Annals of Forensic Research and Analysis

Diagnostic Options for Investigating Viral Respiratory Pathogens in Sudden Unexpected Death in Infancy (SUDI) Cases

Review Article | Open Access | Volume 1 | Issue 2

  • 1. Department of Pathology, University of Stellenbosch, South Africa
+ Show More - Show Less
Corresponding Authors
De Beer C, Division of Medical Virology, Department of Pathology, University of Stellenbosch, P O Box 063, Tygerberg, 7505, South Africa,

Sudden and unexpected deaths in infants have occurred for centuries. It has generally been referred to as sudden infant death syndrome (SIDS). A new concept, called sudden unexpected death in infancy (SUDI) was introduced in 1989, which is used for all unexpected deaths in infants and babies, usually during sleep, where fatal injury can be excluded. By definition, cases that remain unexplained after thorough investigation are still classified as SIDS.

Many risk factors have been associated with SUDI, e.g. poor socioeconomic conditions and prenatal care, multiple pregnancies, parental drug use and smoking, gender, low birth weight, recent infection and the sleeping environment. Ultimately, SUDI is most probably a result of a combination of predisposing factors, external stresses and underlying vulnerabilities, although the exact mechanism of death remains unknown.

Viral infections are common in infants and have repeatedly been implicated in SUDI. Respiratory infections occur frequently in infancy and early childhood and inflammatory changes in the respiratory tract in SUDI cases is often found. Different diagnostic approaches for investigating respiratory viruses in SUDI cases have been reported in the literature, but in the absence of standardised SUDI investigation protocols, research from different centres cannot be compared. Viral viability is compromised in post-mortem samples and results should be interpreted with care, as the mere presence of a pathogen does not confirm that to be the cause of death. It is therefore imperative to use a combination of diagnostic approaches in parallel with epidemiological and clinical information in SUDI cases.


de Beer C, la Grange H (2014) Diagnostic Options for Investigating Viral Respiratory Pathogens in Sudden Unexpected Death in Infancy (SUDI) Cases. Ann Forensic Res Anal 1(2): 1007


IMR: infant mortality rate; SIDS: sudden infant death syndrome; SUDI: sudden unexpected death in infancy; DNA: deoxyribonucleic acid; RNA: ribonucleic acid; CMV: cytomegalovirus; IHC: immunohistochemistry; ISH: in situ hybridisation; EM: electron microscopy; IF: immunofluorescence; PCR: polymerase chain reaction.


The infant mortality rate (IMR) is an estimate of the health status of a country and represents the number of infant deaths for every 1000 live births [1,2]. The four leading causes of infant mortality worldwide are congenital abnormalities, sudden infant death syndrome (SIDS), prematurity and low birth weight [2]. Despite a drastic decline in the IMR of many regions, SIDS remains one of the most frequent causes of death in infancy [2-6].

SIDS is defined as the sudden and unexpected death of an infant younger than one year, which remains unexplained after a complete autopsy, death scene examination and review of the clinical history [7,8]. Prior to any medico-legal investigation, sudden and unexpected infant deaths are categorised as sudden unexpected death in infancy (SUDI).

Several risk factors have been identified, including sociodemographics, parental smoking and/or substance abuse, insufficient maternal education, multiple pregnancies, low birth weight, lack of breastfeeding, male gender, recent infections, sleeping position and – environmentand genetics, [3,6,9-14]. More SUDI cases also occur during the colder months of the year, suggesting a seasonal aetiology [15-17].

Acute respiratory infections occur often in infants and can sometimes be fatal [18-21]. Young children can have up to eight respiratory infections annually and although this rate is comparable throughout the world, a much worse prognosis is found in areas such as rural Africa, where patients suffer from poor health, malnutrition, high incidences of prematurity, anaemia and other co-morbidities, including malaria and human immune deficiency virus [19,22].

Pulmonary viral infections are the most frequently defined cause of death in SUDI cases worldwide [17,23,24].Both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) viruses have been detected in these cases, including enterovirus, human adenovirus, Epstein-Barr virus, parvovirus B19, human herpes simplex virus, cytomegalovirus (CMV), human rhinovirus, influenza virus, respiratory syncytial virus, human parainfluenza virus and rotavirus [14,15,25-27]. However, similarviruses often cause mild illness or self-limiting infections in living babies and infants in the general population [28,29]. Positive results should therefore be interpreted with caution and correlated with microand macroscopic autopsy findings [18]


Different diagnostic approaches for investigating viral respiratory pathogens in SUDI cases have been reported in the literature, such as histology, cell cultures, immunohistochemistry (IHC), in-situ hybridisation (ISH), electron microscopy (EM), immunofluorescence (IF), quantitative and qualitative realtime polymerase chain reaction (PCR) [25,27,30,31]. The latest technology is luminex, which have similar or superior sensitivity compared to direct IF, culture and PCR methods [32].

Obtaining viable lung tissue from SUDI cases for diagnostic purposes has proven to be challenging. Low viral load or extended post-mortem intervals may negatively affect virus viability and nucleic acid integrity, resulting in reduced viral detection [33]. Routine samples are usually formalin-fixed after the autopsy, which can also compromise the quality and integrity of nucleic acids present in the tissue [27]. Autolysis and putrefaction start shortly after death and can lead to overgrowth of post-mortem organisms, such as α-haemolytic streptococci, Staphylococcus epidermidis and Escherichia coli,seriously hampering viral detection [34].

The key qualities required for any method used for viral detection are sensitivity and specificity or selectivity. Sensitivity represents the limit of detection of the assay and is particularly important in early detection to aid in conclusive diagnosis and patient management. Specificity or selectivity refers to the measure of accurate determination of a particular virus in the presence of other target organisms [35]. The difference between specificity and selectivity is that a specific reaction will only occur with the substance of interest, while a selective reaction can occur with other substances, but shows a degree of preference for the substance of interest [36].


Standard haematoxylin and eosin stained sections are provide information about trafficking and localisation of inflammatory cells, as well as the extent of necrosis and apoptosis [37]. Thecell nucleus typically stains with haematoxylin, while the cytoplasm absorbs the eosin. Although it is possible to visualise significant changes in the respiratory tract due to infection [38], it is not possible to distinguish between different viruses [18].

The most important histological finding in SUDI cases is interstitial pneumonitis, which can be classified as Grade 1 or very mild (barely present, often focal, and with no overall alteration of the alveolar septa), Grade 2 or mild (relatively diffuse and mild thickening of alveolar septa), Grade 3 or moderate (diffuse involvement and associated with interstitial oedema) or Grade 4 or severe (diffuse infiltration associated with significantly widened alveolar septa) [39].

Histological changes can include oedema, congestion, focal collapse, alveolar debris or haemorrhage, bronchiolitis, bronchopneumonia and pneumonitis. These changes, although suggestive of a viral infection, may also be non-specific and should be supplemented with additional diagnostic tests [18,30].

Cell culture methods

The use of cultured eukaryotic cells in a controlled environment for propagation and isolation of different viruses has been used since the 1960s and is still considered the gold standard for the detection of respiratory viruses [14,28,35,40,41]. Most of the emerging techniques for viral detection are validated against the cell culture technique. Primary (human fibroblast, Rhesus monkey kidney, etc) and continuous cell lines (human lung carcinoma, etc) have the ability to support the replication of a wide variety of clinically relevant viruses. Specimens are inoculated onto these cell monolayers and then monitored by light microscopy for cytopathic effect, which is indicative of viral growth or a positive culture. The presence of a specific virus must then be confirmed by immunofluorescent staining using virusspecific, fluorescently-labelled antibodies. This method is able to demonstrate the viability of a virus, but can take eight to ten days for sufficient viral growth, which limits the diagnostic value of such methods. Modified, centrifugation-enhanced shell-vial cultures can however, greatly shorten the incubation period necessary for successful culture. A cell monolayer is grown on a coverslip in a small vial into which the test specimen is inoculated and incubated. This method is quicker and cheaper than conventional cell cultures, but generally still as sensitive [42]. However, in selected cases such as CMV, it is less sensitive than conventional cell cultures [43,44].

The use of mixed cell lines, such as a combination of Hep-2 and Madin-Darby Canine Kidney cell lines, can further contribute to a shorter turn-around-time [41,45].

The biggest limitation in using this method for investigation of respiratory viruses in SUDI cases is the time delay between death and post mortem examination, where degradation of possible viral particles can render the virus unable to replicate, resulting in false negative results.


This method utilises a monoclonal or polyclonal antibody that binds with high affinity to its corresponding antigen. It is then incubated with a conjugated secondary antibody that will bind to the Fc portion of the primary antibody and when the chromogen is added, it will deposit at sites where the primary antibody is bound and can bevisualised under a light microscope. The diagnosis is based on the distribution of inclusion bodies in tissue and cells, tissue structure and presence of inflammation and other structural changes [26,37,46].

IHC can be used on fresh and unfixed tissue, but formalin-fixed, paraffin-embedded tissue is most commonly used. However, fixation masks the antigenicity of the tissue by reversible crosslinkages of protein amino acid residues by methylene bridges, rendering the antigen invisible and necessitating an antigen retrievalstep. The amount of cross-linking can be controlled by the fixation time, pH or concentration of the fixative.The antigen retrieval step breaks these cross-linkages and can be done with heat (heat-induced epitope retrieval) and enzyme-based methods, radiation or boiling the tissue sections [47-49].

IHC is an expensive method and even though it can confirm the presence of specific antigen in the tissue, it cannot quantify the infection [50]. False negative results occur frequently, possibly due to subjective interpretation. Differentiation between background staining and true positive results is often difficult [51].

In situ hybridisation

ISHuses a non-radioactive RNA probe to detect mRNA in paraffin-embedded tissue sections. A digoxenin-labelled probed is used to hybridise specific sequences of single-stranded celland tissue-bound RNA and DNA with single-stranded labelled probes of complementary sequences. Because every infectious organism has unique segments of DNA or RNA that are not found in other organisms, cells, or tissues, this method can localise single-copy genes and mRNA transcripts in samples with very low number of cells [52].

Both IHC and ISHhybridisation use paraffin-embedded tissue to demonstrate the presence of specific organisms. IHC is the method of choice in active infections where active viral replication is present, whereas ISH is more suitable in cases of latent infections, such as CMV and human herpesviruses. Both methods are useful in epidemiological studies, as the paraffinembedded tissue maintain their structural characteristics for long periods of time. IHC is relatively quick and inexpensive as opposed to other techniques. The main disadvantages are the difficulty to distinguish between background and specific staining and despite high sensitivity and specificity, non-specific binding to tissue components such as neurons, glandular epithelium and collagen can occur [52].

Electron microscopy

Morphological differences between viruses are visible under an electron microscope and a number of new viruses have been described with the aid of EM, including Hendra and Nipah (Henipa) viruses. It does not need any target specific reagents, but instead allows pathogens to be distinguished on morphology and ultra structure. It is possible to view the presence of all viruses, provided that the viral load is high enough.Due to a low sensitivity, it needs approximately 1 million viral particles per millilitre of fluid for a positive result and low positives often are regarded as negative. This is the main reason that EM has limited diagnostic applications [53,54].

EM has been used in the investigation of SUDI cases to successfully detect viral infections, although at a much lower rate than other diagnostic tests [14,30]. Autolysis of cells starts shortly after death and subsequently results in post-mortem tissue that is not always suitable for EM purposes [30].


IFhas often been used in SUDI investigations [14,28,55]. It is a cytochemical or histochemical assay that is used to detect antigen on the surface of intact cells, inside cells or in serum or plasma. An antibody is coupled to a fluorescent dye and when the antibody binds to an antigen, fluorescence can be seen under a fluorescent microscope[56].

Direct IF involves the overlay of fluorescein-conjugated antibodies against immunoglobulins (IgG, IgM and IgA) and complement and is done on tissue sections. Indirect IFuses an unlabelled primary antibody which is specific for the antigen, but then needs a secondary, fluorescently labelled, antibody specific for the primary antibody[56,57].

Direct IF is a faster assay than indirect IF, but often produces a lower signal. Indirect IF is more sensitive than direct IF, because more than one secondary antibody can bind to each primary antibody, which will strengthen the fluorescent signal, but includes the potential for cross-reactivity [57].

Direct fluorescent antibody tests can be performed in an hour and can detect approximately eight of the common respiratory viruses. The specificity of this test is high, but the sensitivity depends on the specific virus or strain and can be as low as 50% in the case of Adenovirus [58].

Conventional and Real-time PCR PCR

or nucleic acid amplification tests have superior diagnostic sensitivity and specificity for detection of respiratory viruses compared to viral culture techniques and are gaining ground to become the new, more sensitive gold standard [58]. Ituses the principle of amplifying target DNA or RNA and detecting the product by means of gel-electrophoresis at the end of the assay or a fluorescent dye, which is cleaved off when the probe binds to the target sequence (conventional PCR). This omits a signal after being cleaved and can be visualised in real-time (real-time PCR). Real-time PCR methods allow single step amplification and analysis of viral targets and although multiplex PCR methods can detect larger range of viruses in a single reaction, it requires an additional step for post-amplification detection [32,59,60].

These methods are useful in detecting highly pathogenic organisms, as well as viruses that do not proliferate in normal cell cultures, such as Hepatitis B and C, and parvovirus B-19. It is also a much faster method than conventional culture methods [14,22,25,26,41,61].

PCR tests are very expensive and laborious and are limited to the amount of fluorophores that can successfully be differentiated [32,59]. It is also not able to differentiate between active and latent viral infections, unless a quantitative viral load test is performed [41].Because of the latent period and periodic reactivation of human herpes viruses such as CMV, a positive PCR result might be the result of a primary infection, a latent virus or a reactivated viral infection [62]. For some other viruses, such as the human rhinovirus, prolonged shedding can last up to three weeks after the initial infection [63,64]. It is thus imperative to interpret positive results together with the clinical history of the patient and evaluation of viral specific immune responses [27,30,65,66].

Although formalin-fixed samples have been used to detect viruses with PCR, significant degradation of RNA and DNA occurs in formalin-fixed cells compared to fresh cells and cells fixed in formalin-free media. Formalin fixation can thus cause fragmentation of RNA and DNA, reducing the nucleic acids suitable for molecular amplification and fresh tissue should be collected and processed for PCR [67,68].


This is a microsphere immune assay based on a universal beadarray. The beads are carboxylated paramagnetic polystyrene, 6.5 µm in diameter and are stained internally with spectrally distinct fluorochromes. Covalent coupling with either proteins, peptides, antibodies, polysaccharides, lipids or oligonucleotides determines the specific application of this method [69].

The viral application is a qualitative nucleic acid multiplex test and can simultaneous detect and identify multiple DNA and RNA viruses in a single reaction. Primers with proprietary universal tags enable a reverse transcriptase PCR step, after which the amplified product is hybridised to a bead array conjugated to specific probes. Streptavidin-R-phycoerythrin conjugate generates a signal for each bead population and enables identification in combination with the virus-specific bead address[32,69].

Similar to Real-time nucleic acid tests, Luminex assays are also superior in terms of sensitivity and turnaround time compared to viral culture methods and its sensitivity has been shown to be comparable to real-time PCR. This method has the potential to accurately detect a wide range of respiratory viruses in a short time in a single reaction[32,59]


This is by no means an exhaustive list of diagnostic modalities available for respiratory viruses.Additional technologies for viral detection are being developed and refined continuously and include, but are not limited to:

Microarrays: uses hybridisation of a nucleic acid sample (target) to a very large set of oligonucleotide probes, which are attached to a solid support, to determine sequence or to detect variations in a gene sequence or expression or for gene mapping [69].

ELISA: forms complexes between viral antigen and antibodies, that requires a conjugated secondary antibody and substrate to produce a colour change, which can be spectrophotometrically measured at a specific wavelength [69].

Isothermal methods: helicase dependent amplification or recombinase polymerase amplification is used to separate the strands of the double-stranded template to enable primer binding, such that amplification can occur without the repeat cycles of denaturation and annealing required for PCR [69].

Loop-mediated isothermal amplification or LAMP assay: uses three pairs of primers (internal, external and loop primers) to provide at least six primer binding sites, which accelerate amplification by priming at the loop regions [69].

Next generation sequencing: applying massively parallel sequencing approaches with subsequent bioinformatics analysis for viral sequences can lead to routine, as well as generic detection of viruses and other pathogens [69].

Microarrays: an array of immobilised oligonucleotides containing the genetic information of the virus of interest is screened against a fluorescently labelled PCR product to identify and quantify specific DNA sequences [35].


SUDI is still the leading cause of death in infants in many developed and developing countries and although infection has been implicated in many studies, the exact cause and mechanisms of death in many cases remain unknown. There is consensus in the literature that the cause of death is probably multifactorial, but the contributing of underlying role of viral and microbial infection cannot be ignored [70].

Detection of viral pathogens in SUDI cases poses unique challenges. In the absence of standardised protocols with regard to the diagnostic tests to be performed or the selection of viruses to be investigated, research from different centres is not comparable. Viral viability is compromised in postmortem samples and limits the value of quantitative analyses. However, qualitative results should be interpreted with care, as the mere presence of a pathogen does not imply that it caused or contributed to death. It is therefore imperative to use a combination of diagnostic approaches in parallel with epidemiological and clinical information in SUDI cases.


1. Reidpath DD, Allotey P. Infant mortality rate as an indicator of population health. J Epidemiol Community Health. 2003; 57: 344-346.

2. Goutas N, Konstantinidou MK, Vlachodimitropoulos D, Konstantinidou A, Kontogiannis T, Papadodima T,et al. Trends in infant and child mortality. Open Forensic Sci J. 2011; 4: 1-11.

3. Spencer N, Logan S. Sudden unexpected death in infancy and socioeconomic status: a systematic review. J Epidemiol Community Health. 2004; 58: 366-373.

4. Moon RY, Horne RS, Hauck FR. Sudden infant death syndrome. Lancet. 2007; 370: 1578-1587.

5. Hauck FR, Tanabe KO. International trends in sudden infant death syndrome: stabilization of rates requires further action. Pediatrics. 2008; 122: 660-666. 6. Athanasakis E, Karavasiliadou S, Styliadis I. The factors contributing to the risk of sudden infant death syndrome. Hippokratia. 2011; 15: 127-131. 

7. Willinger M, James LS, Catz C. Defining the sudden infant death syndrome (SIDS): deliberations of an expert panel convened by the National Institute of Child Health and Human Development. Pediatr Pathol. 1991; 11: 677-684.

8. Beckwith JB. Defining the sudden infant death syndrome. Arch Pediatr Adolesc Med. 2003; 157: 286-290.

9. Byard RW1, Krous HF. Sudden infant death syndrome: overview and update. Pediatr Dev Pathol. 2003; 6: 112-127.

10. Samuels M. Viruses and sudden infant death. Paediatr Respir Rev. 2003; 4: 178-183.

11. Vennemann M, Bajanowski T, Butterfass-Bahloul T, Sauerland C, Jorch G, Brinkmann B, et al. Do risk factors differ between explained sudden unexpected death in infancy and sudden infant death syndrome? Arch Dis Child. 2007; 92: 133-136.

12. Highet AR. An infectious aetiology of sudden infant death syndrome. J Appl Microbiol. 2008; 105: 625-635.

13. Adams SM, Good MW, Defranco GM. Sudden infant death syndrome. Am Fam Physician. 2009; 79: 870-874.

14. Weber MA, Hartley JC, Ashworth MT, Malone M, Sebire NJ. Virological investigations in sudden unexpected deaths in infancy (SUDI). Forensic Sci Med Pathol. 2010; 6: 261-267.

15. An SF, Gould S, Keeling JW, Fleming KA. Role of respiratory viral infection in SIDS: detection of viral nucleic acid by in situ hybridization. J Pathol. 1993; 171: 271-278.

16. du Toit-Prinsloo L, Dempers JJ, Wadee SA, Saayman G. The medicolegal investigation of sudden, unexpected and/or unexplained infant deaths in South Africa: where are we--and where are we going? Forensic Sci Med Pathol. 2011; 7: 14-20.

17. du Toit-Prinsloo L, Dempers J, Verster J, Hattingh C, Nel H, Brandt VD, et al. Toward a standardized investigation protocol in sudden unexpected deaths in infancy in South Africa: a multicenter study of medico-legal investigation procedures and outcomes. Forensic Sci Med Pathol. 2013; 9: 344-50.

18. Bajanowski T, Vege A, Byard RW, Krous HF, Arnestad M, Bachs L,et al. Sudden infant death syndrome (SIDS)--standardised investigations and classification: recommendations. Forensic Sci Int. 2007; 165: 129- 143.

19. O’Callaghan-Gordo C, Díez-Padrisa N, Abacassamo F, Pérez-Breña P, Casas I, Alonso PL, et al. Viral acute respiratory infections among infants visited in a rural hospital of southern Mozambique. Trop Med Int Health 2011; 16: 1054-1060.

20. Ghani AS, Morrow BM, Hardie DR, Argent AC. An investigation into the prevalence and outcome of patients admitted to a pediatric intensive care unit with viral respiratory tract infections in Cape Town, South Africa. Pediatr Crit Care Med. 2012; 13: 275-281.

21. Kwofie TB, Anane YA, Nkrumah B, Annan A, Nguah SB, Owusu M. Respiratory viruses in children hospitalized for acute lower respiratory tract infection in Ghana. Virol J. 2012; 9: 78.

22. Tregoning JS, Schwarze J. Respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology. Clin Microbiol Rev. 2010; 23: 74-98.

23. Weber MA, Ashworth MT, Risdon RA, Hartley JC, Malone M, Sebire NJ. The role of post-mortem investigations in determining the cause of sudden unexpected death in infancy. Arch Dis Child. 2008; 93: 1048- 1053.

24. Côté A. Investigating sudden unexpected death in infancy and early childhood. Paediatr Respir Rev. 2010; 11: 219-225.

25. Bajanowski T, Rolf B, Jorch G, Brinkmann B. Detection of RNA viruses in sudden infant death (SID). Int J Legal Med. 2003; 117: 237-240.

26. Dettmeyer R, Baasner A, Schlamann M, Padosch SA, Haag C, Kandolf R, et al. Role of virus-induced myocardial affections in sudden infant death syndrome: a prospective postmortem study. Pediatr Res. 2004; 55: 947-952.

27. Alvarez-Lafuente R, Aguilera B, Suárez-Mier MA, Morentin B, Vallejo G, Gómez J, et al. Detection of human herpesvirus-6, Epstein-Barr virus and cytomegalovirus in formalin-fixed tissues from sudden infant death: a study with quantitative real-time PCR. Forensic Sci Int. 2008; 178: 106-111.

28. Urquhart GE, Grist NR. Virological studies of sudden, unexplained infant deaths in Glasgow 1967-70. J Clin Pathol. 1972; 25: 443-446.

29. Williams AL, Uren EC, Bretherton L. Respiratory viruses and sudden infant death. Br Med J. 1984; 288: 1491-1493.

30. Fernández-Rodríguez A, Ballesteros S, de Ory F, Echevarría JE, AlvarezLafuente R, Vallejo G, et al. Virological analysis in the diagnosis of sudden children death: a medico-legal approach. Forensic Sci Int. 2006; 161: 8-14.

31. Dettmeyer R, Sperhake JP, Muller J, Madea B. Cytomegalovirus-induced pneumonia and myocarditis in three cases of suspected sudden infant death syndrome (SIDS): Diagnosis by immunohistochemical techniques and molecular pathologic methods. Forensic Sci Int 2008; 174: 229-233.

32. Pabbaraju K, Tokaryk KL, Wong S, Fox JD. Comparison of the Luminex xTAG respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections. J Clin Microbiol. 2008; 46: 3056-3062.

33. Weber MA, Hartley JC, Brooke I, Lock PE, Klein NJ, Malone M, et al. Post-mortem interval and bacteriological culture yield in sudden unexpected death in infancy (SUDI). Forensic Sci Int. 2010; 198: 121- 125.

34. Prtak L, Al-Adnani M, Fenton P, Kudesia G, Cohen MC. Contribution of bacteriology and virology in sudden unexpected death in infancy. Arch Dis Child. 2010; 95: 371-376.

35. Cella LN, Blackstock D, Yates MA, Mulchandani A, Chen W. Detection of RNA viruses: current technologies and future perspectives. Crit Rev Eukaryot Gene Expr. 2013; 23: 125-137.

36. Vessman J. Selectivity or specificity? Validation of analytical methods from the perspective of an analytical chemist in the pharmaceutical industry. J Pharm Biomed Anal. 1996; 14: 867-869.

37. Flaño E, Jewell NA, Durbin RK, Durbin JE. Methods used to study respiratory virus infection. Curr Prot Cell Biol 2009; 26.3.1–26.3.28.

38. Wittekind D. Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain. Biotech Histochem. 2003; 78: 261-270.

39. Krous HF, Nadeau JM, Silva PD, Blackbourne BD. A comparison of respiratory symptoms and inflammation in sudden infant death syndrome and in accidental or inflicted infant death. Am J Forensic Med Pathol. 2003; 24: 1-8.

40. Ieven M. Currently used nucleic acid amplification tests for the detection of viruses and atypicals in acute respiratory infections. J Clin Virol. 2007; 40: 259-276.

41. Leland DS, Ginocchio CC. Role of cell culture for virus detection in the age of technology. Clin Microbiol Rev. 2007; 20: 49-78..

42. McAdam AJ, Riley AM. Developments in tissue culture detection of respiratory viruses. Clin Lab Med. 2009; 29: 623-634.

43. Mazzulli T, Rubin RH, Ferraro MJ, D’Aquila RT, Doveikis SA, Smith BR, et al. Cytomegalovirus antigenemia: clinical correlations in transplant recipients and in persons with AIDS. J Clin Microbiol. 1993; 31: 2824- 2827.

44. Pedneault L, Anglow M, Alfieri C, Rubin E.Diagnosis of cytomegalovirus (CMV) infection in pediatric transplant patients by the antigenemia, shell vial, and conventional culture assays performed on blood: Correlation with CMV disease. Clin Diagn Virol 1996; 6: 51-61.

45. Mahony JB. Detection of respiratory viruses by molecular methods. Clin Microbiol Rev. 2008; 21: 716-747.

46. Linnoila I, Petrusz P. Immunohistochemical techniques and their applications in the histopathology of the respiratory system. Environ Health Perspect. 1984; 56: 131-148.

47. Shi SR, Cote RJ, Taylor CR. Antigen retrieval techniques: current perspectives. J Histochem Cytochem. 2001; 49: 931-937.

48. Sompuram SR, Vani K, Messana E, Bogen SA. A molecular mechanism of formalin fixation and antigen retrieval. Am J Clin Pathol. 2004; 121: 190-199.

49. D’Amico F, Skarmoutsou E, Stivala F. State of the art in antigen retrieval for immunohistochemistry. J Immunol Methods. 2009; 341: 1-18.

50. Goyal A, Martin TA, Mansel RE, Jiang WG. Real time PCR analyses of expression of E-cadherin, alpha-, beta- and gamma-catenin in human breast cancer for predicting clinical outcome. World J Surg Oncol. 2008; 6: 56.

51. Leake R. Detection of the oestrogen receptor (ER). immunohistochemical versus cytosol measurements. Eur J Cancer. 2000; 36 Suppl 4: S18-19.

52. Segalés J, Ramos-Vara JA, Duran CO,Porter A, Domingo M. Diagnosing infectious diseases using in situ hybridization. Swine Health Prod 1999; s7: 125–128.

53. Goldsmith CS, Miller SE. Modern uses of electron microscopy for detection of viruses. Clin Microbiol Rev. 2009; 22: 552-563.

54. Jeffery K, Aarons E. Diagnostic Approaches. In: Zuckerman AJ, Banatvala JE, Griffiths P et al. Eds. Principles and Practice of Clinical Virology. Chichester, West Sussex Hoboken, NJ: John Wiley & Sons 2009;1-27.

55. Zink P, Drescher J, Verhagen W, Flik J, Milbradt H. Serological evidence of recent influenza virus A (H 3 N 2) infections in forensic cases of the sudden infant death syndrome (SIDS). Arch Virol. 1987; 93: 223-232.

56. Odell ID, Cook D. Immunofluorescence techniques. J Invest Dermatol 2013;133: e4. doi: 10.1038/jid;2012: 455.

57. Robinson JP, Sturgis J, Kumar GL. Immunofluorescence. In: Kumar GL, Rudbeck L (Eds). IHC Staining Methods. 5th Edition. Dako North America, Carpinteria, California. 2009; 61-65.

58. Ginocchio CC, McAdam AJ. Current best practices for respiratory virus testing. J Clin Microbiol 2011; 49: S44-S48.

59. Gadsby NJ, Hardie A, Claas EC, Templeton KE. Comparison of the Luminex Respiratory Virus Panel fast assay with in-house real-time PCR for respiratory viral infection diagnosis. J Clin Microbiol. 2010; 48: 2213-2216.

60. Loeffelholz M, Chonmaitree T. Advances in diagnosis of respiratory virus infections. Int J Microbiol; 2010: 126049.

61. Lee JH, Chun JK, Kim DS, Park Y, Choi JR, Kim HS. Identification of adenovirus, influenza virus, parainfluenza virus, and respiratory syncytial virus by two kinds of multiplex polymerase chain reaction (PCR) and a shell vial culture in pediatric patients with viral pneumonia. Yonsei Med J. 2010; 51: 761-767.

62. Bhatia P, Narang A, Minz RW. Neonatal cytomegalovirus infection: diagnostic modalities available for early disease detection. Indian J Pediatr. 2010; 77: 77-79.

63. Hendley JO, Wenzel RP, Gwaltney JM Jr. Transmission of rhinovirus colds by self-inoculation. N Engl J Med. 1973; 288: 1361-1364.

64. Gwaltney JM Jr. Rhinovirus infection of the normal human airway. Am J Respir Crit Care Med. 1995; 152: 36-39.

65. Blood-Siegfried J. Sudden infant death syndrome: a toxic response. AACN Clin Issues. 2000; 11: 300-308.

66. Howatson AG. The autopsy for sudden unexpected death in infancy. Curr Diagn Pathol 2006;12: 173–183.

67. Gazziero A, Guzzardo V, Aldighieri E, Fassina A. Morphological quality and nucleic acid preservation in cytopathology. J Clin Pathol. 2009; 62: 429-434.

68. Desmons A, Terrade C, Boulagnon C, Giusti D, Nguyen Y, Andreoletti L, etal. Post-mortem diagnosis, of cytomegalovirus and varicella zoster virus co-infection by combined histology and tissue molecular biology, in a sudden unexplained infant death. J Clin Virol. 2013; 58: 486-489.

69. Boonham N, Kreuze J, Winter S, van der Vlugt R, Bergervoet J, et al. Methods in virus diagnostics: from ELISA to next generation sequencing. Virus Res. 2014; 186: 20-31.

70. Weber MA, Klein NJ, Hartley JC, Lock PE, Malone M, Sebire NJ. Infection and sudden unexpected death in infancy: a systematic retrospective case review. Lancet. 2008; 371: 1848-1853.

de Beer C, la Grange H (2014) Diagnostic Options for Investigating Viral Respiratory Pathogens in Sudden Unexpected Death in Infancy (SUDI) Cases. Ann Forensic Res Anal 1(2): 1007.

Received : 24 Jul 2014
Accepted : 28 Sep 2014
Published : 30 Sep 2014
Annals of Otolaryngology and Rhinology
ISSN : 2379-948X
Launched : 2014
JSM Schizophrenia
Launched : 2016
Journal of Nausea
Launched : 2020
JSM Internal Medicine
Launched : 2016
JSM Hepatitis
Launched : 2016
JSM Oro Facial Surgeries
ISSN : 2578-3211
Launched : 2016
Journal of Human Nutrition and Food Science
ISSN : 2333-6706
Launched : 2013
JSM Regenerative Medicine and Bioengineering
ISSN : 2379-0490
Launched : 2013
JSM Spine
ISSN : 2578-3181
Launched : 2016
Archives of Palliative Care
ISSN : 2573-1165
Launched : 2016
JSM Nutritional Disorders
ISSN : 2578-3203
Launched : 2017
Annals of Neurodegenerative Disorders
ISSN : 2476-2032
Launched : 2016
Journal of Fever
ISSN : 2641-7782
Launched : 2017
JSM Bone Marrow Research
ISSN : 2578-3351
Launched : 2016
JSM Mathematics and Statistics
ISSN : 2578-3173
Launched : 2014
Journal of Autoimmunity and Research
ISSN : 2573-1173
Launched : 2014
JSM Arthritis
ISSN : 2475-9155
Launched : 2016
JSM Head and Neck Cancer-Cases and Reviews
ISSN : 2573-1610
Launched : 2016
JSM General Surgery Cases and Images
ISSN : 2573-1564
Launched : 2016
JSM Anatomy and Physiology
ISSN : 2573-1262
Launched : 2016
JSM Dental Surgery
ISSN : 2573-1548
Launched : 2016
Annals of Emergency Surgery
ISSN : 2573-1017
Launched : 2016
Annals of Mens Health and Wellness
ISSN : 2641-7707
Launched : 2017
Journal of Preventive Medicine and Health Care
ISSN : 2576-0084
Launched : 2018
Journal of Chronic Diseases and Management
ISSN : 2573-1300
Launched : 2016
Annals of Vaccines and Immunization
ISSN : 2378-9379
Launched : 2014
JSM Heart Surgery Cases and Images
ISSN : 2578-3157
Launched : 2016
Annals of Reproductive Medicine and Treatment
ISSN : 2573-1092
Launched : 2016
JSM Brain Science
ISSN : 2573-1289
Launched : 2016
JSM Biomarkers
ISSN : 2578-3815
Launched : 2014
JSM Biology
ISSN : 2475-9392
Launched : 2016
Archives of Stem Cell and Research
ISSN : 2578-3580
Launched : 2014
Annals of Clinical and Medical Microbiology
ISSN : 2578-3629
Launched : 2014
JSM Pediatric Surgery
ISSN : 2578-3149
Launched : 2017
Journal of Memory Disorder and Rehabilitation
ISSN : 2578-319X
Launched : 2016
JSM Tropical Medicine and Research
ISSN : 2578-3165
Launched : 2016
JSM Head and Face Medicine
ISSN : 2578-3793
Launched : 2016
JSM Cardiothoracic Surgery
ISSN : 2573-1297
Launched : 2016
JSM Bone and Joint Diseases
ISSN : 2578-3351
Launched : 2017
JSM Bioavailability and Bioequivalence
ISSN : 2641-7812
Launched : 2017
JSM Atherosclerosis
ISSN : 2573-1270
Launched : 2016
Journal of Genitourinary Disorders
ISSN : 2641-7790
Launched : 2017
Journal of Fractures and Sprains
ISSN : 2578-3831
Launched : 2016
Journal of Autism and Epilepsy
ISSN : 2641-7774
Launched : 2016
Annals of Marine Biology and Research
ISSN : 2573-105X
Launched : 2014
JSM Health Education & Primary Health Care
ISSN : 2578-3777
Launched : 2016
JSM Communication Disorders
ISSN : 2578-3807
Launched : 2016
Annals of Musculoskeletal Disorders
ISSN : 2578-3599
Launched : 2016
Annals of Virology and Research
ISSN : 2573-1122
Launched : 2014
JSM Renal Medicine
ISSN : 2573-1637
Launched : 2016
Journal of Muscle Health
ISSN : 2578-3823
Launched : 2016
JSM Genetics and Genomics
ISSN : 2334-1823
Launched : 2013
JSM Anxiety and Depression
ISSN : 2475-9139
Launched : 2016
Clinical Journal of Heart Diseases
ISSN : 2641-7766
Launched : 2016
Annals of Medicinal Chemistry and Research
ISSN : 2378-9336
Launched : 2014
JSM Pain and Management
ISSN : 2578-3378
Launched : 2016
JSM Women's Health
ISSN : 2578-3696
Launched : 2016
Clinical Research in HIV or AIDS
ISSN : 2374-0094
Launched : 2013
Journal of Endocrinology, Diabetes and Obesity
ISSN : 2333-6692
Launched : 2013
Journal of Substance Abuse and Alcoholism
ISSN : 2373-9363
Launched : 2013
JSM Neurosurgery and Spine
ISSN : 2373-9479
Launched : 2013
Journal of Liver and Clinical Research
ISSN : 2379-0830
Launched : 2014
Journal of Drug Design and Research
ISSN : 2379-089X
Launched : 2014
JSM Clinical Oncology and Research
ISSN : 2373-938X
Launched : 2013
JSM Bioinformatics, Genomics and Proteomics
ISSN : 2576-1102
Launched : 2014
JSM Chemistry
ISSN : 2334-1831
Launched : 2013
Journal of Trauma and Care
ISSN : 2573-1246
Launched : 2014
JSM Surgical Oncology and Research
ISSN : 2578-3688
Launched : 2016
Annals of Food Processing and Preservation
ISSN : 2573-1033
Launched : 2016
Journal of Radiology and Radiation Therapy
ISSN : 2333-7095
Launched : 2013
JSM Physical Medicine and Rehabilitation
ISSN : 2578-3572
Launched : 2016
Annals of Clinical Pathology
ISSN : 2373-9282
Launched : 2013
Annals of Cardiovascular Diseases
ISSN : 2641-7731
Launched : 2016
Journal of Behavior
ISSN : 2576-0076
Launched : 2016
Annals of Clinical and Experimental Metabolism
ISSN : 2572-2492
Launched : 2016
Clinical Research in Infectious Diseases
ISSN : 2379-0636
Launched : 2013
JSM Microbiology
ISSN : 2333-6455
Launched : 2013
Journal of Urology and Research
ISSN : 2379-951X
Launched : 2014
Journal of Family Medicine and Community Health
ISSN : 2379-0547
Launched : 2013
Annals of Pregnancy and Care
ISSN : 2578-336X
Launched : 2017
JSM Cell and Developmental Biology
ISSN : 2379-061X
Launched : 2013
Annals of Aquaculture and Research
ISSN : 2379-0881
Launched : 2014
Clinical Research in Pulmonology
ISSN : 2333-6625
Launched : 2013
Journal of Immunology and Clinical Research
ISSN : 2333-6714
Launched : 2013
JSM Biochemistry and Molecular Biology
ISSN : 2333-7109
Launched : 2013
Annals of Breast Cancer Research
ISSN : 2641-7685
Launched : 2016
Annals of Gerontology and Geriatric Research
ISSN : 2378-9409
Launched : 2014
Journal of Sleep Medicine and Disorders
ISSN : 2379-0822
Launched : 2014
JSM Burns and Trauma
ISSN : 2475-9406
Launched : 2016
Chemical Engineering and Process Techniques
ISSN : 2333-6633
Launched : 2013
Annals of Clinical Cytology and Pathology
ISSN : 2475-9430
Launched : 2014
JSM Allergy and Asthma
ISSN : 2573-1254
Launched : 2016
Journal of Neurological Disorders and Stroke
ISSN : 2334-2307
Launched : 2013
Annals of Sports Medicine and Research
ISSN : 2379-0571
Launched : 2014
JSM Sexual Medicine
ISSN : 2578-3718
Launched : 2016
Annals of Vascular Medicine and Research
ISSN : 2378-9344
Launched : 2014
JSM Biotechnology and Biomedical Engineering
ISSN : 2333-7117
Launched : 2013
Journal of Hematology and Transfusion
ISSN : 2333-6684
Launched : 2013
JSM Environmental Science and Ecology
ISSN : 2333-7141
Launched : 2013
Journal of Cardiology and Clinical Research
ISSN : 2333-6676
Launched : 2013
JSM Nanotechnology and Nanomedicine
ISSN : 2334-1815
Launched : 2013
Journal of Ear, Nose and Throat Disorders
ISSN : 2475-9473
Launched : 2016
JSM Ophthalmology
ISSN : 2333-6447
Launched : 2013
Journal of Pharmacology and Clinical Toxicology
ISSN : 2333-7079
Launched : 2013
Annals of Psychiatry and Mental Health
ISSN : 2374-0124
Launched : 2013
Medical Journal of Obstetrics and Gynecology
ISSN : 2333-6439
Launched : 2013
Annals of Pediatrics and Child Health
ISSN : 2373-9312
Launched : 2013
JSM Clinical Pharmaceutics
ISSN : 2379-9498
Launched : 2014
JSM Foot and Ankle
ISSN : 2475-9112
Launched : 2016
JSM Alzheimer's Disease and Related Dementia
ISSN : 2378-9565
Launched : 2014
Journal of Addiction Medicine and Therapy
ISSN : 2333-665X
Launched : 2013
Journal of Veterinary Medicine and Research
ISSN : 2378-931X
Launched : 2013
Annals of Public Health and Research
ISSN : 2378-9328
Launched : 2014
Annals of Orthopedics and Rheumatology
ISSN : 2373-9290
Launched : 2013
Journal of Clinical Nephrology and Research
ISSN : 2379-0652
Launched : 2014
Annals of Community Medicine and Practice
ISSN : 2475-9465
Launched : 2014
Annals of Biometrics and Biostatistics
ISSN : 2374-0116
Launched : 2013
JSM Clinical Case Reports
ISSN : 2373-9819
Launched : 2013
Journal of Cancer Biology and Research
ISSN : 2373-9436
Launched : 2013
Journal of Surgery and Transplantation Science
ISSN : 2379-0911
Launched : 2013
Journal of Dermatology and Clinical Research
ISSN : 2373-9371
Launched : 2013
JSM Gastroenterology and Hepatology
ISSN : 2373-9487
Launched : 2013
Annals of Nursing and Practice
ISSN : 2379-9501
Launched : 2014
JSM Dentistry
ISSN : 2333-7133
Launched : 2013
Author Information X