Malaria diagnosis among HIV-positive patients is uncommon in Nigeria despite the high burden of both diseases.
We evaluated the performance of a malaria rapid diagnostic test (MRDT) against blood smear microscopy (BSM) among HIV-positive patients in relation to anti-retroviral treatment (ART) status, CD4+ count, fever, cotrimoxazole prophylaxis and malaria density count.
A cross-sectional study involving 1521 consenting randomly selected HIV-positive adults attending two ART clinics in Kano, Nigeria, between June 2015 and May 2016. Venous blood samples were collected for testing with MRDT, BSM, and CD4+ T cells count by cytometry. Biodata and other clinical details were extracted from patient folders into an Excel file, cleaned, validated, and exported for analysis into SPSS version 23.0. Sensitivity, specificity, predictive values of MRDT were compared with BSM with a 95% confidence interval.
Malaria parasites were detected in 25.4% of enrollees by BSM and 16.4% by MRDT. Overall sensitivity of MRDT was 58% and specificity was 97%. Cotrimoxazole prophylaxis and fever status did not affect MRDT sensitivity and specificity. Unexpectedly, the sensitivity was highest at parasite density count of less than 500 cells/µL. At CD4+ T cells count over 500 cells/µL the sensitivity was higher (62.4%) compared to 56% at less than 500 cells/µL. In the non-ART group sensitivity was higher (65%) compared to those on ART (56%) but the specificity was similar. All differences were significant for all variables (
Although the MRDT specificity was good, the sensitivity was poor, requiring further evaluation for use in malaria diagnosis among HIV-malaria co-infected persons in these settings.
Malaria remains a major public health problem in Nigeria with 97% of the population at risk.
A key factor for effective management of malaria is early and accurate diagnosis. The global impact of malaria has prompted an increase in the development of diagnostic strategies. The updated World Health Organization (WHO) recommendations indicate that all suspected cases of malaria must be confirmed with a laboratory diagnostic test before treatment.
Although microscopy is the current gold standard for the diagnosis of malaria, it is time consuming and labour intensive.
Data from reviewed literature showed that very few studies have evaluated the validity of RDT for malaria diagnosis in the general population in Nigeria.
The objective of the study was to evaluate the performance of a commercially available MRDT (based on
The protocol was reviewed by Kano State Hospitals Management Board local ethical committee and written approval was given (study approval number: HMB/GEN/488/1 of 17/04/2015). Participant consent was obtained from all respondents.
This was a cross-sectional study carried out from June 2015 to May 2016 at two health facilities in Kano City, Nigeria, a malaria endemic area.
The population of interest in this study were HIV-positive adult participants who routinely visit the two health facilities to access ART services. They consisted of male and female (non-pregnant) patients (on ART and non-ART) aged 18 years and older enrolled in an ART programme who had not been on an antimalarial drug in the past 14 days and who willfully consented to participate in the study.
A total of 1521 consenting participants were randomly selected and blood specimens were collected and analysed for the presence of malaria parasite using both RDT and microscopy techniques and density of malaria parasites was determined. Additionally, relevant socio-demographic and clinical data including clients’ code, age, gender, use of co-trimoxazole prophylaxis, ART status (receiving or not receiving treatment), and episodes of fever were retrieved from both patients’ folders and Lafiya Management Information System (LAMIS®) software with the support of trained data entry clerks. We ensured data confidentiality through participant coding and restricting access to the data to only research team members.
From 1521 consenting HIV-positive participants, 4 mL venous blood sample was aseptically collected using sterile vacutainer needle and holder into an ethylenediaminetetraacetic acid (EDTA) anticoagulant tube. The blood samples were mixed properly to avoid clotting before laboratory investigations.
We used the MRDT kit (Code 05FK30) as described by the manufacturers (Standard Diagnostics Bioline Korea, 2013).
For microscopy slide method, both thin and thick smear slides were prepared and examined. The microscopy procedure adopted was described by WHO.
Malaria parasite density for each positive smear was calculated using individual white blood cell count according to this formula:
Data were reviewed, cleaned and validated in a Microsoft 2013 Excel file (Microsoft Corp., Redmond, Washington, United States) and analysed using Statistical Package for Social Scientists (SPSS version 23.0; IBM, Armonk, New York, United States). The sensitivity, specificity, positive predictive value and negative predictive value of MRDT were estimated against blood smear microscopy (BSM), the gold standard, at 95% confidence interval in relation to (1) malaria parasite density count, (2) fever (presence or absence), (3) CD4+ T cells count, (4) co-trimoxazole prophylaxis use, and (5) ART status (currently on ART or not taking ART). In addition, differences in performance of MRDT against BSM were analysed (overall and for sub-groups of the five variables mentioned above) based on level of ‘equality of marginal positives’ in disease classification between the two using McNemar tests and significance level was set at
This study included a total of 1521 HIV-positive participants consisting of 1074 (70.6%) women and 447 (29.4%) men. The mean age was 37.20 with a standard deviation (SD) of 10.41 years.
Data on clinical information showed that the majority (84.7%) were on ART. An almost equal proportion of the participants (
The prevalence of malaria was 25.4% with BSM technique (gold standard method) and only 16.4% with MRDT. It is worth nothing that participants were recruited at routine ART follow-up visits, with or without symptoms of malaria. Almost all (99.2%) malaria infections were
Prevalence of malaria parasite among HIV-positive patients determined by malaria rapid diagnosis test and standard blood smear microscopy.
Variables | Malaria positive |
Percentage | Malaria negative |
Percentage |
---|---|---|---|---|
RDT MP parasite species |
250 | 16.4 | 1271 | 83.7 |
Prevalence of malaria & HIV | 386 | 25.4 | 1135 | 74.6 |
BSM MP parasite specie |
383 | 25.2 | – | - |
BSM MP parasite species |
3 | 0.2 | – | - |
Overall |
383 | 99.2 | – | - |
Mixed infection prevalence | 3 | 0.8 | – | - |
BSM, blood smear microscopy significant difference; MP, Malaria parasites;
Overall performance of malaria rapid diagnosis test in comparison with standard blood smear microscopy among HIV positive participants.
RDT result | Standard BSM |
||||||||
---|---|---|---|---|---|---|---|---|---|
Positive | Negative | Sensitivity | Specificity | PPV | NPV | LR +ve | LR -ve | McNemar test (P value) | |
Positive | 222 | 28 | 57.5 (52.41–62.50) | 97.3 (96.45–98.35) | 88.8 (84.48–92.03) | 87.1 (85.73–88.35) | 21.29 | 0.44 | < 0.001 |
Negative | 164 | 1107 | - | - | - | - | - | - | - |
HIV, human immunodeficiency virus; BSM, blood smear microscopy; LR, likelihood ratio; NPV, negative predictive value; PPV, predictive positive value, RDT, rapid diagnostic test.
Overall, there was significant difference in MRDT performance compared to BSM (
The MRDT showed a sensitivity of about 72% among HIV-positive patients with malaria density count between 0 and 499 cells/µL and greater than 1000 cells/µL. Unexpectedly, a low sensitivity of about 34% was observed when density was between 500 and 999 cells/µL (
Performance of malaria rapid diagnostic test in comparison with standard blood smear microscopy at different malaria parasite density, fever status, cotrimoxazole prophalaxis,CD4+ count and ART status
RDT result | Standard BSM |
||||||
---|---|---|---|---|---|---|---|
Positive | Negative | Sensitivity (95% CI) | Specificity (95% CI) | PPV (95% CI) | NPV (95% CI) | McNemar test ( |
|
Positive | 145 | 28 | 72.5 (65.76–78.56) | 97.5 (96.45–98.35) | 83.8 (78.06–88.29) | 95.3 (94.14–96.18) | 0.004 |
Negative | 55 | 1107 | - | - | - | - | - |
- | - | - | - | - | |||
Positive | 50 | 0 | 33.8 | NA | 100.0 | NA | < 0.001 |
Negative | 98 | 0 | - | - | - | - | - |
- | - | - | - | ||||
Positive | 27 | 0 | 71.1 | NA | 100.0 | NA | < 0.001 |
Negative | 11 | 0 | - | - | - | - | - |
- | - | - | - | - | |||
Positive | 23 | 1 | 54.8 (39.72–69.84) | 98.4 (95.31–99.99) | 95.8 (87.83–99.97) | 76.5 (67.34–85.81) | < 0.001 |
Negative | 19 | 62 | - | - | - | - | - |
- | - | - | - | - | |||
Positive | 199 | 27 | 57.8 (52.63–63.14) | 97.5 (96.53–98.42) | 88.1 (83.80–92.32) | 87.8 (86.04–89.72) | < 0.001 |
Negative | 145 | 1045 | - | - | - | - | - |
- | - | - | - | - | |||
Positive | 97 | 18 | 57.7 (49.89–65.31) | 97.0 (95.33–98.22) | 84.3 (77.05–89.64) | 89.2 (87.36–90.79) | < 0.001 |
Negative | 71 | 586 | - | - | - | - | - |
- | - | - | - | - | |||
Positive | 125 | 10 | 57.3 (50.48–63.99) | 98.1 (96.56–99.09) | 92.6 (87.00–95.89) | 84.9 (82.76–86.73) | < 0.001 |
Negative | 93 | 521 | - | - | - | - | - |
- | - | - | - | - | |||
Positive | 164 | 22 | 56.0 (50.08–61.74) | 97.0 (95.56–98.14) | 88.2 (82.99–91.93) | 84.9 (83.12–86.45) | < 0.001 |
Negative | 129 | 723 | - | - | - | - | - |
- | - | - | - | - | |||
Positive | 58 | 6 | 62.4 (51.72–72.21) | 98.5 (96.68–99.43) | 90.6 (81.14–95.60) | 91.6 (89.41–93.45) | < 0.001 |
Negative | 35 | 384 | - | - | - | - | - |
- | - | - | - | - | |||
Positive | 178 | 27 | 56.0 (50.33–61.51) | 97.2 (95.98–98.16) | 86.8 (81.78–90.64) | 87.1 (85.61–88.41) | < 0.001 |
Negative | 140 | 943 | - | - | - | - | - |
- | - | - | - | - | |||
Positive | 44 | 1 | 64.7 (52.17–75.92) | 99.4 (96.67–99.98) | 97.8 (86.08–99.68) | 87.2 (83.20–90.41) | < 0.001 |
Negative | 24 | 164 | - | - | - | - | - |
- | - | - | - | - |
BSM, blood smear microscopy; CI, confidence interval; CTX, cotrimoxazole; RTD, rapid diagnostic test; ART, antiretroviral therapy; NA, Not available; LR, likelihood ratio; NPV, negative predictive value; PPV, predictive positive value.
MRDT performance was significantly different from BSM findings, regardless of malaria parasite density count of participants (
The MRDT showed slightly lower but similar sensitivity in cases with and without fever, but similar specificity for both groups of participants. However, positive predictive value was higher (95.6%) among clients that complained of fever compared to those without (88.1%) fever (
MRDT performed similarly in sensitivity and specificity in both groups, although higher positive predictive value was observed among participants who were not on co-trimoxazole (
MRDT revealed higher (sensitivity 62%, specificity 99%, positive predictive value 91% and negative predictive value 91%) diagnostic accuracy among participants with CD4+T cells count greater than 500 cells/µL. This was lower (sensitivity 56%, specificity 97%, positive predictive value 88% and negative predictive value 85%) among participants with CD4+T cells count under 500 cells/µL (
The MRDT revealed a higher (65%) sensitivity among HIV participants who were not on ART compared (56%) to those on ART. A similar pattern of higher positive predictive value (98%) was observed among the non-ART group compared to 87% among the ART group (
We detected malaria parasites in 25.4% of participants by BSM and 16.4% by MRDT. Overall sensitivity of MRDT was 58% and specificity was 97%. The sensitivity and specificity were similar irrespective of co-trimoxazole and fever status. At malaria parasite density count of under 500 cells/µL, sensitivity was 73% and between 500 and 999 cells/µL, sensitivity was 34%. At CD4+ T cell count over 500 cells/µL the sensitivity was higher (62.4%) compared to 56% at under 500 cells/µL. In the non-ART group sensitivity was higher (65%) compared to those on ART (56%) but the specificity was similar.
Although blood smear microcopy still remains the gold standard for malaria diagnosis, several limitations ranging from infrastructure and equipment to competence still exist in endemic areas like Nigeria. These justify the need for an alternate method like MRDT.
The prevalence of malaria in HIV-positive patients in this study was 25.4% based on microscopy compared to 16.4% based on MRDT method. This was similar to a Tanzanian study reporting a malaria prevalence of 23.8% using microscopy method among HIV-positive patients compared to 17.5% with MRTD
MRDT overall sensitivity and specificity reported in this study was similar to a 55.4% sensitivity reported in Lagos, Nigeria, but varied in specificity (90.3%).
The 2.5% false positive results observed with the use of MRDT may be an indicator of residual antigenemia. The average time that histidine rich protein-2 (pFHRP-2) remains positive after resolution of parasitaemia is about 2 weeks, although it has been shown that the protein can take over a month to clear.
The 27.55% false negative results observed might be partly due to deletion or mutation of HRP2 gene in the malaria parasite, which is the most common target for MRDT.
Unexpectedly, with the MRDT, we observed a lower sensitivity of 34% among HIV-positive participants with a malaria density count between 500 cells/µL and 999 cells/µL compared to a 73% sensitivity when the malaria density count was less than 500 cells/µL. However, the specificity at between 500 cells/µL and 999 cells/µL was 97.5%. This was contrary to a previous study in Lagos, Nigeria, that observed an increase in sensitivity from 90.9% at parasite densities greater than 200 cells/µL to 97.6% at densities greater than 500 cells/µL.
Besides this study, and to the best of our knowledge, only one other study has assessed performance of MRDT among HIV-positive patients based on febrile status in Africa.
MRDT performance was similar in terms of sensitivity, specificity and positive predictive value regardless of whether a participant was on co-trimoxazole prophylaxis or not. Information on similar studies is not available; however, with microscopy a relatively higher prevalence has been reported on those without co-trimoxazole prophylaxis compared with those on it.
MRDT performance revealed a higher sensitivity among participants with a CD4+ T cell count greater than 500 cells/µL compared to a lower sensitivity among participants with a CD4+ T cell count under 500 cells/µL. Although information on similar studies is not available, it has been reported in a similar setting that HIV-positive participants with CD4+ T cell counts 250 cells/µL or higher are significantly less likely to have patent parasitaemia.
The MRDT revealed a higher sensitivity among HIV-positive participants who were non-ART compared to those on ART. Lower incidence of malaria among HIV-positive adults on ART have been reported in different settings
Based on other reports in the general population, the use of MRDT method as an alternative to BSM in malaria endemic areas is recommended for epidemiological studies. However, the performance varied depending on species of the malaria parasite, level of parasitaemia, and immunity.
This study was sufficiently powered having a large sample size of 1521 HIV-positive participants across the two health facilities. Apparently, this may be the first study to assess the performance of MRDT in an African setting of significantly high HIV and malaria co-morbidity in association with variables like immune status, parasitaemia and some clinical (co-trimoxazole prophylaxis, fever and ART status) factors.
The cross-sectional design of this study was a limitation as it was difficult to determine how the potential antimalarial effect of co-trimoxazole prophylaxis and ART could have affected MRDT performance over time. Note that we observed better diagnostic accuracy of MRDT among participants who were neither on co-trimoxazole prophylaxis nor on ART. Secondly, we did not measure individual body temperature to correlate it with participants’ reports of fever.
As observed in this study, the sensitivity of SD Bioline, which is based on
It is worth speculating that a similar observation with the MRDT could have been observed in non–HIV-positive persons.
The data reported in this current article reflect findings of work done on evaluation of standard diagnostic Bioline rapid test kits for malaria diagnosis among HIV patients in Kano, Nigeria, by our research team members who all participated in the study design, execution, collection, analysis of data and report writing.
The research outcomes presented in this report were from the diagnostic evaluation of standard diagnostic Bioline rapid test kits for malaria diagnosis among HIV-positive participants in Kano, Nigeria, at Infectious Diseases Hospital and Murtala Mohammed Specialist Hospital Kano. Although previous studies have reported on the evaluation of rapid malaria diagnosis against gold standard microscopy methods with a focus on the general population, it is worth noting that data among HIV-positive persons under different conditions are not readily available. Hence, this study sought to address paucity of data on performance evaluation of standard diagnostic Bioline among HIV-positive patients in relation to various factors such as fever status, CD4+ cell count, ART status, co-trimoxazole prophylaxis and malaria density.
The outcomes of this study and proposed recommendations could be useful in public health programme planning in such settings where HIV and malaria burden is high. Apart from disease prevalence data reported, malaria density count and performance of rapid test kits was also evaluated in addition to other clinical and useful variables. This may serve as a baseline upon which comparison may be made in future.
The authors wish to express their sincere appreciation to all the health care workers of both facilities especially the laboratory staffs for various support provided during the study and ensuring a conducive working atmosphere for success.
The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article and its content is solely the authors’ responsibility and views expressed do not necessarily represent the official views of the study facilities and any affiliated institution listed.
None.
H.A.M and T.I.O were the project leaders. F.E.J, T.I.O and S.A.A were responsible for the experimental and project design. F.E.J and S.A.A performed most of the experiments. H.A.M, F.E.J and T.I.O made conceptual contributions. F.E.J and S.A.A performed some of the experiments. F.E.J and T.I.O prepared the samples and data analysis. Data interpretation was performed by S.A.A, F.E.J, H.A.M and T.I.O. F.E.J and S.A.A drafted the manuscript. H.A.M and T.I.O reviewed the manuscript for scientific soundness. All authors reviewed and approved the final version of the manuscript.