Conceived and designed the experiments: LWW EL MAK MKN HN WM RFB BO. Performed the experiments: GKK LWW MKN. Analyzed the data: JMW EL MAK LWW. Contributed reagents/materials/analysis tools: MAK MKN RFB. Wrote the paper: LWW MAK EL MKN JMW. Collected clinical specimens and managed clinical data: HN WM. Coordinated field activities: BO WM.
The authors have declared that no competing interests exist.
Understanding shedding patterns of 2009 pandemic influenza A (H1N1) (pH1N1) can inform recommendations about infection control measures. We evaluated the duration of pH1N1 virus shedding in patients in Nairobi, Kenya.
Nasopharyngeal (NP) and oropharyngeal (OP) specimens were collected from consenting laboratory-confirmed pH1N1 cases every 2 days during October 14–November 25, 2009, and tested at the Centers for Diseases Control and Prevention-Kenya by real time reverse transcriptase polymerase chain reaction (rRT-PCR). A subset of rRT-PCR-positive samples was cultured.
Of 285 NP/OP specimens from patients with acute respiratory illness, 140 (49%) tested positive for pH1N1 by rRT-PCR; 106 (76%) patients consented and were enrolled. The median age was 6 years (Range: 4 months–41 years); only two patients, both asthmatic, received oseltamivir. The median duration of pH1N1 detection after illness onset was 8 days (95% CI: 7–10 days) for rRT-PCR and 3 days (Range: 0–13 days) for viral isolation. Viable pH1N1 virus was isolated from 132/162 (81%) of rRT-PCR-positive specimens, which included 118/125 (94%) rRT-PCR-positive specimens collected on day 0–7 after symptoms onset. Viral RNA was detectable in 18 (17%) and virus isolated in 7/18 (39%) of specimens collected from patients after all their symptoms had resolved.
In this cohort, pH1N1 was detected by rRT-PCR for a median of 8 days. There was a strong correlation between rRT-PCR results and virus isolation in the first week of illness. In some patients, pH1N1 virus was detectable after all their symptoms had resolved.
The 2009 pandemic influenza A (H1N1) (pH1N1) virus has been circulating worldwide since the initial cases were detected in the United States in April 2009
Much of the information on pH1N1 virus shedding is from studies conducted in North America, Asia, and Europe
The first case of pH1N1 in Kenya was detected in June 2009
This study was approved by both the Institutional Review Board of CDC-Atlanta and the Ethical Review Committee of the Kenya Medical Research Institute (KEMRI).
Informed written consent was obtained from all participants. For children, written consent was obtained from parents or guardians.
We conducted the study in a large informal urban settlement in Nairobi, in an existing population-based surveillance system that has been previously described
An ILI case was defined as a patient with fever ≥38°C and cough or sore throat for all ages. For SARI, the definitions varied by age group. In children <5 years old, SARI was defined as cough or difficulty breathing along with a danger sign; unable to drink or breast feed, lethargic or unconscious, vomiting everything, convulsions, nasal flaring, grunts, chest indrawing, stridor in a calm child or oxygen saturation ≤90%. In people ≥5 years, SARI was defined as fever ≥38°C plus cough or shortness of breath or chest pain or oxygen saturation ≤90%.
Patients who came to Tabitha Clinic between October 14, 2009, and November 25, 2009, who had ILI or SARI and NP/OP specimens that tested positive for pH1N1 were recruited for the study. Patients who were positive for pH1N1 were contacted at their homes by field workers and requested to return to the clinic for follow up every 2 days. During the initial visit and subsequent visits, a trained clinician recorded signs and symptoms and collected both NP and OP specimens. Patients who had 2 consecutive rRT-PCR negative specimens were released from the study. A patient was considered to have pH1N1 RNA if a specimen collected on that day was positive for pH1N1 by rRT-PCR. Patients were not followed over the weekend.
Specimens were obtained according to the following procedure: for OP specimens, a sterile nylon-tipped plastic-shafted OP swab touched the back of the oropharyngeal mucosal membrane for 3–5 seconds and then was placed into a cryovial containing 1 mL of viral transport media (VTM). VTM was prepared at the KEMRI/ CDC-K laboratory using the standard WHO protocol that includes bovine serum albumin and veal infusion broth supplemented with amphotericin B (
The rRT-PCR testing was performed using the CDC pH1N1 testing protocol
Later, in order to evaluate the concordance between rRT-PCR testing and viral culture for pH1N1, influenza virus isolation in Mardin-Darby Canine Kidney (MDCK) cells was attempted for the first rRT-PCR-positive specimen, the last rRT-PCR-positive specimen, and the first rRT-PCR-negative specimen from each enrolled patient. Confluent monolayers of MDCK cell line growing in T25 cell culture flasks were used. Media were removed from the flasks. Each flask was inoculated with 100 µL of specimen and inoculum was allowed to adsorb on the cells for 30 min at 37°C. Following adsorption, 6 mL of Viral Growth Medium [DMEM supplemented with HEPES buffer, bovine serum albumin, L-Glutamine, trypsin, and antibiotic-antimycotic (Sigma-Aldrich, Inc., St. Louis, MO, USA)] was added and cultures were observed daily for cytopathic effect (CPE) for up to 6 days, at which point all the cultures were harvested. Cultures that showed observable CPE by microscopy were subjected to hemagglutination assay using guinea pig red blood cells. Supernatants from cultures that showed no CPE were subjected to a second passage, following which cultures with observable CPE were subjected to hemagglutination assay. Cultures that had no observable CPE after the second passage were considered negative by viral isolation.
Data were collected in three ways. First, information related to the patient's initial visit was recorded on computers by clinicians at Tabitha Clinic. Second, during the first follow-up clinic visit and subsequent visits, information about signs and symptoms was recorded on paper questionnaires and entered into a Microsoft Access database. The signs and symptoms recorded were fever, temperature ≥38°C, cough, sneezing, runny nose, vomiting, diarrhea, chest pain, and difficulty breathing for all patients. In addition, the following symptoms were recorded for patients ≥5 years; earache, sore throat, headache, chills, muscle pain, and abdominal pain. Third, laboratory data were recorded into Freezerworks software (Dataworks Development, Inc.). Data were combined into a central database for analysis.
We considered the date of onset of illness to be the date on which the patient reported the first symptom associated with the illness, according to the history taken during the patient's initial clinic visit. For possible initial symptoms, we considered fever, cough, and difficulty breathing for patients <5 years old. In patients ≥5 years old, these symptoms and sore throat were considered as possible initial symptoms. Patients were considered to have no detectable virus RNA once they had two consecutive specimens that tested negative for pH1N1 by rRT-PCR. We defined the last day of virus detection as the midpoint between the dates of collection of the last rRT-PCR pH1N1-positive specimen and the first rRT-PCR pH1N1-negative specimen. We were unable to follow all patients until they had two negative rRT-PCR tests because some did not return to the clinic for the necessary follow-up visits. The time to cessation of pH1N1 virus detection for these patients was right censored at their last pH1N1 rRT-PCR-positive test date. Thus, the duration of pH1N1 virus detection for these patients was only known to occur after their drop out time and was treated accordingly in the survival analysis below.
Comparisons of duration of pH1N1 virus detection between groups, described by the median and mean, were conducted using the log-rank test. A multivariable Cox regression model was used to determine the association between gender and age with duration of pH1N1 virus detection. A Kaplan-Meier plot was used to determine duration of pH1N1 virus detection. Odds ratios were calculated to determine the odds of isolating pH1N1 virus from rRT-PCR-positive specimens collected over time. All analyses were conducted with SAS version 9.1. Findings were considered statistically significant if the resulting p-value was <0.05.
A total of 481 patients with respiratory illness were seen at Tabitha Clinic between October 14, 2009 and November 25, 2009; 175 (36%) had ILI and 306 (64%) had SARI. Specimens were collected from 285 (59%) of these patients, and 140 (49%) of these were positive for pH1N1 by rRT-PCR. Of the 140 pH1N1-positive patients, 106 (76%) consented to participate in the study; 85 (80%) completed the study (
The median age of the study population was 6 years (Range: 4 months–41 years); 60 (57%) were females (
Characteristics | rRT-PCR Results | |
Age in years | pH1N1-positiveN (%) | pH1N1-negativeN (%) |
<5 | 43 (40) | 90(62) |
5–14 | 41 (39) | 32(22) |
≥15 | 22 (21) | 23(16) |
|
||
Male | 46(43) | 71(49) |
Female | 60(57) | 74(51) |
|
||
HIV | 2(13) |
3(14) |
Asthma | 2 (2) | 0 |
Treated with oseltamivir | 2(2) | N/A |
*16 people were tested for HIV.
From the 106 patients enrolled to the study, 449 specimens were collected including the initial specimen from which pH1N1 was detected; 200 (44%) were positive for pH1N1 by rRT-PCR. Virus isolation was attempted in 262 of the 449 (58%) specimens collected. The specimens cultured included the 106 initial rRT-PCR-positive specimens, 56 final rRT-PCR-positive specimens (fifty patients had only one rRT-PCR-positive specimen), and100 first rRT-PCR-negative specimens from the enrolled patients. No rRT-PCR-negative specimens were obtained from 6 of the patients. Of the 162 rRT-PCR-positive specimens, pH1N1 virus was isolated from 132 (81%) specimens. Of rRT-PCR-positive specimens taken on day 0–3 after illness onset, 81/85 (95%) were culture-positive, as were 37/40 (93%) taken on day 4–7, 11/20 (55%) taken on day 8–10, and 3/17 (18%) taken ≥11 days after illness onset (
The median number of days pH1N1 virus RNA was detectable in patients specimens 8 days (95% CI: 7–10 days) after symptom onset. There was no statistically significant difference in the duration of pH1N1 virus detection between children <5 years old and those 5 to 14 years or persons ≥15 years. There was no difference in the median duration of pH1N1 virus detection between males and females. In the majority (58%) of patients, pH1N1 virus RNA was detected for ≥7 days, and in 16% of patients for ≥14 days (
There were 200 patient visits that were associated with pH1N1-positive rRT-PCR results. We were able to link laboratory results with clinic visit data on symptoms for 186/200 patients. From the linkable patient visits, there were 69/186 pH1N1-positive rRT-PCR results from patients <5 years old and 117/186 from patients ≥5 years old. The most common signs and symptoms in the pH1N1-positive patients <5 years old were cough [52/69 (75%)], runny nose [47/69 (68%)], reported fever [44/69 (64%)], and temperature ≥38°C [40/69 (58%)]. The most common signs and symptoms in the patients ≥5 years old were cough [83/117 (71%)], temperature ≥38°C [63/117 (54%)], reported fever [61/117 (52%)], and runny nose [60/117 (51%)] (
Patients <5 yrs | Patients ≥5 yrs | |||||||||
Days after symptom onset | Days after symptom onset | |||||||||
Symptom | 0–3 | 4–7 | 8–11 | 12–20 | 0–3 | 4–7 | 8–11 | 12–20 | ||
Total Patient visits | 69 | 36 | 15 | 14 | 4 | 117 | 50 | 29 | 19 | 19 |
Reported Fever (%) | 44(64) | 35(97) | 7(47) | 2(14) | 0(0) | 61(52) | 45(90) | 16(55) | 0(0) | 0(0) |
Temp (≥38) (%) | 40(58) | 34(94) | 6(40) | 0(0) | 0(0) | 63(54) | 49(98) | 14(48) | 0(0) | 0(0) |
Cough (%) | 52(75) | 33(92) | 12(80) | 5(36) | 1(25) | 83(71) | 45(90) | 23(79) | 9(47) | 6(32) |
Sneezing (%) | 24(35) | 19(53) | 4(27) | 1(7) | 0(0) | 31(18) | 20(40) | 9(31) | 2(11) | 0(0) |
Runny nose (%) | 47(68) | 31(86) | 8(53) | 6(43) | 2(50) | 60(51) | 32(64) | 17(59) | 7(37) | 4(22) |
Vomiting (%) | 17(25) | 13(36) | 3(20) | 1(7) | 0(0) | 9(8) | 6(12) | 3(10) | 0(0) | 0(0) |
Diarrhea (%) | 8(12) | 6(17) | 2(13) | 2(14) | 0(0) | 5(4) | 2(4) | 1(3) | 1(5) | 1(5) |
Chest pain (%) | 0(0) | 0(0) | 0(0) | 0(0) | 0(0) | 23(20) | 13(26) | 7(24) | 3(16) | 0(0) |
Difficulty Breathing (%) | 6(9) | 4(11) | 2(13) | 2(14) | 0(0) | 7(6) | 4(8) | 1(3) | 2(11) | 0(0) |
Ear problem (%) |
6(5) | 5(10) | 0(0) | 1(5) | 0(0) | |||||
Sore throat (%) |
34(29) | 22(44) | 8(28) | 3(16) | 1(5) | |||||
Headache |
48(41) | 30(60) | 15(52) | 2(11) | 1(5) | |||||
Chills |
22(19) | 14(28) | 7(24) | 1(5) | 0(0) | |||||
Joint pain |
15(13) | 8(16) | 6(21) | 1(5) | 0(0) | |||||
Muscle pain |
10(9) | 4(8) | 5(17) | 1(5) | 0(00 | |||||
Abdominal pain |
8(7) | 5(10) | 2(7) | 1(5) | 0(0) |
*This information was not obtained from patients <5 years old.
Eighteen (17%) pH1N1 enrolled patients who were initially symptomatic and pH1N1-positive continued to be rRT-PCR-positive after all of their signs and symptoms had resolved for a median of 9 days (interquartile range 7–10 days) after all signs and symptoms had resolved. Viable pH1N1 virus was isolated from specimens obtained from 7/18(39%) of these patients.
This study describes the duration of pH1N1 virus RNA detection, the correlation between rRT-PCR-positive results and virus isolation, and clinical symptoms associated with pH1N1 virus detection in patients living in a densely populated community with low socioeconomic status in sub-Saharan Africa. Pandemic H1N1 RNA was detected from respiratory specimens by rRT-PCR for a median duration of 8 days but up to 17 days after symptoms onset. This duration of virus RNA detection is similar to the median of 6 and 8 days reported in studies from China and Hong Kong, respectively
Cough, fever, and runny nose were the most common clinical symptoms associated with pH1N1 virus RNA detection. This is similar to studies carried out in Korea
Of the 2 HIV-positive patients in the study, the patient who was not on antiretroviral therapy (ART) had detectable pH1N1 virus RNA for 16 days, much longer than the median duration for the study population. This is slightly longer than was shown in a study of HIV-positive school-aged children carried out in Germany in which the median time from symptoms onset to first negative rRT-PCR result was 9 days (Range, 5–14 days) and cultures become negative after 6 days (Range 3–11 days)
PCR, which detects viral nucleic acid instead of infectious viral particles, is more sensitive than virus culture in detecting influenza
Our study had some limitations. First, our study population was mainly comprised of persons <14 years, and the oldest patient was 41 years old. Thus, we were not able to evaluate viral shedding patterns in elderly individuals. Second, since few people in our study had known underlying medical conditions, we were not able to determine the impact of co-morbidities, including HIV infection, on duration of pH1N1 viral shedding. Some patients whose HIV status was unknown may have been HIV-positive; this could explain the slightly longer duration of rRT-PCR positive pH1N1 results in our study compared to other studies. Third, there was an overlap between the patients who presented with ILI and SARI, thus it was difficult to analyze data for each of the syndromes separately. Finally, 20% of patients enrolled in the study did not fully complete the study. Therefore, we have little information on these patients regarding shedding. We used right-censoring in our analysis for these patients. These results were comparable to those obtained from the 85 patients who completed the study.
In this study, we show that pH1N1 shedding patterns in an impoverished, densely populated urban community in Nairobi, Kenya, are similar to those described in studies in more affluent countries in temperate and subtropical areas of the world. Because of unique co-morbidities in sub-Saharan Africa compared to other areas of the world
The authors wish to express gratitude to Leonard Cosmas Otieno, Regina Waithaka, Fredrick Nindo, Dennis Odhiambo, Beatrice Wambui, Irene Omwenga, and Erin Russell for their excellent technical support.