Respiratory viral infections in Western Australians with cystic fibrosis.

BACKGROUND: Viral respiratory infections (VRI) in people living with Cystic fibrosis (CF) is less well understood than respiratory bacterial infections, particularly adults with CF and few studies have compared children with adults. This study evaluated the frequency of respiratory viruses in patients with cystic fibrosis (CF) in Western Australia (WA). We determined the VRI in CF and compared them with non-CF patients. Further, we compared CF patients that were hospitalised with those that were not. PATIENTS/METHODS: Nucleic acid from sputum of 157 CF and 348 non-CF patients was analysed for influenzavirus A (Flu A) and B, (Flu B), respiratory syncytial virus (RSV), human metapneumovirus (hMPV), human rhinovirus (RV), and parainfluenza viruses (PIV 1-3) by RT-PCR, during the 2016 winter respiratory season. RESULTS: No significant difference in the frequency of respiratory virus detection between CF and non-CF patients was found. RV was the most frequently detected virus in CF patients, and in hospitalised CF. RSV and hMPV were found less frequently in CF patients and RSV was not found in any hospitalised CF patient. A trend for fewer influenzavirus detections in adult CF patients was observed, however the trend was opposite for paediatric patients. RV and Flu A were the most common viruses detected in hospitalised CF patients. CONCLUSION: There was no significant difference in VRI between CF and non-CF patients. RV and influenza A were most commonly found in hospitalised CF patients, suggesting that infection with these viruses may contribute to hospitalisation for CF respiratory exacerbations.

Human Papillomavirus Associated Cancers of the Head and Neck: An Australian Perspective.

Human papillomavirus (HPV) associated head and neck squamous cell carcinomas (HNSCCs), have become a serious global health problem. Despite decreases in HPV-negative HNSCCs, the prevalence of HPV-positive HNSCCs has significantly increased. HPV-positive cancers are associated with superior survival outcomes when compared to HPV-negative cancers, which appears likely to be associated with differences in the molecular pathogenesis of the two diseases. While therapies are still problematic, the current HPV vaccine programs hold a promise for the primary prevention of HPV-related HNSCCs and since Australia was the first to introduce a nationwide HPV vaccine program, it is in a unique position to observe the effects of the vaccine on HNSCCs. This review discusses the epidemiological trends associated with HPV in HNSCC, with reference to the differences between HPV-positive and HPV-negative HNSCCs and the prevention potential of HPV vaccines.

Primary acute dengue and the deletion in chemokine receptor 5 (CCR5Δ32).

Dengue virus is a significant arboviral pathogen that is continuing to spread due to human travel and invasion of the mosquito vectors into new regions. Chemokine receptor 5 (CCR5) has a truncated 32 base pair deletion form (CCR5Δ32), which has been associated with resistance to HIV but increased severity in some flaviviral diseases. If CCR5Δ32 is associated with dengue, European carriers of this mutation may be at increased risk. In a Western Australian population with the same frequency of CCR5Δ32 (0.08) as that found in southern Europe there was no significant difference in CCR5Δ32 allele frequency between returned travellers with and without dengue (p = 0.82, OR = 0.86, 95% CI = 0.35-2.1).

A rapid DNA extraction method suitable for human papillomavirus detection.

Infection with oncogenic human papillomavirus (HPV) genotypes is necessary for the development of cervical cancer. Testing for HPV DNA from liquid based cervical samples can be used as an adjunct to traditional cytological screening. In addition there are ongoing viral load, genotyping, and prevalence studies. Therefore, a sensitive DNA extraction method is needed to maximize the efficiency of HPV DNA detection. The XytXtract Tissue kit is a DNA extraction kit that is rapid and so could be useful for HPV testing, particularly in screening protocols. This study was undertaken to determine the suitability of this method for HPV detection. DNA extraction from HeLa and Caski cell lines containing HPV 18 and 16 respectively together with DNA from five liquid based cervical samples were used in a HPV PCR assay. DNA was also extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) as a comparison. DNA extracts were serially diluted and assayed. HPV DNA was successfully detected in cell lines and cervical samples using the XytXtract Tissue kit. In addition, the XytXtract method was found to be more sensitive than the QIAmp method as determined by a dilution series of the extracted DNA. While the XytXtract method is a closed, the QIAamp method uses a spin column with possible loss of DNA through DNA binding competition of the matrix, which could impact on the final extraction efficiency. The XytXtract is a cheap, rapid and efficient method for extracting HPV DNA from both cell lines and liquid based cervical samples.

Human papillomavirus, high-grade intraepithelial neoplasia and killer immunoglogulin-like receptors: a Western Australian cohort study.

BACKGROUND: Human papillomavirus (HPV) is the causative agent in cervical cancer and HPV genotypes 16 and 18 cause the majority of these cancers. Natural killer (NK) cells destroy virally infected and tumour cells via killer immunoglobulin-like receptors (KIR) that recognize decreased MHC class I expression. These NK cells may contribute to clearance of HPV infected and/or dysplastic cells, however since KIR controls NK cell activity, KIR gene variation may determine outcome of infection. METHODS: KIR gene frequencies were compared between 147 patients with a history of high-grade cervical intraepithelial neoplasia (CIN) and a control population of 187, to determine if any KIR genes are associated with high-grade CIN. In addition a comparison was also made between cases of high grade CIN derived from 30 patients infected with HPV 16/18 and 29 patients infected with non-16/18 HPV to determine if KIR variation contributes to the disproportional carcinogenesis derived from HPV 16/18 infection. RESULTS: High-grade CIN was weakly associated with the absence of KIR2DL2 and KIR2DS2 (p = 0.046 and 0.049 respectively, OR 0.6; 95% CI 0.4 - 0.9) but this association was lost after correction for multi-gene statistical analysis. No difference in KIR gene frequencies was found between high-grade CIN caused by HPV 16/18 and non-16/18. CONCLUSION: No strong association between KIR genes, high-grade CIN and HPV genotype was found in the Western Australian population.

Can HPV DNA testing on FNA material determine anogenital origin in metastatic squamous cell carcinoma?

AIMS: Currently there are no diagnostic techniques that can precisely determine the primary site of a metastatic squamous cell carcinoma (SCC). Anogenital SCC has a high prevalence of high-risk (HR) human papillomavirus (HPV) DNA, particularly in the cervix where the value approaches 100%, whereas non-anogenital SCC generally has a low prevalence. The aim of this study was to examine whether the finding of HR HPV DNA in a fine needle aspiration (FNA) of metastatic SCC could be used to determine a likely anogenital origin. METHODS: Polymerase chain reaction (PCR) and viral sequencing were used to identify HR HPV DNA in cell block and needle rinse material derived from FNA samples in a series of metastatic SCC. RESULTS: HPV DNA was detected in nine of 12 (75%) metastatic anogenital SCC, and type 16 was sequenced in seven. HPV DNA was detected in only one of 18 (5.6%) metastatic SCC from other sites such as lung, oral cavity and skin. The positive control case was an oral cavity tumour and type 33 was sequenced. CONCLUSIONS: Although reservations remain, particularly in relation to the prevalence of HPV DNA in non-anogenital sites, HPV DNA testing by PCR: (1) can be successfully performed on FNA material, and (2) in the correct clinical context, can guide clinicians in assigning a site of origin. HPV DNA detection was the major indicator to the primary site in one occult tumour and several where the previous diagnosis of anogenital cancer was not known at presentation.

Multiplex nested PCR (MNP) assay for the detection of 15 high risk genotypes of human papillomavirus.

BACKGROUND: Human papillomavirus (HPV) is now recognized as the causative agent in cervical cancer. The HPV genotypes that infect the genital region have been classified into high and low risk types according to their oncogenic potential. There is still uncertainty regarding rare HPV genotypes, however the types considered high risk in this study are: HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 70. OBJECTIVES: We have set out to develop a multiplex nested PCR (MNP) assay with primers directed at the early region of the HPV genome to detect 15 high risk HPV (HRHPV) genotypes. Since it is known that the late region of HPV is lost on integration into the host cell genome, the primers are directed at the early region of the HPV genome so as to ensure the detection of integrated virus, in the absence of the episomal form of the virus. STUDY DESIGN: Primers were designed to detect specifically the high risk HPV in the MNP assay. The MNP assay was compared to a generic mucosal HPV nested PCR and another nested HRHPV PCR assay. DNA sequencing was carried out on the samples tested and matched with the PCR results. RESULTS: The MNP assay demonstrated that it was able to detect all 15 HRHPV types and was positive for more CIN1, CIN2 and CIN3 cases than the other nested HRHPV PCR. Further to this, the PCR product sizes differ for most of the HRHPV types detected in this system, so it is possible to type most of these HRHPV by the molecular size of the PCR products. CONCLUSION: The MNP assay detects 15 currently recognized HRHPV and could be very useful, in conjunction with the Pap smear, as a screening assay or to help manage Pap smears of uncertain cytology.

Human papillomavirus genotypes and their association with cervical neoplasia in a cohort of Western Australian women.

Human papillomavirus (HPV) is known to be the cause of almost all cervical cancers. The genotypes have been classified into high and low risk types according to their oncogenic potential. However, data for many of the genotypes are limited and some (HPV-26, 53, and 66) have no agreed status. A study was undertaken to determine the HPV genotype distribution in women of Western Australia and the association with cervical neoplasia. Liquid based cervical samples from a cohort of 282 Western Australian women were tested for HPV DNA by PCR followed by DNA sequencing to determine HPV genotypes. HPV-53 and HPV-16 were the most common genotypes found in this population. In addition 86 archived liquid based cervical samples from women with cervical intraepithelial neoplasia grades 1-3 (CIN 1-3) were tested for HPV DNA. Also 32 archived paraffin biopsy samples from women with squamous cell carcinoma were also tested. HPV-16 was the most common genotype found in these samples. Of the cohort of Western Australian women tested, 27% were found to contain HPV and approximately half of these contained known high-risk HPV genotypes, but only 30% of these were types 16 or 18. The data from this study indicate that HPV-53 is not oncogenic based on an R value and odds ratio (OR) of zero. The data also suggest that HPV-73 may be oncogenic, while HPV-66 is unlikely to be. Two high-risk HPV genotypes that are associated with the Asian region (HPV-52 and HPV-58) were found in Western Australian women suggesting a possible epidemiological link between women in these countries.

Validation of assembled nucleic acid-based tests in diagnostic microbiology laboratories.

Medical microbiology and virology laboratories use nucleic acid tests (NAT) to detect genomic material of infectious organisms in clinical samples. Laboratories choose to perform assembled (or in-house) NAT if commercial assays are not available or if assembled NAT are more economical or accurate. One reason commercial assays are more expensive is because extensive validation is necessary before the kit is marketed, as manufacturers must accept liability for the performance of their assays, assuming their instructions are followed. On the other hand, it is a particular laboratory's responsibility to validate an assembled NAT prior to using it for testing and reporting results on human samples. There are few published guidelines for the validation of assembled NAT. One procedure that laboratories can use to establish a validation process for an assay is detailed in this document. Before validating a method, laboratories must optimise it and then document the protocol. All instruments must be calibrated and maintained throughout the testing process. The validation process involves a series of steps including: (i) testing of dilution series of positive samples to determine the limits of detection of the assay and their linearity over concentrations to be measured in quantitative NAT; (ii) establishing the day-to-day variation of the assay's performance; (iii) evaluating the sensitivity and specificity of the assay as far as practicable, along with the extent of cross-reactivity with other genomic material; and (iv) assuring the quality of assembled assays using quality control procedures that monitor the performance of reagent batches before introducing new lots of reagent for testing.

The value of HPV DNA typing in the distinction between adenocarcinoma of endocervical and endometrial origin.

AIMS: Distinguishing between adenocarcinomas of endocervical and endometrial origin histologically can be difficult, particularly in small biopsies. Most endocervical adenocarcinomas contain human papillomavirus (HPV) deoxyribonucleic acid (DNA) of 'high-risk' (HR) types, whereas this has not been consistently demonstrated in endometrial adenocarcinomas. The aim of this study was to determine whether HPV DNA testing could aid in this differential diagnosis. METHODS: The frequency of HPV DNA in paraffin-embedded tissue samples from 50 endocervical and 50 endometrial adenocarcinomas was investigated using polymerase chain reaction (PCR) amplification techniques involving (i) a screening HPV test followed by HPV DNA sequencing, and (ii) a test designed to detect HR genotypes 16, 18, 31, 33, 35, 45 and 58. Control specimens included cervical intraepithelial neoplasia (CIN) III lesions, squamous cell carcinomas (SCCs) of the cervix and lung, and colonic adenocarcinomas. Measures to minimise cross-contamination were implemented. RESULTS: The screening test followed by HPV DNA sequencing had the highest sensitivity. By this test HR HPV DNA was detected in 11 of 11 (100%) cervical intraepithelial neoplasia (CIN III) lesions, nine of 10 (90%) cervical SCCs, none of 10 (0%) colorectal adenocarcinomas and none of 10 (0%) SCCs of the lung. Thirty-nine (78%) endocervical adenocarcinomas contained HR HPV DNA, compared to one (2.0%) endometrial adenocarcinoma. CONCLUSIONS: The results suggest that HPV DNA testing could be a useful adjunct in distinguishing between endocervical and endometrial adenocarcinomas in curettings or small biopsy specimens.