Photobiomodulation as an Adjunct Therapy in Wound Healing.

"Light amplification by stimulated emission of radiation" or more commonly known as Laser has become very popular in the field of dermatology and aesthetic medicine over the past decades. For the treatment of wound healing, a combination of different wavelengths for laser therapy has been introduced which includes 660, 800, and 970 nm. The aim of this study was to note wound healing utilizing photobiomodulation as an adjunct therapy by measuring the wound size in terms of length and width (area measurement). Study participants were selected randomly from a pool of patients who were attending for their routine follow-up visits in the Wound Care Unit in Hospital Kuala Lumpur. Eleven patients with chronic wounds of different etiologies, ie, diabetic foot ulcer and nonhealing ulcer, were recruited for this study . Wound assessment was done prior to cleansing using distilled water and followed by debridement if necessary. Subsequently, the laser technician and patients used protective goggles before applying a super intense continuous flow of laser with 3 wavelengths, ie, 660, 800, and 970 nm with 30 kJ of energy with the handpiece over a 3 min period whereby it is focused on the wound milieu and then rotated around the periwound area. There were 9 diabetic foot ulcers and 2 nonhealing ulcers treated with photobiomodulation as an adjunct therapy. All wounds were managed with the standard of care. Three wounds ie, 3 diabetic foot ulcers and 1 nonhealing ulcer were closed completely. Meanwhile, the other 7 ulcers are at 68.2% to 99% in terms of wound area reduction and new granulomatous tissue was present indicating high healing potential. Therefore, the photobiomodulation was effective as an adjunct in the management of diabetic foot and nonhealing ulcers in this case series. A larger sample size would be able to show the significance of this finding.

Hirudotherapy in Wound Healing.

Leeches are hermaphrodite, bloodsucking parasitic worms usually found in places with fresh water. Leech therapy existed 3000 years, and it is being used at a different scope. Several species of leeches have been used in medicine, and the most common species used is Hirudo medicinalis. Leeches suck the excess blood, reduce the swelling in the tissues, and promote healing by allowing fresh oxygenated blood to reach the area until normal circulation can be restored. Pain relief from leech therapy is rapid, effective, and long-lasting in many conditions. The aim of this study was to evaluate the efficacy and duration of healing utilizing sterile medicinal leeches, Hirudinaria manillensis, in the management of pain and wound healing. Leech was taken out from its sterile tube by using a pair of non-tooth sterile plastic forceps and gloved hands. Each leech was left in place for as long as it was feeding. Leeches were removed only after they became detached from the patient. The specimen jars containing the used leeches were sealed in either a biohazard bag or in a small yellow clinical waste bin liner securely fastened with a cable tie. The leech was killed by using 70% alcohol prior to disposal into a yellow hazard bin, which undergoes incineration. All 3 patients had improvements in their condition, especially in terms of reduction in the pain and improvement in their sense of balance. All the wounds healed well. Therefore, leech therapy is effective in reducing pain and increasing perfusion to allow the wounds to heal quickly. However, a more robust trial is needed to show significance as the sample size is small.

Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5' untranslated region (5' UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

Pandemic Planning in Homeless Shelters: A Pilot Study of a Coronavirus Disease 2019 (COVID-19) Testing and Support Program to Mitigate the Risk of COVID-19 Outbreaks in Congregate Settings.

We tested 104 residents and 141 staff for coronavirus disease 2019 who failed daily symptom screening in homeless shelters in Hamilton, Canada. We detected 1 resident (1%), 7 staff (5%), and 1 case of secondary spread. Shelter restructuring to allow physical distancing, testing, and isolation can decrease outbreaks in shelters.

Maggot Debridement Therapy in Malaysia.

Maggot therapy, also known as maggot debridement therapy, larval therapy, biodebridement, or biosurgery, is a type of biotherapy involving the intentional application of live, disinfected fly larvae or maggots into the nonhealing wound of a human or animal to debride the necrotic wound, reduce bacterial contamination of the wound as well as enhance the formation of healthy granulation tissue and stimulate healing in nonhealing wounds. In addition, van der Plas et al reported that the use of the medicinal larvae as natural remover of necrotic and infected tissue had prevented amputation in 11 selected patients. In Malaysia, Aaron et al had demonstrated prevention of amputation in 25 patients.

Comparison of four COVID-19 screening strategies to facilitate early case identification within the homeless shelter population: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: 1. To compare the effectiveness of four different surveillance strategies in detecting COVID-19 within the homeless shelter population. 2. To assess the participant adherence over time for each surveillance method. TRIAL DESIGN: This is a prospective cluster-randomized study to compare the effectiveness of four different surveillance regimens across eight homeless shelters in the city of Hamilton. PARTICIPANTS: Participants will include both residents of, and the staff working within, the homeless shelters. All participants aged 18 or older who consent to the study and are able to collect a swab sample (where relevant) are eligible for the study. The study will take place across eight homeless shelters (four men-only and four women-only) in the City of Hamilton in Ontario, Canada. INTERVENTION AND COMPARATOR GROUPS: The comparator group will receive active daily surveillance of symptoms and testing will only be completed in symptomatic participants (i.e. those who fail screening or who seek care for potential COVID-19 related symptoms). The three intervention arms will all receive active daily surveillance of symptoms and testing of symptomatic participants (as in the comparator group) in addition to one of the following: 1. Once weekly self-collected oral swabs (OS) regardless of symptoms using written and visual instructions. 2. Once weekly self-collected oral-nares swab (O-NS) regardless of symptoms using written and visual instructions. 3. Once weekly nurse collected nasopharyngeal swab (NPS) regardless of symptoms. Participants will follow verbal and written instructions for the collection of OS and O-NS specimens. For OS collection, participants are instructed to first moisten the swab on their tongue, insert the swab between the cheek and the lower gums and rotate the swab three times. This is repeated on the other side. For O-NS collection, after oral collection, the swab is inserted comfortably (about 2-3 cm) into one nostril, parallel to the floor and turned three times, then repeated in the other nostril. NPS specimens were collected by the nurse following standard of care procedure. All swabs were placed into a viral inactivation medium and transported to the laboratory for COVID-19 testing. Briefly, total nucleic acid was extracted from specimens and then amplified by RT-PCR for the UTR and Envelope genes of SARS-CoV-2 and the human RNase P gene, which is used as a sample adequacy marker. MAIN OUTCOMES: 1. PRIMARY OUTCOME: COVID-19 detection rate, i.e. the number of new positive cases over the study period of 8 weeks in each arm of the study. 2. SECONDARY OUTCOMES: Qualitative assessment of study enrollment over 8 weeks. Percentage of participants who performed 50% or more of the weekly swabs in the intervention arms in the 8 week study period. RANDOMIZATION: We will use a computer-generated random assignment list to randomize the shelters to one of four interventions. Shelters were stratified by gender, and the simple randomization scheme was applied within each stratum. The randomization scheme was created using WinPEPI. BLINDING: This is an open-label study in which neither participants nor assessors are blinded. NUMBERS TO BE RANDOMIZED (SAMPLE SIZE): Since we are including our total sample frame, a sample size estimation at the cluster level is not required. However, if we succeed to enroll 50 participants per shelter from 8 shelters (n=400), and the detection rate is 3 times higher in the intervention groups (0.15) than in the comparator groups (0.05), we will have 90% power to detect a statistically significant and clinically important difference at a type I error rate of alpha=0.05 (one tailed), assuming an intraclass correlation of ~0.008. These computations were done using WinPEPI, and informed by conservative estimates from other studies on respiratory illness in the homeless (see Full protocol). TRIAL STATUS: The protocol version number is 3.0. Recruitment began on April 17, 2020 and is ongoing. Due to low numbers of COVID cases in the community and shelter system during the initial study period, the trial was extended. The estimated date for the end of the extended recruitment period is Feb 1, 2021. TRIAL REGISTRATION: The trial was registered with ClinicalTrials.gov on June 18, 2020 with the identifier NCT04438070 . FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Validation of Harikrishna Periwound Skin Classification for wound assessment.

OBJECTIVE: To validate the accuracy and reliability of Harikrishna Periwound Skin Classification (HPSC) for wound assessment. METHOD: Post-basic students (staff nurses and medical assistants) were given real life pictures showing the wound and periwound area. The students were asked to classify all pictures according to the HPSC at zero months (before attachment) and after two months of attachment. The images were the same but the answers were never given or discussed after the first test. RESULTS: A total of 30 post-basic students participated in the study, assessing wound 30 images. The results showed that there was an increase of 25.42% in accuracy of wound assessment using the HSPC after two months of clinical attachment compared to pre-attachment. The reliability of the HPSC in wound assessment 79.87%. CONCLUSION: Health professionals have to be able to assess and classify wounds accurately to be able to manage them accordingly. Assessment and classifications of the periwound skin are important and need to be validated and integrated as a part of a full wound assessment. With experience and adequate training, health professionals are able to comprehensively assess wounds using the validated tool, to enable effective wound management and treatment, accelerating wound healing and improving the quality of life for patients.

Electrical Tweezer for Droplet Transportation, Extraction, Merging and DNA Analysis.

Droplets of aqueous solutions distributed in an immiscible oil phase are increasingly used and investigated as a means to handle and assay small volumes of samples. The primary attraction of this method is that surface interactions are kept to a minimum, and changes in sample concentration, especially due to adsorption to the walls, are avoided. Microfluidic methods to generate, transport, merge, split and perform reactions in droplets were developed recently. These methods depend on the continuous flow of the two phases involved inside closed microfluidic channels. Alternatively, an electrowetting phenomenon was also exploited to control the movement of droplets between two solid substrates. However, there are some situations where small volume sample transport and assaying are required in open systems. Here, we demonstrate a simple electromechanical probe (tweezers) that is capable of manipulating a small aqueous droplet in a bi-layer oil phase. The tweezer consists of two needles positioned close to each other and uses polarization of the aqueous droplet in an applied electrical field to confine the droplet between the needles with minimal solid contact. Mechanical motion of the tweezer can be used to transport the droplet to various positions. Operations such as aliquoting, merging and transport are demonstrated. Finally, this method was used to perform a DNA amplification assay where droplets of the sample and the amplification mixture are aliquoted separately, mixed and amplified using an in-situ heater. This electromechanical tweezer is of interest in low-throughput, small-volume biological and chemical assays where the investigator requires direct and open access to the samples.

Immunization with chlamydial type III secretion antigens reduces vaginal shedding and prevents fallopian tube pathology following live C. muridarum challenge.

Chlamydia trachomatis infections in women are often asymptomatic and if left untreated can lead to significant late sequelae including pelvic inflammatory disease and tubal factor infertility. Vaccine development efforts over the past three decades have been unproductive and there is no vaccine approved for use in humans. The existence of serologically distinct strains or serovars of C. trachomatis mandates a vaccine that will provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of both structural and effector proteins which is an essential virulence factor for infection and intracellular replication. In this study we evaluated a novel fusion protein antigen (BD584) which consists of three T3SS proteins from C. trachomatis (CopB, CopD, and CT584) as a potential chlamydial vaccine candidate. Intranasal immunization with BD584 elicited serum neutralizing antibodies that inhibited C. trachomatis infection in vitro. Following intravaginal challenge with C. muridarum, immunized mice had a 95% reduction in chlamydial shedding from the vagina at the peak of infection and cleared the infection sooner than control mice. Immunization with BD584 also reduced the rate of hydrosalpinx by 87.5% compared to control mice. Together, these results suggest that highly conserved proteins of the chlamydial T3SS may represent good candidates for a Chlamydia vaccine.

Evaluation of anatomically designed flocked rectal swabs for molecular detection of enteric pathogens in children admitted to hospital with severe gastroenteritis in Botswana.

Two-hundred eighty matched bulk stool and anatomically designed flocked rectal swab samples were collected from children admitted to the hospital with acute diarrhea in Botswana. Their parents were asked about the acceptability of the swab collection method compared with bulk stool sampling. All samples underwent identical testing with a validated 15-target (9 bacterial, 3 viral, and 3 parasite) commercial multiplex PCR assay. The flocked swabs had a 12% higher yield for bacterial pathogen targets (241 versus 212; P = 0.003) compared with that of stool samples, as well as similar yields for viral targets (110 versus 113; P = 0.701) and parasite targets (59 versus 65; P = 0.345). One hundred sixty-four of the flocked swab-stool pairs were also tested with separate laboratory-developed bacterial and viral multiplex assays, and the flocked rectal swabs had a performance that was similar to that seen with commercial assay testing. Almost all parents/guardians found the swabs acceptable. Flocked rectal swabs significantly facilitate the molecular diagnosis of diarrheal disease in children.

Co-circulation of multiple genotypes of human rhinovirus during a large outbreak of respiratory illness in a veterans' long-term care home.

BACKGROUND: Human rhinoviruses (HRVs) are a well-recognized cause of long-term care home (LTCH) outbreaks of respiratory illness. However, there are limited data on the molecular epidemiology of the HRV types involved. OBJECTIVES: To determine whether a large respiratory outbreak in a LTCH was caused by a single type of HRV, and to describe the clinical impact of the outbreak. STUDY DESIGN: Nasopharyngeal swabs were collected from residents with one or more of the following: fever, cough, rhinitis, or congestion. Specimens were interrogated by multiplex PCR using the ResPlex II assay. Samples positive for HRV were then submitted for genotyping by partial sequence analysis of the 5' untranslated (UTR) and viral protein (VP) 1 capsid regions. RESULTS: Of 71 screened, 56 residents were positive for a HRV during an outbreak that lasted 5.5 weeks; 27 healthcare workers also had respiratory symptoms. Three residents were transferred to hospital and 2 died. Seven units in two wings of the LTCH were affected, resulting in 3152.5 resident unit closure days. Three different HRV genotypes were identified, although HRV-A1 dominated. CONCLUSIONS: This large outbreak of HRVs among residents and healthcare workers in a LTCH was associated with substantial resident and staff morbidity as well as significant unit closures. Multiple types of HRV were implicated but an HRV-A1 type dominated, warranting further investigation into viral determinants for virulence and transmission.

Multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of influenza A/H1, A/H3 and influenza B can provide a specimen-to-result diagnosis in 40 min with single genome copy sensitivity.

BACKGROUND: Rapid isothermal amplification methods have recently been introduced and they offer significant advantages over PCR. OBJECTIVE: To develop a rapid and sensitive M-LAMP assay for the detection of influenza A (H1 and H3) and B that does not require RNA extraction. STUDY DESIGN: We designed six primers targeting the matrix genes of influenza H1 and H3 and the NS1 gene of influenza B and developed a M-LAMP assay using a commercially available Master Mix and a real time fluorometer (Genie II, Optigene, UK) that displays real time amplification, time to positivity and amplicon annealing temperature (Tm). M-LAMP was evaluated against PCR by testing 202 nasopharyngeal (NP) specimens. RESULTS: Optimized M-LAMP was rapid with a mean amplification time of 12 min (compared with 90-120 min for PCR), had an analytical sensitivity of 1 genome equivalent (ge), and could distinguish influenza A including subtypes A/H1 and A/H3 from influenza B by Tm. M-LAMP detected 26/28 influenza A/H1, 27/27 influenza A/H3 and 39/39 influenza B specimens and had a combined sensitivity and specificity for detecting influenza (A and B) of 97.9% (92/94) and 100% (108/108), respectively. The rapid amplification time of LAMP coupled with a novel 10-min specimen preparation procedure consisting of vortexing and heating in M-Swab diluent (Copan Italia) provided a rapid result. CONCLUSIONS: M-LAMP had excellent sensitivity and specificity for detecting influenza A and B in NP specimens and when used together with a rapid specimen processing method provided a specimen-to-result diagnosis in 30 min.

Development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in 30 minutes.

Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature (Tm). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81/81) and specificity of 100% (194/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98°C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min.

Use of an improved quantitative polymerase chain reaction assay to determine differences in human rhinovirus viral loads in different populations.

Human rhinoviruses (HRV) frequently cause acute respiratory infections and chronic respiratory disease exacerbations. However, testing is not generally offered. We developed a modified HRV quantitative polymerase chain reaction (qPCR) assay to assess viral loads in the community and hospital patients. The assay had a lower limit of detection of 2 log(10) viral copies/mL and displayed linearity over 5 log(10) viral copies, with a lower limit of quantitation of 4 log(10) viral copies/mL. Mean viral loads (95% confidence interval) for hospitalized children, university students, and institutionalized elderly, were 7.08 log(10) viral copies/mL (6.7-7.5), 6.87 log(10) viral copies/mL (6.5-7.2), and 7.09 log(10) viral copies/mL (6.9-7.3), respectively (P = 0.67). Serial specimens of 14 university students showed a decrease of mean viral loads from 6.36 log(10) viral copies/mL on day 1 to 2.32 log(10) viral copies/mL 7 days past symptom onset (P < 0.001). Using an HRV qPCR, we showed that viral loads did not differ between the community and hospitalized populations and significantly decreased following symptoms onset in healthy individuals.

A confirmatory factor analytic approach on perceptions of knowledge and skills in teaching (PKST).

This paper reports the cross-validation of the factor pattern of the Perceptions of Knowledge and Skills in Teaching (PKST) survey, which was used to assess the self-perceived pedagogical knowledge and skills of pre-service and beginning teachers. The sample comprised 323 pre-service teachers enrolled in a 1-yr. post-graduate teacher education program in Singapore. The survey had 37 items distributed across six scales: student learning, lesson planning, instructional support, accommodating diversity, classroom management, and care and concern. A confirmatory factor analysis (CFA) was used to cross-validate the survey's factor pattern. The results showed that the model was an acceptable fit to the data. The PKST survey can thus be adapted by different teacher education programs to assess pre-service and beginning teachers' progress in developing their pedagogical knowledge and skills.

Evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses.

The clinical and public health importance of influenza and other respiratory viruses has accelerated the development of highly sensitive molecular diagnostics, but data are limited regarding preanalytical stages of diagnostic testing. We evaluated CyMol, an alcohol-based transport medium, for its ability to maintain specimen integrity for up to 21 days of storage at various temperatures; for its ability to inactivate virus; and for its compatibility with antigen- or nucleic acid-based diagnostics for respiratory viruses in clinical samples. In mock-infected samples, both universal transport medium (UTM-RT) and CyMol maintained equivalent viral quantities for at least 14 days at room temperature or colder, whereas a dry swab collection maintained viral quantities only if refrigerated or frozen. CyMol inactivated influenza virus within 5 min of sample immersion. UTM-RT- and CyMol-collected nasal swab specimens from 73 symptomatic students attending a campus health clinic were positive for a respiratory virus in 56.2% of subjects by multiplex PCR testing, including influenza A and B viruses, rhinovirus/enteroviruses, coronaviruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, and adenovirus. Detection by PCR was equivalent in UTM-RT- and CyMol-collected specimens and in self- and staff-collected swabs. Direct fluorescent antibody (DFA) testing was substantially less sensitive (23.3%) than multiplex PCR, and DFA testing from UTM-RT-collected swabs was more sensitive than that from CyMol-collected swabs. These data indicate that an alcohol-based transport medium such as CyMol preserves respiratory virus integrity, rapidly inactivates viruses, and is compatible with PCR-based respiratory diagnostics.

Expectant management versus immediate treatment for low-grade cervical intraepithelial neoplasia : a randomized trial in Canada and Brazil.

BACKGROUND: The optimal management strategy for women with low-grade biopsy-proven cervical intraepithelial neoplasia (CIN) is not clear. Our objective was to compare the effectiveness of regular colposcopic follow-up and treatment of progressive disease only versus immediate treatment. METHODS: Data were accrued between November 2000 and March 2006 for a noninferiority randomized clinical trial of 415 women with biopsy-proven grade 1 CIN from 8 Canadian and 2 Brazilian colposcopy clinics. Subjects were randomly assigned to either undergo immediate treatment with a loop electrical excision procedure (LEEP) or receive regular colposcopic follow-up for 18 months. The primary outcome was progression of disease to CIN 2 to 3 was based on histology obtained during 18 months of follow-up. Treatments were compared using differences of proportion with a 9% noninferiority margin. Analysis was conducted on the basis of intention-to-treat. RESULTS: An initial LEEP was performed on 179 women. Disease progression was found in 32. Easily controlled vaginal bleeding occurred in 16 (8.9%). During follow-up, disease progression was identified in 3 (1.7%) women in the immediate treatment arm and 9 (4.4%) in the colposcopic follow-up arm-a tolerable difference of 2.7% with 1-sided 95% confidence interval (CI) upper limit of 6.0%. Compliance with all 3 follow-up visits was 61% overall, but significantly worse in women ≤30 years of age (P < .05). CONCLUSIONS: The risk of progression to CIN grade 2 or 3 or cancer over 18 months was similar in the 2 treatment groups. In Canada and Brazil, follow-up for 18 months is a reasonable management strategy for women with persistent low-grade cytology who are found to have grade 1 CIN on referral for colposcopy and cervical biopsy.

Comparative evaluation of AMPLICOR HPV PCR and Linear Array assays on SurePath liquid-based Pap samples for the detection of high-risk HPV genotypes.

BACKGROUND: Persistent cervical infection with high-risk [HR] HPV is a causative factor for cancer. Liquid-based [L-Pap] Pap samples are convenient for HPV testing and SurePath samples have been least studied. Most HPV tests have multiple step protocols and testing laboratories experience large volumes of samples. OBJECTIVES: Using SurePath L-Pap residual samples the objectives were as follows: [1] to test the performance of AMP-HPV and LA-HPV. [2] To perform an agreement study between two laboratories for the AMP-HPV test and [3] to compare agreement of results between AMP-HPV and LA-HPV and HC2. STUDY DESIGN: Samples from 657 women were tested for Pap cytology then assayed for HR-HPV using AMP-HPV and LA-HPV tests. AMP-HPV performance was compared between 2 laboratories and agreement studies were conducted between AMP-HPV, LA-HPV and HC2. RESULTS: HR-HPV genotypes were associated with L-Pap readings as follows: HSIL 92% [23/25], LSIL 73.6% [162/220], ASCUS 70.4% [131/186], normal 31.9% [72/226]. More women less than 30 were infected with HR-HPV and multiple genotypes regardless of the L-Pap reading. AMP-HPV and LA-HPV testing had an overall raw agreement with each other of 84.2% [Kappa 0.66] and each had agreement of 94% with HC2 testing of 133 samples [Kappa 0.86/0.87]. AMP-HPV agreement between two laboratories was better at 93% [Kappa 0.84] compared to 76.1% [Kappa 0.40] when extraction was standardized. CONCLUSION: It is feasible to perform AMP-HPV and LA-HPV on SurePath samples to detect HR-HPV genotypes. HC2, AMP-HPV and LA-HPV showed strong agreement. The extraction component of the AMP-HPV assay needs careful attention to yield consistent results.

Development of a novel bead-based multiplex PCR assay for combined subtyping and oseltamivir resistance genotyping (H275Y) of seasonal and pandemic H1N1 influenza A viruses.

BACKGROUND: The identification of influenza A virus subtypes in clinical specimens is becoming increasingly important for clinical laboratories since seasonal H1N1, H3N2 and pandemic H1N1 influenza A viruses can have defined antiviral resistance patterns and subtyping can be used as a surrogate for antiviral resistance testing. OBJECTIVES: To develop a novel multiplex PCR (M-PCR) assay for the combined identification of influenza A subtype and oseltamivir resistance (H275Y) genotype in a combined assay format using Luminex xMAP™ technology. STUDY DESIGN: The M-PCR assay employed five degenerate primers to amplify the hemagglutinin (HA) and neuraminidase (NA) genes and eight tagged primers in a target specific primer extension reaction (TSPE). Products were analysed using xTAG™ beads containing specific anti-tag oligonucleotides. RESULTS: M-PCR correctly identified the subtype for 54/54 specimens that were influenza A positive, including 13/13 seasonal H3N2, 17/17 seasonal H1N1 and 24/24 pandemic H1N1 for both HA and NA genes. For oseltamivir resistance the M-PCR assay correctly identified 41/41 H1N1 viruses as oseltamivir sensitive (H275) or resistant (H275Y). Analysis of sequential specimens from two immunocompromised patients revealed the appearance of the H275Y allele at earlier time points after infection compared with Sanger sequencing. CONCLUSIONS: The combined M-PCR assay correctly subtyped seasonal and pandemic influenza A viruses and accurately detected the H275Y oseltamivir resistance allele. This assay should provide useful information to clinicians for appropriate patient management.