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.

Clinical and laboratory predictors of cholelithiasis in patients with sickle cell anaemia.

Cholelithiasis is a common clinical condition in patients with sickle cell disease and there are conflicting reports on laboratory indices useful in predicting those patients who are likely to have gallstones. There is however lack of similar studies from Kenya. We therefore studied the role of clinical (Body Mass Index), haematological (reticulocyte count, haemoglobin level), and biochemical (serum bilirubin: direct and indirect, serum alkaline phosphatase, serum transaminase) indices in predicting sickle cell anaemia patients likely to develop gallstones. A cross sectional descriptive study was conducted from October 1993 to December 1994 on consecutive male and female patients of all ages with homozygous sickle cell disease (HbSS) confirmed by cellulose acetate paper electrophoresis. A total of 64 patients aged between three and 37 years were recruited into the study. They were classified into two groups: stone formers and non-formers. The difference in the two groups with respect to clinical, haematological and biochemical indices were determined by Chi-square contingency test. Body mass index (BMI), reticulocyte count and alkaline phosphatase were found to have a significant positive association with increased likelihood of gallstone formation at p values of 0.004, 0.007 and 0.007, respectively. The rest of the study indices had no association. The cut-off points were reticulocyte counts above ten per cent and alkaline phosphatase levels above 13 K.A. units. Though sickle cell anaemia patients with BMI > 20 had significant increased likelihood of cholelithiasis, we could not determine its cut-off value.

Fibrinolytic activity in adult Kenyan patients with sickle cell disease.

The sickling of erythrocytes increases viscosity and reduces the rate of both local circulation and arterio-venous transit time. This causes occlusion of capillaries by "microthrombin". The occlusion is implicated in the multiplicity of vaso-occlusive complications of both acute and chronic nature. Whether or not anticoagulant therapy is warranted in these states has remained debatable. There is no clear evidence that there is an inherent coagulation disorder. Earlier studies indicate that fibrinolysis is normal in steady state sickle cell disease but decreased during sickle cell crisis. We studied fibrinolytic activity or euglobulin clot lysis time (ECLT) in 47 subjects, twenty six of them with homozygous sickle cell (HbSS) disease and 21 healthy controls of whom eighteen had the HbAA and three had the HbAS genotypes. The sex distribution was sixteen males to ten females for the HbSS and 13 males to eight females for the controls. Age range was 17-35 years for the HbSS and 25-35 for the controls. Means for basic haematologic parameters including platelets were also performed. Mean clot lysis time (MCLT) was 3.75 hours for the HbSS and 1.91 hours for the controls (normal range 1 1/2-4 hours). The difference in ECLT between patients and controls was statistically significant (p < 0.001). Fifty three and a half per cent of the HbSS fell above the upper limit of normal MCLT. All the 21 controls fell within normal range. There were also statistically higher values (p < 0.001) in HbSS as compared to the controls with regard to MCV, WBC count, and platelet count.

Upper gastrointestinal mucosal lesions in dyspeptic patients with homozygous sickle cell disease in Kenya.

A case controlled study comprising 51 patients with homozygous sickle cell (HbSS) disease who complained of dyspepsia and 41 age and sex matched non-HbSS control dyspeptic patients was carried out, to look at upper gastrointestinal mucosal lesions associated with dyspepsia. Upper gastrointestinal tract (UGIT) endoscopy was performed with gastric control biopsy taken for histology. Thirty two (62.3%) of the HbSS or sickle cell anaemia (SCA) patients had upper gastrointestinal pathology at endoscopy as compared to 17 (41.5%) of controls. The difference was significant at p = 0.042. Bile reflux (47%) was the predominant abnormal morphological finding in SCA patients while duodenal ulcer was the most common morphological finding in dyspeptic controls. The prevalence of duodenal ulcer in controls (22%) though higher than in SCA patients (9.8%), was not statistically significant p = 0.18. Gastric ulcer was not found in SCA patients. Duodenal ulcer was commoner in males than females in both cases and controls with a ratio of 4:1 and 3.5:1 respectively. Only four (7.8%) SCA patients and one (2.4%) of controls had normal mucosa at histology, the rest had evidence of histological gastritis. We could not draw any correlation between non-steroidal anti-inflammatory drugs (NSAIDS) use and UGIT findings. Since the proportion of SCA cases with UGIT abnormalities was significantly high, we recommend that dyspeptic SCA patients undergo UGIT investigations including endoscopy to maximise their clinical care.

Higher resistance to Plasmodium falciparum infection in patients with homozygous sickle cell disease in western Kenya.

Sickle haemoglobin (HbS) is considered to be protective against malaria. Malaria is fatal in homozygous sickle cell (HbSS) disease. In a cross-sectional survey by alkaline. Hb-electrophoresis of 766 residents of Western Kenya near Lake Victoria, 20 were found to have HbSS disease, 120 sickle cell trait (HbAS) and 626 the normal genotype (HbAA). Blood slides for malarial parasites (MPs) were performed in 728 cases, i.e. 592 HbAAs, 116 HbASs and all 20 HbSSs. Malaria parasites were found in 261 (35.8%) HbAAs, 42 (5.8%) HbASs and 4 (0.5%) HbSSs. Malaria prevalences per genotypic group were 44.1% (261 out of 592) in HbAAs, 36.2% (42 out of 116) in HbASs, and 20% (4 out of 20) in HbSSs. The relative risk of malarial infection was 0.33 in the HbSSs compared to both HbAAs and HbASs. It seems that the protection conferred by HbS against malaria is more marked in HbSS disease than in HbAS and is HbS-content related, and that the balanced polymorphism in the HbS-malaria relationship is maintained-by higher mortality risk of HbAAs due to malaria and high mortality risk of HbSSs caused by complications of HbSS.

Platelet function in patients with sickle cell anaemia in Nairobi.

From July 1990 to January 1991 we studied platelet functions in 55 indigenous Kenyan patients (23 males and 32 females) with sickle cell anaemia (SCA) in steady state (SCASS) and in 20 (11 males and 9 females) SCA patients in vaso-occlusive sickle cell crisis (VSCC). A control group of 50 healthy (23 males and 27 females) individuals matched for age and sex was also studied. Platelet aggregation time to ADP in SCASS (57.2 +/- 39.1) and in VSCC (31 +/- 11.1) were more prolonged (p < 0.05) compared to controls (12.7 +/- 5.2). It was also significantly more prolonged (p < 0.05) in VSCC than in SCASS. Platelet adhesiveness time was 31.1 +/- 13.7 seconds in SCASS, 30.9 +/- 11.1 in VSCC, and 37.7 +/- 13.0 in controls and was significantly lower in both SCA groups (p < 0.05) but there was no significant difference between the two SCA groups themselves. Clot retraction was 52.8 +/- 6.9 in SCASS, 53.6 +/- 10.7 in VSCC, and 45.9 +/- 8 in controls and was significantly higher in both SCA groups than in controls (p < 0.05). There was no significant difference between the two SCA groups themselves. We conclude that platelet function is deranged in indigenous Kenyan patients with SCA.

Sensorineural hearing loss in patients with sickle cell anaemia in Kenya.

The auditory function of sixty two Kenyan sickle cell anaemia patients aged seven to thirty years was compared to age-matched fifty five healthy controls with haemoglobin AA. Sensorineural hearing loss of 30 db and above was demonstrated in 25 (40%) of sickle cell anaemia patients and in three (5.5%) control subjects. Both sexes were equally affected. Bilateral lesion registered in 16%. Hearing threshold level was normal in 97% of the sickle cell group. High frequencies were commonly affected with hearing loss of 30-40 db. Two cases with severe unilateral deafness at all frequencies had severe recruitment suggestive of cochlea lesion. There were no cases of acoustic reflex decay in all study patients. The hearing loss was of slow onset. The high risk of deafness in Kenyan sickle cell anaemia patients may be a reflection of the severe course of the disease due to specific Kenyan haematological profile (haplotype 20 with low Hb F level), the level of medical care available and the geographical distribution in the tropics together with other factors.

The presence of sickle cells in the peripheral blood film. Specificity and sensitivity of diagnosis of homozygous sickle cell disease in Kenya.

The final diagnosis of sickle cell disease (SCD) is established by haemoglobin (Hb) electrophoresis. The test, however, is expensive and absent in most hospitals in Kenya. We studied sensitivity, specificity and cost-effectiveness of the peripheral blood film (PBF) in diagnosing sickle cell anaemia (SCA), the most common type of sickle cell disease (SCD) in Kenya. The PBF can be done even in dispensaries. The study was performed in SCA endemic western Kenya in 767 subjects during 12 months. Hb level, WBC count, PBF, sickle cell test (SCT) and cellulose acetate paper electrophoresis (CAPE) were performed. In the PBF, presence of sickle cells was pathognomonic for SCA. SCA was found in 21, sickle cell trait in 120, and normal genotype in 616 subjects. Sensitivity of the PBF versus SCT and CAPE to detect SCA was 76% with a specificity of 99.7%. The PBF was cheaper than both methods by 31.1%.

Survey of sickle disease in Kenya.

In Kenya and most of Eastern Africa, sickle haemoglobin (HbS) is the pre-dominant beta-globin chain abnormality; homozygous sickle cell disease, (SCA), is the predominant form of sickle cell disease. Although the prevalence of sickle cell trait (SCT) in Kenya was known, the magnitude of SCA was yet to be established. We performed a national survey in all hospitals from November 1987 to May 1990 and found 3605 cases with SCA. Age was recorded for 2821 patients. Seventy-seven per cent of these patients were below the age of fifteen. The oldest patient was a 50-year-old female. The paediatric to adult ratio was 3:1. More than 80% of the patients were of Luo or Luhya ethnic origin (Luo 58.4%, Luhya 23.9%). There was a discrepancy between SCT rate (SCTr) and the percentage distribution of SCA patients per province. The Kambes of the Mijikenda group in the Coast Province with the highest SCTr (35%) constituted only 8.5%, but the Luos with a SCTr of 28%, 58% of the total SCA patient population in Kenya. We found reports of SCA in the Somali and Turkana, in whom no SCT had been found previously.

Neurological complications of sickle cell anaemia at KNH: a five year retrospective study.

In a five year retrospective study of 360 patients with homozygous (SS) sickle cell disease, eighteen (5%) were found to have neurological complications. Their ages ranged from 7 months to 21 years with a mean of 11.1 +/- 6 years. Of those with neurological complications, twelve (67%) of the patients had cerebrovascular accident, six (33.3%) convulsions, three visual disturbance; one sensorineural deafness, one cerebellar degeneration and the last one confusion and hallucinations. Four of the patients had multiple neurological complications. There was only one patient with recurrence of neurological complications. Two patients were hypertransfused and up to the end of the study period none of them had any recurrence. The pattern of neurological complications are similar to that observed in other studies. However, in this study, there were fewer recurrences of neurological complications.

Management of sickle cell disease: a review.

The treatment of sickle cell disease is still mainly symptomatic. However, there have been major advances in understanding the pathophysiology of sickling syndromes. This in turn has led to a proper approach in the management of the sickle cell patient. This review summarises current treatment regimens in sickle cell disease.

The efficacy and safety of a controlled release formulation of salbutamol in the management of patients with asthma in Nairobi, Kenya.

The treatment of asthma in Africa is influenced by cultural and environmental factors as well as the availability of drugs. Poor compliance with regard to long-term maintenance treatment of chronic asthma is also a problem in Africa. The present study reports on 45 patients from 2 centres in Nairobi treated with 8 mg twice daily of an oral controlled release formulation of salbutamol ("Volmax"). The treatment produced a significant improvement in lung function measured by the PEFR compared with baseline data on previous therapy. Controlled release salbutamol was rated as effective or very effective by 84.2% of patients and for 81.6% of patients their physicians preferred this preparation to the therapy used before the study. Side effects were infrequent and usually occurred during the first few days of treatment. The study which is the first report in Africa to assess this novel formulation of salbutamol in a group of African patients, demonstrates that controlled release salbutamol 8 mg administered twice daily is safe and effective, offering benefits over current therapies in the treatment of asthma.

Short course chemotherapy for pulmonary tuberculosis in routine service in Kenya.

The results are reported of the application of a short-course regimen for pulmonary tuberculosis in routine service in three districts in Kenya. All patients were treated in hospital for one month receiving Streptomycin, Isoniazid, Rifampicin and Pyrazinamide daily and followed by Rifampicin and Isoniazid daily on outpatient basis for five months. There was no failure of chemotherapy among 194 patients with pre-treatment sensitive strains at the end of chemotherapy. Six (3.9%) had a doubtful status but none relapsed during follow-up. Among 123 patients assessed after completion of follow-up only one (0.8%) relapsed and 16 of 22 patients with pretreatment resistant strains had a favourable response at the end of chemotherapy and follow-up. The therapeutic results achieved are excellent under programme conditions though of necessity with some amount of supervision that is not routinely available. Considering the advantages of short course regimens, a case for the choice of an appropriate short regimen to be used nationwide in the near future is made.

Maternal and foetal outcome of pregnant patients with sickle cell anaemia at Kenyatta National Hospital Nairobi. A retrospective study.

In a retrospective study we assessed pregnancy outcome in relation to sickle haemoglobin (HbS) and anaemia at Kenyatta National Hospital, Nairobi (KNH) from 1981-1986. There were 36% maternal and 45% foetal losses in sickle cell disease (SCD) pregnants and 7% foetal losses in sickle cell trait (HbAS) from 26 HbS-related pregnancies. 11 had homozygous sickle cell (SS) disease and 15 had HbAS. Age ranges for both groups were comparable. Mean haemoglobin-level for SS disease patients was 7.8 gm/dl (SD +/- 1.68), for AS patients 7.8 gm/dl (SD +/- 2.1). These maternal and foetal losses are quite high. Anaemia alone does not satisfactorily account for the higher losses in SS pregnancies. Other contributory factors need elucidation and intervention.

An effective traditional medicine for bronchial asthma: clinical demonstration and preliminary toxicological evaluation.

This clinical paper reports the investigations conducted on a traditional African anti-asthmatic formulations and evaluates its toxicological and phyto-chemical effects.