannot be interpreted as causing the central nervous system disease. Currently the definitive way to establish a diagnosis of herpes simplex encephalitis is by direct demonstration of the virus in a brain biopsy or by polymerase chain reaction (PCR) on CSF. In neonates, however, isolation of the virus from any site should raise the possibility of severe infection.
Isolation of adenoviruses, herpes simplex virus, varicella-zoster virus, and some enteroviruses from the cornea and conjunctiva in patients with inflammatory disease at these sites usually establishes the etiology of the infection.
DETECTION OF VIRAL ANTIGENS
Antibodies can be used as sensitive tools to detect, identify, and quantitate the presence of viral antigen in clinical specimens or cell culture. Monoclonal or polyclonal antibodies prepared in animals may be used. Viral antigens on the cell surface, within the cell, or released from infected cells can be detected by IF, EIA, RIA, and latex agglutination (LA). IF detects and locates cell-associated antigens, whereas RIA or different variations of enzyme-linked immunosorbent assay (ELISA) are used to detect and quantitate soluble antigens. LA is a rapid, easy assay for antigen; viruses or viral antigens in a sample cause the clumping of latex particles coated with specific antibody.
Virus-infected tissue or cell cultures can be detected by IF or EIA. By attaching a fluorescent signal to an antiviral antibody, and reacting it with the sample, viral antigen can be detected; this is called direct IF. A modification of this technique is the use of unlabeled antiviral antibodies and then a second antibody with a fluorescent label that will bind to IgM or IgG antibodies. EIA uses a second antibody conjugated to an enzyme, such as horseradish peroxidase or alkaline phosphatase, which releases a chromophore to mark the presence of antigen.
Direct IF assay is especially useful for (1) respiratory viruses (eg, respiratory syncytial virus or influenza A), (2) varicella-zoster virus and herpes simplex virus antigen in lung and visceral biopsies, and (3) cytomegalovirus in leukocytes from blood or CSF.
Soluble antigen can be quantitated by ELISA, RIA, and LA. The basis for these procedures is the separation and quantitation of antibody-bound and free antigen. Many of the ELISA and RIA techniques use an antibody immobilized to a solid support to capture soluble antigen and a labeled antibody to detect captured antigen.
Influenza, parainfluenza, and togaviruses produce a glycoprotein that binds erythrocytes. This property allows detection of free virus produced in cell culture by agglutination of erythrocytes, a process termed hemagglutination. The infected cells also adsorb erythrocytes to the surface by a process referred to as hemadsorption.
Detection and assay of characteristic enzymes can identify and quantitate specific viruses. For example, reverse transcriptase in cell culture is used as an indicator of infection by retroviruses.
SEROLOGIC TEST METHODS
Introduction
Serology can be used to determine whether an infection is primary or a reinfection and if acute or chronic. The first antibodies to be produced by the immune system are directed against antigens on the virion or infected cell surfaces and are best detected by, eg, IF. Later in the infection, when cells have been lysed by the infecting virus or the cellular immune response, antibodies are directed against the cytoplasmic viral proteins and enzymes and can be detected by, eg, complement fixation. Seroconversion is characterized by a change from negative to positive antibody between serum taken during the acute phase of disease and that taken = 2-3 weeks later; it is the best serologic marker of recent infection. A fourfold or greater rise in antibody titer in paired sera may also be significant but fluctuations in antibody titer do occur naturally. Detection of virus-specific IgM is theoretically associated with recent infection, but technical difficulty in measuring specific IgM responses may lead to false-positive and false-negative results.
For many viruses, culture or antigen detection is the best diagnostic test. However, certain viruses (eg, HIV, hepatitis A and B viruses, rubella virus, Epstein-Barr virus, measles virus, coronaviruses, and togaviruses) are difficult to isolate in cell culture, and infections are diagnosed most easily by serologic techniques. When a virologic workup is planned for a patient, it is generally useful to obtain = 2-3 ml of serum during the acute phase of disease and store it at -20oC. This may become valuable, particularly if virus detection subsequently fails or if the significance of an isolate is uncertain. In these instances a convalescent-phase serum specimen may be requested 2-3 weeks later, and both the acute and convalescent sera may then be tested against appropriate viral antigens. In general, if a virus is isolated, the antibody titers need not be measured to confirm infection; for example, if a patient has aseptic meningitis and an enterovirus is recovered from the throat, that agent is probably responsible for the illness. Similarly, if influenza virus is recovered from the throat of a patient who has clinical influenza, no serologic confirmation of the etiology is necessary.
Complement fixation is a standard but technically difficult serologic test. The serum is first reacted with the suspected viral antigen and complement, and the residual complement is assayed by lysis of indicator antibody-coated erythrocytes. If the complement is used in the first reaction and is therefore not available for the second reaction, it indicates the presence of antibody to the suspected virus. Antibodies measured by this system generally develop slightly later in the course of an illness than those measured by other techniques. This delayed response is useful for documenting seroconversion when the initial serum specimen is collected late in the clinical course. Members of some virus groups (eg, enteroviruses) do not possess group-specific antigen and must be tested individually.
The neutralization test is essentially a protection test. When a virus is incubated with homologous type-specific antibody, the virus is rendered incapable of producing infection in an indicator cell culture system. A neutralization antibody response is virus type specific, with titers rising rapidly and persisting for long periods.
The hemagglutination inhibition test can be performed with a variety of viruses that can selectively agglutinate erythrocytes of various animal species (eg, chicken, guinea pig, or human). The hemagglutination capacity of a virus is inhibited by specific immune or convalescent sera. Hemagglutination-inhibiting antibody develops rapidly after the onset of symptoms, plateaus, declines slowly, and may last indefinitely at low levels. This test is useful for both the detection of acute viral infection and the determination of immunity.
For the indirect fluorescent antibody test, virus-infected cells are placed in prepared wells on microscope slides and then fixed in cold acetone and dried. Patient serum is applied and, after incubation, anti-human globulin conjugated with fluorescein is added. If fluorescence is observed, it indicates the presence of specific antiviral antibody.
Interpretation of Serologic Results
Virus-specific IgM antibody usually rises during the first 2-3 weeks of infection and persists for several weeks to months. Thus an elevated titer of specific IgM antibody suggests a recent primary infection, which may be further supported by demonstrating a fall in IgM antibody in subsequent sera. Detection of specific IgM has been used with success in the diagnosis of infections caused by cytomegalovirus and rubella virus and is currently the procedure of choice to establish a recent or acute infection from hepatitis A or B.
Several limitations of interpretation must be remembered. It is now recognized that IgM-specific antibody responses are not always restricted to primary infections. Reactivation or reinfection may result in IgM responses, particularly in herpes virus infection. In addition, patients may continue to produce IgM-specific antibody to rubella virus or cytomegalovirus for many months after a primary infection. Heterotypic IgM responses may also occur. For example, antibody responses to cytomegalovirus may develop in Epstein-Barr virus infections and vice versa. Other pitfalls include falsely low or negative IgM titers caused by competition from high-titer IgG antibody for antigen-binding sites and false-positive reactions resulting from rheumatoid factor. Both types of errors appear to occur most frequently in solid-phase assays with IF.
The serologic diagnosis of most viral infections is based on demonstration of a seroconversion or a rise (fourfold or greater) of IgG antibody. However, significant antibody titer rises may result from cross-reactions to related antigens; for example, an antibody rise to parainfluenza virus may actually result from infection with mumps virus. Furthermore, seroconversions may not be seen with some patient populations (eg, infants and immunocompromised patients) or when the initial serum is collected late in the course of disease.
A serum specimen can also be used for screening an infant's blood for certain antibodies of the IgG class. Antibodies to Toxoplasma spp., rubella virus, cytomegalovirus, and herpes simplex virus (known as the "TORCH" screen) may be measured to determine possible congenital infection with these agents. However, the value of these tests must be understood. They are useful in excluding a possible infection but not in proving an etiology. For example, if rubella antibody is absent, an infant almost certainly does not have congenital rubella infection. To diagnose active rubella infection in such a baby, viral cultures are required. Screening of blood supplies for cytomegalovirus antibody is used to eliminate transmission of antibody-positive blood to seronegative babies and other immunocompromised patients.
Serologic Panels
Selection of several antigens for testing with paired sera in cases in which a virus is suspected can usually be made based on clinical syndrome, the known local epidemiology of particular viruses, and the patient's age. This has led to the concept of serologic batteries of panels. Some examples of possible panels are included in Table 6-10.
Antigens from mumps, western equine encephalitis, eastern equine encephalitis, St. Louis encephalitis, and California encephalitis viruses—and perhaps lymphocytic choriomeningitis virus, Epstein-Barr virus, and HIV—may be included in a panel of tests for central nervous system diseases. Although herpes simplex antigen is sometimes included in such a panel, a rise in antibody titer is not sufficient to diagnose herpes encephalitis. Many viral central nervous system illnesses, especially aseptic meningitis, are caused by the enteroviruses; however, the many serotypes and the cumbersome serologic methods necessary for their diagnosis usually make it impractical to include them in a panel. When one or two enteroviruses have been shown to be epidemic in an area in one summer, one can pick up some additional cases by performing neutralization tests on paired sera by using only those specific enteroviruses that are endemic in the community.
The viral antigen panel for testing respiratory syndromes might include influenza A and B; respiratory syncytial virus; parainfluenza types 1, 2, and 3; and adenoviruses.
To test for viral causes of exanthems, the panel would include measles and rubella. If the disease is vesicular, herpes simplex virus and varicella-zoster virus should be included, although the herpes viruses are best diagnosed by culture or antigen detection.
Antigens from group B coxsackie virus types 1-5 and perhaps influenza A and B viruses could make up the panel for myocarditis and pericarditis. Although numerous viruses have been implicated in inflammatory diseases of the heart and its covering membranes, the group B coxsackieviruses are believed to account for almost one half of the cases. Unfortunately, much of the clinical illness is expressed late in the infection, at the time when standard methods of virus detection are likely to fail and it is too late to demonstrate seroconversion or significant antibody titer rises.
MOLECULAR TEST METHODS
In recent years a variety of assays have been developed to detect viral nucleic acid—either DNA or RNA. The best known of these is PCR, an amplification technique that allows the detection and selective replication of a targeted portion of the genome. The technique uses special DNA polymerases that initiate replication in either the 3' or 5' direction. The specificity is provided by primers that recognize a pair of unique sites on the genome so that the DNA between them can be replicated by repetitive cycling of the test conditions. Because each newly synthesized fragment can serve as the template for its own replication, the amount of DNA doubles with each cycle. The amplification power of PCR offers a solution for the sensitivity problems inherent in the direct application of probes. Although the nucleic acid segment amplified by PCR can be seen directly on a gel, the greatest sensitivity and specificity are achieved when probe hybridization is carried out after PCR. A probe is a fragment of DNA that has been cloned or otherwise recovered from a genomic or plasmid source. In some cases the probe is synthesized as a single chain of nucleotides (oligonucleotide probe) from known sequence data. The probes are labeled with a radioisotope or other marker and used in hybridization reactions either to detect the homologous sequences in unknown specimens or in gel electrophoresis.
The diagnostic use of DNA probes is to detect or identify microorganisms by hybridization of the probe to homologous sequences in DNA extracted from the entire organism. A number of probes have been developed that will quickly and reliably identify organisms that have already been isolated in culture. The application of probes for detection of infectious agents directly in clinical specimens such as blood, urine and sputum is more difficult.
Recently, the branched DNA (bDNA) a rapid assay for direct quantification of viral nucleic acid has been developed for hepatitis B, hepatitis C, and HIV infections. Because the bDNA assay measures viral nucleic acids at physiological levels by boosting the reporter signal, rather than by amplifying target sequences, it is not subject to the errors inherent in the amplification steps of PCR-based methods. Inherently quantitative and amenable to routine use in a clinical setting, the bDNA assay may be useful in the management of patients with chronic viral diseases. Recent studies have illustrated the potential clinical utility of the bDNA assay in determining the prognosis and in therapeutic monitoring of infection. Additional nucleic acid tests include hybrid capture and nucleic acid sequence-based amplification.
It is becoming increasingly important for the practitioner to perform viral diagnostic studies because prognostic, epidemiologic, and therapeutic considerations may be greatly influenced by knowledge of the specific virus causing a given illness. Even if no therapy is available, the establishment of a definite diagnosis of viral infection is often beneficial in (1) epidemiologic monitoring, (2) educating physicians and patients, (3) defining the disease process, and (4) evaluating therapeutic implications, both positive and negative. Moreover, identification of a virus as the cause of a patient's illness may be cost effective, because expensive diagnostic procedures and antibiotic therapy may be avoided or discontinued.
The virology laboratory can confirm the suspected diagnosis by cytologic examination of clinical specimens; attempting to isolate the virus; detecting the presence of viral antigens, or nucleic acids or evaluating the patient's immune response to the virus (serology).
CYTOLOGY
The simplest technique for viral diagnosis is cytologic examination of specimens for the presence of characteristic viral inclusions, but this approach is insensitive and applicable to only a few viruses, especially herpes viruses. These intracellular structures may represent aggregates of virus within an infected cell or may be abnormal accumulations of cellular material resulting from the virus-induced metabolic disruption. Papanicolaou (Pap) smears may show these inclusions in single cells or in large syncytia (aggregates of cells containing more than one nucleus), as in a patient with herpes simplex infection of the cervix. Cytology can be used to detect infections with herpes simplex virus, varicella-zoster virus, cytomegalovirus, human papillomavirus, and adenoviruses. Rabies infection may also be detected by finding Negri bodies (rabies virus inclusions) in brain tissue.
CULTURE-BASED METHODS
Introduction
The historical "gold standard" of viral diagnosis is recovery of the agent in tissue culture, embryonated eggs, or experimental animals. Embryonated eggs are still used for the growth of virus for some vaccines but have been replaced by cell cultures for routine virus isolation in clinical laboratories. Likewise, the use of experimental animals rarely occurs in most clinical laboratories. Just as multiple media are used in bacteriology, several different types of tissue culture cells (eg, monkey kidney, human fetal lung, human amnion, or human cancer cells) are inoculated with each viral specimen.
Most clinically significant viruses can be recovered in at least one of these cell cultures, but several clinically important viruses are not isolated in these cells. For example, specimens submitted for the identification of viruses such as human immunodeficiency virus, coxsackie A virus, and rubella virus require, respectively, cocultivation with normal human peripheral blood mononuclear cells, inoculation of suckling mice, and the use of specialized cell cultures, which are not generally available. Therefore infections caused by these and several other viruses are most frequently diagnosed serologically or by detection of virus-specific antigens or nucleic acids.
Detection of the growth of a virus is by observation of changes in the cell culture monolayer [cytopathic effect (CPE)]. Characteristic CPEs include changes in cell morphology, cell lysis, vacuolation, syncytia formation, and presence of inclusion bodies. Inclusion bodies are histologic changes in cells caused by the presence of viral components or changes in cell structures. With experience a technologist can distinguish CPE characteristics of the major virus groups. The observation of which cell culture exhibits CPEs and the rapidity of viral growth can be used for the presumptive identification of many clinically important viruses. This approach for identifying viruses is similar to bacterial identification based on growth and morphology of colonies on selective, differential media. Some viruses do not readily cause CPEs in cell lines typically used in clinical virology laboratories. However, some of these can be detected by other techniques, such as (1) erythrocyte hemadsorption onto cells infected with paramyxoviruses or mumps virus or (2) interference with the replication of other viruses (eg, picornaviruses cannot replicate in cells previously infected with rubella virus; this is known as heterologous interference).
In contrast with the intrinsic delay of antibody studies, the results of viral culture can be surprisingly rapid. Almost 50% of all viral isolates can be reported within 3 to 4 days of culture, with herpes simplex virus and influenza A virus usually detected within 1 to 3 days (Table 6-6).
The selection of the appropriate specimen for viral culture is complicated because several different viruses may cause the same clinical disease (Table 6-7). For example, several types of specimens should be submitted from patients with viral meningitis to enhance the recovery of the possible etiologic agents: CSF (enteroviruses, mumps virus, and herpes simplex virus), throat swabs and washings (enteroviruses), and stool or rectal swabs (enteroviruses). Also, serum should be collected as an acute-phase specimen in case subsequent serologic tests are indicated (eg, acute and convalescent sera for mumps virus or arbovirus infections). Many considerations, however, allow the physician to select the most appropriate specimens (Table 6-8). For example, during the summer, when enteroviral meningitis is prevalent, CSF, throat, and stool specimens should be submitted. On the other hand, the development of encephalitis in children after being bitten by mosquitos in wooded areas endemic for California encephalitis virus suggests that a serum specimen for antibody testing would be preferred. Central nervous system disease after parotitis would suggest collection of CSF and urine for the isolation of mumps virus. The specimens that should be collected for other viruses are summarized in Table 6-7.
Timing of Specimen Collection
Proper timing of specimen collection is essential for adequate recovery of viruses. Specimens should be collected early in the acute phase of infection. Studies with respiratory viruses indicate that the mean duration of viral shedding may be only 3-7 days. Also, herpes simplex virus and varicella-zoster virus may not be recovered from lesions beyond 5 days after onset. Isolation of an enterovirus from the CSF may be possible for only 2-3 days after onset of the central nervous system manifestations.
Transport to Laboratory
The shorter the interval between collection of a specimen and its delivery to the laboratory, the greater is the potential for isolating an agent. When feasible, all specimens other than blood, feces, urine, and tissue, which need special processing, should be inoculated directly onto cell cultures at the patient's bedside (Table 6-9). These should then be transported to the laboratory promptly.
For specimens that cannot be inoculated onto cell cultures immediately, several types of transport media have been used. It is generally believed that protein (serum, albumin, or gelatin) incorporated into a transport medium enhances survival of viruses.
Improper storage of specimens before processing can also adversely affect viral recovery. Significant losses in infective titer occur with enveloped viruses (eg, herpes simplex virus, varicella-zoster virus, or influenza virus) after specimens have been frozen and then thawed. This is not observed with nonenveloped viruses (eg, adenoviruses or enteroviruses). Therefore, when it is impossible to process a specimen immediately, it should be refrigerated but not frozen and packed in shaved ice for delivery to the laboratory if delays in transit are anticipated. Storage of specimens for the recovery of viruses at 4oC is far superior to storage at ambient temperature.
Interpretation of Culture Results
In general the detection of any virus in host tissues, CSF, blood, or vesicular fluid can be considered highly significant. Recovery of viruses other than cytomegalovirus in urine may be diagnostic of significant infection. For example, both mumps virus and adenovirus type 11 (associated with acute hemorrhagic cystitis) may be recovered in urine and indicate acute infection. However, the presence of cytomegalovirus in urine is difficult to interpret because this may reflect asymptomatic virus replication long after infection or indicate a significant active infection in the patient. In the newborn, viruria (isolation of virus in urine) in the first 3 weeks of life establishes a diagnosis of congenital cytomegalovirus infection, whereas the onset of viral excretion after 3-4 weeks of life reflects intrapartum or postpartum infection. Diagnosis of acquired cytomegalovirus in older patients usually requires a combination of findings, including positive cultures, illness compatible with cytomegalovirus disease, reasonable exclusion of other potential etiologic agents, and support by specific serologic or histologic data.
The significance of viruses isolated in upper respiratory tract, vaginal, or fecal specimens varies greatly. At one extreme, isolates such as measles, mumps, influenza, parainfluenza, and respiratory syncytial virus are significant because asymptomatic carriage and prolonged shedding of these viruses are unusual. Conversely, other viruses can be shed continually or intermittently without symptoms for periods ranging from several weeks (enteroviruses in feces) to many months or years (herpes simplex virus or cytomegalovirus in the oropharynx and genital tract; adenoviruses in the oropharynx and intestinal tract). Herpes simplex virus, cytomegalovirus, varicella zoster virus, and Epstein-Barr virus may remain latent for long periods and then become reactivated in response to a variety of stressful stimuli, including other infectious agents. In this setting their detection may not be significant, may merely represent a secondary problem complicating the primary infection (eg, herpes simplex virus "cold sores" in patients with bacterial sepsis), or may be associated with significant disease, especially in the immunocompromised patient.
Based on the epidemiology of adenovirus infections and observed serologic responses, the simultaneous isolation of these viruses from throat and feces is significantly associated with febrile respiratory disease. Isolation of viruses from the throat alone is less frequently associated with disease, and isolates from feces alone are probably nondiagnostic in a patient with respiratory disease.
Enteroviruses are generally found in infants and children, particularly during the late summer and early autumn. A knowledge of the relative frequency of virus shedding among various age groups in a particular locale is extremely helpful in assessing the significance of results of throat or stool cultures. For example, the peak prevalence of enteroviruses in the stools of toddlers during the late summer may range from 5% in temperate zones to > 20% in subtropical climates. Even in temperate areas, rates may approach 30% in infants during periods of enterovirus activity. Shedding of enterovirus in the throat usually occurs for 1-2 weeks, whereas fecal shedding may last 4-16 weeks. Thus, in a clinically compatible illness, isolation of an enterovirus from the throat supports a stronger temporal relationship to the disease than does an isolate from only the feces.
Herpes simplex virus is unusual in a fecal culture. In such cases it usually represents either severe disseminated infection or local infection of the perianal areas. Detection of herpes simplex virus in the upper respiratory tract may mean nothing other than nonspecific reactivation of virus caused by fever unless typical vesicles or ulcers are also present. Because of the fever-related phenomenon, isolation of herpes simplex virus in the throat or mucocutaneous lesions of patients with encephalitis cannot be interpreted as causing the central nervous system disease. Currently the definitive way to establish a diagnosis of herpes simplex encephalitis is by direct demonstration of the virus in a brain biopsy or by polymerase chain reaction (PCR) on CSF. In neonates, however, isolation of the virus from any site should raise the possibility of severe infection.
Isolation of adenoviruses, herpes simplex virus, varicella-zoster virus, and some enteroviruses from the cornea and conjunctiva in patients with inflammatory disease at these sites usually establishes the etiology of the infection.
DETECTION OF VIRAL ANTIGENS
Antibodies can be used as sensitive tools to detect, identify, and quantitate the presence of viral antigen in clinical specimens or cell culture. Monoclonal or polyclonal antibodies prepared in animals may be used. Viral antigens on the cell surface, within the cell, or released from infected cells can be detected by IF, EIA, RIA, and latex agglutination (LA). IF detects and locates cell-associated antigens, whereas RIA or different variations of enzyme-linked immunosorbent assay (ELISA) are used to detect and quantitate soluble antigens. LA is a rapid, easy assay for antigen; viruses or viral antigens in a sample cause the clumping of latex particles coated with specific antibody.
Virus-infected tissue or cell cultures can be detected by IF or EIA. By attaching a fluorescent signal to an antiviral antibody, and reacting it with the sample, viral antigen can be detected; this is called direct IF. A modification of this technique is the use of unlabeled antiviral antibodies and then a second antibody with a fluorescent label that will bind to IgM or IgG antibodies. EIA uses a second antibody conjugated to an enzyme, such as horseradish peroxidase or alkaline phosphatase, which releases a chromophore to mark the presence of antigen.
Direct IF assay is especially useful for (1) respiratory viruses (eg, respiratory syncytial virus or influenza A), (2) varicella-zoster virus and herpes simplex virus antigen in lung and visceral biopsies, and (3) cytomegalovirus in leukocytes from blood or CSF.
Soluble antigen can be quantitated by ELISA, RIA, and LA. The basis for these procedures is the separation and quantitation of antibody-bound and free antigen. Many of the ELISA and RIA techniques use an antibody immobilized to a solid support to capture soluble antigen and a labeled antibody to detect captured antigen.
Influenza, parainfluenza, and togaviruses produce a glycoprotein that binds erythrocytes. This property allows detection of free virus produced in cell culture by agglutination of erythrocytes, a process termed hemagglutination. The infected cells also adsorb erythrocytes to the surface by a process referred to as hemadsorption.
Detection and assay of characteristic enzymes can identify and quantitate specific viruses. For example, reverse transcriptase in cell culture is used as an indicator of infection by retroviruses.
SEROLOGIC TEST METHODS
Introduction
Serology can be used to determine whether an infection is primary or a reinfection and if acute or chronic. The first antibodies to be produced by the immune system are directed against antigens on the virion or infected cell surfaces and are best detected by, eg, IF. Later in the infection, when cells have been lysed by the infecting virus or the cellular immune response, antibodies are directed against the cytoplasmic viral proteins and enzymes and can be detected by, eg, complement fixation. Seroconversion is characterized by a change from negative to positive antibody between serum taken during the acute phase of disease and that taken = 2-3 weeks later; it is the best serologic marker of recent infection. A fourfold or greater rise in antibody titer in paired sera may also be significant but fluctuations in antibody titer do occur naturally. Detection of virus-specific IgM is theoretically associated with recent infection, but technical difficulty in measuring specific IgM responses may lead to false-positive and false-negative results.
For many viruses, culture or antigen detection is the best diagnostic test. However, certain viruses (eg, HIV, hepatitis A and B viruses, rubella virus, Epstein-Barr virus, measles virus, coronaviruses, and togaviruses) are difficult to isolate in cell culture, and infections are diagnosed most easily by serologic techniques. When a virologic workup is planned for a patient, it is generally useful to obtain = 2-3 ml of serum during the acute phase of disease and store it at -20oC. This may become valuable, particularly if virus detection subsequently fails or if the significance of an isolate is uncertain. In these instances a convalescent-phase serum specimen may be requested 2-3 weeks later, and both the acute and convalescent sera may then be tested against appropriate viral antigens. In general, if a virus is isolated, the antibody titers need not be measured to confirm infection; for example, if a patient has aseptic meningitis and an enterovirus is recovered from the throat, that agent is probably responsible for the illness. Similarly, if influenza virus is recovered from the throat of a patient who has clinical influenza, no serologic confirmation of the etiology is necessary.
Complement fixation is a standard but technically difficult serologic test. The serum is first reacted with the suspected viral antigen and complement, and the residual complement is assayed by lysis of indicator antibody-coated erythrocytes. If the complement is used in the first reaction and is therefore not available for the second reaction, it indicates the presence of antibody to the suspected virus. Antibodies measured by this system generally develop slightly later in the course of an illness than those measured by other techniques. This delayed response is useful for documenting seroconversion when the initial serum specimen is collected late in the clinical course. Members of some virus groups (eg, enteroviruses) do not possess group-specific antigen and must be tested individually.
The neutralization test is essentially a protection test. When a virus is incubated with homologous type-specific antibody, the virus is rendered incapable of producing infection in an indicator cell culture system. A neutralization antibody response is virus type specific, with titers rising rapidly and persisting for long periods.
The hemagglutination inhibition test can be performed with a variety of viruses that can selectively agglutinate erythrocytes of various animal species (eg, chicken, guinea pig, or human). The hemagglutination capacity of a virus is inhibited by specific immune or convalescent sera. Hemagglutination-inhibiting antibody develops rapidly after the onset of symptoms, plateaus, declines slowly, and may last indefinitely at low levels. This test is useful for both the detection of acute viral infection and the determination of immunity.
For the indirect fluorescent antibody test, virus-infected cells are placed in prepared wells on microscope slides and then fixed in cold acetone and dried. Patient serum is applied and, after incubation, anti-human globulin conjugated with fluorescein is added. If fluorescence is observed, it indicates the presence of specific antiviral antibody.
Interpretation of Serologic Results
Virus-specific IgM antibody usually rises during the first 2-3 weeks of infection and persists for several weeks to months. Thus an elevated titer of specific IgM antibody suggests a recent primary infection, which may be further supported by demonstrating a fall in IgM antibody in subsequent sera. Detection of specific IgM has been used with success in the diagnosis of infections caused by cytomegalovirus and rubella virus and is currently the procedure of choice to establish a recent or acute infection from hepatitis A or B.
Several limitations of interpretation must be remembered. It is now recognized that IgM-specific antibody responses are not always restricted to primary infections. Reactivation or reinfection may result in IgM responses, particularly in herpes virus infection. In addition, patients may continue to produce IgM-specific antibody to rubella virus or cytomegalovirus for many months after a primary infection. Heterotypic IgM responses may also occur. For example, antibody responses to cytomegalovirus may develop in Epstein-Barr virus infections and vice versa. Other pitfalls include falsely low or negative IgM titers caused by competition from high-titer IgG antibody for antigen-binding sites and false-positive reactions resulting from rheumatoid factor. Both types of errors appear to occur most frequently in solid-phase assays with IF.
The serologic diagnosis of most viral infections is based on demonstration of a seroconversion or a rise (fourfold or greater) of IgG antibody. However, significant antibody titer rises may result from cross-reactions to related antigens; for example, an antibody rise to parainfluenza virus may actually result from infection with mumps virus. Furthermore, seroconversions may not be seen with some patient populations (eg, infants and immunocompromised patients) or when the initial serum is collected late in the course of disease.
A serum specimen can also be used for screening an infant's blood for certain antibodies of the IgG class. Antibodies to Toxoplasma spp., rubella virus, cytomegalovirus, and herpes simplex virus (known as the "TORCH" screen) may be measured to determine possible congenital infection with these agents. However, the value of these tests must be understood. They are useful in excluding a possible infection but not in proving an etiology. For example, if rubella antibody is absent, an infant almost certainly does not have congenital rubella infection. To diagnose active rubella infection in such a baby, viral cultures are required. Screening of blood supplies for cytomegalovirus antibody is used to eliminate transmission of antibody-positive blood to seronegative babies and other immunocompromised patients.
Serologic Panels
Selection of several antigens for testing with paired sera in cases in which a virus is suspected can usually be made based on clinical syndrome, the known local epidemiology of particular viruses, and the patient's age. This has led to the concept of serologic batteries of panels. Some examples of possible panels are included in Table 6-10.
Antigens from mumps, western equine encephalitis, eastern equine encephalitis, St. Louis encephalitis, and California encephalitis viruses—and perhaps lymphocytic choriomeningitis virus, Epstein-Barr virus, and HIV—may be included in a panel of tests for central nervous system diseases. Although herpes simplex antigen is sometimes included in such a panel, a rise in antibody titer is not sufficient to diagnose herpes encephalitis. Many viral central nervous system illnesses, especially aseptic meningitis, are caused by the enteroviruses; however, the many serotypes and the cumbersome serologic methods necessary for their diagnosis usually make it impractical to include them in a panel. When one or two enteroviruses have been shown to be epidemic in an area in one summer, one can pick up some additional cases by performing neutralization tests on paired sera by using only those specific enteroviruses that are endemic in the community.
The viral antigen panel for testing respiratory syndromes might include influenza A and B; respiratory syncytial virus; parainfluenza types 1, 2, and 3; and adenoviruses.
To test for viral causes of exanthems, the panel would include measles and rubella. If the disease is vesicular, herpes simplex virus and varicella-zoster virus should be included, although the herpes viruses are best diagnosed by culture or antigen detection.
Antigens from group B coxsackie virus types 1-5 and perhaps influenza A and B viruses could make up the panel for myocarditis and pericarditis. Although numerous viruses have been implicated in inflammatory diseases of the heart and its covering membranes, the group B coxsackieviruses are believed to account for almost one half of the cases. Unfortunately, much of the clinical illness is expressed late in the infection, at the time when standard methods of virus detection are likely to fail and it is too late to demonstrate seroconversion or significant antibody titer rises.
MOLECULAR TEST METHODS
In recent years a variety of assays have been developed to detect viral nucleic acid—either DNA or RNA. The best known of these is PCR, an amplification technique that allows the detection and selective replication of a targeted portion of the genome. The technique uses special DNA polymerases that initiate replication in either the 3' or 5' direction. The specificity is provided by primers that recognize a pair of unique sites on the genome so that the DNA between them can be replicated by repetitive cycling of the test conditions. Because each newly synthesized fragment can serve as the template for its own replication, the amount of DNA doubles with each cycle. The amplification power of PCR offers a solution for the sensitivity problems inherent in the direct application of probes. Although the nucleic acid segment amplified by PCR can be seen directly on a gel, the greatest sensitivity and specificity are achieved when probe hybridization is carried out after PCR. A probe is a fragment of DNA that has been cloned or otherwise recovered from a genomic or plasmid source. In some cases the probe is synthesized as a single chain of nucleotides (oligonucleotide probe) from known sequence data. The probes are labeled with a radioisotope or other marker and used in hybridization reactions either to detect the homologous sequences in unknown specimens or in gel electrophoresis.
The diagnostic use of DNA probes is to detect or identify microorganisms by hybridization of the probe to homologous sequences in DNA extracted from the entire organism. A number of probes have been developed that will quickly and reliably identify organisms that have already been isolated in culture. The application of probes for detection of infectious agents directly in clinical specimens such as blood, urine and sputum is more difficult.
Recently, the branched DNA (bDNA) a rapid assay for direct quantification of viral nucleic acid has been developed for hepatitis B, hepatitis C, and HIV infections. Because the bDNA assay measures viral nucleic acids at physiological levels by boosting the reporter signal, rather than by amplifying target sequences, it is not subject to the errors inherent in the amplification steps of PCR-based methods. Inherently quantitative and amenable to routine use in a clinical setting, the bDNA assay may be useful in the management of patients with chronic viral diseases. Recent studies have illustrated the potential clinical utility of the bDNA assay in determining the prognosis and in therapeutic monitoring of infection. Additional nucleic acid tests include hybrid capture and nucleic acid sequence-based amplification.
15 comments:
Am Joyce Benson, I want to testify about how Dr.Ekpiku help me to cure my herpes virus. It was in my brain causing constant pounding and messing with my sight. It tingles at the base of my spine all the time and messes hugely with my bowel function. My right leg was numb and I can not sleep at night because of the symptoms. This was hell on earth. I have tried everything to get off the virus but nothing comes out good on it. I saw so many testimony on how Dr. Ekpiku the herbs medicine man help to cure so many people herpes. So i contact him for help and tell him my herpes problem. He assure me that i will be negative after he finish casting the spell for 48 hours. He cast the spell and asked me to go for check-up after three days of casting the spell, Luckily for me were tested herpes negative, now I believe all these Testimonies about him on the internet, he is truly a great man, I am now free from herpes all thanks goes to Dr. Ekpiku for what he did in my life...If you are out there have herpes problem or any other disease you have to seek for his help because he is going to help you cure your herpes or any other sickness. email him on: Ekpikuspellhomeofgrace@gmail.com or Ekpikuspellhomeofgrace@hotmail.com or call him +2348073673757) THESE ARE THE THINGS Dr. Ekpiku. . HERPES . HIV/AIDS . CANCER...
Hello everyone I’m here to share a testimony on how my HIV was cure by a herbal doctor with the help of herbal medicine and herbal soap, As we all know medically, there is no solution or cure for HIV and the cost for Medication is very expensive. Someone introduced me to a man (Native Medical Practitioner) I showed the man all my Tests and Results and I told him i have already diagnosed with HIV and have spent thousands of dollars on medication. I said I will like to try him cause someone introduced me to him. He asked me sorts of questions and I answered him correctly. To cut the story short, He gave me some medicinal soaps and some herbs(have forgot the name he called them) and he thought me how am going to use them all. At first I was skeptical but I just gave it a try. I was on his Medication for 2 weeks and I used all the soaps and herbs according to his prescription. That he will finish the rest himself. And I called him 3 days after, I arrived and I told him what is the next thing he said, he has been expecting my call. He told me to visit my doctor for another test. Honestly speaking, i never believe all he was saying until after the test when my doctor mention the statement that am, HIV negative and the doctor started asking me how come about the cure, And I make a promise to dr osas that if I’m heal I will testify his good work in my life, if there is anyone out there who may need the help of dr osas you can email him via his email address drosasherbalhome@gmail you can reach him via phone calls or WhatSAPP number +2349035428122 or email him via drosasherbalhome@gmail.com
Dr Bello herbal cure is 100% Guarantee percent sure to cure your (HEPATITIS B AND ANY OTHER DISEASE) ,He is only person that i can boldly say he can cure any types of Disease.my wife was having HEPATITIS B for more than 5 year when i met Dr Bello online how and how he has cured so many people and how greatly he has helped many individuals online,so i contacted him and explained my wife situation to him and behold my wife was cure with his herbal medicine and now we are living happily, so to anyone issue on herpes challenges i advised that you contact him for help and he will send you the medication and you will be fine and OK : adeyemibello1990@gmail. com he can also cure any disease such as HIV/AIDS HEPATITIS B,DIABETICS,CANCER,HERPES E.T.C HE is the great herbalist man called Dr.Bello i must say a big thanks for curing my wife disease, i owe you in return. Thanks and be blessed sir.his Mobile number: 09035594773 or what’sapp him on 08107996445"
Though I haven't met DR UTU but I have being hearing and seeing his wonderful deeds in people's life.. This made me contacted him because I was also diagnosed of Herpes Virus, When I contacted Him This man proved to me that he is truly a savior that God sent to this modern age to redeem man kind as he's commonly know. without wasting time, he started His Miraculous work in my Life, He prepared and sent me herbal medicine through DHL. Within two days of using this herbal medicine I started noticing changes in my body system. I used this herbal medicine for just 8days before going to the lab for test where I was confirmed HERPES negative. I am happy and Glad to say that I am now cured after using His herbal Medicine.. Don't be deceived by anyone nothing is impossible in this modern world you just need the right medicine, herbal medicines have been helping mankind from the beginning of the world and still very active even till now. Are you tired of your ailments and need a cure? Email him on;
drutuherbalcure@gmail.com or dr.utu@outlook.com.
whatsapp; +2347032718477
i was scam several time not until i meant this doctor called Dr. Yakubu who cured me completely, some times we just need to give a try of herbal Medicine to see for our self. I am very happy today that i do not listen to what people say if not i would have been a dead person by now. I was infected with HERPES SIMPLEX VIRUS in 2015, i went to many hospitals for cure but there was no solution, so I was thinking how can I get a solution out so that my body can be okay. One day I was in the river side thinking where I can go to get solution. so a lady walked to me telling me why I am so sad and i open up all to her telling her my problem, she told me that she can help me out, she introduce me to a doctor who uses herbal medication to cure HERPES SIMPLEX VIRUS and gave me his email, so i mail him. He told me all the things I need to do and send me the herbal medicine through courier service and also give me instructions to take, which I followed properly. Before I knew what is happening after 3 to 4 days the HERPES SIMPLEX VIRUS that was in my body got vanished and blister dried off . I went to the hospital for a test and it was negative. this man is real and he is the solution to herpes virus. so if you are also heart broken and also need a help, you can also email him dryakubuherbalhealingclinic@gmail.com
I am bold enough among many others to state that there is now a potent cure to this sickness but many are unaware of it. I discovered that I was infected with the virus 3 months ago, after a medical check-up. My doctor told me and I was shocked, confused and felt like my world has crumbled. I was dying slowly due to the announcement of my medical practitioner but he assured me that I could leave a normal life if I took my medications (as there was no medically known cure to Herpes). I went from churches to churches but soon found that my case needed urgent attention as I was growing lean due to fear of dying anytime soon. In a bid to look for a lasting solution to my predicament, I sought for solutions from the herbal world. I went online and searched for every powerful trado-medical practitioner that I could severe, cos I heard that the African Herbs had a cure to the Herpes syndrome. It was after a little time searching the web that I came across one Dr Itua(A powerful African Herbal Doctor), who offered to help me at a monetary fee. I had to comply as this was my final bus-stop to receiving a perfect healing. My last resolve was to take my life by myself, should this plan fail. At last it worked out well. He gave me some steps to follow and I meticulously carried out all his instructions. Last month, to be precise, I went back to the hospital to conduct another test and to my amazement, the results showed that negative,Dr Itua Can As Well Cure The Following Desease…Cancer,Hiv,Herpes, Hepatitis B,Liver Inflammatory,Diabetis,Fribroid,,Non Hodgkin Lymphoma,Skin Cancer,Uterine Cancer,Prostate Cancer Dercum,Infertility,fibromyalgia,Get Your Ex Back,Als,SYPHILLIS,Genetic disease,Epilepsy, Parkinson's disease..You can free yourself of this Herpes virus by consulting this great African Herbal Doctor via this e-mail: drituaherbalcenter@gmail.com or call and whatsapp him on +2348149277967 He will help you and his herb medication is sure. he has the cure on all disease .You can talk to me on INSTAGRAM..tashamoore219....
I have being on blog Sites for a while now and today I felt like I should share my story because I was a victim too. I had HIV for 6 years and i never thought I would ever get a cure I had and this made it impossible for me to get married to the man I was supposed to get married to even after 2 years of relationship he broke up with me when he finds out I was HIV positive. So I got to know about Dr. Itua on Blog Site who treated someone and the person shared a story of how she got a cured and let her contact details, I contacted Dr. Itua and he actually confirmed it and I decided to give a try too and use his herbal medicine that was how my burden ended completely. My son will be 2 soon and I am grateful to God and thankful to his medicine too.Dr Itua Can As Well Cure The Following Disease… Cancer, HIV, Herpes, Hepatitis B, Liver Inflammatory,Diabetes,Fibroid, Get Your Ex Back, If you have (A just reach him on drituaherbalcenter@gmail.com Or Whatsapp Number.+2348149277967)He can also advise you on how to handle some marital's issues. He's a good man.
I'm Rachel and I own a herbal Therapy Center. What I am about to share with you is based on actual fact and I am not a socially irresponsible person. I am a woman of character and I believed that we have only one purpose for existing and that is to help our fellow humans. And everyday millions of other people are joining me and waking up to the understanding that love is the only thing that matters in this life.
For the past 10+ years I have dealt with a lot of people who have come to me asking if herbal remedies will get rid of herpes, usually genital. I always tell people from what I have observed and heard about NATURAL REMEDY and I suggest that they try it for themselves. I have never told anyone that herbal remedy would cure their herpes.
I have never told anyone this because I have never had any physical proof to back that up... until this day.
I have always told anyone who would listen the mystical work herbal remedy do for me when ever I'm in depression, I could say that because if it did that for me, it would do the same for anyone else.
I have also always told people how herbal remedies got my doctor to successfully cured DIABETES, INFERTILITY, CANCERS, PARALYSIS, STDS & INFECTIONS.
And I tell anyone about how herbal remedy got me off the Lysinopril I was taking for my high blood pressure That also normalized within 2months. And I am thankful it did because Lysinopril has since been recalled for turning the livers of hundreds of thousands of people into mush!
And with all the good things herbal remedy has done for my body, I still resisted the urge to tell anyone suffering with herpes that herbal remedy would cure them... but now I am here to tell you differently because I finally came in contact with Dr Utu an African Traditional Herbal Practitioner.
Few days ago I received an email from a young lady and she told me that her doctor has told her that her body is 100% free of any HSV virus. That her herpes has been cured! I could feel her bouncing off the walls through her email... and my heart was just as happy for her.
I have asked her to please post a comment on my YouTube Channel.
I don't know how long she had been infected before she purchased Dr Utu African Traditional Herbal Cure and I do not know anything about the severity of her outbreaks. All I know was that she promised me that she was following the guidelines that Dr Utu gave her to the letter.
I know there are a lot of people who will think this is too good to be true... but I have always believed that herbal remedy was a viable cure for herpes - but without any proof I felt it would be seriously irresponsible to tell anyone that it would.
Please feel free to leave a comment to me but please no negative ones. My company is my life and I have spent over 10 years nurturing a good reputation for myself and my business. I am putting everything on the line here, so please bear with me until we know more.
Why am I risking so much? Why don't I just wait for her? - Are you serious? This is the BIGGEST health breakthrough in my entire lifetime! I'm over 60 and there have been NO CURES for anything since the day I was born! The big pharmaceutical companies all know there is no money in a cure. The money is in teaching you and me to 'live with' and 'manage' deadly, body-wasting and disgusting diseases for our entire lives.
Imagine the joy all over the world when her story breaks! I invite you all to share in that joy with her and with me.
You can also contact Dr Utu for yourself via e-mail; drutuherbalcure@gmail.com
Hi there everyone, i want to give my testimony on how i get cured of herpes virus, i want to thank God for sending Doctor abaka that cured me of herpes virus, I have been with herpes virus for 7 years, i thank God that i am healed from this virus, I was 28 when I started seeing symptoms. I really don’t know where to begin… But I use to think that I will grow old with this virus in my body, because i have use so many different type of drugs, believing that i will get cured of this virus, but it was not cured, i do not know what else to do, i keep on believing in God that one day i will be cured of this deadly virus called herpes, i was Praying for healing over every part of my body, thanks to Doctor abaka for curing me of herpes virus that i have sick for 7 years, so if you are sick of any kind of disease and you need cure, you can contact him on email: drabakaspelltemple@gmail.com or call his phone number +2349063230051 May the Lord that sent Doctor abaka to cure my disease will also cure your disease, thanks to you all God bless you.
Herpes Finally Gone, thanks to Dr. Utu for his Herbal Treatment.
I had a sudden onset of cold sores and a new one was appearing each day. I was on primary immunosuppressant I noticed my body just can't fight the virus on its own. After five days of treatment and no success I suspected herpes I went for test and it was confirmed. God was my strength I met a doctor for treatment. He gave me Dr Utu's contact. I contacted him immediately, This medication was far amazing that within 24-48 hrs the blisters were crusting over. The swelling and redness was decreasing as well. I was on four weeks herpes medication which I completed before going for test. I went for herpes test and then again back to my doctor who confirmed me herpes negative it was really like a dream. What a relief! Feel Free to contact Dr Utu Email:- drutuherbalcure@gmail.com OR WhatsApp :- +2347032718477
I have been suffering from (HERPES) disease for the last four years and had constant pain, especially in my knees. During the first year, I had faith in God that I would be healed someday.This disease started to circulate all over my body and I have been taking treatment from my doctor, a few weeks ago I came on search on the internet if I could get any information concerning the prevention of this disease, on my search I saw a testimony of someone who has been healed from (Hepatitis B and Cancer) by this Man Dr. Silver and she also gave the email address of this man and advise we should contact him for any sickness that he would be of help, so I wrote to Dr. Silver telling him about my (HERPES Virus) he told me not to worry that I was going to be cured!! hmm i never believed it,, well after all the procedures and remedy given to me by this man few weeks later I started experiencing changes all over me as the Dr. assured me that I have cured, after some time i went to my doctor to confirmed if I have been finally healed behold it was TRUE, So friends my advice is if you have such sickness or any other at all you can email Dr. Silver (drsilverhealingtemple@gmail.com) sir I am indeed grateful for the help I will forever recommend you to my friends!!! with your lovely Email Address ( drsilverhealingtemple@gmail.com
Just wanna say a big thank you lucas mason for introducing me to Dr okosun the great HERBALIST that helped me prepare home remedies that cured my herpes simplex virus (HSV1&2)
I was infected with herpes for the past four years and i was not happy all day so i was desperate to get a cure so that i can live normal and get healthy.One day i was less busy so i decided to make latest research on herpes cure and i found a site were everyone was talking about Dr okosun and has ability to cure Herpes, HIV, HPV, Cancer, ALS, COPD, Anthrax, genital wart, cold sore, Arthritis skin tag and all manners of diseases and virus.So i discussed with lucas and he explained to me that its very easy working with Dr okosun so i contacted Dr okosun via email drokosun55@gmail.com and he helped me just as he has helped others now I am now cured from herpes am very happy now.and i can also assure you that he can also help you. so if you need the service of DR okosun kindly contact him drokosun55@gmail.com or whatsapp +2348124363791
Living with Herpes for the past six years,it has been hell to me and I have
tried all possible means to get cure but all to no avail, luckily for me
one day as i was browsing through the Internet searching for remedy on
Herpes, and i saw comment of people talking about how Doctor BIYA cured
them,so I contacted him base on the testimonies i have been seeing about
him on the internet,Lo and behold friends,that was the end used
omen(Herpes) now i'm free no more matters that touches the heart. Dr words
is just not enough to express what you have done for me,thanks to you....
You are indeed a light to those living with Herpes virus and may you remain
bless for your good work... Dr Biya have cure for any types of diseases,
Herpes HIV DIABETES CANCER Asthma STROKE INFERTILITY HEPATITIS B HEART
DISEASE and you will be also cured Gmail: drbiyajatto@gmail.com WhatsApp
+2348111856507 The almighty God shall continue to bless you Dr Biya.
Herpes is a serious and recurring disease which can't be cured through drugs or injections by the American doctors but the best way to deal with Herpes is by taking natural herbs medicine for it, I have read about DR JAMES the great herbalist doctor who cure me from herpes with his powerful herbal medicine. I contacted him to know how he can help me and he told me never to worry
that he will help me with the natural herbs from God!
After 2 days of contacting him, he told me that the cure has been ready and
he sent it to me via UPS SPEED POST and it got to me after 3 days!
i used the medicine as he instructed me (MORNING and EVENING) and i was
cured!
it's really like a dream but I am so happy! for the people suffering from the following disease Alzheimer’s disease,Bechet’s disease,Crohn’s disease,Parkinson's disease,Schizophrenia,Lung Cancer,Breast Cancer,Colo-Rectal Cancer,Blood Cancer,Prostate Cancer,siva.Fatal Familial Insomnia Factor V Leiden Mutation ,Epilepsy Dupuytren's disease,Desmoplastic small-round-cell tumor Diabetes ,Coeliac disease,Creutzfeldt–Jakob disease,Cerebral Amyloid Angiopathy, Ataxia,Arthritis,Amyotrophic Lateral Sclerosis,Fibromyalgia,Fluoroquinolone Toxicity
Syndrome Fibrodysplasia Ossificans ProgresS sclerosis,Seizures,Alzheimer's disease,Adrenocortical carcinoma.Asthma,Allergic diseases.Hiv_ Aids,Herpe ,Copd,Glaucoma., Cataracts,Macular degeneration,Cardiovascular disease,Lung disease.Enlarged prostate,Osteoporosis.Alzheimer's disease,
Dementia.Lupus.
,Cushing’s disease,Heart failure,Multiple Sclerosis,Hypertension,Colo_Rectal Cancer,Lyme Disease,Blood Cancer,Brain Cancer,Breast Cancer,Lung Cancer,Kidney Cancer, HIV, Herpes,Hepatitis B, Liver Inflammatory,Diabetes,Fibroid,
should contact him for his herbal medicine because i am a living testimony and i was cured of herpes and his medicine is legit. I sent him what he requested and he sent me his medicine which I took for 2 good weeks and today I am out here with a negative result. When I went for the test I was so happy after taking his herbal medication I paid homage to his country to celebrate with him on his African festival which he told me it usually happens every year. you can reach him through VIA E-mail drjamesherbalmix@gmail.com or whatsapp number:+2348152855846
I'm from Paris, I was diagnosed with second-stage liver cancer and brain fog following a scheduled examination to monitor liver cirrhosis. I had lost a lot of weight. A CT scan revealed three tumors; one in the center of my liver in damaged tissue and two in healthy portions of my liver. No chemotherapy or radiotherapy treatment was prescribed due to my age, the number of liver tumors. One month following my diagnosis I began taking 12 (350 point) Salvestrol supplements per day, commensurate with my body weight. This comprised six Salvestrol Shield (350 point) capsules and six Salvestrol Gold (350 point) capsules, spread through the day by taking two of each capsule after each main meal. This level of Salvestrol supplementation (4,000 points per day) was maintained for four months. In addition, I began a program of breathing exercises, chi exercises, meditation, stretching and stress avoidance. Due to the variety of conditions that I suffered from, I received ongoing medical examinations. Eleven months after commencing Salvestrol supplementation But all invalid so I keep searching for a herbal cure online that how I came across a testimony appreciating Dr Itua on how he cured her HIV/Herpes, I contacted him through email he listed above, Dr Itua sent me his herbal medicine for cancer to drink for two weeks to cure I paid him for the delivering then I received my herbal medicine and drank it for two weeks and I was cured until now I'm all clear of cancer, I will advise you to contact Dr Itua Herbal Center On Email...drituaherbalcenter@gmail.com. Www.drituaherbalcenter.com If you are suffering from Diseases listed below,
Cancer
HIV/Aids
Herpes Virus
Bladder cancer
Brain cancer
Colon-Rectal Cancer
Breast Cancer
Prostate Cancer
Esophageal cancer
Gallbladder cancer
Gestational trophoblastic disease
Head and neck cancer
Hodgkin lymphoma
Intestinal cancer
Kidney cancer
Leukemia
Liver cancer
Lung cancer
Melanoma
Mesothelioma
Multiple myeloma
Neuroendocrine tumors
Non-Hodgkin lymphoma
Oral cancer
Ovarian cancer
Sinus cancer
Skin cancer
Soft tissue sarcoma
Spinal cancer
Stomach cancer
Testicular cancer
Throat cancer
Thyroid Cancer
Uterine cancer
Vaginal cancer
Vulvar cancer
Hepatitis
Chronic Illness
Lupus
Diabetes
Fibromyalgia
enteroviruses
Men/Women Infertility
Menstrual Cramp.
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