Reverse transcriptase -polymerase chain reaction and DNA enzyme immunoassay for diagnosis of dengue fever and for dengue virus typing

In this study the use of commercially prepared reagents for detection and typing of dengue viruses from clinical samples using reverse transcriptase- polymerase chain reaction [RT- PCR] was evaluated on local dengue strains isolated in Jeddah Saudi Arabia over the period from February 1994 to July 1999. Total of 80 dengue culture confirmed cases, and 30 culture negative and IgM negative non dengue cases, and additionaly dengue virus infected, 20, and non infected, 20, tissue culture supernatants from C6/36 cell line, were used. All dengue culture positive cases were detected by RT- PCR followed by gel electrophoresis and or DNA enzyme immunoassay [DEIA], except 1 sample, which was negative by RT-PCR but was positive in culture in 1994 epidemic, with a sensitivity of 98.7% and specificity of 100%. All positive cases were correctly typed using gel electrophoresis following nested PCR and also by DEIA from first amplification product, without the need of nested PCR. This study showed that the commercially availabe reagents for extraction, RT- PCR, and detection are effective methods for the rapid and sensitive as well as specific diagnosis of dengue virus infection and are also effective in rapid typing of the infecting serotype, and that the use of DEIA in detection and typing from first amplification products is as effective as nested PCR using specific primers followed by gel electrophoresis or culture followed by antigen detection using polyclonal and monoclonal antibodies in an indirect immunofluorescent assay

Enteroviral infections of neonates and children: clinical utility of different methods used in the diagnosis

To determine the clinical utility of TaqMan real time PCR, nested polymerase chain reaction [PCR] assay and virus culture of different samples for rapid diagnosis of Enteroviral infections among neonates and children, with suspected enterovirus infections. Samples were collected from neonates and children suspected of having enterovirus infections, over a period of three years. The samples included CSF, nasopharyngeal aspirates, throat swabs, skin vesicular swabs, blood, stool, conjunctival swabs. The samples were tested by virus cultures, nested RT-PCR, and TaqMan real time PCR. Enteroviruses isolates from tissue cultures were typed using molecular techniques based on VP-1 region sequences. A total of 1020 specimens obtained from 518 patients, were tested. Enterovirus isolates were obtained from 182 [35%] of patients. All culture positive specimens were also positive by Real time PCR and nested PCR, In addition real time PCR and nested RT-PCR gave an additional 05 positive specimens. The mean time required for virus culture is about one week, for nested RT-PCR is one day for Real time PCR is 4-5 hours. The real time PCR showed a sensitivity of 100% and specificity of 98.5% compared to virus culture. The Real time PCR is of great value to the clinician and the patients, in providing rapid diagnosis within 4-5 hours from sample receiving and minimizing unnecessary hospital stay, minimizes extra diagnostic tests or use of antibiotics in cases of confirmed enterovirus infections

Detection of human metapneumovirus in children hospitalized for acute respiratory tract infections

Human metapneumovirus [hMPV] is a newly recognized paramyxovirus associated with acute respiratory tract infections [ARTIs] mainly in infants and children. To evaluate the role of hMPV infection in children hospitalized with RTD and to analyze the virologic and clinical features of hMPV infection. Real time Reverse transcription-polymerase chain reaction [RT-PCR] was used to detect N gene of hMPV in 282 nasopharyngeal aspirates that were collected over the period from December 2004 to June 2006, and were also tested for other common respiratory viruses by indirect immunofluorescent assay. Twenty six samples [9.2%] were positive for hMPV, most of them detected during winter months. Respiratory Syncytial Virus [RSV] was detected in 99[35.1%] specimens. RSV Co-infection was detected in 3/26 [11.5%] of hMPV positive specimens. Sequencing of 10 of the 26 hMPV positive samples showed type A predominance [90% vesrus 10% type B]. Culture was performed on 14 of the 26 real time PCR positive specimens and 10 isolates were detected [71%]. Nineteen of the 26 [73%] hMPV positive cases were aged 4 to 12 months whereas 66 of the 99 [66.6%] RSV positive cases occurred mostly during the first 6 months of life. Similar to other viral ARTIs cough, fever, dyspnea and wheezing were the most common symptoms. hMPV was detected in 9.2% of children with ARTIs, mainly in winter months; and mostly at the age of 4-12 months. Both type A and B were detected but the former was more common. Symptoms are similar to those of RSV

Herpes simplex 1 and 2 infections: common clinical presentation and comparison of two methods for virus typing in tissue culture

Herpes simplex serotypes 1 and 2 were isolated in tissue culture from 80 patients, 26 females and 54 males, aged from new born to 56 years. The herpes isolates included 36 Herpes simplex type 1 and 44 isolates of Herpes simplex type 2. The clinical data commonly seen in patients with primary HSV-1 infection were gingivostomatitis of children associated with systemic symptoms in the form of fever, malaise, generalized body aches, which were resistant to antibacterial therapy. Recurrent herpes labials was the next common presentation of HSV-1 infection. HSV-1 was isolated from pharyngeal swabs of 4 patients, with clinically and serologically confirmed measles infection. One HSV-1 isolate was isolated from a genital vesicular eruption in a female. Herpes simplex type 2 infected patients presented mainly with recurrent vesicular or ulcerative genital or extra genital or extra genital [on buttocks and legs] lesions. One new-born showed vesicular eruption on the skin that was caused by HSV-2. Typing of the virus isolates front tissue culture was done using two methods, namely, Dako herpes simplex ELISA, and Syva fluorescene labelled type specific monoclonal antibodies. The two tests gave more or less identical results. Typing with the Syva monoclonal kit was less laborious, more rapid, [completed in less than one hour], cultures could be tested one by one, which is suitable for laboratory with few nubmer of samples. The Dako ELISA kit, required preparation of a number of buffers, test was completed in 4.5 hours, must be done in groups of 16, there was always background reading in the negative serotype which in cases of HSV-2 made interpretation with naked eye impossible and required use of ELISA reader. In conclusion the clinical presentation of herpes types 1 and 2 in a private clinic are as described elsewhere, in adition to the isolation of herpes 1 from measles cases. Typing of HSV isolates by Syva monoclonal antibodies test is easier, more rapid, and more specific than use of Dako ELISA kit

Dengue infections in Jeddah; virological and serological findinges in clinically diagnosed cases

Laboratory diagnosis of dengue fever, using virus isolation on C6/36, and serology by Ig[M] capture ELlSA and hemagglutination inhibition [HI] test, was carried out on 540 clinically diagnosed dengue fever cases in Jeddah, Saudi Arabia, during the period from February 1994 to February 1995. Dengue infections were confirmed in 294 [54.44%] of cases, dengue 1and 2 viruses were isolated from 182 [33.70%] cases, and Ig[M] caputre ELlSA was positive in 112 [20.74%] culture negative cases. Primary and secondary dengue infections were confirmed in 42 [14.30%] and 56 [19.00%] of confirmed cases respectively. More than 90% of virus isolates were from samples taken during the first 5 days of symptoms, while Ig[M] positive cases were more after the 5[th] day of symptoms. Virus isolation and Ig[M] capture ELISA were positive in more than 50% of samples taken around the fifth day. It is evident from this study that dengue 1 and 2 circulates in Jeddah Saudi Arabia, and that virus isolation and Ig[M] capture ELISA are very efficient tools in surveillance for dengue infection among suspeted cases. The use of HI test is not of much benefit in diagnosis of dengue infection in our study, since it requires convalescent blood sample which is not obtained in the majority of cases. It is important to maintain surveillance for dengue infection using Virus Isolation to monitor dengue activity and to detect introduction of other dengue types or even other arboviruses

Viral lower acute respiratory infections: efficacy of antigen detection

Two hundred and twenty infants and children with fever and respiratory distress below three years of age [8.9 +/- 2.6 months] were examined for the diagnosis of viral etiology. Nasopharyngeal aspirates [NPA] was used for viral isolation by tissue culture and compared to the demonstration of viral antigens in the specimens by indirect immunoflourescent test using the Baxter Bartel respiratory screening and identification kit. In a series of 220 NPAs tested. 139 [63.1%] respiratory viral agents were isolated on tissue culture. Respiratory syncetail virus [RSV] was the most commonly isolated agent particularly among infants below 1 year of age with a peak below 6 months of age. Other isolated virsus included influenza A and B, Parainfluenza 1, 2 and 3 and adnoviruses. Direct antigen detection by immunofluorescent technique gave a high level of sensitivity and specificity compared to the culture as a standard measure, Viral ARI is a common pediatric problem. RSV is a common infectious agent of ARI below 1 year. The rapid, cheaper, sensitive and specific immunoflourescent test for antigen detection is of great help for paediatricians to take a rapid decision regarding the etiology and need of antimicrobial agents in ARI