DNA Polymerase Chain Reaction Testing Research Articles

Molecular Identification of Kocuria Species Isolated from Pediatric Teaching Hospital in Sulaymaniyah, Iraq

Abstract Background: Kocuria species is now increasingly being recognized as an emerging human pathogen, suggesting that this genus has mostly been neglected or misidentified. Objective: This study attempts to use molecular techniques to correctly identify Kocuria species isolated from inpatient children, and to compare between the identification results obtained from two methods: VITEK 2 and 16S rRNA gene sequencing. Materials and Methods: VITEK 2, an automated system, was used to identify clinical isolates. Isolates identified as Kocuria spp. were submitted to DNA extraction and polymerase chain reaction testing. A partial sequence of the 16S rRNA gene was constructed and compared to GenBank sequences of the 16S rRNA gene. To confirm the strains’ identity, a phylogenetic tree including all Kocuria species was constructed. Results: Three isolates of Kocuria spp. were identified; the first two isolates were confirmed as K. kristinae by VITEK 2, pairwise 16S rRNA sequencing, and phylogenetic tree. However, the third isolate was identified as K. rosea, contradicting the pairwise 16S rRNA sequencing and phylogenetic tree identification as K. turfanensis. Conclusion: It is crucial to utilize molecular techniques for correctly identifying Kocuria species. In this study, it is found that K. turfanensis emerges as a pathogenic species and the first known cause of meningitis in humans and K. kristinae is identified as the first known cause of pediatric urinary tract infections.

Evaluation of the optimal sampling approach for HPV genotyping in circumcised heterosexual men with genital warts

BackgroundHuman papillomavirus (HPV) causes a serious socioeconomic burden globally. However, there is currently no consensus on the optimal sampling method for HPVDNA genotyping in circumcised heterosexual men. This study aimed to determine the diagnostic efficacy of 6 different anatomic sampling sites in HPV DNA polymerase chain reaction (PCR) testing of circumcised heterosexual men with genital warts. MethodsThe study included circumcised heterosexual men who presented to our clinic with complaints of genital warts. Swab samples were obtained from the penile shaft (PS), scrotum, coronal sulcus (CS), and external urethral meatus (EUM). First-void urine (FVU) and genital wart biopsy (GWB) were also tested for HPV DNA by PCR. ResultsA total of 32 patients (mean age: 36.9 ± 6.9 years) were included. None of the six samples studied was sufficient on its own to reveal all HPV types detected in a patient. When the samples were analyzed individually, GWB detected an average of 49.5% of total HPV types in a patient. This rate was 50.5% for PS, 40.4% for CS, 31.6% for scrotum, 26.3% for EUM, and 15.8% for FVU samples. The detection rate increased to 75.8% with combined testing of GWB and PS samples, 83.2% with GWB/PS/CS, 90.5% with GWB/PS/CS/scrotum, and 98.9% with GWB/PS/CS/scrotum/EUM samples. ConclusionNo single anatomic region or sample type can detect all HPV types present in circumcised heterosexual men by PCR assay. The detection rate approaches 99% when wart biopsy is combined with swab sampling of the penile shaft, coronal sulcus, scrotum, and external urethral meatus.

Human papilloma virus-related oral and oropharyngeal squamous cell carcinoma: An assessment of histomorphological features

ABSTRACT Background: Human papillomavirus (HPV) has emerged as an important cause of oral and oropharyngeal squamous cell carcinomas (OSCC and OPSCC). Cancers with HPV as a causative agent are seen to exhibit certain specific histomorphological features. Aim: This study aims to describe the histomorphology of oral and oropharyngeal cancers and discuss their association with HPV. Material and Methods: Hospital-based prospective study done over 31 months (December 2018–April 2021), including a total of 90 cases of OSCC and OPSCC. They were subjected to detailed histopathological evaluation, DNA polymerase chain reaction testing for testing of HPV association, followed by the analysis of data by statistical methods. Results: Out of a total of 90, 73 cases were OSCC and 17, OPSCC. A total of 15 cases were HPV positive. HPV status was found to be significantly associated with purely nonkeratinizing tumors and hybrid/mixed squamous cell carcinomas, pushing the pattern of invasion, absence of perineural invasion, presence of tumor infiltrating lymphocytes, moderate-to-dense host lymphocyte response, loose stromal response, absence of tumor budding, and high mitotic rate (>20/10 hpf). Conclusion: HPV-positive OSCC and OPSCC have distinct histomorphological features and careful analysis of the same can assist in identifying these types of cancers better and thus help in prognostication and treatment.

Immunohistochemical Evaluation of p16 and p53 in Oral and Oropharyngeal Squamous Cell Carcinoma with Special Regard to Human Papillomavirus Status.

Incidence of human papillomavirus (HPV)-associated oral and oropharyngeal squamous cell carcinomas (OSCC and OPSCC) is on a rising trend globally and has specific therapeutic implications. HPV-related tumors have a distinct pathogenetic mechanism targeting p16 and p53 both. However, there are limited studies evaluating p16 and p53 expression in combination. The aim of the study is to evaluate p16 and p53 immunohistochemical expression pattern in OSCC and OPSCC, with special reference to HPV association. This was a hospital-based prospective study done over 22 months (September 2018 to June 2020), including a total of 54 cases of OSCC and OPSCC. They were subjected to clinicopathological evaluation, p16 and p53 immunohistochemistry, and DNA polymerase chain reaction testing for testing of HPV association, followed by analysis of data by statistical methods. Out of 54, 43 cases were OSCC and 11 cases were OPSCC. A total of nine cases were HPV positive. HPV association was found to be significant with tonsil as primary site, age range between 40 and 60 years, and absence of tobacco or alcohol habit. Presence of HPV infection was also significantly associated with p16 overexpression, in combination with p53 negativity. The findings indicate that p16 overexpression combined with a negative p53 expression can be used for HPV detection and the former alone may be used as diagnostic marker in OPSCC only. HPV-associated OSCC and OPSCC are a unique subset of cancers, and using combination of molecular biomarkers could help in diagnosis and prognosis.

Long-Term Lacticaseibacillus rhamnosus GG Storage at Ambient Temperature in Vegetable Oil: Viability and Functional Assessments.

Vegetable oils with varying saturated fat levels were inoculated with Lacticaseibacillus rhamnosus GG (LGG), subjected to different heat treatments in the absence and presence of inulin and stored for 12 months at room temperature. After storage, the heat-treated probiotics actively grew to high concentrations after removal of the oils and reculturing. The bacterial samples, regardless of aerobic or anaerobic conditions and treatment methods, showed no changes in their growth behavior. The random amplified polymorphic DNA-polymerase chain reaction, antimicrobial, morphology, and motility tests also showed no major differences. Samples of LGG treated with a higher antioxidant content (Gal400) showed reduced inflammatory and anti-inflammatory properties. These findings have been confirmed by metabolite and genome sequencing studies, indicating that Gal400 showed lower concentrations and secretion percentages and the highest number of single nucleotide polymorphisms. We have shown proof of concept that LGG can be stored in oil with minimum impact on probiotic in vitro viability.

An Early Warning Mobile Health Screening and Risk Scoring App for Preventing In-Hospital Transmission of COVID-19 by Health Care Workers: Development and Feasibility Study.

BackgroundHospitals have been identified as very high-risk places for COVID-19 transmission between health care workers and patients who do not have COVID-19. Health care workers are the most at-risk population to contract and transmit the infection, especially to already vulnerable patients who do not have COVID-19. In low-income countries, routine testing is not feasible due to the high cost of testing; therefore, presenting the risk of uncontrolled transmission within non–COVID-19 treatment wards. This challenge necessitated the development of an affordable intermediary screening tool that would enable early identification of potentially infected health care workers and for early real time DNA–polymerase chain reaction testing prioritization. This would limit the contact time of potentially infected health care workers with the patients but also enable efficient use of the limited testing kits.ObjectiveThe aims of this study are to describe an early warning in-hospital mobile risk analysis app for screening COVID-19 and to determine the feasibility and user-friendliness of the app among health care workers.MethodsThe primary result of this research project was the development of a mobile-based daily early warning system for in-hospital transmission of COVID-19. Overall, the Early Warning System for In-Hospital Transmission of COVID-19 (EWAS) mobile app was found to be feasible, with over 69% of the health care workers having logged more than 67% of the required times. Over 93% of the participants reported that the tool was easy to use.ResultsThe primary result of this research project was the development of a mobile-based daily early warning system for in-hospital transmission of COVID-19. Overall, the Early Warning System for In-Hospital Transmission of COVID-19 (EWAS) mobile app was found to be feasible, with 69% of the health care workers (69/100) having logged more than 67% of the required times. Of the 100 participants, 93 reported that the tool was easy to use.ConclusionsThe EWAS mobile app is a feasible and user-friendly daily risk scoring tool for preventing in-hospital transmission of COVID-19. Although it was not designed to be a diagnostic tool but rather a screening tool, there is a need to evaluate its sensitivity in predicting persons likely to have contracted COVID-19.

Evaluating maternal characteristics and DNA polymerase chain reaction birth testing of neonates born with HIV in a KwaZulu-Natal referral hospital – missed opportunities?

Background. Congenital HIV infection has declined in South Africa with an expanded programme to decrease perinatal HIV transmission. Understanding the challenges within this programme can identify opportunities for improvements. There is an opportunity with birth HIV DNA polymerase chain reaction testing to expedite very early treatment of neonates born with HIV in referral hospitals. Objectives. This study was aimed at describing the characteristics of mothers of neonates born with HIV as well as the evaluation of the access and availability of birth HIV DNA PCR results within a referral hospital. Method. This study was a retrospective chart review of all documented neonates born with HIV delivered between 1 January 2017 and 30 June 2019 at King Edward VIII Hospital, Durban, KwaZulu-Natal. The birth HIV DNA PCR results provided through institutional programmes were analysed, and the clinical characteristics of mothers of neonates born with HIV were described. Results. Review of hospital-based records, during the 30-month study period, identified 6 902 (40.02% of 17 243 total live births) birth HIV DNA PCR tests having been done. During the sample period, documented positive, rejected or indeterminate results were analysed. These results indicated only 37 (0.54%) of known HIV-exposed neonates who were tested, to have a documented positive birth HIV DNA PCR result. Fifty-nine neonates had either indeterminate or rejected results. Negative HIV DNA PCR results could not be verified. Analysis of the mothers of neonates with documented HIV-positive results indicated high mean viral loads of these mothers (52 343 copies/mL) and most were diagnosed in the current pregnancy or at delivery. Conclusion. Review of the characteristics of mothers of neonates born with HIV within a public referral hospital reiterates that late HIV diagnosis is common in this high-risk group. Birth HIV DNA PCR results may fail to adequately identify all positive neonates timeously for very early initiation of antiretroviral treatment.

A Study on the Prevalence and Source of Staphylococci and Methicillin-resistant Staphylococcus aureus Causing Superficial Incisional Surgical Site Infection

Staphylococcus aureus and coagulase-negative staphylococci (CoNS) are common causatives of superficial incisional surgical site infection (SSI). The source of the pathogens is still not fully diagnosed whether it is endogenous or exogenous particularly with regard to the extent of its resistance to antibiotics. Therefore, this study is designed to determine the rate of infection, the source of pathogens, and the extent of their resistance to antibiotics. For this purpose, pre-, intra- and post-operative swabs from the nasal and skin of patients undergoing surgeries and samples from the hospital environment have been collected and processed for isolation and identification of staphylococci. Bacterial analysis and antibiotic susceptibility profiles of the isolates are assessed by unweighted pair group method with arithmetic mean (UPGMA) analysis based on random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and disc diffusion test for antibiotics susceptibility profile. The microbiological and PCR results indicate that SSIs are found in 113/512 (22.07%), Staphylococcus spp. rated 67/512 (13.09%) of infections. Further analysis indicates that S. aureus, CoNS, and both of them were causes SSI with different rates 41/67 (61.2%), 23/67 (34.3%), and 3/67 (4.5%), respectively. Results of RAPD-PCR for 70 isolates reveal that 52/70 (74.28%) of SSIs are from endogenous source, followed by 10/70 (14.29%) and 8/70 (11.43%) from hospitals acquired and undetermined sources, respectively. Moreover, results of antibiotic susceptibility test reveal that 24/44 (54.5%) of isolates belong to methicillin-resistant Staphylococcus aureus; from both endogenous and exogenous sources with 13/24 (54.17%) and 11/24 (45.83%), respectively.

Correlation of p16 immunostaining in cell-blocks with corresponding tissue specimens for squamous cell carcinomas of the oropharynx.

The goal of this study was to evaluate the performance of p16 staining in cell-blocks vs tissue specimens as a surrogate marker for human papillomavirus (HPV) status in oropharyngeal squamous cell carcinomas. Head and neck squamous cell carcinoma cases presenting as a neck mass with a p16 result on cytology and corresponding tissue specimens (1 January 2014 to 30 June 1920) were included in the study. The following were assessed from cell-block material: number of tumour clusters, percentage of tumour cells with p16 staining, and presence of staining in clusters vs single cells. Results were compared to tissue p16 status. Results of any other ancillary HPV testing were also noted. Forty-two head and neck squamous cell carcinoma neck metastases (35 oropharyngeal, five non-oropharyngeal, and 2 unknown primaries) were identified. The p16 staining pattern in cell-blocks was seen in single cells (27.6%), clusters (44.8%), or both (27.6%). The percentage of tumour cells staining for p16 in cell-blocks was much lower than in corresponding tissue specimens. There were four false negatives and one false positive (concurrent HPV DNA polymerase chain reaction testing was positive in cytology and surgical material). Compared to tissue, the cut-off for p16 interpretation in cell-blocks is substantially lower and staining may be present in single cells or clusters. In 96.9% of cases, any p16 staining in cell-blocks correlated with positive p16 staining in surgical specimens. However, a negative or discrepant p16 result on cell-block should prompt confirmatory HPV studies, as false negative p16 staining in cell-blocks is high.

Functional Medicine-Based Dentistry: A Clinical Conversation with Mary Ellen S. Chalmers, DMD, and Robert Rountree, MD

Functional Medicine-Based Dentistry: A Clinical Conversation with Mary Ellen S. Chalmers, DMD, and Robert Rountree, MD

Inclusion of an E7 DNA Amplification Test Improves the Robustness of Human Papillomavirus-Associated Oropharyngeal Squamous Cell Carcinoma Diagnosis.

BackgroundThe rise in human papillomavirus (HPV) infection rates over the last few decades in the USA has contributed to a significant increase in the overall incidence of patients diagnosed with squamous cell carcinoma of the head and neck. These head and neck carcinomas develop in the oropharynx, with more than 90% of them caused by infection with high-risk HPV type 16. Patients diagnosed with HPV-induced oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis and treatment response than those diagnosed with head and neck cancers caused by alcohol consumption and tobacco use. To identify patients with HPV-positive OPSCC, new guidelines recommend positive staining of oropharyngeal tissues for p16 INK4a (p16) by immunohistochemistry (IHC). Herein we discuss the testing algorithm that was adopted to address discrepant results between p16 IHC and a DNA in situ hybridization (ISH) test used routinely to diagnose HPV-positive OPSCC patients.MethodsA DNA polymerase chain reaction (PCR) test that amplifies HPV16 and HPV18 E7 was developed to aid in the diagnosis of HPV-positive OPSCC in a subset of patients. Specimens from these patients stained positive for p16 by an IHC test, but negative for high-risk HPV by a commercial DNA ISH test. Moreover, these results did not match the histopathological characteristics of the specimens, nor the clinical presentations of the patients.ResultsOf 21 patients’ specimens that were tested for p16 by IHC, 11 specimens showed concordant results with the high-risk HPV 16/18 DNA ISH test. Whereas, in eight p16 IHC positive specimens, HPV viral DNA was not detected by HPV16/18 DNA ISH, and two specimens were not tested by DNA ISH. When these eight p16 IHC positive specimens with discrepant p16 IHC and DNA ISH results were further tested by DNA PCR, six specimens showed concordance with p16 IHC with positive results for HPV16 E7, while two specimens were negative for HPV16 E7 by DNA PCR. All tested specimens were negative for HPV18 E7 by DNA PCR. Thus, the addition of the HPV16 and HPV18 E7 DNA PCR test identified a significant number of false negative test results by the HPV16/18 DNA ISH test and likely several false positive results by p16 IHC.ConclusionsInclusion of an HPV16 E7 DNA PCR test improved the robustness of HPV-associated OPSCC diagnosis in patients with discrepant results from p16 IHC staining and a DNA ISH test, and identified patients for proper management with less misclassification.

2334. Saliva Screening for Congenital Cytomegalovirus Infection in the Neonatal Intensive Care Unit: Beware!

BackgroundCongenital cytomegalovirus (cCMV) infection is the most common cause of non-genetic sensorineural hearing loss in infancy. Screening of newborns for cCMV infection has been performed utilizing saliva due to ease of collection and high sensitivity. Positive saliva screens for CMV DNA by polymerase chain reaction (PCR) testing has been reported to occur secondary to breast milk feeding without signifying congenital infection. The NICUs of Nationwide Children’s Hospital recently began universal saliva screening of all admissions. We report 3 neonates whose saliva CMV screen was positive yet the urine CMV PCR test was negative in order to inform CMV screening strategies.MethodsRetrospective review of the electronic health records of neonates admitted to the neonatal intensive unit (NICU) at Nationwide Children’s Hospital, Columbus, OH who had CMV detected by PCR from saliva specimens but not from urine. Pertinent demographic and clinical data were obtained.ResultsThree female neonates had a positive saliva CMV DNA PCR test but urine CMV PCR was negative. The first infant (gestational age [GA] 34 weeks, birth weight [BW]1790 Grams) was a monochorionic diamionic twin gestation and born vaginally with unknown duration of rupture of membranes (ROM). At 16 days of age, the infant had a positive saliva CMV PCR but a negative urine CMV PCR test. The infant received maternal milk. The twin’s CMV PCR tests of saliva and urine were negative. The second infant (GA 38 weeks, BW 2952 grams) was born vaginally after 9 hours of ROM. On the first day of age, the infant had a positive saliva CMV PCR test that was followed by a negative urine CMV PCR on the third day of age. The infant had not been breastfed. The third infant (GA 33 weeks, BW 1762 grams) was born by C-section delivery with ROM at delivery. Saliva CMV PCR screen was positive on the second day of age but urine PCR was negative twice (days 5 and 7). All 3 infants had no signs/symptoms of cCMV infection and passed the newborn hearing screen.ConclusionTesting of saliva for CMV DNA by PCR is not always confirmatory for cCMV infection as contamination of saliva specimens with CMV could result from exposure to maternal milk and possibly vaginal secretions. Definitive diagnosis of cCMV infection requires additional confirmatory testing preferably with urine.Disclosures All authors: No reported disclosures.

Adapting the HIV Infant Tracking System to Support Prevention of Mother-to-Child Transmission of HIV in Kenya: Protocol for an Intervention Development Pilot Study in Two Hospitals.

BackgroundDespite progress to expand access to HIV testing and treatment during pregnancy in Kenya, gaps still remain in prevention of mother-to-child transmission of HIV (PMTCT) services. This study addresses the need for effective and scalable interventions to support women throughout the continuum of care for PMTCT services in low-resource settings. Our research team has successfully implemented the HIV Infant Tracking System (HITSystem), a Web-based, system-level intervention to improve early infant diagnosis (EID) outcomes.ObjectiveThis study will expand the scope of the HITSystem to address PMTCT services to bridge the gap between maternal and pediatric HIV services and improve outcomes. This paper describes the intervention development protocol to adapt and pilot an HITSystem version 2.0 to assess acceptability, feasibility, and preliminary PMTCT outcomes in Kenya.MethodsThis is a 3-year intervention development study to adapt the current HITSystem intervention to support a range of PMTCT outcomes including appointment attendance, antiretroviral therapy (ART) adherence, hospital deliveries, and integration of maternal and pediatric HIV services in low-resource settings. The study will be conducted in 3 phases. Phase 1 will elicit feedback from intervention users (patients and providers) to guide development and refinement of the new PMTCT components and inform optimal implementation. In Phase 2, we will design and develop the HITSystem 2.0 features to support key PMTCT outcomes guided by clinical content experts and findings from Phase 1. Phase 3 will assess complete PMTCT retention (before, during, and after delivery) using a matched randomized pilot study design in 2 hospitals over 18 months. A total of N=108 HIV-positive pregnant women (n=54 per site) will be enrolled and followed from their first PMTCT appointment until infant HIV DNA Polymerase Chain Reaction testing at the target age of 6 weeks (<7 weeks) postnatal.ResultsFunding for this study was received in August 2015, enrollment in Phase 1 began in March 2016, and completion of data collection is expected by May 2019.ConclusionsThis protocol will extend, adapt, and pilot an HITSystem 2.0 version to improve attendance of PMTCT appointments, increase ART adherence and hospital-based deliveries, and prompt EID by 6 weeks postnatal. The HITSystem 2.0 aims to improve the integration of maternal and pediatric HIV services.Trial RegistrationClinicalTrials.gov NCT02726607; https://clinicaltrials.gov/ct2/show/NCT02726607 (Archived by WebCite at http://www.webcitation.org/78VraLrOb)International Registered Report Identifier (IRRID)DERR1-10.2196/13268

Tracking progress on the health status and service delivery outcomes for neonates and children in the Metro West geographic service area of the Cape Metropole, 2010 - 2015

Background. Monitoring the health status of populations of children is one of the building blocks of the health system. The provision of an indicator dashboard with disaggregated data that are collected over time can be used to gauge the performance of the health system, guide the allocation of resources and prioritise health interventions within districts. Objectives. To determine neonatal and child mortality, morbidity and health service outcomes over a 6-year period in the Metro West geographic service area (GSA) of the Cape Town metropole. Methods. A dashboard with key indicators was developed using existing data. Results. From 2010 to 2015, there was a decrease in the perinatal mortality rate from 31.7 to 24.8 per 1 000 deliveries, and the early neonatal and neonatal mortality rates from 7.8 and 8.6 to 7.0 and 8.2 per 1 000 live births, respectively. The main obstetric causes of early neonatal deaths were antepartum haemorrhage (22 - 24%) and unexplained intrauterine death (13 - 16%); the main neonatal causes were immaturity (17 - 34%), congenital abnormalities (23 - 29%) and hypoxia (23 - 26%). Under-five mortality decreased in 2013 from 25 to 22 per 1 000 live births, with the main causes being neonatal conditions (32%), pneumonia (25%), congenital abnormalities (9%), injuries (8%) and diarrhoea (8%). Fifty percent of child deaths were out of hospital, with pneumonia and diarrhoea accounting for more than half of these. There was an improvement in health service coverage rates in 2015: immunisation <1 year old (99%); measles second dose (85%), pneumococcal third dose (100%) and rotavirus second dose (100%); maternal antiretroviral coverage (90%); HIV testing in mothers (93%); HIV DNA polymerase chain reaction testing in babies (97%); and a decrease in HIV transmission (2%). Exclusive breastfeeding coverage rates at 14 weeks, and vitamin A supplementation at 12 - 59 months, were only 30% and 44%, respectively, across the GSA. Conclusion. There was a decrease in perinatal, early neonatal, infant and under-five mortality in Metro West over the 6 years. Further reductions in under-five mortality will require focusing on interventions to reduce neonatal and out-of-hospital deaths across the service delivery platform. Home visits to at-risk mothers and infants by community health workers could prevent out-of-hospital deaths and improve exclusive breastfeeding and vitamin A coverage. This will require increasing the number of community health workers and broadening their scope of practice.

Caregiver socioemotional health as a determinant of child well-being in school-aged and adolescent Ugandan children with and without perinatal HIV exposure.

Caregiver socio-emotional attributes are major determinants of child well-being. This investigation in vulnerable school-aged Ugandan children estimates relationships between children's well-being and their caregiver's anxiety, depression and social support. Perinatally HIV-infected, HIV-exposed uninfected and HIV-unexposed Ugandan children and their caregivers were enrolled. Perinatal HIV status was determined by 18months of age using DNA-polymerase chain-reaction test; status was confirmed via HIV rapid diagnostic test when children were 6-18years old. Five indicators of child well-being (distress, hopelessness, positive future orientation, esteem and quality of life (QOL)) and caregivers' socioemotional status (depressive symptoms, anxiety and social support) were measured using validated, culturally adapted and translated instruments. Categories based on tertiles of each caregiver psychosocial indicator were defined. Linear regression analyses estimated percent differences (β) and corresponding 95% confidence intervals (CI) for child well-being in relation to caregiver's psychosocial status. As per tertile increment, caregiver anxiety was associated with 2.7% higher distress (95%CI:0.2%, 5.3%) and lower self-esteem/QOL (β=-1.3%/-2.6%; 95%CI: -5.0%,-0.2%) in their children. Child distress/hopelessness increased (β=3.3%/7.6%; 95%CI:0.4%, 14.7%) and self-esteem/QOL decreased 2.3% (β=-2.3%/-4.4%; 95%CI: -7.2%, -1.3%) as per tertile increment in caregiver depression. Higher caregiver social support was associated with lower distress and higher positive outlook (β=3%; 95%CI:1.4%, 4.5%) in their children. HIV-infected/exposed children had most caregiver depression-related QOL deficit (β=-5.2%/-6.8%; 95%CI: -12.4%, -0.2%) and HIV-unexposed children had most caregiver social support-related enhancements in positive outlook (β=4.5%; 95%CI:1.9%, 7.1%). Caregiver anxiety, depressive symptoms and low social support were associated with worse well-being in school-aged and adolescent children. Improvement of caregiver mental health and strengthening caregiver social support systems may be a viable strategy for improving well-being of vulnerable children and adolescents in this setting.

Infant HIV testing at birth using point-of-care and conventional HIV DNA PCR: an implementation feasibility pilot study in Kenya

BackgroundInfant HIV diagnosis by HIV DNA polymerase chain reaction (PCR) testing at the standard 6 weeks of age is often late to mitigate the mortality peak that occurs in HIV positive infants’ first 2–3 months of life. Kenya recently revised their early infant diagnosis (EID) guidelines to include HIV DNA PCR testing at birth (pilot only), 6 weeks, 6 months, and 12 months postnatal and a final 18-month antibody test. The World Health Organization (WHO) approved point-of-care (POC) diagnostic platforms for infant HIV testing in resource-limited countries that could simplify logistics and expedite infant diagnosis. Sustainable scale-up and optimal utility in Kenya and other high-prevalence countries depend on robust implementation studies in diverse clinical settings.MethodsWe will pilot the implementation of birth testing by HIV DNA PCR, as well as two POC testing systems (Xpert HIV-1 Qual [Xpert] and Alere q HIV-1/2 Detect [Alere q]), on specimens collected from Kenyan infants at birth (0 to 2 weeks) and 6 weeks (4 to < 24 weeks) postnatal. The formative phase will inform optimal implementation of birth testing and two POC testing technologies. Qualitative interviews with stakeholders (providers, parents of HIV-exposed infants, and community members) will assess attitudes, barriers, and recommendations to optimize implementation at their respective sites. A non-blinded pilot study at four Kenyan hospitals (n = 2 Xpert, n = 2 Alere q platforms) will evaluate infant HIV POC testing compared with standard of care HIV DNA PCR testing in both the birth and 6-week windows. Objectives of the pilot are to assess uptake, efficiency, quality, implementation variables, user experiences of birth testing with both POC testing systems or with HIV DNA PCR, and costs.DiscussionThis study will generate data on the clinical impact and feasibility of adding HIV testing at birth utilizing POC and traditional PCR HIV testing strategies in resource-limited settings. Data from this pilot will inform the optimal implementation of Kenya’s birth testing guidelines and of POC testing systems for the improvement of EID outcomes.Trial registrationClinicalTrials.gov, NCT03435887. Registered 26 February 2018.

Assessment of the Use of PCR as an Early Diagnostic Indicator of Bovine Tuberculosis in Dairy Farms

Background: Zoonotic tuberculosis has been mainly associated with the consumption of bovine dairy products and its control has been prioritized by the Food and Agriculture Organisation. Objective: As such, the aim of this study is to assess whether the polymerase chain reaction (PCR) can be used as an early diagnostic indicator of Mycobacterium bovis infection in bovine dairy farms. Methods: Milk samples (n=78) were collected from all the animals older than 8 years of age, in all (n=4) bovine dairy farms located in a specific agricultural region of Greece, with high prevalence in bovine tuberculosis. Detection of DNA belonging to slow growing members of the genus Mycobacterium was conducted in pooled samples using two PCR assays targeting 16S-rRNA and 65-kDa heat shock protein (hsp65). Randomly selected PCR products were submitted to sequence analysis for confirmation of the specificity of the amplification process. DNA isolation and PCR testing were conducted in compliance with ISO17025 accreditation requirements. Results: The overall percentage of positivity was 47.7%, and ranged among farms from 0% to 76.9%. PCRpositive animals were detected in both farms that were at the time of investigation positive with the tuberculin skin test (TST), whereas the only farm with a negative TST record tested also negatively with PCR. Interestingly, one farm that was negative with TST since 2012 but had a long prior record of high level TST-positivity, tested positively with PCR. Conclusion: In conclusion it can be stated that PCR can be used for the detection of mycobacteria in pooled samples of milk collected from the older animals of a dairy farm, as an early and sensitive diagnostic indicator. This can support TST monitoring for the control of bovine tuberculosis, and improve detection of farms, in which routine monitoring should be intensified. The specific approach offers significant practical benefits that compensate for the additional cost of PCR.

Evaluation of the diagnostic utility of the new clinical case definition of pertussis - experience from sentinel and hospital-based pertussis surveillance

Introduction/Objective. Global surveillance systems use different clinical case definitions of pertussis. The aim of this study was to identify sign and symptom combinations with best relation with laboratory confirmed pertussis. Methods. A one-year prospective observational study, proposed by the Global Pertussis Initiative (GPI) for three age groups (0?3 months, four months to nine years, and ? 10 years) was performed in Novi Sad to evaluate the performance of the clinical case definition of pertussis. Laboratory confirmation of B. pertussis infection was obtained using the DNA polymerase chain reaction (PCR) or ELISA serology tests. Results. From October 1, 2013 to September 30, 2014, 103 (32.3%) out of 319 participants with suspected pertussis had laboratory-confirmed pertussis. Combined whooping, post-tussive emesis, and worsening of symptoms at night was the best predictor of pertussis in outpatients aged four months to nine years (positive likelihood ratio (LR+) 11.6), while among inpatients of the same age group it was apnoea (LR+ 13.5). The LR+ in outpatients aged ?10 years for combinations of apnoea and post-tussive emesis, or a combination of whooping and sweating episodes between paroxysms and post-tussive emesis was 16.8, while among in-patients LR+ was &lt; 2.3 for all combinations in the same age group. Conclusions. The GPI case definitions for pertussis are good predictors for laboratory-confirmed pertussis and are useful for the purpose of pertussis surveillance.

Prevention of Mother-to-Child Transmission and Early Real-Time DNA Polymerase Chain Reaction Results Among HIV-Exposed Infants in Bujumbura, Burundi

Prevention of mother-to-child transmission (PMTCT) programmes aim to both eliminate vertical transmission of HIV and optimise the health and survival of infants born with HIV. Therefore, early infant diagnosis (EID) of HIV infection via DNA polymerase chain reaction (PCR) testing is a key component of PMTCT programming. We assessed the effectiveness of EID and PMTCT interventions at health-care facilities in Bujumbura, Burundi.This was a prospective analytical study of infants born to HIV-positive mothers on antiretroviral therapy (ART), who were followed from December 2016 to March 2017 at 3 centres providing PMTCT services in Bujumbura. Babies enrolled in this study received once-daily nevirapine from birth through to 6 weeks of life, after which HIV DNA PCR testing was conducted.Of 122 HIV-exposed infants, 60 were boys and 62 were girls. The mother-to-child transmission rate at 6 weeks of life was 0.9%. Eighty-three (68%) of the women had commenced ART before pregnancy and 39 (32%) during pregnancy. The mean CD4 lymphocyte count was 653±308 cells/μl. Ninety-two (75.4%) of the pregnancies were planned, and 98 (80%) of the births were via spontaneous vaginal delivery. After birth, 111 (91.0%) infants were exclusively breastfed, and 11 (9.0%) infants received exclusive replacement feeding.There was a low rate of transmission of HIV from women taking ART to children who were given nevirapine for the first 6 weeks of life. Infants of HIV-positive women can live healthy lives free from HIV infection if their mothers participate in PMTCT programmes.

Cotransmission of Malaria and HIV to Infants of Mothers Coinfected With Malaria and HIV in the HAART Era in Benin City, Nigeria.

Malaria and HIV are vertically transmitted to infants. In an era where specific interventions are available to reduce the burden of malaria in pregnancy and vertical transmission of HIV, we examined the transmission and cotransmission of HIV and malaria to infants of mothers coinfected with malaria and HIV. A cross-sectional analytic study performed on 101 HIV/malaria-coinfected mothers and their infants for whom DNA polymerase chain reaction results were available. Blood film for malaria parasites was performed on cord blood and peripheral blood on days 1, 3, and 7 in the newborns. Maternal peripheral blood film for malaria parasite was performed at delivery, and placental tissue was obtained for confirmation of placental malaria by histology. All infants received DNA polymerase chain reaction testing for HIV at 6-8 weeks of life. Cotransmission of malaria and HIV occurred in 2 (1.98%) infants. Vertical transmission of HIV occurred in 7 (7%) babies, whereas congenital malaria was present in 32 (31.7%) babies. The transmission of HIV and presence of congenital malaria were not significantly associated with each other P = 1.000. Of the 88 babies of mothers who commenced highly active antiretroviral treatment (HAART) before pregnancy, 5 (5.7^) had HIV, which was significantly less than 2 (40%) of 5 babies of mothers who commenced HAART during pregnancy P = 0.04. Among mothers coinfected with malaria and HIV, vertical transmission of malaria and HIV was not associated with each other. HAART reduces vertical transmission of HIV especially if started before pregnancy.