CDC Critique of Sapi et al Blood Culture for Borrelia in Human Blood:Many Mistakes by the CDC critique methods and False Conclusions

CDC Critique of Sapi et al Blood Culture for Borrelia in Human Blood:Many Mistakes by the CDC
critique methods and False Conclusions:
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CDC Analysis of the DNA sequencing Data for the Advanced Labs Blood culture isolates:
by Dr. Barbara Johnson et al.
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An Official CDC critique and an Official CDC accusation of alleged Flawed Advanced Laboratory techniques
resulting in Only 3 genovars instead of 60 + Genovars of Borrelia as reported by Sapi
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The CDC Analysis is flawed for the reasons posted below and the CDC manuscript must be 
withdrawm
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The Reader of the CDC manuscript will infer that The CDC had in their possession
aliquots of each of the isolates from Advanced Labs
If this is NOT the case.

The CDC may be required to issue a Retraction of theirmanuscript
to clarify this point for the reader and to address the
BLASTn data which follows for a single isolate .
Advanced Laboratories and the CDC agreed had 21 nucleotide mismatches with B31 were present
in the exemplary Isolate. [Isolate JX867376}


CDC analysis of isolate JX86 7376 detected 21 base mismatches with respect to B31
and the Sapi Paper also reported 21 base mismatches with respect to B31 [97% identity with B31]
I did a BLASTn search on isolate JX867376 against all borrelia.

A 100% match was obtained with JX867376 itself as deposited in GenbBank.This is expected
and this 100% match indicates that the 603 bases were correctly entered into the NCBI BLASTn
Search Engine.
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B31 borrelia complete DNA sequence produced a result with 21 mismatches against Sapi deposit JX867376 for a congruence percentage of 97%
It is interesting that the BLASTn search also revealed 97% matches for the additional Borrelia on Deposit in GenGank:
N40, CA382 ZS7 and multiple numbered haplotypes of Borrelia spp.
A 96% match was noted for multiple genotypes of B. garinii and B. Bavariensis.

So if the truth be told, based on a BLASTn search of all Borrelia in GenBank, it
is very very misleading to state that isolate JX867376 was UNIQUE for B31 
as is posted at position 49 in the CDC Table.....AND .....
and to omit any mention of the following borrelia genotypes
which also yielded 97% matches with JX867376 according to BLASTn search.
ST12
N40
ST8
CA382
ST4
ST51
ST56
St1
ZS7
Each and every one of the above genotypes should have been posted at postion 49 in the CDC table.
To post only B31 is very misleading to the reader who does not use the BLASTn tool
for sequence analysis and borrelia genotyping.
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Correction to the CDC Table of Results for (97%) [21 base mismatches against reference strain borrelia burgdorferi B31] against Advanced Labs Isolate JX867376.

" This Advanced labs isolate may indicate ANY of the following Borrelia species: 
B31, N40,ZS7,ST! ST4, ST8ST12,,ST51,ST56, CA382,
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Mismatches of Bases by BLASTn analysis should now be initiated for Each and Every Advanced Lab nucleotide sequences
and the original manuscript should be withdrawn and re-written
to accommodate BLASTn search data for each of the Advanced Lab PyrG nucleotide sequences.

I look forward to CDCreconciliation of BLASTn data with the observed Advanced Lab dep[osited nucleotide Sequences
with the hope that the issue of Contamination of Patient Recovered Borrelia from blood cultures
may be viewed in the full context of BLASTn search Data to embrace all possible borrelia species with
closely matching nucleotide sequences.

The maximum number of polymorphic sites in CDC analysis of the Sapi Data was 21.
CDC speciated only 3 strains of borrelia in Their analysis :
B31 burgdorferi [N=22], 
B023 afelii [N=2}, and 
Fuji P1 garinii [ n=27]
These assignments conflict with the Sapi paper as follows:
In the Sapi manuscript Table 3 :
Sapi et al reported 
5 isolates of Garinii - [ no identical garinii genotypes noted by Sapi }
1 isolate of afzelii Pko [consistent with European type afzelii]
3 kurtenbachii [ all genotypically distinct]
1 americanum and
6 burgdorferi [B31=1] , [ZS7=1} , [ N40=1], { ST1 =1], { ST11 =1}, { St8=1}.

For the remaining: "JX86***** series isolates I will consult the GenBank accessions
as I have for isolate JX867376....
and perform my own Clustal analysis. of nucleotide Base alignments

CDC group registered the following genovars [ your results in parentheses]
among the JX86**** series:
7424 51 polymorphic sites [Called Afzelii B023 by CDC group]
7398 49 polymorphic sites [called Afzelii B023 by CDC group]
7394 43 polymorphic sites { Not classified by CDC group}
7417 42 polymorphic sites [Called Garinii Fuji P1 by CDC group}
7380 42 polymorphic sites {called Garinii Fuji P1 by CDC group}
7378 42 polymorphic sites { Called Garinii Fuji P1 by CDC group}
7379 42 polymorphic sites Called Garinii Fuji P1 by CDC group}

Twenty one isolates with 41 polymorphic sites EACH [ isolates not collated here]
7393 39 polymorphic sites [Called garinii Fuji P1 by CDC group}
7376 21 polymorphic sites [[See above - called B31 burgdorferi by CDC group}}
7419 2 polymorphic sites [ Called burgdorferi B31 by CDC group}

CDC also indicate that CDCS equence analysis yielded
27 Identical isolates of Garinii 
2 i dentical isolates of Afzelii. 
22 isolates of B31 { with only 2 cases #48 and 49 called B31 burgdorferi with "mismatched" bases : #48 n=1 miusmatch and #49 with 21 mismatches]
So instead of 60+ unique Borrelia genovars, the CDC reduced the Sapi/Advanced Labs results to just 3 genovars
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I insist the the genotyping of the Advanced Labs GenBank deposits
speaks for heterogeneity of genovars, and reinforces the concept that So called 
European type borrelia may very well be now recognized as vectored by USA Ixodid ticks.
Mexico, Central America and Brazil are geographies in which GARINII has been proven
to be vectored by ticks. To make matters more complex, the Amblyomma family of ticks (Amblyomma cajenesse)
are competent vectors for Garinii infections in the Western Hemisphere.
We need not look to Sea bird migrations to explain USA cases of Ixodid scapulairs
and Amblyomma spp. vectored human infections.

The uniqueness of the Sapi report of Advanced Labs Human Blood culture Borrelia strains
is therefore CORRECT.
No Laboratory Contamination of patient material with any living borrelia ever occurred
European Strains of borrelia were recovered from the blood of USA patients in the
Advanced Labs study of Borrelia blood cultures

Respectfully submitted,
Alan B. MacDonald MD FCAP FASCP
August 16,2013
http://www.lymeneteurope.org/forum/viewtopic.php?f=6&t=4545&start=40#p36709