Multistix 10 sg

Venous blood was collected into three vaccutainer tubes, labelled and transported to the MRC Unit’s Clinical Diagnostic Laboratory Services for laboratory measurements. Renal function tests (blood in a plain serum tube) and fasting blood glucose (blood in a Sodium fluoride tube) were measured using the Clinical chemistry analyser, Cobas Integra 400 plus (Roche Diagnostics). The blood in an EDTA tube was used for: (1) CD4 cell counts measurements using either the FACSCount or FACSCalibur machine (Becton–Dickinson, USA), and (2) Plasma HIV-1 RNA load measured using the COBAS Ampliprep/Taqman V2.0 HIV-1 viral load assay (Roche Molecular Diagnostics (RMD), NJ, USA. All participants (apart from women in their menses) provided a fresh midstream urine specimen that was portioned in two plastic centrifuge tubes, one plain for urine creatinine and the other acidified for urine phosphates measurements; and both were measured using the Clinical chemistry analyser, Roche Integra 400 plus (Roche Diagnostics). Proteinuria was measured using a Siemens Multistix 10SG urine dipstick strip test that was read using Clinitek Status Analyzer (Siemens Healthcare Diagnostics).

Urine was collected by midstream collection (to eliminate potential contamination of urine by bacteria in the urethra) or urinary bladder catheterization (UBC, less prone for contamination than a midstream urine) at the outpatient clinic. The minimum sample volume was 6 mL. Urinalysis was performed using the dipstick method (Multistix^® 10 SG on a Clinitek Status+^® analyzer, Siemens Healthcare, The Hague, The Netherlands). Abnormal urinalysis was defined as a positive result for nitrite and leukocytes [17 (link)]. If an abnormal urinalysis was found the remaining urine was divided into three separate test tubes, one for urine culture and two for eNose analysis. All samples for eNose diagnostics were marked, directly frozen, and stored at −20 °C until further handling.

Patients were asked to monitor urinary ketosis using the urine strips provided at the time of enrolment (Multistix 10SG, Siemens Healthcare Diagnostics, Inc., Tarrytown NY). The presence of ketosis was assessed at weekly intervals from T0 to T8 and then at T12. Patients were instructed about the correct usage of the strips and to send a picture of the results to a dedicated email address for evaluation by one of the investigators. During the study period, patients were not allowed to use medications potentially responsible for unreliable urinary ketone results such as valproic acid, captopril, levodopa, ascorbic acid or nitrates (30–32 (link)).

Urine samples from patients and controls were analysed within 4 h of collection for nitrite and leukocyte esterase using Multistix® 10SG (Siemens) according to the manufacturer’s instructions. Urines with positive nitrite and leukocyte results, were regarded as having urinalysis suggestive of an UTI. Semi-quantitative urine culture was performed according to current UK National Standard Methods of Investigation, using both 1 μl and 10 μl aliquots. Urine samples were plated onto CPS ID 3 (CPS3) or CPS Elite chromogenic agar plates (bioMérieux), incubated at 37 °C, in room air for 18–24 h. The presence of presumptive E. coli was noted, and the bacterial counts enumerated. The remaining urine was filtered using a 0.45 μm filter (GE Healthcare Life Sciences), and aliquots stored at − 80 °C for cytokine analyses.
Single colony isolates of E. coli were typed using a multi-locus sequence typing scheme [23 (link)]. Genomic DNA was isolated using the standard procedure from the Bacterial Genome Kit (Sigma). PCR products generated using published primers, at the recommended temperatures, were purified using commercial PCR Purification kits (Sigma) prior to sequencing (SourceBioscience). Sequence results were processed using the pubmlst.org website by choosing the Achtman database to identify the allele number of each gene and the corresponding sequence type.

Vaginal pH was measured during the speculum examination by pressing commercial pH strips (pH Fix 3.6–6.1, Machery-Nagel) against the vaginal wall.
Detection of prostate-specific antigen (PSA), a marker for sexual intercourse within the past 24 hours [31 (link)] in vaginal swab fluid was performed using a chromatographic immune assay (the Seratec® PSA SemiQuant Cassette Test, Seratec, Gottingen, Germany) according to the manufacturer’s instructions. Pregnancy was assessed by testing urine with a rapid hCG test (QuickVue One-Step hCG Test (Kigali, Johannesburg) or Unimed First Sign hCG test (Mombasa)). Leucocytes and erythrocytes in urine were detected using dipsticks according to the manufacturer’s instructions (Siemens Multistix 10 sg in Kigali, Mission® urinalysis strips in Mombasa, and Neotest 4 Urine Dipstick in Johannesburg).