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Citation: Ajobiewe OJ et al. Level of Correlation of Opportunistic Mycobacterium Spp. Infection with Western Blot Confirmed
Positive HIV-1 Infection in GOPD and S.T.C. Patients Attending National Hospital Abuja F.C.T. Nigeria. Sch J App Med Sci, 2021
Sept 9(9): 1362-1366.
1362
Scholars Journal of Applied Medical Sciences
Abbreviated Key Title: Sch J App Med Sci
ISSN 2347-954X (Print) | ISSN 2320-6691 (Online)
Journal homepage: https://saspublishers.com
Level of Correlation of Opportunistic Mycobacterium Spp. Infection with
Western Blot Confirmed Positive HIV-1 Infection in GOPD and S.T.C.
Patients Attending National Hospital Abuja F.C.T. Nigeria
Ajobiewe JO.1*, Yashim NA.1, Sidi II1, Ajobiewe HF.2, Alexander P.2, Umeji L.3, Ogundeji AA.4, Dangana A.5,
Oguji C.5
1National Hospital Abuja, Plot 132 Garki Central District, Nigeria
2Bingham University Kodape Km 5 Nasarawa State of Nigeria
3Defence Reference Laboratory, Asokoro, Abuja, Nigeria
4United State Department of Defence, Walter Reed Program-Nigeria US Embassy Nigeria
5University of Abuja Teaching Hospital, Gwagwalada, Nigeria
DOI: 10.36347/sjams.2021.v09i09.009 | Received: 28.07.2021 | Accepted: 02.09.2021 | Published: 08.09.2021
*Corresponding author: Ajobiewe OJ
Abstract
Original Research Article
The sole aim of this study is to monitor the level of correlation between GOPD and STC Patients whose samples (sera
and sputa) were tested and confirmed with Western Blot, for HIV-1 and Ziehl Nielsen, Z N Stain/ Lowenstein Jensen,
L J slope culture for Sputa respectively. These samples were screened for HIV- 1 using three test principles-
Determine, Capillus and Genie. Positive samples were confirmed with the Western Blot assay. Sputa samples of the
same patients were collected and tested for Mycobacterium spp. infection using the Ziehl Nielsen staining technique
and the Lowenstein Jensen Slope cultural technique. The Spearman’s rank correlation coefficient was 0.466 (P<0.1)
showing a relatively high level of concordance between Western Blot confirmed positive HIV-1 infection and
Mycobacterium spp. infection in the same patients.
Keywords: Mycobacterium, Ziehl Nielsen Western Blot Lowenstein Jensen Slope.
Copyright © 2021 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (CC BY-NC 4.0) which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original
author and source are credited.
STUDY BACKGROUND
Mycobacterium spp. the etiologic agent of
Mycobacterium tuberculosis disease has been found
associated with HIV-1 infection in several studies along
this line of reasoning. Critical assessment of this sort of
association is of utmost necessity in this context---as a
sort of swift clinical intervention for Koch’s disease [1]:
Tuberculosis was popularly known as
consumption for a long time. Scientist knows it as an
infection caused by M. tuberculosis in 1882, the
microbiologist Robert Koch1: discovered the tubercle
bacillus, at a time when one of every seven deaths in
Europe was caused by TB. Because antibiotics were
unknown, the only means of controlling the spread of
infection was to isolate patients in private sanitoria or
hospitals limited to patients with TB—- [2] a practice
that continues to this day in many countries including
Nigeria. The net effect of this pattern of treatment was
to separate the study of tuberculosis from mainstream
medicine. Entire organizations were setup to study not
only the disease as it affected individual patients, but its
impact on the society as a whole. At the turn of the
twentieth century morethan 80% of the population in
the United States were infected before age 20, and
tuberculosis was the single most common cause of
death. By 1938 there were more than 700 TB hospitals
in some countries [3].
Mycobacterium tuberculosis which might be
opportunistic in HIV-1 infection [1]. Screening for
HIV- 1 could be done using three test principles-
Determine, [4] Capillus [5] and Genie [6]. Positive
samples were confirmed with the Western Blot assay.
Sputa samples of the same patients were collected and
tested for Mycobacterium spp. infection using the Ziehl
Nielsen staining technique and the Lowenstein Jensen
Slope cultural technique. And the Lowenstein Jensen
Slope cultural technique [7].
Control Samples: (HIV-1 Assay) Control sera-
both positive and negative controls as well as the
internal controls were supplied by the kit manufacturers
and were applied and used as they instructed.
Microbiology
Ajobiewe OJ et al; Sch J App Med Sci, Sept, 2021; 9(9): 1362-1366
© 2021 Scholars Journal of Applied Medical Sciences | Published by SAS Publishers, India
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Control Samples: (for Mycobacterium spp.
Assay) Negative controls were obtained from known
and pooled negative sputa of Patients while positive
controls were obtained from known and pooled positive
sputa of patients-using the ZN/L J Mycobacterium spp.
Assays [7].
TEST PRINCIPLES (HlV-1 SCREENING ASSAYS)
Determine: Determine HIV-1/2 is an immune
chromatographic test for the qualitative detection of
antibodies to HIV-1 and HIV-2. Sample is added to the
sample pad. As the sample migrates through the
conjugate pad, it reconstitutes and mixes with the
selenium colloid-antigen conjugate. This mixture
continues to migrate through the solid phase to the
immobilized recombinant antigens and synthetic
peptides at the patient’s window site.
If antibodies to HIV-1 and / or HIV-2 are
present in the sample, the antibodies bind to the antigen
— selenium colloid at the patient window, forming a
red line at the patient’s site.
If antibodies to HIV-l and / or HIV-2 are
absent, the antigen selenium colloid flows past the
patient window and no red line are formed at the
patient’s window site. To insure assay validity, a
procedural control bar is incorporated in the assay
device.
MATERIALS AND METHODS
Mycobacterium spp. infection may be
associated with HIV-1 infection. Critical assessment of
this sort of association is of utmost necessity in this
context---as a sort of swift clinical intervention for
Koch’s disease: which might be opportunistic in HIV-1
infection. A total of 211 Patients had their blood
samples prospectively collected between Januarys to
October, 2003: while a total of 443 Patients had their
blood samples similarly collected between Januarys to
October 2004. These samples were screened for HIV- 1
using three test principles- Determine, Capillus and
Genie. Positive samples were confirmed with the
Western Blot assay. Sputa samples of the same patients
were collected and tested for Mycobacterium spp.
infection using the Ziehl Nielsen staining technique and
the Lowenstein Jensen Slope cultural technique. A total
of 211 Patients had their blood samples prospectively
collected between Januarys to October, 2003: while a
total of 443 Patients had their blood samples similarly
collected between Januarys to October 2004. These
samples were screened for HIV- 1 using three test
principles- Determine, Capillus and Genie. Positive
samples were confirmed with the Western Blot assay.
Sputa samples of the same patients were collected and
tested for Mycobacterium spp. infection using the Ziehl
Nielsen staining technique and the Lowenstein Jensen
Slope cultural technique. The total duration for the
work was 20 months.
Control Samples: (HIV-1 Assay) Control sera-
both positive and negative controls as well as the
internal controls were supplied by the kit manufacturers
and were applied and used as thy instructed.
Control Samples: (for Mycobacterium spp.
Assay), Negative controls were obtained from known
and pooled negative sputa of Patients while positive
controls were obtained from known and pooled positive
sputa of patients-using the ZN/L J Mycobacterium spp.
assays
TEST PRINCIPLES (HlV-1 SCREENING ASSAYS)
Determine: Determine HIV-1/2 is an immune
chromatographic test for the qualitative detection of
antibodies to HIV-1 and HIV-2. Sample is added to the
sample pad. As the sample migrates through the
conjugate pad, it reconstitutes and mixes with the
selenium colloid-antigen conjugate. This mixture
continues to migrate through the solid phase to the
immobilized recombinant antigens and synthetic
peptides at the patient’s window site.
If antibodies to HIV-1 and/or HIV-2 are
present in the sample, the antibodies bind to the antigen
— selenium colloid at the patient window, forming a
red line at the patient’s site.
If antibodies to HIV-l and/or HIV-2 are absent,
the antigen selenium colloid flows past the patient
window and no red line are formed at the patient’s
window site. To insure assay validity, a procedural
control bar is incorporated in the assay device
Cappillus Assay Principles
The majority antigens from the envelope
proteins of HIV-1 and HIV-2 have been identified and
cloned using recombinant DNA technology. These
HIV-1 and HIV-2 proteins have been expressed and
purified. The Trinity Biotech Capillus, HIV- I and HIV-
2 employs these two proteins bound to polystyrene latex
beads to form the basis of a direct latex aggregation
assay for the detection of antibodies to HIV-I and HIV-
2 in the human serum, whole blood and plasma. The
assay is performed on a patented capillary slide.
Genie Assay Principle
The (Gennie II HIV-1/HIV-2 is a dual
recognition enzyme — immunoassay (EIA), based upon
the specific detection of antibodies HIV-1 and HIV-2
antibodies by antigens that bind both antibody binding
sites. The test incorporates a combination of immune —
chromatography and immunoconcentration. The
reaction device contains two ports, a circular specimen
Port A for the addition of specimen: and a larger
elliptical reaction Port B. Antigens derived from HIV-I
and HIV-2 are immobilized in two separate spots on the
reaction zone of port B. A third spot serves as the
internal Control for monitoring the performance of the
test.
Ajobiewe OJ et al; Sch J App Med Sci, Sept, 2021; 9(9): 1362-1366
© 2021 Scholars Journal of Applied Medical Sciences | Published by SAS Publishers, India
1364
The procedure followed was as given by BlO-
RAD 3 Boulevard Raymond Poincare: 92430
MARNES COQUETTE-FRANCE [6].
Western blot confirmation assay
(New-Lav Blot-1) [8] Principle: The test is
based on indirect ELISA technique on a nitrocellulose
strip containing all the HIV — I constituent proteins
and an internal anti IgG control. The band
corresponding to the internal control is localized on the
strip end without any number, before the P-18 reaction
and allows validating the addition of the sample and
reagents as well as the correct progress of the
procedure. Inactivated HIV- 1 proteins are separated
according to their molecular weights by polyacrylamide
gel electrically transferred onto a nitrocellulose
membrane sheet.
Reagents are first stabilized at room
temperature for 30 mm. Strip is dehydrated by adding
2m1 of the reconstituted buffer solution into each cell
for 5mm. 2Oul of each sample or control serum is
added to the corresponding cell for 2hrs incubation and
continuous slow shaking. The contents of each cell are
completely drained using a vacuum pump. Each strip is
washed 3times with the reconstituted buffer according
to man.inst.2m1 of conjugate is dispensed into each cell
and then incubated for 1hr. at room temperature. They
were washed as stated above. 2m1 pf color
development solution was dispensed into each cell for
5mm incubation at room temperature under slow
shaking. The reaction is stopped by removing the
development solution and rinsing the strips three times
with distilled water. Strips are dried between 2 sheets of
absorbent paper at room temperature. The bands on the
trips are then validated and interpreted according to the
manufacturer’s instruction.
Negative Control Strip
Ziehl- Nielsen staining Procedure [7]. The
reagents used were Carbol Fuchin: a saturated solution
of basic fuchin( 3g of basic fuchin in 100ml of 95%
ethyl alcohol),5% aqueous solution of phenol,3% acid
alcohol, 0.3% aqueous; the detailed procedure followed
was-a smear (1cm x 2cm) was prepared on a labeled,
grease-free slide. The slide was placed on a hot plate at
85°C for 15 mins. in a p.3 safety cabinet. The smear
was flooded with Carbol fusion and was allowed to
stand for 15 mines at room temperature. It was steamed
gently with flame from the underside for some minutes
(* but it wasn’t allowed to boil).The stain was allowed
to stand for 5min. on the slide and then washed with
distilled water and tilted to drain. The stained slide was
completely decolorized with 3% acid alcohol. The slide
was rinsed with distilled water and tilted to drain. The
slide was flooded with methylene blue for one minute.
The slide was rinsed and allowed to air dry. The slide
was then examined under X100 oil immersion lens.
For clarity and future safety of those using this Journal, the procedure would be clearly outlined
Ajobiewe OJ et al; Sch J App Med Sci, Sept, 2021; 9(9): 1362-1366
© 2021 Scholars Journal of Applied Medical Sciences | Published by SAS Publishers, India
1365
Ajobiewe OJ et al; Sch J App Med Sci, Sept, 2021; 9(9): 1362-1366
© 2021 Scholars Journal of Applied Medical Sciences | Published by SAS Publishers, India
1366
Rs = 0.466 (P<O.1)
The Spearman’s rank correlation coefficient
was 0.466(P<0.1) showing a relatively high level of
concordance between Western Blot confirmed positive
HIV-1 infection and Mycobacterium spp. infection in
the same patients.
CONCLUSION
Mycobacterium spp. which may be reliably
tested in Sputa samples of HI V-I positive patients
using the above techniques-appears to be a strong
indicator of opportunistic infection in HIV- I infected
patients. It is suggested that early diagnosis of this, shall
boost
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Medicine @ 2002 by McGraw Hill
2. Ushananthini, A. S. (2012). Assessment of DNA
Damage in Pulmonary Tuberculosis Patients by
Single Cell Gel Electrophoresis/Comet
Assay (Doctoral dissertation, Swamy
Vivekanandha College of Pharmacy,
Tiruchengode).
3. Koch, R. (1881). "Zur Ätiologie des Milzbrandes"
On the etiology of anthrax]. Mittheilungen aus dem
Kaiserlichen Gesundheitsamte. (2010). Berlin:
Robert Koch-Institut, 174–206
4. Abott, L. (2000). ABOTT Laboratories Diagnostic
Division SEPT.2000, 100, Abott Park IL60064
USA
5. Trinity Biotech; Trinity Biotech 2005 www.Trinity
biotech.com
6. Bio, R. (2000). Biorad 3 Boulevard Raymond
Pointcare 92430 MARNES COQUETTE
FRANCE.
7. Monica, C. (2001). Regional or
Central Microbiology Laboratory for pathogen
identification. The 3rd edition, 2001; Principles of.
Medicine in Africa.3. 462 pages
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8. Mahmood, T., & Yang, P. C. (2012). Western blot:
technique, theory, and trouble shooting. North American journal of medical sciences, 4(9), 429.