Added Value of Next Generation over Sanger Sequencing in Kenyan Youth with Extensive HIV-1 Drug Resistance.

HIV-1 drug resistance testing in children and adolescents in low-resource settings is both important and challenging. New (more sensitive) drug resistance testing technologies may improve clinical care, but evaluation of their added value is limited. We assessed the potential added value of using next-generation sequencing (NGS) over Sanger sequencing for detecting nucleoside reverse transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations (DRMs). Participants included 132 treatment-experienced Kenyan children and adolescents with diverse HIV-1 subtypes and with already high levels of drug resistance detected by Sanger sequencing. We examined overall and DRM-specific resistance and its predicted impact on antiretroviral therapy and evaluated the discrepancy between Sanger sequencing and six NGS thresholds (1%, 2%, 5%, 10%, 15%, and 20%). Depending on the NGS threshold, agreement between the two technologies was 62% to 88% for any DRM, 83% to 92% for NRTI DRMs, and 73% to 94% for NNRTI DRMs, with more DRMs detected at low NGS thresholds. NGS identified 96% to 100% of DRMs detected by Sanger sequencing, while Sanger identified 83% to 99% of DRMs detected by NGS. Higher discrepancy between technologies was associated with higher DRM prevalence. Even in this resistance-saturated cohort, 12% of participants had higher, potentially clinically relevant predicted resistance detected only by NGS. These findings, in a young, vulnerable Kenyan population with diverse HIV-1 subtypes and already high resistance levels, suggest potential benefits of more sensitive NGS over existing technology. Good agreement between technologies at high NGS thresholds supports their interchangeable use; however, the significance of DRMs identified at lower thresholds to patient care should be explored further. IMPORTANCE HIV-1 drug resistance in children and adolescents remains a significant problem in countries facing the highest burden of the HIV epidemic. Surveillance of HIV-1 drug resistance in children and adolescents is an important public health strategy, particularly in resource-limited settings, and yet, it is limited due mostly to cost and infrastructure constraints. Whether newer and more sensitive next-generation sequencing (NGS) adds substantial value beyond traditional Sanger sequencing in detecting HIV-1 drug resistance in real life settings remains an open and debatable question. In this paper, we attempt to address this issue by performing a comprehensive comparison of drug resistance identified by Sanger sequencing and six NGS thresholds. We conducted this study in a well-characterized, vulnerable cohort of children and adolescents living with diverse HIV-1 subtypes in Kenya and, importantly, failing antiretroviral therapy (ART) with already extensive drug resistance. Our findings suggest a potential added value of NGS over Sanger even in this unique cohort.

Characteristics of HIV-infected children seen in Western Kenya.

OBJECTIVES: To describe the characteristics and outcomes of children registered for care in a large HIV care programme in Western Kenya. DESIGN: A retrospective descriptive study. SETTING: USAID-AMPATH HIV clinics in health centres; district and sub-district hospitals; Moi Teaching and Referral Hospital in Western Kenya. SUBJECTS: HIV-infected children below age of 15 years seen in a network of 18 clinics in Western Kenya. INTERVENTIONS: Paediatric HIV diagnosis and care including treatment and prevention of opportunistic infections and provision of combination antiretroviral therapy (CART). MAIN OUTCOME MEASURES: Diagnosis, clinical stage and immune status at enrollment and follow-up; hospitalisation and death. Descriptive statistical analyses and chi square tests were performed. RESULTS: Four thousand and seventeen HIV-infected children seen between June 2002 and April 2008. Median age at enrollment was four years (0-14.2 years), 51% girls, 25% paternal orphans, 10% total orphans and 13% maternal orphans. At enrollment, 25% had weight-for-Age Z scores (WAZ) > or = -1 and 21% had WAZ scores < or = 3. Orphaned children had worse WAZ scores (p=0.0001). Twenty five per cent of children were classified as WHO clinical stage 3 and 4, 56% were WHO clinical stages 1 and 2 with 19% missing clinical staging at enrollment. Cough (25%), gastroenteritis (21%), fever (15%), pneumonia (10%) were the commonest presenting features. Twenty six per cent had been diagnosed with tuberculosis and only 25% started on cotrimoxazole preventive therapy (CPT). Median CD4% at enrollment was 16% (0-64%); latest recorded values were 22% (0-64). Sixty four per cent were on cART (cART+), median age at start was 5.4 (014.4 years). The median initial CD4% among cART+ was 13 (0-62) compared to 24 (0-64) for those not on ART (cART-). Median CD4% for cART+ improved to 22% (0-59); whereas cART- was 23% (0-64) at last appointment. During the period of follow-up, one fifth (19%) of children on cART were lost to follow-up compared to slightly over one third (37%) for those not on cART. Thirty four percent were hospitalised; 41% diagnosed with pneumonia. Six per cent of 4017 were confirmed dead. CONCLUSIONS: HIV-infected children were enrolled in care early in childhood. Orphanhood was prevalent in these children as were gastroenteritis, fever, pneumonia and advanced immuno-suppression. Orphans were more likely to be severely malnourished. Only a quarter of children were put on cotrimoxazole preventive therapy. Children commenced on cART late but responded well to treatment. Loss to follow-up was less prevalent among those on cART.

'Triangulating' AMPATH: demonstration of a multi-perspective strategic programme evaluation method.

Clinical programmes are typically evaluated on operational performance metrics of cost, quality and outcomes. Measures of patient satisfaction are used to assess the experience of receiving care, but other perspectives, including those of staff and communities, are not often sought or used to assess and improve programmes. For strategic planning, the Kenyan HIV/AIDS programme AMPATH (Academic Model Providing Access to Healthcare) sought to evaluate its performance in 2006. The method used for this evaluation was termed 'triangulation', because it used information from three different sources--patients, communities, and programme staff. From January to August 2006, Indiana University external evaluators and AMPATH staff gathered information on strengths, weaknesses and suggestions for improvement of AMPATH. Activities included in-depth key-informant semi-structured interviews of 26 AMPATH clinical and support staff, 56 patients at eight clinic sites, and seven village health dialogues (mabaraza) at five sublocations within the AMPATH catchment area. Data sources included field notes and transcripts of translated audio recordings, which were subjected to qualitative content analysis. Eighteen recommendations for programme improvement emerged, including ten from all three respondent perspectives. Three recommendations were cited by patients and in mabaraza, but not by staff. Triangulation uncovered improvement emphases that an internal assessment would miss. AMPATH and Kenyan Ministry of Health leadership have deliberated these recommendations and accelerated strategic change actions, including rural satellite programmes, collaboration with village-based workers, and door-to-door village-based screening and counselling.

Nasopharyngeal Streptococcus pneumoniae among under-five year old children at the Moi Teaching and Referral Hospital, Eldoret, Kenya.

OBJECTIVES: To determine the prevalence, risk factors and antibiotic sensitivity of streptococcus pneumoniae carried in the upper respiratory tract of children. DESIGN: A cross-sectional study on consecutive clients. SETTING: Maternal Child Health Clinic (MCH) at Moi Teaching and Referral Hospital (MTRH) in western Kenya. SUBJECTS: Seventy eight of children attending Maternal Child Health Clinic between March 10th 2003 and July 11th 2003. MAIN OUTCOME MEASURES: Upper airway carriage status, ventilation, housing, age, illness, sensitivity patterns. RESULTS: Fifty six percent were boys; the median age was six months (range 1-42 months). Streptococcus pneumoniae carriage rate was in 28 (35.9%) cases. Fifty two percent of S. pneumoniae were resistant to penicillin, 25% to ampicillin and 78% to cotrimoxazole. There was significant association between the type of floor with pneumococcal carriage (p = 0.009) with people living in earth floor houses being five times more likely to be pneumococcal carriers as compared to those living in cement floor houses. CONCLUSIONS: A significant resistance of S. pneumoniae to penicillin, ampicillin and cotrimoxazole was found. Earth floored houses may increase susceptibility to upper airway S. pneumoniae carriage. RECOMMENDATION: Similar studies should be conducted in other parts of Kenya in order to learn about susceptibility patterns and associated risk factors, including floor type, in the country and tailor better treatment regimens.

Prevalence of asthma, allergic rhinitis and dermatitis in primary school children in Uasin Gishu district, Kenya.

OBJECTIVE: To establish the relative increase in the prevalence of asthma, allergic rhinitis and eczema in primary school children aged 13-14 years over a six year interval. DESIGN: Cross sectional comparative study. SETTING: Primary schools in three rural divisions at Uasin Gishu district in the Rift Valley Province of Kenya. METHODS: Three thousand two hundred and fifty eight children aged 13-14 years from seventy two primary schools in Uasin Gishu district were studied using the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. All children in the selected schools in this age range whose parents gave consent were included. RESULTS: There were 47.4% males and 52.6% females as compared to 48.7% and 51.3% respectively in 1995. The cumulative prevalence for wheezing, rhinitis, itchy eyes and dermatitis was 23.6%, 43%, 24.1% and 28.5% respectively which were higher than for 1995 which were 21.2%, 32.4%, 11.8% and 13.8% respectively (p = 0.001). The period prevalence rates for wheezing, rhinitis and eczema were 13.8%, 31.4% and 21.3% respectively as compared to 10.2%, 25.3% and 14.4% respectively for 1995 (p = 0.001). The prevalence of asthma, allergic rhinitis and eczema was 12.6%, 38.6% and 28.5% respectively in 2001 compared to 6.6%, 14.9% and 13.9% respectively in 1995 (p = 0.001). CONCLUSION: There was a significant increase in the prevalence of asthma, allergic rhinitis and eczema in children in the study population over the last six years.