INTRODUCTION
Adenoviruses play an important role in the etiology of severe acute lower respiratory infection, especially in young children and immunocompromised individuals1. Human adenoviruses (HAdV) spread rapidly in closed environments, and often cause epidemic disease in crowded communities2.
Health care workers (HCWs) are in direct contact with HAdV infected patients and this population represent a susceptible group to the virus infection, acting as an infection source in outbreaks. Furthermore, there is a lack of studies among this population, with little knowledge about HAdV circulation compared to community subjects3.
In immunocompromised patients, specially stem cell and solid organs transplanted patients, the HAdV could cause severe infection, with case-fatality rate ranging from18-83%, depending on the immunossupression nature4-8. In Brazil there is a lack of sufficient information in immunocompromised population, with detection ranging from 0-3%3,9.
Furthermore, it is difficult to clinically distinguish adenovirus infection from other viral or bacterial respiratory infections10. Rapid diagnostic methods avoid inadequate antimicrobial treatment and support the infection control interventions during nosocomial infection. Molecular methods demonstrate greater sensitivity when compared to conventional assays for detecting adenovirus in respiratory samples11,12.
Brazilian studies have described frequencies from 3 to 7.1%, according to different diagnostic techniques13-16. Outbreaks reports have been frequent among young patients17.
The aim of the present study was to assess the HAdV detection, through different methods, such as direct fluorescence assay (DFA) and nested-polymerase chain reaction (nested-PCR) from samples collected from different risk groups, during six years.
METHODS
Population of study
In the present study four different populations were studied from 2001 to 2010.
Adults attended in an Emergency Room Patients (ERP): from 2001 through 2003, 139 adults patients were evaluated by general practitioners from the general community attended in São Paulo Hospital emergency room.
Health care workers (HCWs): from 2001 through 2003, 205 adult’s health care workers were evaluated by general practitioners in the center of support to the health of the Sao Paulo Hospital employees.
Renal tranplant outpatients (RTO): from 2001 through 2003, 69 adult’s outpatients of the renal transplant clinic were evaluated.
Patients in a hematopoietic stem cell transplantation (HSCT) program: between 2008 and 2010, 230 adults patients attended in a hematopoietic stem cell transplantation program of hematological service of the São Paulo Hospital were evaluated. All patients had sample collected by medical staff, usually an infectious disease physician. Of the 230 studied patients in this group, information about transplantation was available for 221 (96%): 55.2% (122/221) of these patients were undergoing transplantation at the moment of study enrollment and 44.8% (99/221) were non-transplanted.
Inclusion criteria
Adults (> 12 years old) were eligible, after a physician evaluation, if presented acute respiratory infection (ARI) of probably viral etiology, with up to 7 days of symptoms onset until the collection. Respiratory symptoms assessed were: coryza, cough, sore throat, or nasal congestion; systemic symptoms were: fever, headache, malaise, chills, or fatigue.
Sample collection
Nasal wash (NW) specimens were collected from patients presenting with acute respiratory symptoms referred by physicians from São Paulo Hospital to the Clinical Virology Laboratory, São Paulo, Brazil18.
Adenovirus detection
All samples were maintained at 4oC and transported immediately to laboratory. An aliquot (1mL) was separated for molecular analysis and stored at -70oC. The remaining specimen volume was evaluated, on the same day, by direct fluorescent assay, according to previous study19. One aliquot from each stored sample had deoxyribonucleic acid (DNA) extracted by QIamp DNA Blood extraction kit (Qiagen, USA), according to manufacturer’s instructions; it was followed by nested-PCR for detection of all adenovirus serotypes, as previously described20.
Direct immunofluorescence assay: All studied samples were evaluated by direct immunofluorescence technique (DFA). The tests were performed using the kit Simulfluor Respiratory Screen and Panel(Chemicon Int., EUA), in accordance with the manufacturer’s instructions.
Nested-PCR assay: For the first amplification reaction (PCR product – 301bp), 5μL of extracted DNA were placed in a tube with a reaction mixture consisting of 2.5μL of 10X buffer (200mM Tris-HCl, pH 8.4, 500mM KCl ), 3.5mM of MgCl2, 0.5μM of primers Hex1deg and Hex2deg20 (Hex1deg – 5´- GCC SCA RTG GKC WTA CAT GCA CAT C – 3´, Hex2deg – 5´- CAG CAC SCC ICG RAT GTC AAA – 3´), 1μL of dNTPs mixture containing 20mM of each nucleotide, 2.5U of Platinum(r) TaqDNA Polymerase (Invitrogen, Brazil) and autoclaved MilliQ water to a final volume of 25μL.
For the second amplification reaction (PCR product – 171bp), 2μL of first reaction amplicon were placed in a tube with a reaction mixture consisting of 2.5μL of 10X buffer (200mM Tris-HCl, pH 8.4, 500mM KCl ), 3.5mM of MgCl2, 0.5μM of primers Nehex3deg and Nehex4deg20 (Nehex3deg – 5´- GCC CGY GCM ACI GAI ACS TAC TTC – 3´, Nehex4deg – 5´- CCY ACR GCC AGI GTR WAI CGM RCY TTG TA – 3´), 1μL of dNTPs mixture containing 20mM of each nucleotide, 2.5 of Platinum(r) TaqDNA Polymerase (Invitrogen, Brazil) and autoclaved MilliQ water to a final volume of 25μL.
Positive controls (Adenovirus Serotype 3) and redundant negative control (autoclaved MilliQ water) were included in each series. Positive results were considered when amplicon were visualized after 2% agarose gel electrophoresis.
The sensibility of the reaction were standardized and showed a detection limit of 10-4TCID50/mL .
RESULTS
Samples from 643 symptomatic patients were analyzed (40.4% females and 59.6% males). The demographic characteristics of all studied patients are showed in Table 1.
TABLE 1 Demographic data of studied population.
| Samples | Gender male | Age (years) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| n | % | n | % | mean±SD | median | range | |||
| HCWs | 205 | 31.9 | 154 | 75.1 | 36.2±11.7 | 36.0 | 16-68 | ||
| ERP | 139 | 21.6 | 62 | 44.6 | 32.7±13.8 | 29.0 | 12-83 | ||
| RTO | 69 | 10.7 | 35 | 50.7 | 38.8±11.7 | 38.5 | 15-60 | ||
| HSCT | 230 | 35.8 | 132 | 57.4 | 43.7±15.2 | 47.0 | 18-80 | ||
| Total | 643 | 100.0 | 383 | 59.6 | 38.3±14.12 | 37.0 | 12–83 | ||
HCWs: health care workers; ERP: emergency room patients; RTO: renal transplant outpatients; HSCT:patients in program of hematopoietic stem cell transplant; SD: standard deviation.
Among the 643 samples included during the study period, 85 (13.2%) were positive for at least one test: 7/205 (3.4%) from HCW, 6/139 (4.3%) adults from community ERP, 4/69 (5.8%) from RTO, and 68/230 (29.6%) from hematological patients. Table 2 shows differential detection between assays according to different studied populations. Overall, 24 (3.8%) of 643 samples were DFA positive (10 had insufficient material for performed DFA) and 64 (10%) of 643 samples were nested-PCR positive (one sample had insufficient volume in aliquot).
TABLE 2 Adenovirus detection by direct immunofluorescent assay and nested-PCR assay among different studied populations from São Paulo during 2001-2010.
| Number of samples |
DFA positive |
Nested-PCR positive |
Total positivity |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| n | % | n | % | p | n | % | ||||||
| HCWs | 205* | 0 | 0.0 | 7 | 3.4 | 0.015 | 7 | 3.4 | ||||
| ERP | 139 | 0 | 0.0 | 6 | 4.3 | 0.030 | 6 | 4.3 | ||||
| RTO | 69 | 0 | 0.0 | 4 | 5.8 | 0.12 | 4 | 5.8 | ||||
| HSCT | 230** | 24 | 10.4 | 47*** | 20.4 | 0.005 | 68 | 29.6 | ||||
| Total | 643 | 24 | 3.8 | 64 | 10.0 | <0.001 | 85 | 13.2 | ||||
PCR: polymerase chain reaction; DFA: direct fluorescence assay; HCWs: health care workers; ERP:emergency room patients; RTO: renal transplant outpatients; HSCT: patients in program of hematopoietic stem cell transplant.
*eight samples the DFA were not accomplished;
**in one sample DFA were not accomplished;
***three samples were positive in both tests, and were counted as positive for nested-PCR
Nested-PCR presented a higher detection, statistically significant, among all studied populations, except for renal transplanted outpatients group. When the entire studied population was analyzed, molecular method shown higher detection rate when compared to DFA (p < 0.001). The higher detection rate was found in HSCT patients (p < 0.001). The positive samples distribution along the period of study is show in ( Figure 1 ).
Time of symptoms onset until the sample collection was analyzed and showed a statistical difference between DFA and nested-PCR ( Table 3 ). Nested-PCR compared to DFA, detected a higher number of cases in both situations, in samples collected within five days of symptom onset and after. Among HSCT group nested-PCR assay detection was higher in patients with more than five days of symptoms onset until the sample collection (p<0.001).
TABLE 3 Time of symptoms onset until the sample collection according to direct fluorescence assay and nested-PCR assay.
| Days | |||||||
|---|---|---|---|---|---|---|---|
| < 5 | > 5 | ||||||
| positive | % | total | positive | % | Total | ||
| DFA | 17 | 3.4 | 501 | 7 | 4.9 | 142 | |
| Nested-PCR | 42 | 8.4 | 501 | 22 | 15.5 | 142 | |
| p-value | <0.01 | <0.01 | |||||
PCR: polymerase chain reaction; DFA: direct fluorescence assay.
HSCT patients presented 12 times (p<0.001; CI: 5.37-26.9) more chance to acquired hADV infection when compared to HCWs.
DISCUSSION
In Brazil few studies have investigated adenovirus viral infection among immunocompetent non-hospitalized adults. The detection rates among the adult population (emergency room – 4.3% and health care workers – 3.4%) were similar to those described in the literature 2.5% – 12.8%21,22. Lessa et al.22described that 19% of studied HCWs were infected with HAdV, in comparison to 5.3% among non-HCWs, in a military hospital.
Human adenoviruses infections are a important cause of morbidity and mortality among stem cell transplant recipients23,24. Hospitalized patients’ presenting hematological diseases, with prolonged hospitalizations stay, were at high risk of severe HAdV infection and represents a potential source of nosocomial outbreaks25. Studies described a rate ranging from 2 to 21% of HAdV infected patients, with a mortality rate of 10-60% related to HAdV infection, depending on the immunossupressive regimen26.
Evaluating the two used tests many questions raised of importance for both clinicians and hospital administrators dealing with which is the better test; up to which day is better for sample collection, is there a need to test repetition, which is the ideal number of samples for laboratory routine flow, and which is the meaning of negative and positive test in high risk patients? Some of these aspects could be evaluated in the present study. HAdV was detected in 13%, by at least one of the used test. The HAdV occurred throughout the year, with higher detection rates, obtained by DFA, during 2008 March and April. This occurrence could be explained by an HAdV outbreak that occurred in the same period in hematological ward, and also by the clinical re-evaluation in which these patients were submitted, in other words, patients with more than one sample collected in the same month could overestimated the virus occurrence.
The evaluation of patient´s symptom onset and used test showed that molecular method displayed always more sensibility than DFA, and this difference was more evident after the fifth day of symptoms onset. Another studies described similar results, with molecular methods presenting higher sensitivity when compared to DFA, in adults’ patients27,28. Lower detection of DFA was related to low viral load in respiratory samples29.
The higher detection rates among patients with more days of symptom onset were expected, due to higher nested-PCR sensibility30,31. DFA test successful needs some key factors as good sample collection, experienced laboratory staff and higher viral load29,32, while nested-PCR, based on nucleic acid amplification, can detect a very low number of viral particles20. Among HSCT patients nested-PCR presented higher sensibility after the fifth day, suggesting that among patients with prolonged infection history the molecular method is best for the HAdV detection.
Human adenoviruses can produced asymptomatic33,34 and prolonged34 infections. These two parameters must be in consideration when the diagnostic technique is going to be selected. Carraro18 described no difference of influenza virus detection between DFA when compared to RT-PCR, up to the fifth day of symptoms onset, probably indicating a reasonable performance of serological test in an acute phase of the disease. On the other hand, positive result of molecular method, not necessarily, indicates a symptomatic infection, mainly due to high sensibility of this test20. The use of quantitative PCR (real time PCR), could address this issue, from the moment when the viral load can indicate the presence or absence of disease11.
All patients enrolled in the present study had acute respiratory infection, which reduces the possibility of asymptomatic infection by adenovirus. Moreover, one of the inclusion criteria was symptoms onset less than 7 days, thus not allowing analysis of viral shedding duration.
A limitation of our study was the lack of analysis of possible co-infections of HAdV with other respiratory viruses. However, data from several studies showed low rates, ranging from 1.04% to 3.5%35-37.
Based on data obtained in the present evaluation, we concluded that HAdV detection frequency by nested-PCR assay detection was higher, mainly among high-risk patients (hematopoietic stem cell transplant). However, the inclusion of molecular methods for HAdV detection in routine diagnosis should be considered, evaluating the economic reality of each specific health service and the demand for HAdV testing.
