Most of the neonatal infections are diagnosed with isolation of the microorganisms from different sites as blood, urine, cerebrospinal fluid (CSF), peritoneal, and pleural fluids. Different techniques as blood culture for bacterial organisms; viral isolation techniques for viruses like cytomegalovirus (CMV), rubella, herpes, human immunodeficiency virus (HIV) etc have been employed. These remain the gold standard for diagnosis of the microbial infection. However these methods of detection are time consuming and require inputs from sophisticated bacteriological and virological laboratories.Microbiologists, molecular biologists and infectious disease personnel have been interested in simplifying disease diagnosis with non-culture techniques like antigen detection methods by ELISA, immunofluorescence, immunobloting technique, fluorescent immunohybridization sequencing (FISH) and other newer innovative methods. In last two decades rapid advances in the field of molecular biology have developed and refined newer innovative tools like polymerase chain reaction (PCR) and nucleic acid sequence base amplification (NASBA).This text will deal with some of these methods that are useful in diagnosis of neonatal infections like bacterial sepsis, herpes, varicella, HIV, hepatitis B, C, CMV, toxoplasmosis, rubella, syphilis etc. Some of the conventional and newer molecular biological methods discussed in this text are immunofluorescent stains like acridine orange and auramine-rhodamine stains, contercurrent immunoelectrophoresis, latex agglutination, enzyme immunoassays, immunofluorescence, DNA hybridization and PCR (<xref ref-type="bibr" rid="bib001">1</xref>–<xref ref-type="bibr" rid="bib003">3</xref>).<sec id="sec002"><title>Staining Methods for Antigen Detection

Most of the neonatal infections are diagnosed with isolation of the microorganisms from different sites as blood, urine, cerebrospinal fluid (CSF), peritoneal, and pleural fluids. Different techniques as blood culture for bacterial organisms; viral isolation techniques for viruses like cytomegalovirus (CMV), rubella, herpes, human immunodeficiency virus (HIV) etc have been employed. These remain the gold standard for diagnosis of the microbial infection. However these methods of detection are time consuming and require inputs from sophisticated bacteriological and virological laboratories.Microbiologists, molecular biologists and infectious disease personnel have been interested in simplifying disease diagnosis with non-culture techniques like antigen detection methods by ELISA, immunofluorescence, immunobloting technique, fluorescent immunohybridization sequencing (FISH) and other newer innovative methods. In last two decades rapid advances in the field of molecular biology have developed and refined newer innovative tools like polymerase chain reaction (PCR) and nucleic acid sequence base amplification (NASBA).This text will deal with some of these methods that are useful in diagnosis of neonatal infections like bacterial sepsis, herpes, varicella, HIV, hepatitis B, C, CMV, toxoplasmosis, rubella, syphilis etc. Some of the conventional and newer molecular biological methods discussed in this text are immunofluorescent stains like acridine orange and auramine-rhodamine stains, contercurrent immunoelectrophoresis, latex agglutination, enzyme immunoassays, immunofluorescence, DNA hybridization and PCR (<xref ref-type="bibr" rid="bib001">1</xref>–<xref ref-type="bibr" rid="bib003">3</xref>).<sec id="sec002"><title>Staining Methods for Antigen DetectionGram stain and Acid-fast stain

Gram stain and acid fast bacilli stain are one of the earliest antigen detecting methods which stain the bacterial cell cytoplasm and represent the bacterial presence when seen under a microscope. However they represents a whole array of antigens present within the cytoplasm of the bacteria. Gram stain has been able to differentiate between Gram positive and negative organisms and the morphological shapes of the microorganisms. These staining procedures can be done for different body fluids like CSF, sputum, pleural and peritoneal fluid, urine, stool and blood. However interpretation of a Gram-stained smear requires some skill and practice. The information is only clue to the likely organism and needs to be confirmed with culture techniques.

The fungal elements in a stained specimen can be demonstrated with special stains as peroxide acid –Schiffs (PAS) stain and Gomori methanamine silver stain. The PAS stains the material as red while as the latter stains it as black.

Acridine orange stain

This technique employs the use of a fluorochrome dye acridine-orange, which has the ability to stain the nucleic acids of the bacteria. The stained bacteria are seen in the yellow-green dark background when viewed under a fluorescent microscope. Intracellular organisms are better seen within the cells with acridine-orange staining as compared to Gram-stain (1). This technique has been proved useful for staining bacteria in buffy coat samples of blood in neonates with septicemia (2). It has been also found to be useful in detecting malaria parasites in children with malaria. Prior to the development of immunochromatographic methods for detection of malarial antigens this technique was used for diagnosis of malarial parasites within the red blood cells.

Counter Current Immunoelectrophoresis

This technique used previously employed low voltage direct current passed in an agar plate (3). Positive or negative charged polysaccharides and immunoglobulins will migrate towards the cathode or anode respectively. This is useful in detecting polysaccharide antigens of various organisms as H. influenzae b, S. pneumoniae, N. meningitis and group B. streptococci. Thus if an antibody is directed to a specific antigen a line of precipitation forms between the antigen-antibody wells. However with the advent of simpler antigen detection method as latex agglutination this is no longer used in most microbiological laboratories.

Latex Agglutination

Latex agglutination technique is the most commonly employed technique for diagnosis of infections in CSF like H. iinfluenzae type B, S. pneumoniae, E.coli, Group B Streptococci and Cryptococcus Neoformans (4). The test is based on detection of bacterial or fungal antigen, which bind on to monoclonal murine or rabbit antibody coated latex beads and thus form a precipitate due to agglutination of antigen-antibody complex. Numerous latex agglutination kits have been made available commercially for immediate diagnosis. Both negative and positive controls are run along with the test samples. CSF is the best sample for this test due to lack of non-specific antibodies, which are otherwise present in sera and urine samples. The test can be false positive due to presence of non-specific antibodies that may be present in normal individuals. Various strategies are employed to avoid to get rid of these antibodies in the serum or urine samples in laboratories or in the commercial kits. The sensitivity of this method for detection of group B streptococcal infection in neonates in CSF is greater than countercurrent immunoelectrophoresis (5).

Neonates with culture negative septicemia and meningitis can be diagnosed by demonstration of bacterial antigens in CSF samples and urine samples respectively when antibiotics have been initiated for two to three prior to collection and testing of the samples (6).

Enzyme Immunosorbent Assays

Enzyme immunoassays (EIA) form the backbone for the diagnostic array of molecular diagnostic investigations. They are the most frequently used immunoassay due to their easy technique, less time consumption, low cost and easy reproducibility. These immunoassays can be used to detect the presence of specific antigen or antibody in the serum of the patient.

The principle underlying immunoassays detects the presence of antigen and antibody complex with a second antibody which is bound to a chemical dye which can be visualized colorimetrically (Figure 1). Antibody to a specific antigen is bound to a solid phase, such as a polyvinyl microtitration plate or plastic beads. Unbound antibody is removed. Figure 1Principles of enzyme immunoassay (EIA).

The procedure involves the specimen (serum, CSF, urine or other body fluid) which contains the antigen in question is placed in the wells of microplate and incubated for a variable period, during which the antigen binds to the solid phase antibody. The wells are washed with a buffer solution and the enzyme-labelled antibody is added to the same wells (direct method). Conversely in the indirect immunoassay, unlabelled antibody from an animal species, murine or rabbit different from solid-phase antibody is used. The enzyme linked antibody directed against the globulin of the second antibody is applied. The plates are washed, and the enzyme from the substrate is added. The linked enzymes catalyze the substrate, and the rate of reaction is monitored by a color reaction with a spectrophotometer. This depends on the enzyme-linked antibody, which is related directly to the quantity of the antigen present in the specimen. Alkaline phosphatase and horseradish peroxidase are enzymes that are used in most of the immunoassays as colorimetric agents. A whole array of bacterial, viral and fungal antigens can be detected by ELISA methods.

The TORCH screen for detection of various antibodies was developed for diagnosis of important perinatally transmitted infections like Toxoplasma, other infections, Rubella, Cytomegalovirus and Herpes simplex infection. These serological tests can be used for detection of IgM or IgG antibodies in the serum. Various commercially available kits employing enzyme linked immunosorbent assay can detect these antibodies. However to interpret these tests one needs to understand the exact kinetics of the humoral immune response in the fetus and the newborn infant. Figure (2) given below illustrates the maternal and fetal IgG and IgM antibody response. Maternal response to viral and bacterial antigens can be seen as expression of IgG antibodies that can cross transplacentally to the fetus and thus can be detected in the fetal or cord blood specimens. These antibodies decline to undetectable levels in the infant by 6–12 months of age detected by various ELISA systems, depending on the sensitivity of the system. Thus mere presence of IgG antibodies against there viral pathogens cannot be pathogmonic of infection. However maternal IgM antibodies formed against the microbial antigens do not cross the placenta and thus cannot be detected in the fetus or newborn infant. The fetus has the ability to mount an IgM response to various antigens if it is challenged. This specific IgM response can be detected in the cord or neonatal blood for various antigens. In addition the fetus mounts a total IgM response to various transplacental infections. Thus this approach has been used to make a serological diagnosis of viral infections in the newborn infant. Though this approach seems theoretically sound, but it can lead to false positive and false negative results. The diagnosis is limited to various viral infections. Detection of IgM antibodies against HIV infection by ELISA or immunofluorescent techniques have been fraught with failures. Various non-specific antibodies in the serum like IgM rheumatoid factor antibodies binding to Fc fraction of IgG can give false positive results. Similarily false negative results occur in perinatal CMV infection where IgM antibodies can be detected in 70% of affected neonates as compared to other techniques as isolation of CMV from urine samples. Figure 2Trends of fetal and neonatal immunoglobulins.

Immunofluorescence

With the advent of fluorescent microscope and fluorscein conjugate dyes this method has been used for several years for rapid detection of antigens and antibodies from the serum, tissue samples like skin, lungs, kidney and pleural fluid. The principle underlying this assay is similar to ELISA, however the antigen-antibody complex is detected by the fluorecent conjugates which are visualized under the fluorescent microscope. This procedure needs an experienced microscopist to see the specimens and is thus observer dependent. However this has been used in both clinical and research settings for rapid diagnosis of selected bacterial and viral infections. Various infections like Bordetella pertussis, Leigonella, P. carinii, Shigella dysenteriae and Giardia lamblia can be detected with this technique.

DNA Hybridization

Various molecular biological techniques as DNA hybridization, polymerase chain reaction (PCR), nucleic acid sequence base amplification (NASBA) and Gene-Probe assay have been developed over last decade for rapid identification of numerous microorganisms. These molecular methods employ the use of certain specific sequences of DNA containing nucleotides or messenger RNA to detect the microorganisms. Most of these techniques need a good molecular biology laboratory with expertise.

DNA hybridization probes are available for detection of certain microbes like N. gonorrhoeae, C. trachomatis, Mycoplasma pneumoniae, Mycobacterium tuberculosis and Listeria monocytogenes. The principle involved in this technique is based on detecting a short segment of microbial DNA with a single stranded cloned DNA probe, which has been labeled with biotin. Homologous DNA-DNA binding occurs in this process.

The exact method involves extracting DNA/RNA (nucleic acids) from the clinical specimens. The extracted DNA is placed on nitrocellulose paper and allowed to dry. Then this is incubated with the specific labeled DNA probe and subjected to autoradiography (a X-ray detection system) for detection of these probes. Radio-labeled probes can be detected by other detector systems.

Polymerase Chain Reaction

This molecular biological technique had caught the fancy attention of microbiologists, molecular biologists and the clinicians alike in last two decades. Originally designed as an innovative technique by Saiki et al. (7), it has been modified and used by different laboratories to amplify short segments of microbial DNA. Today virtually any organism can be detected by the PCR technique. The principle involved in this process is the amplification of a short DNA segment whose nucleotide sequence is known (Figure 3). This is called as ‘primer’. The primer can bind or pair with the microbial DNA (DNA extracted from microbial specimens). This paired DNA sequence can be amplified to a million copies with the help of thermal cyclers used in this process. Thus the microbial amplified DNA can be detected on nitrocellulose paper. This technique is very sensitive and any cross contamination with other specimens run at the same time may lead to false positive results. This method is becoming the gold standard for detection of various infections like HIV, herpes simplex virus, hepatitis B, hepatitis C virus, tuberculosis, N. meningitides, C. trachomatis, T. gondii, and T. pallidum in neonates. Figure 3Polymerase chain reaction.

PCR technique has been used to detect toxoplasma DNA in the amniotic fluid, placenta, CSF, fetal and infant blood.

Newer Nucleic Acid Amplification Techniques

PCR technique led the path to development of newer innovative techniques as nucleic acid sequence based amplification technique (NASBA). This technique is similar to PCR, however the single stranded messenger RNA is amplified in this process instead of the conventional DNA. In this method the DNA that is complimentary to RNA is synthesized first. This DNA is then used as template for transcription. Reverse transcriptase enzyme is used for synthesizing DNA from RNA. Newer innovative molecular biological methods for detection of microbial DNA/RNA have been developed. Ligase chain reaction (LCR), b-DNA PCR assay, nested PCR, real time PCR and Gene-Probe assay are some of the methods that are being used for diagnosis of various infection like HIV infection, tuberculosis, chlamydial infections and other neonatal infections. However the interpretation of these tests needs to be evaluated in the clinical scenario, prior to initiation of targeted antiviral or microbial chemotherapeutic agents. A close coordinated effort between the molecular biology laboratories and the clinicians can result in better diagnostic results for early detection of microbial infections.

Antigen detection & molecular biological methods in diagnosis of neonatal infections