With the recent increase in dengue outbreaks in various parts of the world, DENV infection continues to be a major public health problem. According to a WHO report, an estimated 500,000 people with DHF require hospitalization each year and about 2.5% of those affected die, a very large proportion of whom are children.6
Fatalities can be higher in some countries caused by inadequate disease management facilities and without proper treatment DHF fatality rates can exceed 20%.6
In addition, fatality rates reported in hospitalized patients can reach up to 50–60% in dengue patients with complications (acute renal failure, fulminant hepatitis, liver failure, and encephalopathy).32–34
The increased prevalence of dengue infections worldwide in recent decades and the high mortality caused by DHF and DSS highlights the need for more sensitive and specific diagnostic assays such as RT-PCR for the detection and typing of DENV in fatal cases. Diagnosis of DENV infection in fatal cases often can be challenging because of the unavailability of serum and fresh or frozen specimens. Furthermore, in patients who die during the first week of illness, serology may have limited use because of low levels of IgM antibodies that cannot be detected by serological assays.35,36
On the other hand, in the early viremic stage of DENV infections patients may have higher viral loads.37–39
Therefore, RT-PCR analysis of FFPE tissues, often the only specimens available for fatal cases, can be a useful adjunct to conventional diagnostic techniques, particularly in patients who die relatively soon after the disease onset. However, the application of RT-PCR for the detection of DENV in FFPE tissues has been very limited because of difficulties in extracting good quality RNA.
In the current study, we recovered RNA from archived FFPE autopsy tissues (some of which had been stored for more than 15 years) and showed that RT-PCR was a sensitive and valuable diagnostic tool for the detection and serotyping of DENV in FFPE tissues of fatal cases. Although RT-PCR and sequencing detected DENV in 60 (49%) cases of this series, IHC was able to detect DENV antigens only in 49 of these PCR-positive cases. Thus, in 11 (18%) cases dengue diagnosis was confirmed by RT-PCR only. The IHC negative results of these 11 cases may be due to less sensitivity of assay or clearance of viral antigens by the host immune response.40
Therefore, this study underscores the importance of postmortem tissue analysis by combination of RT-PCR with IHC for the diagnosis of DENV and shows that this approach improves the detection of DENV in fatal cases.
Our data also showed that 15 out of 60 PCR-positive cases were negative by DENV RT-PCR but positive by flavivirus RT-PCR assay. Interestingly, 14 of these cases were recent DENV-1 cases from Puerto Rico. This may indicate that the flavivirus RT-PCR that targets the more conserved NS5 gene was able to detect this more recent and maybe variant strain of DENV-1, whereas DENV RT-PCR that targets the capsid region was not. Similarly, DENV antibody, which is also a broad spectrum polyclonal antibody and reacts with different dengue strains, also detected DENV in 12 of the dengue RT-PCR negative cases. Previous studies have shown that many variant strains exist within each DENV serotype.41,42
Furthermore, DENV, being a positive-strand RNA virus, has a high potential for mutation, resulting in nucleotide differences between genotypes and also within a genotype.42
Therefore, to reduce the rates of false negatives, we recommend using more than one gene target for the PCR assays and selecting primers in the conserve regions of the viral genome for the detection of DENV. Additionally, using pan-flavivirus RT-PCR may also be useful for the identification of other flaviviruses in the samples, particularly from the countries where multiple flavivirus circulate.
Infecting serotype was identified for 95% (57 of 60) of DENV tissue RT-PCR-positive cases in our series. In 45% of the cases (27 of 60), DENV-2 was identified to be the cause of fatality. One possible reason for this may be that 13 out of 27 (48%) DENV-2 cases were from Puerto Rico where DENV-2 was the most prevalent serotype before the recent epidemic. On the other hand, some previous studies have also indicated that DENV-2 can cause more severe disease outcome.43,44
Moreover, the genotypes originating in Southeast Asia and the Indian subcontinent, which belongs to serotype 2 and 3, respectively, have been identified to cause more outbreaks of severe dengue disease.41,45–47
Although the host immune status can also play an important role in determining the outcome of infection,35
the determination of the virus nucleotide sequences in the fatal cases can help to identify the origin and serotype of the infecting virus and its association with severe disease and fatality. Furthermore, we also observed that out of 36 dengue-positive cases from Puerto Rico, all the cases that occurred from 1998 to 2003 were DENV-3 cases. Other studies also reported the re-introduction of DENV-3 in 1998 in Puerto Rico after a 20-year absence.48
Interestingly, all the cases that occurred from 2009 to early 2010 were DENV-1 cases and in more recent 2010 cases, DENV-4 was identified. This shows that even though the epidemiologic impact of retrospective tissue analysis may not be immediate and direct, such as for the identification of the circulating serotype during the outbreak, this approach can help to link some of the previously undiagnosed cases to the particular outbreaks and may be helpful to assess the true burden of the outbreak, and to a certain extent, in the phylogenetic analysis.
In this study, we detected both dengue viral antigens and RNA predominately in the liver and in lung, spleen, and kidney. Several previous studies also recognized liver as the major target organ in DENV pathogenesis.17,49,50
Interestingly, in 28% (17 of 61) of DENV-negative cases, organism-specific IHC and/or PCR detected other pathogens (IDPB, CDC, unpublished data) including Leptospira
, Streptococcus pneumoniae
, Staphylococcus aures
, 2009 pandemic H1N1 influenza A virus, and West Nile virus, which shows that clinical differential diagnosis of dengue-like syndrome can be extremely difficult and emphasizes the importance of postmortem tissue analysis of cases suspect to have DENV infection by histopathologic evaluation and other tissue-based techniques. However, availability of postmortem tissue for analysis is usually very limited because autopsies are not often performed, particularly in dengue-endemic regions, because of religious and cultural beliefs and bio-safety issues.
In conclusion, the data presented in this study shows that RT-PCR is a more sensitive and specific assay than IHC for the detection of dengue viruses in formalin-fixed tissue of fatal cases and provides sequence information that can be useful to facilitate typing and phylogenetic analysis, and may be also helpful to better characterize the pathogenic potential of distinct DENV serotypes. The RT-PCR on FFPE tissues can be a particularly valuable diagnostic tool in patients who die relatively soon after disease onset and for whom serology may be negative, and also when the FFPE tissues are the only specimens available. Thus, this approach can have important implications for dengue diagnosis and epidemiologic studies. In addition, combination of RT-PCR and IHC analysis of pathological specimens allows determination of tissue tropism and provides an insight into the pathogenesis of severe disease outcome.